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FOOD SCIENCE AND TECHNOLOGY
HANDBOOK OF SEAFOOD
QUALITY AND SAFETY MAINTENANCE

AND APPLICATIONS
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HANDBOOK OF SEAFOOD
QUALITY AND SAFETY MAINTENANCE

AND APPLICATIONS
SMAIL YKSEL GEN

EDUARDO ESTEVES
AND
ABDULLAH DILER
EDITORS
New York
Copyright 2016 by Nova Science Publishers, Inc.
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Library of Congress Cataloging-in-Publication Data
Names: Gen, smail Yksel, editor. | Esteves, Eduardo, editor. | Diler,

Abdullah, editor.
Title: Handbook of seafood : quality and safety maintenance and applications
/ smail Yksel Gen, Eduardo Esteves, and Abdullah Diler (Suleyman
Demirel University Fisheries Faculty, Fishing and Processing Technology
Department, pChunhur, Isparta, Turkey, and others), editors.
Description: Hauppauge, New York : Nova Science Publishers, Inc., [2016] |
Series: Food science and technology | Includes bibliographical references
and index.
Identifiers: LCCN 2016030869 (print) | LCCN 2016040679 (ebook) | ISBN
9781634858236 (hardcover) | ISBN 9781634858526
Subjects: LCSH: Seafood--Quality control--Handbooks, manuals, etc. | Fishery
processing--Quality control--Handbooks, manuals, etc.
Classification: LCC TX385 .H365 2016 (print) | LCC TX385 (ebook) | DDC
664/.94--dc23
LC record available at https://lccn.loc.gov/2016030869
Published by Nova Science Publishers, Inc. New York

CONTENTS
Preface vii
Chapter 1 General Introduction to Seafood Quality and Safety
Maintenance and Applications 1
Eduardo Esteves, Abdullah Diler and smail Yksel Gen
Chapter 2 Microbiology of Fish and Fish Products and Its Implications
on Public Health 13
Regine Helena Silva dos Fernandes Vieira,
Francisca Gleire Rodrigues de Menezes
and Oscarina Viana de Sousa
Chapter 3 Relating Sensory and Instrumental Analyses of Well-Known
and Emerging Fish and Seafood Products 31
Eduardo Esteves
Chapter 4 Measurement of Visual Attributes of Fresh and Processed Seafood 65
Murat O. Balaban and Zayde Ayvaz
Chapter 5 Proteomics and Application in the Seafood Industry 87
Jinru Zhou, Linglin Fu, Yan Zhang and Yanbo Wang
Chapter 6 Quality Changes in Freshwater Fish and Crustacean Species 99
Irineu Batista and Carla Pires
Chapter 7 Quality Changes in Crustaceans during and after Processing 127
Aygl Kkglmez, Mehtap Baykal, Ali Eslem Kadak
and Mehmet elik
Chapter 8 Trace Elements and Stable Isotopes Analysis as Seafood
Quality Indicators 139
Jaime Anbal and Cristina Veiga Pires
Chapter 9 Safety and Quality Issues in Global Fish Trade 151
Shalini Amnee Neeliah, Dayawatee Goburdhun
and Harris Neeliah
vi Contents
Chapter 10 Elimination and Control of Pathogens by Novel

and Hurdle Technologies 175
Alex Augusto Gonalves and Adriene Rosceli Menezes de Oliveira
Chapter 11 Packaging Technologies and Material Type for the Maintenance
of Seafood Safety 191
Ana Augusto, Maria Manuel Gil and Susana Filipa Jesus Silva
Chapter 12 Quantitative Risk Assessment in Seafood 209
Violeta Trinidad Pardo Sedas, Karla Mara Lpez Hernndez
and Argel Flores Primo
Chapter 13 Computer-Based Applications for Monitoring the Quality
and Safety of Seafood 223
smail Yksel Gen and Eduardo Esteves
Chapter 14 Rapid Detection of Foodborne Bacterial Pathogens in Seafood 247
Kitiya Vongkamjan, Siyun Wang and Andrea I. Moreno Switt
Chapter 15 Application of Natural Antimicrobials in Seafood 259
Celso Alves, Susete Pinteus, Rui Pedrosa and Maria Manuel Gil
Chapter 16 Biological Hazards and Natural Antimicrobials for
Seafood Preservation 275
Susete Pinteus, Celso Alves, Rui Pedrosa and Maria Manuel Gil
Chapter 17 HACCP Economics in Seafood Processing Plants 303
Aurora Zugarramurdi, Mara Amelia Parin
and Hctor Mateo Lupin
Chapter 18 Hygiene and Sanitation Applications in Seafood Industry 315
Abdullah Diler and Frantiek Vcha
Chapter 19 Basics of Seafood Quality Indices 333
Frantiek Vcha and Abdullah Diler
About the Editors 343
Index 345
PREFACE
During the last decade, the consumption of seafood and related products increased as they
are increasingly considered one of the recommended food items by health organizations due
to their nutritional composition. In this context, the need to assure the quality and safety of the
seafood and related products is equally important compared the other food products
categories (meat, dairy and poultry products).
Quality is considered the main parameter determining seafood acceptability by
consumers. Marine and freshwater systems are quite diverse both in terms of environmental
conditions as well as the number and diversity of aquatic organisms. Thus, the observable
changes in the quality characteristics of seafood processed and/or stored under various
conditions (temperature, processing and packaging procedures, additives and/or preservatives,
etc.) are also different. The quality assessment method is expectedly species- or group-
specific. The quality loss in seafood is initially caused by autolytic enzymes and later thru
microbial-induced changes. New and emerging methodologies, and hurdle and traditional
technologies to retain the quality as well as methodologies to assess the changes observed in
several seafood products under different conditions (chilled fresh, packaged under different
conditions, with added preservatives, etc.) are reviewed and discussed in chapters 2 to 11.
Moreover, the assurance of safety is increasingly more important as seafood may contain
several chemical compounds and biological agents, such as biotoxins, biogenic amines, heavy
metals, pathogenic bacteria, and virus that are reasonably likely to cause illness or injury in
the absence of control. Qualitative and quantitative detection methods, application areas and
risk assessment methodologies pertaining to fishery products are compiled and reviewed in
chapters 11 to 19.
This book, edited by 5 editors affiliated to institutions in 3 different countries, is expected
to constitute a reference manual for academia and industry. Contributors are experts in their
fields of research and are from more than 10 countries in 3 continents, thus providing
comprehensive perspectives on the subjects. The books detailed chapters on a varied set of
topics dealing with seafood quality and safety applications, with an intended focus on less-
known or undervalued species from different locales, constitute an excellent and notable
reference for university libraries, seafood processing technology departments, industry
assessors and professionals, and government inspectors.
Finally, the editors would like to thank Nova Science Publishers for the opportunity to
publish this handbook and evidently the contributors for their peerless work and patience.
This book is the outcome of their excellent work and self-devotion to this editorial project.
We wish them all the best in their academic and professional life.
In: Handbook of Seafood ISBN: 978-1-63485-823-6
Editors: . Yksel Gen, E. Esteves and A. Diler 2016 Nova Science Publishers, Inc.
Chapter 1
GENERAL INTRODUCTION TO SEAFOOD QUALITY

AND SAFETY MAINTENANCE AND APPLICATIONS
Eduardo Esteves1,*, Abdullah Diler2 and smail Yksel Gen2

1
Instituto Superior de Engenharia (ISE DEA),
Universidade do Algarve and CCMAR Centro de Cincias do Mar,
Faro, Portugal
2
Suleyman Demirel University Fisheries Faculty,
Fishing and Processing Technology Department, Isparta, Turkey
ABSTRACT
The combined world fishery and aquaculture production has been steadily rising
since the 1950s and reached 158 million tons in 2012. Just over 86% of this produtction
is used for direct human consumption. The world average consumption of seafood
products in 2013 was estimated at nearly 19 kg/capita/yr. A number of characteristics,
both ambient and species-specific make seafood a very perishable food product with a
limited shelf life. While sensory evaluation still remains the most satisfactory and
important method for freshness assessment in the fish sector, other forms of
determination and maintenance of quality and safety are gaining significance in terms of
productive utilization of aquatic sources.
Herein, a brief and introductory account is given on relevant issues in seafood
quality and safety, providing a framework and anticipating the more in-depth reviews
carried out in subsequent chapters in the handbook.
Keywords: seafood production and consumption, quality and safety
*
Corresponding author: E-mail: eesteves@ualg.pt.
2 Eduardo Esteves, Abdullah Diler and smail Yksel Gen
SEAFOOD PRODUCTION AND UTILIZATION

Fisheries and Aquaculture
In 2012, the combined world fishery and aquaculture production reached 158 million
tons. The production has been steadily rising since the 1950s when production was ca. 20-25
million tons [1]. In the latest top-18 ranking of producer countries (for 2012) listed by FAO
(2014), several countries, such as China (with ca. 14 million tons), Indonesia (5.4 million
tons), the USA (5.1 million tons), Peru (4.8 million tons), Russian Federation (4 million tons),
Japan (3.6 million tons), India (3.4 million tons), Chile (2.6 million tons), Viet Nam (2.4
million tons), Myanmar (2.3 million tons), the Philippines (2.1 million tons) or Norway (2.1
million tons) exceeded the 2 million tons/year and in total they represented about 76% of
world total.
Adapted from [2].
Figure 1. Graphs of catches by commercial (top) and functional (bottom) groups in the global ocean
plotted from reconstructed data that combine official reported data and reconstructed estimates of
unreported data (including major discards).
General Introduction to Seafood Quality and Safety Maintenance and Applications 3
The rising world catches are fairly diverse in terms of commercial and functional groups
[2] (Figure 1) but dominated by perch-like, herring-like, cod-like fishes, tunas and billfishes
and anchovies, and consisting mostly of pelagics, small and medium demersals and large
benthopelagics, respectively. Worldwide, fishery catches in oceans and seas represent ca.
90% of total catches [1].
In decreasing order, Anchoveta (Engraulis ringens) with 4.7 million tons, Alaskan polock
(Theragra chaclchogramma) with 3.3 million tons, skipjack tuna (Katsuwomus pelamis) with
2.8 million tons, Sardinellas spp. (2.3 million tons), Atlantic herring (Clupea harengus) with
1.8 million tons, chub mackerel (Scmober japonicas) with 1.6 million tons, Scads
(Decapterus spp.) and yellowfin tuna (Thunnus albacares) with 1.4 million tons each,
Japanese anchovy (Engaulis japonicas) with 13 million tons and largehead hairtail
(Trichiurus lepturus) with 1.2 million constitute the top-10 species most fished worldwide
[1]. On the other hand, aquaculture production already represents (in 2012) more than 40% of
worldwide seafood production with China (ca. 65%) and other Asian and Pacific countries
(26%) representing about 90% of total aquaculture production [1].
The remaining 10% is produced in Europe (ca. 4.5%), Latin America and the Caribbean
(about 3.5%), Africa (ca. 2%), North America (1%) and Near East (0.5%). World
aquaculture production continues to grow, albeit at a slowing rate since the 1950s [1]. Apart
from crustaceans (ca. 45%) and marine fishes (<5%), the production of all other groups of
fish and seafood products considered by FAO, namely aquatic plants, freshwater and
diadromous fishes, molluscs and other aquatic animals, comes primarily (>50%) from
aquaculture since 2008 [3].
Only 15 countries, mostly from Asia (China (ca. 62%), India, Viet Nam, Indonesia,
Bangladesh, Thailand, Myanmar, Philippines, Japan, Republic of Korea) but also from
Europe (Norway), the Americas (Chile, Brazil, USA) and Africa (Egypt), are responsible for
almost 93% of total aquculture production in the world [1].
Adapted from [3].
Figure 2. Percentage contribution of aquaculture to total production (between 1950 and 2008) per
selected, major species groups. The horizontal, continuous red line is at the 50% level.
Fish and Seafood Products Utilization
In 2012, more than 86% of world fish production, i.e., 136 million tons, was utilized for
direct human consumption (Figure 3). The remaining amount (21.7 million tons) was destined
to non-food uses, mostly reduction to fishmeal and fish oil (75%) but also utilized as
ornamental fishes, as fingerlings/fry for culture purposes, as bait, for pharmaceutical uses and
as raw material for feeds (14%). Edible seafood products are primarily consumed live, fresh
or chilled (ca. 40%), then in frozen form (about 29%) and less so in cured (dried, salted,
smoked or other forms; 12%) and prepared or preserved forms (13%) [1].
Adapted from [1].
Figure 3. Utilization and processing methods of fish and fishery products: (top) by year; (bottom)
breakdown by form of consumption.
Utilization and processing methods show marked continental, regional and national
differences with marked differences between developed and developing countries markets.
The former favouring frozen and other processed forms while in the later fish is
commercialized mainly live or fresh soon after landing or harvesting, or processed using
traditional preservation methods, such as salting, drying and smoking. Nevertheless,
developing countries have experienced a growth in the share of fish production utilized as
frozen products (from 13% to 24% in the 1992-2012 decade) [1].
Considering food supply quantity (kg/capita/year), i.e., food available for actual human
consumption, the world average consumption of seafood products in 2013 was estimated at
nearly 19 kg/capita/yr [4]. Seafood consumption is diverse between (and many time even
within) countries. Differences reflect, among others, geography, consumers preferences,
availability, or prices/household budget. These are echoed in the variation among estimates of
food supply (in 2011) that range from <1 kg/capita/yr, for countries such as Afghanistan,
Tajikistan, Ethiopia, Mongolia, Uzbekistan and Lesotho, to values of 50-70 kg/capita/yr in
China-Hong Kong SAR, Kiribati, Micronesia, Antigua and Barbuda, China-Macao SAR,
Republic of Korea, Malaysia, Portugal, Myanmar, Japan and Norway, to 90 kg/capita/yr in
Iceland, to estimates as high as 165.7 kg/capita/yr in the Maldives [5].
Social and Economical Importance
The social significance and economical value of fisheries and aquaculture are evident.
According to FAO [1], in 2012, 58.2 million people worked in capture fisheries and
aquaculture (of which 37% are full time and 23% part-time). Most (84%) of all people
employed in the fisheries and aquaculture sector were in Asia, followed by Africa (>10%).
Employment in the sector has grown faster than the worlds population. Overall, fisheries and
aquaculture assure the livelihoods of 1012% of the worlds population.
In 2012, about 200 countries reported exports of fish and fishery products [1]. The fishery
trade is especially important for developing nations (in some cases accounting for more than
half of the total value of traded commodities). In addition, fish exports are a valuable source
of foreign exchange for many developing countries, which export more than they import.
Fishery exports declined slightly but still represented 129.2 billion USD in 2012 while
aquaculture production peaked at 144.4 Billion USD. Together, they are equivalent to the
gross domestic product of a developed country such as Finland (ranked 40th in the world) [1].
SEAFOOD QUALITY
Quality characteristics of fish and seafood products are comprehensively presented in a
number of books [1, 6-13] only an introductory, brief account is given here.
In terms of nutritional composition, fish and fishery products have a very high water
content (50-85%), are rich in protein (12-24%) but poor in carbohydrates (0.1-3%), and their
lipid content is quite variable (0.1-22%). Besides, fish and fishery products constitute
important sources (0,8-2%) of minerals (K>P>Na>Mg>Ca>Zn>Cu) and vitamins (B that is
water soluble, and A, D and E that are fat-soluble, thus occurring in fatty fish and molluscs).
Most of the proteins, 80-90%, constitute the muscle while the remaining are non-protein,
nitrogenous compounds, such as volatile bases (ammonia, methylamine, dimethylamine and
trimethylamine), trimethylamine oxide (TMA-O), creatine, free amino acids (AA),
nucleotides, purine bases and urea in the case of cartilaginous fish, that influence the sensory
characteristics and are important in the process of fish and fishery products deterioration. On
the other hand, lipid content is quite variable even in the same species, depending on
reproductive cycle stage/sexual maturity, growth, water temperature, food abundance and
quality, stress, etc.
Moreover, lipid content sustains the classification of fish and fishery products into
categories: lean, if [lipids]<5% (e.g., sole, cod, hake and crustaceans); semi-fat, if [lipids]
range 5-10% (e.g., turbot and scabbard fish); and fat when [lipids]>10% at least during a part
of the year (e.g., sardine, tuna and salmon). Fat (or blue) fish are rich in long-chain,
polyunsaturated fatty acids (PUFA), that are nutritionally valuable (e.g., eicosapentaenoic
acid, EPA, 20:5n-3; docosapentaenoic acid, DPA, 22:5n-3; and docosahexaenoic acid, DHA,
22:6n-3) but highly susceptible to hydrolysis and oxidation (leading to rancidity), that
produce a number of by-products (aldehydes and ketones) that have characteristic smell and
flavor.
All these characteristics make fish and fishery products highly prone to post-mortem
deterioration due to autolithic (A), microbiological (M) and chemical (Q) phenomena. A
number of signs e.g., development of unpleasant tastes and smells (due to A, M, Q), the
formation of mucous and production of gas (M), the changes in color/abnormal coloration (A,
(M), Q) and changes in texture (A, (M)).
Species-related factors, such as anatomy (size, skin thickness, etc.), physiology (enzymes,
pH, etc.) and habitat (e.g., water quality, pollution), and the manipulation of fish and seafood,
e.g., capture (fishing gear/method), production (feed, water quality, slaughter, etc.),
transportation (maritime and in-land), processing (on-board or in-land), affect its quality loss
and spoilage [9, 11].
Seafood products are marketed and consumed in a wide spectrum of forms (chilled fresh,
modified atmosphere packed, marinated, salted, canned, etc.) in order to fulfill consumers
demands. Emerging technologies (i.e., high-hydrostatic pressure, ionizing radiation, chitosan
coating, etc.) and novel packaging forms that have positive effects on the utilization of raw
fishery products and contribute to the quality and safety of both raw and processed products
are becoming widely used. The increased demand for fishery products in recent decades has
been accompanied by growing awareness of quality and safety, and nutritional aspects as well
as attention to waste reduction and valorization of by-products. Due to the nutritional
composition, weak connective tissue, and high moisture content, fishery products are very
perishable foods.
After harvesting or catch, seafood is prone to spoilage through microbial growth,
chemical change and breakdown by endogenous enzymes and can rapidly become improper
for Human consumption and possibly dangerous to health. In this context, following the good
hygienic/manufacturing practices, proper handling, processing, preservation, packaging and
storage measures from sea to dish (Figure 4) are essential to improve fishery products shelf-
life, guarantee its safety, preserve its quality and nutritional attributes and avoid waste and
losses [1].
The methods used to assess the freshness (and/or quality) of seafood can be divided into
sensory and instrumental [6].
Adapted from [14].
Figure 4. Illustration of a supply chain of fish and fishery products. Even if it describes the situation in
Portugal, it probably depicts what happens in several countries.
The former, that included the Torry scale, the EU scheme or the Quality Index Method,
are also deemed (more) subjective, while the later are considered (more) objective and
include numerous (bio)chemical (e.g., K-value, TVB-N, and TBARS), physicochemical (e.g.,
colorimeter, Torrymeter, texture profile analysis, e-nose, and Vis-NIR spectroscopy) and
microbiological methods (e.g., total viable counts, coliforms, and specific spoilage
organisms). Nevertheless, the increased demand for fish products in recent decades imposed
the adoption of increasingly stringent hygiene measures, at national and international trade
levels, to account for food safety and consumer protection. Various parameters (not only
those mentioned above) and methodologies, both traditional and more technologically
demanding, are presented in the next chapters of this handbook, particularly for undervalued
and/or less studied species or locales.
SEAFOOD SAFETY
Seafood is rich in terms of nutritional composition, making seafood a preferable when
trying to maintain a healthy life. However, due to habitat, species or group-specific (e.g.,
finfish, mollusk, crustacean) biological characteristics, fishing grounds and season, there are
hazards, biological and chemical, that might have serious health effects (causing illnesses,
sometimes fatal) after consumption, particularly of raw (fish and shellfish) and contaminated
seafood. These include virus, bacteria, parasites and biotoxins that already occur in seafood at
pre-harvest [6, 9, 10]. Moreover, there is no reliable and accurate preventive method to
determine the risks level during harvesting [30]. However, during processing and/or handling
there are established, demonstrated methods to control and maintain the quality and safety
and to prevent (re-)contamination of seafood products such as pre-requisite programs (good
hygiene practices (GHP), good manufacturing practices (GMP)) and the HACCP system [15,
16]. Additionally, controlling the growth of pathogenic microorganisms in seafood, that
eventually limit the shelf life of the product, is also necessary not to [17, 18]. The main
parameter that affects the growth of spoilage and pathogenic microorganisms which
contaminate and/or have re-contaminated the product is temperature. Thus, proper handling,
processing and application of preservatives plays a significant role in controlling and
maintaining the safety of seafood [19]. A number of risk assessment models for biological
hazards [e.g., 20-23] and detection methodologies for chemical hazards [e.g., 24-27] have
published in the literature. In the next sections, existing biological and chemical hazards
together with their detection and prevention methods are compiled and discussed.
Biological Hazards
Public health problems can be caused by many factors such as environmental conditions,
climate change, and tobacco and health equity. However, most of the reports regarding public
health issues showed that the main problem is coming from the consumption of contaminated
food. Seafood as a very perishable food poses a high level of risk and can harbour a wide
range of biological agents (i.e., bacteria, virus, and parasites). Once unfit or contaminated
seafood is consumed, symptoms can arise in 1 to 7 days. Some symptoms are very mild (i.e.,
abdominal cramps and low-temperature fevers). In contrast, there are some severe symptoms
depending on the type of biological hazard that need to be treated in the hospital (i.e., bloody
diarrhea, haemolytic uremic syndrome caused by E. coli O157:H7, liver disease by V.
parahaemolyticus, enteric fever, urinary tract infections by Salmonella serovars, toxic
megacolon, bacteremia, Reiters syndrome by Shigella species, acute, symmetric, descending
flaccid paralysis by Clostridium botulinum, diarrhea, vomiting, nausea, abdominal cramps,
and sometimes headaches, myalgias, and low-grade fever by norovirus [28].
As the vegetative cells and spores of the microorganisms are widely spread in the aquatic
environment, contamination is very likely before harvesting or at the final preparation of the
product (i.e., processing). Growth or survival of the pathogens is also depending on the
processing methodologies (application of non-thermal technologies such as ionizing radiation,
high-hydrostatic pressure, thermal technologies, packaging such as MAP, salting, freezing,
marinating), storage and transportation temperatures, and hygienic procedures.
On the other hand, regardless the contamination, re-contamination of lightly preserved
seafood and/or undercooked or raw products also poses health risks to the consumers. To
control the contamination level, authorized agencies play a very significant role from
harvesting area to the retail level (from sea to dish).
Chemical Hazards
Occurrence of chemical hazards in seafood is generally due to improper conditions of the

catch area which are contaminated by marine toxin producers (i.e., dinoflagellates and
diatoms). The toxins produced by these aquatic organisms accumulates in filter feeding
shellfish, namely mussels, oysters, scallops and clams. The shellfish is not affected by the
toxins, however, the higher the concentration of the toxin in the edible portion of the shellfish,
the higher the risk of (chemical) poisoning after consumption. Depending on the accumulated
quantity of toxin the symptoms vary. Notwithstanding, a number of health conditions arise:
amnesic shellfish poisoning (ASP), paralytic shellfish poisoning (PSP), neurologic shellfish
poisoning (NSP), diarrhetic shellfish poisoning (DSP), azaspiracid shellfish poisoning (AZP),
spirolides and gymnodimines (cyclic imines) [29]. In the period 1992-1996, 5-28% of
reported seafood-borne disease outbreaks were by caused by biotoxins [30].
Another type of seafood-borne toxin that can be poisonous is scombrotoxin. Compared to
biotoxins the prevalence of scombrotoxin poisoning is higher (51% of the cases in 1992-
1996) [30]. Scombrotoxin (or histamine) poisoning is the result of decarboxylation of free
histidine by bacteria such as Morganella morganii, Klebsiella pneumonuae, K. oxytoca,
Plesiomonas shigelloides, Enterobacter intermedium, Serretia mercescens, S. plymuthica and
S. fonticola in the fish species that belongs to Scombroid family [31-32].
The preventive measures and determination methods and applications are reviewed in the
section of seafood safety part in this book.
CONCLUSION
Determining and maintaining the quality and safety of seafood has become more reliable
due to the developments in methodologies and technologies in recent years. Unlike
traditional quality (e.g., TVB-N, TBARS, peroxide values, total viable count) and safety
parameters (absence/presence of pathogenic microorganisms, concentration of heavy metals
that constitute chemical hazards, etc.), recent developments in instrumental techniques (e.g.,
High-Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass
Spectrometry (GC-MS), Reverse Transcription-Polymerase Chain Reaction (RT-PCR),
measurements of dielectric properties) allows professionals and researchers to monitor the
changes in seafood quality (e.g., abundance of spoilage organisms, concentration of volatile
compounds, fatty acids content) and safety (e.g., quantifying and determining pathogenic
organisms, marine toxins, or biogenic amines) more reliably and accurately.
Briefly, we feel that the chapters in this handbook critically review, update and/or report
findings, current or emerging developments, technologies, methods and approaches to fish
and seafood processing and quality/safety particularly of undervalued and/or less studied
species and locales that are more interesting and complement the published literature.
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Chapter 2
MICROBIOLOGY OF FISH AND FISH PRODUCTS

AND ITS IMPLICATIONS ON PUBLIC HEALTH
Regine Helena Silva dos Fernandes Vieira1,2,3,*,

Francisca Gleire Rodrigues de Menezes1
and Oscarina Viana de Sousa2,3
1
Department of Fisheries Engineering (DFE),
Federal University of Cear (UFC)
2
National Council for Scientific and Technological Research (CNPq/Brazil)
3
Institute of Marine Sciences (LABOMAR/UFC)
ABSTRACT
This chapter reviews bacteria that deteriorate fish; those that may harm the
consumer; fish contaminant bacteria; the risk ranking for consumers; limited bacteria in
some legislations and worldwide recommendations; methods and techniques to detect
spoilage and pathogens in fish.
Keywords: seafood, bacteria, legislations, human pathogens
INTRODUCTION
The world production of fish and fishery products in 2013 was approximately 160 million
tons, and its consumption is estimated at 20 kg per inhabitant per year [1]. Although fish is
one of the most sought sources of muscle meat, with high market value, its consumption in
Brazil is considerably below the world average. Nevertheless, in the past years an increase of
around 14.5% in demand has been observed [2]. Cultural globalization, price reduction,
*
Corresponding author: Institute of Marine Science- Federal University of Cear- Fortaleza -Brazil. Email:
reginevieira@terra.com.br.
14 R. H. Silva dos Fernandes Vieira, F. G. Rodrigues de Menezes and O. V. de Sousa
diversity of marketed products, and an ever-growing desire for a healthier diet contributed to
a worldwide increase in fish consumption [3].
Due to its high amount of water and the richness of its chemical constituents, fish
provides an excellent culture medium for bacteria, which make hygiene and temperature parts
of the binomial important for its conservation and acceptance. In addition, time (i.e., fast and
accurate decision-making) is another factor that, added up to the binomial in our equation,
strengthens it, and tends to keep the product from spoilage and decay.
The deterioration process is caused, mainly, by psychrophilic and psychrotrophic bacteria
but, above all, the quality of fishery products is measured by the risk of diseases a customer is
under by consuming it. Thus, many countries adopt laws and other legislation in order to
control fish quality and its products, aiming to protect consumers from any harm they may
cause.
BACTERIAL DECOMPOSITION IN FISH

It is possible to describe deterioration as the set of simultaneous autolytic and microbial
reactions in an animal right after its death, which originates undesirable compounds/by-
products with unpleasant smells and tastes.
Fish might be contaminated with the broadest and most diverse group of microorganisms,
through contaminated or polluted waters from estuaries and fishing grounds. The living
animal presents bacterial contamination mainly in the skin, gills and scales; and it may spread
through other tissues after its death. Therefore, an improper handling and the non-compliance
with proper hygienic measures during transportation, handling and storage processes may
promote the development of pathogens present in the fish itself or in the environment [4].
Given the difficulty in identifying the source of unpleasant taste and odor in decayed fish, it is
interesting to establish standards for fish freshness so that, by making use of a table of
attributes, trained panelists are able to analyze products through sensory signals and features
In a parallel analysis, there should be physicochemical tests and bacteriological counts at
different temperatures. After these tests, a proper identification of the bacteria responsible for
the deterioration should be less arduous. In crustaceans, for example, Vieira et al. [5]
described a typical sequence of decomposition while studying lobsters of the genus Panulirus
White, 1847, in Fortaleza, Cear State, Brazil. During the first six days of the samples in ice
storage, no changes in color, odor and texture of meat were observed. Bacterial counts (log
CFU/g) in the muscle at 5 and 25C did not change considerably. However, on the 13th day of
the experiment, some changes were already noticeable. From that day on, meat turned flacid
and for the content of total volatile bases exceeded 25 g/100g; trimethylamine reached values
above 2.28 mg/100g and panelists rejected the crustaceans. On that day, the Standard Plate
Count (SPC) at 5 and 25oC reached 4 108 and 8 108 CFU/g, respectively, confirming the
deterioration by psychrophilic and psychrotrophic bacteria.
Generally speaking, specific spoilage microorganisms (SSO) [6, 7] produce metabolites
related to certain smell and tastes of this process. In species from temperate waters,
Shewanella putrefaciens is a typical dominant microorganism in this particular stage. Bacteria
from the Vibrionaceae and Enterobacteriaceae families are also related to spoilage. In tropical
waters, Pseudomonas have been described as dominant [8]. According to Simmonds and
Microbiology of Fish and Fish Products and Its Implications on Public Health 15
Lamprecht [9], freshness of ice-stored fish correlates well with sensory analysis, when the
counting occurs at 20oC. At this temperature, Pseudomonas, Acinetobacter and Moraxella
predominate.
BACTERIA THAT MAY CAUSE HARM TO THE CONSUMER

Seafood is colonized by natural and/or pathogenic microbiota, mostly depending on
where it is captured. Some factors that may contribute to an infection by pathogenic bacteria
are the presence of sewage and storm water runoffs in the aquatic environment. Diseases
related to seafood consumption can be caused by biological, chemical or physical agents.
Biological pathogens are represented by a vast amount of bacteria, viruses and parasites [10].
According to Huss et al. [11], pathogenic bacteria detected in seafood and its related products
can be divided into three groups: 1) those normally present in the habitat of the species:
belonging to the Vibrio genus (V. parahaemolyticus, V. cholerae, V. vulnificus), Clostridium
botulinum Non-proteolytic serotype E, Plesiomonas shigelloides and Aeromonas spp.; 2)
those generally present in the environment: Listeria monocytogenes, Clostridium botulinum
proteolytic type A and B, Clostridium perfringens and Bacillus spp.; 3) those that usually
have man and other warm-bloodied, terrestrial animals as their natural habitat (Salmonella
spp, Shigella spp, Escherichia coli, Campylobacter jejuni and Staphylococcus aureus). In
order to control these bacteria and prevent them from contaminating products derived from
fish, it is of utmost importance to know their origin, biology, physiology and ecology; as well
as their survival and growth within the products related to the diseases with which they are
associated [10].
In the following sections are described relevant microorganisms; types of fishery
products, where they have been most frequently observed/found are pointed out; and
incidents in Brazil and/or Latin America are detailed.
Group 1: Bacteria Normally Present in Fish Habitats
Vibrio parahaemolyticus is part of the Vibrionaceae family. They live only in brackish or
seawater and are known to cause gastrointestinal illness in humans. V. parahaemolyticus is a
natural inhabitant of coastal warm waters; in countries with colder weather they are present in
higher concentrations during summer; it is a halophilic organism (i.e., organisms that thrive in
environments with very high concentrations of salt). Moreover, as a mesophilic organism it is
eliminated from seafood by exposing it to heat. When seafood is served raw (e.g., oysters,
mussels, sushi, sashimi and/or Carpaccio), its consumers are under risk of infection [8]. The
illness symptoms usually start within 24 hours of ingestion. Illness is commonly self-limited
and known to last for up to 3 days. Severe disease is rare, occurring with higher frequency in
people with weakened immune systems. V. parahaemolyticus may cause infection on the skin
when an open wound is exposed to warm, contaminated seawater [12]. According to Boutin
et al. [13] V. parahaemolitycus intrudes and invades with considerable impact and may spread
throughout the body via the circulatory or lymphatic system, causing septicemia. Mahmud et
al. [14] stated that the consumption of seafood products may pose risks to public health, since
toxigenic strains of V. parahaemolyticus (O3:K6) have been isolated from those sources,
demonstrating pandemic potential. In Fortaleza, Cear State, 26 individuals were involved
with an outbreak of gastroenteritis after eating raw crab claws in a restaurant of a hotel where
they were hosted. V. parahaemolyticus O3:K6 (Kanagawa positive) was confirmed in six
samples among nine sent to The Oswaldo Cruz Foundation (Rio de Janeiro) [15]. Despite the
low number of reported cases involving V. parahaemolyticus, detection of pandemic clones
with high potential for virulence in northeastern Brazil and the high temperatures of the
region are factors that favor the spread of these bacteria [16]. V. parahaemolyticus produces
an enterotoxin similar to the one produced by V. cholerae, which is able to inflamate the
epithelial lining of the small intestine. In almost 1/3 of the cases, the diarrhea has bloody form
[17]. Vieira et al. [18] while working with oysters purchased at a local restaurant in Fortaleza,
Brazil, identified urease-positive and also tdh and trh positive V. parahaemolyticus,
concluding that oysters served at restaurants in State of Cear may cause gastroenteritis to the
costumers. In Brazil, V. parahaemolyticus was recorded as etiologic agent in several cases of
intestinal infection associated with the consumption of, crustaceans and shellfish [10, 19].
The first reference to the isolation of V. parahaemolyticus (O5:K17 sorotype; Kanagawa-
positive) in Brazil was made by Hofer [20] in diarrheal stools from a six-year old child in
Cascavel (State of Cear).
Vibrio cholerae and V. vulnificus, as V. parahaemolyticus, also belong to the group of
species that cause vibriosis naturally lives in brackish or marine waters but there is no
mention to them in the legislation, neither in Brazil nor the European Union. They may infect
fish and if product is consumed raw or not properly cooked, may also be transmitted to the
consumer. In fact, if the Vibrio cholerae belongs to the serotype groups O1 and O39 it might
transmit cholera, an epidemic disease characterized by abundant diarrhea.
Vibrio vulnificus is homologous to V. parahaemolyticus, differing only on the ability to
ferment lactose. It may cause septicemia and eventually death, via ingestion or the
bloodstream - reaching through the intestinal tract or open wounds exposed to contaminated
marine environment [21].
C. botulinum is widely distributed in the soil and aquatic environments worldwide. It has
the ability to produce the most lethal toxin, known as botulinum toxin (BoNT). Seven types
of this toxin, from A to G, are known [22]; toxin E is usually associated with the consumption
of marine fish and fish/seafood products [23] because its spores tend to be more confined to
water, especially seawater [24]. Spores of C. botulinum -non-proteolytic type E can germinate
at temperatures below 3C and are often found in association with stocked marine fish [25].
The first botulism outbreak on record in Brazil was in 1958 in the State of Rio Grande do Sul:
nine people died after having ingested fish using homemade preservation techniques [26]. In
2007, an isolated case of botulism - again for the same reason - occurred in city of So Paulo
[27].
Clostridium botulinum serotypes E, A and B, proteolytic and non-proteolytic, are not
contemplated by current guidelines (or regulations or legislation) in Brazil, only sulphite-
reducing clostridia (Resolution 12 by the Brazilian Health Surveillance Agency, Ministry of
Health [28]). The authors decided to include them all in one item only (Tabela 1). Clostridium
botulinum is cited, but not limited, by FDA and CODEX.
Proteolytic types A and B of C. botulinum were found in samples from areas above
Cheasapeake Bay (USA) and in samples of fresh blue crab meat (also in the USA) [29].
During 2007, in Brazil, a case of botulism linked to the consumption of homemade canned
fish was reported in So Paulo State. The case was confirmed and related by the Center of
Epidemiological Surveillance of So Paulo [30]. Zadeh et al. [31] reported a case of a 12-year
old that developed symptoms of weakness and diplopia six hours after the ingestion of
barbecued caviar, a fish roe product. Immediately, serum, gastric and stool samples were sent
to the Pasteur Reference Laboratory (France) for botulinum toxin detection. Based on the
clinical report, the patient received three monovalent antitoxins: A, B and E; and
progressively evolved to better health state within 10 days. In Argentina, 21% of the cases of
botulism recorded between 1980 and 1989 were related to the consumption of fish and fish
products [32].
Plesiomonas shigelloides belongs to the Enterobacteriaceae family, with its primary
habitat being warm water environments and fish from both freshwater and seawater. P.
shigelloides is a Gram-negative bacillus with a positive oxidase test [33]. Ingestion of P.
shigelloides may not always be a cause of diseases to its host, but the microorganism may
remain as a non-infectious, transitory member of the intestinal microbiota. The disease caused
by P. shigelloides is gastroenteritis, which is usually self-limiting, and yields fever, chills,
abdominal pain, nausea, diarrhea or vomiting. The bacteria may be invasive and produce
toxins [34]. They are not considered in any global regulation. This bacterium has been
isolated from the tissue of fresh and pre-cooked mussels from a farm in Niteroi, Brazil [35].
Infections have been associated with travel to or residence in tropical and subtropical
countries, with the consumption of raw seafood or with exposure to amphibians or reptiles
[36, 37].
Aeromonas spp. are Gram-negative microorganisms, rod in shape but with a slight
resemblance to cocci straight but with rounded ends. They are catalase and oxidase positive,
reduce nitrate to nitrite and ferment D-glucose with acid/acid and gas production. In addition,
they are mesophilic and resistant to the vibriostatic agent 2,4-diamino-6,7-diisopropyl-
pteridine O/129. They are found in aquatic environment, both clean and/or contaminated with
waste and disposal from sewer systems. After ingestion, they may cause intra and
extracellular infection. In Brazil, a case of mild diarrhea was reported after the consumption
of a shrimp cocktail, and A. hydrophila was isolated from both the incriminated food and the
patient's stool samples. Both isolates had identical ribotypes [38]. Aeromonas are frequently
isolated from fish [39] and mollusks [40]. They usually contaminate fish due to their ubiquity
in aquatic environments, with several genospecies described as pathogens of fish and humans
[41].
Group 2: Bacteria Generally Present in the Environment
Listeria monocytogenes is a mobile, Gram-positive bacterium which grows at 37oC but at

the same time is both psychro and halotolerant [42]. L. monocytogenes has been isolated from
processed marine products (cooked and frozen), marinated fish, surimi, sushi and smoked fish
and it is able to rapidly grow in brined shrimp and cold smoked fish. The bacterium is of little
importance in semi-preserved fish products when using 2.5% acetic acid. The freezing
process eliminates these bacteria, and certain levels of acid and NaCl prevent its growth [43].
However, Hofer and Ribeiro [44] studied and found Listeria in samples of frozen Penaeus
subtilis and Xiphopenaeus kroyeri shrimps in Brazil. From a total of 45 frozen shrimps, L.
innocua serotype 6a was found in three samples and L. monocytogenes in four; 1/2a in three
samples and 4b in one. Furthermore, Laciar et al. [45] reported the presence in 50 samples of
fish, 22 samples of squids and shellfishes, the isolation of L. monocytogenes in a sample of
squid and L. innocua in a sample of mollusk. No fish sample showed the presence of Listeria
spp.
Clostridium perfringens is an anaerobic, Gram positive, mesophilic, spore-forming
bacterium distributed in the environment; in soil, it is found at levels of 102-104/g. If high
levels of vegetative cells are ingested, they are likely to reach the intestine and sporulate,
producing an enterotoxin which may result in abdominal pain, nausea, diarrhea and vomiting
in about 8 to 24 hours after ingestion. In the USA, approximately 7 out of 200 reported cases
of infection per year are related to consumption of marine fish [46]. Virulent and toxigenic
strains of C. perfringens were also isolated in unprocessed fishes from freshwater randomly
obtained from local sources in Tamil Nadu, India [47]. Poisoning outbreaks caused by C.
perfringens are especially common in institutions where food is prepared in advance before
serving, and preparation conditions favor bacterial multiplication. It is possible to detect C.
perfringens in foods of animal origin, such as meat and meat products, meat dishes, stews,
soups, gravies, and milk. Occasionally, poultry products, pork, lamb, fish, shrimp, crab,
legumes (beans), potato salad, and macaroni and cheese may contain C. perfringens. These
foods at improper storage temperatures provide risk of infection and disease from C.
perfringens [48]. Since there is no law on mandatory reports of infections by C. perfringens
in Brazil, there are no cases on record involving fish consumption.
The Bacillus species typically responsible for infections, including symptoms of diarrhea
and vomiting, is B. cereus. Fish, meat, and vegetables are commonly associated with this type
of disease transmitted by food. In industrial fish farms in Germany, B. cereus was identified
in fish from Clupea genus after the addition of contaminated seasoning ingredients. The
bacilli sporulated after incorrect freezing, a subsequent process that follows pasteurization
procedures [46]. In Netherlands, a higher prevalence of psychrophilic strains of B. cereus
have been reported in meat and meat products (20.8%, n = 24) and in fish and fish products
(40%, n = 40) [49]. In Brazil and other Latin American countries, the habit of consuming
dishes based on raw fish, with its handling and preparation based on techniques brought from
Eastern cultures (e.g., sushi and sashimi) has caused frequent events of intoxication. Rice is
an ingredient of sushi, which without proper acidification control introduces the risk of toxin
formation by B. cereus [50].
Group 3: Bacteria that Usually Have Man and Other

Warm-Bloodied, Terrestrial Animals as Their Natural Habitat
Salmonella, Shigella and Escherichia species are fecal bacteria. They belong to the
Enterobacteriaceae, having humans and terrestrial, warm-bloodied animals as hosts. These
microorganisms can contaminate the living animal (incl. fish), depending on the capture site.
Further contamination may occur during processing. They may cause serious harm depending
on contamination levels but proper cooking eliminates harm. A special attention must be
directed to cross-contamination, as well as contamination in work areas (production lines in
industrial facilities for seafood processing, kitchens), and the transfer of microbial pathogens
into products that are to be consumed raw [51].
Standards set by Brazilian legislation for Salmonella in food is that the organism should
be absent in 25 g [28], due to its pathogenicity. However, outbreaks of salmonellosis are not
of mandatory notification (except if the case involves typhoid fever), a factor that
compromises proper monitoring and investigation of this pathogen. In a study that analyzed
the occurrence of Salmonella in fish in northeastern Brazil, this enterobacteria was detected in
4% of the samples of fish and shellfish (shrimp and lobster) assessed in the experiment [52].
Campylobacter jejuni are Gram-negative rod-shaped, S-shaped (or of curved
morphology) non-spore-forming bacteria that might present a spherical or coccoid form in old
cultures. They move by a single polar flagellum at one or both ends [53]. C. jejuni and C. coli
are the most common species of Campylobacter, and are usually associated with diarrhea
[54]. Although less frequently found when comparing fish to other types of meat, the low
infectious dose (500 cells), makes campylobacteriosis one of the most typical zoonotic
diseases in human beings [55].
Staphylococcus aureus belongs to the Micrococcocaceae. They are Gram-positive round
cells, which are found grouped in the shape of grape clusters. They cause food intoxication
and are transmitted due to low hygiene standards, during its use and handling. Albuquerque et
al. [56] while investigating S. aureus in ice, water, benches and in the body parts of sellers
and fishmongers (nose, mouth and hands) at a fish fair in Fortaleza, Brazil, identified the
bacteria in all surveyed sites, including 100% of the fair shrimp vendors. It was then
suggested that there should be more information on adequate hygiene standards and proper
handling of the ice for fish refrigeration.
OTHER CONTAMINANT BACTERIA IN FISH AND FISHERY PRODUCTS

In addition to the previously mentioned bacteria, Gelli [57] lists microorganisms able to
release histamine (a decarboxylation of histidine), a characteristic that involves mainly fish
from the Scombridae family (tuna and bonito). Some histamine-producing bacteria are
members of the Enterobacteriaceae, along with Vibrio sp., Clostridium and Lactobacillus
spp. The strongest histamine producers are Morganella morganii, Klebsiella pneumoniae and
Hafnia alvei [58]. In general, biogenic amine-producing bacteria are not part of the marine
fish microbiota, their origin is due to contamination by inadequate hygiene habits during
capture or associated with a contaminated aquatic environment [59]. Thus, it is possible to
affirm that the presence of histamine is also an indicator of bad bacteriological quality [60].
In reference to fermented fish-based products, Staphylococcus spp. and Tetragenococcus
spp. were listed as histamine-producing [61]. Oliveira et al. [59] investigated the presence of
histamine in samples of canned tuna and sardines purchased in retail stores in Fortaleza,
Brazil, which were analyzed by high performance liquid chromatography (HPLC) in reverse
phase. All samples showed the presence of histamine. Levels above 100 mg histamine/kg
were detected in 55% of the tuna samples and 13.33% in sardines. The authors concluded that
it is necessary to improve the quality of the raw material, and the amounts found in the
samples would be able to cause symptoms of poisoning in consumers of fish.
RISK CLASSIFICATION IN A DIETARY FISH CONSUMPTION

Considering aspects of seafood microbiology (afore-mentioned), Huss et al. [62]
classified the fish by the risks they might present to its consumers. Accordingly, the highest
ranked products in terms of health risks would be the mollusks (fresh and frozen mussels,
oysters and sururu i.e., Charru mussel Mytella charruana) and raw fish (e.g., sushi, sashimi)
or if ingested with no proper cooking, followed by crustaceans and fish, fresh or frozen,
which should be properly prepared. Finally, the fish and fish products presenting the lowest
risk levels include lightly preserved (salted, marinated, smoked or fermented), semi-preserved
(caviar), with proper addition of food preservatives, such as sorbate, benzoate and nitrite, and
heat-processed fish, that need to be sterilized and properly disposed into strongly sealed cans.
BACTERIA WITH SPECIFIC RESTRICTIONS

AND RECOMMENDATIONS WORLDWIDE
Table 1 presents a comparison of the bacteria used as a standard to assess the

microbiological quality in fresh fish and fish products under national and international codes
of best practice and legislation. It allows us to compare and analyze the similarities in laws
from different countries and their views on bacteria that may pose health risk to the consumer.
Table 1. Bacteria listed in different laws and guidelines concerning the microbiological
quality of fish and its by-products
Source
Bacteria listed in Codex
Brazil1 FDA2 EU3
laws and guidelines Alimentarius4
Fecal coliform X X
Escherichia coli X X Xa
Thermotolerant coliform X
Coliforms at 45C X
Staphylococcus aureus X X Xb
Coagulase positive staphylococci X X
Salmonella spp. X X X Xa
Bacillus cereus X
Sulphite-reducing Clostridium X
Clostridium botulinum X Xb
Vibrio parahaemolyticus X X Xa
c
V. cholerae X Xa
V. vulnificus X Xa
Listeria monocytogenes X Xb
Shigella Xa
Aerobic mesophilic bacteria X
1
[28]; [63]; [64]; [65]; pre-harvest and harvest hazards in incoming fish and shellfish; b the post-
2 3 4 a
harvest and further processing of fish and shellfish; c food for raw consumption.
In general, laws and codes use information related to different stages and conditions in
the production, processing and marketing phases: bacteria with pathogenic potential, how
they relate to their environmental origins and the quality of the water; bacteria directly related
to poor hygienic conditions and their involvement in degradation and loss of food quality.
Overall, Salmonella is a consensus in the regulations due to the severity of the diseases to
which it is related.
DETECTION METHODS FOR PATHOGENS

AND SPOILAGE AGENTS IN FISH
In the international market offish products, quality is focused on two primary aspects:
food safety and sensory quality; both, closely related to microbiological parameters. Several
methods are used in order to determine and set the standards of freshness and/or quality.
Those can be categorized in sensory and instrumental. The latter group encompasses physical,
chemical and microbiological methods [66].
Unlike other characteristics, the microbiological parameters do not provide information
as to the freshness or palatability of the fish. The purpose of these analyses is to provide a
clearer image of the hygienic quality of the product, the hygiene standards during processing
and preparation, and the possible presence of bacteria or other microorganisms of importance
to public health [67]. Spoilage and deterioration by microorganisms are important sources of
disease outbreaks and economic losses. Every year, about one-third of the worlds food
production is lost as a result of microbial deterioration [68].
In the next sections, culture-dependent and rapid methods are briefly described for further
reference on methodologies to assess microbiological contamination in seafood products
Culture-Dependent Methods
Microbiological methods can be divided into detection and enumeration techniques based
on classic microbiology procedures, involving selective culture media and incubation periods.
Among those classic techniques, it is possible to cite direct counts of culturable bacteria
(Standard Plate Count), estimation of bacterial populations (Most Probable Number),
counting and detection of bacterial groups and species involved in deterioration processes of
fishery products, such as psichrophilic bacteria (e.g., Mol et al. [69]) and histamine-forming
bacteria (e.g., Bjornsdottir et al. [70]). Despite (or because of) being classical, these
techniques constitute national and/or international standards. As a result, they are still often
used in quality control (QC), research and development (R&D). For instance, Lin et al. [71]
used culture-dependent methods to isolate and quantify histamine-forming strains of salted
fish samples. Moreover, Dalgaard [72] employed quantitative and qualitative tests using
culture media to characterize the degrading activity of the microbiota of fish products.
In the case of bacterial pathogens, monitoring may be done by a series of different
detection methods. Some are only capable of a qualitative presence/absence confirmation of
the pathogen, while others allow the quantification of the bacterial load [73]. For pathogens
with zero tolerance criteria in food, there is no purpose in quantifying the bacterial load, but
in case of pathogens that are tolerated below an established limit; the later approach is
extremely useful. In this case, conventional plating techniques and cultivation of pathogenic
bacteria are still the primary choice to assess the degree and extent of contamination in food
products, including fish [74].
There are well-established protocols for enumeration and detection of human pathogens
related to fish, such as Salmonella spp., Vibrio spp., Staphylococcus aureus and Clostridium
spp. In general, the protocol includes a nonselective pre-enrichment step, followed by
enrichment in selective medium and a subsequent plating on selective agar plates. Detection
of suspicious strains requires further isolation and confirmation by biochemical and
serological tests [75].
Quantitative microbial techniques also have the advantage of being adaptable to meet the
specific needs of each sample, as well as requirements of target microorganisms [76]. As an
example, the use of culture media of different composition and the incubation at various
temperatures was the strategy used by Uddin et al. [77] in order to assess the bacterial
microbiota of frozen fish imported from Denmark.
However, in spite of the sensitivity of these cultivation methods and their well-
established, standardized protocols, they are time consuming and often expensive, requiring
specific technical skills to be run. These characteristics make them unsuitable for quick
inspections in the case of fish and fish products [78, 79]. Another disadvantage is the
difficulty that stressed, i.e., viable but non-culturable bacteria have to grow in the
conditions created during the tests.
Rapid Methods
Rapid detection of pathogens and analysis of the deterioration processes in food samples
is essential to ensure fish and fish-products safety and quality. Rapid microbiological methods
have been developed to meet this specific demand from the global food market. In the case of
fish and fish-based products, the high perishable nature of these products should be taken into
consideration as a determining factor in choosing techniques that offer faster and accurate
responses.
The two main groups of rapid methods to detect microorganisms are based on
immunological reactions (Enzyme-Linked Immunosorbent Assay-ELISA, monoclonal
antibodies) and genetic engineering techniques (Polymerase Chain Reaction-PCR, DNA
probes, etc.) [80]. The first group provides faster response while the second group has a
higher specificity to detect microorganisms in fish and fish products [81]. Thus, genetic
techniques are used more often.
Molecular-based methods, especially those based on polymerase chain reaction (PCR) or
microarray have proven accuracy both in quantification and detection of pathogens in fish-
based food [82-87]. Research has proven the efficacy of these techniques in detecting
pathogens in fish and in tracking sources of contaminants bacteria. Already in 1996, Destro et
al. [88] used molecular-typing methods, namely random amplified polymorphic DNA
analysis (RAPD) and pulsed-field gel electrophoresis (PFGE), to track the dissemination of
Listeria monocytogenes in shrimp processing plants. Rrvik et al. [89] characterized Listeria
isolates using multi-locus enzyme electrophoresis and restriction enzyme analysis of total
DNA to establish the environmental sources of this pathogen in marine fish and industry.
Similarly, Mauffret et al. [90] tested real-time PCR to track microbial sources in samples of
bivalve mollusks. Shimizu et al. [91] used the Fluorescence In Situ Hybridization (FISH)
method to rapidly quantify Salmonella enterica in food samples, including fish.
These rapid techniques may also assist in monitoring and quantifying bacteria that
produce metabolites responsible for off-flavors and cause sensory rejection of fish and fish-
based products. Off-flavours are the main indicators used by consumers to evaluate fish
freshness [92]. Compounds that produce such characteristic odors, for example
trimethylamine (TMA), various nitrogen and sulfur compounds, aldehydes, ketones and esters
are produced by a variety of microorganisms during the process of deterioration of fish [93,
94].
CONCLUSION
To conclude, there is a major need from all the stakeholders involved in the process of
producing, processing and exporting fish and fishery products to focus on the quality of the
products, for only awareness and education will make the food that gets to the consumer a
harmless one.
Progress in rapid detection methods for foodborne pathogens, advancements in the
fishing gear, dynamic forms on which we are able to communicate and share information in
the present days - all these elements foster a standardization and a much more balanced
comparison of the different regulations at a worldwide level.
There are, however, two major needs in order to improve the quality and safety of the
currently marketed fish and fish products: discipline to comply with the regulations, and a
huge effort from governmental agencies. This is particularly true in countries where the level
of industrialization of the fishing and processing of fish or the subsequent regulations are
newer and/or less consumer-oriented, or little worried about environment issues and
sustainability.
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189, 153163.
Chapter 3
RELATING SENSORY AND INSTRUMENTAL

ANALYSES OF WELL-KNOWN AND EMERGING
FISH AND SEAFOOD PRODUCTS
Eduardo Esteves*
Instituto Superior de Engenharia (ISE DEA), Universidade do Algarve
and CCMAR Centro de Cincias do Mar, Faro, Portugal
ABSTRACT
Seafood is a very perishable food with a limited shelf life. Sensory evaluation
remains the most satisfactory and important method for freshness evaluation in the fish
sector. Nonetheless, multiple analyses may be required to define acceptability and
quality. Instrumentally-determined physicochemical parameters have gained increased
interest considering that the need for robust, rapid, and non-destructive or non-invasive
analytical techniques to measure seafood quality and freshness is ever-increasing.
Moreover, multivariate statistical techniques (usual in Chemometrics and Sensometrics)
have shown their effectiveness to extract information from complicated data sets of
sensory and physicochemical parameters. In this chapter, the most commonly used
sensory, biochemical and instrumental-based methods for the evaluation of the freshness
and quality of fish and fish products are briefly presented. In addition, an account is given
of selected, recently published papers wherein results from sensory analysis and
instrumental methods of fish and fish products are found to be correlated both in the case
of well-known species or products as well as regarding emergent species or products.
Keywords: sensory analysis, instrumental methods, multivariate statistical techniques,

correlation, seafood products
INTRODUCTION
* Corresponding
author: Email: eesteves@ualg.pt
32 Eduardo Esteves
Even though freshness is the major contributor to describe the quality of seafood
products, no single method is reliable enough for assessment of their freshness and quality
[1]. Other than (bio)chemical and microbiological methodologies, such as adenosine 5-
triphosphate (ATP) breakdown compounds, K and related values, trimethylamine (TMA),
total volatile base-nitrogen (TVB-N), thiobarbituric acid-reactive substances (TBARS), and
biogenic amines, or total viable counts (TVC) and other microbiological parameters (cf.
Vieira et al., this book), instrumentally-determined physicochemical parameters, e.g.,
dynamic/static headspace analyzer-gas chromatography-mass spectrometry (D/SHA-GC-
MS), gas chromatography-olfactometry (GC-O), solid-phase micro-extraction (SPME), and
electronic noses and tongues, near-infrared (NIR), mid-infrared (MIR), image analysis and
color (cf. Balaban and Aliek, this book), or texture analyzes, have gained increased interest
[1], while sensory evaluation remains the most satisfactory and important method for
freshness evaluation in the fish sector [1-5]. Alasalvar et al., [6], Rehbein and Oehlenschlager
[7], Nollet and Toldr [8], Nollet [9], and Boziaris [10], are reference manuals that present
and discuss methods and applications. Only a brief introduction is given next.
In this chapter, the most commonly sensory, biochemical and instrumental-based
methods for the evaluation of the freshness and quality of fish and fish products are succinctly
presented. Expectedly, the findings from different methods are related, since seafood
freshness and its deterioration dynamics are complex events of sensory, biochemical, and
microbial factors, and multiple analyses may be required to define acceptability and quality
[11]. Thence, an account is given of selected, recently published papers wherein results from
sensory analysis and instrumental methods of fish and fish products are found to be correlated
both in the case of well-known species or products as well as regarding emergent species or
products.
METHODS TO ASSESS SEAFOOD FRESHNESS AND QUALITY

Sensory Analysis
The evaluation of freshness and quality and spoilage during storage of (raw) fish by
sensory methods has been actively researched in recent decades [12] and several methods
have been developed and implemented; most notably the US NOAA Seafood Inspection
Program [13] the EU (or EC) grading scheme described in the Council Regulation (EC) No.
2406/96 [14] for raw whole fish/seafood products, the Torry scale (for cooked samples) [5],
and the Quality Index Method or QIM [15]. Sensory evaluation is applied in research, in
quality control, in product development and consumer studies. Seafood freshness is of special
interest as seafood is a very perishable food with a limited shelf life [16].
US Seafood Inspection Program

In the United States, a voluntary but paid inspection service is offered to seafood
producers and processors by the US Department of Commerce (USDC), National Oceanic
and Atmospheric Administration (NOAA) (under the authority of the Agricultural Marketing
Act of 1946). Products inspected and certified under the USDC Seafood Inspection Program
that comply with all of the requirements and criteria specified have the US Grade A seal of
Relating Sensory and Instrumental Analyses of Well-Known and Emerging Fish 33
approval. The standards for grading are grouped into categories such as whole or dressed fish,
fish steaks and fillets or shellfish [17]. The Seafood Inspection Program is expected to help
firms ensure compliance with applicable federal food regulations, including the US Food and
Drug Administrations (FDA) seafood HACCP regulations, for which FDA as published a
guidance [18]. Parsons et al., [19] used contingent behavior analysis to study survey data
wherein consumers were informed that the seafood inspection program would
(hypothetically) become mandatory. The authors found that consumers were quite responsive
to seafood inspection programs, having demonstrated confidence in such programs.
EU Grading Scheme
In the European Union, Council Regulation (EC) No. 2406/96 [14] laying down common
marketing standards for certain fishery products (firstly introduced as Council Regulations
(EC) No. 103/76, for fish, and No. 104/76, for crustaceans) is commonly accepted and
recommended for freshness grading of market fish and generally carried out by trained
personnel in first auctions and/or the competent authority (inspection body). There are
different schemes for whitefish, bluefish, selachii, cephalopods and crustaceans (Table 1 for
bluefish). Whole and gutted fish are assessed in terms of appearance of skin, eyes, gills,
surface slime, belly cavity, odor, and texture of fish. Fish are categorized into one of four
levels of decreasing quality: E (extra), A (good quality), B (satisfactory quality), and below
level B (named unfit or C) is the level where fish is discarded or rejected for human
consumption. Several drawbacks of this scheme have been pointed out in recent years: trained
and experienced persons are required, since the scheme uses only general parameters for iced
fish; it does not take differences between species into account; it mixes subjective and
objective sensory methods in the assessment scheme; and it does not provide information on
the remaining shelf-life of fish [2, 3, 20-23]. Howgate et al., [24], suggested the renewal and
extension of the EU scheme via in their Multilingual Guide to EU Freshness Grades for
Fishery Products in which special schemes for some fish species (e.g., whitefish, dogfish,
herring, and mackerel) were developed. A few of the disadvantages mentioned above are
shared with the USDC Seafood Inspection Program.
Quality Index Method

Developed in the late 1970s and early 1980s at the Tasmanian Food Research Unit of the
Commonwealth Scientific and Industrial Research Organization (CSIRO) of Australia, the
QIM is an attribute scoring methodology that allows: the evaluation of typically 10 to 15
attributes, that change during storage of a particular species; the use of short scales, usually
from a minimum of 0 to maxima of 1 to 3; and the sum of the scores to give and overall index
that is (cor)related with the (remaining) shelf life of fish and other seafood [2, 3, 20-23]. A
few examples of QIM schemes are presented in Tables 2 to 6 from which its species-
specificity is evident. It is expected that the QIM will turn out to be the leading reference
method for the quality assessment of fresh fish within the European Union [25], since it
addresses the downsides of the current EU grading scheme (described in the Council
Regulation (EC) No. 2406/96, see above) namely the fact that it uses only general parameters
for groups of species and so it does not account for differences between species, it confounds
subjective and objective sensory methods, it needs trained and experienced assessors, and it
does not give information on the remaining shelf life of fish [2, 3, 20-22].
34 Eduardo Esteves
Table 1. Freshness ratings established in the Annex of Council Regulation (EC) No.
2406/96 [14] that apply to bluefish, namely albacore or longfinned tuna, bluefin tuna,
bigeye tuna, blue whiting, herring, sardines, mackerel, horse mackerel, anchovy
Criteria
Freshness category Not admitted(1)
Extra A B
(2)
Skin Bright Loss of lustre Dull, lustreless, Very dull
pigmentation, and shine; duller insipid colors; pigmentation;
bright, shining colors; less skin creased when skin coming
iridescent colors; difference fish curved away from
clear distinction between dorsal flesh(3)
between dorsal and and ventral
central surfaces surfaces
Skin mucus Aqueous, Slightly cloudy Milky Yellowish
transparent gray, opaque
mucus(3)
Consistency Very firm, rigid Fairly rigid, firm Slightly soft Soft (flaccid)(3)
of flesh(2)
Gill covers Silvery Silvery, slightly Brownish and Yellowish(3)
red or brown extensive seepage
of blood from
vessels
Eye Convex, bulging; Convex and Flat; blurred Concave in the
blueblack bright slightly sunken; pupil; blood centre; gray
pupil, transparent dark pupil; seepage around pupil; milky
eyelid slightly the eye cornea(3)
opalescent
cornea
Gills(2) Uniformly dark red Less bright Becoming thick Yellowish;
to purple. No color, paler at discolored opaque milky mucus(3)
mucus edges. mucus
Transparent
mucus
Smell of Fresh seaweed; No smell or Slightly Rotten sour(3)
gills pungent; iodine seaweed. Neutral sulphureous(4)
smell fatty smell, rancid
bacon cuttings or
rotten fruit
Legend:
(1)
This column will apply only until a Commission Decision is taken establishing criteria for fish which
is unfit for human consumption, pursuant to Council Directive 91/493/EEC.
(2)
For herring and mackerel preserved in cool seawater (either chilled by ice (CSW) or refrigerated by
mechanical means (RSW)) complying with the requirements laid down in Directive 92/48/EEC
Annex II, point 8, the following freshness categories apply: criterion A applies for categories
Extra and A.
(3)
Or in a more advanced state of decay.
(4)
Iced fish goes rancid before stale, CSW/RSW fish goes stale before rancid.
Table 2. Quality Index Method (QIM) scheme for (raw) hake (adapted from [26]).
The sum of demerit points (Scores) gives an overall index of freshness/quality
Attribute Description Score

General Surface Gray, bright 0
appearance Gray, less bright 1
Tenuous rosy-gray(1) (pink shade in dorsal region) 2
Tenuous yellowish rosy-gray(1) 3
(pink-yellow shade in dorsal region)
Flesh Firm, elastic 0
firmness Firm, less elastic 1
(dorsal Less firm, much less elastic 2
region) Soft 3
Eyes Clarity Transparent, bright 0
(cornea) Slightly opaque 1
Opaque(2) 2
Opaque, bloodstained 3
Pupils Black, bright 0
Black, grayish, less bright 1
Black, grayish distorted(3) 2
Gray, whitish 3
Shape Plane 0
Slightly sunken 1
Sunken, slightly concave 2
Gills Color Dark or bright red, little translucent mucus 0
Dark or intense red, slightly opaque mucus 1
Discolored red, yellow-brownish mucus 2
Dull red, clear mucus 3
Odor Fresh, seaweedy 0
Fresh, slightly seaweedy 1
Neutral 2
Slightly acid or pungent 3
Acid or pungent or bitter or rancid 4
QIM score 0 19
Legend:
(1)
simply in the original scheme;
(2)
opalescent in the original scheme;
(3)
typical of cataract in the original scheme.
36 Eduardo Esteves
Table 3. Quality Index Method (QIM) scheme for (raw) redfish (adapted from [25]).
Quality parameter Description Score

Appearance Skin Bright, iridescent pigmentation 0
Rather dull, becoming discolored 1
Dull 2
Stiffness In rigor 0
Firm, elastic 1
Soft 2
Very soft 3
Eyes Cornea Clear 0
Opalescent 1
Milky 2
Form Convex 0
Flat, slightly sunken 1
Sunken, concave 2
Pupil Black 0
Opaque 1
Gray 2
Gills Color Blood red 0
Reminds of beef 1
Reddish areas 2
Rusty, dark brown 3
Odor Fresh, seaweedy, metallic 0
Neutral, grassy, musty 1
Yeast, bread, beer, sour milk 2
Acetic acid, sulphuric, very sour 3
Mucus Clear 0
Milky 1
Discolored, rusty, brown, clotted 2
Flesh fillets Color Translucent, bluish 0
Waxy, milky 1
Opaque, yellow, brown spots 2
Viscera Solution Whole 0
Beginning to dissolve 1
Viscera dissolved 2
Quality Index 023
Table 4. Quality Index Method (QIM) scheme for herring product (Maatjes herring)
stored in air and modified atmosphere (adapted from [27]). The sum of demerit points
(Scores) gives an overall index of freshness/quality
Quality parameters Attributes Score

Appearance Skin side White-silver, creamy-white, light brown, shiny 0
Light-gray, light-creamy, light-brown, mat 1
Gray, creamy, some yellow, brownish, mat, light 2
aubergine(1)
Dark gray, yellow, brownish, mat, aubergine, 3
green
Bone side Creamy-white, clear, shiny, translucent 0
(Creamy) white, clear, less shiny, gray 1
Creamy, mat, gray, light-brown, darker margins 2
Green, aubergine, brown, pink, darker margins 3
Color of the Fresh-red 0
blood Red-brown 1
Brown-red 2
Brown 3
Odor Rancidity Not rancid 0
Little rancid 1
Rancid 2
Very rancid 3
Others Marine, fresh seaweedy, fresh, fresh fish 0
Less marine, fresh seaweedy, watery 1
Light sour, prickly, like wet carton, musty, rotten 2
egg
Some as above plus dominating rancidity 3
Taste Rancidity Not rancid 0
Little rancid 1
Rancid 2
Very rancid 3
Others Salty, metal, creamy 0
Light sour, salty, light butter, watery 1
Sour, bitter, like wet paper, light musty, rotten egg 2
Same as above plus dominating rancidity 3
Texture Texture Firm, good bite, tender 0
Grainy, mealy(2), fibrous 1
Soft, musty 2
Aftertaste Aftertaste Marine, metal, creamy 0
Fatty, light bitter, light sour 1
Bitter, sour, salty, rotten flower water 2
Quality Index 025
Legend:
(1)
purple egg-shape tropical fruit a.k.a. egg-plant;
(2)
farinaceous, soft, dry, and friable.
38 Eduardo Esteves
Table 5. Quality Index Method (QIM) scheme for whole raw octopus
(Octopus vulgaris) stored crushed ice (adapted from [28]).
Quality parameters Description Score

Skin Appearance/ Very bright, well-marked colors, white in the 0
Color clearest parts of the body, skin elastic
Bright, less colored, slightly onik in the clearest 1
parts of the body, skin with low elasticity
Less bright, colorless, orange or brown spots, 2
color somewhat orange, rose(1) in the clearest
parts of the body, shrunken skin
Odor Seaweedy, fresh(2) 0
Slightly seaweedy, slightly grassy, neutral 1
Metallic, grassy, acid, intense 2
Mucus Transparent, watery 0
Slightly milky, viscous(3), moderate or absent 1
Flesh Texture Firm, tense 0
Flaccid, soft 1
Eyes Cornea Translucent 0
Slightly opalescent 1
Opalescent 2
Pupil Black, shining 0
Black, dark red, muddy 1
Dark red, opaque, usually bloodstained 2
Mouth Color White, yellowish 0
region Slightly rose 1
Odor Seaweed or neutral 0
Sulphurous, citric, sweet, acid 1
Mucus Clear 0
Milky 1
Yellowish 2
Arms Material in the As a film all over sucker 0
sucker Starting to agglomerate in the sucker center 1
Completely agglomerated in the sucker center 2
QIM score 016
Legend:
(1)
or rose pink i.e., a shade of pink that is saturated (tone) and bright(er);
(2)
sea-like; (3) sticky.
Table 6. Quality Index Method (QIM) scheme for cuttlesh (Sepia ofcinalis) boxed
in crushed ice and stored refrigerated (adapted from [29]).
Parameters Description Score

Supercial Dorsal side Brownish with bright pigmentation; indistinct shell 0
appearance Still brownish, with pink tones; more distinct shell 1
Brown to dark pink; perfectly distinct shell 2
Ventral side Iridescent bright white; at mantle 0
White with less iridescence; slightly sunken mantle, 1
with few stretch marks
Pink without iridescence; sunken mantle with stretch 2
marks
Skin Well adherent to the esh, resistant 0
Slightly fragile but still adherent 1
Fragile, without adhesion 2
Odor Seaweedy, fresh 0
Metallic or neutral 1
Musty or grassy 2
Ammoniacal, sour or rotten 3
Eyes Color Black 0
Purple 1
Lilac(1) 2
White, milky 3
Eyelid Clear, transparent 0
Opalescent, foggy 1
Milky, opaque 2
Head Suckers Well adherent, resistant 0
Slightly detachable (35 per tentacle) 1
Detachable, removable (>5 per tentacle) 2
Tentacles Resistant, doesnt break when pulled away 0
Still resistant, break when pulled away 1
Not resistant, break easily when pulled away 2
Shape Firm head, well dened ocular globe 0
Head and ocular globe slightly sunken 1
Head and ocular globe sunken and liqueed 2
Mantle Odor Seaweedy, fresh 0
cavity Metallic or neutral 1
Musty our slightly sour 2
Ammoniacal or rotten 3
Flesh color Mother-of-pearl or pearly-white 0
Yellowish, ivory-white 1
Greyish, translucent 2
Gills Well dened, creamy color 0
Slightly liqueed, black (from the ink) 1
Liqueed, with only its laments left 2
Ink sac Well dened, liquid ink 0
Hard, thick ink 1
Soft, waxy or gummy ink 2
QIM score 029
Legend: (1) a pale violet tone representing the average color of most lilac flowers [30].
40 Eduardo Esteves
Torry Scale
As pointed out by Howgate [12], the QIMs recent popularity (cf. QIM Eurofish website,
www.qim.eurofish.com) applies just to measurements of freshness in the raw state; since the
Torry scales have been the most frequently used method for evaluation of cooked fish even in
those laboratories where QIM was used for raw fish. Developed at the Torry Research Station
in Scotland, UK [24], the Torry scale is a 10-point scale for the assessment of cooked fish
samples. Scores are given from 10 (very fresh in taste and odor) to 3 (spoiled). Descriptions
below 3 are considered unnecessary since the fish is then not fit for human consumption. An
average score of 5.5 may be used as the limit for consumption. The Torry scale has been
developed for lean, medium fat, and fat fish species [21] (Tables 7 and 8).
Physicochemical and Instrumental Methods
Albeit QIM (as well as other sensory analysis methodologies) is relatively fast and quite
reliable in determining the freshness of seafood, it still requires experts to evaluate the quality
attributes. Alternatively, (changes in) appearance, odor, and taste of seafood during storage
due to autolytic enzymes, microbial activity, or chemical reactions can be gauged by
traditional (sensu [33] and [34]) indices, such as K-value, TVB-N, PV or TBARS, and/or
instrumental methods, e.g., torrymeter, texture profile analysis (TPA), machine vision system
(MVS), electronic nose (e-nose) and electronic tongue (e-tongue) [35], and spectroscopic
techniques. These are succinctly presented in the next subsections, following the overviews
by Howgate [33], Rustad [36], Oehlenschlager [34] and Ozogul [4], and, more recently,
Cheng et al., [37].
Table 7. Torry scoresheet for freshness evaluation of cooked herring

(adapted from [31])
Score Odor Flavor Texture

10 Fresh oil, marine, creamy, Fresh oil, sweet, meaty, Firm, slightly dry
boiled potato, weak odor. creamy, metallic, green plant.
9 Fresh oil, meaty, creamy, Fresh oil, sweet, meaty, Becoming less
boiled clothes, musty, creamy, musty characteristic firm but still
characteristic. quite dry.
8 Oily, musty, burnt, slightly Oily, sweet, meaty, creamy,
brown oil. burnt, neutral.
7 Oily, musty, slightly rancid. Oily, sweet, meaty, creamy,
musty, slightly rancid, slightly
sour.
6 Oily, rancid, cheesy, slightly Oily, sweet, stale meat,
sour, boiled clothes. creamy, rancid, sour.
5 Rancid, sweaty, cheesy, sour, Rancid, sweaty, musty, sour.
malty.
4 Rancid, sweaty, cheesy, sour, Rancid, sweaty, cheesy, sour
stale meat. fruit, slightly bitter.
3 Rancid, sweaty, cheesy, sour Rancid, cheesy, sour, bitter,
stew, ammonia, vinegar. rotten fruit.
Table 8. Torry scoresheet for freshness evaluation of cooked lean fish such as cod,
haddock and Pollock (adapted from [32])
Odor Flavor Score

Initially weak odor of sweet, boiled Watery, metallic, starchy. Initially no 10
milk, starchy, followed by strengthening sweetness but meaty flavors with slight
of these odors sweetness may develop
Shellfish, seaweed, boiled meat Sweet, meaty, characteristic 9
Loss of odor, neutral odor Sweet and characteristic flavors but 8
reduced in intensity
Woodshavings, woodsap, vanilin Neutral 7
Condensed milk, boiled potato Insipid 6
Milk jug odors, boiled clothes-like Slight sourness, trace of off-flavors 5
Lactic acid, sour milk, TMA Slight bitterness, sour, off-flavors, TMA 4
Lower fatty acids (e.g., acetic or butyric Strong bitter rubber, slight sulphide 3
acids), composed grass, soapy, turnipy,
tallowy
K-value
The most important nucleotide in all living organisms is adenosine 5-triphosphate (ATP),
as it functions as the universal carrier of energy (commonly termed the cells energy
currency), transferring energy from chemical bonds to endergonic reactions within the cell.
The key chemical reaction for bioenergetics is the inter-conversion of ATP and ADP
(adenosine-5-diphosphate) that can be symbolized as: ATP ADP + Pi + energy [38]. Rigor
mortis, a phenomenon that occurs in post-mortem muscle tissue and is associated with
stiffness of muscle or flesh, results from breakdown of ATP [4]. In addition, nucleotide
breakdown, that is due to the action of autolytic enzymes and bacteria, is correlated with loss
of freshness [4, 38]. Taking into account the major final products formed from ATP
breakdown, the K-value (originally proposed by Saito and collaborators in 1959) has been
used extensively as a commercial index (particularly in Japan) for estimating fish freshness
[34, 38]:
K-value (%) = [(Ino + Hx) / (ATP + ADP + AMP + Ino + Hx)] 100
where Ino stands for Inosine, Hx for hypoxanthine, and AMP for adenosine-5-
monophosphate. High-performance liquid chromatography (HPLC) is the most reliable
method for the analysis of single or a combination of nucleotide catabolites. A K-value of
20% has been defined by Japanese researchers as the limit for raw fish (sashimi grade)
consumption [34]. A number of alternative indices to the K-value have been proposed since
its original inception, e.g., K1, G, P, H, and Fr, where the determination of ATP, ADP or
AMP is not required [4, 34].
Biogenic Amines
Biogenic amines (BA), namely histamine, putrescine, cadaverine, tyramine, tryptamine,
-phenylethylamine, spermine and spermidine, are produced post-mortem in fish and other
seafood, mainly via the action of exogenous enzymes, resulting from the activity of the
42 Eduardo Esteves
various microorganisms related to seafood and, less so, due to endogenous decarboxylase
enzymes naturally occurring in sh or shellsh tissue [4, 34, 39]. In fact, through
decarboxylation reactions, tyrosine produces tyramine, histidine yields histamine, and
arginine leads to putrescine. Cadaverine is derived from lysine, tryptamine from tryptophan,
and 2-phenylethylamine is derived from phenylalanine. Putrescine is also an intermediate of a
metabolic pathway that leads to spermidine and spermine [39]. Depending on the species, the
concentration of BA has been reported to be a reliable method of evaluating the quality of fish
and two indices have been proposed, the QI and the BAI:
QI = (histamine + putrescine + cadaverine)/(1 + spermidine + spermine)

and
BAI = (histamine + putrescine + cadaverine + tyramine).
HPLC is commonly carried out to determine BA concentrations because of its sensitivity,

reliability, and reproducibility [4, 39].
Total Volatile Basic Nitrogen

In seafood, particularly marine fish, volatile amines such as trimethylamine (TMA, that
are produced by spoilage bacteria), ammonia (which is produced by deamination of amino
acids and nucleotide catabolites), and DMA (produced by autolytic enzymes during frozen
storage) are the characteristic substances responsible for the fishy odor and flavor
encountered after specimens are no longer fresh or fit for human consumption [34]. Even
though the analyses of these indicators are considered unreliable because they reflect only
later stages of spoilage rather than freshness [40], the European Commission (Regulation
(EC) No. 2074/2005 (Annex II), amended by Regulation (EC) No. 1022/2008) [41, 42]
stipulates that if the organoleptic examination displays any doubt as to the freshness of the
fish, total volatile basic nitrogen (TVB-N), should be used as a chemical check [4]. The
principle of TVB-N determination is quite straightforward [33]: a suspension of fish muscle
or an extract of fish muscle is made alkaline and the free bases are distilled, usually at boiling
point at atmospheric pressure, collected, and estimated using standardized acid or alkali. For
example, in the European Union [41], the reference method to be used for checking the TVB-
N limit involves distilling a sample extract deproteinised by perchloric acid that after
alkalinisation undergoes steam distillation and the volatile base components are absorbed by
an acid receiver. Then, the TVB-N concentration is determined by titration of the absorbed
bases. Instead, routine methods, e.g., micro diffusion method described by Conway and Byrne
[43], may be used to check the TVB-N limit. In addition, relatively inexperienced analysts
using standard laboratory glassware or equipment are able to carry out the measurements. The
result is conventionally expressed on a nitrogen basis, thus the TVBN, because the amines
comprising total volatile bases contain one atom of nitrogen per molecule. Although the
principles of the analytical procedure can be simply stated, and though the method has been in
use for almost a century, there seems not to exist an accepted standardized practical procedure
for its measurement [33]. Notwithstanding, there are national standardized methods, e.g.,
Portuguese standard NP 2930 [44] describes the Conway method [43].
Lipid Oxidation
Due to the high content of long-chain, highly unsaturated and labile PUFAs, marine
lipids are very susceptible to oxidation [36, 40, 45]. Lipid oxidation is the most important
factor limiting the shelf life of marine oils as well as being an important factor determining
the shelf life of seafood products, even at low temperatures, except when microbial processes
limit the shelf life [36, 45, 46]. Reaction products from lipid oxidation have a negative effect
on the sensory properties of fish products. In the early stages of oxidative deterioration,
hydroperoxides are formed, which are essentially odorless and flavorless. They are often
detected chemically before any rancidity is organoleptically noticeable [40]. One of the
classical methods, both one of the oldest and one of the most used methods for determination
of oxidative status is the peroxide value (PV) an abbreviated designation of
(hydro)peroxides. For determination of PV in foods (incl. seafood), the lipids can be extracted
before analysis using, for example, the Bligh and Dyer the method, e.g., [47,48], and further
quantified using one of several analytical procedures, e.g., a simple titration method where
the sample is dissolved in chloroform-acetic acid (or isooctane-acetic acid), potassium iodide
is added (this is oxidized by the hydroperoxides or other components present in the sample),
and the liberated iodine is titrated with sodium thiosulfate with starch as an indicator [36]. For
example, Portuguese standards NP 3142-1/2 [48, 49] describe in detail (versions of) the
procedure. Increase in the PV is most useful as an index of the earlier stages of oxidation [4].
Peroxides are unstable and are rapidly transformed into secondary oxidation products,
aldehydes and ketones [4, 36, 40], which have a very disagreeable fishy or rancid odor
and taste [40] the off-taste and off-odor resulting from lipid oxidation are usually defined as
rancidity [4]. The determination of thiobarbituric acid-reactive substances (TBARS) or
andanisidine (AnV) values measure the secondary products of lipid oxidation. TBARS is a
common index but there are many published methods to determine TBARS, but as for the
determination of PV, different methods give different results. In Portugal, for example, there
is a (Portuguese) national standard NP 3356 [50] describing the methodology to be followed.
All the methods are based on the pink color absorbance formed by the reaction between TBA
and oxidation products of polyunsaturated lipids [36]. Unfortunately, according to Huss [40],
neither PV or TBARS correlates well with sensory assessment of rancidity.
Torrymeter, Fischtester, Freshtester

Dielectric properties of fish can be used for determination of freshness, since they are
altered in a systematic way during spoilage tissue components degrade [4, 34, 50]. These
changes that occur at a microscopic level are linearly related to (macroscopic) alterations in
appearance, odor, texture, and flavor during spoilage, e.g., in cod, Baltic herring, hake, and
blue whiting. These features are the basic principles of the Torrymeter (Distell Ind. Ltd., UK;
originally developed at Torry Research Station in Aberdeen, Scotland) (Figure 1), the
Intellectron Fischtester VI (Intelectron Intern. Electronics, Germany) or the RT-Freshtester
(RT Rafagnataekni, Iceland), viz. measuring the electric properties (resistance R, conductivity
k, and capacitance C) of the fish flesh. Readings (that combine those properties) from all
instruments decrease with storage time [4,51]: immediately after catch the resistance
measured in fresh fish is about 2000 while spoiled fish has only 50 of resistance left; on
the other hand, the conductivity of fresh fish is approximately 500 S whereas that of spoiled
fish is 20 000 S [34]. In the Torrymeter, these changes are quantitatively assessed by a
composite measure, the electrical Q-factor defined as Q = 2fCR, where f is the frequency of
measurement (2 kHz). These and other aspects are discussed more in depth in [52].
44 Eduardo Esteves
Figure 1. Image depicting operation of Distell Torrymeter to assess whole fish freshness.
Texture Analysis
Szczesniak [53] defined texture as the sensory and functional manifestation of the
structural and mechanical properties of foods detected through the senses of vision, hearing,
touch, and kinesthetic in her review of the state of knowledge and specific research areas
that could constitute new significant breakthroughs of texture research. In addition, she
emphasizes that realizing that texture is a sensory property gives proper orientation to facets
of texture research [53], since texture is one of the most important parameters determining
the overall perception of fish quality [54, 55], namely hardness [4] and/or firmness [56].
Notwithstanding the problems recognized in the analysis of fish samples, e.g., shape of
the whole fish and fillets, the complex, heterogeneous structure of fish muscle, and the
slippage of the myotomes upon cooking [53, 56], that make many instrumental methods
difficult to apply in the case of fish [57], the literature shows that there has been an effort to
design instrumental methods that could be correlated with either the oral or non-oral sensory
texture [57]. Most of the reported data on fish flesh texture for quality assessment are based
on mechanical tests that are empirical (instrumental parameters correlated with texture
measured by sensory analysis) or imitative (tests that mimic the conditions suffered by food
material in practice) [57]: e.g., the Kramer test, Warner-Braztler, puncture, tensile and
compression tests, texture profile analysis (TPA), and viscoelastic methods such as stress
relaxation, creep and oscillatory measurements [34, 55, 58]. Succinctly, TPA consists of
compressing a sample twice in a reciprocating motion, mimicking the action of the jaw. The
resulting forcedeformation curve is analyzed to determine several texture parameters,
originally dened as hardness, cohesiveness, elasticity, adhesiveness, brittleness, chewiness
and gumminess (these have been examined and updated over the years) [53, 55, 58]. Various
equipments with a variety of knives, blades, cells or probes attached have been used, e.g., the
Instron Universal Testing Machine (Instron, USA), the TA.XT2 Texture Analyzer (Texture
Technologies Corp., USA) or the LFRA Texture Analyzer (Brookfield Engineering
Laboratories, USA) (Figure 2). Seemingly, there is no perfect texture measurement equipment
or system that can be universally recommended [4, 54, 56] and (single) instrumental analysis
cannot entirely simulate the overall experience of texture [54]; it may be necessary to
combine some methods [34].
Figure 2. The LFRA Texture Analyzer system (left) set with the spherical steel probe used for the
compression test and connected to a PC (right) running the LFRA software that enables the remote
operation of the device including the recording, plotting and analysis of data.
Machine Vision System

Humans perceive the world using the five senses - vision, hearing, touch, taste, and
smell-, but the sense of vision is usually used first in detecting events and objects. Visual
quality of seafood includes appearance attributes, such as size, shape, and color. These have a
direct influence on the seafoods value and acceptance [35, 59]. One of the methods of
measuring them is by using a machine vision system (MVS) [35], which consists of a digital
camera to acquire images, an illumination system, and computer software to analyze the
image, i.e., carrying out its segmentation followed by feature extraction and finally by
classification/matching [59]. MVS is a rapid, objective, repeatable, and non-destructive
method, and has been recognized as the most promising approach to objective evaluation of
visual quality of seafood, with many successful applications [35]. For the industry, the
implementation of an on-line inspection system can increase speed, efficiency, and accuracy
along with cost reduction [35]. The authors elaborated on the background and principles of
image analysis and concludes that it is capable of investigating particular aspects of quality
assessment in the case of fishery products. Dowlati et al., [60] reviewed the use of machine
vision and imaging technologies for fish-quality assessment while Balaban and Aliek (this
book) present various applications of this technology.
Electronic Nose and Tongue

Odor is the main indicator of fish freshness [4, 61] and is one of the most significant
features of volatile compounds, which can be used to evaluate fish freshness [62]. The most
important chemicals involved in fresh fish odors are long-chain, C6-C9 alcohols and
carbonyls, bromophenols, and N-cyclic compounds [4, 37, 61]. Microbial activity and
endogenous enzyme decompositions in seafood are able to produce volatile compounds
related to nitrogen, amine, ammonia, alcohols, sulfur-containing compounds and others [37,
61]. In fact, a variety of chemicals are produced by microbial activity and lipid oxidation
during storage of fish, namely short-chain alcohols and carbonyls, amines, sulfur compounds,
46 Eduardo Esteves
and aromatic, N-cyclic, and acid compounds. The concentrations of these compounds are
related to the degree of spoilage [4]. Consequently, monitoring and determination of the
freshness or spoilage stage of fish can be based on the valuable measurements of those
volatile compounds [37].
Odor has been analyzed by sensory panel or gas chromatography (GC) [4]. In the first
case, the use of human nose as a smell assessment instrument has limitations, namely the fact
that our sense of smell is subjective (thus panelists need extensive training), gets weary easily,
and is therefore difficult to use [63]. In the second, the analysis of odors, viz. the investigation
of gaseous samples containing volatile compounds, is a typical subject of analytical chemistry
where several methods are available to isolate and concentrate the volatile compounds from
the headspace/food matrix, such as the solid phase micro-extraction (SPME) [64], and to
separate mixtures in individual compounds and analyze them qualitative and quantitative,
e.g., gas chromatography (GC) or gas chromatography/mass spectrometry (GC/MS) [34, 35,
61, 64]. Individual components could also be correlated to sensorial perception using GC-
olfactometry (GC-O) [35, 64]. These kinds of analyses are both time-consuming and
expensive [4] and no single index is expected to cover all the complex changes that occur
during spoilage [35].
Electronic-noses (e-noses) were developed in order to analyze a gaseous mixture without
separation [34] while mimicking the function of human nose and bypassing the
abovementioned limitations [63]. Notwithstanding, the currently available electronic noses
are still based on an oversimplified olfaction model taking into consideration very little of the
complexity of the natural olfaction [34]. An array of e-noses (e.g., electrochemical gas, metal
oxide, conducting polymer sensors, etc.) with different, but carefully selected types of sensors
(metal oxide semiconductors (MOS), conducting polymer, surface/bulk acoustic wave
(S/BAW) devices, metal oxide field effect transistors (MOSFET), electrochemical, and
GC/MS-based; cf. [65]) coupled with different signal extraction and data processing methods
(most frequently multivariate data analysis, e.g., principal component analysis (PCA),
discriminant function analysis (DFA), or partial least-squares regression (PLSR), and
artificial neural networks (ANN)), have been employed for freshness assessment and other
quality parameters of fish and other seafood [4, 35, 63].
Liking for the taste of a seafood product is another factor positively related to its
consumption [66]. The sense of taste in mammalians is organized in a similar way to olfaction
(but it is less developed) and the perception is carried out by non-specific taste buds, situated
on the papillae of the tongue. Overall, taste is correlated with a combination of basic tastes
and taste sensations (bitterness, saltiness, sourness, sweetness, umami, metallic, astringency,
spicy, and cooling effects) [35, 61]. Because taste and odor are often perceived
simultaneously, the term flavor is widely used to describe their combination, especially
when speaking about food [61, 67, 68]. Recently, the same principle of the e-nose was also
applied to sensors working in environments for the classification of liquids, wherein a sensor
array is combined with pattern recognition tools (for signal extraction and data processing) to
detect and identify/quantify tastes of food samples, particularly liquids. Most of the e-tongues
reported so far consist of a combination of electrochemical methods based on potentiometric
or amperometric sensors [35, 61]. The e-tongue has emerged as a tool for rapid assessment of
complex liquids [63].
Cosio et al., [70] present e-noses and e-tongues in a well-described and well-illustrated
manner. Loutfi et al., [71] reviewed very recently this topic mentioning fish applications.
Vis/NIR Spectroscopy
Cnovas et al., [71] showed that there has been an increasing growth of both new and
(more) efficient methods of online and at-line control that are able to provide information
about the internal quality of foods, besides the commonly monitored external properties (e.g.,
weight, size, color, etc.), particularly in the seafood sector. Those methods employ (quality)
sensors, i.e., devices that can respond to some physical or chemical property or properties of
food and transform the response(s) into a signal, often an electric signal. This signal provides
direct information about the (internal and external) quality factor(s) to be measured or may
have a known relation to the quality factors. Moreover, using sensors circumvents the need
for off-line destructive and time-consuming procedures (see above) without producing
permanent effects on the food while making it possible to apply to the product under
development the necessary corrective measures while it is still in the manufacturing line.
These requirements compel the use of elastic (sonic) waves such as ultrasounds [71] or
nonionizing electromagnetic radiation, such as radio frequency (RF), microwave, near-
infrared (NIR) and/or visible (Vis) spectroscopy [71-73], and Raman spectroscopy [72] or
hyperspectral imaging [74]. Nilsen and Heya [75], Cheng et al., [76] and Hassoun and Karoui
[63] reviewed the applications of non-destructive spectroscopic techniques for fish quality
and safety evaluation and inspection, also noting the advantages and limitations of these
techniques and presenting some perspectives about the current work (Figure 3).
Understandably, a very brief account is given herein.
Since fish muscle absorbs different components of light differently, depending on the
composition and state of the muscle (the presence of different organic molecules and the
degree of hydration and coagulation), thence the spectra change contingent upon the level of
spoilage during chilled or frozen storage [62]. The operating principle of the Vis/NIR
spectroscopy (as well as other spectroscopic techniques) is the illumination of the sample
with broad-banded light and then measuring the light coming from the sample at different
wavelengths (either via transmission or reflection). The wavelength region of the light
used names the method, e.g., visible (400700 nm) and near infrared (7002500 nm)
regions [34, 75].
While visible spectroscopy allows only the surface of the sample to be examined and NIR
is limited by its low penetration (ca. tenths of mm) [75], mid-infrared (MIR, 250025000 nm)
and Raman spectroscopy, have high structural selectivity, particularly in the case of Raman
spectroscopy which allows identification of changes in relevant food components (proteins,
lipids and water) which are implicated in the loss of quality of the meat and fish due to
handling, processing and storage [72]. In the late 1990s-early 2000s, a new technique referred
to as imaging spectroscopy or hyperspectral imaging (HSI) has been developed. In addition to
the spectral data, this technique also gives spatial information of the sample, i.e., a full
spectrum (in the range 450-1000 nm) is recorded at each location at the sample or spatially-
resolved scattering profiles (sensu [75]). This can be illustrated as simultaneously recording
information about shape and color. This implies that this technique is a powerful tool for
segmentation and classification and that it may also map the chemical composition into the
spatial domain. HSI can be used for transmission, reflection as well as transflection
measurements [75, 78].
48 Eduardo Esteves
Figure 3. Uses of NIR spectroscopy and imaging techniques for assessment of fish quality (adapted
from [73]).
Figure 4. Analysis and use of Vis/NIR spectra. Firstly, multivariate data analysis is used to model the
recorded spectra and reference measurement. Then, the fitted model is used to predict the pursued
parameter using the recorded spectra from new samples (adapted from [75]).
Spectroscopy applied to fish or fish products is not a direct technique, in the sense that
further analysis of the recorded spectrum is required. Commonly, multivariate (data) analysis
methods, also referred to as Chemometrics1 (e.g., response surface methodology (RSM),
1 Chemometrics is the area consisting of versatile mathematical and statistical techniques, such as experimental
design, pattern recognition, and calibration, to conduct chemical experiments efficiently and extract useful
information from multidimensional chemical data (cf. [79]).
principal component analysis (PCA), linear discriminant analysis (LDA), partial least square
(PLS) regression and soft independent modelling of class analogies (SIMCA) [80], are used
[75]. In practice, measured spectra (X) and pertinent quality parameters (Y) are analyzed and
their relationship(s) modelled using multivariate techniques; the resulting model is used to
predict quality (Y^) from spectra obtained from new samples (Figure 4). The NIR
spectroscopy has been used in the contexts of chemical composition assessment, fish
freshness documentation and storage time estimation, food authenticity and adulteration,
safety determination in fish for human consumption, (potentially) in the evaluation of texture
and detection of bruises and even in sensory quality, namely appearance and texture, of
cooked fish [75].
Multi-sensor data fusion and the Artificial Quality Index

Olafsdottir et al., [62] proposed an interesting multisensory approach to overcome the
disadvantages of each rapid technique for investigating quality issues in seafood products.
Since each physicochemical, instrumental technique is particularly valuable at measuring
certain quality attributes (e.g., electronic nose for odor analysis, colorimeter measurements,
and texture analyzer for texture analysis), the authors suggested the combination of the
outputs of complimentary sensors and calibrating them with sensory scores of the QIM for
attributes like appearance, smell and texture, to give an Artificial Quality Index (AQI) (Figure
5), that was originally referred to by Di Natale [81].
Figure 5. Diagrammatic construct of the Artificial Quality Index (AQI). After calibration with sensory
data (QIM), instrumental readings are combined into (artificial quality scores (adapted from [81]).
50 Eduardo Esteves
The AQI is based on the same principles and can be as accurate and precise as the QIM
sensory score [1, 55, 59, 62]. The outcome provides a basis for the construction and industrial
exploitation of multi-sensor-devices for defining the quality of fish [62]. Snchez-Alonso et
al., [55] asserts that the replacement of trained sensory panels by a combination of
instrumental methods that mimic human senses is a promising approach. Nevertheless, not
(all) instrumental techniques considered in the AQI are necessarily measuring precisely the
same alterations as the sensory evaluation, e.g., the electrical testers and VIS spectroscopy
have no clear sensory relation (albeit they can be calibrated against the skin appearance), but
showed an excellent correlation to the total QIM score for the iced fish and similarly were
highly correlated with days in ice [62].
RELATIONSHIPS BETWEEN INSTRUMENTAL AND SENSORY TRAITS

Since 2010 a number of studies have been published, wherein sensory analysis
assessments of fish and fishery products freshness and quality were related to results
obtained using instrumental methods. In this section, a brief account of selected studies is
given.
Successes in Well-Known, Recognized Species
In rainbow trout fillets, stored at super chilling (-3C) and chilling (+3C) temperatures,
the correlation coefficients between TVB-N and other freshness indicators, namely total
aerobic count (TAC), K value and sensory score, were relatively low but the K value-related
H value yielded higher correlation coefficients with other freshness indicators, and thus were
considered better freshness indicator [82].
On the other hand, in a number of experiments, Fischtester [34] and Torrymeter [4]
readings were compared with those obtained with QIM for several species, e.g., cod, Baltic
herring, hake, blue whiting, flounder, mackerel, whole, iced gilthead sea bream, and farmed
Senegalese sole [4], and strong and significant linear correlation coefficients, |r| > 0.95, were
found [34].
In a study to compare differences in physical, chemical and sensory post-mortem changes
between wild and farmed gilthead sea bream (Sparus aurata), Simat et al., [83] found that
changes in pH and dielectric properties, the later measured using Distell Torrymeter, were
influenced by differences in lipid content, while changes in total volatile base nitrogen and
trimethylamine showed high correlation with sensory assessment and storage time. On the
other hand, high correlations were found between sensory attributes, viz. oystery, fishy, and
fired pork odor, and alcohols (1-penten-3-ol), aldehydes (propanal, butanal) and pryrasines,
respectively, in commercial brand oyster sauces [84]. The volatile compounds were extracted
and detected by headspace-solid phase micro-extraction (HSPME) and gas chromatography-
mass spectrometry (GC-MS) while the sensory evaluation was carried out by 11 trained
panelists. The objective of the study by Wang [85] was to develop a set of systematic
methods for quality evaluation of live eastern oysters (Crassostrea virginica) including
textural analysis (e.g., hardness, gumminess and chewiness), free amino acids (FAA) analysis
(using HPLC) and consumer sensory evaluation and preferences, such as texture, flavor and
overall likeability. Seemingly, flavor had a stronger effect on oyster consumption than
texture. In fact, sweet FAA of body correlated with flavor likeability and overall likeability,
while sulfurous FAA of adductor muscle was negatively associated with flavor likeability. In
a paper published in 2013, Liu et al., [73] reviewed the application of NIR spectroscopy and
imaging techniques for the evaluation of chemical composition (fat, protein, and moisture),
presence of parasites (nematodes), microbiological (freshness, and spoilage), and sensory
(flavor, texture, and color) attributes of fish and fishery products as well as their usefulness
for fish authentication and classification. PSLR models satisfactorily predicted descriptive
sensory traits, such as muddy/earth aroma, fresh flavor, muddy/earthy flavor and
muddy aftertaste, from Vis-NIR data (0.54 < R < 0.73 with 3.2 < SECV < 5.0) in farmed
Australian barramundi (Lates calcarifer) [86]. Ritthiruangdej and Suwonsichon [87] applied
PCA to describe the differences and relationships among sensory attributes and NIR spectra
of fish sauce samples. The first three principal components, identified therein as the fishy
flavor component (PC1), the sweet component (PC2), and the bitterness component (PC3),
respectively, described well the investigated data and the samples.
When comparing the effects of different processing methods on raw Atlantic salmon
(Salmo salar) fillets, Vaiseth-Kent et al., [88] analyzed the results from their experiment
using PCA and found that the breaking force values from texture analyzer gives a good
estimate of sensory perceived tenderness and hardness and the values obtained using a
colorimeter conveys assessments of perceived color. Instrumental measurements of color and
texture measured (with a Minolta colorimeter and TA.XT2 Texture Analyzer, respectively)
and sensory evaluation (via QDA) of intensity and overall liking of properties such as color,
texture, flavor and aroma, were conducted by Larsen et al., [89] on farmed New Zealand King
Salmon (Oncorhynchus tshawytscha) that was prepared according to common consumer
techniques, namely poached, steamed, microwaved, pan fried (no oil), oven baked (no oil)
and deep fried (in sunflower oil). The instrumental texture measurements of the cooked King
Salmon were closely linked with the texture ratings from the sensory panel using PCA. In a
study to assess the texture of fillets of farmed Atlantic salmon (S. salar), Isaksson et al., [90]
successfully used Vis-NIR reflectance spectroscopy to predict Kramer shear force
measurements based upon cross-validated correlation coefficients of 0.94 and 79% correct
classification using linear discriminant analysis (LDA). In another study, Wu et al., [89]
obtained PLSR models that described the relationship between spectral signatures (in the
4001,000 nm range) of salmon fillets and their corresponding TPA parameters (hardness,
cohesiveness, and adhesiveness). Correlation coefficients of 0.665, 0.555, and 0.606 and
RMSECV of 4.09, 0.067, and 0.504 were obtained for hardness, cohesiveness, and
adhesiveness, respectively. Similarly, Wu et al., [92] were able to predict the color of salmon
from effective spectra selected in long-wavelength NIR spectral region (9641,631 nm) using
a successive projections algorithm. After establishing the correlation between the
concentration of astaxanthin (one of dietary carotenoids deposited in the muscle that is
responsible for color of salmon) and the spectral response, the final multiple linear regression
(MLR) prediction model resulted in correlation coefficients of 0.869, 0.728, and 0.805 for L*,
a*, and b* color values, respectively.
Recently, Cheng and Sun [74] reviewed the basic knowledge and the current research and
potential industrial applications of hyperspectral imaging (HSI) on quality inspection and
evaluation of fish and other seafood. Therein, a number of successful (R2 > 0.8) applications
52 Eduardo Esteves
of spectroscopic techniques (Vis/NIR spectroscopy) are listed, e.g., detection of astaxanthin

content (PLSR, R2 = 0.92) and texture analysis (PLSR, R2 = 0.85) in Atlantic salmon, fat
content (PLSR, PLS-DA, R2 = 0.97) in sea bass, evaluation of freshness (PCR, R2P = 0.83) in
swordfish and in cod (PCA, PLSR, R2 = 0.97), and protein (PLSR, R2 = 0.97), fat (PLSR, R2 =
0.97) and glycogen (PLSR, R2 = 0.94) content in oysters. HSI has been implemented as an
alternative to traditional analytical methods and has proved its potential for a number (of
other) tasks, namely the concurrent quantitative and qualitative determination of nematode
contamination, measurement and visualization of physical and chemical constituents,
recognition of fresh and otherwise treated samples besides the detection of microbial spoilage
and seafood products adulteration.
Using multiple factorial analysis (MFA) to explore potential relationships between
sensory attributes and nutritional content properties between the raw and cooked mussels
(Mytilus sp.) from the north-west coast of Portugal and Spain (Minho and Galicia,
respectively) and the new offshore production site of Armona (Algarve, south Portugal),
Oliveira et al., [93] found that some nutritional components were related to specific sensory
sensations. Free amino acids were greatly correlated to the firmness of mussels meat while
conversion of lipids (mainly PUFA) into volatile compounds resulted in the variation of the
specific characteristics of flavor in cooked mussels.
Applications Pertaining to Less-Know, Emerging Species
In a study of quality deterioration of tray-packed tilapia fillets, obtained from a

genetically improved farmed tilapia strain of Oreochromis niloticus, stored at 0C, Liu et al.,
[94] found that Pseudomonas counts, total volatile basic nitrogen (TVBN), cadaverine and K-
value were highly correlated (r > 0.90) with storage time and, more importantly, sensory
acceptability. Moreover, the measured hardness decrease tested using a Stevens QTS texture
testing instrument was consistent with texture softening of fillets observed in the sensory
evaluation.
Ariyani et al., [95] studied the changes in sensory and chemical parameters of estuary
grouper (Epinephelus tauvina) kept in flake ice for 21 d. The authors found strong but
nonlinear, quadratic polynomial relationships between traditional indexes, TVB-N, TMA
and K value, and sensory scores, namely total demerit point scores (sensu QIM).
The quality of vacuum-packaged finfish fillets stored at 4C for 5 days of a number of
species, blue-spotted emperor (Lethrinus sp.), saddletail (Lutjanus malabaricus), crimson
snapper (Lutjanus erythropterus), barramundi (Lates calcarifer), and Atlantic salmon (Salmo
salar), was studied by Fuentes-Amaya et al., [96]. They found strong relationships between
total viable counts (TVC) and sensory scores obtained using the Torry scheme (r = -0.72, p <
0.001) and between TVC and QIM scores (R2 0.94) for Atlantic salmon, saddletail snapper
and crimson snapper but not for the other traditional parameters, viz. pH, TMA and TVB-N,
total psychotropic organisms, and H2S-producing bacteria, or species studied.
Harikedua et al., [97] used Pearson correlation analysis and partial least-squares
regression (PLS-R) to analyze the data obtained thru quantitative descriptive (sensory)
analysis (QDA) and traditional physicochemical analysis of the attributes of a traditional
Indonesian fermented fish product named bakasang. A number of gustative sensations such
as salty, umami, bitter and bitter aftertaste, meaty, overripe cheese or sweaty showed an
excellent correlation to moisture content, water activity (aW), salt content, or TVB-N.
While investigating the changes in biogenic amines and their relation to total volatile
base nitrogen (TVB-N), microbiological and sensory score of silver carp
(Hypophthalmichthys molitrix) fillets stored at 0, 3 and 15C, Shi et al., [98] found that
putrescine concentration was significantly correlated with TVB-N, total aerobic counts,
sensory scores, tryptamine and phenylethylamine and proposed it to be a good index of silver
carp fillets in the cold chain.
Using only traditional indexes, positive correlations between TMA and TVB-N contents
and microbial counts (TVC) of small spotted grunter Pomadasys commersonnii, an important
fish species in Nigerian waters, stored in ice for 22 days were found [99]. In addition, the
authors observed that sensory evaluation of cooked fish and TVC were positively and
strongly correlated (r > 0.96).
The results obtained by Zhang et al., [100] on freshwater grass carp (Ctenopharyngodon
idella) showed that traditional freshness indexes, such as total aerobic count (TAC), K value,
and TVB-N values, increased during storage time at chill temperature, while others, namely
the impedance change ratio value (Q value) and sensory assessment (SA) decreased. In
addition, authors found that that were good relationships between Q value and TAC, K value,
TVB-N, and SA (p < 0.01), with the correlation coefficients greater than 0.940. They
concluded that that the Q value can be used as a valid index and a fast and nondestructive
method for freshness evaluation stored freshwater grass carp.
Vardanis et al., [101] attempted to demonstrate the use of chromaticity parameters,
namely lightness (L*), hue (Hab) and entire color index (ECI; a combination of skin hue
and chroma, C*ab), and dielectric properties (measured with a Torrymeter) as reliable and
convenient approaches to quality assessment of cultured red porgy (Pagrus pagrus)
slaughtered in ice slurry and stored in melting ice for 7 d. There were statistically significant
(p < 0.01) correlations between the dielectric properties, the sensorial analysis score (using
the EU freshness rating scheme for finfish), the polyamines (spermidine, spermine and
putrescine) and the ECI.
In a shelf-life study of raw bogue (Boops boops), Bogdanovic et al., [102] developed a
quality index method (QIM) scheme and used multivariable analysis to identify the most
prominent variables during spoilage, including pH, dielectric properties (measured using
Distell Torrymeter), thiobarbituric acid (TBA) index, and volatile amine changes (TVB-N).
The authors found high correlations, |r| > 0.88, of these parameters with storage time and,
particularly, sensory assessment.
Mallick et al., [103] studied the quality of thermally processed Indian white shrimp
(Fenneropenaeus indicus) in curry medium. The instrumental texture parameters, derived
from texture profile analysis (TPA) (e.g., cohesiveness and springiness) and shear force test
(e.g., hardness) measured using the Universal Testing Machine, correlated well (|r| > 0.95)
with sensory-derived textural parameters, chewiness succulence, toughness, and fibrosity,
derived from a 10-person trained panel. In addition, the CIELAB L*, a* and b* measured
with a Hunter lab MiniScan spectrocolorimeter correlated significantly (|r| 0.895) with the
sensory color parameters.
The results obtained by Zhu et al., [104] indicate that during post-mortem storage of
bighead carp (Aristichthys nobilis) heads at 0-3C, the Q value measured with a voltammetry
method is significantly (p < 0.05) related to pH value and texture indexes (hardness,
54 Eduardo Esteves
adhesiveness, springiness, cohesiveness, and resilience) determined via TPA performed using
a TA-XT2i Texture Analyzer (Stable Micro System, UK). Moreover, the correlation
coefficients between Q and K values, TVB-N, total aerobic counts (TAC), drip loss, and
sensory assessments of both raw and cooked samples exceeded 0.95, indicating significant (p
< 0.05) linear relationships.
Li et al., [105] studied the changes in textural and sensory characteristics of large yellow
croaker (Larimichthys crocea; authors incorrectly used the genus Pseudosciaena) stored
frozen at -20C. Using PCA and stepwise regression analysis, the authors were able to
generate prediction equations that significantly (p < 0.05) and accurately (>77%) describe the
changes in sensory parameters, such as appearance, smell, taste and texture, from TPA
indicators, viz. hardness, springiness, cohesiveness, gumminess, chewiness, adhesiveness and
resilience. Notwithstanding, multicollinearity existed among sensory indicators, and also
between TPA-derived indexes.
The texture changes in whole and peeled shrimps (Penaeus vannamei) during iced
storage (for 7 days) determined thru texture profile analysis (TPA), namely hardness,
springiness index, chewiness and cohesiveness, and the total sensory scores were found to
strongly agree [106]. In another study of partially frozen storage of shrimp (Penaeus
vannamei) samples, Li-jie et al., [107] found that according to the results of TVB-N and
sensory scores, textural properties determined via TPA, viz. hardness, springiness, shear
force, chewiness, adhesiveness and gumminess, could be used to estimate quality changes in
shrimps even though showing distinct dynamics.
Ochrem et al., [108] reported a research wherein changes in physical (pH, and electrical
resistance and conductivity measured using a Consort C931 device) and dielectrical
properties (measured with a Torrymeter) as well as sensory evaluation (thru a developed
QIM scheme) of gutted and ungutted carp (Cyprinus carpio) muscle during 10-days storage
under refrigerated conditions were addressed. Torrymeter readings and QIM scores were
significantly (p < 0.0001) and inversely correlated (r < -0.80). Similarly, electrical resistance
(ER) was significantly (p 0.002) and also inversely correlated (r -0.481) with Torrymeter
readings. In contrast, ER and QIM scores were positively correlated (r = 0.776, p < 0.0001).
Thus, authors propose that consumers and traders are able to monitor the quality of fish fillets
during refrigeration storage using Torrymeter readings and QIM (quality index method), and
measurements of electrical resistance.
The results reported by Xu et al. [109] suggest that hardness, springiness and resilience
(determined via TPA), sensory attributes, summarized by the QIM demerit points, TVB-N, K-
value, and TVC combined with some volatile compounds (determined via SPME GC-MS),
using PCA could more completely reflect the quality changes of turbot (Psetta maxima) fillets
stored at 4C for 16 days.
Sullivan Ritter [110] investigated an alternative method to assess oxidation in fish oil
supplements, which many consumers avoid due to fishy tastes and odors despite its many
health benefits, using solid-phase micro-extraction (SPME) and gas chromatography-mass
spectrometry (GC-MS). Using principal component analysis (PCA) and linear regression in
combination with sensory panel scores, the authors identified eight key volatile compounds,
primarily aldehydes and ketones, that they suggest allow monitoring of oxidation in fish oils
without the use of a sensory panel. In addition, they found that the peroxide value (PV) and
anisidine value (AV) that are typically used to assess fish oil quality have little relationship
with sensory properties. In another study [111], an alternative method using SPME in
conjunction with GC-MS was studied to monitor volatile oxidation products in fish oil
supplements, since current methods used to assess oxidation have little correlation with
sensory properties of fish oils [110, 111]. The former authors used stepwise discriminant
function analysis (DFA) to classify oils characterized as acceptable and unacceptable based
on sensory analysis; a 100% cross-validated success rate was achieved the function that
included 14 variables, mostly aldehydes and ketones, as significant discriminators.
Ji et al., [112] studied the volatile compounds present in edible parts of steamed (male)
Chinese mitten crab Eriocheir sinensis from the Songjiang district in Shanghai which were
extracted using monolithic material sorptive extraction (MMSE) method and analyzed by
GC-MS/Olfactometry (GC-MS/O), the E-nose, and sensory evaluation. Authors identified
different important odor compounds (IOC) in the various parts analyzed, e.g., ethylpyridine
(fishy odor) was found in all four parts (abdomen, claws, leg meat, and gonads),
benzaldehyde (almond odor) and trimethylamine (fishy odor) were not IOC in abdomen meat,
and 3-methyl-2-thiophenecarboxaldehyde (chocolate odor) and 2-acetylthiazole (roast meat
odor) were found exclusively in abdomen meat and gonads, respectively. Moreover, sensory
evaluation results showed that meaty aroma was the prominent aroma of abdomen meat and
claw meat while leg meat had moderate aroma, and ammonia-like, fishy, grassy and fatty
aromas were correlated with the gonads. The relationships between volatile compounds and
sensory descriptors were studied using PCA.
CONCLUSION
Besides, the high correlations found between traditional methods and sensory assessment
in both recognizable and emerging species, several studies have confirmed the usefulness of
instrumental methods, individually or in combination, to assess freshness and quality of fish
and seafood products, analogous to sensory evaluation. Expectedly, more studies on well-
known species employed high-end, complex instrumental methods, e.g., GC-MS and Vis-
NIR/HSI, and thus depended upon more complex (statistical) procedures to analyze data,
compared to studies on less-known species. Most of the latter relied on traditional methods.
Notwithstanding, quite a few reported the successful use of texture profile analyzers and the
Torrymeter (or similar devices to measure dielectric properties) to evaluate quality and
freshness, comparable to sensory analysis.
Still sensory evaluation is considered the most effective technique to measure fish
freshness and quality. But although it is fast and reliable it still requires experts for the
assessment. Moreover, since seafood freshness and its deterioration are complex events with
sensory, (bio)chemical and microbial facets, numerous traditional, (bio)chemical,
microbiological, as well as other instrumental methods have been (and are still being)
developed and used for freshness and quality evaluation.
Admitting that traditional methods of measuring quality have a very limited place in
current practices of quality assurance of fishery products, the new physicochemical methods
implemented via instrumental techniques have the advantage of being non-invasive, objective
and rapid, allowing them to be potentially used to monitor the properties of foods during
processing real time. Notwithstanding, there is a need to purchase and maintain complex and
(at present) expensive equipment that often need calibration contingent on sample
56 Eduardo Esteves
preparation, season, fishing grounds, and fish-handling procedures. In addition, to be useful in

quality control, responses of instrumental methods should be causally related to sensory
changes in seafood or they should at least correlate with sensory analyses.
In this chapter, the compilation of selected, recent published papers that report
correlations between sensory attributes and results from instrumental techniques in well-
known species as well as emergent species, supports the more widespread, practical
application of instrumental methods beyond research settings. Nonetheless, issues related to
fish-handling procedures and/or sample preparation (e.g., when measuring dielectric
properties), to the complex nature of phenomena (e.g., volatile compounds and
odor/aroma/flavor) and to demonstration of repeatability and reproducibility of results,
require further developments in instrumentation, understanding of underpinning science, and
application of contemporary statistical techniques (e.g., Chemometrics). One interesting
approach to overcome the disadvantages of each rapid technique for investigating quality has
been considered by Olafsdttir et al., [62], that proposed a multisensor approach (the AQI).
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Chapter 4
MEASUREMENT OF VISUAL ATTRIBUTES

OF FRESH AND PROCESSED SEAFOOD
Murat O. Balaban1,* and Zayde Ayvaz2

1
Chemical and Materials Engineering, University of Auckland,
Auckland, New Zealand
2
Faculty of Marine Sciences and Technology,
anakkale Onsekiz Mart University, anakkale, Turkey
ABSTRACT
Machine vision and image analysis are emerging and quickly spreading technologies
that can quantify the visual attributes of seafood (color, size, shape, visual texture,
defects), can identify different fish species automatically for sorting, can estimate weight
from morphological attributes, can prepare seafood for processing by orientation, and can
perform all of these in a fast, repeatable, objective and flexible manner. The evidence of
successful applications of both machine vision and image analysis to raw and processed
seafood is increasing in the scientific literature, and in the processing and distribution
sectors of seafood. Examples of various applications of this technology are presented in
this chapter.
Keywords: visual attributes, machine vision, seafood, image analysis
INTRODUCTION
Importance of Color and Appearance
The visual impression of a product has a powerful influence on the judgment and
response of consumers, and is strongly related to convention and personal notions of taste [1].
*
Corresponding author: Engineering and CyberSolutions, Gainesville, Florida USA.
Email: MuratKodiak@gmail.com.
66 Murat O. Balaban and Zayde Ayvaz
For example, Alfnes et al. [2] found that the red color of salmon has a strong influence on
consumers decision to buy and their willingness to pay. The redder the color, the stronger
was the assumption of better quality, and the higher was the price consumers were willing
to pay.
The visual attributes of foods in general, and of seafood in particular involve color, size,
shape, visual texture, shine, and defects [3]. Since product consistency is very important,
there have been many attempts to standardize and quantify the visual attributes of seafood.
For example, a reference color card has been developed to subjectively match the color of
aquacultured salmon fillets fed different amounts of astaxanthin [4]. This is useful to a
degree, but since the color of a salmon fillet is not homogeneous, the judgment on the overall
color becomes less certain when addressed by subjective means.
Using color meters such as Minolta and Hunter can eliminate the subjectivity of these
evaluations. The instrument flashes a light of known properties (color temperature, color
rendition index) at a given angle to the surface of the sample, and reads the reflected light.
After calibration this method results in consistent readings. However, the reading port of
these devices is relatively small (in the order of a few cm in diameter), and this poses a
problem of selection of sampling location for large samples such as a fillet of fish. The
reverse is also true: for small samples that do not cover the whole reading port (such as small
shrimp), several samples are blended together to cover the opening and blending tends to
reduce the color information. In addition, the surface characteristics of the sample can affect
the colorimeter readings that may be different from the real color of the sample [5].
Machine Vision
Another alternative is to use Machine Vision (MV) to capture an image of the seafood,
either as a single sample, or as aggregates of several samples in a repeatable and
representative manner. Then, by using image analysis methods useful information about the
sample can be extracted and acted upon, such as sizing, sorting, and defect detection [6, 7]. In
MV-image analysis, the method of image acquisition is important. The properties of the
illuminant (color temperature, color retention index, distribution uniformity, consistency, etc.)
are critical in obtaining accurate information regarding the visual attributes of the sample.
An important step in the analysis of images is segmentation, or in this case, the
separation of the samples of interest from the background. Traditional segmentation of a color
image can be difficult if the colors of the background and the object are similar. For example,
Kim et al. [8] tried to detect the mussel meat in a half-shelled mussel. Since the colors were
very similar, the separation was not possible by color analysis. In this case, the two-image
method can be of value [9]. First, the backlighted image of the object is taken. The objects
silhouette can be easily separated from the background. This silhouette is then used in
defining the object in the image taken by the traditional way, i.e., front lighting. The only
restriction is not to move the object and the camera between taking back and front lighted
images. This method allows the segmentation of objects with very similar colors to that of the
background.
Measurement of Visual Attributes of Fresh and Processed Seafood 67
VISUAL ATTRIBUTES
Machine vision and image analysis can determine several visual attributes of seafood:
Color
An image captured by a color camera consists of pixels with red, green and blue
components (RGB). However, unless the camera has 3 separate sensors to capture the image,
one for each color, the resulting image consists of interpolated values of colors generally
following the Bayer pattern. In this case, each pixel of the single sensor sees the intensity of
one color (Figure 1). For example, the pixel in the upper left corner of Figure 1 measures the
intensity of blue color. The red and green components of this pixel are obtained by proper
interpolation of its green and red neighbors intensity values. Despite this guesswork
modern single sensor cameras have a high resolution (many pixels) and result in an
acceptably accurate description of the colors.
A very important step in the quantification of color is to use a reference color standard to
correct minor changes in the lighting conditions (both illuminance and color temperature) and
inaccuracies in the camera settings. Typically, this is done by including the color reference in
the picture to be taken, and then by software correcting its average color to the known value.
This is also applied to the rest of the image, and therefore color is corrected. This method will
not be effective in correcting large deviations from the real color of the reference material.
Other methods of calibration exist (e.g., Costa et al. [10]). There are Neural Network based
techniques to obtain accurate colors from non-ideal illumination conditions.
The color of many seafood species has been determined by MV and image analysis. For
example, Aliek and Balaban [11] measured the colors of raw and cooked tiger prawns
(Paneaus monodon) using MV. The advantage was to analyze several prawns at the same
time, without the need to orient the prawns. The average colors (e.g., average L*, average a*
and average b*) can be measured, similar to a colorimeter. In addition, color distributions can
be computed and used to determine the area that specific colors represent as a percentage of
the view area of the sample. Since the color of most agricultural materials in general, and
seafood in particular, is non-homogeneous, the measurement of average color may be less
meaningful. In this case, the non-homogeneity of color can be quantified by the color change
index or CCI [12]. A higher CCI implies more change in color, both in terms of space, and
also in the intensity of color. The quantitative measurement of non-homogeneity can be very
useful, because in general humans have difficulty quantifying the colors in a multi-colored
sample, not only in terms of the correct color, but also in terms of the amount of that color
[13].
Salmon is an important and internationally traded fish. The changes in the skin and fillet
color of Atlantic salmon affected by pre-mortem stress, rigor mortis, and conditions of
storage on ice have been measured by MV [14]. Salmon fillets can also be sorted easily by
MV according to their color [15]. Bekhit et al. [16] measured optical properties of fish roes
from several commercial species from New Zealand, including their color (L*, a*, b*) both
raw and after karasumi-like processing (salting and drying). They quantified the color
changes resulting from the processing operation. Roes with intense pigments (e.g., salmon
and warehou) had lower a*-values after processing. Hosseinpour et al. [17] used MV color
measurement for on-line monitoring of shrimp drying using superheated steam and hot air.
The effects of drying temperature and the drying medium velocity on the color parameters
were investigated. The advantage of the method was to analyze dozens of shrimp
simultaneously.
Stien et al. [18] used automated image analysis to quantify the color and composition
(amount of fat) of rainbow trout cutlets. The color data was used in a principal component
analysis (PCA) and correlated relatively well (r = 0.78) with fat measurement using mid-
infrared transmission spectroscopy. Stien et al. [19] also estimated the fat content of salmon
fillets by color image analysis. They emphasized the speed of the method. The white
mycommata of the flesh measured by image analysis correlated well (r = 0.84) with fat
content measured by chemical means.
Yaz et al. [20] measured the a* value of Atlantic salmon by MV, and correlated this to
the amount of astaxanthin in the flesh. When they irradiated the fish at different doses, the a*
values and the corresponding axtaxanthin levels decreased.
Tokuolu and Balaban [21] determined the changes in the color of oysters during
storage using MV. Certain colors decreased steadily during storage. However there was also
significant oyster-to-oyster variation in color, therefore the color of an individual oyster did
not give an indication of its length of storage time.
Figure 1. Bayer pattern for color interpolation with single sensor cameras.
Shine
Piironen [22] mentioned the differences in the images of wet and dry fish, and
experimented with polarized light to remove specular reflection. Indeed, the perceived color
of shiny surfaces can be different than their real color by the interference of specular
reflection. Wolff [23] also mentioned the polarization in the perception of images. One
example is given in Figure 2, which shows the images of same flounder taken with and
without polarized light, and when the surface is dry or wet [24].
The analysis of color at the surface is shown in Table 1. By inspecting Figure 2 and
comparing the values in Table 1, it can be seen that for the dry images, the surface color looks
slightly different and there is a clear reflection in the non-polarized image. The average L*
value of the non-polarized image is higher (lighter) because of reflection. Also, the average a*
value of the polarized image is lower (greener). For the wet surface, the shine is also more
apparent in the non-polarized image, as can also be seen from the higher L* value. The
polarized images of both the dry and wet fish give close color parameters, indicating that as
long as polarized light is used the color reading will not be affected by wet or dry surface
condition.
Figure 2. Images of the same flounder taken with and without polarized light, when the surface is wet,
or dry [24].
Table 1. Color analysis of the skin surface of the flounder shown in Figure 2
Dry, Dry, Wet, Wet,

non-polarized polarized non-polarized polarized
Average L* 38.61 13.53 35.6 12.78 40.05 14.13 36.16 12.71
Average a* -3.24 2.93 -10.63 4.19 -3.21 2.72 -7.86 2.90
Average b* 14.01 5.99 16.76 6.72 13.39 5.90 16.57 6.60
Average S 10.11 10.73
Averages and standard deviations refer to all of the pixels of a given fish image.
Figure 3 shows the pixel-by-pixel differences in the L*, a* and b* values of the polarized
(P) and non-polarized (NP) images, quantified by the S value:
S = [(L*P - L*NP)2 + (a*P - a*NP)2 + (b*P - b*NP)2]1/2 (1)
The average S values for the images are given in Table 1. The wet images have slightly
larger S values, as expected. This is an area that requires attention. For example, in the
machine vision analysis of fish eyes to quantify freshness, reflection can introduce artifacts
and therefore affect the accuracy of the results. The same can be said for gills analysis by
MV.
Length, Width
Since MV does not need to contact the seafood, with proper calibration it can be used to
determine various geometrical and physical parameters by image analysis. This requires the
experimental determination of e.g., length-weight (W-L) relationships for a given species,
under certain conditions. The W-L relationship is commonly of the form W = a (L)b wherein
the b value is the allometric coefficient. Petrakis and Stergiou [25] determined the weight-
length relationship from 33 fish species from Greek waters and that the b value ranged from
2.32 to 3.5, with a median of 2.987. Dulcic and Kraljevic [26] determined the weight-length
relationships for 40 species of fish from the Eastern Adriatic. Experimental data showed that
the b value was very close to 3. This is reasonable, since weight is related to volume, and the
units of volume are (length)3. Gonalves et al. [27] measured the weight-length relationships
for 31 selected fish species from the south of Portugal. They used the ln-transformed
equation, ln(Weight) = a + b ln(Length), and the b value was close to 3 for most species.
Similar studies reported weight-length relationships for 33 species of demersal fish from
Azores [28], and 50 fish species from the Algarve coast of Southern Portugal [29].
Figure 3. E values between the polarized and non-polarized images for dry and wet fish surfaces.
Once these data are available, then the estimation of weight of the fish by MV is possible
because length can easily be determined by image analysis [30]. Also, the condition of the
fish can be assessed by using a modified weight-length relationship [31]. The weight
estimation of the fish is not restricted to after-harvest. Harvey et al. [32] used an underwater
stereo-video system to measure the length of the fish, and therefore estimate the weight.
Martinez-Palacios et al. [33] also used a video technique to monitor the growth of juvenile
fish by remotely measuring their length, and therefore calculating their weight.
The measurement of length is not restricted to fish. In Alaska Pollock roe grading the
uneven skeins are evaluated as a defect. The length of the two skeins can be measured and
compared using image analysis [34], by determining the length of the median line.
Shape is another visual attribute that is important for consumer acceptance and quality
control. Shape representation and description is important in computer science and machine
vision fields [35]. An advantage of recognizing fish shape is to determine overlapping fish in
images and separate them from each other using image analysis [36]. Costa et al. [37] used
shape analysis to differentiate populations of clams relative to their geographical locations.
Merz and Merz [38] used morphological features from image analysis to identify
Chinook salmon sex during fish passage through fish ladders. They found that a good
predictor for gender of handled fish was the snout length to fork length ratio (96% accurate).
Also, the adipose fin length to fork length was a good gender predictor from video images
(86%). Combining this ratio with head length increases gender prediction accuracy to 92%.
Jeong et al. [39] measured the morphometric characteristics (total length, width and
height) of flatfish with speeds of 900 fish/hr. Misimi et al. [40] measured the pre- and post-
rigor changes in the size and shape of Atlantic salmon fillets as a result of perimortem
handling stress. Costa et al. [41] used external shape analysis of cultured seabass to sort by
size, sex, and skeletal abnormalities at a rate of about 10 fish per second. Since female
seabass grows faster, farmers screen for the sex of the fish. Elliptic Fourier analysis combined
with multivariate techniques (partial least squares) helped in developing techniques to
estimate the size (from weight, r = 0.977), by sex (82% accurate) and by malformations,
mostly ordosis (88.2% accurate). Loy et al. [42] tested outline fitting methods and compared
geometric morphometrics to determine fish shape variability for the sharpsnout seabream. To
replace the manual determination of landmarks on the perimeter, they tried Elliptic Fourier
Analysis and Bezier functions. The former was more accurate and could be automated. The
Bezier method performed poorly.
Ling and Searcy [43] developed a MV based shrimp deheader. Kassler et al. [44] reported
on an automatic grading and packing system for prawns at speeds of 20 prawns per second.
The system recognized the prawns head, as well as legs, back and tail.
There is much work completed in the area of the shape analysis of oysters. The two shells
are joined at the hinge by a complex, elastic ligament. The irregular shape is quite different
from oyster to oyster and presents challenges in MV. Diehl et al. [45] measured the geometric
and physical properties of raw oyster meat for grading purposes. Lee et al. [46] reported on an
automated system to analyze the shape of oysters for grading. Li [47] and Li and Wheaton
[48] developed methods to detect oyster hinge line using image processing. This is a
preparation for automated oyster shucking. The hinge is usually pointed and narrow when the
oyster is observed from above. Little et al. [49, 50] reported on automated oyster shucking, by
correctly orienting the oyster for an automated device. This device conveyed, oriented and
transferred the oyster to the next process stage without losing orientation. So and Wheaton
[51] developed software for the detection of oyster hinge lines: it contained code that was
both commercially available and also developed by the authors. It used circularity,
rectangularity, aspect ratio, and Euclidian distance to distinguish the hinge. Depending on the
geographical origin, of the oyster there was a 4 to 24% misclassification rate. Tojeiro and
Wheaton [52] also worked on oyster orientation by machine vision. They used a black-and-
white camera, and a mirror to collect both the top and side views of the oyster at the same
time. The software that they developed calculated two width-to-thickness ratios taken 1.5 cm
from each end of the principal axis. They obtained a correct orientation rate of 98%. Xiong et
al. [53] developed a method to characterize the shape of oysters based on detecting the
contour of the oyster, and placing 50 points on this contour. Then the turn angle cross-
correlation method was used to categorize the oysters to 2 different groups. This method
allows for the grading of oysters not only by diameter and weight, but also based on shape.
Area and Volume, Weight
View area (or shadow area, projected area) is another easily measured parameter from
machine vision. After segmentation, the number of pixels in an object can be corrected by a
size reference of known number of pixels, and therefore the view area can be reported in units
of area, e.g., cm2. There is advantage in measuring view area. Length (L) measurement can be
difficult for bent objects, but area is not affected by this. Area (A) has the units of length
squared (L2). Since volume has units of length cubed, or L3, then the estimation of volume
from area requires that area is raised to the power of 3/2. Once volume is estimated, weight
can also be estimated for most seafood.
Stien et al. [54] measured the contraction of rainbow trout fillets during rigor using image
analysis. Rigor development in parts of the fillet differed in magnitude from that of the whole.
Isolated muscle strips showed a similar contraction to that of the same muscle part measured
by image analysis of the whole fillet (r = 0.70, p < 0.0001).
Balaban et al. [55] measured the view area of shrimp (intact, deheaded, deheaded and
peeled, and meat only) and correlated this information to the weight (W). Linear (weight = a
+ b area), Power (weight = a exp(b area)) and forced power (weight = a (area)b where the
exponent b was taken as 3/2 = 1.5) functions were tested. The lowest R2 was 0.93 for all
forms of shrimp tried. Once the weight is accurately estimated, then the count (number of
shrimp per kg) and uniformity ratio (weight ratio of top 10% to bottom 10% shrimp) can be
calculated accurately. Recently, Pan et al. [56] performed a similar study to predict the weight
of shelled shrimp. Instead of using area only, they included the area, perimeter, length and
width of shrimp into their model to estimate weight. An artificial neural network was used for
prediction and an average relative error of 2.67% was achieved.
Gm and Balaban [57] measured the view areas of rainbow trout from 3 different farms
in Turkey by image analysis, measured the weight of the fish, and developed correlations to
predict the weight of the fish from the measured view area. Linear, power, and second order
polynomial functions were tried. Power correlation resulted in R2 = 0.99, with the fitted value
of the coefficient b in the neighborhood of 1.43 (theoretical value = 1.5). The same method
was used by Balaban et al. [58] to predict the weight of Alaska salmon of different species
(pink, red, silver and chum salmon). Linear, exponential, logarithmic, power, and other
expressions were used to correlate the view area to the weight. The power equation resulted in
the highest R2 values for silver salmon, where the lowest R2 was 0.948 for pink salmon. Since
pink salmon males tend to develop a hump, which does not exist in females, this may explain
the lower R2 for this species.
Balaban et al. [59] used the same method to correlate the view area of Alaskan Pollock to
its weight. Again, the power fit gave the best R2 (0.99). Since the fins and the tail are very
different from the body in thickness, they may affect the correlation of view area vs. weight.
To evaluate this, the images of the Pollock were used as is, or with the tails erased, or with
both fins and tails erased and the equation fitting to weight was repeated. Interestingly,
although the parameters were different as expected, the R2 values for all power fits were 0.99.
Therefore, the existence of fins and/or tail did not affect the accuracy of the weight
prediction. However, the b value changed from 1.47 for intact fish, to 1.51 for the fish with
tails and fins removed.
Alaskan Pollock roe weight could be estimated from image analysis [60] by using the
power fit to weight vs. view area data. A R2 = 0.97 was obtained. In addition, the image
analysis program could identify single and double roes.
Mathiassen et al. [7] used a 3D laser triangulation system at speeds of 1 m/s to estimate
the weight of whole herring. View area, length, width, middle cross-sectional area, maximum
cross-sectional area, volume, and thickness were used as parameters in the weight estimation
model using a linear combination of the parameters. They reported that this resulted in a mean
square error of 4.6 g for fish weighing an average of about 290 g.
Volume can also be estimated by image analysis. In this case a top view image and a side
view image are necessary. The cubic spline method to calculate cross-sectional areas from
these two images and their integration to calculate the volume has been discussed by Damar
et al. [61] to estimate the volume of oysters. R2 values of 0.92 were obtained. Aliek and
Balaban [62] also used the same method to calculate the volume of green-shelled mussels,
and obtained a R2 value of 0.97. Balaban et al. [63] used the cubic spline method to calculate
the volume of whole Alaskan pollock fish. A R2=0.99 was obtained between measured and
predicted volume.
Lee et al. [64] used laser triangulation method to obtain the 3D shape of oyster meat and
combined it with the 2D description of area projection to reconstruct the volume. They found
that the 2D approach resulted in an average volume error of 2.55 cm2, while the 3D methods
average error was 1.4 cm2, an error reduction of 41%.
Defects, Grading, Quality
A distinct advantage of MV and image analysis is the flexible detection and

quantification of visible defects. The product can be sorted and graded based on different
criteria that can be programmed into the MV system.
Hatano et al. [65] worked on the standardization of the fall chum salmon quality by
image processing. One of the main quality attributes of wild Pacific salmon is
watermarking during the last stages of its migration back into fresh water. Flesh redness of
the fish was estimated from the spawning coloration of the skin, by separating the surface to
different sections. Oliveira et al. [66] reported on a MV based grading of watermarked pink
salmon by skin color. A specific region on the fish image was taken to evaluate the average
L* value, which was used to grade the fish into bright, semi-bright and dark categories. These
are the current categories that are used in manual grading of watermarked salmon.
Roth et al. [67] reported that blood residue due to improper exsanguination of farmed
turbot affected the visual appearance of the fillets. Blood tended to accumulate in the lower
fillet due to gravity, and caused more redness and this effect could be quantified by MV.
Marty-Mahe et al. [68] quantified the quality traits of brown trout cutlets by image
analysis. They measured flesh color, visible fat, and myomera. The results were compared
with lipids measured by Soxhlet and NMR. The correlation coefficient between % lipids and
L* value was 0.77. Misimi et al. [15, 69] used computer vision to quality-grade Atlantic
salmon fillets according to their color level. They found no significant difference between
visual evaluations of fillets by human inspectors using the Roche SalmoFan standard and
the colors measured by machine vision.
Korel et al. [70, 71] evaluated the quality of raw tilapia fillets and raw and cooked catfish
using MV and electronic nose. The storage time (up to 12 days) was well correlated with the
changes of specific colors, and the combination of e-nose and visual attributes resulted in the
best correlation.
Kohler et al. [72] used machine vision to evaluate the color of salted cod fillets to grade
them into two categories. Experienced workers first selected representative samples from both
categories. The soft independent modeling of class analogies (SIMCA) method was used. It
was found that the red color variance histogram and the gray level histograms showed
promise and had low misclassification rates.
Croft et al. [73] introduced an intelligent herring roe grading technology to a practice that
was done manually and subjectively. The grade of the roe is influenced by shape, texture,
color and weight. An intelligent decision making system was developed by first acquiring
knowledge from grading experts and off-line experiments. Then fuzzy logic rules and model
matching procedures were developed. A prototype grading machine developed achieved a
grading accuracy of 85 to 95%.
Parr et al. [74] used MV to grade raw oyster meat into three weight categories. Their
system achieved 88% accuracy, and could evaluate one sample every 2 seconds.
Timmermans et al. [75] used a MV system to evaluate the color of crawfish shells. It is
known that their color changes as molting approaches. By focusing on a strip of the tail
section, and by using the average red/green and red/blue ratios, they developed a method that
predicted molting to within 3 days, and reached accuracies of 80%. Notwithstanding, they
also noted that there was a wide natural variation in the shell color of crawfish.
Species
Zion et al. [76] developed an image-processing algorithm to sort three species of fish
based on moment-invariants together with geometrical considerations. The method was
insensitive to fish size, orientation, and location of the camera relative to the fish. Carp, St.
Peters fish and grey mullet were pictured in a light box under different lighting conditions
and at different positions and orientations. Based on moment invariants, the identification of
grey mullet was 100% accurate, those of carp and St. Peters fish were 98 and 96% accurate,
respectively. They reported that fish weight and length could be obtained from images with
accuracies better than 0.95.
Strachan and Nesvadba [77] worked on the recognition of the fish species by shape
analysis of images obtained from MV. A data bank of shapes from pictures was created for 7
fish species. They tested 3 different methods to discriminate between the shapes. The
invariant moments method sorted the fish correctly 73% of the time. The optimization of
mismatch method was accurate at 63%. Shape descriptors method worked the best at 90%
accuracy. They recommended more work to make this method commercially acceptable.
Arnarson [78] listed the difficulties of sorting the fish by computer vision: the fast speeds
required, the multitude of fish species to be sorted and the variations in the form, size and
shape of the species, the batch flow of the processing lines, the harsh environment in the
factories (wet, vibrations, uneven lighting, etc.), variations in the optical characteristics of the
fish, and the elastic nature of the fish. He listed the required level of complexity of the vision
system (Table 2) and mentioned that in the industry practical levels of sorting included levels
2-4. Arnarson [78] also pointed out to the inverse relationship between the cost of the feeding
system and the cost of the MV system as the complexity of the MV system increased.
Storbeck and Daan [79] used a neural network to recognize fish species by machine
vision. The widths and heights of the fish at various locations were measured perpendicular to
a conveyor belt using a camera-laser system. These and the information regarding species
were input to a neural network. After training and optimization of the neural network they
obtained correct recognition rates of 95%.
White et al. [80] described a MV system (The CatchMeter) where the fish pass on a
conveyor belt under a camera. By processing the images, the system can determine the
orientation of the fish using moment-invariant methods, can differentiate between a flatfish
and a round fish with 100% correctness, measure the length of the fish with a standard
deviation of 1.2 mm, and can recognize and sort 7 species of fish with 99.8% correctness.
They estimated that the system could process up to 30 thousand fish per hour.
Kuo and Tewfik [81] developed a method to classify rock sole subspecies using the
contour and stripes of the fish. The contour was transformed to a one-dimensional shape
signature as a first criterion. Then, a pattern-enhancing algorithm transformed the pattern to
the spatial frequency domain. Their method correctly recognized all 15 southern rock soles,
and 12 out of 13 northern rock soles.
Table 2. Required level of complexity in a vision sorting system

(according to Arnarson [78])
Complexity Multi-
Feeding system to MV sorting
level processing
1 Objects oriented and there is a minimum distance between No
objects
2 Objects not oriented and there is a minimum distance No
between objects
3 Objects oriented, no minimum distance, not overlapping No
4 Objects random and not overlapping Yes
5 Objects random and overlapping Yes
Williams et al. [82] developed segmentation methods to automatically detect salmon in

video images taken underwater, in fish farm cages. The original image was histogram-
equalized, edge-detected (Sobel filter) and background removed, and segmented. An active
shape model was applied by creating a salmon shape model. The performance analysis of the
system revealed that of the 1122 fish identified by eye from the video, only 125 (11%) could
be automatically detected by the system.
McCarthy [83] reported on the development of a fish sorting system using MV. A
prototype system was installed in a fish processing plant. It included a conveyor belt, a light
table, and a system to control chutes and gates to direct the fish to different filleting lines
based on species and size. The contour of the fish was taken, its length and weight estimated,
and was routed to the filleting line. The system could handle 6 tonnes of cod per 8 h shift.
Lee et al. [84] noted that for fish recognition shape is a very important characteristic.
Although finding critical landmark points on fish shape using curvature function analysis has
been satisfying, the main difficulty of this approach is in accurately locating these landmark
points. Therefore, they used whole shape matching for fish recognition. Shape descriptors,
such as Fourier descriptors, polygon approximation and line segments, were tested. A power
cepstrum (inverse Fourier transform of the log of the estimated spectrum) technique was
developed to improve the categorization speed using contours represented in tangent space
with normalized length. They found that Fourier descriptors using bend angle function had
the highest recognition accuracy of 64%
Han and Tewfik [85] developed a method to evaluate the shape of two crab species and
their hybrids. Morphologic characteristics were determined by empirical covariance matrices
of the two species and their hybrid. They concluded that for the crab classification the
graphical classifier that uses the bias estimator and the scaled principal components performs
better than the original Eigen image classifier.
APPLICATIONS TO RAW SEAFOOD

Skin and Meat
Tuckey et al. [86] looked at the effect of storage temperatures (4oC and 0.3oC) on the
muscle biochemistry and color (skin and fillet) of snapper. Tissue biochemistry significantly
correlated with fillet color. Redness (a*) and yellowness (b*), which decreased rapidly during
the first 24 hours, could be used as an indicator of muscle biochemistry. They also used a
novel laser penetration method to evaluate translucency and therefore muscle ultrastructure.
In another experiment, changes in skin color of snapper and gurnard were followed
during storage for up to 12 days in refrigerated temperatures. For both fish the a* and b*
values decreased. The L* value of snapper did not change significantly, while that of gurnard
increased slightly [87].
Eyes, Gills
The colors of eyes and gills are often used to evaluate the freshness of fish, e.g., in the
Quality Index Method. These can be measured by MV; however, the accuracy of the results
depends on the method of measurement. Since both eyes and gills are very reflective, using
polarized light should eliminate the biasing of the color by specular reflection. Aliek and
Balaban [88] used polarized lighting in a lightbox to measure the increase of L* over time for
the eyes using a circular region of interest (ROI), and the decrease of a* of the gills using a
polygonal ROI.
Dowlati et al. [89] used a MV system to capture the images of stored gilthead seabream.
They developed software to automatically select the appropriate ROI for gills and eyes. The
L* value of eyes increased, and the a* value of the gills decreased. Regression and neural
networks correlated gill color change better than eye color change with storage, maybe
because of the effect of specular reflection on the eyes.
APPLICATIONS TO PROCESSED SEAFOOD

Cooked
Kong et al. [90] cooked pink salmon in a specially designed heating cell and looked at
many aspects of the heating induced changes, including color and shrinkage. Color and
shrinkage were measured by image analysis. The L* value increased sharply and the a* and
b* values were reduced drastically. Area shrinkage ratios reached 25% after heating at 121C
for 120 min.
Omar and da Silva [91] applied the optimal portion control methodology that they
developed to an industrial fish canning process. They presented an example on a batch of
salmon, and discussed filling accuracy and meeting regulatory requirements. Water-
immersion and a laser-based position transducer measured the cross-section of fish to be
placed in the cans.
Morioka and Ueda [92] described a cooking support system using cameras and
projectors. They give an example of cutting a fish into 3 fillets. The system recognized the
fish (position, size, direction), and directed the user to where the fish should be cut based on
the cooking method selected, by projecting the suggested cutting onto the fish on the counter.
Minced Fish
Kse et al. [93] measured the color of whiting burgers containing pre-cooked minced
product, with other ingredients added. Differences detected by machine vision between
average surface colors of samples with different preparations and different additives were
significant after mincing, but decreased after addition of ingredients, and after cooking.
Salted and Smoked
Salting and smoking change the color and surface characteristics of fish and other
seafood. Aliek and Balaban [94] measured the polarized and non-polarized color of hot
smoked salmon heads and quantified the shine that developed as a result of processing.
When fish such as salmon is dry brined, its color darkens as a result of water loss. Liquid
smoke application further changes the color. Aliek and Balaban [95] dry brined, liquid
smoked and High Hydrostatic Pressure (HHP) treated mussel meat, and measured the color
changes. They also reported changes in the view area as a result of water loss during brining.
CO Treatment
Carbon monoxide binds to the iron in the hemoglobin and myoglobin molecules strongly,
and results in a cherry-red color [96]. This has been used in fixing the color of fish where red
color is desired, such as tuna [97, 98]. The red color can actually increase over and above the
level for fresh fish. There is also the possibility of enhancing the color of already browned
fish to red by CO treatment [99]. These color changes have been quantified by the use of
machine vision and image analysis. For fish such as tilapia that has a red center stripe in the
fillet, the retention of the red color has been achieved by CO [100]. It was also demonstrated
that euthanasia of tilapia by CO had the advantages of humane treatment, even distribution of
CO to the flesh without filleting, and self-limiting the level of CO exposure because when the
fish dies the respiration of CO and its distribution into the flesh stop [101].
High Hydrostatic Pressure
HHP is used increasingly in seafood. Oyster and mussel shucking by HHP has been
commercially applied, with a side-benefit of reducing microorganisms in the final products.
An advantage of this method is the minimal shrinkage in the meat, which renders the
appearance of HHP-treated shellfish more appealing for half-shelled products. Kim et al. [8]
measured the percent area of the mussel shell covered by meat using image analysis, and
found that while heat-treated products had a meat area of about 41% of the shell, that of HHP
treated mussels reached 80%.
HHP causes protein denaturation and therefore affects color and appearance. Yaz et al.
[102] quantified the color change in HHP treated rainbow trout and mahi mahi. L* values
increased significantly with pressure, while a* values decreased significantly. Yaz et al. [5]
also studied the color changes in Atlantic salmon treated with HHP using image analysis.
Again, as pressure increased, the L* and b* values increased, and the a* values decreased.
CONCLUSION
As more data is accumulated on the morphometric, geometric and other physical
properties of seafood, as the speed and capabilities of the hardware for image acquisition and
processing is increased, as the sophistication and capabilities of software for image analysis is
enhanced, and as more evidence of the reliability, objectiveness, speed and economic
advantages of using machine vision/image analysis become more evident, it is expected that
this technology will spread more into the industry and will be studied more in the research
fields.
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Chapter 5
PROTEOMICS AND APPLICATION IN

THE SEAFOOD INDUSTRY
Jinru Zhou1, Linglin Fu1, Yan Zhang2 and Yanbo Wang1,*

1
Key Laboratory for Food Microbial Technology of Zhejiang Province, Food Quality
and Safety Department of Zhejiang Gongshang University, China
2
Hebei Food Inspection and Research Institute, Shijiazhuang, China
ABSTRACT
In the last decade, proteomics technologies have been applied and discussed
primarily in the biomarkers investigation about diseases of human physiology,
reproduction toxicity mechanism in medicine fields. However, the application of
proteomic tools for the investigation of seafood and other marine products has been
scarce. In the present review, the state-of-the-art and future trends of the application of
proteomics in the seafood industry are presented carefully. Based on the current related
studies, the lack of completed genetic information on most fish species has been the
major drawback for a more general application of the different proteomic technologies. In
addition, this review also describes the future status of the proteomics technologies
development related with the seafood industry.
Keywords: Proteomics, seafood industry, fish, shellfish
INTRODUCTION
Proteomics is a research field focused on the analysis of protein fractions expressed by
organisms, tissues and cells, contributing to a better knowledge of the biochemistry and
physiology of organisms challenged by a given environmental stimulus. Diagnostic,
predictive, and prognostic biomarkers are required in measuring the progress of disease and
*
Corresponding author: 18, Xuezheng road, Xiasha University Town, Hangzhou, 310018, China. Tel.: +86-571-
28008963. E-mail: wangyb@mail.zjgsu.edu.cn (Prof. Wang).
88 Jinru Zhou, Linglin Fu, Yan Zhang et al.
the effects of treatment for better clinical outcomes in patients. The proteome is considered a
rich source of biomarkers. Therefore, sizable time and funding have been spent in proteomics
to develop biomarkers [1]. Proteomic tools also are used in food toxicology. Zhang et al. [2]
showed that differential proteomics had the potential to understand reproduction toxicity
mechanism in marine molluscs through the Hg-contaminated food chain. Besides, proteomic
tools are widely used in the drug industry. Targeted proteomics is now emerging as a superior
method to quantify proteins, including membrane transporters. Prasad et al. [3] showed the
quantification of drug transporters in tissues and cells by MRM Proteomics.
Moreover, the application of proteomic tools for the investigation of seafood and other
marine products has been scarce. Some authors have pointed out that the fish production in
2020 will have to multiply 7-fold to satisfy consumer demand across the planet [4]. It is
evident that because human eating habits will be significantly based on products of aquatic
origin, seafood safety will be a major challenge to be faced by humankind in the new century
[5]. The application of proteomics brings the opportunity to develop new methods in order to
ensure the safety during food production, storage, delivery and consumption. Several
approaches to achieve this goal have been used in the seafood industry at the molecular level,
including biochemistry, molecular biology and more recently genomics.
ART OF PROTEOMICS
The definition of proteomics is that it is a large-scale analysis of proteins in a particular
biological system at a particular moment in time [6]. Not only is proteomics a study of
structure and function of proteins, but also proteomics is the analysis of protein modifications,
the interactions between them, their intra-cellular location and the quantification of their
abundance. The proteome technologies are associated with many different disciplines of
science and include mass spectrometry (MS), electrophoresis.
Two-Dimensional (2D) Gel Electrophoresis
Two-dimensional gel electrophoresis (2-DE) is the most common proteomics technique

in which proteins are first separated along a linear gel according to isoelectric point, and then
separated on a polyacrylamide slab gel according to molecular weight. This technique can
separate and detect hundreds (and sometimes thousands) of different proteins [7]. The two
classical methods, coomassie brilliant blue (CBB) and silver staining, are used in 2-DE to
enable estimation of protein quantity by scanning 2-DE gels in the visible range.
However, 2-DE is a time-consuming and labor-intensive process. The two inherent
limitations of it concern hydrophobic and alkaline proteins, which are therefore often
underrepresented in Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).
Besides, this technique has low dynamic range and gel-to-gel variability. Recently, the
development of multiplex 2-DE (dubbed difference gel electrophoresis or DIGE), which
instead involves tagging the protein samples with different fluorophore prior to 2-DE, not
only allows several samples to be run on a single gel, but also significantly improves gel-to-
gel variability by providing a common reference channel across all gels of an experiment [8].
Proteomics and Application in the Seafood Industry 89
Another development is immobilized pH gradients (IPGs) technique. It overcomes the

limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling,
resolution and separation of very acidic and/or basic proteins [9].
Free-Flow Electrophoresis
Free-flow electrophoresis (FFE) has gained a role as a useful preparative and analytical
tool for proteome analysis. FFE is a liquid-based, matrix-free separation technology. An
electric field is applied perpendicular to an aqueous fluid flowing laminarily through a gap
formed by two plates. On one end of the gap, a sample solution is injected continuously into
the carrier buffer as a narrow band. In the electric field, sample components are separated
laterally according to their surface charge and collected when leaving the gap at the other end
[10]. Several FFE instruments such as the Elphor VAP (Germany), the ACE710 (USB), the
Octopus FFE system (Hong Kong) are developed to resolve difficulties, as for instance, the
accumulation of Joule heat [11, 12], sample wall contacts during injection [13], sample
precipitation, dispersion, and penetration of the electrode reaction products into the separation
chamber. However, gel-based methods still have some limitations, such as the separation of
hydrophobic and poorly soluble proteins and the limited sensitivity of the available detection
methods [14]. Nevertheless, we believe that both the gel-based and gel-free methods will
continue to prove useful in the long term.
Mass Spectrometry
Mass spectrometry (MS) has been widely used as an analytical technique in the life
sciences and has played a critical role in the high-throughput identification of proteins. Mass
spectrometry consists of three basic components (an ion source, a mass analyzer and an ion
detector) and measures, with extremely high sensitivity, the mass to charge ratios (m/z) of
gas-phase ions [15].
Liquid chromatography (LC) (coupled to mass spectrometry (LC-MS)) is a powerful
fractionation method. Actually LC can be compatible with any type of mass spectrometers.
The LC-column is used so sensitive that large amounts of analytes can be separated on it. The
sorbent materials of the column are various, and have distinct physical, chemical, and
immunological properties. The peptides in the sample are eluted and separated at different
moments in time depending on their separation characteristics, when the sample moves
through the column [16, 17].
LC-MS/MS technology is a good choice for quantitative proteomics [18]. Shotgun
proteomics strategies are developed on the basis of this approach. In the shotgun proteomics
strategies, several on-line or off-line multidimensional chromatographic steps are used to
provide the separation power required for whole proteome analysis by MS. In LC-MS/MS,
the most frequently used chromatographic method is ion exchange chromatography combined
with a reversed-phase chromatographic separation.
For LC-MS/MS quantification, isobaric tags for relative and absolute quantitation
(iTRAQ, Applied Bio-systems) have several advantages over other labeling methods. Firstly,
coverage of the proteome is increased because peptides are labeled on the amine group, and a
larger number of peptides can be simultaneously labeled, processed, and statistically

compared across groups. Secondly, multiple samples can be labeled and analyzed in the same
experiment allowing direct comparison of samples in the same experiment. Only iTRAQ has
been used to quantify proteome differences, in this case to determine the effects of endocrine
disruptors on the fish liver proteome [19].
Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass
spectrometry recognized as an indispensable tool for proteomics studies [20] allows for the
transfer of large, polar, thermally labile biomolecules into the gaseous phase for mass analysis
without prior derivatisation. It has made mass spectrometry a viable tool in biology,
biomedicine and molecular medicine [21].
Ionization by MALDI is connected with a protein suspended or dissolved in a crystalline
structure (the matrix) of small, organic, and UV-absorbing molecules. The energy used to
ionize the protein comes from the crystal which absorbs energy at the same wavelength of the
laser. The laser energy strikes the matrix to cause rapid excitation of the matrix, and then the
matrix and analyte ions are subsequently passed into gaseous phase. The principal ion
detected using threshold laser intensity for MALDI is, although signals for multiply charged
ions and oligomeric forms of the analyte may be seen, especially for large proteins. The
ionized protein is accelerated by an electrostatic field and expelled into a flight tube. As it
exits the flight tube, the mass analyzer is encountered. The analyzer is often a time-of-flight
(TOF) analyzer [22, 23]. Two TOF systems were constructed. The first system utilized a
digital wave memory and accumulation circuits. This system could accumulate the spectrum
data of 8 K words within 1 ms. In the first place, a "one shot" TOF spectrum was stored into
the wave memory, in the subsequent accumulating the spectrum was accumulated in
sequence. The second system utilized a constant fraction discriminator (CFD) and a multi-
stop time-to-digital converter (TDC).The time intervals between start and stop pulses
were measured with a time resolution of 1 ns [24].
MS imaging has good potential to become one of the invaluable tools in aquaculture
research. This technology involves the direct digestion of histological samples fixed to a
suitable support, followed by direct MS/MS (Two stage mass spectrometry) analysis (for
example, by applying a matrix solution on the sam and doing Matrix-assisted laser
desorption/ionization-time Mass spectrometry (MALDI-MS) all over the surface, point by
point). This technology (MS imaging) uses immunocytochemistry methods and provides
information on a key variable: location. MS imaging doesnt necessarily require protein
identification: using computational methods (dimensionality reduction methods, classifiers,
neural networks and similar statistical machine learning techniques) its possible to map all
the MS and MS/MS information obtained for each point in space as a pixel, where color
information is defined so that it reflects the similarity relations between proteomes. So, MS
imaging can provide useful information on proteome distributions over any tissue (regardless
of source), distinguish between sub-populations of cells with different proteome profiles and
pinpoint exactly where proteome changes occur [25].
Data Compression
Data compression is an essential technique, because the raw mass spectrometry data are
getting larger and larger. The general file compression algorithms and/or programs do not
provide the optimal compression ratio while allowing the direct access to individual spectrum
of the compressed data. Nowadays, there have been efforts on developing better compression
tools specifically for mass spectrometry data [26, 27]. Some tools support both lossy and
lossless compression, such as vector quantization [28], transform-based methods [29], and
fractals [30]. It is reasonable to omit the less important ones and encode the rest for lossy
compression. This technique preserves the quality of the reconstructed image as much as
possible. The proposed compression method has three independent levels for lossy
compression, each of which further increases the compression ratio. We may use only a
number of these levels depending on the desired compression ratio [31].
Table 1. Research and development (R&D) applications of proteomics in fish
Aim and/or Conclusion

Author Species
Scomber The higher lability of sarcoplasmic proteins under high-pressure

Pazos et al. [35] processing treatment enhances the sensory quality
scombrus
Zebrafish proteomic approaches can aid in our understanding of proteins
Gebriel et al. [36] Danio rerio central to important neuronal processes and neurodegenerative disorders
Paralichthys The expression levels of 82 proteins involved in immune responses and

Cha et al. [37] other cellular activities were altered by the bacterial pathogens
olivaceus
A 96-h exposure to 1 mg perfluorooctane sulfonate (PFOS) per L
significantly altered the activity of mitochondrial abundance (CS and
Dorts et al. [38] Cottus gobio
CCO); Gills from the control fish group and tissues from fish exposed for
96 h to either 0.1 or 1 mg PFOS per L were all compared using 2-DE
The acquired protein expression data partially confirmed the existing data
Groh et al. [39] Danio rerio on mRNA expression in the zebrafish gonads for several genes, including
three novel transcripts
Proteins identified in gill of GBD-affected fish are related to oxidative
Salas-Leiton et Solea
alteration of cytoskeleton structure/function, motility, or regulatory
al. [40] senegalensis
pathways and showed the central role of gill in oxygen exchange
The changes in the testis proteome of wild-caught males and F1 males
Solea
Forne et al. [41] were investigated in order to identify proteins potentially involved in the
senegalensis
low production and fertilization capacity of the sperm
Xiphophorus The proteome has been analyzed towards the search for proteins involved
Lokaj et al. [42] in the malignancy of tumors produced and/or induced in the present fish
sp.
Identify intact and/or proteolytic fragments of muscle specific gene
Perca
Reddish et al.[43] products that may be involved in muscle growth using proteomic analysis
flavescens
methods
The normalized volumes of different proteins showed three statuses
Schiavone et al. Sparus (skeletal alpha-actin and tropomyosin were not affected; myosin light
[44] aurata chain 3 and major histocompatibility complex (MHC) class II beta 1
increased; Sec 13-like and par-albumin decreased)
Some of the identified proteins may play a role in homolog processes
Zupanc et al. [45] Salmo salar occurring in mammals
Kjaersgrd et al. Gadus Eleven protein spots with significant differences were found within the
[46] morhua partial proteome in post-mortem cod muscle
APPLICATIONS OF PROTEOMICS IN THE SEAFOOD INDUSTRY

Proteomics methodologies have been recently proposed as faster, more sensitive and
higher-throughput approaches for the assessment of the authenticity and traceability of
species in fishery products [32-34].
Table 2. Research and development (R&D) applications of proteomics in shellfish
Author Species Aim and/or Conclusion

Sun et al. [47] Chlamys farreri The comparative proteomics analysis revealed the
modulation of inducible nitric oxide on the immune
response of scallop
Fernndez-Cisnal et al. Procambarus clarkii Specific cysteine residues were found for the evidence of
[48] reversible oxidation and could be used as markers of
exposure using redox proteomics methods
Zhang et al. [49] Crassostrea The proteins including 14-3-3 protein, GTP binding
angulata protein, arginine kinase and heat shock connate protein are
all potential biomarkers of reproduction toxicity to oysters
and of Hg contamination to humans and other mammals
after bioaccumulation of Hg through the food chain.
Campos et al. [50] Mytilus Proteins identified in the 2-DE gels are involved in energy
galloprovincialis production and carbohydrate metabolism, metal transport,
chaperones and stress response, cell signaling and
regulation, proteolysis and protein transduction
Zhu et al. [51] Saccostrea cucullata Serine/threonine protein kinase are promising indicative
proteins for the reproductive dysfunction evaluation and
might be treated as the biomarkers for Cd contamination in
mammals and human by food chain.
Silvestre et al. [52] Penaeus monodon The haemolymph protein expression are overwhelmed by
the effects of the conditions encountered in different
oxygen and nitric concentrations using a differential
proteomic approach
Furey et al.[53] Mytilus edulis Azaspiracid poisoning (AZP) is more dangerous than the
other known shellfish toxins and can cause serious tissue
injury
Cao et al. [54] Crassostrea gigas The susceptibility to bonamiosis is associated with
haemolymph protein expression patterns of the resistant
Wang et al. [55] Artemia franciscana Dehydrated cysts actually store more proteins, in both type
and amount, than developing cysts
Chongsatja et al. [56] Penaeus vannamei The C-terminal and N-terminal haemocyanin fragments
may have differential roles in haemocytes using proteomic
analysis methods
Martinez and Friis [57] Pandalus borealis The markers for post-mortem muscle deterioration in
shrimps has been reported and some spots can be used as
markers to estimate the freshness using proteome analysis
Yu et al. [58] Penaeus monodon A novel allergen, designated Pen M 2, was found using
proteomics and immunological analysis and could be used
in allergy diagnosis and the treatment of crustacean-
derived allergic disorder
Fish
Proteomics approaches are easily applied to model or non-model species in the main
areas of research with impact in aquaculture, namely welfare, nutrition, health, quality or
safety. Research and development (R&D) applications of proteomics in fish dealing with
those areas above is shown as Table 1.
Shellfish
Proteome techniques have been used occasionally to study the changes and variety in
protein expression between shellfish populations from different environments, especially
biotoxin contamination [49, 51, 53]. Besides, the interest in the study of shellfish proteomics
has been prompted by the need for better control of diseases in shellfish aquaculture and the
ecological importance of shellfish in marine ecosystems. Table 2 shows a number of research
and development (R&D) applications of proteomics in shellfish.
CONCLUSION
The growing application and importance of proteomics technologies in the seafood
industry is discussed in the present review. Proteome techniques have been used in the study
of a variety of subjects such as physiological function relevant molecules and mechanisms,
the biomarkers for aquatic organism welfare, and the tracking of quality changes and allergies
for seafood. Proteome techniques are also used for evaluations between aquatic organisms
and environmental pollution. Moreover, the proteomics technologies are very useful in the
assessment of the quality and safety of fish products. Especially, technologies used in
proteomics have been used to identify and detect pathogenic and spoilage bacteria.
However, to date the use of proteomics in the seafood industry has been limited. As for
the technology of proteomic, such as LC-MS/MS, MALDI-TOF, MS imaging, the integration
will definitely be the future research direction although it has its limitations. The limitations
of proteomics technology include the influence of sample preparation methods, the user
factor, the difficulty of ionizing certain peptides and other instrumental and analytical
limitations. Thus, the increasing automation, improvements in instrument quality and a
decrease in user intervention throughout the workflow would hopefully overcome the above
difficulties. In fact, the development of more cost-effective and sensitive technologies is
necessary to solve the above problems. For example, meta-proteomics and multi-dimensional
liquid chromatography will further enhance the value of proteomics to the field of seafood in
general and the seafood industry sector in particular. In addition, the use of new technologies
like MALDI imaging or protein array/protein chip approaches will greatly develop the
understanding of the biological processes that are pertinent to seafood industry. In addition,
lab-on-chips and protein arrays based on micro-fluid devices offer a promising area in
modern food science, wherein the proteomics can be implemented for routine control,
diagnosis and monitoring of fish products.
In short, the road ahead appears full of challenges as the databases of proteomic
information increase and more efficient and updated proteome techniques should become
available. This will require interdisciplinary collaborations between a broad range of sciences,
including those of physiology, cell biology and computer sciences, as well as from the
aquaculture and food industries.
ACKNOWLEDGMENTS
This work is supported by the National Science and Technology Support Program
(2015BAD17B01), the National Natural Science Foundation of China (No. 31571913 and
No. 31571770) and the Zhejiang Provincial Natural Science Foundation of China
(LY14C200001 and LZ15C200001).
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Chapter 6
QUALITY CHANGES IN FRESHWATER FISH

AND CRUSTACEAN SPECIES
Irineu Batista* and Carla Pires

Instituto Portugus do Mar e da Atmosfera (IPMA), Lisbon, Portugal
ABSTRACT
The current production of freshwater species shares a significant proportion of the
global world fisheries. In many regions, they are an important source of animal protein
and also add to their overall economy. Despite the large number of existing freshwater
species, the vast majority of their world production relies on a limited number of species.
This review deals with the research and development of the post mortem changes
occurring in the main freshwater species during refrigerated storage. The preservation
techniques used in different fish species and recent physical methods and chemical
indicators for fish freshness evaluation are presented and discussed. Special attention is
given to the most recent humane slaughter procedures developed for certain species such
as common carp, rainbow trout and Atlantic salmon.
Keywords: freshwater species, slaughtering, handling, preservation, freshness methods
INTRODUCTION
The total of the worlds fisheries accounted for 157.9 million tons in 2012, of which 91.3
million tons came from the capture fisheries and 66.6 million tons were from aquaculture
production [1]. The capture fisheries included an inland production of 11.6 million tons
whereas that from aquaculture was responsible for 41.9 million tons of inland production.
These figures highlight the current importance of aquaculture in the total fish supply and,
moreover, that the freshwater aquaculture accounts for about 63% of its total production. The
distribution of world captures of freshwater fish species by inland fishing area is shown in
* Corresponding author: Instituto Portugus do Mar e da Atmosfera (IPMA), Rua Alfredo Magalhes Ramalho, 6,
1495-006 Lisbon, Portugal; Email: irineu@ipma.pt.
100 Irineu Batista and Carla Pires
Figure 1a. The large majority of catches come from Asia representing about 68% of total
catches followed by Africa (ca. 23%) [1].
Table 1. Production of the main freshwater finfish and crustaceans in 2012 [1]
Species Output (tonnes) Value, 000 USD

Finfish
Cyprinidae Grass carp 5,028,661 6,464,586
(Ctenopharyngodon idella)
Silver carp 4,189,578 5,540,946
(Hypophthalmichthys molitrix)
Common carp 3,791,913 5,207,971
(Cyprinus carpio)
Bighead carp 2,898,816 3,723,608
(Hypophthalmichthys nobilis)
Catla 2,761,022 5,488,405
(Catla catla)
Crucian carp 2,451,845 2,674,406
(Carassius carassius)
Rohu 1,555,546 2,934,143
(Labeo rohita)
Wuchang bream 705,821 1,164,605
(Megalobrama amblycephala)
Black carp 495,074 1,148,527
(Mylopharyngodon piceus)
Mrigal carp 396,476 666,907
(Cirrhinus mrigala)
Cichlidae Nile tilapia 3,197,330 5,260,695
(Oreochromis niloticus)
Salmonidae Atlantic salmon 2,066,561 10,095,957
(Salmo salar)
Trout 855,982 3,631,521
(Oncorhynchus mykiss)
Channidae Northern snakehead 480,854 588,275
(Channa argus)
Siluridae Amur catfish 413,350 547,741
(Silurus asotus)
Ictaluridae Channel catfish 394,179 638,748
(Ictalurus puntactus)
Pangasidae Pangasius catfish 285,089 360,342
(Pangasianodon hypophthalmus)
Synbranchidae Asian swamp eel 321,006 837,781
(Monopterus albus)
Percichthyidae Mandarin fish 281,502 2,620,784
(Siniperca chuatsi)
Crustaceans
Varunidae Chinese mitten crab 714,392 4,972,498
(Eriocheir sinensis)
Cambaridae Freshwater crayfish 598,289 2,808,462
(Procambarus clarkii)
The distribution of freshwater aquaculture production by continent shows (Figure 1b) that
Asia production represents 93% of total production [2]. Nevertheless, it has also to be stressed
that China production contributes to about 65% of world freshwater production. As referred
to in that FAO report, finfish inland aquaculture is the most important sub-sector of
Quality Changes in Freshwater Fish and Crustacean Species 101
aquaculture production in terms of volume. It is also the source of protein food of reasonable
quality in many developing countries.
In 2010, the number of freshwater species with statistics in the FAO capture database
attained 190 [3]. However, the majority of the total aquaculture production relies on a limited
number of species. The dominant freshwater species of aquaculture production include
various carp species and other cyprinids, tilapias, salmons and trouts, pangasius catfish and
freshwater crayfish. The production and value of the main freshwater finfish and crustaceans
are presented in Table 1.
Figure 1. Distribution of world captures of freshwater fish species by inland fishing area (a) [1] and
aquaculture production by continent (b) in 2012 [2].
Freshwater species as their marine fish species counterparts undergo a variety of post
mortem metabolic, structural, and physical changes. The main metabolic changes include the
consumption of ATP, depletion of creatine-P and glycogen, lactate accumulation, pH fall, and
Ca+2 release. The contraction of muscle actomyosin, breakdown of high molecular weight
components and partial destruction of cellular compartimentalization are important structural
changes. As physical changes it may be mentioned the increase in brightness of muscle,
decrease in intensity of color, increase of water-holding capacity and tenderization of muscle.
These degradative processes are basically due to the action of different proteases. After the
autolytic phase a progressive bacterial growth takes place leading to the formation of the
typical fish smell until the total fish spoilage. This chapter intends to present the most recent
data published on the post mortem changes, slaughter methods and preservation techniques
used in the main freshwater fish and crustacean species.
POST MORTEM QUALITY CHANGES

Cyprinidae
Grass Carp (Ctenopharyngodon idella)

Grass carp is the most important freshwater fish species in terms of its catches (Table 1).
Several works on the chemical composition of this species have been published [4-9]. It was
observed that the supplementation of diet with lysine and methionine led to an increase of
whole body moisture and muscle protein content and to a decrease of lipid content of juvenile
grass carp [10].
The utilization of chlorinated ice in the preservation of grass carp reduced the mesophilic
and psychrotrophic bacterial counts as well as pH and TVBN [11] and could extend the shelf
life by around three days. Two slaughter methods (immersion in ice-water slurry and
electrical stunning followed by ice slurry asphyxiation) of grass carp tested by Scherer et al.
[12, 13] had no effect on the shelf life of this species. Wild grass carp lipids [9] were less
prone to oxidation than farmed fish such as also observed in silver carp. The combined effect
of sodium acetate and nisin treatments on the preservation of refrigerated grass carp slices
exhibited the highest efficacy in retardation of microbial growth [14]. The antilisterial effect
of nisin was enhanced with the increasing concentration of sodium acetate [15] and the
combination of these preservatives could be used to keep the freshness under refrigeration
storage of grass carp fillets. It was possible to predict the quality changes of grass carp stored
between -3 and 15C based on kinetics models [16]. The good relationship between
impedance change ratio (Q value) and traditional freshness allowed concluding that Q value
could be used as a valid index for freshness evaluation [17]. Niani et al. [18] observed that the
application of gelatin coating to refrigerated grass carp slices had a beneficial effect on the
preservation of EPA and DHA levels during storage. The superchilling preservation at -3C of
grass carp filets delayed the microbial spoilage and proteolytic degradation of fillets stored
when compared with those stored at 0C [19] but tissue structures were damaged. Biogenic
amines in several commercial fish and fish products, including grass carp, have been
determined [20]. Fresh grass carp samples presented low levels of total biogenic amines but
lightly cured samples had a mean total content of 116.77 mg/kg where cadaverine represented
75% of total amines. Grass carp fillets brined in the highest salt concentration (10% NaCl)
showed the lowest TVBN formation and also the lowest accumulation of some biogenic
amines such as tryptamine, putrescine and cadaverine [21].
Silver Carp (Hypophthalmichthys molitrix)

The production of silver carp has gained increasing importance in several countries
(Figure 2). A number of works on its chemical composition have been published [7, 22-26]
and a comparison of the nutritional value of wild and farmed silver carp was done [5]. The
effect of different culinary methods on the chemical composition of silver carp was also
studied by several researchers [27-33].
Figure 2. Silver carp (Hypophthalmichthys molitrix).
Notwithstanding, a limited number of recent works on the early post mortem changes of
silver carp is available. The treatment of iced stored silver carp with tea polyphenols led to
retention of the good quality characteristics for longer period of storage time and an increased
shelf life [31]. A study on the formation of biogenic amines in silver carp stored at three
temperatures (0, 3 and 15C) showed that low temperature could effectively inhibit the
production of these amines and putrescine was regarded as a good quality marker during
refrigeration storage [32]. Khidhir et al. [33] showed that farmed silver carp had higher
thiobarbituric reactive substances (TBARS) content, peroxide values and free fatty acids than
wild fish, which could be related with farming conditions, including nutrition factors. High
correlations were obtained between electrical conductivity (EC) and several physical and
biochemical parameters of silver carp after 4 hours post mortem [34]. These authors
concluded that EC could be used as a rapid indicator to evaluate the quality of fish stored at
0C for 4-72 hours. The effect of salt and sucrose on the rigor mortis changes in silver carp
was studied [35]. It was concluded that fish sprinkled with salt or a mixture of salt and
sucrose, packaged and stored in refrigerated incubators at 4C had an improved quality during
the post mortem process. Predictive models of the quality changes of silver carp stored at
different refrigerated temperatures indicated that EC, total aerobic count (TAC), and total
volatile base nitrogen (TVBN) followed a first-order reaction equation but sensory score
followed a zero-order reaction equation [36]. Chitosan coatings applied to super-chilled silver
carp could keep the good quality characteristics and extend the shelf life during storage at -
3C [37]. The higher TBARS values recorded in freeze-thawed silver carp fillets when
compared with fresh fillets allowed concluding that the refrigeration storage of freeze-thawed
fillets of this species could not be recommended [38]. The structural changes of silver carp
myofibrillar proteins during frozen storage were recently studied [39] and it was shown that
the pre-treatment with ascorbic acid and citric acid reduced lipid changes and improved the
water holding capacity of proteins [40].
Common Carp (Cyprinus carpio)

A wild population of common carp in the Danube is assumed to be originally a European
species and was introduced throughout the world and nowadays it can be found in all
continents. An extensive review of the main aspects related to growth and quality of common
carp dates back to the middle of the 1990s decade [41] Additionally, the fish lipid content of
wild common carp caught in Lake Naivasha (Kenya) was studied [42] Other studies on the
chemical composition of this farmed species were also published in recent years [7, 8, 43-47].
The methods currently practiced in the European Union for stunning and killing common
carp are asphyxia followed by percussion, percussion and whole body electrical stunning in
water. A scientific opinion [48] on the welfare aspects concluded that taking out carp of water
(asphyxia) before stunning results in poor welfare. Conversely the application of an adequate
electrical current density rendered carp immediately unconscious. It is also concluded that the
application of such current, in combination with chilling, prevented the recovery of
consciousness allowing for a humane slaughter procedure.
Fauconneau et al. [41] reviewed the changes occurring in tissues and flesh after death and
post mortem storage of common carp. The possible role of biological characteristics and
tissues of this fish species on the quality was also analyzed. The application of an electrical
current to individual common carps in combination with chilling in ice water was considered
an effective procedure for slaughter [49]. Slaughtering of common carp by percussion was
considered more adequate than asphyxia because the onset of rigor was delayed for ~24 hours
and fish presented better texture and other attributes [50]. Rahmanifarah et al. [51] also
compared different slaughtering methods on the flesh quality of this fish species. The clove
oil stunning was considered the less aversive pre-slaughtering method and also provided a
higher product quality. Common carp fillets dipped in a solution of 0.5% carvacrol and 0.5%
thymol delayed bacterial growth and extended the shelf life from 4 to 12 days during storage
at 5C [52]. The preservation of common carp fillets in electrolyzed NaCl solutions
containing 0.5% carvacrol and 0.5% thymol at room temperature did not affect the quality of
fillets and could be an alternative to synthetic preservatives [53]. Regarding the quality
differences between wild and farmed common carp, it was concluded that wild fish lipids
were more stable than those of farmed fish [33]. A study to evaluate reduction potential (Eh)
and pH during storage at two temperatures of four tropical fish species, including common
carp, concluded that Eh values of freshwater fish were higher than marine fish and also
demonstrated a relationship between Eh and pH [54]. The application of Purac, a
preservative agent based on lactic acid produced by Corbion, Amsterdam, The Netherlands,
to common carp halves and mince could extend the shelf life of these products [55]. In this
work, putrescine concentration was proposed as a chemical indicator of carp meat quality.
Putrescine and cadaverine also showed the best correspondence with the sensory and
microbial analyses of vacuum and non-vacuum-packed carp samples stored at 3 and 15C.
Vacuum packaging at 3C extended the shelf life by about 3-5 days [56]. The shelf life of
common carp fillets stored under modified atmosphere packaging (MAP) (69% N2/25%
CO2/5% O2/1% CO) was extended until 7 days [57]. On the other hand, the application of
high pressure at low temperature to preserve common carp fillets induced lipid oxidation and
negative color changes [58] attesting the importance of establishing the best treatment
conditions according to the fish species. The physicochemical changes occurring in freeze-
thawed common carp fillets were more pronounced than in fresh fillets [38] which did not
recommend the cold storage of freeze-thawed fillets.
Bighead Carp (Hypophthalmichthys nobilis)

This fish species has not been so widely studied as the previous ones and thus a limited
number of works was published. The available data on the chemical composition of bighead
carp were published together with other fish species and particular attention was given to the
fatty acid profile [9, 59] The brine treatments of bighead carp fillets improved their quality
and safety during chilling storage [60]. Subsequently, a model based on the global stability
index (GSI) was established and could effectively predict the freshness of bighead carp heads
[61]. In a second work these authors [62] derived predictive models for bighead carp fillets
based on sensory score and several chemical parameters that could predict the freshness
indicators in the range of -3 to 15C. Good correlations between the Q value and different
biochemical parameters of refrigerated bighead carp heads were established and Q value
could be used as a fast non-destructive method to estimate the quality of this species stored at
0 and 3C [63]. A study on the effect of refrigerated storage time of bighead carp before
freezing concluded that the fish stored in ice until 4 days allowed to maintaining the quality of
frozen fish for 4 weeks [64]. The effect of different freezing treatments of bighead carp heads
on the quality changes during ice storage allowed the conclusion that previous freezing at -
40C for 12 h and then storage at -18C was advantageous for the preservation of these food
products [65].
Other Carp Species

Rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) are the dominant
fish species cultured in several Asian countries (Figure 3). The proximate chemical
composition of the flesh or the roe of these fish species was reported previously [66-73]. An
evaluation of the rigor progress in rohu and mrigal specimens with different sizes may
conclude that the duration and resolution of rigor mortis increased with the size of the fish
[74]. The microbial quality of farm-reared rohu was evaluated by Jeyasekaran and Ayyaappan
[75] and the quality changes occurring in ice storage and followed by different methods
advocated a maximum shelf life for this species stored in ice of 17 days [76].
Sankar and Ramachandran [77] studied the thermal stability of myofibrillar proteins from
three Indian carps. In addition, the changes of functional properties of proteins from these
carp species during ice storage were also followed [71, 78]. A decrease of the different
functional properties was generally observed, in particular, the gel-forming ability was
negatively affected. In line with these studies, a decrease of the hardness of the skin and flesh
of rohu iced-stored was observed [79]. Similarly, the degradation of myofibrillar proteins of
rohu was evaluated in unfrozen and frozen storage and the importance of the post mortem
storage temperature on the protein degradation was emphasized [80]. A combination of
coating rohu steaks with a gel dispersion and gamma irradiation at 1 kGy enhanced the shelf
life to 42 days [81].
Figure 3. Catla (Catla catla).
Crucian carp (Carassius carassius) is one of the main freshwater fish species in China. A
kinetic model based on EC and freshness indicators of crucian carp was developed and it
could predict early freshness of this fish species based on TAC [82]. Natural preservatives
(tea polyphenols and rosemary extract) were successfully used to preserve chilled crucian
carp and to extend the shelf life from 7-8 days for untreated fish to 13-14 days for tea
polyphenols treated fish and 15-16 days for rosemary extract treated fish [83]. Moreover,
Zhai et al. [20] detected a low level of biogenic amines in crucian carp commercialized in
southern China.
A very limited number of reports on the post mortem changes of black carp
(Mylopharyngodon piceus) muscle were published. A study on the effects of low salt and
sugar dry-curing treatments on the quality of black carp fillets stored at 4C concluded that
they were safe, healthy, and convenient for preservation [84].
A study on the chemical composition and post mortem changes of wuchang bream
(Megalobrama amblycephala) concluded that it is better to preserve this fish species at -3C
for short-term storage and consume it within 8-24 h post mortem [85]. In another work, a GSI
model was developed to follow the quality changes of wuchang bream which could
effectively predict the quality deterioration of this fish species during chilled storage [86].
Cichlidae
Tilapia (Oreochromis niloticus)

The Nile tilapia is a fish native to Africa but is currently one the most popular cultivated
freshwater fish in the world. The chemical composition of this species (wild or farmed) has
been reported in various works [87-89]. On the other hand, it was shown that welfare
protection of tilapia at slaughter could be obtained with electro-stunning followed by killing
the stunned fish in ice water [90]. The study on the post mortem changes occurring in iced
stored tilapia allowed concluding that the recommended limit of acceptability was up to 16
days [91]. The changes occurring in whole ungutted or gutted and filleted tilapia stored at 5C
were followed and the storage life was estimated at 12, 10, and 6 days, respectively [92]. The
quality evaluation of tray-packed tilapia fillets stored at 0C concluded that shelf life was
approximately 10-12 days [93]. In another study tilapia stored in ice was considered
unsuitable for consumption after 10 days based on the microbiological analysis [94]. The
study of lipid changes in the skin of tilapia stored in ice showed that the development of fishy
odor was mostly due to lipid oxidation by autoxidation or induced by lipoxygenase [95].
Salmonidae
Atlantic Salmon (Salmo salar)

The production of farmed Atlantic salmon has increased exponentially since the middle
of the 1980s when it was about 44000 tons and attained over 1.7 million tons in 2011. This
boosted the research and thus the number of studies published (Figure 4).
Figure 4. Atlantic salmon (Salmo salar).
A study on the lipid distribution [96] within salmon fillets showed that its content varied
with position from 2.4% to 18.6% and a strong correlation between lipid and moisture
contents was found.
Live-chilling of salmon before slaughter was adopted as a method to increase the time for
onset of rigor mortis and the resolution of rigor. Skjervold et al. [97] showed that the live-
chilling method can prevent some of the negative effects on fillet quality caused by crowding
stress. A later study on the effect of crowding stress on the salmon muscle quality showed
that long-term stress had significant negative effects thru lowering pH, softening fillets and
increasing percentage of myofibre-myocommata detachments [98]. The starvation of salmon
for five weeks could improve the resistance to acute stress prior to slaughtering and also
hamper the rigor development [99]. Concerning the humane slaughtering of salmon, the
Norwegian Food Authority established that farmed fish must be rendered unconscious and the
death is ensured by cerebral ischemia. It is also generally agreed that cerebral concussion
results in an instantaneous diminution or loss of consciousness without gross anatomical
changes in the brain [100]. Salmon stunning with carbon dioxide (CO2) before slaughter was
a common practice. However, several studies demonstrated that hypoxia caused by CO2
induced secondary stress responses leading to a reduction of the shelf life and losses of
quality. The flesh of salmon anaesthetised with CO2 was softer, slightly redder and more
yellowish than that of salmon sedated with iso-eugenol [101]. Electricity was considered
efficient for stunning market-sized salmon but it should be applied for less than 1.5 s at field
strengths ranging from 125 to 150 V/m to avoid injuries [102]. It was also concluded that
electrical stunning can cause immediate loss of consciousness in salmon and could be applied
prior to slaughter [103]. In another study it was shown that sinusoidal alternating current
(AC) inflicted less rate of injuries in salmon than square-wave AC and frequencies between
500 and 1000 Hz are recommended at field strengths exceeding 50 V/m for 10 s [104].
Percussive stunning of salmon by a hammer shaped cylinder promoted both welfare and
efficiency in industrialized slaughter [105]. Lambooij et al. [100] concluded that combined
AC and direct current (DC) can be a recommended source for dry electrical head to body
stunning. Erikson [106] compared three stunning methods of farmed salmon and concluded
that only isoeugenol fulfilled all the established criteria related to fish welfare and stress.
Regarding the effect of stunning and slaughter on the quality of flesh salmon it was
concluded that the most severe consequences of electrical stunning could be reduced by
manipulating current frequency [107]. However, the application of electricity for long periods
(15-30s) resulted in earlier onset of rigor mortis, higher tensions, softer texture, higher drip
and colour loss [108, 109]. A later study [110] indicated that optimum electrical stunning
performs equally well as percussive stunning and also that pumping and crowding had a
significant effect on quality. Harder salmon muscle was obtained when percussion followed
by bleeding were applied as compared with CO2 stunning and bleeding [111]. It was shown
that continuous washing of wounds in combination with pre rigor filleting reduced the
incidence of blood spotting in salmon flesh as compared to traditional bleed-out, gutting and
filleting procedures [112].
Many works have tackled the assessment of biochemical changes occurring during
chilled storage of salmon. For instance, it was shown that pre-rigor fillets of salmon presented
better quality than post-rigor fillets [113, 114] and the characteristics of actomyosin during
storage at 4C were followed [115, 116]. During ice storage fish became softer, fillet color
turned out lighter and redder and an increasing number of fish displayed high gaping [117]
and the effect of endogenous enzymes on muscle proteins and texture was also reported [118,
119]. It was recognized that seawater temperature influences the storage life of raw salmon
and that hypoxanthine is a valuable proxy of sensory quality evaluation [120]. The effects of
ante and post mortem temperature regimes on the rigor process in salmon were evaluated
[121]. The rigor process was always delayed when post mortem storage temperature was
reduced. The effect of ante mortem temperature was studied in fish either kept at constant
temperature (4 or 12C) or changed from 12 to 4C 2 h before slaughter. In the latter case a
more rapid rigor process occurred than in fish kept at a constant temperature (4C) before
slaughter. Veiseth-Kent et al. [122] reported that the positive effects of pre-rigor filleting on
the quality can be reduced or even eliminated if the fillets are restricted from contraction
during rigor mortis development. In a study on the possible role of collagen in texture of
salmon was demonstrated that muscle firmness was related with higher collagen stability
[123].
The effect of rigor mortis [124-126] and fat content [127] on the salt uptake in salmon
fillets was also studied. The application of super-chilling to preserve salmon was studied by
several authors. For example, it was reported that salmon fillets superchilled for 9 days
behaved as salmon stored on ice for 2 days [128]. It was also shown that the shelf life of
vacuum packed salmon fillets stored at -1.4C and -3.6C doubled the storage time of ice-
chilled fillets [129]. In another work [130], authors stressed the need for optimisation of this
technique to avoid the formation of ice crystals in order to obtain high quality products. The
quality of gutted salmon superchilled in seawater slurry and continuously stored in slurry did
not present advantages over traditional ice stored fish after 4 days [131]. No significant
differences were found in drip loss between 1 and 14 days of storage for superchilled salmon
muscle samples [132] and also no differences in texture between superchilled and ice stored
salmon at the end of the storage period were observed [133]. The combination of high
pressure and MAP to preserve salmon fillets allowed extending the shelf life for at least 5
days [134]. MAP and superchilling were also combined to preserve salmon fillets [135]
having been shown that fillets maintained a good quality for more than 24 days of storage.
Similarly, a combination of MAP (90% CO2/10% N2) and superchilling in salmon fillets
increased their shelf life from eleven to 22 days [136]. Notwithstanding, it was also
demonstrated that Photobacterium phosphoreum dominated the spoilage of salmon fillets
preserved in MAP [137].
Rainbow Trout (Oncorhynchus mykiss)

The chemical composition and fillet quality of rainbow trout has been reported in several
works [7, 26, 138, 139]. The welfare aspects of stunning and killing farmed rainbow trout
were addressed by a Panel on Biological Hazards [48]. Electrical stunning of rainbow trout
was tested and a mathematical model of the electric field in the stunning tank was
constructed; a two-stage approach to stunning was proposed [140-143]. The electro-
stimulation of rainbow trout after death reduced the rigor duration and affected the flesh color
[144]. This fish species stunned by percussion and stored in MAP showed lower K values
than the fish slaughtered according to the ice slurry method [145]. It was also observed that
stress before slaughter decreased fillet lightness and initial pH and induced a softer flesh
[146] and affected flesh quality and accelerated the onset of rigor mortis [147]. Percussion
slaughtering and pre-rigor filleting of rainbow trout was considered more adequate than the
asphyxiation slaughtering to obtain a product with better quality [50]. A comparison between
beheading and asphyxia in air of rainbow trout showed that fish slaughtered by asphyxia had
faster glycolysis and resolution of rigor mortis and also higher cooking losses and a softer
texture [148].
The quality characteristics, including liquid leakage (LL) and mechanical properties
within rainbow trout fillets during ice storage varied among fillet sections. LL increased in
the cranial-caudal direction and dorsal compressing and puncturing resistance increased
towards the tail. A pronounced softening of the tail-half upon freezing was also observed
[149]. A study on the changes occurring in iced whole and filleted rainbow trout indicated
that their shelf life was 15-16 and 10-12 days, respectively [150]. In another study on the
quality assessment of whole rainbow trout during ice storage it was estimated a shelf life of 9
to 11 days based on the microbiological and sensory data [151]. The same shelf life period
was reported in another work [152] but a delay in icing for 4 and 8 hours was found to
shorten the shelf life about 5-7 and 1-3 days, respectively. Chytiri et al. [153] proposed a
putrescine value of 13-14 mg/kg and a spermidine value of 7 mg/kg for both the whole and
filleted ice stored trout after 12 and 9 days, respectively, as the upper limit for spoilage
initiation. Furthermore, a good linear correlation between putrescine content and
Pseudomonas spp. and psychrotrophic counts in whole ice stored rainbow trout was obtained
[154]. These results led to the conclusion that monitoring putrescine levels may have a great
potential for evaluating freshness of this fish species. Rainbow trout fillets preserved with a
combination of salt, oregano oil and vacuum packaging and stored at 4C had a shelf life of
16-17 days [155] Chitosan coatings enriched with cinnamon oil could maintain refrigerated
trout fillet shelf life until 16 days without significant loss of texture, odor, color or microbial
growth [156]. Edible films prepared with quince seed mucilage (QSMF) and incorporated
with oregano or thyme oil were tested in the preservation of refrigerated rainbow trout fillets
[157]. The incorporation of 2% of oregano or thyme oil in QSMF resulted in a shelf life
extension of 9 and 11 days respectively as compared to the control samples. Raeisi et al. [158]
also used carboxymethylcellulose (CMC) edible coating containing Zataria multiflora
essential oil (ZEO) or grape seed extract (GSE) to preserve refrigerated rainbow trout meat.
The authors concluded that CMC edible coating enriched with ZEO and GSE was effective in
reducing undesirable chemical reactions during refrigerated storage of fish meat. The
application of MAP to preserve filleted rainbow trout allowed to concluding that sensory
quality deterioration was delayed together with a decrease of chemical indices [159]. The
composition of the best atmosphere was: 10% O2 + 50% CO2 + 40% N2/Ar. Similarly,
Arashiara et al. [160] observed that mixtures with higher CO2 concentrations increasingly
depressed bacterial growth and the atmospheres with higher O2 concentration promoted lipid
oxidation. A shelf life of 10 to 12 days was recorded for refrigerated whole rainbow trout
ozonized and vacuum-packaged as compared to a shelf life of 8 days for non-ozonized fish
[161]. On the other hand, gamma irradiation (2kGy) of whole salted vacuum-packaged
rainbow trout allowed extending the shelf life (at 4C) to 28 days [162}. Moreover, the
application of high pressure to vacuum packed rainbow trout fillets extended shelf life to
about 21-28 days [163]. The shelf life [164] and the protein changes [165] of gravad rainbow
trout were studied.
Other Freshwater Species
Northern snakehead (Channa argus) is a fish species native to China, Russia and Korea
but it was introduced in other Asian countries. Alginate-calcium coating incorporated with
nisin or cinnamon was efficiently applied to snakehead fillets to enhance the fish quality [166,
167].
The production of channel catfish (Ictalurus puntactus) in the four major commercial
producing states of this fish species in U.S.A. represented around 89% of total worldwide
catches of this species in 2011 [168]. It was shown that increasing dissolved oxygen levels
during transport could improve channel catfish fillet quality [169]. The effects of AQUI-STM
sedation during harvest of catfish were studied [170]. The muscle of sedated fish had higher
pH, lower lactate and higher ATP levels and also lower drip losses. These results
demonstrated that the utilization of this anesthetic may improve the catfish meat quality.
Pangasius catfish (Pangasianodon hypophthalmus) (Figure 5) has a great aquaculture
potential owing to its ability to grow, omnivorous feeding habit and resistance to common
diseases [171]. The chemical composition and quality attributes of conventionally and
organically farmed pangasius catfish was analyzed [172]. The spoilage microbiota profile of
pangasius fillets processed in three factories was studied and the results obtained could be
used to improve Good Manufacturing Practices for processed pangasius fillets and to select
effective practices to extend the shelf life of thawed fillets [173]. Heat treatments of
pangasius fillets slightly reduced PUFA levels but according to Domiszewski et al. [174]
fillets are not a valuable source of n-3 PUFA The quality changes of pangasius stored in ice
were followed in a study by Azam et al. [175] and only slight quality differences between fish
caught in the Summer and Winter were found. Moreover, pangasius slices treated with tannic
acid and stored at 4C in MAP had a shelf life of 15 days based on microbiological
acceptability limit (107 cfu/g) whereas that of fish stored in air was only 3 days [176].
Asian swamp eel (Monopterus albus) is native to East and Southeast Asia where it is
farmed in polyculture rice fields. The nutritional composition of swamp eel was reported
[177, 178] and a combination of chitosan coating and MAP was effective to preserve fish
fillets [179].
Figure 5. Pangasius (Pangasianodon hypophthalmus).
Mandarin fish (Siniperca chuatsi) is native to the Amur River basin and other rivers in
China. The amino acid profile and proteomic pattern of mandarin fish were evaluated [24]
and a method for the determination of volatile amines to monitor freshness was developed
[180]. A study on the level of nitrogenous compounds in a large number of fish species
showed that mandarin fish contained relatively high level of trimethylamine oxide (TMAO)
[181]. The aroma compounds in fermented mandarin fish were identified and the results could
be used to characterize the quality of this product [182].
Chinese mitten crab (Eriocheir sinensis) is native to the coastal rivers and estuaries of the
Yellow Sea but nowadays is spread throughout Europe and California (Figure 6). The
proximate chemical composition and nutritional quality [183] as well as the content of non-
volatile taste active components [184, 185] in the meat of this crab have been evaluated. The
production of biogenic and volatile amines in this crab stored at two temperatures (4 and
20C) was studied [186]. Histamine was the main biogenic amine formed reaching a level of
91.2 mg k-1 and could be used as a safety index for crab.
Figure 6. Chinese mitten crab (Eriocheir sinensis).

The freshwater crayfish (Procambarus clarkii), native to north-eastern Mexico and south
central USA, is nowadays distributed throughout several European countries. Post mortem
changes in freshwater crayfish tails stored in three different conditions (aerobic and vacuum
packaging and MAP) were studied [187]. Different physical and biochemical changes were
recorded in crayfish tails stored in the three types of packaging used. These findings
suggested that the physicochemical mechanisms involved in post mortem alteration of
crayfish muscle depended on the packaging systems studied [187].
CONCLUSION
Information on different fish slaughter methods is given as well as on the application of
recent techniques used in fresh fish preservation. Physical methods and chemical indicators to
evaluate quality changes occurring during refrigeration are also described. Despite the large
volume of work developed on fish slaughter there is still the need of introducing and testing
different methods in a number of fish species. The introduction of new preservation methods
to increase the fish shelf life is another great challenge to face the increasing demand of fresh
seafood. As a consequence, rapid analytical methods to evaluate quality and freshness are
also necessary and particularly physical methods may play a significant role.
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Chapter 7
QUALITY CHANGES IN CRUSTACEANS DURING

AND AFTER PROCESSING
Aygl Kkglmez*, Mehtap Baykal,

Ali Eslem Kadak and Mehmet elik
Cukurova University, Faculty of Fisheries,
Department of Fishing and Fish Processing Technology,
Adana, Turkey
ABSTRACT
In this chapter, the quality changes of the main crustacea species like shrimp, prawn,
crab, lobster and crayfish are discussed during and after processing. Some undesirable
structural changes occur during the fresh consumption and processing of crustaceans
which are widely used in the world as nutrition and food supplements. The causes of
these changes and the measures needed to be taken are highly significant for the seafood
processing technologies to be employed. In this chapter, studies conducted on chemical,
physical, microbial and sensory changes which occur after the harvest, during and after
the processing of most-processed and most-consumed crustacean species in the world are
examined. By researching the proper processing methods and the quality changes of
crustaceans post-mortem and during processing, it will be possible to have fresher
products with longer shelf-life.
Keywords: quality, crustaceans, processing
INTRODUCTION
Seafood is the first food to be preferred in many countries today. As a whole, seafood
products have been lauded for their health promoting characteristics. Seafood is nutritionally
valuable source of polyunsaturated fatty acids, essential amino acids, minerals and vitamins.
*
Corresponding author: Cukurova University, Faculty of fisheries, Department of Fishing and Processing
Technology, Adana, Turkey. E-mail: akucukgulmez@cu.edu.tr.
128 Aygl Kkglmez, Mehtap Baykal, Ali Eslem Kadak et al.
It was reported in various researches that polyunsaturated fatty acids have a protective effect
especially against cardiovascular diseases, hypertension, diabetes, brain development in
infants, cancer, depression, autoimmune diseases, anemia and allergy and dermatological
diseases [1-4]. Crustaceans which have an important place among seafood are also highly
important for human nutrition. A large majority of crustaceans (Order Decapoda, Class
Crustacea) have economic importance worldwide. The most common decapod crustaceans
which are processed and offered for consumption after being cultivated economically are
shrimp, prawn, crayfish, lobster and crab in order of commercial importance [5]. The capture
and aquaculture production of these species in general in the world are shown in Table 1.
Crustacean shellfish are highly perishable, generally spoil faster than other muscle foods
because of their biological composition and they are also more sensitive to post mortem
texture deterioration than other meats. Spoilage and limited shelf-life occur primarily due to
tissue enzymes and microbial activities [7]. As it is known, the general course of the events is
as follows; first of all, early systematic enzymatic breakdown leads to lost of freshness; then,
fast growing starts in bacteria and finally spoilage occurs [8]. Predominant bacteria during the
low-temperature storage of crustacean shellfish are generally pseudomonads and
Moraxella/acinetobacters. The pathogenic bacteria of most concern in shellfish are Vibrio
parahaemolyticus and other pathogenic bacteria are: Salmonella, Clostridium, Escherichia
coli, Campylobacter, Aeromonas and Listeria monocytogenes [9]. Bacteria, viruses, parasites
and naturally occurring toxins in fresh and processed crustacean shellfish can cause food-
borne illnesses. Besides it can also be contaminated by materials introduced into environment
through animal, human and agricultural pollution. Other factors that can increase the risk of
illness are the environmental conditions in the growth environment, the method of harvest,
processing methods and the handling and marketing conditions [10].
The degree of freshness loss and spoilage of shellfish can be assessed quantitatively using
biochemical and microbiological methods. In addition, alterations occur in sensory attributes
as well, as a result of biochemical and microbiological changes [11, 12]. In this chapter, the
changes that occur during and after processing of crustaceans and their effects on quality
paramaters are described according to species.
QUALITY CHANGES IN CRUSTACEANS

Shrimp
Shrimp is the most important shellfish species in the world seafood market.
Table 1. Capture production and aquaculture of shellfish in the world [6]
Species Capture production (ton) Aquaculture (ton)

Shrimp, prawn 3.353.661 4.327.520
Crabs 1.472.759 289.499
Lobster 293.823 2.035
Freshwater crustaceans 447.805 1.827.313
Quality Changes in Crustaceans during and after Processing 129
The origin of approximately 75 percent of shrimp production, both cultured and wild
caught, is developing countries; however, around 70 to 75 percent of global shrimp
consumption takes place in developed countries [10]. The shrimp meat has very low fat
content and is rich in protein and highly rich in terms of the essential amino acids it contains.
It is among the easily digestible foods because it is poor in terms of connective tissue.
Either as cooked or raw, peeled or unpeeled, with or without breading or other coatings,
shrimp can be marketed in different ways. In addition, it can be consumed frozen, dried,
smoked, marinated and brined. Chitin and chitosan are produced from shrimp shell in many
countries and these materials are used as raw material in many industries [13, 14].
The shrimp meat is one the most sensitive foods and spoilage can occur very quickly
during processing. Spoilage generally accelerates in cases when there is insufficient cooling
and poor storage and when distribution and marketing conditions are inadequate. The
spoilage process in shrimp results in changes in flavor, texture, appearance and in changes in
many biochemical components in the shrimp meat [15]. The most important two changes in
the shrimp meat are bacterial activities and colour changes.
Bacterial activity occurs primarily in the head (cephalothorax) of the shrimp where most
viscera lie because of the bacteria in the environment. Free amino acids and non-nitrogenous
substances in the shrimp are used as nutrients for microbial growth. Microbial growth is
responsible for the spoilage of the shrimps which are not sufficiently iced and stored in proper
conditions [10, 15, 16].
One of the most important problems of catching or processing shrimp is the color
changes. After the shrimp has been caught, color changes occur especially in the head with
the influence of environmental factors (sunlight, temperature etc.). In this context, late
beheading after the fishing, insufficient cooling or no cooling at all of the processed products
as well as the environmental factorsaccelerate the color changes, called melanosis or "black
spot" [13]. An enzymatic oxidation in phenolic precursors causes formation of irresolvable
black pigments (melanin) within the internal shell surface; this process is called as
melanogenesis. It assumed that the dark discoloration is related to the enzyme polyphenol
oxidase and starts at the cephalothorax, generally within 2 to 4 d following catch, and extends
through the abdomen, pereipods, and tail during ice storage [17]. Despite the fact that black
spots might be considered as harmless for consumers, they dramatically reduce the market
value of the product and the acceptability by the customers which are all leading to serious
financial loss. Various studies related to the prevention of melanosis have been conducted to
date [18, 19]. These are both cooling processes and preservative-adding applications. Sodium
metabisulphite application is the leading application of additives and is widely used.
Studies on the effect of different processing methods on the quality of shrimp are
gradually increasing in number [20]. In a research conducted on the effect of shrimp
processing methods on the final product's microbial quality, correlation analysis was made
between the final microbial quality (coliforms, E. coli, Salmonella spp., coagulase-positive
staphylococci, Clostridium spp., Listeria monocytogenes and aerobic plate count) and the
peeling, cooking and tail-removing processing methods. According to the authors, the total
contamination of shrimp samples was reported as less in cooked, peeled, tail-on samples
compared to raw, unpeeled and tail-off products. Besides, statistically significant correlations
were reported between cooking, peeling and tail-keeping processes for the manufacturing
technology and coliforms counts about shrimp products which lead to an improved
microbiological quality of the final product. As suggested by Hossain et al. [16], processed
frozen shrimps at a fish processing plant were investigated and they were found qualified
enough for exportation and cooked IQF shrimp was found much better compared to other
shrimps according to a microbiological perspective.
Jayasinghe et al. [21] found that the shrimp packed in styrofoam box showed
significantly lower bacterial growth. It was reported that barrier properties of styrofoam
packages were very effective; they prevent bacterial contaminations and decrease the
bacterial multiplication within the package. In many retail seafood markets, crustaceans are
generally stored in ice before being sold. Mushiness occurs in the muscle because of the
diffusion of proteolytic and collagenolytic enzymes from hepatopancreas as a result of storage
of freshwater prawn (Macrobrachium rosenbergii) in ice. In fact, such tissue deterioration can
be eliminated by beheading the prawns before storage. There are few studies on this
subject [22].
Crab
Among crustaceans, crab is an indispensable seafood in several world cuisines because

its meat is highly rich in terms of nutritional composition. Crab meat contains many nutrients
and is an excellent source of high quality proteins, vitamins and minerals. Many curative
properties are attributed to crab meat, referred to treat asthma and chronic fever [23-25]. Blue
crab (Callinectes sapidus), snow crab (Chionocetes opilio), stone crab (Menippe mercenaria),
Alaska king (Paralithodes camtschaticus) and Dungeness crab (Cancer magister) species are
widely distributed around the world.
The main types of products from crab are cooked, ready-to-eat refrigerated and
pasteurized crab meat. Out of value-added products (e.g., crab cakes and deviled crabs),
frozen crab meat has been a conventional type. Market interest towards not only live but also
frozen (cooked) whole crab, crab portions and vacuum-packed crab meat has recently rised
[26, 27].
Like other shellfish, during processing, crab has a critical cooking process and it loses its
characteristics in high-temperature heat treatments (in especially pressure cooking). Cooking
duration and method affects the shelf-life of the cooled product [27]. While no hazardous
microbial activities are observed in its natural microbiota, very fast spoilage and
microbiological activitiy are observed when it is not stored under proper conditions.
Therefore, it is stipulated that crab meat and extremities to be processed in vapour pressure
should be processed in line with sterilization rules according to the international
standards [28].
There are various studies on the quality changes that occur during the preservation and
processing of crab meat with different methods. Zamir et al. [29] reported that the
deteriorative changes occurred in the nutritive quality of crab meat during storage at
refrigerator temperature (7 2C). The authors recommended that the quality of crab meat
can be accepted until one day of storage at refrigerator temperature; after this period of time it
becomes deteriorated. Another study assessed quality features of fresh blue crab meat held at
0-4C in tamper-evident containers. This study suggested that blue crab processors could
safely use the new tamper-evident packaging since it has either little or no effect on the
quality of product or shelf life [30]. According to Rebach et al. [31], the Jonah crab species
was not much used in the mid-Atlantic region, and the use of the whole, frozen crab would
enrich marketing options for this seafood product. It was observed by the authors that there
were evident quality changes in crabs cooked as a whole and held in a common freezer for up
to 50 weeks.
In their study, Hong and Flick [32] detected the impact of cooking period, cooking
technique, season, and storage condition on the basis of microbiological quality, sensory
features, and shelf-life of crabmeat under commercial processing conditions. Accordingly, it
was found that crab meat cooked by boiling showed higher microbial count and shorter shelf-
life in comparison with 10 and 12 min retort cooked meat.
In another study, Gates et al. [33] in which the crab meat was pasteurized, pasteurization
and storage of blue crab meat in steel, aluminum, plastic cans, and nonbarrier and barrier
pouches were investigated. The sensory and microbiological quality and also shelf-life of
meat which had been pasteurized in plastic and aluminum cans were found more enhanced
compared to the meat packed in steel cans. Similarly, packaging materials had no improving
effect on the microbiological shelf-life of crab meat which was cooked fresh. Vacuum skin
packaging offered improved sensory qualities of freshly cooked and pickled meat [33].
Chen et al. [34] reported that irradiation effectively reduced spoilage bacteria, which as a
result extended shelf-life by more than 3 days compared to control crab samples. During
storage, total scores for acceptability by a sensory panel of irradiated crab samples were
found better compared to control samples throughout 14-days of ice storage.
In addition to all these studies, it is known that the conditions of commercial crab
processing factories have an effect on the quality changes of the crab meat [35]. Ray et al.
[36] reported that less amounts of total anaerobes and psychrotrophs were found in crab meat
which was produced under appropriate sanitary conditions; shelf-life of these products were
found to be relatively longer.
Lobster
Lobster is another important member of the Crustacea in terms of its high economic
value. The two most commonly marketed species are: European Lobster (Homarus
gammarus) and American Lobster (Homarus americanus). Besides these, there are other
various lobsters which are categorized as different species in the seas of the world [37].
Lobster meat is very sensitive in terms of processing. Quality changes vary according to
the environment, food, season, physiological conditions at the time of catching, biochemical
reactions which occur post mortem and the condition of the raw lobster [11].
It is highly important that lobsters be processed under proper conditions and transported
to the consumer after catching. Freezing technology is placed a top among lobster processing
methods. Various studies have been conducted on the quality changes which occur during
freezing lobster products with different preservative substances in different ways after
catching and the storage of these products. Work et al. [38] recently demonstrated that
cryogenically freezing lobsters before frozen storage could retain high-quality texture and
flavor attributes of whole lobster. The excellent textural qualities of both hard-shell and soft-
shell lobsters were assumed to be correlated with the high freezing rate. Another, similar
study reported that the addition of low concentrations of sodium tripolyphosphate may extend
the shelf-life of whole cooked lobster which is cryogenically frozen, may decrease lipid
oxidation over frozen storage time, maintain texture, color, and flavor attributes, increase
yield, and reduce drip loss [39]. Perez-Won et al. [40] studied instrumental and sensory
textural features of frozen blue squat lobster (Cervimunida johni) tails stored at -22C.
According to their results, frozen storage did not affect the textural quality of the blue squat
lobster tails and using instrumental analysis may be useful as an alternative to sensory
analyses with the purpose of assessing the textural features of blue squat lobster tails.
Bremner and Veith [41] analysed taste panel evaluation, yield measurements and
analytical tests were conducted on the frozen stored (-18C) lobster tail The results of taste
panel did not show any difference in the organoleptic quality of frozen lobster flesh subjected
to various holding periods with subsequent frozen storage at -18C for up to 40 wk. Chevalier
et al. [42] compared pressure shift freezing (PSF) of a whole Norway lobster (Nephrops
norvegicus) with air-blast freezing and also with pressurized samples without freezing to
detect its effect on the quality of texture, structure, water, and salt soluble protein
extractabilities. Those authors reported that for the pressurized Norway lobster meat either
with PSF or without freezing, toughness raised; on the other hand, salt soluble protein
extractability decreased and air-blast freezing did not have any impact upon the textural
quality of the meat. In addition, studies on the quality changes which occur during storing the
lobster under different conditions have been conducted. For this purpose, Albalat et al. [12]
observed the quality deterioration of Norway lobster tail meat during ice storage. They
suggested a precautionary principle which offers that maximum 4 hours should elapse
between catching and icing, regardless of the ambient temperature, with the purpose of
preserving the tail meat in prime condition. Power et al. [43] conducted a study on the
irradiation of cooked lobster (Homarus americanus) meat by means of one dosage of gamma
radiation.
Researchers reported that higher levels of irradiation led to an immediate loss of quality
because of toughening and loss of flavor and the growth of trimethylamine-producing bacteria
were inhibited over 10 days on irradiated samples than on the unirradiated iced control.
Crayfsh
There are quite many species of crayfish occurring in all the continents except in Africa
but especially in America. In particular, Astacus leptodactylus (lake lobster) have a wide
distribution area in the world and are important in Turkey. Its nutritional value is quite high
and it is important in terms of its protein content and quality. It is a high-priced seafood
product [44].
As in other shellfish, the quality changes in crayfish also occur very quickly after
catching. Therefore, the crayfish should be subjected to a proper processing method after
catching. Generally, the crayfish which are processed alive, fresh by freezing or canning are
evaluated as especially cooked tail. However, cooked tails have a limited shelf-life because of
oxidative processes, such as lipid and protein oxidation, microbial spoilage and endogenous
proteolytic processes. All these cause the market value of cooked crayfish tails to decrease
[45].
Freezing which usually provides longer preservation periods is also widely used in the
crayfish processing sector. Various studies related to the quality changes which occur during
the frozen preservation of different crayfish species have been conducted until today. Tseng et
al. [46] reported that there is a relationship between repeated freezing-thawing (F/T, six
cycles) treatment and muscle quality of Australian red claw crayfish (Cherax
quadricarinatus).
The results indicated that the freezing-thawing method should not be repeated more than
three times in terms of the quality of the meat. Another, analogous study assessed textural
quality of quick frozen and conventionally frozen whole crayfish. It was reported that:
toughness increased significantly as a result of freezing; toughness diminished in all treatment
groups after the 16th week of frozen storage; that the individually quick frozen samples were
softer than the conventionally frozen samples [47]. Tseng et al. [22] investigated the quality
changes occurring in shell-on tails of Australian red claw crayfish, C. quadricarinatus, sealed
plastic freezer bags and stored on ice (at 0C).
According to their results, red claw crayfish muscle was sensitive to protein denaturation
and lipid oxidation, and these chemical changes may be responsible for decreased cooking
yield and reduced tenderness of meat during extended refrigeration storage.
In addition, studies on the effect of different packaging conditions on the quality of the
crayfish have been conducted. Wang and Brown [48] studied how elevated levels of CO2
affect the storage quality of cooked freshwater crayfish stored at 4C.
Following 28 days of storage, the concentrations of ammonia and trimethylamine and
total plate counts were found lower in crayfish which was stored under carbon dioxide
compared to samples stored in air. The authors reported that the shelf-life of the samples
stored after being packaged with CO2 atmosphere were considerably longer than others.
It is assumed that the protease activity during storage has a significant impact on
decreased shelf-life of fresh seafood. In order to investigate this perception, three batches of
red swamp crayfish (Procambarus clarkii) tails, placed on trays, were packed with a
polyvinyl chloride film, under vacuum or a modified atmosphere.
Afterwards, proteolytic activity was measured during storage at 2C. The results
indicated that other physicochemical mechanisms were involved in post-mortem
diversification within the form of crayfish muscle under the packaging systems investigated
[49].
CONCLUSION
The quality changes of different fish species and fish products during catching,
processing and storage have been researched in great detail but are still being researched
widely. Notwithstanding, there is limited information about the changes that occur post-
mortem in crustaceans which have (often high) commercial value. More and detailed
information about those changes will lead to the development of procedures and
methodologies in important stages such as catching, handling, processing and storage and
thus result in more shellfish and shellfish products that are fresher and have longer shelf-life.
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8658-8663.
Chapter 8
TRACE ELEMENTS AND STABLE ISOTOPES ANALYSIS

AS SEAFOOD QUALITY INDICATORS
Jaime Anbal1,2,* and Cristina Veiga Pires1,3

1
CIMA-Centro de Investigao Marinha e Ambiental,
Universidade do Algarve, Portugal
2
School of Engineering, University of Algarve, Portugal,
3
Faculty of Science and Technology, University of Algarve, Portugal
ABSTRACT
The aim of this chapter is to discuss the use of trace elements and stable isotopes
analyses as seafood quality indicators, due to their capacity to assess hazards that are
prior to capture and can originate problems afterwards; progressive loss of freshness from
the moment of capture/death to the time of consumption; and determine authentication
and traceability of acquired sea products. Trace elements can be generally defined as
chemical elements that are present in minute amounts. In living organisms, trace elements
can be essential for many physiological and biochemical processes, although they can
also result from the exposure to toxic conditions. Consequently, trace elements can be
used to evaluate the toxicity, especially heavy metals, and geographic origin in seafood
and seafood products, with a main focus always in the quality assurance perspective.
Stable isotopes are chemical elements having the same atomic number between them, but
having a different atomic mass, and showing no tendency to undergo radioactive
breakdown. However, stable isotopes are dependent on kinetic effects related to
biological processes, and equilibrium effects between different matter phases, making
them good proxies to follow metabolic or physiological pathways and authenticity issues
related to seafood and seafood products.
Keywords: trace elements, stable isotopes, seafood quality, heavy metals, freshness,
authentication and origin
*
Corresponding author: Universidade do Algarve, Campus da Penha, Instituto Superior de Engenharia,
Departamento de Engenharia Alimentar, 8005-139, Faro, Portugal. Email: janibal@ualg.pt.
140 Jaime Anbal and Cristina Veiga Pires
INTRODUCTION
In 2012, the worlds fish production was around 158 million tonnes, from which 86%
was used for human consumption [1]. Alongside with fish, several species of crustaceans,
molluscans, and seaweeds, as well as microalgae, are used as food for humans. Developing
strategies for full utilization of seafoods and their by-products to produce value-added novel
products (e.g., omega-3 fatty acids, specialty enzymes, protein hydrolysates, peptides,
chitin/chitosan, glucosamine, squalene, collagen, carotenoids, etc.) is of great interest [2].
From the consumers point of view, there are three main issues that deeply impact
seafood quality: 1) hazards that are prior to capture and can originate problems afterwards
(e.g., bacteria contamination, pollution toxins, and parasites); 2) progressive loss of freshness
from the moment of capture/death to the time of consumption (e.g., spoilage odors, biogenic
amines production and rancidity); and 3) authentication and traceability of purchased sea
products (e.g., geographic origin and authentication of species). Trace elements are more
associated to the first issue, stable isotopes analysis can be related to the second, and both
methods can be used to access the third issue.
Throughout their life cycles, seafood species are subjected to considerable environmental
changes and fluctuations in the availability and compositions of their feed, which can affect
their morphological and chemical compositions [3]. Despite their recognized benefits, fish
and seafood may represent a risk for human health since they can accumulate contaminants
from aquatic environment and magnify them up the food chain [4, 5]. Fish can contribute
significantly to dietary human exposure to environmental pollutants [6], and, in many studies,
fish species have been employed as bioindicators of environmental contamination [7]. Water
pollution is the main cause of seafood contamination with toxic metals, from many sources,
e.g., industrial and domestic waste water, natural runoff and contributory rivers [8]. Heavy
metals discharged into the marine environment can damage both marine species diversity and
ecosystems, due to their toxicity and accumulative behavior [4, 5]. In the sea, pollutants are
potentially accumulated in marine organisms and sediments, and subsequently transferred to
man through the food chain [9]. For this reason, determination of trace elements in aquatic
organisms, particularly the contents of heavy metals in seafood is extremely important for
human health [10].
From a nutritional point of view, seafood is considered an important source of high-
quality protein, minerals and essential polyunsaturated fatty acids [6, 11]. Those same groups
of molecules are also the cause for rapid deterioration, leading to serious food safety issues.
The condition named spoilage isnt clearly defined in objective terms, but it is related with
post-mortem conditions. Seafood spoilage is the set of sensory changes, resulting from the
production of off-odors, off-flavors, slime, gas, discoloration and changes in texture. These
signs result in products being unacceptable for human consumption, and are caused by
chemical autolytic changes, bacterial metabolism or oxidative reactions [2]. The rate and
relative importance of each cause depends on the group of species being studied (e.g., lean or
fat fishes, crustaceans, bivalves). Besides the current practice in the commercial and industry
sectors of evaluating seafood freshness based on sensory attributes, seafood spoilage can be
assessed through chemical indexes (e.g., nitrogen- or biogenic amines-based indices) and
microbiological counts (e.g., TVC, Escherichia coli, Listeria monocytogenes) [2]. Although
these indicators support public health decisions regarding the consumption of seafood, they
Trace Elements and Stable Isotopes Analysis as Seafood Quality Indicators 141
usually lack precision in defining spoilage biochemical pathways. For instance, several
chemical indicators of spoilage can be originated by autolytic processes or by microbiota
metabolism, and microbiota presence doesnt always mean the occurrence of spoilage. In the
future, stable isotopes might be a key resource to unveil these issues, because they allow
assessing and following the biochemical processes leading to spoilage.
Food authentication can be defined as the process by which a food is verified as
complying with its label description [12]. In the last decades the growing awareness of
consumers to this issue has led the European Union, United States of America and Japan to
create specific legislation related to authentication and traceability of seafood products.
Species identification, production method and geographic origin are the main issues
addressed by this legislation [13]. The necessity to confirm the geographic origin of seafood
lies in the fact that some areas are considered to be pristine and others polluted,
especially for the content of some metals (e.g., mercury, cadmium, lead), radioactive elements
(e.g., cesium-137), or environmental pollutants (e.g., pesticides, polychlorinated biphenyls,
dioxines) [14, 15]. Costumers also tend to prefer seafood species that are traditionally
consumed in their geographic area, even if they have to pay higher prices for it. These
concerns make the determination of the geographic origin of seafood a paramount issue, in
order to avoid false labels, which are occasionally found in markets, and discourage the
offering of seafood species from less attractive areas labeled as their more expensive
counterpart [13, 16]. Techniques aimed at identifying the geographic origin, or authentication
of samples, make use of the different distribution of isotopes in different geographic regions.
The isotopes may be from the most common elements making up the organic material such as
H, C, O, N, S, or isotopes of trace elements that nonetheless are either essential for normal
functioning of organisms, such as zinc, selenium, magnesium, manganese, or contaminants
picked up from the environment such as mercury, cadmium, lead, and so on [13].
The aim of this chapter is thus to discuss the usage of trace elements and stable isotopes
analysis as seafood quality indicators.
TRACE ELEMENTS AS TOXICITY AND GEOGRAPHIC

ORIGIN INDICATORS
Trace elements can be generally defined as chemical elements that are present in minute
concentrations, in an order of magnitude around ppm (parts per million). In living organism,
trace elements can be essential for many physiological and biochemical processes (e.g.,
minerals, enzymatic cofactors), or result from the exposure to toxic conditions (e.g., heavy
metals). Heavy metals can be classified as potentially toxic (arsenic, cadmium, lead, mercury,
nickel, etc.), probably essential (vanadium, cobalt) and essential (copper, zinc, iron,
manganese, selenium) [17]. Toxic elements can be very harmful even at low concentration
when ingested over a long time period. The essential metals can also produce toxic effects
when the metal intake is excessively elevated [18].
Trace element signatures are usually analyzed by mass spectrometry, using several
different techniques. Trace elements determinations in seafood start with a sample digestion
using several approaches, being the most commons microwave digestion [6], and dry ashing
with addition of concentrated HNO3, Mg(NO3)2 [11] or hydrogen peroxide [19]. After sample
digestion, several analytical techniques are available for trace element determination in
seafood samples such as inductively coupled plasma optical emission spectrometry (ICP-
OES), inductively coupled plasma mass spectrometry (ICP-MS), graphite furnace atomic
absorption spectrometry (GFAAS), or flame atomic absorption spectrometry (FAAS) [20,
21]. In some studies different analytical techniques are chosen depending on the quantified
element. Medeiros et al. [11] determined Al, Zn, Fe and Mn using ICP-OES, and Co, Cu, As,
Se, Cd, Ba, Pb and Bi through ICP-MS. This last technique is a type of mass spectrometry
which is capable of detecting metals and several non-metals at concentrations as low as one
part in 1012 (part per trillion). This is achieved by ionizing the sample with inductively
coupled plasma and then using a mass spectrometer to separate and quantify those resulting
ions. ICP-OES, also referred to as inductively coupled plasma atomic emission spectroscopy
(ICP-AES) is a type of emission spectroscopy that uses the inductively coupled plasma to
produce excited atoms and ions that emit electromagnetic radiation at wavelengths
characteristic of a particular element. The intensity of this emission is indicative of the
concentration of the element within the sample [22]. Due to analytical specifications, mercury
(Hg) is determined by different methods than other trace elements. Hg can be quantified
through an ICP-coupled hydride generator using argon as carrier gas and a 25% SnCl2 (m/v)
solution as reductant [19], by cold vapor atomic absorption spectrometry (CV-AAS), or cold
vapor atomic fluorescence spectrometry (CV-AFS) [23].
In recent decades, much attention has been paid to the study of essential and toxic trace
element content in foodstuffs, as a result of a growing concern about the health benefits and
risks of food consumption. The evaluation of risks and benefits of the consumption of fish
and other seafood has been particularly controversial [6]. On one side, nutritionists consider
these products to be an important source of high-quality proteins, minerals and essential fatty
acids [3]. The particular composition of their lipid fraction, rich in essential omega-3
polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA), and poor in cholesterol makes them a primer food [24]. On the
other side, toxicologists tend to regard seafood as a major vector for toxic substances such as
metal trace elements and persistent organic pollutants. A reconciliation of these two
contradictory viewpoints requires that we take into consideration both nutritional and toxic
substances contained in food products and also consumer behavior with regard to these
products. As a safeguard for human health, guidelines and regulations stipulate maximum
permissible levels of cadmium, lead and mercury in fish and seafood in order to limit dietary
exposure of consumers to toxic metals [25]. These compounds are of great concern due to
their toxicity, persistence, bioaccumulation and biomagnification in the food chain [24].
Metals and metalloids are naturally present in the environment reaching aquatic environments
via various geochemical processes. Additionally, anthropogenic sources such as industrial
wastes, agricultural and urban sewage and mining of metals create a potential source of heavy
metals pollution in the aquatic environment [26]. The contamination chain of heavy metals
almost always follows the recurring order: industry, atmosphere, soil, water, phytoplankton,
zooplankton, fish and human. Heavy metals can be accumulated by marine organisms through
a variety of pathways, including respiration, adsorption and ingestion and often reach the
human body by ingestion [26]. The adverse human health effects associated with exposure to
heavy metals, even at low concentrations, are diverse and include, but are not limited to,
neurotoxic and carcinogenic actions [24].
Moreover, fish have been found to be good indicators of heavy metal contamination in
aquatic systems because they occupy different trophic levels and are of different sizes and
ages [27, 28]. As an example, tuna is especially well known to accumulate substantial
amounts of mercury compared to some other fish species. Furthermore sediments can also act
as sinks for contaminants, which can persist in the aquatic environment for decades. Since
elevated concentrations of some trace elements have been reported in sediments, changes in
sedimentary fauna activity and water chemistry allow these persistent contaminants to enter
food chain, and thus into fish tissues [29]. Heavy metals such as Hg, Cd, Pb, As, and Cr react
with diffusing ligands, macromolecules, and ligands present in membranes, which mostly
provide bioaccumulation and biomagnification properties in the food chain, persistence in the
environment, and disorders in the metabolic processes of living organisms. Bioaccumulation
and biomagnification transform a concentration considered normal into a toxic concentration
for a different biota species as well as human beings [19].
Trace elements are particularly interesting to discriminate between products from
different small-scale geographic regions. However, the use of a single element as a marker is
not enough to produce good results; instead several trace elements, which are characteristic
for the local water, soil, air, and feed should also be assayed simultaneously [30]. Secor et al.
[31] used trace elements signatures in otoliths to distinguish between Atlantic and
Mediterranean tuna, whereas Campana et al. [32] used the same approach to discriminate
different spawning aggregations in cod. The principle behind these approaches is that trace
elements are integrated into otoliths in direct proportion to their availability in surrounding
environment or food. However, physiological factors, temperature and genetics may also
affect the uptake of specic elements into otoliths [33]. Most of the work on this subject has
been based on the analyses of otholites and/or scales [34, 35], but several authors have shown
that the approach is also valid when applied to soft tissues. Yamashita et al. [36] applied trace
element (Se, Hg, Zn, Cu, Mn and As) analyses to eels muscle to identify their origin. The use
of techniques with high sensitivity (e.g., ICPMS) would allow the assessment of certain rare
trace elements such as uranium, lead, cadmium and vanadium, which may be particularly
useful for discriminating the geographic origin of sh. Interestingly, some of these very same
elements are also highly relevant from the point of view of food safety, for example
cadmium, mercury, lead or arsenic. If, in addition, the accumulation of some elements
presents a species-specic pattern, as shown for mercury in the muscle of tuna and alfonsino
[37], that would give the potential to identify the species, as well as determining the
geographic origin and the potential detection of toxic levels of certain elements in seafood
[13] in one sampling moment and using one analytical technique.
STABLE ISOTOPES AS INDICATORS OF SEAFOOD QUALITY

Stable isotopes are chemical elements having the same atomic number but a different
atomic mass, and showing no tendency to undergo radioactive decay. Traditionally stable
isotopes have been used in geochemistry to assess the chemical processes controlling the
lithosphere, hydrosphere and atmosphere [38], and in ecology and environmental sciences to
trace the flow of organic matter in food webs [39, 40]. More recent approaches use stable
isotope analysis in archaeology to assess human dietary preferences in the past [41, 42] and in
food sciences to authenticate food products [43], traceability [44] and geographic origin [45].
Due to their difference in atomic mass, stable isotopes studies are based on the relation
between the quantities of the heavy and light isotopes of the studied element, which are
differently discriminated during chemical and/or biological processes. Characterizing such
differentiation, which is also known as fractionation, allows thus to have insights on the
processes that are occurring in a defined environment. The terminology used for reporting
stable isotopes results is stable isotope composition of an element X, expressed in permil
and noted as follows [46]:
nX = [(Rsample / Rstandard) -1]
where nX is the heavy isotope of the element X, R is the ratio between the heavy isotope and
the light isotope of the sample or the standard. Examples of standards are: the air for N
isotopes and the Pee Dee Belemnite (PDB) for C isotopes [46].
The two main techniques used to determine the isotope ratios of natural products are
isotope ratio mass spectrometry (IRMS) and site-specic natural isotope fractionation
analyzed by nuclear magnetic resonance (SNIF-NMR), each one having specific advantages
and disadvantages. For instance, IRMS has the advantage of being able to analyze almost all
elements, and SNIF-NMR has the advantage of allowing the precise and accurate
quantication of the natural abundance of hydrogen isomers (atomic nuclei that have the
same atomic number and the same mass number but different energy states [47]). SNIF-NMR
is also used to determine unique and distinctive isotopic ngerprint for a variety of
substances. The ngerprint is created on the basis of the isotopic composition of
biomolecules, because different isotopes of the same element (e.g., hydrogen, carbon,
nitrogen or oxygen) occur at characteristic relative quantities; these quantities and the
proportion of each isotope or the relative position of each isotope in a given molecule will
vary depending on the geographic origin and the processing and production techniques
applied to the sample [13]. For example, carbon isotopes are usually studied to differentiate
among sources of organic matter and to clarify carbon flow pathways, whereas nitrogen
isotopes provide information on trophic level as well as on organic matter origin [48, 49].
Likewise, the ratios of the stable isotopes of oxygen and hydrogen can give indication on
environmental conditions [50]. In fact, stable isotopes are dependent on kinetic effects, which
are irreversible and predominantly due to biological processes/mechanisms, such as
respiration, but also on equilibrium effects between different matter phases, such as
evaporation, being thus reversible. This is why their analysis for spoilage evaluation could be
of interest, besides the fact that only a very small quantity of sample is needed for such
analysis.
Stable isotopes composition and fractionation can be related to several processes leading
to food spoilage. For instance, metabolic starvation can create conditions for the muscle to be
catabolized in order to produce energy, originating a decrease in nitrogen content with
ammonia formation and elimination. These reactions can be related to smaller 15N values
that are associated with a loss in the heavier 15N isotope in relation to 14N [51].
In a later phase, after the death of the organism (animal), microbiota can develop and
start to colonize the tissues, increasing the total nitrogen content. Because this newly formed
microbiota is also sampled and quantified along with the organism's flesh, this will originate
the enrichment in heavy isotopes, reflecting the addition of microorganisms that were
developing onto the isotopically heavier excreted tissue fluids [52].
Finally, during microbiota-induced spoilage reactions, equilibrium is established between
the microbiota proliferation and the nitrogen loss by the formation of volatile compounds
(which can be attested by TVB-N determinations). This process can originate a decrease in
15N values and can also be linked either to the continuation of progressive deamination [51]
or to bacterial growth adding 15N-depleted biomass to the deteriorating tissues/organism [53].
It is noteworthy that, during seafood deterioration, the carbon content may not show
significant variations because spoilage processes are normally nitrogen related. However, the
C isotopic compositions can reflect a 13C depletion, while the organism/tissues are surviving
on their own resources, probably reflecting a selective preservation of 13C-depleted organic
compounds resulting from the release of 13C enriched CO2, during cellular respiration [53].
Furthermore, during the first phase of post-mortem spoilage, the possible subsequent increase
of 13C values may be originated by the incorporation of bacterial cells, due to natural
spoilage proliferation. This can also be linked to a decrease in C/N, due to equilibrium
isotopic effects inducing a preferential loss of light carbon [51]. In posterior spoilage phases,
a possible decrease in 13C can be induced again by a 13C preferential release, but this time
due to bacterial aerobic respiration [53].
Regarding the use of isotopes for authentication, studies on sh have mostly been carried
out on oil extracts. The stable isotopes signatures were used to differentiate Atlantic salmon
from different sources [54-57]. The ngerprint that resulted from the chemical shift
position and peak height of 13C of lipids can be used to identify the species and origin of
puried marine oils [57]. Aursand et al. [56] were able to correctly identify samples of wild
and farmed salmon from Norway and Scotland according to both the geographic origin and
the production method using stable isotopes in sh lipids extracts. A similar study was carried
out by Moltenkin et al. [58] using a combination of fatty-acid and 15N composition to
identify organically-farmed salmon, and differentiate their geographic origin (Norway or
Ireland). Furthermore, food authentication requires the construction of large and
representative databases containing ngerprints of all the relevant products, either authentic
or fraudulent, in order to satisfactorily apply stable isotopes analysis as an origin indicator.
This is because identication is supported by multivariate data analysis classication
techniques, which require the processing of the ngerprint obtained from an unknown sample
with as many ngerprints as possible (usually thousands) of samples representative for all the
possible species, tissues, origins, products, and production and processing conditions [13].
CONCLUSION
In our world, two statements can be applied to all living organisms: we are the product
of our environment and we are what we eat. These statements also apply to seafood
quality. On one hand, trace elements can be used to evaluate the toxicity, especially due to
heavy metals, and geographic origin of seafood and seafood products, mainly focusing on the
quality assurance perspective. On the other hand, stable isotopes are suitable for assessing the
metabolic pathways of spoilage and authenticity issues related to seafood. Regarding
spoilage, stable isotopes analysis does not seem to be a good method for immediate quality
control assessment, because it is not a rapid method and requires very expensive equipment
that is difficult to master technically. Nevertheless, stable isotopes analysis might play a
future role in setting-up or verifying quality assurance programs, where promptness and costs
are less important.
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Chapter 9
SAFETY AND QUALITY ISSUES

IN GLOBAL FISH TRADE
Shalini Amnee Neeliah1,*, Dayawatee Goburdhun2

and Harris Neeliah1
1
Sustainable Agri-Food Systems, Quatre-Bornes, Mauritius,
2
University of Mauritius, Reduit, Mauritius
ABSTRACT
Total world trade of fish and fishery products has undergone huge evolution in the
last three decades. Given this rapid expansion, food safety and quality have become
increasingly important. Consumers are becoming more demanding in respect of quality.
National and international regulatory frameworks have been established to ensure food
safety systems that function across national borders are well-established. For fish safety
and quality issues, the Sanitary and Phytosanitary (SPS) and the Technical Barriers to
Trade (TBT) Agreements and the Food Standards of the Codex Alimentarius
Commission are mostly relevant. Codex standards have been recognized as the key
reference point for the development of national technical regulations in the area of food
safety and quality. New WTO rules require national authorities to implement and enforce
government technical regulations, including SPS measures, for different product areas.
Even after the ratification of SPS and TBT Agreements, differences among various
national standards and inspection systems may maintain or create new non-tariff trade
barriers. Indeed, countries and regions have put in place national and regional regulations
to control seafood entering or exiting their territories. This diversity in control systems
has given rise to a new set of problems. Developing exporting countries often complain
that they are penalized by the complexity of sanitary regulations of importing countries,
by the disparities among importing countries regulations and enforcement mechanisms.
A second school of thought emerging from this literature review is that some countries
developed systems to conform to the stringent SPS requirements to exploit the developed
countries markets. In parallel to public control systems governing fish safety, the private
sector has also developed a number of private standards and codes of practice. Private
*
Corresponding author: Sustainable Agri-Food Systems, Quatre-Bornes, Mauritius Email: san.ahiscons
@gmail.com.
152 Shalini Amnee Neeliah, Dayawatee Goburdhun and Harris Neeliah
standards and related certification schemes are becoming important aspects of

international fish trade and marketing. The compliance costs connected with certification
to a private standard represent another controversial issue. It is foreseen that private food
safety standards will not diminish the role of regulatory food safety requirements and can
be mutually reinforcing.
Keywords: trade, fish safety and quality, private standards, regulatory, SPS
INTRODUCTION
This chapter considers relevant issues pertaining to the safety and quality of fish and fish
products in global fish trade, including mandatory and voluntary frameworks. The
methodology adopted is an extensive literature review, which is structured and systematically
presented into the salient safety and quality themes surrounding the global fish trade. These
are: International framework governing safety and quality of fishery products, Codex
standards governing fish and fishery products, SPS measures and fish trade and Private
standards in fish trade.
THE IMPORTANCE OF THE FISH INDUSTRY

Fish is the most widely consumed animal protein worldwide, with about one billion
people consuming it as a primary source of animal protein [1, 2]. It is well known that fish,
particularly oily fish, are an important source of long chain fatty acids (LC n-3
polyunsaturated fatty acid or PUFA), reducing the risk of cardiovascular diseases, as well as
having beneficial effects on fetal development [3]. Fish and other marine organisms are a
source of food and cash income, and frequently women are major beneficiaries of this activity
[4]. More than 70% of the total fish catch is used for direct human consumption. The
remainder is mostly used for the production of fishmeal and oil. The complexity of fish
production, utilization and marketing stems from the number of species marketed, the
technology used for harvesting and processing, and historical and cultural considerations [5].
For these reasons total world trade of fish and fishery products has undergone huge
development in the last three decades, increasing from USD 8 billion in 1976 to USD 126
billion in 2011 [6]. Given the rapid expansion in global fish trade, food safety has become a
topic of increasing importance and has taken centre stage.
The Global Trade in Fish and Fishery Products
Total world production of fish (by capture and aquaculture) amounted to some 156
million tons in 2011 [2, 6]. There has been an increase of more than 200% in the total fish
catch from 1960 to 2009 [7] and fish has emerged as one of the largest export commodities in
the world [1]. Fish is a highly traded commodity, with a third of global output by value traded
across international borders. The growth of the global fish trade has been substantial in recent
decades, providing a rare food-trade success story for several developing countries [8]. Fish
Safety and Quality Issues in Global Fish Trade 153
imports rose 79% from 2000 to 2008, reaching a new record of over USD 108 billion.
Developed countries accounted for about 80% of imports, in value terms [9].
The EU, Japan and the USA account for about 73% of world fishery product imports,
therefore controlling the market, both in terms of prices and quality requirements [10]. Since
2002, China has been the worlds largest exporter of fish and fishery products. In 2008 its
exports reached USD 10.3 billion. In the same year, other major exporters were Norway
(USD 7.0 billion), Thailand (USD 6.5 billion), Viet Nam (USD 4.6 billion), the USA (USD
4.5 billion) and Chile (USD 4.0 billion) [9]. On the other hand, developing economies with
their hold over key fishing grounds are playing an increasingly important role in the global
fish industry. In 2010, their exports represented 49% (USD 42.5 billion) of world fish exports
in value and 59% (31.6 million tons live weight equivalent) in volume. For most of the
developing countries, the revenue from these exports is an important source of foreign
currency. The EU imported USD 23 billion worth of fish and fisheries products from non-EU
suppliers in 2007 [11].
African countries in particular have seen a net increase of 250% in the value of export of
fishery products in real terms from 1980 to 2000 [12]. Although Africa is a huge continent,
with an enormous coastline, the continent only accounts for 8 million tons or 5.1% of the
worlds total fish production (capture plus aquaculture in 2007). The largest producers in
Africa include Egypt (just over 1 million tons in 2007), Morocco (894,000 tons) and South
Africa (683,000 tons) [11]. Fisheries are a key source of employment, export revenue and
food security for many ACP countries [13].
Commercially Important Fish Species
The type of species caught depends on consumer demand. Demersal fish such as cod are
preferred in Northern Europe and North America and cephalopods are generally consumed in
several Mediterranean and Asian countries [14]. The bulk of the world fish catch (some 30%)
is made up of pelagics of relatively small size, with a high lipid content; white fish, such as
hake, Alaska pollack and haddock constitute 13% of the total catch. Tuna, bonito, mackerel
and Thyrsites atun (snoek), which are especially fatty species but larger and in greater
demand commercially, represent 9% of the catch. Skipjack is by far the main tuna species
caught [15].
SAFETY AND QUALITY ISSUES IN GLOBAL FISH TRADE

Market quality is a complex issue, reflecting the sophistication and variety of products
and of markets. Fishery products are at the forefront of food safety and quality improvement,
as they are among the most internationally traded food commodities [14]. Moreover, fish is
highly perishable and can be contaminated by naturally occurring pathogenic marine bacteria,
by viruses and bacteria of human origin, by toxins produced by marine plankton, by heavy
metals that accumulate through the food chain [16]. Some areas of safety concern in seafood
also include parasites, decomposition (e.g., biogenic amines, histamine, putrescine,
cadaverine), environmental contaminants, pesticides and aquaculture drugs [17]. The quality
of fish and fish products relies principally on safe, hygienically-produced products [18].
Examples of safety and quality concerns linked to tuna consumption include histamine and
methyl mercury contents [19]. Other concerns which have acted as technical barriers /sanitary
barriers to trade, according to Macri and Lucangeli [20], are:
Country Of Origin Labelling in USA (COOL)

Turtle Excluding Devices for catching shrimp
Animal welfare or tuna-dolphin issue: the certification that dolphins are not killed
while fishing for tuna
Trade description of scallops: Saint-Jacques against ptoncle
Australia: banning imports of salmon from Canada due to possible fish disease
agents
Trade description of sardines: Peru versus EU Sardinella pilchardus vs. Sardinops
sagax.
Many Asian countries (China, Thailand, Indonesia, India, Philippines, Vietnam,

Bangladesh) had problems with antibiotics (chloramphenicol) in shrimp exported to the EU,
leading to the destruction of the product and to a temporary ban on shrimp from the above
countries [21].
International Framework Governing Safety and Quality of Fishery Products
Consumers are becoming more aware of possible food hazards and are more demanding
in respect of quality. Action to regulate fish trade at international level is felt necessary
because of the [22]:
increasing demand for fishery products,

development in international fish trade,
globalization of the economy,
development of regional economic groupings and
need to ensure fish safety and fair trade practices.
National and international regulatory frameworks have been established to ensure food
safety systems that function across national borders are well-established and have been
described in Washington and Ababouch [23]. The joint Food and Agriculture Organization
(FAO)/World Health Organization (WHO) Codex Alimentarius Commission (CAC) is the
international reference for national food safety and quality strategies. However, fish exporters
still have to deal with safety and quality-control regimes that vary from one jurisdiction to the
next, as well as standards being increasingly imposed by the private sector [23]. In addition to
their firm-specific product and process specifications, many large retailers, commercial brand
owners and food service industry firms demand that their suppliers of processed fish and
seafood be certified to a national or international food safety management scheme (FSMS),
and for aquaculture products to be certified to one or other scheme that mixes quality and
safety with environmental protection, animal health and even social development.
Role of WTO in Fish Trade

The World Trade Organization (WTO) was established in 1995 as the successor to the
General Agreement on Tariffs and Trade (GATT). Fishery products fall in the category of
industrial goods and therefore do not fall under the WTO Agreement on Agriculture. Instead,
fishery products are discussed in the Non-Agricultural Market Access (NAMA). The Uruguay
Round produced major improvements in market access for NAMA products in the developed
country markets, as tariff averages were reduced from 6.3% to 3.8%. In the case of
developing countries, the most important contribution was made in the form of new tariff
bindings. Despite the significant improvements in market access for NAMA products that
previous GATT rounds and the Uruguay Round produced, tariffs remain an important barrier
to world trade, as tariff peaks, high tariffs, and tariff escalation persist [24].
Fishery products are regulated via a number of multilateral and bilateral agreements. The
WTO Agreements of relevance for fisheries are the following [15]:
Sanitary and Phytosanitary Measures (SPS) Agreement

Technical Barrier to Trade (TBT) Agreement
Agreement on Subsidies and Countervailing Measures
Agreement on Import Licensing Procedures
Agreement on Anti-Dumping
Agreement on Rules of Origin
The Agreement on Technical Barrier to Trade (TBT) and the Agreement on the
Application of Sanitary and Phytosanitary Measures (SPS), deal mostly with the preparation,
adoption and application of technical regulations and stipulate the responsibility of Central
Government of WTO member countries to regulate the national market in a transparent way.
For fish safety and quality issues, the SPS and TBT Agreements and the Food Standards of
the CAC are most relevant [22]. To this end, a number of national structures and systems
have to be established [25, 26]. New WTO rules require national authorities to implement and
enforce government technical regulations, including SPS measures, for different product
areas.
The main objective of the SPS Agreement is to promote the harmonization of standards.
Another objective is the protection of human, animal and plant health in all WTO Member
Countries, through the drawing of multilateral trade rules and disciplines to guide the
development, adoption and enforcement of SPS measures and mitigate the negative effects on
trade. WTO has assigned this rule-making responsibility in the field of food safety to the
CAC. With respect to animal and plant life or health, international standards made by the
World Organization for Animal Health (OIE) and the International Plant Protection
Convention (IPPC) serve as reference [27].
The SPS Agreement is composed of 14 articles stipulating procedural and substantive
requirements and three annexes with the definitions and additional details on the procedural
requirements. The SPS Agreement introduces new disciplines that underpin trading practices
at the international level. It sets out the rights and responsibilities of WTO Members desiring
to take action to restrict imports in order to protect human, animal or plant life or health [28],
within the territory of the member country from risks of diseases, pests and disease-carrying
organisms [27].
Under the SPS Agreement, WTO members have certain rights and obligations. Article 2
stipulates the use of scientific principles by members to take measures and that members must
not be arbitrary or unjust in their application of measures on other members where similar
conditions prevail. Although members can deviate from the use of international standards,
they must do so based on a risk assessment (Article 5) and sound science and avoid the use of
measures that are more trade-restrictive than required to achieve the desired level of
protection [29]. Neeliah et al. [30] discusses the impact of the different principles of the
Agreement on Members.
The SPS Agreement is supplemented by the TBT Agreement, also part of the Uruguay
Round Agreements. The objective of the TBT Agreement is to ensure that technical
regulations and standards, including packaging and conformity assessment procedures do not
act as trade barriers. It covers all types of standards and also quality requirements for foods,
except those under SPS Agreement. The TBT Agreement contains many measures geared
towards consumer protection against deception and economic fraud. Together, the SPS and
TBT Agreements encompass all aspects of food standards, including food safety and quality
and additional concerns relating to labelling and consumer fraud. The aspects of food
standards that TBT requirements specifically cover are quality provisions, nutritional
requirements, labelling, packaging and product content regulations, and methods of analysis
[6].
The SPS Measures are among the most relevant for fish trade [31] as they may prove to
be the preferred means of protectionism for importing countries. The relevant provisions of
the SPS Agreement for trade in fish and fish products are:
to use harmonization principles, i.e., to establish national sanitary and phytosanitary

rules reflecting standards agreed in the relevant international institutions such as the
Codex Alimentarius Commission for fish products and OIE for live fish;
when international standards do not exist or harmonization is not appropriate, to use
the alternative equivalence principle whereby the importing country accepts that SPS
measures in the exporting country achieve an appropriate level of health protection,
even though they differ from the measures used in the importing country;
to provide either scientific evidence or appropriate risk assessment if a country
intends not to rely on harmony or equivalence but rather on its own domestic
standards.
National Regulatory Systems

Even after the ratification of SPS and TBT Agreements, differences among various
national standards and inspection systems may maintain or create new non-tariff trade barriers
[17]. The public health significance of seafood-borne illnesses depends on the likelihood and
the severity of the illness [6]. Following the WTO SPS Agreement, the concept of risk
analysis is by default the method for setting tolerable levels of hazards in foods in
international trade and, subsequently, within national jurisdictions. In the current international
food safety management arena, the risk is expressed as food safety objectives in order to
achieve what is called an appropriate level of protection for populations [6]. For
international fish trade, countries and regions have put in place national and regional
regulations to control seafood entering or exiting their territories. As more than 70% of
seafood trade is targeted towards three main markets (the European Union [Member
Organization], the United States of America, and Japan), these markets are important
regulatory reference points.
Demands for improved quality and safety in the major markets of the EU, USA and
Canada have resulted in the creation of fish quality legislation [21]. In the early 1990s, the
major fish producing, exporting or importing countries have initiated a renovation of fish
inspection regulations for the implementation of systems based in the Hazard Analysis and
Critical Control Points (HACCP) system, in conformity with the guidelines of the CAC.
HACCP-based regulations are being increasingly used worldwide in the majority of
developed countries and in developing countries exporting to developed countries. These
have influenced the fish processing industry to a large extent [22]. Since 1992, Canada has
applied a Quality Management Program (QMP), the first mandatory food inspection program
in the world based on HACCP principles.
The USA has a decentralized system for food safety and quality regulation. There are 17
federal government agencies involved in food regulation. The two main ones are the Food
and Drug Administration of the Department of Health and Human Services, which regulates
all food except meat and poultry, and the Food Safety Inspection Service of the Department
of Agriculture, which is primarily responsible for meat and poultry. The Food Safety
Modernization Act (FSMA) of 2011 is now the leading legislation for enhanced food safety in
the USA [6]. In recent years, regulatory control has been reinforced by better cooperation
among the various agencies and by the FSMA of 2011.
The National Oceanic and Atmospheric Administration (NOAA) is responsible for
fisheries management in the United States. The NOAA Seafood Inspection Program offers
inspection services for fish, shellfish, and fishery products to the industry. The NOAA
Seafood Inspection Program offers a variety of professional inspection services on a fee-for-
service basis which assure compliance with all applicable food regulations. The Program
offers sanitation inspection, laboratory analyses, export certification as well as system and
process auditing in facilities, on vessels, or other processing establishments in order to be
designated as participating establishments. Product quality evaluation, grading and
certification services are available on a product lot basis. The program is the competent
authority within the U.S. Government for delivery of health certificates for export of fish and
fishery products to foreign countries [32].
The FDA and the Seafood Inspection Program have been working more closely on
seafood issues through a renewed memorandum of understanding that was completed in 2009
[6]. The USA adopted mandatory seafood HACCP regulations in December 1997. In June
2002, the USA passed the Public Health Security and Bioterrorism Preparedness and
Response Act of 2002 (the Bio Terrorism Act). The law includes specific provisions that
protect US citizens from food imports that are dangerous to human health [33].
In the European Union, following a white paper on food safety published in 2000, the
regulatory approach has been to detach aspects of food hygiene from animal health and to
harmonize food control across the member countries. A crucial aspect of the legislation is that
all food and feed business operators, from farmers and processors to retailers and caterers,
have the responsibility for assuring that food placed on the market in the European Union
meets the required food safety standards [6]. The HACCP system has been adopted in 1994 as
a Directive 491/93 EEC and Decision 94/356/EEC and still forms part of the Hygiene
Package that is composed of Regulations (EC) 852/2204, 853/2004 and 854/2004.
Third countries have been allowed to export to the EU since January 1999 if approved by
the European Commission. Approval is subject to an assessment of the ability of the
countrys competent authority to guarantee the standards of the operators [34]. Products
imported from third countries must adhere to the same provisions that govern products made
in the EU for the EU market. In the case of non-harmonized products, national rules from
Member States can be applied in addition to the EU legislation. The incoming consignments
are subjected to border inspections, which are carried out in approved and listed border
inspection posts. The requirements on the sanitary control system of third countries have
given way to two categories of countries (Commission Decision 2004/359/EC for fishery
products), taking into account the:
third country legislation,

organization and powers of the third countrys Competent Authority,
inspection services, and
actual health conditions.
Countries included in List I are harmonized or approved countries, that is, their
legislation requirements are at least equivalent to those governing the EU domestic
production. This is after an EU inspection team has audited the Competent Authority and has
found it to satisfy EU requirements. A specific decision has been adopted for each of those
countries fixing specific import conditions, including the official recognition of the
Competent Authority, a specific model of health certificate and a list of approved
establishments. Import from non-harmonized countries into the EU is not allowed. Import
controls at border level are done through [35]:
a documentary check: examination of the health certificate;

an identity check: visual inspection to confirm consistency between documents and
products, verification for the presence of required sanitary marks (country of origin,
approval number);
a physical check on the product itself (organoleptic control, packaging, temperature),
it may include sampling and laboratory testing.
Products imported from harmonized countries are subject to these checks at the
approved border inspection post at the first point of entry into the EU territory. When such a
consignment satisfies EU requirements, it is then considered as an EU product.
The new EU food safety and hygiene framework (commonly designated hygiene
package) in force since January 2006 covers all foodstuffs from farm-gate to retail and
requires a traceability system to be in place (Table 1). Special provisions/chapters/annexes
apply to fisheries products, some coming from previous fishery-specific regulations. With the
implementation of the new hygiene package, third countries require health and sanitary
regulations at least equivalent to the ones required within the EU. Competent authorities
should also be present to guarantee effective implementation of the relevant regulations
through inspection, monitoring and sanctioning systems. Food business operators need to
apply specific sanitary and health practices in catching, handling, processing and packaging
fishery products, and a system of risk management based on HACCP. Regulations pertaining
to contaminants and pesticide residues have been updated (Regulations (EC) 1881/2006 and
396/2005, respectively).
Japan has enacted the Food Safety Basic Law (enacted in 2003), a complete law to ensure
food safety to protect public health [6]. Through the development of the basic law and other
related laws such as the Food Sanitation Law, the Abattoir Law, the Poultry Slaughtering
Business Control and Poultry Inspection Law, Japan has introduced a risk analysis approach
to the national food safety control program. While the Food Safety Basic Law gives
responsibility for risk assessment, the Food Sanitation Law and related legislation identify
those responsible for risk management. The risk assessment is, in practice, conducted by the
Food Safety Commission established under the Food Safety Basic Law [6]. The use of
HACCP-based systems is voluntary for domestic production but mandatory for exports to the
EU and the United States [5, 22].
Table 1. EU Directives, Regulations and Decisions affecting fishery products trade
Directives Commission Regulations Commission Decisions

Directive 91/493/EEC: health Regulation (EC) No 178/2002- Decision 93/140 - parasites: checks
conditions for the production general principles and requirements are carried out visually on shore or on
and placing on market of of food law board factory vessels at all stages of
fishery products- most Regulation (EC) No 852/2004 on the production on a representative
provisions incorporated in the the hygiene of foodstuffs number of samples
General Hygiene Package Regulation (EC) No 853/2004- Decision 98/140/EC: detailed rules
Directive 92/48/EEC: hygiene rules for food of animal concerning on-the-spot checks
minimum hygiene rules origin (with effect from the 1st (veterinary)
applicable to fishery products January 2006) Decision 93/351-list of species which
caught on board certain Regulation (EC) No 854/2004: must be frequently sampled
vessels specific rules for the organisation of Decision 2001/183/EC-sampling
Directive 2001/22/EC: official controls on products of plans and diagnostic methods for
sampling methods / methods animal origin intended for human detection and confirmation of certain
of analysing for lead, consumption fish diseases
cadmium, mercury and 3- Regulation (EC) No 882/2004: on Decision 95/249/EC - total volatile
MCPD official controls performed to ensure basic nitrogen (TVB-N) limit values
the verification of compliance with for certain fishery products and
feed and food law, animal health analysis methods
and animal welfare rules Decisions 93/25 and 97/275 and
Regulation (EEC) No 3703/85- recently Regulation 2073/2005:
detailed rules for applying the microbiological criteria for various
common marketing standards for commodities
certain fresh or chilled fish Decision 94/356 to implement an
Regulation (EC) No 1250/2008 own-check system (HACCP)
(amending Regulation (EC) No Decision implementing Regulation
2074/2005)- amending Regulation (EU) No 1420/2013 on the common
(EC) No 2074/2005 as regards organisation of the markets in fishery
certification requirements for import and aquaculture products
of fishery products, live bivalve
molluscs, echinoderms, tunicates
and marine gastropods intended for
human consumption
Regulation (EC) No 2377/90:
maximum residue limits of
veterinary medicinal products
Microbiological criteria for
foodstuffs
Regulation 2065/2001 on labelling
Table 1. (Continued)
Directives Commission Regulations Commission Decisions

maximum levels for certain
contaminants in foodstuffs amended
by Regulation (EC) No 78/2005 for
fishery and aquaculture products
maximum levels are set for mercury,
cadmium and lead
Regulation (EU) No 1379/2013 on
the common organisation of the
markets in fishery and aquaculture
products
(Source: [17, 37, 38])
Other countries with a well-developed regulatory system for fish safety are New Zealand
and Australia. The food regulatory and legal systems in these two countries have been
harmonized through a joint food standards system covered by the Australia New Zealand
Food Standards Code [36]. Furthermore, these two countries have rigorous quarantine,
biosecurity and safety provisions to protect their disease-free status and their consumers. The
safety of the imported food supply and the prevention of the spread of non-indigenous
diseases are of high concern to their governments. Therefore, both countries have broad,
science-based requirements relating to the import of seafood [6].
Features in Fish Safety Management

There is increasing evidence that the implementation of HACCP-based systems has
contributed to improving fish safety and quality and there is a growing awareness of the
importance of an integrated, multidisciplinary approach to safety and quality, considering the
entire fish food chain. Ryder et al. [6] recommends that in fisheries and aquaculture, there are
five broadly defined needs on which a strategy supporting the food chain approach to food
safety should be based:
Fish safety and quality from a food chain perspective should incorporate the three
fundamental components of risk analysis, that is, risk assessment, risk management
and risk communication;
Tracing techniques (traceability) from the primary producer through post-harvest
treatment, processing and finally distribution to the consumer must be enhanced,
Harmonization of fish quality and safety standards,
Equivalence (a principle under the WTO SPS Agreement) in food safety systems
must be further developed,
Increased emphasis on preventative approach including development and
dissemination of good aquaculture practices (GAPs), Good Manufacturing Practices
(GMPs) and safety and quality assurance systems, i.e., HACCP, are necessary to
complement the traditional approach to fish safety and quality management based on
regulation and control.
Assessment and Management of Seafood Safety and Quality

It is worthwhile mentioning that recently, the FAO released a technical paper [6]
compiling the state of knowledge on seafood safety and quality, covering topics on emerging
issues such as new pathogens, the impact of climate change on seafood safety, the
developments in safety and quality systems and the changing regulatory framework. The
hazards of public health concern in fish and fish products, covering biological (pathogenic
bacteria, histamine, viruses, parasites and biotoxins), chemical (veterinary drugs, industrial
organic contaminants, environmental inorganic contaminants and allergens) and physical
hazards are reviewed in detail therein. The implementation and certification of seafood safety
systems covering risk mitigation and management tools, with a detailed description of the
requirements for the implementation of good hygiene practices and good manufacturing
practices; the HACCP system; and the monitoring programs to control biotoxins, pathogenic
bacteria and viruses and chemical pollutants, on private labelling and certification schemes
are also covered. Ryder et al. [6] also delves into the international framework, covering the
WTO, the CAC, the FAO Code of Conduct for Responsible Fisheries, and the OIE. It then
showcases the regulatory frameworks governing seafood trade in the European Union, the
United States of America, Japan, Australia and New Zealand. The FAO paper [6] is certainly
an interesting reference manual in seafood quality and safety.
Codex Standards Governing Fish and Fishery Products
The CAC was established in 1962 by the FAO and the WHO to set out food standards.
Since its establishment, Codex standards have been recognized as key references for the
development of national technical regulations in the area of food safety and quality. Thus, in
1985, United Nations Resolution stipulated that governments should adopt standards from the
Codex as far as possible when formulating national food policies [39]. With the setting up of
the WTO in 1995, CAC standards, guidelines and recommendations have been recognized as
the international benchmark in the area of food safety and thus in any SPS dispute raised
within WTO jurisdiction. Therefore, the standards developed by the Codex Commission
should be viewed as providing countries with necessary protection. Higher levels of
protection must be justified with sound science and the use of appropriate risk assessment
techniques.
This new status has heightened the profile of the activities of the Codex Commission.
Although this could be seen as a positive step, it has already led to a review of standards
drafting and standards acceptance procedures at meetings of the Commission [40]. Another
spillover effect of the WTO Agreements is a major reorganization of the activities of Codex
Committees, moving the focus from commodity specific standards relating to quality
characteristics to more general aspects linked to safety, like food hygiene. Decisions have
been based on votes cast [40] and not on consensus as was the case before 1995. Moreover,
with the emphasis placed on risk assessment in the SPS Agreement, Codex Commission has
worked on the required guidelines and has initiated action to develop a consistent science-
based approach to its future recommendations in this area. A number of institutional
innovations including the adoption of a fast track approval procedure for some standards,
creation of more working groups to address new issues, and an increase in the number of its
meetings to speed the adoption process, have followed [41].
The Codex Commissions Committee on Fish and Fishery products has prepared a code
of practice for fish and fishery products [42]. This code integrates Codex Alimentarius
general hygiene principles and adapts them to the fish industry. It is intended for all those
engaged in the handling, production, storage, distribution, export, import and sale of fish and
fishery products. It is also an attempt to harmonize food safety regulations in the fish trade. It
contains information from previous codes as well as new material pertaining to aquaculture
products, HACCP, frozen surimi distribution and retail display of fish and fishery products.
In addition, the code of practice contains guidance on prerequisite programs, the use of
HACCP and a similar systematic approach referred to as defect action point (DAP)
analysis. The latter has been applied to essential quality, composition and labelling
provisions of the appropriate Codex product standards [42]. However, DAP analysis is
optional. The code is expected to be a user-friendly guide to assist all those who are in the
business of handling and production of fish and fishery products, or are concerned with their
storage, distribution, export, import and sale in producing safe and wholesome products that
can be sold on national or international markets and meet the requirements of the Codex
standards [42].
SPS Measures and Fish Trade
One of the most serious difficulties faced by fish exporters is that distinct importing
countries impose different standards. Developing exporting countries often complain that they
are penalized by the complexity of sanitary regulations of importing countries, by the
disparities among importing countries regulations and enforcement mechanisms. Moreover,
diverse border control systems are used by importing countries. These variations are further
complicated by differences in the type of tests to which samples are subjected and to the
methods of analysis applied. Certificate requirements of different countries cause
inconvenience and transaction costs to both exporter and responsible government regulatory
agency. Use of different forms and languages often results in confusion [43]. All these have a
negative impact on the free flow of trade as exporters have to master several (or more)
systems in order to get their products to market. This wastes time, adds cost and leads to
mistakes [17].
Effect of SPS Measures on the Fish Export Sector

Many studies have highlighted the effect of SPS measures on the fish sector. Although
some were qualitative in nature, others provided a quantitative estimate of the negative effects
of SPS measures. Table 2 provides a summary.
Table 2. Past studies on the impact of SPS measures on the fish sector
(adapted from Neeliah et al. [44])
Exporting country Nature of SPS

Source Impact and remedial measures
and destination measure
Wilson and East African Ban on fish exports Impact at macro and micro levels: Reduced
Abiola [49], countries (Uganda) in 1999 because of returns of USD 36.9 million; 3 out of the 11
Balagadde to the EU incapacity of factories closed and related industries like
[50] Uganda's packaging, transport and the economy in

Competent general affected. Implementation of HACCP
Authority (UNBS) and GMP (USD 100 million to comply with
to guarantee fish quality requirements); training, equipment
safety due to purchase, certification, resulted in lifting of
inadequate testing ban and increase in exports. Capacity of
facilities Competent Authority strengthened and
inspection improved.
Henson India to the EU Sanitary problems A 9% decline in total exports by value; other
et al. [51], (shrimp peeling markets targeted; official control revisited;
Mehta and sheds); deficient improvements made by plants to comply with
Georges [52], official system of the EU requirements costing some US$
Henson inspection in 1997 174,000 and 220,000; training on HACCP.
et al. [53] Seafood Exporters Association of India spent
USD 25 million to upgrade facilities. Costs of
compliance ranged from USD
51,400 to USD 514,300. As a proportion of
turnover in a single year (199798), these
costs ranged from 2.5% to 22.5%.
Henson Vietnam to the EU Problem with Initiatives taken by Government to improve
et al. [51] microbiological sanitary conditions; implementation of
content of seafood HACCP by affected company.
products in 1998
Henson Ghana to the EU Introduction of EU Suspension of fresh and frozen fish to the EU
et al. [51] regulations relating at the initiative of the Ghana Standards
to fish in 1997 Bureau; bilateral negotiation with the EU;
technical assistance from the EU for HACCP
implementation.
Henson Kenya, Uganda, Salmonella in Nile GBP 20 Million of trade lost by Kenya during
et al. [51], Tanzania to the EU Perch ban on ban; part of exports directed towards other
Henson exports in 1997 markets such as United Arab Emirates, Israel,
et al. [34] Japan; 37% decrease in exports; legislative
changes; reform of procedures for approval of
plants for export to the EU and for health
certificates; investments in upgrading of
processing facilities; improvement in fresh
fish supply management
Cato and Bangladesh to the Problems in plants Increased export to alternative markets such
Lima dos EU and at level of as US and Japan; average of USD 7,584 lost
Santos [54] control by per firm where product destroyed. Total
competent authority estimated lost revenue due to ban: US D 14,
detected: Ban in 665 million; on subsequent inspections after a
1997 year, ban lifted for 11 companies; competent
authority recognized.
Wilson and Mozambique to the Ban on fishery Loss of about USD 60,000 a month in hard
Abiola [49] EU products (1998) currency earnings. Authorities of
because of a Mozambique tried to resolve problems
Cholera outbreak in through consultations (bilateral level and ACP
Mozambique. level) with the EU.
Wilson and Nigeria to the USA In 2001 and 2002, Information not available
Abiola [49] products rejected
(smoked fish and
sardines)
Cato and Bangladesh to the In 1997, ban on Cost to the Bangladesh shrimp-processing
Subasinghe EU fishery products due sector: USD 15 million in lost revenues. By
[55] to serious 1997, the Bangladesh shrimp processing
deficiencies in industry had invested USD17.6 million in
infrastructure/ plant upgrades, the government had invested
hygiene in USD382,000 in laboratory and personnel

upgrades, and outside partners had invested
establishments and USD 72,000 in training programs in
in government Bangladesh. By 2002, out of 65 plants
inspection system. licensed for export by the government, 48
plants had EU approval.
CTA [56], Kenya to the EU Import restrictions This ban resulted in a 68% decline in the
Abila [57] for fish from Lake value of fish exports.
Victoria (1997 and
1999) due to
concerns about
hygiene standards
in supply chain
Nanyaro [58] Tanzania to the EU Between 1996 and Loss of foreign exchange earnings (about
1999 it suffered USD 90 million for the 1999 ban). Collapse
three major bans, of ancillary industries leading to massive
the worst being in unemployment, collapse of stakeholders
1999, which lasted incomes. Total fishery products export fell by
11 months 40%. Around USD 8 million was reinvested
by the Government and the industry to
address the perceived hygienic non
compliances
Neeliah Mauritius to EU Following a mission At the level of the public sector, a consultant
et al. [48] of the Food and was appointed for reviewing activities of the
Veterinary Office of competent authority; government also
EU, a number of undertook legal reform and reallocated
deficiencies with responsibilities for fish control. A one-stop
respect to the shop where different ministries collectively
implementa-tion of provided a service to the exporters for rapid
the EU hygiene delivery of export-related certificates was set
norms (2004) were up. Government also recruited an expert to
highlighted in local assist in restructuring the supply chain and
processing plants increasing control over primary production.
that could affect Public and private stakeholders also
exports to EU. collaborated to enhance compliance of the
fishery export sector by setting up a Seafood
Hub Committee.
Many developing countries face various problems associated with meeting SPS/TBT
measures. This not only applies to the fishery sector but also to a number of other export
sectors [33]. Based on a review of past studies, Neeliah et al. [44] found that the progressively
stricter food safety requirements in major industrialized countries have had a negative impact
on exporters of fishery products in developing countries both at the micro and macro levels.
The review of past studies also indicates that in the wake of legislative reform that took place
around year 2004 in the EU, developing exporting countries could face the same problems
unless they adopted a proactive approach.
Several countries, mostly in Africa and Asia, face problems while exporting [44]. Based
on the studies reviewed, the market posing more problems seems to be the EU. Individual
developed country markets have different effects for products subject to detailed SPS controls
[45]. This may be due to the differences in regulatory approaches existing in different
developed countries markets. Indeed, there is great divergence between the food safety
requirements and the related conformity assessment procedures applied to fish and fishery
product imports in the EU, US, Japan and Australia [12, 17]. For example, in both the US and
the EU, imports of fish and fishery products must be processed in premises of equivalent
standards to domestic facilities, including the implementation of HACCP. However, while in
the United States the importer must ensure that imports meet regulatory requirements, in the
EU this is the responsibility of a competent authority in the exporting country. This requires
not only that the exporter complies with EU regulatory requirements, but that the exporting
countrys government puts in place regulations and procedures so as to certify that this is the
case. This may create an additional difficulty [44].
A second school of thought perceived from the literature review is that some countries
developed systems to conform to the stringent SPS requirements to exploit the developed
countries markets [46]. There were also very positive returns in terms of continued and/or
expanded access to high-value markets for those exporters that were able to comply [12, 47,
48].
Findings from EU Food and Veterinary Office Missions to Third Countries

The European Commission has the responsibility for ensuring that Community legislation
on food safety, animal health, plant health and animal welfare is properly implemented and
enforced. The EU Food and Veterinary Office (FVO) fulfills this role by [59]:
promoting effective control systems in the food safety and quality, veterinary and
plant health sectors;
checking on compliance with the requirements of EU food safety and quality,
veterinary and plant health legislation within the EU and in third countries exporting
to the EU;
contributing to the development of EU policy in the food safety and quality,
veterinary and plant health sectors; and
informing stakeholders of the outcome of evaluations.
In this context, the following inspections are carried out by the FVO [59]:
veterinary inspections
plant health inspections
contamination of food and feed materials inspections
food hygiene inspections
food irradiation inspections
genetically modified food inspections
pesticides inspections (inspections of controls on marketing/use of plant protection
products and on pesticides residues in foodstuffs by country)
organic farming inspections.
Building on the findings of food hygiene inspections carried out by the FVO in African
countries exporting fish, it can be concluded that the status of compliance with EU
requirements for the export of fish ranged from acceptable to serious. Examples of
deficiencies include lack of clearly written guidelines and procedures at the level of
inspection; absence of follow-up from the Authority when non-conformances had been noted;
inappropriate laboratory facilities, i.e., not all tests implied by the EU Directives were being
performed: and, if facilities were available, they were not accredited [44].
Rapid Alert System for Food and Feed

The EU operates a Rapid Alert System for Food and Feed (RASFF) as per Regulation
(EC) 178/2002. The role of the system is to provide the control authorities with a means for
exchange of information on measures taken to ensure food safety. Information is categorised
according to risk [60]:
alert notifications are sent when the food or feed presenting the risk is on the market
and immediate actions are needed;
information notifications are sent when the food or feed presenting the risk has not
reached the destined market and the consignments have been tested and rejected at
the external borders of the EU.
The number of notifications transmitted through the RASFF decreased from 7354 in 2007
to 3137 in 2013. In 2013, 9.9% of the alert notifications pertained to fishery products. Of
greater concern in fishery products were notifications pertaining to mercury, histamine,
Salmonella species, pesticide residues and dioxins [60].
Based on the review of studies dealing with the effect of SPS measures on fish exports
from developing countries [44], on findings of FVO missions to African countries and on
RASFF notifications pertaining to fish products [44], it is clear that developing countries face
a number of challenges whilst exporting fishery products to the EU.
PRIVATE STANDARDS IN FISH TRADE

In parallel to public control systems governing fish safety, the private sector has also
developed a number of private standards and codes of practice. This action has been
motivated by the need to address food safety risks, consumer concerns and preferences, but
also to mitigate commercial risks and as a strategy of differentiation [61-63]. It can also be a
way of extracting rent from suppliers. For instance, Tesco has its private standard (Tescos
Nature Choice) and suppliers are audited and certified by Tesco itself against a fee. More
recently, fish exporters are increasingly under pressure to match private quality standards set
by their main costumers, such as processors and leading supermarket chains [64]. It is not
proposed here to dwell at length on private standards: the subject has been amply discussed
by Henson [65] and reviewed by Henson and Humphrey [39] and ITC [66]. But the main
private requirements for fish and fish products are highlighted in Table 3.
Washington and Ababouch [23] examined the two main types of private standards
affecting fish trade and their implications for different stakeholders, in addition to their
overall policy and governance implications. They focused on ecolabels or private standards
and certification schemes related to the sustainability of fish stocks, geared to prompt
responsible fisheries practices and to influence the procurement policies of large retailers and
brand owners, as well as the purchasing decisions of consumers. Private standards and
certifications related to food safety and quality in fish and seafood from both marine capture
and farmed sources are also considered. The authors concluded that the use of such standards
was becoming more common in efforts to ensure food safety, quality and environmental
sustainability in the growing aquaculture industry. Indeed, private certification schemes have
emerged in aquaculture in response to concerns about aquaculture by offering guarantees
related to quality, safety, environmental impacts, social responsibility, traceability, and
transparency of production processes [23]. It was foreseen that impact of private standards
was likely to increase, even in developing countries, although not uniformly across markets,
species or product types.
Private standards relating to food safety reflects the need of buyers to be assured that
good practices have been implemented properly throughout the supply chain, rather than a
lack of confidence in public food safety management systems, including the lack of direct
access to audit reports on individual operators. For developing countries, it is more than clear
that the most important impediment to increased exports is no longer import tariffs by
importing countries but quality- and safety-related import requirements in import markets.
The range of private standards adds to that challenge. For example, Carrefour, the worlds
second largest retailer, procures shrimp directly from farmers in Thailand, which entails
sending their own inspectors to verify that products and farming practices meet their own
standards [23]. Apart from firm-specific product and process specifications, firms might also
request suppliers to be certified as follows [23]:
For processed fish and seafood: a national or international food security management
systems (FSMS), such as the British Retail Consortium (BRC), International Food
Standard (IFS), Safe Quality Food (SQF);
For aquaculture: to one or other of the schemes that merge quality and safety with
environmental protection, animal health and even social development such as those
certified by the Aquaculture Certification Council (ACC);
For wild capture fish and seafood: to an ecolabelling scheme.
Private standards and related certification schemes are becoming important aspects of
international fish trade and marketing. Private standards have the likelihood to result in
positive effects and trigger positive impact both at the producer and at the supply chain level
[66]. They have surfaced in areas where there is a perception that public regulatory
frameworks are not reaching the desired outcomes, such as sustainability and responsible
fisheries management [23]. Adoption of private standards tends to be preferred in contexts
where (i) the type of product has elevated requirements regarding traceability, (ii) in
extractive businesses, (iii) where commodities are identifiable in end-products, or (iv) where
there are shorter supply chains with fewer stakeholders [66]. Furthermore, their use is,
becoming more widespread in efforts to guarantee food safety, quality and environmental
sustainability in the blossoming aquaculture industry [23].
The compliance costs connected with certification to a private standard represent another
controversial issue. These costs are borne disproportionately by those upstream in the supply
chain rather than those downstream where the demands for certification generate [23]. Since
2005 the trade effects of private standards have been raised at the level of the WTO [65]. But
this is still work-in-progress, as discussions revolve primarily on issues of technical co-
operation and strategies for facilitating compliance [67]. Henson [65] and Hobbs [67]
consider that the WTO does not have any jurisdiction over private food safety standards.
Others insist that it is still uncertain whether the SPS Agreement has any legal jurisdiction
over private standardization activities [68, 69]. A major impact of this grey area is that private
standards are still dominating the agro-food trade. The evolution of private food safety
standards has important implications for the WTO, especially for the SPS Agreement and the
role of the CAC within the Agreement [65]. Private food safety standards present both
challenges and opportunities for the CAC [39] and the WTO.
Table 3. Private standards relating to food safety, animal and plant health
Product Technical
Environment Social
category regulations
Fish and Fish Protection of specific Fish welfare in Labelling
products species aquaculture requirements,
Fish catch restrictions packaging standards
(Source: [46])
It is also increasingly difficult to demarcate between private voluntary standards and

public mandatory regulations. It is foreseen that that private food safety standards will not
diminish the role of regulatory food safety requirements and the latter will certainly remain
under the SPS Agreement. Instead they will work side by side as consistent pairs. Research
on the inter-relationships between public regulations and private standards is still new [70].
The change in public regulations has triggered an increased use of private standards, which in
turn has instilled an ongoing debate in the inter-relationships of these two, that is, on their
substitutability and their complementarity. This is a pertinent issue that needs to be addressed
with respect to the role of public and private institutions in enforcing and setting food safety
norms [39] and to both the functioning of markets and the safety in the final market. This
must be done especially in the international trade context, as the effects of market distortions
due to standards may be most severe for producers in developing countries who lack the
capacity to comply [71]. According to Smith [70], the development of private standards
suggests that closer coordination between public and private standards and their related
control mechanisms will lead to economic gains. Based on a review of literature, Smith [70]
concludes that public and private standards are complementary.
Public standards set the minimum requirements of a safe food supply and the results to be
achieved while private standards elaborate on the means to meet and often exceed these
requirements [39, 70]. Private standards may act as a substitute in situations where effective
public measures are absent or if there is a need to differentiate products and facilitate
compliance with public measures. Thus, public regulations and private standards can be
mutually reinforcing, thence resulting in higher quality food being supplied on global markets
[70].
CONCLUSION
This chapter has focused on the international framework that governs safety and quality
of fishery products. Over the years, fish and fish products have become a very important trade
commodity. Along with the globalization of fish trade, there have been rising concerns over
fish safety in different parts of the world. This has led to the development of a series of public
regulations and private standards. Control of fish quality and safety varies with importing
country. While some developing exporting countries have been able to thrive and even
prosper on the global market in this framework, others have seen the stringent safety
requirements as trade barriers due to high cost of compliance.
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Chapter 10
ELIMINATION AND CONTROL OF PATHOGENS

BY NOVEL AND HURDLE TECHNOLOGIES
Alex Augusto Gonalves*

and Adriene Rosceli Menezes de Oliveira
Laboratory of Seafood Technology and Quality Control (LAPESC),
Animal Sciences Department (DCAN), Federal Rural University
of Semi-Arid (UFERSA), Mossor, RN, Brazil
ABSTRACT
As long as humans have existed, microorganisms and their activity in foods have
created challenges because of their ability to cause quality reduction or diseases. Seafood
and seafood products represent high quality nutrients for humans, and for
microorganisms. Due their numbers and activity, bacteria may be responsible for a strong
quality loss during storage. In some cases, bacteria or virus contaminating seafood
products may cause foodborne infectious diseases in humans. An adequate
microbiological evaluation of the quality and safety of seafood and seafood products and
techniques for their reduction or elimination requires skills and experience. In order to
suppress bacterial growth, and thereby retain a high quality and safe product throughout
the shelf life, several preservation techniques may be applied. This chapter presents
traditional and emergent technologies for elimination and control of pathogens in seafood
and seafood products.
Keywords: emergent technology, microorganisms, quality, safety
*
Corresponding author: Alex Augusto Gonalves. Federal Rural University of Semi-Arid (UFERSA), Department
of Animal Sciences (DCAN), Chief of Laboratory of Seafood Technology and Quality Control. Mossor, RN,
Brazil. Phone/Fax: 55 84 3317-8510 r. 1419; Mobile: 55 84 99171-3135. Email: alaugo@gmail.com.
176 Alex Augusto Gonalves and Adriene Rosceli Menezes de Oliveira
INTRODUCTION
Several technologies are now used or being developed to increase shelf life and guarantee
product quality and safety. The methods are very diverse, ranging from ancient ways to
preserve food, such as chilling and sterilizing, through the relatively modern technologies of
food additives, active and modified atmosphere packaging, advanced oxidation processes,
ultra-high pressure, irradiation, biopreservation and combination processes (as hurdle
technology). Thus, this chapter will cover the novel and hurdle technologies that are being
used with a focus on elimination and control of pathogens in seafood and seafood products.
Furthermore, we must take into consideration that the potential of these technologies to be
successful for a product would depend on the technologys ability to control and inhibit the
shelf life deteriorating spoilage reactions (e.g., the bacterial growth of specific bacteria,
oxidative rancidity, and color changes) in the specific product.
GENERAL ASPECTS OF SEAFOOD MICROBIOLOGY QUALITY

AND SAFETY
Fish and other seafood are considered particularly prone to spoilage. Fresh fish are very
perishable with a neutral pH (6.5-7.0), a high protein content and high water activity (aW >
0.95). Enzymes in fish from cold waters are also adapted to low temperatures, and autolytic
processes may easily accelerate post-harvest if the temperature increases [12, 33].
Considering its own physicochemical characteristics, seafood is a food category that can be
contaminated by various foodborne pathogens. Specific spoilage organisms commonly
associated with seafood are Shewanella putrefaciens and Pseudomonas spp.; and pathogens
commonly associated with seafood are Salmonella spp., L. monocytogenes, E. coli, S. aureus,
V. parahaemolyticus, C. botulinum, Bacillus cereus, and others [1, 27, 33, 46].
Appropriate handling of microbial hazards must be based on knowledge of some key
characteristics of relevant organisms such as minimum growth or toxin production
temperature, pH, and water activity (aW) for growth and information on the relationship to
oxygen. Examples [19, 33, 37] of such information about minimal temperature, pH, aW,
aerobic/anaerobic, and typical food item and environmental reservoirs, for some bacteria are
presented follows:
Bacillus cereus: 4C (min), pH 4.3 (min), aw 0.95 (min), facultative (aerobic/anaerobic),

Typical food item and environmental reservoirs (rice, spices, eggs, vegetables, dairy
products, heat-treated fish products);
Clostridium botulinum (mesophilic, proteolytic): 10C (min), pH 4.6 (min), aw 0.93
(min), anaerobic, typical food item and environmental reservoirs (meat, fish, vegetables,
soil, sediments);
Clostridium botulinum (psychrotrophic, non-proteolytic): 3C (min), pH 5.0 (min), aw
0.97 (or 5.5% NaCl), anaerobic, typical food item and environmental reservoirs
(seafood for type E, meat for type B, F);
Clostridium perfringens: 12C (min), pH 5.0 (min), aw 0.95, typical food item and
environmental reservoirs (heat-treated meat and fish products, soil, aquatic sediments);
Elimination and Control of Pathogens by Novel and Hurdle Technologies 177
Escherichia coli: 7C (min), pH 4.4 (min), aw 0.95 (min), facultative (aerobic/anaerobic),

Typical food item and environmental reservoirs (meat and fish products, intestine of
warm-blooded animals, fecal contaminated water);
Listeria monocytogenes: -0.4C (min), pH 4.4 (min), aw 0.92 (min), facultative
(aerobic/anaerobic), Typical food item and environmental reservoirs (seafood, meat,
vegetables, non-pasteurized dairy products, soil, water, sewage drain);
Salmonella spp.: 5.8C(<7) (min), pH 3.8 (min), aw 0.94 (min), facultative
(aerobic/anaerobic), Typical food item and environmental reservoirs (poultry, egg, spices,
animal feed, dried ingredients);
Staphylococcus aureus: 6C (10C for toxin) (min), pH 4.0 (4.5 for toxin) (min), aw 0.83
(0.9 for toxin) (min), facultative (aerobic/anaerobic), Typical food item and
environmental reservoirs (recontaminated heat-treated foods; human and warm-blooded
animals);
Vibrio cholerae: 10C (min), pH 5.0 (min), aw 0.97 (min), facultative (aerobic/anaerobic),
Typical food item and environmental reservoirs (seafood, human intestine, fecal polluted
water);
Aeromonas hydrophila: 0C (min), pH 4.0 (min), aw 0.97 (min), facultative
(aerobic/anaerobic), Typical food item and environmental reservoirs (fish and shellfish,
some red meats (beef, pork, lamb), and poultry);
Vibrio parahaemolyticus: 5C (min), pH 4.8 (min), aw 0.94 - moderately halophilic (min),
facultative (aerobic/anaerobic), Typical food item and environmental reservoirs (seafood,
coast, and brackish water).
GENERAL ASPECTS OF HURDLE TECHNOLOGY

The microbial stability and safety of most foods are based on a combination of several
factors (hurdles), which should not be overcome by the microorganisms present in the foods.
This is illustrated by the so-called hurdle effect and is of fundamental importance for the
preservation of foods, since the hurdles in a stable product control microbial spoilage, food
poisoning, and the desired fermentation processes. Furthermore, some authors acknowledged,
that the hurdle concept illustrates only the well-known fact that complex interactions of
temperature, water activity, pH, redox potential, and preservatives are significant for the
microbial stability of foods [30, 31, 32].
The application of Hurdle Technology (HT - synonymously called combined methods,
combined processes, combination preservation, combination techniques, barrier technology,
or hurdle technology) proved very successful since an intelligent combination of hurdles
secures the microbial stability and safety as well as the sensory, nutritive, and economic
properties of a food. Potential hurdles for use in the preservation of foods can be divided into
physical, physicochemical, microbial-derived and miscellaneous hurdles. More than 50
hurdles of potential use for foods, which improve the stability and/or the quality of these
products, have been identified [13, 40].
When using HT, the effect of each hurdle on product safety and shelf life must be
considered to ensure that no unexpected outcomes occur. Examples of hurdles in marine
products are salt (salted fish products), smoke (cold or hot smoked fish products), acids
(marinated products, pickles), temperature (low or high), fermentative microorganisms
(traditional Asian sauces), and more recently redox potential (vacuum-packed products) [13,
30]. Others examples of seafood and seafood products preserved by hurdle technology
(respectively intensity) [13, 40] are presented follows:
Fish pt: enzymatic inactivation (cooking - T > 60C); water activity (0.90-0.915 by
salt, glycerol), pH (5.8-6.0 by acetic acid), preservatives (potassium sorbate as fungicide),
oxygen reduction (vacuum - optional), refrigeration (0-5C refrigerated storage);
Fish loin: enzymatic inactivation (cooking - T > 60C) in infusion solution (NaCl,
glycerol); water activity (decrease by refrigerated infusion solution - NaCl, glycerol); pH
(5.8-6.0 by acetic acid); preservatives (potassium sorbate as fungicide); oxygen reduction
(anaerobic condition by adding oil - coverage); refrigeration (60-65C 30 minutes);
Marinated fish: water activity (0.95 decreased by salt); salt content (4,5%); pH (4.2
decreased by acetic acid); oxygen reduction (anaerobic condition by adding oil -
coverage); refrigeration (0-5C refrigerated storage);
Smoked salmon slices*: water activity (0.89 decreased by salt); pH (6.3 decreased by
acetic acid); preservatives (fungicide, smoke - active ingredient); oxygen reduction
(vacuum); refrigeration (0-5C refrigerated storage);
Salted fish: salty (16% NaCl); water activity (<0.75); oxygen reduction (vacuum/product
covered with brine); Refrigeration (5 to 10C);
Pressed and salted minced fish: acidification (1-2% citric acid); salted and pressed (aW =
0.75); oxygen reduction (packaging with reduced permeability to O2);
Fish silage: cooking# (120C); competitive flora (3% inoculum); acidification (pH 3.5);
refrigeration# (5-10C)
* In refrigerated vacuum-packed smoked fish, hurdle technology is used to prevent the growth and toxin
formation of Clostridium botulinum, as well as Listeria monocytogenes. The common hurdles or
barriers implemented to inhibit their growth and toxin formation in refrigerated vacuum-packed smoked
fish include: i) water phase salt (3.5% without nitrates and nitrites; ii) nitrates/nitrites; iii) smoke; iv)
thermal processing, and v) refrigeration. For species with high histidine content; # Can be considered
a hurdle because reduces the microbial load, it also softens the hardest structures to facilitate grinding.
NON-THERMAL TECHNOLOGIES
Active Packaging (Antimicrobial Packaging)
Active packaging (AP) can be defined as performing some desired role in food quality or
safety other than to provide an inert barrier to external conditions [41, 50]. AP is the
incorporation of specific compounds into packaging systems that interact with the contents or
environment to maintain or extend product quality and shelf life, and in the food industry,
there are many applications [12]. One of many applications of active packaging is an
antimicrobial packaging and can be defined as the packaging system that is able to kill or
inhibit spoilage and pathogenic microorganisms that contaminate foods [22, 23]. The new
antimicrobial function can be achieved by adding antimicrobial agents in the packaging
system and/or using antimicrobial polymers that satisfy conventional packaging requirements.
When the packaging system acquires antimicrobial activity, the packaging system (or
material) limits or prevents microbial growth by extending the lag period and reducing the
growth rate or decreases live counts of microorganisms [8, 12, 21, 50].
The primary goals of an antimicrobial packaging system are (i) safety assurance, (ii)
quality maintenance, and (iii) shelf-life extension, which is the reversed order of the primary
goals of conventional packaging systems. Nowadays food security is a big issue and
antimicrobial packaging could play a role in food security assurance [12, 21]. There are many
antimicrobial agents that exist and are widely used. Various antimicrobial agents may be
incorporated in the packaging system, which are chemical antimicrobials, antioxidants,
biotechnology products, antimicrobial polymers, natural antimicrobials and gas. Chemical
antimicrobial agents are the most common substances used in the industry and include
organic acids, fungicides, alcohols and antibiotics [12, 55].
Examples of potential antimicrobial agents for antimicrobial food packaging agents [12,
23, 43] and seafood and seafood products application are presented follows: Organic acids:
acetic-, benzoic-, lactic-, citric-, malic-, propionic-, sorbic-, succinic-, tartaric acid, and
mixture of acids (Fish fillets, sausage); Acid salts: potassium sorbate, sodium benzoate; Acid
anhydrides: sorbic anhydride, benzoic anhydride; Para benzoic acids: propyl-, methyl-,
and ethyl paraben; Alcohol: ethanol emitters (semi-moist and dried fish products);
Bacteriocins: nisin, pediocin, subtilin, lacticin (fresh fish, shellfish, sausage); Fatty
acids: laurie acid, palmitoleic acid; Fatty acids esters: glycerol monolaurate; Chelating
agents: EDTA, citrate, lactoferrin (fresh fish, shellfish, sausage); Enzymes: lysozyme, glucose
oxidase, lactoperoxidase (fresh fish, shellfish, sausage); Metals: silver, copper, zirconium;
Antioxidants: BHA, BHT, TBHQ, iron salts (fresh fish fillets); Antibiotic: natamycin;
Fungicides: benomyl, Imazalil, sulfur dioxide; Sanitizing gas: ozone, ClO2, CO, CO2 (fresh
fish fillets); Sanitizers: cetylpyridinium chloride, acidified NaCi, triclosan (fresh fish,
shellfish); Polysaccharide: chitosan (fresh fish fillets); Phenolics: catechin, cresol,
hydroquinone; Plant volatiles/essential oils: allylisothiocyanate, cinnamaldehyde, eugenol,
linalool, terpineol, thymol, carvacrol, pinene (fresh and processed fish - dried fish, nuggets,
ground fish meat shellfish); Probiotics: lactic acid bacteria (L. reuteri) to control E. coli
0157:H7;
Modified Atmosphere Packaging
Modified atmosphere packaging (MAP) is considered another technique available that

consistently delays microbial growth, ensures freshness, and provides a long shelf life for
fresh meat. Nevertheless, shelf life goals can only be achieved with strict temperature control
to have maximum microbial inhibition [5, 7, 38, 45].
The principle of MAP is the replacement of air in the package with a fixed gas mixture.
Once the gas mixture is introduced, no further control of the gas composition is exercised,
and the composition will inevitably change. The three major gasses used in the MAP of foods
are oxygen (O2), nitrogen (N2) and carbon dioxide (CO2). For most food products different
combinations of two or three of these gasses are used, chosen to meet the needs of the specific
product. Usually for non-respiring products, where microbial growth is the main spoilage
parameter, a 30-60% CO2 split is used, the remainder being either pure N2 (for O2 sensitive
foods) or combinations of N2 and O2 [5, 11, 43].
There are four categories of preservative packaging that can be used with raw muscle
foods. These are vacuum packs (VP), high oxygen modified atmosphere packs (High O2
MAP), low oxygen modified atmosphere packs (Low O2 MAP), and controlled atmosphere
packs (CAP) [11], that can be resumed in type; packaging gases; film gas permeability;
atmosphere: i) Vacuum pack: residual air; low permeability; anaerobic (residual O2 consumed
by enzymatic reaction or respiration); ii) High O2 MAP: O2, CO2, and often N2; low
permeability; aerobic (O2 decreases over time); iii) Low O2 MAP: CO2, N2 and residual O2;
low permeability; anaerobic (residual O2 consumed by enzymatic reaction or respiration); iv)
CAP: CO2, N2; impermeable; anaerobic (stable atmosphere; oxygen scavenger may be used).
Modified atmospheres containing CO2 are effective in extending the shelf life of many
food products. However, one major concern is the inhibition of normal aerobic spoilage
bacteria and the possible growth of psychrotrophic food pathogens, which may result in the
food becoming unsafe for consumption before it appears to be organoleptically unacceptable.
Most of the pathogenic bacteria can be inhibited by low temperatures (<7C). At these
conditions, only psychrotrophic pathogens can proliferate [5, 38].
A particular concern is a possibility that psychrotrophic, non-proteolytic strains of C.
botulinum types B, E and F are able to grow and produce toxins under MAP conditions. Little
is known about the effects of modified atmosphere storage conditions on toxin production by
C. botulinum. The possibility of inhibiting C. botulinum by incorporating low levels of O2 in
the package does not appear to be feasible. Toxin production by C. botulinum type E, prior to
spoilage, has been described in fish, at O2 levels of 2% and 4% [39].
EMERGING TECHNOLOGIES
Advanced Oxidation Processes
The advanced oxidation process (AOP) has been described as a set of chemical
treatments procedures designed to remove inorganic and organic materials in wastewater by
oxidation and also, as an alternative for the removal of pollutants and persistent effluents with
high organic load, while conventional treatments do not reach the required efficiency. The
AOP is based on physical and chemical processes that can produce profound changes in the
chemical structure of pollutants and are defined as processes involving the generation and use
of strong oxidizing agents, primarily hydroxyl radicals (OH) [2, 14, 47].
Due to the high oxidizing power of hydroxyl radicals (2.80 mV), surpassed only by
fluorine (3.03 mV), the AOP has been used with increasing interest. The common feature of
all AOP is the use of reactive free radicals, especially hydroxyl radicals, which can be
generated by reactions involving strong oxidizers such as ozone (O3) and hydrogen peroxide
(H2O2), semiconductors such as titanium dioxide (TiO2), zinc oxide (ZnO) and ultraviolet
radiation (UV) at different dosages, sequences, and combinations [14, 34, 38].
Ozone (O3) is an allotropic form of oxygen (O2), and is one of the most powerful
oxidants known, for this reason, it has a strong capacity for disinfection and sterilization [4,
15, 16, 36]. The three major action pathways occur as follows [17]: (i) Direct oxidation
reactions of ozone, resulting from the action of an atom of oxygen, these are typical first
order, high redox potential reactions; (ii) In indirect oxidation reactions of ozone, the ozone
molecule decomposes to form free radicals (OR) which react quickly to oxidize organic and
inorganic compounds; and (iii) Ozone may also act by ozonolysis, by adhering the complete
molecule on double linked atoms, producing two simple molecules with differing properties
and molecular characteristics.
The bactericidal effects of ozone have been studied and documented in a wide variety of
organisms, including Gram-positive and Gram-negative bacteria as well as spores and
vegetative cells. Ozone at low concentration (0.01 ppm) is toxic for Gram-positive and Gram-
negative bacteria and has an oxidation potential 1.5 times greater than that of chlorine. Ozone
may also inactivate microorganisms, especially viruses, by causing damage to their genetic
material. In summary, ozone is generally known for being an effective broad-spectrum
disinfectant, which is characterized by its rapid and irreversible antimicrobial reaction [14,
15, 16, 17, 20].
In the seafood industry, ozone has been tested to disinfect seafood products and to
improve sensory qualities [4, 15]. The bactericidal effect of ozone depends on the ozone
concentration, the contact time, temperature and the production system used. Low ozone
concentrations in solution decrease the half-life of ozone in some cases and the bactericidal
effect is unobserved. Ozone has been reported in numerous studies to extend the storage life
of many perishable foods by slowing decomposition caused by microorganisms, and
Gonalves [15] organized these studies in the recent review.
Electrolyzed Oxidized Water
The concept of Electrolyzed Water (EW) as a sanitizer for several kinds of objects and
surfaces was created in Japan and is currently used as a non-thermal treatment to prevent
contamination in the food industries. EW has shown great effectiveness against bacteria and
virus, as well as moderate fungicidal properties when used in vegetables, fruits, poultry, ready
to eat meals, fish and surfaces such as cutting boards, gloves, and other equipment. EW
consist, basically, of tap water and a mixture of water and a salt (NaCl; KCl or MgCl
usually 1 to 12% solution), which are placed in an electrolysis chamber with positively and
negatively charged inert electrodes separated by a septum (membrane or diaphragm) [25].
Through this chamber passes a current of 8 to 10 amperes, and a voltage of 9 to 10 volts
is applied. Two types of EW may be collected from this procedure: Acidic electrolyzed water
(AEW) or Electrolyzed oxidizing water (EO water); and, Basic electrolyzed water (BEW),
also known as Alkaline electrolyzed water (AlEW) or Electrolyzed reducing water (ER
water). Machines without a septum generate neutral water, pH usually around 6.8, because
the anions from one side neutralize the cations from the other. Authors usually attribute the
antimicrobial effect of EW to OHCl, pH, and oxidation-reduction potential (ORP).
Hypothetically, under high or low ORP there is a destabilization of the cellular membrane of
bacteria and other pathogens by OHCl, which allows these components to disrupt metabolic
processes inside the cell, resulting in cellular death [25].
Acidic Electrolyzed Water (AEW)

The AEW derives from the anode stream, pH around 2.5, ORP over 1,100 mV, and
chlorine concentration between 40 and 90 ppm. This ensures its strong bactericidal effects,
especially important in reducing the growth of pathogens such as Escherichia coli,
Salmonella enteritidis, Listeria monocytogens, Campylobacter jejuni, Vibrio

parahaemolyticus, and others. In recent studies, a group of researchers from China used
weakly acidic electrolyzed water (pH 6.4-6.6, ORP 520-540 mV) ice glazing associated to
modified atmosphere packaging (MAP) to prevent deterioration during frozen storage in
shrimp. Results are very interesting, showing that the combined effect of both technologies
significantly extends shelf life by inhibiting the growth of microorganisms and the increase of
TVNB, TMA and TBARS values [49].
Basic Electrolyzed Water (BEW)

From the cathode stream, BEW is collected, it has pH around 11.5 and an OPR of -795
mV, which indicates strong reducing potential, making it effective to reduce free radicals. It is
common knowledge that AEW has more antimicrobial potential than BEW, probably due to
its higher ORP. However, some studies have been done with BEW as a pretreatment to AEW,
since both solutions are originated in the same equipment; they could be more effectively
used to sanitize surfaces and foods together. Results from this study show that a pretreatment
with BEW at 50C, combined with AEW treatment at the same temperature were successful
in controlling Vibrio parahaemolyticus contamination in shrimp [48].
Electrolyzation of seawater as a new technology with bactericidal and virucidal effect has
been studied earlier and information about the effectiveness of electrolyzed seawater (ESW)
for the disinfection of aquaculture material and depuration system is needed. Elsewhere
electrolyzation is reported to be suitable to disinfect seawater used for seafood sanitation at
fish markets or fish ports [28]. ESW has strong bactericidal effects on most pathogenic
bacteria that are important to food safety [25].
Biopreservation through Antimicrobial Systems
Biopreservation consists on the use of bacteria or other microorganisms, as well as their

metabolites, to inhibit the growth of spoilage inducers and pathogens. Among the most used
and studied biopreservatives are bacteriocins, produced by Gram-positive bacteria, mainly
Lactic Acid Bacteria (LAB); bacteriophages, which are bacteria-specific viruses; and,
endolysins, which damage bacterial cell membrane [10].
LAB are a large group of anaero-aerotolerant, non-sporulating, Gram-positive rods and
cocci, which have lactic acid as the main metabolite derived from their metabolism. The
major genera that comprise LAB are Lactobacillus, Lactococcus, Leuconostoc, Pediococcus,
Streptococcus, and the minor ones Aerococcus, Carnobacterium, Enterococcus, Oenococcus,
Sporolactobacillus, Tetragenococcus, Vagococcus, Weissella and Bifidobacterium [9].
Studies have been done mostly concerning the inhibition of Listeria spp. by LAB, but a
French research group isolated LAB strains from seafood and tried to inhibit target strains of
Listeria monocytogenes, Staphylococcus xylosus, Pseudomonas sp., Serratialiquefaciens, and
14 others Gram-positive and -negative spoiling and pathogenic bacteria. From all the isolates,
Lactobacillus fuchuensis and Leuconostoc gelidum inhibited most of the target strains tested
[35]. However, another researcher also working with LAB in seafood believes the role of
these bacteria in seafood products is complex because it depends on aspects such as the fish
species, treatment, storage, bacteria species and strains, and interactions between bacteria. In
some cases, LAB can cause sensory spoilage [9].
Irradiation
Processing by ionizing radiation is a particular kind of energy transfer: the portion of the
energy transferred per transaction is high enough to cause ionization. This kind of radiation
was discovered because the emitting radioactive material caused ionization in the surrounding
air. From the multitude of atomic particles known, only gamma rays from nuclear
disintegration and accelerated electrons are useful for food processing [6, 42].
The dose absorbed by the irradiated material is the amount of energy absorbed per unit
mass of irradiated material. The unit used is called Gray (Gy), where 1 Gy is equivalent to 1
Joule of energy absorbed per 1 kg of material. Food irradiation is essentially a cold process
since this treatment does not cause an increase in temperature. However, the temperature of
the irradiated product influences the radiation-induced changes, i.e., reactions between free
radicals increase with temperature, affecting the overall rate of radiolysis [6, 42].
In foods, the irradiation is defined by three processes that do not involve heat: i)
Radurization (cold pasteurization/prolonging shelf life) which involves the inactivation of
non-spore forming bacteria using low doses of radiation (<1 kGy); ii) Radicidation
(Reduction of pathogens) using the intermediate levels (2 to 8 kGy) to reduce the number of
viable and non-pathogenic bacteria produce spores, and parasites; iii) Radappertization (cold
sterilization) which ensures the elimination of highly pathogenic bacteria such as Clostridium
botulinum and use higher doses (>10 kGy) [3, 42].
Low dose irradiation has been studied extensively as a method for extending the shelf life
of fresh iced or refrigerated fish. Generally, Gram-negative bacteria are more sensitive to
radiation than Gram-positive bacteria and tend to be mainly responsible for spoilage of fresh
and marine finfish. This means that low dose levels of 1-3 kGy (cold pasteurization) can often
reduce the initial load of potential spoilage microorganisms 1-3 log10 cycles to extend the
shelf life of fresh fish significantly. In some investigations, doses up to 5 kGy were required.
The dose level for shelf life extension must be evaluated for each species to maintain
acceptable flavor, texture, and wholesomeness [42]. A dose of 4 kGy has been found to be
sufficient to eliminate non-spore-forming pathogens in many different kinds of foods,
including frozen seafood. The naturally occurring Vibrio species are relatively sensitive to
low-dose irradiation, and can generally be easily eliminated, as has been determined in vitro
in saline or seafood homogenates and in vivo by inoculation, or seeding, or by natural
contamination of the product.
In vitro reduction or elimination of some potential pathogens in fresh and frozen finfish,
frog legs, shellfish products using low- and medium-dose irradiation [29] are presented
follows (Microorganism: Product(s); Initial contaminationa as CFU/g; Extinction Doseb as
kGy):
Aeromonas (sp. n.id.): frozen blocks of fish; 105; 4.0-5.0;

Aeromonas hydrophila: dried cuttlefish, ground bluefish; 105; 1.5;
Clostridium perfringens: dried cuttlefish; 105; 3.0;
Clostridium botulinum type E: Gulf of Mexico shrimp (inoc.); 104 spores; 5.0 (no toxin;
30 days/60C);
Escherichia coli: dried cuttlefish; 105; 3.0 | Oysters (seeded) (Crassostrea virginica); 104;
1.5 | Shrimp (seeded); 104; > 2.0;
Salmonella (sp. n.id.): frozen blocks of fish; Unknown; 4.0;
S. typhimurium: Frozen shrimp (inoc.); 108; 4.0;

Salmonella enteritidis: Frozen shrimp (inoc.); 108; 4.0 | Live oysters (seeded); 1010; 2.5;
Staphylococcus aureus: Dried cuttlefish; Unknown; 3.0 | Dried smoked mackerel;
Unknown; 5.0;
Vibrio sp.: Oysters, live (inoc.), C. virginica; 108 (inoculated); 1.2;
Vibrio cholerae (unspecified): Crab meat - blue crab (inoc.), C. sapidus; 107; 0.5-1.0 |
Fresh Gulf of Mexico shrimp; 107; 1.0 | Fresh crayfish meat (inoc.), P. clarkia; 107; 1.0 |
Live oysters (seeded) (C. virginica); 104; 1.0 | Oyster meat (inoc.); 107; 1.0;
Vibrio cholerae Ol El Tor: Jurel (T. murphy) and Lisa (Mugil cephalus); 106; 1.0-1.2 |
Fresh frog legs; 7x107 (inoc.); 0.5-1.0 | Clams (inoc.) (A. purpuratus, Garisolida); 106-
108; 1.2 | Sea snails (inoc.) (Thais chocolata); 106-108; 1.2 | Shrimp (inoc.) (Penaeus
vannamei); 106-108; 1.2
Vibrio parahaemolyticus: Fish homogenate; Unknown; 0.4 | Oyster (seeded) (C.
virginica); 104; 1.0 | Gulf of Mexico shrimp (inoculated); 107; 0.3;
Vibrio vulnificus: Shrimp (inoc.) and Crabmeat (inoc.); 106; 0.35 | Oyster, live (natural
contamination), C. virginica; 5x105; 1.5.
Studies on commercial-scale harvesting, processing, packaging, transportation, and

distribution of seafood products treated with ionizing irradiation in bulk at commercial
irradiation facilities are greatly needed to update the vast body of literature on irradiation
processing technology that is available but may not relate to current commercial practices.
Comprehensive commercial evaluation of dose levels and packaging for optimal doses for
both spoilage microorganisms and potentially pathogenic microorganisms in commercial
species is very important [29].
Ultra High Pressure
High pressure processing, HPP (also known as High Pressure Pasteurization, HPP; High
Hydrostatic Pressures, HHP; Ultra High Pressure, UHP) has become established as a
nonthermal technology which is capable of reducing microbial numbers and therefore
extending shelf-life and improving the microbiological safety of ready-to-eat meats and
seafood, and also improving the processing of shellfish and crustaceans [18, 44].
Products are loaded into a vertical or horizontal processing vessel and subjected to high
hydrostatic water pressure. Since pressure is uniformly distributed around the product, the
products shape is preserved. Foods are typically packed for food safety. Meat-from-shell
separation typically is performed without packaging [18]. High pressure interferes with
biological functions important to sustaining bacteria life, selective proteins are denatured, or
the bacteria die. The HPP process can be conducted with products at refrigerated
temperatures, thus retaining the products fresh or as-prepared quality. Pressure is instantly
transmitted throughout the product, and treatment time does not depend on product size. A
large package will need the same processing time as a small product. HPP is environmentally
friendly and consumes less energy than thermal pasteurization [18, 44].
Since high-pressure technology offers advantages in retaining food quality attributes, it
has recently been the subject of considerable interest in the food industry as a non-thermal
unit operation. High-pressure equipment with pressure levels up to 800 MPa and temperatures
in the range of 5 to 90C (on average) for times up to 30 minutes or longer is currently
available to the food industry [26]. In contrast to conventional heat treatment, the main
advantage of HHP is that this technology generally inactivates microorganisms and enzymes
at ambient or low temperatures, without changing most of the organoleptic and nutrient
characteristics of the product [18, 44]. The medium pressure (MPa) required to achieve a 5-
log inactivation ratio for various microorganisms, for a treatment of 15 minutes is: Yersinia
enterocolitica (275), Salmonella typhimurium (350), Listeria monocytogenes (375),
Salmonella enteritidis (450), Escherichia coli O157:H7 (680), and Staphylococcus aureus
(700) [24].
CONCLUSION
The trade value of seafood products in the international market and their social value to
the communities that produce and consume them has highly increased in the past decades;
this makes the issue of seafood safety even more important.
Knowledge in microbiology will enhance the safe production of fish as food, and is a
strict requirement to maintain a high quality of seafood and seafood products for sufficiently
long periods necessary for harvest, processing, transport and storage.
Pathogen elimination and control is a major part of ensuring seafood safety. The amount
of contamination and the kind of microorganisms found in fish and fishery products depend
on several aspects, such as location and method of capture or farming, season, handling,
processing, and storage among others.
The application of any new technology presents significant challenges to food
technologists and food researchers. As consumers become more aware of food safety and
quality concepts, the food industry has to be more welcoming to new and efficient ways to
improve shelf life and guarantee good quality, pathogens-free products. Therefore, hurdle
technologies are an ideal tool to achieve such goal.
Its clear that the hurdle concept will be utilized more and more in food preservation as
minimal processing, and is gradually becoming more popular in food manufacturing. New
hurdles will certainly be found, like antimicrobial agents and active packaging material that
target the surface of the foodstuffs where spoilage occurs.
Packaging foods in atmospheres containing an inert gas and carbon dioxide inhibits both
oxidative reactions and microbial spoilage.
Antimicrobial packaging is a promising form of active food packaging. The antimicrobial
substances incorporated into package materials can control microbial contamination by
reducing the growth rate and maximum growth population and extending the lag period of the
target microorganism.
Ozone has been reported in numerous studies to extend the storage life of many
perishable foods by slowing decomposition caused by microorganisms. Ozonated water
treatment also presents an opportunity to improve product quality by reducing spoilage
bacteria during seafood processing operations.
Electrolyzed Water has shown great effectiveness against bacteria and virus, as well as
moderate fungicidal properties when used in seafood, seafood processing, and cleaning
equipment.
HP processing offers an alternative to thermal processing in the food industry. The

resistance of a microorganism to pressure varies considerably depending on the pressure
range applied, temperature and treatment duration, and type of microorganism.
Comprehensive commercial evaluation of dose levels of ionizing irradiation and
packaging for optimal doses for both spoilage microorganisms and potentially pathogenic
microorganisms in commercial species is very important.
Technologies such as biopreservation, especially when used combined with other
techniques to preserve seafood, such as MAP or HPP, are extremely efficient, but
underutilized, demonstrating the need to expand research in this field and continue to study
these practices as well as try to implement them in a commercially viable way in the seafood
industry.
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Chapter 11
PACKAGING TECHNOLOGIES AND MATERIAL TYPE

FOR THE MAINTENANCE OF SEAFOOD SAFETY
Ana Augusto, Maria Manuel Gil and Susana Filipa Jesus Silva*
MARE Marine and Environmental Sciences Centre, ESTM,
Instituto Politcnico de Leiria, Peniche, Portugal
ABSTRACT
This chapter discusses packaging technologies, materials and novel strategies for the
maintenance of seafood quality throughout storage. Seafood is a valuable source of
protein, fatty acids, minerals and vitamins, and in some cultures it represents the main
protein source in feeding. Seafood is a highly perishable product, which, unlike meat
products, starts decomposition immediately after post-mortem. In addition, seafood is a
rich nutrient matrix providing a suitable environment for the growth of pathogenic and
spoilage microorganisms. The increased demand for high quality fresh seafood has
intensified the research for new methods and technologies for more efficient preservation
methods. Improvements in the cold distribution chain has made international trade of
perishable foods possible, but refrigeration process alone cannot assure the quality and
safety of all perishable foods. Most of them are stored at low temperature and sometimes
are packaged under modified atmosphere (MAP) in order to extend their shelf-life. The
use of specific packaging allows the control of quality and security of seafood increasing
shelf-life of the products.
Seafood packaging has historically been passive and employed as a physical barrier
to gas and moisture transfer and microbial contamination. The main objective of food
packaging is to contain in the product in an environmentally sustainable cost-effective
way that satisfies both industry requirements and consumer desires, ensuring food safety.
Therefore, in the last years some new packaging strategies have evolved, such as MAP,
edible films and coatings and, more recently, active and intelligent packaging systems.
The aim of this chapter is to compile relevant information concerning technologies
and materials type used for the maintenance of seafood safety and quality.
*
Corresponding author: MARE Marine and Environmental Sciences Centre, ESTM, Instituto Politcnico de
Leiria Campus 4 | Edificio CETEMARES, Avenida do Porto de Pesca, Peniche 2520 630 Portugal. Email:
susana.j.silva@ipleiria.pt.
192 Ana Augusto, Maria Manuel Gil and Susana Filipa Jesus Silva
Keywords: seafood packaging, vacuum packaging, modified atmosphere packaging, edible

films, edible coatings, active and intelligent packaging
INTRODUCTION
Seafood
Over the last years, consumer purchasing behavior towards fish and seafood products has
been capturing the interest of researchers internationally for political and economic reasons
related to aspects of nutrition and diet, food safety, sustainability and business of the fish
industry. Seafood is a valuable source of protein, important fatty acids, minerals and vitamins.
In some developing countries it represents the main protein source in nutrition. World per
capita fish consumption has increased from an average of 9.9 kg (live weight equivalent) in
the 1960s to 19.2 kg in 2012 [1]. In recent years, the nutritional benefits of aquatic organisms
have mainly been associated with their exceptionally advantageous fatty acids profile
characterized by a high level of long chain polyunsaturated fatty acids (LC-PUFAs or
Omega-3) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which
provide various health benefits such as decrease in the risk of getting cardiovascular diseases
[1]. According to the International Society for the Study of Fatty Acids and Lipids, an intake
of about 500 mg of EPA+DHA per day would be expected to significantly reduce the risk for
death from coronary heart diseases in healthy adults.
These well-known nutritional benefits combined with its exceptional gastronomic value
and diversity of species, make this food category very attractive to consumers in developed
countries over the world. However, seafood is also highly perishable being prone to rapid
post-mortem bacterial and enzymatic changes which may result in a short shelf-life [2, 3].
PUFAs are highly susceptible to oxidation, and oxidative degradation of lipids in foods and
foodstuffs during processing and subsequent storage directly affects the quality of products,
including flavor, color, texture and nutritional value [3-5]. Seafood is highly susceptible to
bacterial degradation due to its low glycogen levels, slow post-mortem pH decrease, high
moisture content and appreciable quantities of non-protein nitrogen [2].
During storage, fish quality is quickly reduced as chemical and enzymatic reactions lead
to quality degradation and microbial spoilage leads to shelf-life expiration. The increased
demand for high quality fresh seafood has intensified the search for new methods and
technologies for a more efficient fish preservation. Freezing is a widely spread and efficient
method of fish seafood conservation that allows for a prolonged shelf-life with minor changes
in product quality. But even below freezing temperatures, some undesirable such as protein
denaturation, weight loss, freeze burning and lipid oxidation can occur. The control of these
degradative processes requires the use of appropriate packaging [6].
The application of traditional processes like salting, smoking and canning applied to fish
and seafood has decreased in favor of mild technologies involving the application of lower
salt concentrations, lower heating temperature and packaging under vacuum (VP) or under
modified atmosphere (MAP) which brings about new challenges in the selection and
development of appropriate packaging systems [7].
Packaging Technologies and Material Type 193
The global supply of safe, convenient and environmentally sustainable seafood products
demands more than the traditional passive systems used to date making the application and
development of novel packaging solutions essential.
Packaging Technologies
Advances in food processing and food packaging play a primary role in assuring the
supply of safe food around the world. Nowadays, packaging attempts to make the daily lives
of consumers easier in many ways, increasing convenience and communication to consumers
by providing information about the ingredients and nutritional characteristics of the foods
contained therein [8, 9]. The main role of food packaging is to protect food products from
environmental conditions acting as a barrier to light, oxygen, humidity and microbial
contamination [10, 11]. An efficient packaging system will also reduce food waste and
spoilage during distribution meeting pollutants and solid waste disposal regulations whilst
presenting itself as appealing and convenient to consumers, contributing towards sales and
marketing efforts [5, 12]. Food is the only product class typically consumed 3 times per day
per person. Consequently, food packaging accounts for almost two-thirds of total packaging
waste by volume. Moreover, food packaging is approximately 50% (by weight) of total
packaging sales, therefore the need of accounting for its environmental footprint in the
process of food packaging systems development.
The degree of protection required by a food product is a key factor in selecting packaging
materials and design. Food protection is defined in terms of variety of factors that can impact
the quality attributes of the food product from the time the product is placed in the container
to the expected time of consumption [11].
Protection from 3 major classes of external influences is required: chemical, biological
and physical [10, 11]. Chemical protection minimizes compositional changes triggered by
environmental parameters such as exposure to oxygen, nitrogen, carbon dioxide, water vapor
or aromas and light (visible, infrared, or ultraviolet). Biological protection provides a barrier
to microbial contamination (pathogens and spoiling agents), insects, rodents, and other
animals, thereby preventing disease and spoilage. Biological barriers can also maintain
conditions to control senescence (ripening and aging). Such barriers function via a
multiplicity of mechanisms, including preventing access to the product, preventing odor
transmission, and maintaining the internal environmental conditions. Physical protection
shields food from mechanical damage and includes cushioning against the shock and
vibration encountered during distribution [11].
The quality of the packaged food is determined by the food matrix attributed and
physico-chemical properties of the packaging materials. Transfer phenomena, such as
moisture absorption, oxygen incursion through the package material, flavor loss, undesirable
odor absorption, and the migration of packaging components into the food can deteriorate the
food product. These transfers can occur between the food and the atmosphere, between food
and packaging materials, or amongst the heterogeneous ingredients in the food product itself
[8, 10]. The development of new packaging technologies and materials requires the
understanding of mass transfer phenomena effects on the migration of material components
and food ingredients, the absorption and desorption of volatile ingredients, flavors, and
moisture, gas permeation and the kinetics of ingredients degradation.
There are three broad categories of packaging systems passive, active and intelligent
packaging. A passive packaging system is a system that serves as a physical barrier between
the product and the environment surrounding the package. An active packaging system is a
system that detects or senses changes within the package environment, followed by
modification of package properties in response to the detected change. An active packaging
system responds in some manner to changes occurring in the environment surrounding the
food product. Active packaging materials are defined as materials that aim at extending the
shelf-life or at maintaining or improving the condition of package food with its protection and
preservation [11, 13]. A packaging system that senses changes in the environment and
responds with corrective action is defined as an intelligent or smart packaging system. With
this system, it is possible follow the food quality along the manufacturing and distribution
chain until the consumers consumption [11].
Among the many technological developments in food processing, packaging innovation
has remarkable, leading to higher standards of regulation, hygiene, health, safety, and to the
commercial availability of new materials. Environmental sustainability and food security
concerns amongst producers, regulators and consumers are turning the use of biodegradable
packaging materials (to reduce the environmental impact of food packaging) and the
development of active packaging applications (in order to minimize food losses) the greatest
technological challenges in the food packaging area nowadays.
Together these trends will lead to number of challenges for seafood producers with
respect to new product development, processing and packaging technologies that ensure the
product has maintained its nutritional and sensory quality by the time it reaches the consumer.
The development of new seafood packaging technologies implies new materials and new
packaging designs, and packaging can be expected to take on new roles when, for example,
new functional packaging materials are used.
Seafood Packaging Technologies
Processed seafood products are commonly packaged and kept at low temperatures by
overwrapping in plasticized cling-wrap film of polymer polyvinyl chloride (PVC) with high
water vapor transmission rate (WVTR) and low oxygen permeability [14]. This approach
protects the product from further contamination; however, it is not as efficient in delaying
deterioration of already contaminated products as fish maintenance in melting ice. Shelf-life
extension of these products demands in most cases an additional barrier to physical, chemical
and microbial degradation, thus temperature control and the physical barrier of wraps or
thermally sealed plastic films is in most cases combined with the modification of the package
internal atmosphere composition either by vacuum application (vacuum packaging) or by the
injection of gas with a determined composition (modified atmosphere packaging). Recent
innovations in seafood packaging have created an array of new terms associated with the role
of packaging in the improvement of safety, shelf-life and convenience of the food product.
Presently research efforts are being directed towards active and intelligent packaging, and the
search for new biodegradable or even edible packaging materials.
Vacuum and Modified Atmosphere Packaging

Vacuum packaging (VP) of seafood can be applied individualized (e.g., refrigerated
smoked products) or for individual pieces contained in a modified atmosphere master pack
(e.g., packages of frozen seafood products). This process consists in placing the product in a
package, evacuating air and sealing the package. Vacuum skin packaging is used for delicate
products such as smoked or pickled fish and soft-shell crabs. Whereas the vacuum packed
parcels are to be contained in a MAP master pack, the film should have a higher permeability.
When compared to MAP, VP has the advantage of minimizing package volume [8, 14].
A substantial shelf-life extension of foods can be achieved by modifying the gas
atmosphere in the immediate environment of a food product during storage. A commonly
applied process is modified atmosphere packaging (MAP) combined with refrigeration
temperature storage, which, based on average data from fish and shellfish, can increase these
products shelf-life in 30-100% for raw fish and 100-200% for cooked shell fish [15, 16]. The
basis for this technology dates back to 1930, when it was first reported that the shelf-life of
muscle foods could be extended in CO2 rich atmospheres. MAP is traditionally used to
preserve the freshness of fresh produce, meats, and fish by controlling their biochemical
metabolism [8].
Typically, under vacuum and modified atmosphere packaging, the spoilage microflora
shifts from Gram (-) to Gram (+) bacteria. Historically, it was thought that this would
represent a shift from Pseudomonads to Lactobacilli. Using CO2 in the gas mixture has long
been known to hydrate and form carbonic acid, which was thought to favor the shift to lactic
acid organisms. Fish fillets stored under MAP typically show lower bacterial numbers and
extended shelf-life when compared against counterparts stored in air [2]. In order to
contribute for a better utilization of these species, the preparation of different types of
products has been tried, like fillets and smoked products as well as minces and surimi.
Modified atmosphere packaging including vacuum-packaging, along with refrigeration, have
become increasingly popular preservation techniques, which have brought major changes in
storage, distribution, and marketing of raw and treated products to meet consumer demands
[17]. MAP solutions for wet foods are however hampered by the high gas volume to product
volume ratio (g/p) necessary in order to obtain the appropriate dissolution of CO2 and to
ensure CO2 availability and thereby inhibition of bacterial growth. The g/p ratio is often as
high as 2 or 3, implicating a package size 3-4 times the size of the product. The consequences
are increased distribution costs, space necessary in retail display cabinets, and increased
amount of plastic packaging materials and waste. Too low g/p could induce package collapse
by the inevitable volume contraction/pressure decrease caused by the diffusion of CO2 from
the gas phase into the water and fat phase of the product [18].
In MAP, the product is placed in a barrier packaging material, such as polyamide (PA) or
polyethylene terephthalate (PET). The air is then removed from the package and replaced by
a mixture of gases (usually O2, CO2, N2). Table 1 shows the gas percentages used in MAP of
seafood products. In passive MAP, the product is placed usually in a low-barrier packaging
material, such as polyethylene, polypropylene perforated or not, polyvinylchloride,
polystyrene, or ethylene vinyl acetate and left to attain equilibrium conditions during storage
[19]. Carbon dioxide is the principal antimicrobial factor in MAP. It is both bacteriostatic and
fungistatic. MAP functions principally through the inhibition of fast-growing aerobes that
otherwise would quickly spoil perishable products. Most packaging materials used in MAP
applications should provide ease and quality of seal. Laminated or coextruded multilayer
materials are the most used materials nowadays for seafood MAP. Such multilayered
structures contain polyethylene as the inner thermal sealing layer, whereas the outer layer is
PET and PA. In cases in which a truly high-barrier material is required, either polyvinylidene
chloride or ethylene vinyl alcohol are used. Such materials have an oxygen permeability (PO2
< 10 cm3 O2 24 h-1 m-2atm-1) compared with low density polyethylene (LDPE) (PO2 > 5000
cm3 O2 24h-1 atm-1). In less demanding MAP applications, PA or PET (PO2 = 50-150 cm3 O2
24h-1 m-2 atm-1) may be used. PCO2 is usually four to six times higher than PO2 for a given
material [19]. In the United States, MAP and VP are tightly regulated due to concerns
regarding temperature abuse and toxin production by Clostridium botulinum type E [14].
In fish storage studies involving MAP, the development of various sensory attributes over
time are quite different from those that develop for the same fish species stored in melting ice.
Studies found that the odor associated with Atlantic mackerel (Scomber scombrus L.) held
under MAP conditions changed dramatically over storage time (from seaweed, fishy and
rancid in day 0 to seaweed, sour, fishy, metallic and rancid in day 21) [8, 25]. If MAP fish is
held at high refrigeration temperatures (>10C) no strong spoilage sensory signals develop
before C. botulinum toxin production.
MAP application could offer enhancement in fish and fishery products shelf-life with
minimal quality defects [17]. MAP containing CO2 with refrigeration are effective in
extending the shelf-life of many foods. CO2 atmospheres extend the lag phase and generation
time of aerobic bacteria decreasing the growth rate and extending shelf-life. The inhibition of
bacterial growth in food package with CO2 increases as the storage temperature decreases. It
has been found that it exhibits an inhibitory effect, mainly against Gram negative
microorganisms. Generally, spoilage flora is replaced probably to a large extent, by CO2
resistant organisms. The use of gas packaging, specifically elevated CO2 levels have been
shown to inhibit normal spoilage bacteria such as pseudomonads in fish from cold and
temperate waters and thus double or triple shelf life [26].
Table 1. Modified atmosphere composition applied to different types

of seafood products
Product % O2 % CO2 % N2 Reference

Seabass 10 80 10 [20]
Sardine 0 60 40 [17, 21]
5 60 35
Mackerel 0 60 40 [22]
Whiting 30 40 30 [22]
Salmon 0 60 40 [22]
Pacific white shrimp 20 30 50 [23]
Seabream 30 40 30 [24]
Edible Films and Coatings

One of the possibilities of new packaging technology is the use or application of edible
films and coatings. An edible coating can be defined as a thin layer of edible material applied
directly to the product surface, providing a semipermeable barrier to moisture, oxygen, and
solute movement from the food [27, 28]. Edible films and coatings are a good alternative for
the partial or total substitution of plastic packaging due to their properties: they are
biodegradable, non-toxic, environmental-friendly and, in many occasions, made with by-
products of the food industry [29]. These new materials reduce or control permeability of
agents that could impact the safety or shelf-life of the food product within the container [8,
10, 11]. The use of edible films and coatings can be application of active food packaging, as
the edibility and biodegradability of the films are extra functions not present in conventional
packaging systems and it can be applied for the continuous delivery of active compounds and
additives to the food matrix [8, 30]. The functional characteristics required depend on the
product matrix (low to high moisture content) and major deterioration processes to which the
product is subjected [28].
Recently, considerable research has been conducted to develop and apply bio-based
polymers produced a variety of undervalued agricultural commodities and/or of food waste
products. Table 2 shows the possible interactions between foodstuff, polymer films and the
environment and their adverse consequences.
Several compounds can be applied as edible coatings in frozen fish with the main
objective of delaying lipid oxidation and prevent superficial drying of the product. Proteins
(caseins, whey, soy and egg proteins) are able to form coatings which can delay lipid
oxidation and decrease moisture losses from the product [6]. The application of proteins in
edible package formation demands a thermal treatment in order to denature them and obtain
insoluble edible packages with improved mechanical and oxygen barrier properties through
cross-linking between the protein molecules. The thermal treatment is also usually applied to
the protein solutions for frozen fish coatings, even though they never form a solid film.
Protein denaturation can have the added advantage of originating the exposure of antioxidant
amino acid groups [6, 32]. Several studies with Atlantic salmon (Salmo salar) have observed
that the sonication of whey protein based coating applied in frozen fillets resulted in reduced
lipid oxidation of frozen fish without addition of chemical additives, extending shelf-life of
this product [6, 29].
Table 2. Possible interactions between environment, package film and foodstuff, possible
migration substances and adverse consequences for the foodstuff. Adapted from [11, 31]
Interface between food, package and environment

Substances Direction Adverse consequences
Permeation Oxygen Environment to Oxidation, microbial and mold growth, color,
Water vapor Food flavor and aroma changes, respiration,
Carbon dioxide texture changes and stickiness.
Other gases
Aroma
Oxygen Food to Dehydration, decarbonation, respiration and
Water Vapor Environment texture changes.
Carbon dioxide
Other gases
Migration Monomers Environment to Off-flavor and safety problems.
Additives Food
Package component Package to Food Aroma and flavor changes, toxicity.
Absorption Aroma compounds Food to Changes of aroma intensity, development of
Fats Environment unbalanced flavor profile and damage to the
Organic acids package.
Pigments
Light transmition Environment to Color and flavor changes, nutrient
Food degradation.
Table 3. Edible coatings applied to different seafood products
Seafood product Coating type Temperature and time Reference

of storage
Frozen Atlantic Whey protein, chitosan -10C for 4 months [6, 29, 34, 35]
salmon (Salmo salar) and 6 months
Red fish (Sciaenops Nano SiOx chitosan 4C for 20 days [36]
ocellatus) CaCO3chitosan [37]
Chitosan with grape seed extract [38]
and tea polyphenols
Shrimp (Penaeus Chitosan and shrimp protein-lipid 5C for 17 days [39, 40]
vannamei, concentrate [41]
Litopenaeus Water-based nano-sized chitin and
vannamei) chitosan products Cold storage or 10 [42]
Chitosan with pomegranate peel days
extract
Rainbow trout Carboxymethyl cellulose-based 4C for 20 days [43]
(Oncorhynchus coatings with Zataria multiflora
mykiss) essential oil and grape seed extract
Chitosan with cinnamon oil
4C for 16 days [44]
Silver carp Chitosan and chitosan 4C for 12 days [45]
(Hypophthalmicthys nanoparticles
molitrix)
Lingcod (Ophiodon Chitosan with fish oil 2C for 3 weeks [46]
elongates) -20C for 3 months
Large yellow croaker Tea polyphenol and rosemary 4C for 20 days [47]
(Pseudosciaena extract combined with chitosan
crocea)
Several biopolymers have received increased attention for their food applications, more
specifically regarding its functionalities in the preparation of antimicrobial edible films and
coatings. Chitosan plays an important role due to its well-documented antimicrobial
properties [30]. This polymer is derived from chitin (biopolymer that can be found in the
exoskeleton of crustaceans and in fungal cell walls). Chitosan properties such as antimicrobial
activity and film-forming properties depend on its deacetylation degree. The potential of
chitosan to act as a food preservative of natural origin has been widely reported on the basis
of in vitro trials as well as through direct application on real complex matrix foods. Chitosan
is also an excellent film forming material, having a selective permeability to gases (CO2 and
O2) and good mechanical properties [33]. Studies investigated the effect of chitosan solutions
on frozen salmon preservation comparing to water glazing, having observed that chitosan
coatings efficiently protect frozen fish from deterioration [34]. Microbial growth, assessed by
total viable counts, and total volatile nitrogen were maintained below the maximum limits
recommended which are 5 x105 CFU7g and 35 mg nitrogen/100 g fish, respectively. Table 3
shows different studies of edible coatings applied in seafood.
Active and Intelligent Packaging Systems

The terms active packaging and intelligent packaging are closely related, though
there is an important distinction between them. The Framework Regulation on Food Contact
Materials (1935/2004) offers the following definition: Active materials and articles are
defined as materials and articles that are intended to extend the shelf-life or to maintain or
improve the condition of package food; they are designed to deliberately incorporate
components that would release or absorb substances into or from the packaged food or the
environment surrounding the food.
Yam et al. [48] also defined intelligent packaging as: A packaging system that is capable
of carrying out intelligent functions (such as detecting, sensing, recording, tracing,
communicating, and applying scientific logic) to facilitate decision making to extend shelf
life, enhance safety, improve quality, provide information, and warn about possible problems.
Oxygen scavenging materials can be used to delay oxidative deterioration, but specific
smart polymers can be designed to selectively remove potential decomposition substances. A
common problem during commercialization of fish fillets is the drip of tissue fluid, resulting
in water inside the package, causing the quality perception of the product to deteriorate and
flavoring the growth of food-borne pathogenic microorganisms. In Table 4 it is possible to
observe some examples of active packaging systems for fish. There are some smart packaging
technologies that could be used to remove malodorous compounds (e.g., activated carbon)
and moisture (e.g., moisture absorber sachets of silica-gel) from packed fish and fish products
[49]. Another active packaging solution with antimicrobial effect is based on carbon dioxide
generators. Increased CO2 levels (10-80%) are desirable for seafood preservation, because it
can efficiently reduce the surface microbial growth and thus prolong the shelf-life of the
product. This type of active packaging is frequently associated with modified atmosphere
systems in order to balance out CO2 losses due to dissolution into the seafood and permeation
through the packaging material, CO2FreshPads (CO2 Technologies) are used for meat,
poultry, and seafood packaging [50]. Drip losses from muscle foods are absorbed into pads
and react with citric acid and sodium bicarbonate present in the pad resulting in the
generation of carbon dioxide [51]. Paper Pak Industries have launched UltraZapXtendaPak
pads, a more evolved version of CO2 generators. It is designed as an absorbent pad for fresh
fish that has a double antimicrobial effect due to the incorporation of a CO2 emitter and an
antimicrobial substance [52]. According to the information contained in the patent, the
antimicrobial agent used would mainly consist of a mixture of citric and sorbic acids. A
recent CO2 emitter application has been developed for fish fillets by a Norwegian company,
Vartdal Plastindustri AS, also available for meat packaging [53]. The SUPERFRESH system
consists of a coated expanded polystyrene box with a CO2 emitter. Reported system
advantages are prolonged shelf life, reduced transport volume, less environmental impact and
no bulging or vacuum effect [53].
Time-temperature Indicators or Integrators (TTIs) are simple, cost-effective and easy to
use devices for monitoring, recording and cumulatively indicating the overall influence of
temperature on quality, from manufacturing to the end consumer and are therefore applied to
various food products [52, 54]. Consumers can easily check the quality of food using TTIs,
which are usually expressed as a visible response of irreversible color development, that can
be mechanical, chemical, electrochemical, enzymatic, or microbiological, that matches or
correlates to the shelf life of a food stuff at a target temperature [54, 55].
Food packages featuring TTIs are examples of intelligent packaging, due to their use of a
system that monitors the conditions of the food in real time, informing consumers about the
conditions of transport and storage of these products and establishing the actual parameters of
food quality and safety before consumption. Thus, a modern security system that monitors
and controls critical parameters for the quality of a food product, such as storage temperature,
should ensure good product quality during its life cycle with simplicity and efficiency. Due to
its simplicity, low cost and efficiency, TTIs have been widely applied to establish, monitor
and evaluate the storage shelf-life at a certain temperature of many of many chilled and
frozen food products, such as fish products and seafood [54, 56].
The OnVuTMTTI from BIZERBA North America (Ciba Specialty Chemicals and
Freshpoint, Basel, Switzerland; Patent No. WO/2006/048412) are designed and supplied as
either printing inks or labels that may be affixed to the inner or outer packaging to monitor
the accumulated effects of time and temperature on perishable chilled products (e.g.,
processed fish). This nontoxic, printable timetemperature indicator relies on the properties
of photochromic colorants (dyes or pigments) that change color over time, depending on
temperature fluctuation. The Fresh-Check Indicator from TEMPTIME Corporation (Figure
1a) is an example of a TTI fresh-check indicator. It is a self-adhesive device that is
specifically formulated to match the shelf-life of the food products to which it is affixed.
When the Active Center Circle is lighter than the oval, the product is OK to use. The Active
Center darkens irreversibly- faster at higher temperatures and slower at lower temperatures-
and when the Active Center matches the oval, the product should be used soon. In the end of
shelf-life the Active Center is darker than the oval and the product should not be used [57].
The label or printed display contains a reference color as a ring around the photochromic spot
on the label. Activation of the label is performed just before application to the package by a
specially developed automated ultraviolet light source (often a light-emitting diodes, known
as LEDs). Once activated, the TTI first develops a dark blue color which then gradually
becomes lighter with increasing temperature as time passes. When the activated color of the
label has the same shade as the reference color, it indicates that the product has reached the
end of its shelf life. To keep the performance consistent, a special kind of paper is usually
required for the labels [9].
Vitsab L5-8 Smart TTI Seafood Label from Vitsab (Figure 1b) is a smart label
formulation well adapted to Clostridium botulinum and its toxin formation in the temperature
range between +1 to 25C conforming to the Food and Drug Administration requirements of
packed seafood products imported to the USA. This CheckPoint Indicator is a simple
adhesive that contain two main compartments: one for lipase solution and pH indicator dye
and other for the substrate. When activated, the ingredients form the two compartments are
mixed and as the reaction proceed a pH change results in a color change. Initially, the center
is green color, and becomes progressively yellow or orange as product reaches the end of
shelf-life. It is an irreversible reaction and depends of the temperatures changes [55, 58].
In an enclosed food package, as the fish product spoils, a pH increase occurs over time
within the headspace, which can be detected with an appropriate pH indicating sensor. The
fundamental characteristic of pH indicator dyes that change color when placed in an acidic or
basic environment is the key element of this sensor. This is due to the fact that when fish
spoils, it releases a variety of basic volatile amines which are detectable with appropriate pH
indicating sensors (show Figure 2). Practically, these could be prepared by entrapping within
a polymer matrix a pH sensitive dye (e.g., bromocresol green) that responds, through visible
color changes to the spoilage volatile compounds that contribute to a quantity known as total
volatile basic nitrogen [56].
Table 4. Selected examples of active packaging systems for fish. Adapted from [49]
Active packaging system Mechanisms

Carbon dioxide Iron oxide/calcium hydroxide
Scavengers/emitters Ferrous carbonate/metal halide
Calcium oxide/activated charcoal
Ascorbate/sodium bicarbonate
Preservative releasers Organic acids
Silver zeolite
Spice and herb extracts
BHA/BHT antioxidant
Chlorine dioxide/sulphur dioxide
Ethanol emitters Encapsulated ethanol
Moisture absorbers PVA blanket
Activated clays and minerals
Silica gel
Flavour/odor absorbers Cellulose triacetate
Acetylated paper
Citric acid
Ferrous salr/ascorbate
Activated carbon/clays/zeolites
Temperature control packaging Non-woven plastics
Doubled-walled containers
Hydrofluorocarbon gas
Quicklime/water
Ammonium nitrate/water
Calcium chloride/water
Super corroding alloys/salt water
Potassium permanganate/glycerine
Figure 1. (a) Reading Fresh-Check Time Temperature Indicator. Adapted from [57]. (b) Vitsab L5-8
Smart TTI Seafood Label from Vitsab Adapted from [58].
Figure 2. TTI color response at different pH conditions. Adapted from [59].
The interest of intelligent food packaging application has long been recognized, but its
commercialization is still in the starting stages. Although the many benefits of this innovative
technology are well known in the food industry, those in the industry are still reluctant to
apply it and its costs still limit the broad transfer of these technologies.
CONCLUSION
Food science and packaging technologies are linked to both engineering developments
and consumer studies. Research and development of materials possessing high barrier
properties is a continuing trend in the development of new materials for application as food
packaging. Once seafood is a high perishable food product that requires specific care with its
handling and conservation, the use of specific packaging it is a good alternative to maintain
and increase the quality of fresh seafood at rigor mortis stage or during storage until
consumption. Seafood packaging is being employed as a physical barrier to gas and moisture
transfer and microbial contamination. The increasing research interest in this topic is directed
towards an environmental sustainable cost-effective package that satisfies consumers
requirements whilst ensuring food safety Edible films and coatings and active and intelligent
packaging systems are the most promising technologies in complying with these objectives.
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Chapter 12
QUANTITATIVE RISK ASSESSMENT

IN SEAFOOD
Violeta Trinidad Pardo Sedas *,

Karla Mara Lpez Hernndez and Argel Flores Primo
Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana,
Colonia Unidad Veracruzana, Veracruz, Veracruz, Mxico
ABSTRACT
Seafood is a nutrient-rich portion in a healthy and balanced human diet.
Nevertheless, this advantageous health perception is troubled by the potential risks of
eating polluted seafood. Contaminated seafood is a frequent aetiology of diseases
contracted from the ocean-to-fork continuum, including both chemical and infectious
hazards. Changing global patterns of food production, international trade, technology,
public expectations for health protection and many other factors have created an
increasingly demanding environment in which food safety systems operate. The increase
of food-borne hazards has posed risks to human health and negatively impacted the
international food trade. These risks must be assessed and managed to meet increasingly
complex sets of objectives. Quantitative risk analysis is a systematic, science-based
approach able to link data from food and the various data on human disease to provide
clear estimation of the impact of contaminated food on human public health. It is actually
one the most powerful key tool available today applied for determining the risk of the
hazard/product/process combination and consumption of food in order to achieve safe
operation and major hazard control, and to predict the effects of interventions proposed to
mitigate the risk. Quantitative risk assessment provides numerical expressions of risk and
may include a numerical description of uncertainty, offering a systematic approach for
meeting many of the current challenges arising from foodborne hazards and valuable
improvement in seafood safety.
Keywords: food-borne hazards, risk assessment, seafood safety, public health
*
Corresponding author: Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, Avenida Miguel
ngel de Quevedo s/n esquina Yez, Colonia Unidad Veracruzana, Veracruz, Veracruz, Mxico. Email:
vpardio@yahoo.com.mx and vpardio@uv.mx.
210 V. Trinidad Pardo Sedas, K. Mara Lpez Hernndez and A. Flores Primo
INTRODUCTION
Seafood is one of the most important food commodities contributing to food security and
to the economies of many countries in the world, as fish is the most traded food among the
food commodities in worldwide. The term seafood used here encompasses wild and farmed
fish, shellfish, mollusks and their eggs both from marine and freshwater origin.
Seafood is generally regarded as a wholesome and nutritious food. However, this
perception is troubled by the potential risks of eating contaminated seafood. The safety of
seafood products varies considerably and is influenced by a number of factors. Moreover,
risks to consumers health associated with seafood products may differ from region to region
and vary according to the method of production, management practices and environmental
conditions. Thus, there is a need to estimate the risk to human health and to identify possible
interventions to reduce or eliminate these risks. In this context, international agencies and all
levels of government are increasingly relying on risk assessments for public health protection,
international trade, decision-making and cost-effective resource allocation [16, 20]. In view of
all this, risk assessment is important throughout all aspects of the seafood industry for
companies, national governments and for international regulators.
Risk assessment, a scientifically based process, is the qualitative and/or quantitative
evaluation of the likely intake of biological, chemical and physical agents via food as well as
exposures from other sources. A risk assessment that provides numerical expressions of risk
and indication of the attendant uncertainties is considered as Quantitative Risk Assessment
(QRA). In a QRA, it is vital to define the statement of purpose to reach at the beginning, as
full QRA can be achieved if the distributions of the factors in the system that contribute to the
risk are known. Approaches that use all of this information, the so-called stochastic, or
probabilistic treatments, are the preferred option for risk assessment. Applied to food
safety, this methodology estimates the probability and severity of a human health disturbance
as a consequence of consumption of food [33]. Since the mid-1990s, several methods for
microbial food safety and quantitative risk assessments have been developed and released [9,
10, 11, 12, 13, 15].
Taking these aspects into consideration, this work offers an insight into the basics of risk
assessment with an emphasis on the quantitative approach with a seafood application. This
overview will provide utility for individuals starting out in QRA and who need to be aware of
the essential elements that underlie this methodology. Finally, the applicability of quantitative
microbiological risk assessment in seafood is briefly addressed.
Food Safety and Public Health Risks Associated with Seafood
The Food and Agriculture Organization (FAO) has recommended a food-chain approach
in order to protect public health and facilitate international food trade. Risk analysis is the
preferred discipline to assess possible links between hazards in the food chain and actual risks
to human health, taking into account inputs to decision-making on appropriate control
measures for reducing food-borne illness and strengthening food safety systems [30]. A food-
borne hazard is defined by the FAO/WHO Codex Alimentarius Comission (CAC) as a
biological, chemical or physical agent in, or condition of, food, with the potential to cause an
Quantitative Risk Assessment in Seafood 211
adverse health effect [12]. Food-borne risks to human health can arise from hazards that are
biological, chemical or physical in nature. Chemical and microbiological hazards require
different approaches to risk analysis. A variety of food-borne hazards of current concern are
described below.
Seafood-Borne Microbiological Hazards
In countries with higher seafood consumption, foodborne illnesses are generally reported
from poorest areas where fish and seafood are traditionally eaten raw, marinated, or
undercooked [1]. Pathogenic bacteria associated with seafood can be categorized into five
general groups shown in Table 1: 1) bacteria which are normal components of the marine or
estuarine environment (indigenous bacteria), 2) enteric bacteria due to faecal contamination
(nonindigenous bacteria), 3) bacterial contamination during processing, 4) viruses, norovirus,
a human calicivirus recognized as a leading cause of non-bacterial acute gastroenteritis
associated with consumption of raw shellfish, especially oysters, and 5) parasites that include
nematodes, trematodes, cestodes, and protozoa.
Chemical Hazards
Seafood may harbor several chemical hazards, being the most common: food additives,
persistent organic pollutants such as pesticides, PCBs, PHAs and dioxins, heavy metals, and
marine biotoxins which pose health risks of long-term adverse effects [16, 20, 22]. The
accumulation of biotoxins in shellfish is a serious health concern in many parts of the world;
they have been classified into eight groups based on chemical structure: azaspiracid, cyclic
imines, pectenotoxin, yessotoxin, being heat stable brevetoxin, okadaic acid, saxitoxin, and
domoic acid. Hence, live shellfish consumption will cause disease whether the shellfish are
cooked or not [24]. Some examples of chemical hazards studies in seafood are shown in
Table 2.
GENERAL PRINCIPLES OF THE RISK ANALYSIS

Risk Analysis has emerged as a structured model for improving food control systems,
with the objectives of producing safer food, reducing the numbers of foodborne illnesses and
facilitating domestic and international trade in food. According to the CAC, risk is a function
of the probability of an adverse health effect and the severity of that effect, consequential to a
hazard(s) in food [15].
Risk Assessment Process
The Codex Committee on Food Hygiene has proposed a framework for conducting risk
analysis consisting of three components shown in Figure 1: risk assessment, risk management
and risk communication.
Table 1. Microbiological hazards commonly isolated in seafood

[1, 14, 19, 25, 31]
Table 2. Chemical hazard, tolerances and critical limits in seafood
For a better understanding, the following definitions used in risk analysis are cited here to
facilitate the understanding of certain phrases used in this chapter [21, 23]:
Risk assessment - A process that estimates of the likelihood and severity of known or
potential adverse health effects resulting from human exposure to foodborne hazards, includes
four steps: hazard identification, hazard characterization, exposure assessment and risk
characterization.
Risk Management - The process of weighing policy alternatives in the light of the
results of risk assessment and, if required, selecting and implementing appropriate control
options, including regulatory measures.
Figure 1. Information flow for the components in a risk analysis process [7].
Risk Communication - The interactive exchange of information and opinions

concerning risk and risk management among risk assessors, risk managers, consumers,
industry, the academic community and other interested parties.
Outputs of a risk assessment may be sought in non-numerical (qualitative) or numerical
(quantitative) form. Non-numerical risk estimates provide a less definitive basis for decisions
but are adequate for several purposes, such as evaluating relative impacts on risk reduction of
different control measures. Numeric estimates of risk can take one of two formats [30]: 1)
point estimate, which is a single numerical value representing for example the risk in a worst
case scenario, and 2) probabilistic risk estimates, which include variability and uncertainty
and are presented as a distribution reflecting more real-life situations. To date, point estimates
have been more common outputs of chemical risk assessments while probabilistic outputs are
the usual product of microbiological risk assessments.
There are several types of risk assessment that fall under three broad categories [7, 15,
21, 23]:
1. Qualitative, risk is described as the likelihood of illness (high, medium or low). It

may be performed where data are inadequate to make numerical estimates. This is
the simplest and quickest to do, but it can be rather subjective.
2. Semi-quantitative, the level of risk is compared with some other risk, ranking the risk
on a scale of from 0 to 100, or an estimate of the number of illnesses in the
population of interest per year, or in which the risks from different sources are put in
order of severity. In semi-quantitative risk assessment, a numerical risk estimate
based on a mixture of qualitative and quantitative data is obtained.
3. Quantitative, the risk is expressed as the predicted number of illnesses, and reported
on a per-serving basis or the number of people in a defined population who are likely
to become ill from the pathogencommodity/product combination. Quantitative risk

assessments can be categorized in two models: 1) deterministic model, where the
effects of chance are ignored and all parameters have a fixed value; the end result is
one point estimate; 2) stochastic model, all events are considered as variable and are
represented by probability distributions. Stochastic risk assessment is usually
undertaken using computer simulation software, providing a full representation of the
risk estimate including the average value of the estimated risk, as well as risk
estimates that correspond to different levels of confidence. The probability
distributions used in stochastic risk models may represent uncertainty as well as
variability. In this context, uncertainty represents the lack of perfect knowledge of a
parameter value, which can be reduced by further measurements. Variability,
represents a true heterogeneity of the population that is a consequence of the physical
system and irreducible by further measurements. Subsequently, stochastic models are
usually constructed to fully account for variability and uncertainty in the most critical
stages. One method employed to analyze uncertainty in risk assessment is Monte
Carlo simulation techniques of probabilistic models.
Quantitative Microbiological Risk Assessment (QMRA)
Risk characterization consider the reported health outcome used in developing the dose-
response relationship. A quantitative model uses probability to describe this randomness,
leading to results such as the probability of a randomly individuals being infected in a given
year, or a probability distribution of the number of illnesses in a future period. The model also
describes the uncertainty of the exact values of parameters that would describe the proposed
risk pathways with uncertainty distributions, determined by various statistical methods. The
risk can be characterized as the probability of illness per serving, taking into account the
amount of that food that an individual might consume within a defined period. The risk per
serving measure provides an easy comparison of the risk from direct consumption of different
food products [11].
The QMRA two main objectives are: to determine the factors that contribute to the risk of
becoming ill from the consumption of contaminated food, and to evaluate the likely public
health impact of different control measures, including the effectiveness of current and
alternative microbiological standards. The use of probabilistic quantitative risk assessment
(QRA) methodologies in the modelling of food-borne bacterial pathogens in primary food
production is fairly limited. Models assessing the risk of human exposure to fish/seafood are
more limited in number [7]. In the seafood area, four QMRAs have been conducted for
specific purposes:
a. Listeria monocytogenes in smoked fish in Sweden [18].

b. Listeria monocytogenes in a range of seafoods in the United States [8].
c. Vibrio parahaemolyticus in oysters in the United States [13].
d. Vibrio parahaemolyticus in seafood (raw oysters, bloody clam and finfish in to
estimate risk of illness from this pathogen due to consumption of oysters in Australia,
Canada, Japan and New Zealand [10].
Recently, a quantitative risk assessment was developed for Vibrio parahaemolyticus in

raw oysters produced and consumed in So Paulo State, Brazil and built according to the U.S.
FDA framework for risk assessment [6]. Unlike previous studies on estimated risks, this
model used local data on V. parahaemolyticus density in oysters as a function of water
temperature, distribution of oyster weight and the rate of pathogenic to total V.
parahaemolyticus. Here, an overview of the key concepts is presented, using it as an
illustrative example. For this purpose, the risk assessment model was composed of three
modules, comprising harvest, post-harvest and consumption steps, shown in Table 3.
Table 3. Conceptual model for the quantitative risk assessment

for Vibrio parahaemolyticus in oysters
Hazard identification. Vibrio parahaemolyticus is a Gram-negative, halophilic marine

bacterium that occurs naturally in estuaries and commonly found in many types of seafood. It
has been recognized as a cause of gastroenteritis linked to the consumption of seafood,
particularly oysters consumed raw or inadequately cooked or contaminated after cooking.
Despite the large length of the Brazilian coast, little is known on the occurrence of infections
caused by V. parahaemolyticus, because notification is not mandatory. The average annual
oyster production in Brazil is approximately 142.4 tons. In the southeast coast of So Paulo
State, oyster production is concentrated in the Cananeia lagoon estuarine region, which was
responsible for 98.5% of the production from 2005- 2010.
Hazard characterization. Due to the lack of data in Brazil, the doseresponse model
used in this work was the same BetaPoisson model used by FDA [13], including the
distribution of uncertainty of parameters alpha and beta. The set of estimated means for the
probability of illness per serving was used to characterize the distribution of uncertainty on
the risk of a person to become ill.
Risk characterization. The doseresponse function was combined with the output of
exposure assessment to estimate the probability of illness per serving of raw oysters.
Sensitivity analysis was performed to identify and quantify the relative importance of
variables of the model on the likelihood of occurrence of illness. This is done to determine the
parameters that contribute most to the total uncertainty of the risk assessment output.
Statistical analyses. The statistical analyses were performed by using SAS (SAS
Institute Inc., Cary, NC, USA, v. 9.0), Microsoft Excel and simulations by @Risk softwares
(Palisade Corporation, version 4.5), with a significant level of 5% of probability.
The results showed that the depuration processes used in the processing plants did not
reduce the V. parahaemolyticus density in the oysters. In general, the geometric mean density
of total V. parahaemolyticus predicted by the model was similar to the one obtained in
samples collected at retail level [4]. The highest estimated risk of illness per serving of raw
oysters contaminated with V. parahaemolyticus occurred during summer season (6.0 x 10-4)
when the mean seawater temperature was 27.0C. However, the estimated risk per serving
obtained by using the model could not be validated due to the lack of epidemiological data in
the country. This estimated risk was higher than that reported in Canada (7.5 1010 to 1.1
106) but similar to that in Japan (1.2 104) [10].
Sensitivity analysis was performed to identify and quantify the relative importance of
variables of the model for the likelihood of occurrence of illness. Using the crude sensitivity
analysis, the variables that most influenced the risk of illness were in descending order of
importance: the abundance of total V. parahaemolyticus at harvest, the temperature of
transportation to retail, the relative prevalence of pathogenic V. parahaemolyticus, the time of
storage at retail, the effect of depuration process, the amount of oysters consumed, and the
time between harvest and arrival to the depuration plant. The seasons did not significantly
affect the likelihood of occurrence of illness. Thus, focus of future studies should be to collect
data on these variables to reduce the uncertainty in the estimation of the variability of V.
parahaemolyticus levels in oysters, and the uncertainty in the estimation of the prevalence or
the proportion of virulent strains. This work showed that the abundance of V.
parahaemolyticus in oysters was higher at the consumption level than at the harvest level
indicating that the current practices are not enough to avoid growth of V. parahaemolyticus in
oysters along the production chain.
CONCLUSION
Quantitative Risk Assessment is the most powerful tool available nowadays and a unique
scientific approach able to link data along the harvestprocessconsumption route and the
cases on human diseases to provide an estimation of the impact of contaminated food on
public health, and to assess the efficacy of each possible mitigation strategy. The principal
outcome of a QRA is a prediction of the numbers of illnesses, the severity of illness outcome,
or both. In the light of the seafood safety, QRA may provide a sensible approach to increase
the safety of fish and seafood consumption, and an understanding of the relative importance
and interactions among the factors influencing the associated risk. QRA provides clear
information to lower the risk associated with food-borne pathogens at national and
international levels.
To ensure the development of a high-quality risk assessment, experience, perspective,
and most importantly, careful planning is required. QRA assist risk managers and the seafood
industry in designing and implementing food safety plans useful for a systematic evaluation
of strategies to minimize the impact on public health. To gain a comprehensive insight into
this issue, the public health risk should be better predicted and managed, especially in regions
where the consumption of raw or undercooked shellfish is common, the pollution of coastal
habitats is acting on marine ecosystems, and the prevailing levels of sanitation and rapid and
accurate identification for active diagnosis are limited, especially to improve product safety
and overall public health.
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Chapter 13
COMPUTER-BASED APPLICATIONS
FOR MONITORING THE QUALITY
AND SAFETY OF SEAFOOD
smail Yksel Gen1,* and Eduardo Esteves2

1
Department of Fishing and Processing Technology,
Faculty of Fisheries, University of Suleyman Demirel, Isparta, Turkey
2
Departamento de Engenharia Alimentar, Instituto Superior de Engenharia,
Universidade do Algarve and CCMAR, Centro de Cincias do Mar,
Universidade do Algarve, Faro, Portugal
ABSTRACT
The main concern of predictive microbiology is to transfer the usage of the
developed models to users both researchers and non-researchers (i.e., industry,
inspection, teachers, etc.). Tertiary modeling, namely computer-based applications allows
users: i) to predict shelf-life of the product (considering spoilage bacteria), ii) evaluate
specimens in terms of their safety (growth of pathogens), and iii) development of new
(food) product (effects of environmental factors on spoilage and pathogenic bacteria). In
this chapter, available computer-based applications were evaluated and categorized in
terms of their area of usage (e.g., shelf-life predictors, safety and risk assessment
modules, fitting tools) together with the specified effect of environmental factors (i.e.,
pH, salt, temperature, aw, organic acids, atmosphere, phenolic compounds). This chapter
is a useful guide for available software to utilize both in research and non-research
applications.
Keywords: predictive microbiology, software applications, tertiary modeling, seafood,

spoilage, thermal inactivation, risk assessment
* Corresponding author: Suleyman Demirel University Fisheries Faculty Fishing and Processing Technology
Department Turkey; Email: ismailgenc@sdu.edu.tr and ismailygenc@gmail.com.
224 smail Yksel Gen and Eduardo Esteves
INTRODUCTION
For maintaining the quality and safety of seafood, numerous traditional (salting, freezing,
drying, marinating etc.) and novel (high-hydrostatic pressure, ionizing radiation, modified
atmosphere packaging, etc.) methods are applied to commercially important mollusk,
crustacean and fish species [1-8]. These mentioned methods are being effective in terms of
either inhibition of specific spoilage organisms (SSOs) or elimination of food borne
pathogens. Whatever method is used, determination of the number of microorganisms is
essential for estimating the shelf-life of the product or deciding whether or not the product is
safe for consumption. Nevertheless, generating the large numbers of data for assessing the
number of microorganisms is both time-consuming and labor-intensive. During the last
decade predictive microbiology has been playing a significant role for the determination,
monitoring and prediction of various food quality and safety, particularly of seafood [9-12].
Additionally, predictive microbiology allows researchers and industrial users to estimate the
numbers of microorganisms for untested intervals. However, one of the disadvantages of
using the developed predictive models is the difficulty in utilization by non-researchers. In
this context, computer-based applications provide user-friendly framework in usage of the
developed models by taking into account the specified cell numbers at a specific time.
Computer-based applications, namely tertiary modeling, is the last step in predictive
microbiology (primary and secondary models are described in more detail in Part III, Chapter
31). Mathematical models developed for SSOs, in particular Photobacterium phosphoreum
[13], Shewanella putrefaciens [12], Pseudomonads [14], Brochothrix thermosphacta [15],
Lactic Acid Bacteria (LAB) [16] and for pathogens such as Escherichia coli [17], Listeria
monocytogenes [18-20], Salmonella spp. [21], Shigella spp. [22], Yersinia enterocolitica [23],
Aeromonas hydrophila [24], and Vibrio parahaemolyticus [25], are widely used for shelf-life
studies and safety decisions in academic and industrial settings. Even though, the number of
specific microorganisms that can be estimated precisely enough by the models already
developed, there is still a gap for their utilization due to the complex nature of usage of
models wherein remarkable knowledge of mathematics for predictive microbiology is
required for their implementation. As a result of this complexity, the usage of specific models
is limited, particularly by the industrial users. However, as being a final part in predictive
microbiology, tertiary modeling in the context of the developed models is employed in
available software programs (online, standalone and commercial) to increase the ease of use
for the practical applications. In this context, various software with kinetic-type models for
SSOs (e.g., Food Spoilage and Safety Predictor (FSSPTM), or Combase) and with inactivation
models for pathogenic microorganisms and tools for risk assessment (e.g., Pathogen
Modeling Program (PMP), Food MicroModel, or Risk Ranger, MicroHibro) are available and
distributed via internet. There are also some other software tools (i.e., commercial included)
for laboratory or facility use only. More details can be found in Tenenhaus-Aziza and Ellouze
(2015) [62]. The present paper describes the usage of some of above mentioned available
software programs as well as their comparison of practical applications by using available
data from the literature.
Computer-Based Applications for Monitoring the Quality 225
SOFTWARE AND APPLICATIONS

IN PREDICTIVE MICROBIOLOGY
Food Spoilage and Safety Predictor (FSSP)
Food Spoilage and Safety Predictor (FSSP), extended and updated version of the
original Seafood Spoilage and Safety Predictor (SSSP) originally from January 1999, is a
user-friendly software that was developed by the Predictive Microbiology Group, National
Food Institute (DTUFood), Technical University of Denmark. The software is available at
http://fssp.food.dtu.dk/ [13, 16, 30] and includes kinetic, generic, interaction, relative rates of
spoilage models as well as growth/boundary models for Listeria monocytogenes in food
systems (i.e., seafood, meat and cottage cheese) and was released in July 2014.
Predictive Models in FSSP

The available models of the software are shown in Figure 1. Compared to other software
programs, FSSP contains a significant amount of easy-to-use predictive models that can be
utilized in shelf-life prediction, product development, quality assurance and product safety
studies.
Figure 1. Image of the initial screen and available models implemented in FSSP.
Product-specific models are implemented in the Relative Rate of Spoilage (RRS) models
module. In RRS models, a single known shelf-life of the product at a known, constant
temperature should be entered manually at first and then the software calculates the
Equivalent shelf-life at 0C in days. Following this, software allows the prediction of the
shelf-life at constant and varying temperatures. In constant temperature profile, shelf-life can
be predicted by the software. Users should enter specific storage temperature (from the study
performed) and click on calculate button. To create a dynamic temperature profile, the
Series of constant temperatures tab should be clicked and a dialog box will allow users to
enter measured temperatures manually. The button adds temperature and related storage time,
while button discards the selected time-temperature profile (Figures 2 and 3). Furthermore,
the RRS models with user-defined temperature characteristics module allows to make
comparisons between the models (i.e., Arrhenius, Exponential and Square-root). Compared to
RRS models, Microbial Spoilage Models (MSM) enable the prediction based on the number
of SSOs, such as P. phosphoreum and H2S-producing Shewanella (Figure 4).
Figure 2. FSSPTM dialog box for helf life predictions using Square-root spoilage model under constant
temperature.
Figure 3. Screenshot of FSSPTM shelf life predictions using Square-root spoilage model under dynamic
temperatures.
In some fish species (herring, sardine, anchovy) and particularly fish which belong to
Scombroid family (i.e., mackarel, tuna fish, bonito) biogenic amine formation occurs as a
result of decarboxilation of free amino acids (e.g., histidine). Histamine is one of the most
important biogenic amines that causes histamine poisoning (HP) after consumption of fish
that contains high level of histidine such as tuna [26]. The toxic concentration of histamine
has been determined to be 500-1000 mg histamine/kg. In histamine formation, mesophilic
(Morganella morganii, Klebsiella pneumoniae) and psychrotrophic bacteria (i.e., Ph.
phosphoreum, Morganella psychrotolerans) play a significant role. Among histamine-
producing bacteria M. morganii and M. psychrotolerans are presumably the most important
ones [27]. Compared to M. morganii, the growth intervals of M. psychrotolerans are wider
and thereby can produce significantly high levels of histamine even under chilled conditions
[28].
Figure 4. Microbial spoilage models together with the model comparisons.
Histamine formation models implemented in the software explain the growth,

physiological state (i.e., lag time), and time to histamine formation at different levels as a
function of storage time (Figure 5). Models can predict and plot the variables based on the
temperature (constant and varying), and initial levels of bacteria and histamine concentrations
(Figure 6). The critical point about histamine formation model is that the temperature range
for which the model is valid is above 7C because of the mesophilic characteristics of M.
morganii.
One of the expanded specifications of FSSP is the module of generic growth that
enables the users to process the data obtained from experimental studies in a flexible frame.
Thereby, existing models could be expanded or product specific models be developed. It has
to be noted that the rate of the cardinal parameters (ref, Tref, Tmin and pHmin) for the
growth response of the chosen bacteria should be known before developing the secondary
models. The specification of the cardinal parameters either obtained from the available
literature or from experimental data.
Figure 5. Growth of Morganella morganii and formation of histamine under constant temperature.
Figure 6. Growth of Morganella morganii and formation of histamine under fluctuating storage
temperatures.
ComBase
ComBase predictor [29] is an on-line tool and database to forecast the growth of spoilage
bacteria and/or inactivation/survival of food pathogens as function of time, temperature, pH,
salt content (and water activity aw). The tool was developed by Baranyi and Tamplin [29] and
is available from http://modelling.combase.cc/membership/ComBaseLogin.aspx. The need
for developing ComBase is related to the difficulty of accessing raw data that are used for
development of the models and for the predictions. In this context, ComBase provides several
records of raw data for evaluation of the developed model(s) and allows extension of data
between research and non-research users. Moreover, ComBase can predict the growth of
several bacteria that get involved in spoilage of food products (e.g., meat, dairy, fish products
and vegetables). By accessing the growth model module, predictions can be performed for 20
bacteria under constant and dynamic conditions. The predictions of bacterial survival are
valid for seven bacteria for thermal inactivation and two bacteria for non-thermal survival
module. The tool also provides an option to fit experimental data with DMFit that is included
in ComBase. DMFit is an Excel add-in developed by Baranyi and Roberts [31] and available
from http://modelling.combase.cc/DMFit.aspx that can be used to determine the specific
growth rates of microrganisms by using the experimental data provided by the user. This add-
in basically uses the Baranyi models to fit the bacterial growth curves. Additionally, DMFit
allows users to calculate maximum growth, lag time and standard errors of these parameters.
Risk Ranger
Risk Ranger is an Excel template spreadsheet developed by Ross and Sumner [32] that
contains the general steps in food safety risk assessment. Basically, the algorithms included in
the spreadsheet calculate the probability of the risk resulting from the consumption of
pathogen-containing foods. In this context, several steps have to take into account for the
calculation of the risk estimation. Regarding the calculation of the possible risk, the
spreadsheet predictions are based upon three major factors: severity of the hazard, probability
of exposure to food, and probability of food containing infectious dose. The spreadsheet-
format is useful for risk-assessment trainings, food safety decisions and risk probability
studies. Moreover, from the industrial point of view this tool could be used effectively due to
it easy-to-use framework whereby intensive experience in risk assessment and risk probability
calculation background is not necessary. This software is freely available from
http://www.foodsafetycentre.com.au/riskranger.php.
Microbial Responses Viewer (MRV)
Microbial responses viewer (MRV) is a web application that was developed by S. Koseki
[33] and is freely accessible from http://mrviewer.info/. This software contains several
pathogens (i.e., E. coli, C. perfringens, L. monocytogenes, L. innocula, etc.) growth
probabilities in culture medium and certain food systems (i.e., poultry, seafood, meat, egg,
milk, cheese). MRV includes the growth characteristics of the pathogen and spoilage
organisms based on Combase data. The main factors that are used in MRV for the
determination of the growth of the mentioned microorganisms are aw, pH and temperature.
Additionally, the maximum specific growth rate (max) is also presented in the selected
temperature profile in the known culture/food system.
MicroHibro
Compared to other web-based applications, MicroHibro developed by Optimum Quality,

Grupo Hibro and University of Cordoba. The software allows users to choose several type of
models (i.e., primary [31, 34] and secondary [35-37]) for the evaluation of actual data.
Additionally, this software enables users to save their data by using their personal accounts.
Furthermore, users are free to access their data from anywhere via internet. A module is also
available in MicroHibro for the evaluation of developed models in terms of their reliability
and accuracy. The software provides users the possibility of developing their own risk models
as well. Risk model should be constructed in accordance with process steps of the product.
There are three risk models provided by the software, growth [38-44], transfer and reduction
(i.e., deterministic [45] and stochastic). MicroHibro is freely accessible from
www.microhibro.com.
Pathogen Modeling Program (PMP)
The Pathogen Modeling Program (PMP) is a free, standalone software that in its current
version (7.0) includes models for growth, inactivation, survival, cooling, irradiation, time to
turbidity and time to toxin production (in fish). The software was developed by research
scientists from Eastern Regional Research Center (ERRC), Agricultural Research Center
(ARS) of the United States Department of Agriculture (USDA) and can be downloaded from
www.ars.usda.gov/services/software/dowload.htm?softwareid=90. PMP is able to calculate
the behavior of the microorganisms under aerobic and anaerobic conditions at pre-determined
pH, NaCI and organic acid/additive concentration (in ppm) (Figure 7) [46].
Figure 7. Growth of E. coli O157:H7 in broth culture under aerobic conditions.

Predictions can be selected regarding to type of model (Figure 8) or type of

microorganism (Figure 9) in a particular food (chicken, beef, fish and turkey) or media
(broth) system. For the modeling, the software uses Gompertz equations [47].
The results of the predictions are presented in .xls or graphical format. References of the
models can be provided by the software from References tab on the diaolog box (Figure 10)
[48].
Figure 8. Model selection in accordance with model type.
Figure 9. Model selection in accordance with bacteria type.

Figure 10. References of the models implemented in PMP.
SOFTWARE APPLICATIONS BASED ON LITERATURE DATA

Obtaining the growth response of the microorganisms under different environmental
conditions is the main concern of predictive microbiology especially for validating a
developed model [29]. In this context some software are developed as databases to provide
data sets for comparison and evaluation of a model. ComBase and MRV are the online tools
to be used as databases and contains numerous data sets for specific microorganisms and
particular food matrices. The growth or inactivation of spoilage or pathogen microorganisms
is simply calculated by the software tools under specified environmental conditions. Growth
of particular microorganisms can be predicted by FSSPTM, MicroHibro and ComBase. On the
other hand, some software tools such as ComBase and PMP provide possible inactivation
calculations in broth or specific food matrix (meat, seafood and vegetables). Risk analysis and
modeling the risk factors are emerging approaches for predictive microbiology nowadays.
However, only a limited number of the software tools provide risk analysis tool such as
MicroHibro and Risk ranger.
Predictions of Growth for Spoilage Microorganisms in FSSP
As previously mentioned, numerous models have been developed for specific spoilage
organisms. A simple software application is demonstrated here by using FSSP and the
model parameters shown in Table 1.
Table 1. Model parameters for growth of pseudomonads

under different temperatures
Temperature (C) pH Aw min Tmin b Reference

0-30 5.0 0.95 -7.70 0.020 [49, 50]
5-10 6.1 0.95 -10.65 0.021 [51]
By using the kinetic parameters of pseudomonads, the growth of the microorganism can
be predicted in accordance with the models implemented in FSSP. To this respect, growth
prediction of pseudomonads is calculated based on square-root type model module.
Illustrations of model parameters are shown in Figure 11. Consequentially, all parameters (b,
Tmin (C), % CO2 max, aw min, aw reference, pH min and pH reference value) of
microorganism in concern have to be entered manually to make complete shelf-life prediction
and for estimation of growth rate under studied temperatures. Following the complete
introduction of the parameters simply click on Apply button for the shelf life prediction.
Under the diolog box of Prediction calculated shelf-life (days) and maximum growth rate
(max, 1/h) regarding to the entered kinetic parameters and product characteristics can be
seen. The software also allows making elementary model comparisons by checking the make
comparison box. By clicking on the box model, two dialog box will be activated to enter the
parameters of second model to be evaluated.
Figure 11. Illustrating the kinetic parameters of pseudomonads to be used in the square-root type model
module in FSSP.
The results of the predictions can be presented in graphical format by clicking on Series
of constant temperatures tab (Figure 12). Storage temperature (C) and time (hours) should
be adjusted regarding the experimental design in concern. Graphic button allows the
visualization of the results of predictions graphically. Growth of the bacteria (Figure 13a) and
remaining shelf-life of the product under different temperatures (Figure 13b) are presented in
the graph. In accordance with the results, the shelf-life of the product with the specified
product characteristics (Figure 11) were predicted as 15, 5 and 3 days at 0, 5 and 10C,
respectively.
Figure 12. Temperature (C) and storage time (hours) characteristics of the model in FSSPTM.
(a)
(b)
Figure 13. Prediction results obtained from squre-root type model (in FSSPTM) for (a) pseudomonads
growth and (b) shelf-life of the product (a product with user defined specifications; please refer to
square-root type model module, product characteristics dialog box).
Prediction of Heat Inactivation Kinetics for Pathogenic Microorganisms

Using the Pathogen Modeling Program (PMP)
Besides the importance of quality monitoring and maintenance of food, particularly

seafood, safety is also important in terms of public health. While several safety applications
are available in the area of food processing, heat inactivation is one of the most effective tool
for assuring the safety of the product [53]. Before the development of heat inactivation
models, an utmost significant step is to determine the heat resistance of the specific pathogen.
As in spoilage bacteria, several factors affect the heat resistance of pathogenic
microorganisms, such as pH, aw, preservatives, heating method, fat content, etc. [54-55].
Application of thermal inactivation model for E. coli O157:H7 is shown in this section by
using PMP [52]. The variables of the model are temperature, pH, NaCI and sodium
pyrophosphate. It has to be noted that for the calculation of process lethality of the pathogen,
the z-value, Tref and F-values under dynamic temperatures should be necessarily determined.
The example that is given in this section is not proper for direct applications in food matrix
[56]. Model parameters of thermal inactivation process are shown in table 2.
Table 2. Thermal inactivation model parameters for E. coli O157:H7
T (C) pH NaCI Sodium pyrophosphate (% Log10 Reference

(% g/L) g/L) reduction
55 to 62.5 4 to 8 0 to 6 0 to 0.3 1 to 8 [52]
In the heat inactivation model, time to chosen reduction (in minutes or seconds) is
modeled as a function of log decline of bacteria abundance. Moreover, the effects and
interactions of environmental factors on reduction of bacteria are also employed in the model.
The dialog box of Input Conditions allows the user to introduce environmental factors (i.e.,
temperature, pH, NaCI and sodium pyrophosphate) and desired log reduction of the bacteria
(Figure 14).
Figure 14. Input conditions of heat inactivation model in PMP.
Following the integration of input variables, the Calculate time to choosen reduction
button should be clicked to visualize the predictions. A new diolog box that contains the
results of predictions can be seen in graphical and table format (Figure 15).
PMP predicts the 31.2 min (lower confidence level of 24.9 min and upper confidence
38.9 min) are required for 3 log reduction in E. coli abundance at 55C at pH 7 and with 0.5%
NaCI. Interaction effects of environmental factors can also be calculated by adjusting the
input levels of parameters in PMP.
Figure 15. Prediction of log reduction values of E. coli O157:H7 using PMP.
Risk Assessment Models
Compared to growth and inactivation models, risk assessment basically relies on

combination of the two mentioned deterministic models in a particular food process.
Moreover, risk assessment models contain process-oriented models. In each step of the
process, behavior of specific microorganism generates one part of the risk model. During the
development of risk model in MicroHibro, the processing steps of the food have to be clearly
determined. After composing the steps of the process, the behavior of the microorganism has
to be added in each step of the process diagram as a model component (Figure 16).
MicroHibro contains 3 types of model for risk assessment, namely growth, transfer and
reduction models. Reduction models can be stochastic or deterministic. The chosen
components of the risk model allow users to develop their own models by modifying
previously developed and validated models.
An example for the development of risk assessment model is shown for demonstration of
the model and software. Based on literature data, the behavior of L. monocytogenes in
smoked salmon was modeled (Figure 16).
Development of a complete risk model has to involve all steps, transfer and storage
conditions of the material. In this context, the first step is harvesting of salmon. Occurrence of
contamination mainly depends on living environment of the fish, material that are used for the
harvesting (i.e., seine-net, net, containers) and hygiene of the personnel. Based on this, the
harvested material (salmon) is possibly contaminated by approx. 1.0 log of L. monocytogenes
[57]. During transportation, even without handling of the fish, growth of L. monocytogenes is
expected to occurr. Obviously, contaminated samples will transfer the microorganism to non-
contaminated material in processing line (Figure 16) and transfer model will be used for
handling actions. Temperature and smoke components during smoking process will inhibit
the growth of L. monocytogenes and the model component will be of reduction type. After
clear determination of the processs diagram, the risk model can be structured in MicroHibro
(Figure 17).
Users have simply to click on the run button on the bottom-right margin of the
window. Following this, a new dialog box will open with statistical results (Figure 18).
Figure 16. Processing steps of smoked salmon and behavior of L. monocytogenes in accordance with
processing flow (modified from [61]).
Figure 17. Complete model components of Listeria risk model in smoked salmon using MicroHibro.
Figure 18. Statistical results window of the completed Listeria risk model in smoked salmon using
MicroHibro.
The results window contains input and output variables with their statistical results. Input
and output results are showing the frequency of each step of the process in distribution format
(i.e., normal distribution). Mean, median and mode of the frequency with minimum,
maximum values and standard deviations are also presented. Prevalence of the microorganism
is also presented and Probability of occurrence of the bacteria could be added in the model
in accordance with the type of study (i.e., naturally contaminated samples, surveys, transfer or
challenge studies).
MicroHibro allows users to perform sensitivity analysis for the statistical results for each
component. For instance, statistical results of brining as a function of time are shown in
Figure 19. The changes of mean, median and mode are presented together with percentile
levels of these variables during time (h).
Figure 19. Sensivity analysis of model components using MicroHibro.
CONCLUSION
During the last decade, importance of predictive microbiology and software applications
(i.e., tertiary modeling) has been increased in academic, research and industry contexts.
Therefore, the use of software applications has been steadily increasing because: i) they are
effective tools for simulating the predictions; ii) user-friendly softwares are easy to use; iii) no
deep knowledge in mathematics is needed; iv) software allows comparison of previously
developed and new models; v) there is a wide and diverse range of applications (i.e., spoilage,
thermal and non-thermal inactivation, risk assessment, HACCP plans, traceability); and vi) its
relatively easy to calculate the reliability of the model.
This chapter provides the prediction steps, experimental designs and possible applications
either for SSOs or pathogen bacteria or food matrices by using some of the available
software. The predictions presented in this chapter demonstrated only the applications
considering specific bacteria (i.e., pseudomonads for spoilage, E. coli O157:H7 for thermal
inactivation and L. monocytogenes for risk assessment). However, nowadays recent studies
are focusing on predictions that take into account bacteria-bacteria interactions or bacteria-
food (i.e., organic acids, atmosphere etc.) interaction models. Only some of the software have
interaction modules. Further studies of these interactions and their integration in existing or
new software by providing interactions module(s) should be considered thus allowing more
reliable and accurate predictions in a specific food matrix.
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Chapter 14
RAPID DETECTION OF FOODBORNE BACTERIAL

PATHOGENS IN SEAFOOD
Kitiya Vongkamjan1,*, Siyun Wang2

and Andrea I. Moreno Switt3
1
Department of Food Technology, Prince of Songkla University,
Hat Yai, Thailand
2
Food, Nutrition and Health, Faculty of Land and Food Systems, University of British
Columbia, Vancouver, British Columbia, Canada
3
Escuela de Medicina Veterinaria, Facultad de Ecologa y Recursos Naturales,
Republica 440, Santiago, Chile
ABSTRACT
Traditional methods for detection of foodborne pathogenic bacteria in food and
environmental samples are typically time-consuming and require multiple steps for
identification and confirmation of pathogens. Foodborne diseases caused by consumption
of seafood contaminated with bacterial pathogens, e.g., Vibrio spp., Listeria
monocytogenes, and Salmonella spp., are public health concerns worldwide. Rapid,
reliable, and less labor intensive detection methods can simplify the steps for pathogen
detection in seafood and seafood processing plant environments. Monitoring of
pathogens can become more common and effortless to perform. Immediate responses to
potential pathogen contamination in seafood can be one of the most effective ways to
control foodborne outbreaks. This section reviews the principles and characteristics of
some recent rapid detection methods, including nucleic acid-based methods, antibody-
based methods, and new approaches such as phage-based detection systems. These
methods have gained interest for use to detect pathogens in seafood and seafood
processing environments.
Keywords: rapid detection methods, seafood, processing plant environments
*
Corresponding author: Department of Food Technology, Prince of Songkla University, Hat Yai 90112 Thailand,
Email: kitiya.v@psu.ac.th.
248 Kitiya Vongkamjan, Siyun Wang and Andrea I. Moreno Switt
INTRODUCTION
The food industry, government agencies, and food safety authorities have integrated food
safety programs to ensure the delivery of safe foods from farms to consumers dining tables.
An integral part of such programs has been implemented in various sectors of the food
industry, including the seafood industry. According to the data of the most recent 10 years of
outbreaks (20022011) from the Centers for Disease Control and Prevention (CDC), seafood
was responsible for the second-most outbreaks in the United States (n = 602). Seafood is also
more likely to cause illnesses than other food categories such as poultry, beef and dairy
products. Detection and monitoring of microbiological hazards, particularly pathogens, in
seafood products and seafood processing environments is a major part of the program to
ensure product safety. High demand of food products plays a key role for speed production,
and tests carried out in a shift operation are also typically requested to be completed before a
shift ends. Rapid methods and techniques for microbiological testing, involving rapid and
reliable detection and enumeration of pathogens, are therefore desired. Importantly, early
detection and routine monitoring with generation of faster and more reliable results can
potentially facilitate validation and implementation of adequate control measures and further
prevent post-processing contamination in food products. This section describes rapid
detection methods and some new approaches that currently are of interest for some commonly
identified foodborne pathogens associated with seafood products and seafood processing
plant environments, e.g., Vibrio vulnificus, Vibrio parahaemolyticus, Salmonella spp., and
Listeria monocytogenes.
Overall, methods and techniques involved in rapid detection of pathogens in seafood
products and seafood processing plant environments can ultimately facilitate (i) determination
on the presence of specific pathogens in raw materials, finished products, and environmental
samples, (ii) detection of low numbers of pathogens in complex matrices of organic materials
that are loaded with non-pathogenic microorganisms, (iii) monitoring of process control,
cleaning and hygienic practices during manufacture, and (iv) reduction of time and labor.
Table 1 summarizes the main characteristics of some existing rapid detection methods used
for pathogen detection in the seafood industry. The following sections include a brief
discussion of major approaches for detection of foodborne pathogens in seafood.
RAPID DETECTION METHODS

Molecular-Based Techniques
Polymerase Chain Reaction (PCR) and Multiplex-PCR Assays

PCR techniques have been developed for detection and identification of a variety of
pathogens. This section describes the schemes in details that are specific for Vibrio spp.,
Salmonella spp., and L. monocytogenes. PCR schemes for detection of Vibrio spp. have been
demonstrated to be highly specific and less laborious than the traditional methods [12].
Important aspects are the identification of target genes and the ability to detect the pathogens
Rapid Detection of Foodborne Bacterial Pathogens in Seafood 249
in food matrices [17, 18]. One of the first schemes designed to detect Vibrio in seafood was
reported by Bej et al. [19]. V. parahaemolyticus could be detected in shellfish by targeting
genes that encode different hemolysins (e.g., tdh and trh); however, not all the fragments
were amplified efficiently. Another study demonstrated that the PCR method was able to
detect V. parahaemolyticus and V. cholera in approximately 50% of the living bivalve
mollusk samples more than the standard ISO/TS 21872-1 culture method [20]. In multiplex
PCR schemes which have been found to be specific for the three pathogenic species of Vibrio,
genes toxR [21], toxR and vvhA [22], and atpA [23] have been targeted for detection. Hossain
et al. [24] also designed a multiplex PCR scheme based on the amplification of groEL for
detection of three pathogenic Vibrio spp. in artificially inoculated shellfish homogenates,
flounder, and sea water. Approaches that targeted several bacterial pathogens in seafood have
also been conducted. In a previous study, a multiplex PCR assay was designed to detect
Salmonella spp., V. cholerae, V. parahaemolyticus, and E. coli O157:H7 in spiked shrimps
[25].
Detection of Salmonella spp. in seafood by PCR has been evaluated by targeting the invA
gene in different types of seafood, such as mussels [26] and oysters [27]. Approaches from
these work have been able to detect less than 10 cells/ml of homogenate seafood following
pre-enrichment. The gene hns which encodes a DNA binding-protein has also been used for
detection of Salmonella spp. in finfish, clamps, and shrimps [28]. This detection scheme
performed equivalently well when compared with traditional culture-based method.
Sensitivity of invA-based PCR was found to be higher than the enzyme-linked
immunosorbent assay (ELISA) and U.S. Food and Drug Administration Bacteriological
Analytical Manual (FDA-BAM) methods in naturally contaminated fish, crab, clam, mussel,
oyster, squid, cuttlefish, and octopus [29]. Several commercial kits based on PCR for
detection of Salmonella spp. L. monocytogenes and Listeria spp. are available. For example,
the BAX System PCR Assay has been reported its performance which was equivalently well
when compared with the traditional culture-based method for detection of L. monocytogenes
in smoked fish [30], raw fish [31], and blue crabs [32].
Real Time-PCR Assays

Real-time PCR allows for detection and quantification of the amplified DNA during PCR
cycles. This assay utilizes a dual-labeled fluorogenic probe that is measured during the cycles,
resulting in a faster detection and higher throughput than the conventional PCR [33]. Here we
describe in more details of the real time-PCR schemes that are specific for Vibrio spp.,
Salmonella spp., and L. monocytogenes. Takahashi et al. [34]. targeted the toxR gene of V.
parahaemolyticus, and reported that the real time-PCR scheme could successfully detect this
pathogen in artificially contaminated seafood and water. In addition, a number of real-time
PCR schemes have been designed to detect V. parahaemolyticus in oysters, shrimps, and
other seafood [35-37]. Although these schemes could detect low levels of contamination (i.e.,
<10 CFU of detection limit) and were validated with the traditional culture-based methods,
the seafood matrix could inactivate the PCR reaction in some cases [38]. Kin and Lee [39]
reported a multiplex real-time PCR reaction that simultaneously detected V.
parahaemolyticus, V. vulnificus, and V. anguillarum. The detection limit for this assay was 1
CFU/ml in pure cultures and water samples, and 10 CFU/g in fish [39]. The ability of invA-
real-time PCR scheme was investigated for detection of Salmonella spp. and this scheme was
able to detect 2.5 CFU/25 g in artificially inoculated salmon [40]. Commercial assays are also
available for detection of Vibrio spp. in seafood. For example, the BAX System utilizes the
real-time PCR to detect V. cholerae, V. parahaemolyticus, and V. vulnificus in shrimp, tuna,
oysters, scallops and crab. An enrichment step of approximately 20 hours is needed for this
assay, and the detection limit is 104 CFU/ml after enrichment. A number of schemes and
commercial kits are also available for L. monocytogenes detection in different types of foods.
One study determined sample preparation methods for Listeria detection in smoked salmon
using real-time PCR [41]. The purification of DNA with a commercial kit was found to be
optimal for this assay, which could detect L. monocytogenes at 10 CFU/g.
Isothermal Amplification Methods

The nucleic acid amplification, known as loop-mediated isothermal amplification
(LAMP), was developed by Notomi et al. [42]. LAMP assay is based on autocycling strand
displacement DNA synthesis facilitated by four to six primers specific to six to eight regions
of the target DNA sequence in the presence of Bacillus stearothermophilus (Bst) DNA
polymerase. Amplification occurs under isothermal conditions between 60 and 65C,
resulting in 109 copies of target DNA within an hour [42]. Han et al. [43] applied LAMP
technique for detection of the virulence-correlated gene at the C sequence variant (vcgC)
present in virulent V. vulnificus strains in raw oysters. The LAMP assay was able to detect 2.5
x 103 CFU/g of a virulent V. vulnificus ATCC 33815 strain. In addition, the LAMP assay
could detect 1 CFU/g of this virulent strain in seafood sample after enrichment. Yamazaki et
al. [9] developed a LAMP assay for detection of thermolabile direct hemolysin (tlh) on pure
V. parahaemolyticus cultures and artificially inoculated shrimps. Sensitivity of the LAMP
assay for direct detection of this pathogen in both pure cultures and artificially inoculated
shrimps was 5.3 x 102 CFU per ml or g (2.0 CFU per reaction). Sensitivity of the LAMP
assay was 10-fold more sensitive than that of the conventional PCR assay. The time required
for DNA extraction and LAMP reactions was only 27 to 60 min for pure cultures and less
than 80 min for spiked shrimp samples. In Salmonella spp., invA and HisJ genes have been
evaluated by LAMP assays. The invA gene could be detected by this assay in Salmonella
recovered from several food sources, including marine products [44]. The detection limit was
100 fg DNA/tube. Zhang et al. [45] developed LAMP assay that could target the HisJ gene of
72 Salmonella serovars and reported the detection limit of 16 CFU per reaction. For detection
of Listeria spp. and L. monocytogenes, Fortes et al. [10] evaluated a commercial LAMP-
based detection system on environmental samples from retail stores and food processing
facilities, including seafood processing plants. The LAMP-based detection system showed the
overall performance as comparable as the culture-based method (FDA-BAM). Vongkamjan et
al. [46] has also demonstrated that this commercial LAMP-based molecular showed high
sensitivity (87.0%) and specificity (97.6%) when compared with the standard FDA-BAM
method for detection of Listeria spp., including L. monocytogenes on environmental samples
from seafood processing plants (n = 222).
Table 1. Main characteristics of selected rapid detection methods used for seafood pathogen detection
Duration of
Detection
Specificity the assay Pre-
Test method Target limit Availability Ref.
(ability to identity) including enrichment
(CFU/ml)
prep time (h)
ELISA and ELFA antigen 103-4 Identify specific 13 Needed Available [1-3]
bacterial species commercially
Engineered surface receptor 103-4 Identify specific 13 Needed Available [4-6]
bacteriophage bacterial species commercially
Q-PCR and Multiplex- DNA 102-4 Identify specific 13 Needed Available [7, 8]
PCR bacterial strains commercially
Isothermal DNA DNA or rRNA 102-4 Identify specific 13 Needed Available [9, 10]
amplification bacterial species commercially
Microarray DNA 102-4 Identify specific >3 Needed Laboratory trial [11-14]
bacterial strains
FC-FISH DNA or rRNA 103-4 Identify specific 13 Needed Laboratory trial [15]
bacterial species
Bioluminescence ATP 104 Do not differentia-te <1 Not needed Available [16]
between bacterial commercially
species
Abbreviations: Enzyme-linked immunosorbent assay (ELISA); Enzyme-linked fluorescent assay (ELFA); Quantitative real-time PCR (Q-PCR); Fluorescence in
situ hybridization (FISH) combined with flow cytometry (FC).
Antibody-Based Methods
Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme-Linked Fluorescent

Assay (ELFA)
ELISA is a common immunological-based method whose detection system is based on
enzyme-labeled reagents. Honda et al. [47] developed three types of ELISA assays, including
ganglioside ELISA, direct ELISA, and sandwich ELISA, for identification of thermostable
direct hemolysin (TDH) and TDH-related hemolysin (TRH) of V. parahaemolyticus from
clinical samples. Sandwich ELISA showed satisfactory results for detecting TDH in crude
mixtures. Kumar et al. [48] developed monoclonal antibodies against purified TRH
recombination protein. This sandwich ELISA was able to detect V. parahaemolyticus in
41.8% (14 of 34) of the seafood samples analyzed, while 64.7% (22 of 34) of samples could
be detected by PCR assay that targeted toxR gene. The limit of detection of pathogenic V.
parahaemolyticus was 103 cells in monoclonal antibody-based sandwich ELISA, while the
PCR method showed the limit of 10 cells after enrichment. Although this monoclonal
antibody-based sandwich ELISA developed could not differentiate TDH and TRH of V.
parahaemolyticus, this method was useful for detection of pathogenic V. parahaemolyticus in
various seafood products in a routine food product testing. Overall, detection using automated
and robotic ELISA can generate results within 3 hours after enrichment. Fluorescence is a
product generated from enzymatic reaction in ELFA which can be detected by fluorometry. A
previous study evaluated a reliability of applying a shortened enrichment method with ELFA
for detection of Listeria spp. in a variety of raw food products, including raw fish [49].
Among 105 raw fish samples tested, 29 were positive by both methods, while two samples
showed positive results by ELFA only (considered false-positive). However, the ELFA
system could yield reliable results for detection of Listeria spp. after 52 hours of enrichment.
Lateral Flow Devices (LFD)

Lateral flow immunoassay (LFIA), also known as immunochromatographic strip test, has
been developed as typical rapid diagnostic tests (RDTs) for various pathogens. The LFIA
platform is similar to ELISA; the base substrate is immobilized and labeled with antibody or
antigen depending on test design. Labelling commonly applies colored or fluorescent
nanoparticles and more recent novel application such as quantum dots. Detection time for this
method is about 5-10 minutes after application of samples. However, this method requires
high concentration of target organisms (about 107 to 109 CFU) [50], suggesting that
enrichment is typically required.
Phage-Based Detection Systems
A phage-based detection approach for L. monocytogenes has been developed. A

luciferase reporter Listeria-specific phage (A511::luxAB) was constructed for Listeria host
cell detection [5]. The luxAB genes of Vibrio harveyi were inserted downstream of the major
capsid gene in A511 phage. A real-time light emission produced by luciferase in the Listeria
infected cells can be detected. This detection method has been shown to have ability to detect
L. monocytogenes in food and environmental samples after 20 hours of enrichment, wheras

the traditional culture-based method required about 4 days [51]. In various food samples,
including shrimps, as low as one cell per g of food was necessary to produce detectable
bioluminescence signal. Another approach involves with insertion of the celB gene from
Pyrococcus furiosus into the genome of phage A511. The celB gene encodes a thermostable
-galactosidase which can be used with different chromogenic, fluorescent or
chemiluminescent substrates; light emission can be detected upon phage infection [6]. The
detection limit for viable L. monocytogenes in a 96-well plate platform was 7.2 x 102 cells per
well, corresponding to 6 x 103 CFU per ml in suspension. This assay has been evaluated for
detection of L. monocytogenes and could detect about 10 CFU per g or less in spiked salmon.
A commercial kit (VIDAS UP) that utilizes phage-recombinant protein technology has been
introduced for detection of Listeria spp. and Salmonella in food and environmental samples.
Test results can be generated within one hour after applying samples that have been enriched.
CONCLUSION
Rapid detection methods have gained increased interest for detecting foodborne
pathogens in seafood and environments associated with seafood production for validation of
process controls. A number of detection methods have been developed and introduced to the
seafood industry. The process of selecting an appropriate method with regards to sensitivity,
specificity, and time of analysis is essential. However, there are still no specific criteria or
guidance for selecting. Approaches described in this section intend to be developed to
ultimately overcome some drawbacks of traditional microbiological methods as traditional
assays are typically time-consuming and labor-intensive. Identification of specific foodborne
pathogens needs to be rapid in order to generate real-time results as immediate action is
expected once contamination of food products has been encountered. Monitoring
environments associated with seafood production is important; rapid detection approaches
provide robustness and reliability as some nucleic acid-based methods can be extremely
sensitive and specific.
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Chapter 15
APPLICATION OF NATURAL ANTIMICROBIALS

IN SEAFOOD
Celso Alves, Susete Pinteus, Rui Pedrosa

and Maria Manuel Gil
Instituto Politcnico de Leiria,
Peniche, Portugal
ABSTRACT
Although a large research effort has been given to attain safe seafood products, food-
borne diseases are a constant concern both from a public health and/or economic
perspectives. Consequently, the microbial spoilage of these foods and the presence of
pathogens are of major importance to processing industries, and an appropriate control of
growth and/or inactivation of microorganisms is crucial. In order to ensure food safety
and quality, and as a response to consumers concerns about chemical preservatives used
in food, the application of natural active compounds has been the target of many
scientific studies, and new potent natural antimicrobials have already been identified.
Nevertheless, seafood, like other kinds of food products present several scientific and
technological challenges, such as how to incorporate the antimicrobial compounds and
maintaining them active along time to enhance the product shelf-life. Different
techniques have been developed and others are being tested, presenting huge advantages
for food industries regarding food preservation. This chapter compiles relevant
information concerning the usage of natural antimicrobials for the preservation of
seafood, gathering application techniques of active compounds.
Keywords: antimicrobial compounds, seafood preservation, natural compounds,

antimicrobials incorporation
Corresponding author: M.M.Gil, MARE Marine and Environmental Sciences Centre, ESTM, Instituto
Politcnico de Leiria, Peniche, Portugal. Email: maria.m.gil@ipleiria.pt.
260 Celso Alves, Susete Pinteus, Rui Pedrosa et al.
INTRODUCTION
Several new natural active agents from different sources such as plants, marine
organisms, animals and microorganisms were already described and characterized presenting
strong antimicrobial properties (Chapter - Biological hazards and natural antimicrobials for
seafood preservation). However, seafood products, like other kinds of foods present the great
challenge of how to incorporate the antimicrobial compounds maintaining them active along
time. Therefore, different techniques have been developed and others are being developed
presenting huge advantages for food industries regarding food preservation.
Antimicrobials can be directly added into the product formulation, coated on its surface
or incorporated into the packaging material [1-3]. The direct incorporation of active agents
into food results in an immediate but short-term reduction of bacterial populations, while
other antimicrobial approaches can maintain their activity for a long period of time [3].
Active packaging systems based on the application of packaging materials with
incorporated antimicrobial agents provides one of promising trends in food processing. The
direct application or the application through edible coatings and films, and nanoparticles
(Figure 1) are, therefore, the most common procedures used for antimicrobials incorporation
in food systems and will be described below.
The aim of this chapter was to compile relevant information concerning the usage of
natural antimicrobials for the preservation of fish and seafood, gathering application
techniques of active compounds.
Figure 1. Illustrative scheme of the different procedures used for antimicrobials incorporation in food
systems and respectively antimicrobial efficacy along of the time.
Application of Natural Antimicrobials in Seafood 261
SEAFOOD APPLICATIONS OF NATURAL

ANTIMICROBIAL COMPOUNDS
Direct Application of Natural Antimicrobial Compounds
Antimicrobial compounds have been directly applied in food surfaces either in the form
of a powder or a liquid by use of sprays or dips. Numerous examples of this type have been
reported in the literature [4-5]. However, direct surface application of antibacterial substances
onto foods have limited benefits because the active substances may be neutralized by product
constituents on contact, or diffuse rapidly from the surface into the food mass. On the other
hand, incorporation of bactericidal or bacteriostatic agents into food product formulations
may result in partial inactivation of the active substances by product constituents (with
limited solubility in food matrices), and is expected that natural antimicrobials have limited
effects on the surface microflora (Figure 1) [4-5]. As a result, only a few natural
antimicrobials have found practical application in the food industry and their direct
application in foods as preservatives is often limited due to the strong smell and taste they
impart to these foods. For example, several plant essential oils (EOs) have showed
interesting antimicrobial activities against different pathogenic and spoilage microorganisms
[6-8], however, their direct application in food products is conditioned and need to be
optimized, since they have impact on sensory acceptability. If high concentrations are
required to achieve useful antimicrobial activity, unacceptable levels of inappropriate flavors
and odors may result. Nevertheless, several studies have succeeded in increasing the shelf-life
of seafood products by direct application of natural antimicrobial compounds. Garca-Soto
and co-workers [9] developed a new flake icing system containing citric and lactic acid
resulting in an inhibitory effect on all the five microbiological parameters analyzed in the
samples of fresh European hake (Merluccius merluccius). They suggested that the
antimicrobial washing effect provoked by the melting of the ice crystals, containing the
natural organic acid solution, leading to the subsequent reduction of the surface microbial
load and its diffusion towards the muscle may explain the better protection of hake quality
and its extended shelf-life under these conditions. In the same line, Speranza and
collaborators [10] defined a solution containing a great combination of thymol, a grape fruit
seed extract (GFSE) and chitosan that allowed to increase the shelf-life of gilthead sea bream
(Sparus aurata) fillets. Their results showed the capacity of this solution to inhibit growth of
some seafood specific spoilage organisms, namely, Pseudomonas fluorescens, Shewanella
putrefaciens and Photobacterium phosphoperum, which were previous inoculated in the
fillets. The dipping treatment with an optimized solution containing 2% of chitosan and 6,000
ppm of thymol and GFSE, combined with packaging under 5:95 O2/CO2 allowed to maintain
the fillets at the maximum level of microbiological quality for at least 810 days and the
sensory attributes at acceptable levels for about 20 days. This treatment permitted to increase
the shelf-life of gilthead sea bream (Sparus auratus) fillets when compared with control. In
order to increase the shelf-life of seafood products, also essential oils have shown to have
high antimicrobial activity and potential to be used alone or in combination with other
preservation techniques. The utilization of oregano oil in modified atmosphere-packed of cod
fillets allowed to reduce the growth of Photobacterium phosphoreum, which is a specific
organism responsible for the spoilage of this product, extending the shelf-life from 11-12 days
to 21-26 days at 2C [11].
Although, the exclusive use of this technique present some disadvantages, is also true that
when combined with other techniques, as modified atmosphere packaging, can contribute for
a more marked effect in the extension of fish and seafood products shelf-life.
Edible Coatings and Films with Natural Antimicrobial Compounds
Active packaging describes mainly food packaging that interacts chemically or

biologically with its contents or headspace to extend shelf-life. Packaging may be termed
active when it affects one or more attributes of the packaging in a lasting and desired way.
Substances responsible for the active function can be contained in a separate container or can
be directly incorporated in the packaging material. Hence, an important objective here is to
design functional materials that include the active agent in their structure and that this active
substance can act or be released in a controlled manner [1]. Organic acids, herb extracts or
essential oils, fatty acids, enzymes, fruit plants and seed extracts, are examples of active
substances known for their effective antimicrobial properties producing active films [12].
In the last decade there has been a growing interest in the development of new bio-based
packaging from renewable sources. Food-packaging industries have shown an interest in
edible films and coatings containing natural antimicrobials. In addition, this increased interest
has been intensified due to concerns about limited natural resources of the fossil fuel reserve
and the environmental impact caused by the use of non-biodegradable plastic-based
packaging materials and edible films/coatings can be a potential solution [5, 13]. Different
biopolymers have been studied and applied in this field, such as, starches, cellulose
derivatives, chitosan/chitin, gums, proteins (animal or plant-based) and lipids. They have
been found to be effective to obtain thin films and coatings for fresh or processed foods, used
to extend products shelf-life [5, 13]. Edible films and coatings act as barriers to water vapor,
oxygen, and carbon dioxide and as carriers of substances to inhibit pathogenic and spoilage
microorganisms allowing extending the product shelf-life [14]. Unlike the direct application,
which results in an immediate but short-term reduction of bacterial populations, the utilization
of edible films and coatings with antimicrobial proprieties allows the migration of these
compounds to the coating surface providing a continuous antimicrobial activity on the food
for a long period of time (Figure 1) [3, 15].
The incorporation of natural antimicrobial compounds into the suspensions of edible
films and coatings is an interesting way to enhance their functional properties [14].
Nowadays, a wide variety of natural antimicrobials have been added to edible films and
coatings to prevent the microbiological growth and extend product shelf-life, that include
chitosan, polypeptides, and plant oils, extracts, and spices [16]. Antimicrobials used for the
formulation of edible films and coatings must be classified as food-grade additives or
compounds generally recognized as safe (GRAS) by the relevant regulations. International
regulatory agencies are in charge of approving antimicrobials for the use on foods. In the US
those compounds are regulated by the part 21CFR172 enacted by the US Food and Drug
Administration (US FDA, 2009) and in the European Union by the EU Framework Directive
89/107 (EU, 1989) [16].
Literature widely demonstrates that inclusion of natural compounds in edible coatings

and films are effective preservation methods for fish and seafood products (Table 1).
Actually, chitosan is one of the most important compounds from natural source being used as
coating material, as well as antimicrobial compound in fish and seafood products. Chitosan
possesses a high antimicrobial activity, a broad spectrum of activity, a high kill rate, and low
toxicity towards mammalian cells. Although several studies demonstrated the high capacity of
chitosan as antimicrobial, their mechanism of inhibition is not yet fully understood [17].
Moreover, their properties are specially recognized in the field of food preservation and
packaging to avoid the use of chemical preservatives and to produce edible antimicrobial
films due to the good film forming properties of chitosan. As a polymeric ingredient, it does
not migrate easily out of the protecting film and has better barrier properties. For example,
Gomez-Estaca and co-workers [18] reported that a gelatinchitosan-based edible film,
together with refrigeration and high pressure processes, lowered the microbial growth of cold-
smoked sardine in comparison to uncoated samples. Nevertheless, not only chitosan has
showed interesting results, also essential oils have demonstrated high potential as
antimicrobial when incorporated in edible coatings and films. Mastromatteo and co-workers
[19] reported the increase of ready to use peeled shrimps shelf-life, from 5 to 14 days through
the use of active coating containing thymol, an essential oil (characterized to possess
antimicrobial activity) in modified atmosphere. For other side, different studies have
highlighted promising results through the combination of essential oils and chitosan. Ojagh
and co-workers [20] reported the effects of chitosan and cinnamon oil on the quality of
rainbow trout during refrigerated storage and the results suggest that the combined use of
these compounds exerted a higher inhibitory effect on the native flora in comparison with the
use of chitosan alone. Moreover, the application of a coating with chitosan and cinnamon
essential oil improved trout fillet shelf-life (16 days vs. 12 days of the control) and in
particular it enhanced texture, odor, and color. Similar results were also obtained for trout
fresh fillets, but coated with gelatin enriched with cinnamon oil (1%, 1.5%, and 2%).
In fact, the use of edible films and coatings is an environmentally-friendly technology
that offers substantial advantages for shelf-life increase of many food products including fish
and seafood products. The incorporation of different natural compounds in edible films and
coatings such as essential oils, grapefruit seed extract, lactoperoxidase system and chitosan
demonstrate that they are well suited to be utilized as preservatives in fish and seafood and
could be often valid alternatives to synthetic food additives. In the last years, several advances
have been done to development edible coatings and films contained natural antimicrobial
compounds for fish and seafood products. However, some of these studies mainly focused on
the initial screening of newly developed edible films or coatings for antimicrobial activity in
laboratory media and quantifying the bacterial reductions obtained during storage for
different types of packaged food products. Consequently, for commercial application of these
films and coatings will be important to know the variation in antibacterial activity of the
agents when incorporated into the packaging film from its original activity establishing the
levels that need to be incorporated for effective bacterial inhibition.
Nanoparticles and Their Potential Application as Antimicrobials
Nanotechnology is a new frontier of this century and its application in food systems is an
emerging technology [5, 37]. The researchers interest on application of nanotechnology for
improved food safety has increased in the last few years due to its potential for the
development of nanoparticle systems for antimicrobial compound delivery [5, 38]. An
antimicrobial delivery system is designed to release the active at a particular site of action that
can be on the surface or inside the microbial cell which can be controlled according to the
environment surrounding the system (pH, temperature, ionic environment, or enzymatic
activity) [39]. However, the efficacy of some antimicrobial compounds can be conditioned by
interactions with food components (proteins and lipids), inactivation by enzymatic
degradation or uneven distribution of antimicrobial compounds within the complex food
systems [5]. According with this, nanoparticles have unique properties that can provide a
novel direction over the antimicrobial systems having low efficiencies, such as in the direct
application process [38]. Nanoparticles have therefore emerged as novel carriers of these
active ingredients in food [40].
The main advantage of applying nanoparticles, such as liposomes, dendrimers,
polymeric, and solid lipid nanoparticles, in food systems is to increase the antimicrobial
stability, which plays an important role in the inhibition of spoilage and foodborne pathogens.
Although, the nanoencapsulated antimicrobial compounds may observe initially less activity
compared with the nonencapsulated compound, their antimicrobial activity lasts much longer
(Figure 1). Moreover, the use of nanocarriers can modulate the release of antimicrobials,
protecting them from adverse conditions, improving their stability, solubility and
dispersibility directing them to the site of action, thereby decreasing the amount required to
observe an antimicrobial effect [5, 38-39].
The use of nanoparticles is particularly interesting in the case of essential oils (EOs),
since their poor water-solubility make it difficult to incorporate them into foods and reduces
antimicrobial action. Therefore, a higher concentration of EOs is required to achieve higher
antimicrobial efficacy, which could alter the sensory properties of foods. Several studies have
showed that nanoencapsulated EOs were more evenly distributed even at higher
concentrations above the solubility limit than free EOs, thus resulting in higher antimicrobial
efficacy [41-43].
Although the interest of the nanoparticles application (containing natural antimicrobial
compounds) in food systems has increased in the last years, there are few studies about their
application and use in seafood products. Nevertheless, this approach can have great potential
for applications in this type of food, as evidenced by Osheba and co-workers [44]. In this
work different concentrations of chitosan and chitosan nanoparticles, as active coating on
microbiological characteristics of fish fingers during frozen storage at -18C, were tested.
Table 1. Use of natural antimicrobial films and coatings in seafood
(Adapted from Snchez-Ortega et al. [14])
Seafood Coating material Antimicrobial compound Origin Effects in target microorganisms References
Atlantic cod
(Gadus morhua) and Chitosan coatings Chitosan with different molecular Animal Reduced the growth of psychrotrophic
[21]
herring (Clupea weights and viscosities (Crab) microorganisms and TPC
harengus)
Growth inhibition of L. monocytogenes in
Smoked salmon Whey protein isolate (WPI) Lactoperoxidase system (LPO)
smoked salmon at 4 C and 10C for 35 [22]
(Salmo salar) coatings
days and 14 days, respectively.
Oregano extract (OE) Plant OE-G and RM-G films reduced TVC and
Rosemary (RM) (Origanum vulgare and inhibit the growth of H2S-reducing
Cold-smoked
Rosmarinus officinalis) bacteria.
sardine (Sardina Gelatin (G) films [18]
pilchardus) Animal Strong reduction of TVC; H2S-reducing
Chitosan
organisms, luminescent bacteria, and EB
Growth inhibition of TBC, H2S-producers

Cod (Gadus Gelatin and combination Plant
Clove essential oil organisms, luminescent organisms, [23]
morhua) with chitosan films (Syzygium aromaticum L.)
Pseudomonas sp., EB, and LAB
Sea bass slices Gelatin extracted from Plant Growth inhibition of TVC, PC, EB, H2S-
Lemongrass essential oil [25]
(Lates calcarifer) Aluterus monoceros (Cymbopogon citratos) producing bacteria and LAB
Sea bass Growth inhibition of TVC and
(Dicentrarchus Chitosan films CH with vacuum packaging Animal psychrotrophic aerobic bacteria. Shelf life [26]
labrax) of the product was increased in 20 days.
Growth inhibiton of mesophilic
Indian oil sardine
Chitosan (1 and 2% w/v) microorganisms. The shelf life was
(Sardinella Chitosan (1 and 2% w/v) --- [27]
coatings increased in 2 and 4 days for 1% and 2%,
longiceps)
respectively.
Indian oil sardine
longiceps)
respectively.
Indian oil sardine
longiceps)
respectively.
Salmon Growth inhibition of E. coli O157:H7 and
Barley bran protein and
(Oncorhynchus Grapefruit seed extract --- L. monocytogenes along 15 days. [28]
gelatin (BBG) films
sp.)
Cold-smoked Oregano essential oil (OO)
Potato processing waste Coated samples with PPW-OO, reduced
salmon (Salmo 0.97% and 1.92% (185 and 289 --- [29]
(PPW) films significantly Listeria population
salar) mg oil/g film)
Three chitosan solutions (0.25%,
Atlantic salmon
0.50% and 0.75% w/v) applied in TVC was maintained below the maximum
fillets (Salmo Chitosan coating --- [30]
different amounts (6%, 8% and limits.
salar)
11%)
Edible coating solution
Plant
Fresh mullet fish (wheat flour, sodium Thyme and marjoram 2.5 and Growth inhibition of TBC. Strong effects
(Origanum majorana [31]
(Mugil capito) chloride, cumin and 5.0% against the EB growth
L.and Thymus vulgaris L.)
xanthan)
Moderate increase in APC values of
Tilapia
LDPE Chitosan using Tilapia packed in 3% and 5% LDPE/CH
(Oreochromis 1, 3 and 5% of Chitosan (w/w) --- [33]
maleic anhydride grafted films and samples remained acceptable
mossambicus)
even beyond 15 days.
CH+LPO treatment had significantly lower
numbers of Shewanella putrefaciens,
Chitosan 1.5% (w/v) on 1% Pseudomonas fluorescens, psychrotrophic
Ice-chilled trout Lactoperoxidase system (LPO) --- [32]
v/v acetic acid and mesophilic bacteria than did the CH
and control group during the entire storage
period
Significant reduction in TVC and TPC.
Bighead carp filets Plant
Sodium alginate (SA) Horsemint essential oil (HEO) TVC of SA-HEO-0.5% and SA-HEO-0.5%
(Aristichthys (Mentha [35]
coating 0.5 and 1.0% (w/v) did not exceed the limit value during the
nobilis) longifolia L.)
entire storage.
Rainbow trout Plant
CH + Cinnamon oil 8 (C) (2%, Growth inhibition of TVC and PC
(Oncorhynchus Chitosan coating (Cinnamomum zeylanicum [20]
w/v Ch + 1.5%, v/v C) increasing the shelf life in 8 days.
mykiss) L.)
Silver carp
Growth inhibition of TVC increasing the
(Hypophthalmicht Chitosan coating Chitosan (2%) --- [36]
shelf life in 5 days.
hys molitrix)
Thymol (1000 ppm) combined
Shrimp
with modified atmosphere Growth inhibition of TVC. The product
(Palaemon Sodium alginate solution Plant [19]
packaging (MAP) (5% O2; 95% shelf life increased 14 days
serratus)
CO2)
2% Chitosan + 0.75% glycerol + Plant
Snakeheads Growth inhibition of TVC. Extended the
Chitosan (CH) coating 0.5% Tween-80 + 1% (Tylenchorhynchus [34]
(Channa argus) shelf life on approximately 45 days
Thyme essential oil vulgaris)
TVC Total Viable Count; TBC Total Bacterial Counts; EB Enterobacteriaceae; LAB Lactic actid bacteria; APC- Aerobic plate count; PC-
Psychrotrophilic counts; CH Chitosan; LDPE - Low Density Polyethylene; TPC- Total Psychrotrophilic counts.
After six months of frozen storage ( 18C), chitosan nanoparticles treatments showed the
lowest counts of total bacterial, psychrophilic bacteria, coliform bacteria and proteolytic
bacteria. Also, the treatment of silver carp (Hypophthalmicthys molitrix) fillets with
nanoparticles of chitosan during refrigerated storage at 4C was effective in the preservation
of fillets not exceeding the maximal permissible limit of 7.0 log10 cfu/g until the end of
storage period (12 days), while these values were higher than 7.0 log10 cfu/g in controls after
9 days [45]. In line with this, Ramezani and co-workers [46] studied the coating effects of
chitosan and chitosan nanoparticles on the quality of silver carp (Hypophthalmicthys molitrix)
and verified that both chitosan and nanochitosan coatings were effective for the fillets
preservation. However, nanochitosan exhibited higher antimicrobial activity than chitosan
during the storage period. Similar results were observed in postharvest whiteleg shrimp
(Litopenaeus vannamei), where the chitosan nanoparticle coating exhibited better inhibition
against bacteria than carboxymethyl chitosan coating on day 10, maintaining the quality of
shrimp during the storage at 4C [47]. These results suggest that compounds incorporated in
nanoparticles can present a better performance, since the special characteristics of the
nanoparticles, such as the nanoparticle's larger surface area and higher affinity with bacteria
cells, may contribute for the effects observed. Likewise, Arfat and collaborators [48] studied
the changes in sea bass slices wrapped with a biofilm composed by a fish protein isolate
(FPI), fish skin gelatin (FSG)-ZnO, nanocomposite (ZnONP) and basil leaf essential oil
(BEO), during the storage of 12 days at 4C. They observed that the sea bass slices coated
with FPI/FSG-ZnONP-BEO film had the lowest growth of psychrophilic bacteria, lactic acid
bacteria and spoilage microorganisms including Pseudomonas, H2S-producing bacteria and
Enterobacteriaceae, extending the shelflife from 6 to 12 days. Moreover, the sensory
characteristics maintained stable along the 12 days storage.
Although research on nanotechnology has increased rapidly in the last decades, the
application of nanotechnology in food started to raise interest in the scientific community
much more recently. Actually, this technology holds tremendous potential as an effective
antimicrobial delivery system and could be employed to incorporate natural antimicrobials
that result in safer food products. When compared to micro-sized presentations, nanocarriers
provide more surface area, enhance solubility, and improve bioavailability and targetability.
Nevertheless, the availability of materials recognized as GRASS to produce the
nanoencapsulating systems, has limited the research in food areas. Moreover, the use of
nanotechnology in food has raised a number of concerns over their safety to the consumers.
Regarding seafood products, there are not many studies about the application of
nanoparticles; however, the few existing results are very promising, especially in regards to
the chitosan nanoparticles application.
In order to apply the nanocarriers in food industry, future research has to focus on the
design of scalable methods and identification of low-cost ingredients as well as to develop a
more practical and effective delivery system for these antimicrobial compounds in food
products. Furthermore, safety and health risks of natural antimicrobials need to be assessed
before future applications in food products.
CONCLUSION
Currently, there are available a variety of natural antimicrobials from different sources
such as plants, marine organisms, animals and microorganisms that have showing promising
results in the preservation of seafood such as oregano essential oil, chitosan, and bacteriocins,
respectively. However, some of these natural compounds have the disadvantage of
transferring flavor to the food matrix and lose the biological activity as time passes.
Therefore, the incorporation method for preserving the biological activity of these compounds
during long periods of storage continues to be a great challenge. Several techniques such as
the use of organic biofilms the incorporation of the active compound into nanoparticles and
modified atmosphere packaging have revealed promising results in seafood and fish product
preservation. As a result, the combination between different techniques for the application of
natural compounds in seafood and fish products may be a solution for keeping the sensory
and nutritional characteristics of the fresh products.
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Chapter 16
BIOLOGICAL HAZARDS AND NATURAL

ANTIMICROBIALS FOR SEAFOOD PRESERVATION
Susete Pinteus, Celso Alves, Rui Pedrosa

and Maria Manuel Gil*
Instituto Politcnico de Leiria, Peniche, Portugal
ABSTRACT
Since ancient times, natural products have been used for food preservation. With no
refrigeration systems, the addition of specific plants, salts and herbs were the only
available methods for maintaining the products quality as time passed. Nowadays, new
methodologies have been discovered and developed for maintaining food quality during
long periods of time; however, some of the most common strategies to increase the shelf-
life of a food product consists in applying synthetically preservatives, color additives and
antimicrobials. However, consumers concern over the possible adverse health effects of
certain food chemical preservatives has resulted in an increasing pressure on
manufacturers to remove chemically-synthesized additives from processed foods and to
provide more natural alternatives for the maintenance of food safety and shelf-life.
Over the last few years, several new natural active agents from different sources such
as plants, marine organisms, animals and microorganisms were described and
characterized. Some of those revealed strong antimicrobial properties and could be used
in food industry. This chapter compiles relevant information concerning the usage of
natural antimicrobials for seafood preservation and includes a compilation of natural
antimicrobial products, their sources, and the microorganisms susceptible to these
antimicrobials.
Keywords: seafood hazards, natural antimicrobials, emerging antimicrobials, phenolic

compounds, seafood spoilage
*
Corresponding author: MARE Marine and Environmental Sciences Centre, ESTM, Instituto Politcnico de
Leiria, Campus 4 - Santurio Nossa Senhora dos Remdios, Apartado 126, Peniche 2520641. Email:
maria.m.gil@ipleiria.pt.
276 Susete Pinteus, Celso Alves, Rui Pedrosa et al.
INTRODUCTION
Biological Hazards in Seafood
Fish and other seafood are an important part of a balanced diet and contribute to a good
nutritional status, mainly due to its high levels of many important nutrients that are not
commonly found in other food products. For example, seafood products are an excellent
source of proteins, very long-chain omega-3 fatty acids (EPA and DHA), vitamin D, selenium
and iodine [1-2]. As a consequence of this perception, there has been an increased interest for
seafood at the consumer level and consequently thousands of new fish and other seafood
products have been prepared and launched worldwide [3].
Seafood, like many other food products, is perishable by nature and requires protection
from spoilage during its preparation, storage and distribution to give it the desired shelf-life
[4]. Most of the microorganisms are originate from the microflora of the raw material,
inadequate handling practices and improper hygienic procedures during processing, storage
and distribution. Therefore, treatments should be designed to provide an adequate margin of
safety against microbiological risk of food poisoning and food spoilage throughout shelf life,
thus leading to safer products with improved shelf life and quality. According to the US
Center for Science in the Public Interest (CSPI), seafood is more likely to cause food-borne
illness than any other category of food product. This is mainly caused by biological hazards
contamination (which includes pathogenic bacteria, viruses, parasites and biotoxins) due to
improper handling and storage, and by naturally occurring chemical toxins. Furthermore,
besides the low level of initial contamination, bacteria can grow and multiply easily,
producing compounds responsible for fishy odours and flavours, and discolorations
associated with stale seafood. In addition, if pathogen bacteria exist, they can multiply and
cause illness to the consumers. In Table 1, some of the most important bacterial hazards are
presented.
Aquatic environment is the natural habitat of Plesiomonas shigelloides, Vibrio and
Aeromonas species (indigenous bacteria), which are pathogenic to human and animals. Due to
the selective effect of water temperature, the microflora of tropical waters differs from the
temperate waters. In temperate waters psychotropic bacteria, Gram-negative (genera
Pseudomonas, Moraxella, Acinetobacter, Shewanella and Flavobacterium) are the typical
microorganisms present. In the warmer temperate waters mesofilic bacteria can be isolated.
Gram-positive and enteric bacteria are characteristic of tropical waters. Those
microorganisms should be the natural microflora of seafood.
Specific properties of some microorganisms, such as ubiquity and capacity to survive
under chilling temperatures (for example, Listeria) and even under freezing temperatures (for
example, E. coli), make them dangerous contaminants. So, it is of great importance, the
application of adequate processes that allow the conveniently inactivation of bacteria.
Different prevention and control measures have diverse impact in reducing the bacterial level.
Depended on the food product and bacterial contamination, the appropriate control process
should be carefully chosen.
Biological Hazards and Natural Antimicrobials for Seafood Preservation 277
Table 1. Bacterial hazards in fish and seafood products

(adapted from Ghanbari et al. [5])
Bacterial Product identified

Aeromonas spp. Fish, shellfish
Clostridium botulinum Spores on surface, in intestine, on gills (trout, herring, salmon);
Type E vacuum packaged smoked fish products, cans, fermented fish,
salted fish
Cl. perfringens Cod, tuna salad, boiled salmon
Escherichia coli Fresh fish, tuna paste, salted salmon roe, processed seafood
Listeria monocytogenes Ubiquitous, 3-10% human carriers; rarely in seawater or seawater
fish, more frequently in freshwater and aquaculture fish, cold
smoked products, salted fish products, hot smoked products, raw
fish, prawns, mussels, oysters
Salmonella spp. In intestine (tilapia and carp); prawns, mollusks, alaska pollack; eel
and catfish, smoked eel, smoked halibut, dried anchovy
Staphylococcus aureus Contamination from infected persons, fresh fish and fish fillets
(Cynoscion leiarchus), smoked fish
Vibrio Shellfish, crustaceans on the skin, gills, intestine, fish-balls, fried

parahaemolyticus mackerel (Scomber scombrus), tuna (Thunnus thynnus), and
sardines (Sardina pilchardus)
V. cholera Serovar O1 Prawns, shellfish, squid, seafood, uncooked fish marinade seviche
and O139 (Cilus gilberti)
Natural Antimicrobials
Several traditional preservation techniques, such as heat treatment, salting, acidification,

freezing, irradiation and drying have been used in the food industry to control
microorganisms growth. Additionally, a wide range of food grade chemicals has been added
during food manufacture to extend shelf-life by stabilizing chemical change or by preventing
or inhibiting microbial growth.
Since the discovery of penicillin, antimicrobials have made significant contributions to
human health. They are commonly considered in a therapeutic approach; however,
antimicrobials have a much wider use than therapeutics such as agriculture and food
industries. In food industries, antimicrobials are used along the food chain to increase the
safety and quality of the final product. They are used to clean, sanitize or disinfect by
reducing the levels of microorganisms on the environment and in human surfaces thus
preventing cross-contaminations. They are also commonly applied in food formulations to
prevent food spoilage and control pathogens growth [6]. However, during the last decade,
several studies indicate that the intensive use of synthetic antimicrobials is unsafe, mostly due
to a continued rise of antibiotic-resistant infections [7]. Therefore, together with the
consumers conscience of natural products as a safer and healthier option, industries face the
challenge of finding new safer natural antimicrobials with a broad spectrum of antimicrobial
activity. Nowadays, there are already available a variety of natural antimicrobials from
different sources, that may be used in food industry and are classified by source. Several new
natural active agents from different sources such as plants, marine organisms, animals and
microorganisms were already described and characterized presenting strong antimicrobial
properties. A list of natural antimicrobial products, their sources, and the susceptible
microorganisms will be discussed next.
SOURCES OF NATURAL ANTIMICROBIAL ACTIVE COMPOUNDS

APPLIED TO SEAFOOD PRODUCTS
Microorganisms
Since the discovery of penicillin, microorganisms have been one of the main targets of
research for antimicrobial compounds. In Table 2, a list of microorganisms that are producers
of important antimicrobial compounds is presented.
Table 2. Natural antimicrobial products obtained from microorganisms and target

microorganisms (adapted from Choudhary et al. [8] and Gyawali et al. [9])
Organism Antimicrobial class Susceptible microorganisms

Bacillus polymyxa Polypeptides Gram-negative bacteria
Bacillus subtilis Phospholipids Gram-positive bacteria
Fischerella Ambiguine I Isonitriles Bacteria
Micromonospora Macrolides and Peptides Bacteria
Nostoc commune Noscomins Bacteria
Nostoc flagilliforme Nostoflans Virus
Nostoc muscorum Phenolic compounds Bacteria
Oscillatoria nigrovirdis Viridamine A Protozoa
Penicillium B-lactams Gram-positive bacteria
Salinispora arenicola Macrolides Bacteria
Streptomyces erythreus Macrolides Bacteria
Streptomyces griseus Aminoglycosides Bacteria
Streptomyces lincolnensis Lincomycins Bacteria
Streptomyces mediterranei Rifamycins Bacteria
Streptomyces nodosus Polyenes Fungi
Streptomyces noursei Macrolides Fungi
Tetracyclines Bacteria
Streptomyces Pyrrolosesquiterpenes Bacteria
Quinones and Microlides Bacteria and fungi
Streptomyces venezuelae Chloramphenicols Bacteria
Streptomyces psommoticus Polyketides Bacteria and fungi
Lactic acid bacteria
Clostridium spp., Listeria.
Lactococcus lactis Nisin
monocytogenes
Strains of Pediococcus L. monocytogenes, Enterococcus
acidilactici and P. Pediocin faecalis, Staphylococcus aureus, C.
pentosaceus perfringens

Streptomyces natalensis Natamycin Fungi
Lactobacillus curvatus
Lactocin E. coli strains
CRL705
B. cereus, B. subtilis, L. ivanovii,
L. acidophilus Acidophilin
S. aureus
L. monocytogenes, S. aureus, B.
L. bulgaricus Bulgaricin
subtilis, Helicobacter pylori
L. monocytogenes, Clostridium
L. helviticus Helveticin
botulinum
L. monocytogenes, S. aureus, S.
L. plantarum Plantaricin
Typhimurium, E. coli
L. monocytogenes, S. aureus, E.
Lactobacillus reuteri Reuterin
coli O157:H7, C. jejuni
Although none of the antibiotics discovered since penicillin, have had the same notoriety,
many are just as important and many are more commonly used than penicillin. Most have
been bacterial metabolites, or more specifically metabolites from Actinomycetes, which are
mycelial producing bacteria that were once thought to be fungi [10-11].
The genus Streptomyces has afforded numerous metabolites with activity against a wide
range of bacteria, even bacteria that are not affected by penicillin. For instance, it was found
to be effective against Mycobacterium tuberculosis, Klebsiella pneumonia, Shigella
gallinarum and Salmonella spp., one of the causes of food poisoning, among others [12-14].
With the intensive use of antimicrobials, resistant microbes have arisen and the widely
used antimicrobials became no longer efficient in many situations. Synthetic alternatives have
been developed to counteract resistant bacteria and as it presents lower costs and high
production rates, they became widespread not only in pharmaceutical industries but also in
food industries for preventing food spoilage. Nevertheless, much as been discussed as
concerns to synthetic antimicrobials safety since it continuously give rise to multi-resistant
microbes and may promote undesirable side effects [15].
As referred before, the growing consumers demand for foods without chemical
preservatives has focused efforts in the discovery of new natural antimicrobials. Among
alternative food preservation strategies, particular attention has been paid to biopreservation
techniques. Biopreservation, can be defined as the extension of shelf-life and food safety by
the use of natural or controlled microbiota and/or their antimicrobial compounds. Some
bacteria, such as those referred to as lactic acid bacteria (LAB) and which may be naturally-
occurring either in the initial microflora of fermented or other foods (e.g., vacuum packaged
meats) or added as starter cultures, maybe used because they can inhibit the growth of
spoilage and pathogenic bacteria by depleting nutrients and oxygen and producing inhibitory
metabolic substances. In fact, they may act as powerful competitors to contaminating spoilage
microorganisms, by producing a wide range of antimicrobial metabolites such as organic
acids, diacetyl, acetoin, hydrogen peroxide, reuterin, reutericyclin, antifungal peptides, and
bacteriocins [5, 16-17]. LAB constitute a large group of non sporulating Gram-positive,
catalase and oxidize negative rods and cocci that produce lactic acid as the major metabolite
of the carbohydrate fermentation. LAB are anaero-aerotolerant and generally have complex
nutritional requirements especially for amino acids and vitamins. Certain LAB are able to
grow at refrigeration temperatures and are tolerant to modified-atmosphere packaging, low

pH, high salt concentrations, and in the presence of certain additives such as lactic acid, acetic
acid, and ethanol. Because of these benefits, LAB can be used as protective cultures to restrict
the growth of undesired organisms such as certain spoilage and pathogenic bacteria, with the
subsequent benefits in terms of food safety [18].
Table 3. Natural antimicrobial products obtained from plants and

target microorganisms (adapted from Choudhary et al. [8] and Gyawali et al. [9])
Plant Antimicrobial class Susceptible microorganisms

Abrus schimperi Quinones Leishmania donovani
Aeglemarmelos Terpenoids Fungi
Allium cepa Sulfoxides Bacteria, Candida
Allium sativum Sulfoxides and terpenoids Bacteria, fungi, protozoa
Aloe barbadensis, Aloe vera Complex mixture Corynebacterium, Salmonella
Aloysiatri phylla Terpenoids M. tuberculosis, S. aureus
Anacardium pulsatilla Polyphenols P. acnes, bacteria, fungi
Archidendron Jiringa Lectins Bacteria, fungi
Arctium lappa Terpenoids and Polyacetylenes Bacteria, fungi, viruses
Artemisia dracunculus Terpenoids and Polyphenols Viruses
Berberis vulgaris Alkaloids Bacteria and protozoa
Camellia sinensis Flavonoids and Terpenoids Bacteria, fungi, protozoa, virus
Cannabis sativa Alkaloids Bacteria and viruses
Capsicum annuum Terpenoids Bacteria
Carum carvi Coumarins Bacteria, fungi, viruses
Cassia angustifolia Anthraquinones S. aureus
Centella asiatica Terpenoids M. leprae
Citrus sinensis Terpenoids Fungi
Citrus paradisa Terpenoids Fungi
Croton pullei Alkaloids Bacteria, fungi
Crotolaria pallida Peptides Bacteria
Coccinia cordifolia Flavonoids Bacteria
Coriandrum sativum Essential oils Bacteria, fungi
Curcuma longa Terpenoids Bacteria, protozoa
Dorstenia barteri Phenolics Bacteria, fungi
Galium odoratum Coumarins Bacteria, fungi, protozoa, virus
Helichrysum gymnocomum Phenolics Bacteria
Hydrastis canadensis Alkaloids Bacteria, protista, plasmodium
Lawsonia Quinones M. tuberculosis, Bacteria
Mahonia aquifolia Alkaloids Plasmodium, trypanosomes
Malus sylvestris Flavonoid derivatives Bacteria, fungi, protozoa
Medicago sativa Flavonoids Gram-positive bacteria
Mentha piperita Terpenoids Bacteria, fungi, protozoa
Mentha pulegium Essential oils Gram-positive bacteria
Momordica charantia Essential oils Bacteria, fungi, protozoa
Ocimum basilicum Terpenoids Salmonella, Bacteria
Oleaeuropaea Aldehydes Bacteria, fungi, protozoa
Plant Antimicrobial class Susceptible microorganisms

Papaver somniferum Alkaloids and others Bacteria, fungi, protozoa
Panax ginseng Polyacetelenes Bacteria, fungi
Peganum harmala Alkaloids Bacteria, fungi
Phyllanthus mullerianus Essential oils Bacteria, fungi
Piper bte, Piper nigrum Essential oils and Alkaloids Bacteria, fungi, protozoa
Quercus rubra Polyphenols and Flovonoids Bacteria, fungi
Ranunculus laetus Coumarins Bacteria
Ranunculus scleratus Flavonoids Salmonella, Agrobacterium
Rauvolfia serpentina Alkaloids Bacteria, fungi, protozoa
Rhamnus purshiana Polyphenols and Anthaquinones Virus, bacteria, fungi
Ricinus communis Essential oils Bacteria, fungi, protozoa
Rosmarinus officinalis Terpenoids Bacteria, fungi, protozoa
Santolinachamae cyparissus Essential oils Candida, Schitosoma
Satureja montana Essential oils and Terpenoids Bacteria, fungi, protozoa, virus
Schinus terebinthifolius Terpenoids Bacteria, fungi, protozoa
Thymus vulgaris Terpenoids and polyphenols Viruses, bacteria, fungi
Tussilago farfara Essential oils and Flavonoids Bacteria, fungi, protozoa
Vicia faba Thionins Bacteria
Withania somnifera Lactones Bacteria, fungi
The genera comprise the LAB are at its core Lactobacillus, Lactococcus, Leuconostoc,
Pediococcus, Streptococcus as well as the more peripheral Aerococcus, Carnobacterium,
Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, Weissella and
Bifidobacterium genus [5, 16-17]. As for seafood preservation, numerous studies have been
performed in order to evaluate LAB efficiency extending the shelf-life of these products.
Montiel and co-workers [19] evaluated the efficiency of reuterin in inhibiting pathogenic
microorganisms growth in cold-smoked salmon. Reuterin or b-hydroxypropionaldehyde (b-
HPA) is an intermediate compound produced by some strains of Lactobacillus reuteri during
the anaerobic metabolism of glycerol and has presented high antimicrobial activity against a
broad spectrum of food borne pathogens, including Listeria monocytes growth at moderate
temperature abuse (8C), revealing to be a suitable option for application in fish products.
Furthermore, cold-smoked salmon treated with divergicin M35-producing Carnobacterium
divergens M35 was effective inhibiting the Listeria monocytogenes growth, increasing the
shelf-life of this ready-to-eat product [20]. Treating catfish fillets with of 0.50% sodium
acetate, 0.25% potassium sorbate with 2.50% lactic acid culture completely inhibited growth
of Gram-negative bacteria, improving catfish odor and appearance during 13 days of storage
[21].
The use of LAB in the fish industry is not extensively developed, except in Asia for
preparation of fish sauces and traditional food with fermented mixture of fish and vegetable.
Nevertheless, many studies point to the success of LAB for preserving seafood products.
Besides this, the application of LAB is still in its early stages compared to dairy products
[17].
Plants
There are described around 12,000 plant secondary metabolites of antimicrobial

importance including phenols, quinones, flavonoids tannins, terpenoids, alkaloids and other
mixtures [22]. Although many plants continue to be used due to traditional costumes without
scientific basis of their properties, some studies have already described the principal
molecules involved in antimicrobial activities and their mechanisms of action (Table 3).
Plant-derived compounds are mostly secondary metabolites, most of which are phenols or
their oxygen-substituted derivatives. These secondary metabolites possess various benefits
including antimicrobial properties against pathogenic and spoilage microbes [9, 23]. Phenolic
compounds comprise the main antimicrobial components in spices and their derived essential
oils and extracts, and include, for instance, cinnamic aldehyde from cinnamon; thymol from
thyme and oregano; eugenol from clove, allspice and cinnamon; carvacrol from oregano and
anethole from anise [24]. Nevertheless, the antimicrobial activity detected in in vitro studies
is not always reflected in food products due to the matrix complexity. For instance, the high
fat content of some fish, reduces the antibacterial effect of essential oils against various
microorganisms [25]. Therefore, the evaluation of the antimicrobial properties in food
systems is required in order to achieve the most effective antimicrobial specific for each food
product. As for fish and seafood products, several studies with natural products from plants
have been assessed and are presented in Table 4.
Essential Oils
The antimicrobial activity of essential oils has been widely evaluated including against
several foodborne pathogens such as E. coli, S. Typhimurium, S. aureus, L. monocytogenes,
Campylobacter and others [37].
Recently, Donato and collaborators [38] evaluated the antibacterial activity of a Tuscan
plant and found it to be active against E. coli O157, S. enteritidis, S. typhi, Yersinia
enterocolitica and L. monocytogenes, all of which have great significance in foodborne
infections. Abdollahzadeh et al. [28] tested thyme essential oils and bactericins to control L.
monocytogenes in minced fish meat and the combinations of thyme essential oil at 1.2% with
nisin at 500 or 1000 IU/g proved to be an efficient treatment to inhibit L. monocytogenes
growth in minced fish. The essential oil of thyme and oregano at 0.05% (vol/vol) were also
evaluated for their ability to slow the process of fish spoilage. Fish treated with these oils
were still fit for human consumption after 33 days of storage [39].
Table 4. Overview of studies testing the antibacterial activity of essential oils (EO) and
plant extracts or their components in seafood (Adapted from Pezeshk et al. [24])
Seafood EO or plant extract Target microorganism References

TVC*; Pseudomonas spp., lactic
Rainbow trout (Oncorhynchus
Oregano EO acid bactria, H2S producing [26]
mykiss) fillet
bacteria
Mediterranean swordfish
Thyme oil TVC*, H2S producing bacteria [27]
(Xiphias gladius) fillets
Minced fish meat
Thyme oil Listeria monocytogenes [28]
Silver carp
Tea polyphenol TVC* [29]
(Hypophthalmichthys molitrix)
Seafood EO or plant extract Target microorganism References

Thymol extract Fish spoilage mesophilic
Fish hamburger
Grapefruit seed extract microorganisms [30]
Lemon extract psychrotrophic bacteria
Sea bass (Dicentrarchus
Lactic acid bacteria, psychrophilic
labrax) slice Lemongrass EO
bacteria, spoilage microorganisms
[31-33]
Rainbow trout (Oncorhynchus Turmeric extract
mykiss) TVC*, psychrotrophic count
Shallot extract
Rainbow trout (Oncorhynchus
Cinnamon oil TVC*, psychrotrophic count [34-35]
mykiss)
Crucian carp (Carassius Tea polyphenols
TVC* [36]
carassius) Rosemary extract
*TVC Total viable counts
Plant By-Products
In food industry, many by-products such as fruit pomace, seeds, peels, pulps, unused
flesh, and husks are often generated. Most of the times, these products are under-valorised
and are considered waste. Nevertheless, is already demonstrated that these products have an
important biological value, namely as sources of bioactive compounds, including
antimicrobial compounds [9]. In fact, Ayala-Zavala et al. [40] reported that by-products can
have a similar or even higher proportion of bioactive compounds than the usable parts of
produce. Table 5 presents a list of plant by-products with antimicrobial activity and the
specific target organisms.
Table 5. Natural antimicrobial products obtained from plants by-products and target
microorganisms (adapted from Gyawali et al. [9])
By-product Major component Susceptible microorganisms

Pomegranate fruit L. monocytogenes, S. aures, E. coli,
Phenolics and flavonoids
peels Yersinia enterocolitica, P. fluorescens
Pomegranate juice MRSA ATCC 43300 and E. coli ATCC
Phenolics, flavonoids, tannins
byproducts 35218
Apple peels Phenolic compounds S. aureus P. fluorescens
Almond skin extracts Polyphenols S. aureus, L. monocytogenes
Phytochemical including
Coconut husk S. aureus, L. monocytogenes, V. cholera
phenolics and tannins
S. aureus, E. coli, L. monocytogenes,
Green tea waste Tannins
Bacillus coagulans, Shigella flexneri
Acorn, chestnut, S. aureus, E. coli, L. monocytogenes, B.
Tannins
persimmon hull coagulans, S. flexneri
Metabolites such as fatty S. aureus, S. epidermidis, Micrococcus
Tomato seeds acids, carotenoids, saponins, luteus, E. faecalis,
phenolic compounds B. cereus, Candida albicans
Polyphenolic compounds such
S. aureus, P. aeruginosa, E. coli, C.
Quince fruit peel as chlorogenic acid, catechin,
albicans
quercetin and kaempferol
Chlorogenic, caffeic, gallic, Bacteriostatic effect on E. coli and S.
Potato peels
and protocatechuic acids Typhimurium
By-product Major component Susceptible microorganisms

Antioxidants such as phenolic
Walnut green husk compounds tocopherols, squalene, S. aureus, B. cereus, B. subtilis
and different sterol fractions
Phytochemicals: flavonones,
Peels, seeds, and pulp E. coli O157:H7, S. Typhimurium,
polymethoxylated flavones,
of mexican lime Shigella sonnei
tannins
S. aureus, Salmonella, Enterococci,
Phenolic acids, flavonoids, Total aerobic mesophilic and
Grape pomace
stilbenes psychrotrophic bacteria, yeasts and
molds
Phenolic compounds including E. coli O157:H7, S. enterica, L.
Olive pomace oleocanthal, deoxyloganic acid monocytogenes,
lauryl ester and S. aureus
Beet root pomace Phenolics, flavonoids betacyanins, S. aureus, B. cereus E. coli, P.
extract betaxanthins aeruginosa
Gram-positive (B. cereus, S. aureus,
Phenolics, flavonoids,
Enterococcus faecalis) and Gram-
Buckwheat hull antioxidants comprising
negative bacteria (Salmonella
extracts tocopherols, rutin, quercetin
choleraesuis, E. coli and Proteus
derivatives
mirabillis)
Legume hulls (Vigna
radiate, Cicer Polyphenolic compounds,
B. cereus, S. aureus
arietinum and flavonoids
Cajanus cajan)
Phenolic compounds such as
catechins, epicatechin,
Grapefruit seed
epocatechin-3-O-gallate, dimeric, Pseudomonas spp
extracts
trimeric and tetrameric
procyanidins
B. subtilis, E. coli, L.
Phenolic compounds (sinapic acid monocytogenes,
Brassica juncea L.
and several sinapoyl conjugates) Pseudomonas fluorescens, and S.
aureus
Polyphenols such as flavan-3-ols,
Total viable count, coliform, E. coli,
Coffee pulp hydroxycinnamic acids, flavonols
and fungal count
and anthocyanidins
Macrofungi
Less intensively investigated organisms such as the macrofungi seem greatly promising
in terms of compounds with potential biological activities. In recent decades, interesting
compounds of different biogenetic origins have been isolated from Basidiomycota and were
found to have antibacterial, antifungal, phytotoxic, cytostatic, antiviral, and other
pharmacological activities [41]. Mushrooms are rich sources of natural antibiotics. For
instance, the cell wall glucans are not only well-known for their immunomodulatory
properties, but also for their many excreted secondary metabolites of the mycelium to combat
bacteria and fungi. A well-known example of diterpenoid antibiotic pleuromutilin has been
isolated previously which was derived from the fungus Clitopilus passeckerianus (former
name: Pleurotus passeckerianus) (Table 6). Further research led to the discovery of
retapamulin, which is a C14-sulfanyl-acetate derivative of pleuromutilin with improved
pharmacological properties as an antibiotic drug [41].
Table 6. Natural antimicrobial products obtained from macrofungi (Adapted from

Choudhary et al. [8] and De Silva et al. [41])

Fomitopsis lilacinogilva,
Grifolin, pleuromutilin,
Psathyrella spp., Ramaria spp.,
striatins A, B and C, S. aureus, B. cereus,
Hohenbuehelia spp., Agaricus
Oudemansin, Scorodonin, L. monocytogenes, E. coli
spp., Lentinus spp.,
Ganomycins, Retapamulin
Strobilomyces spp.
Lycoperdon perlatum,
S. aureus (ATCC 25923);
Cantharellus cibarius, Clavaria
Total phenols, flavonoid and B. subtilis (ATCC 6633);
vermiculris, Ramaria Formosa
ascorbic acid E. coli (ATCC 25922),
Quel, Marasmius oreades,
P. aeruginosa (ATCC 27853)
Pleurotus pulmonarius
Fatty acids, b-carotene- Micrococcus luteus,
Agaricus spp. linoleic acid, phenols, and Micrococcus flavus, B.
flavonoids subtilis, B. cereus
Lactarius deliciosus, Lactarius Total phenols, flavonoids,
B. cereus, B. subtilis, P.
piperatu, Sarcodon imbricatus, ascorbic acid, b-carotene,
aeruginosa, E. coli
Tricholoma portentosum lycopene
Flavonoid components,
Lentinula edodes palmitic acid, linoleic acid, Micrococcus luteus, B. cereus
ergosterol
C. sinensis, P. australis Polysaccharides, triterpenes B. subtilis, S. epidermidis
P. aeruginosa, S. Enteritidis,
Laetiporus sulphureus (Bull.)
Phenolics and flavonoids E. coli, Y. enterecolitica, S.
Murrill
aureus, B. cereus
Diterpenoid pleuromutilin; Gram-positive and Gram-
Clitopilus passeckerianus
retapamulin negative bacteria
Ganomycin A and ganomycin Gram-positive and Gram-
G. pfeifferi
B negative bacteria
Multidrug-resistant Gram-
Coprinus sp Coprinol
positive bacteria
Corynebacterium xerosis; S.
Coprinopsis micaceus Micaceol
aureus
Animal
Along with plants, antimicrobials from animal sources are the most explored and used in
food industries. Important molecules such as lactoferrin, lysozyme, Protamine, Pleurocidin,
lactoperoxidase and chitosan have been applied successfully in food matrixes [42]. In the
Table 7 a list of antimicrobial molecules from animal sources is presented.
Table 7. Natural antimicrobial products obtained from animal sources

(adapted from De Silva et al. [41] and Gyawali et al. [9])

Magainins, dermaseptins,
Amphibians Bacteria, fungi, protozoa
Buforins and Brevinins
Cattle Indolicidins Bacteria
Hemipteran Thanatins Bacteria, fungi
Human Histatins Bacteria, fungi
Pig and insects Cecropins Bacteria, fungi
Mammals and insect Defensins Bacteria, fungi, virus
Pig Protegrin I Bacteria, fungi
Cystatins, avidins, G2 Globulins Bacteria, fungi
Egg
Lysozyme Bacteria
Honey Complex mixture Bacteria, fungi
Milk Lactoferrins Bacteria, fungi
Enterobacter sakazakii ATCC 12868,
E. coli DPC5063, S. aureus,
Milk protein Casein and whey
L. monocytogenes, S. typhimurium,
B. subtilis
Exoskeletons of
E. coli, S. aureus, Pseudomonas spp.,
crustaceans and Chitosan
E. coli, and L. monocytogenes
arthropods
B. subtilis, L. monocytogenes, S. aureus
ATCC 6538, S. aureus KCTC 1916, and
Fish and shellfish Lipids Enterobacter aerogenes, E. coli, E. coli
O157:H7, P. aeruginosa, S. enteritidis,
and S. typhimurium
Raw milk, colostrum,
Salmonella, E. coli, S. aureus,
saliva and other Lactoperoxidase
L. monocytogenes, Y. entericolitica
biological secretions
Myeloid cells and
mucosal tissues of L. monocytogenes, E. coli O157:H7,
Pleurocidin
many vertebrates and pathogenic fungi
invertebrates
L. monocytogenes, total bacteria,
Salmon Protamine
coliforms
Antimicrobials derived from animals are compounds like proteins and enzymes that are
isolated from animals or are animal derived. Lysozyme has been investigated exhaustively as
a preservative in vegetables, meat and seafood products in Japan and has shown effectiveness
in seafood products such as sushi. Other studies evaluated the antimicrobial potential of
lysozyme as a gelatin dip which revealed to be effective in retarding the microbial growth,
extending the shelf-life of seafood products [43].
Among antimicrobial compounds from animal sources, chitosan is probably the most
recent finding. Chitosan (poly b-(1-4)N-acetyl-d-glucosamine), a deacetylated form of chitin
can be obtained from crustacean shells (crabs, shrimp and crayfishes) and from some fungi
(Aspergillus niger, Mucor rouxii, Penecillium notatum) [44]. Chitosan based polymeric
materials can be formed into fibers, films, gels, sponges, beads or even nanoparticles.
Chitosan films have shown potential to be used as a packaging material for the quality
preservation of a variety of food. Besides this, chitosan has widely been used in antimicrobial
films to provide edible protective coating, in dipping and spraying for the food products due
to its antimicrobial properties [45].
In seafood experiments, chitosan has presented some promising antimicrobial capacity.
Cao et al. [46] evaluated the antimicrobial potential of chitosan in filleted tilapia and showed
wide-spectrum antibacterial activity against several bacteria extending the shelf-life for 12
days (6 days more than the non-treated fillets). Tsai et al. [47] reported that chitosan has
antimicrobial activity in seafood against A. hydrophila, L. monocytogenes, S. typhimurium,
etc. Pretreatment of fish fillets (Oncorhynchus nereka) with 1% chitosan solution for 3 h
retarded the increase in the volatile basic nitrogen content, as well as the counts for
mesophiles, psychrotrophs, coliforms, Aeromonas spp., and Vibrio spp. The shelf-life was
consequently extended from 5 to 9 days. Lpez et al. [48] studied the effect of chitosan
gelatin blends as a coating for fish patties and showed a difference of around 2 log cycles
between the control and the coated batches for total bacterial counts, Pseudomonas and
enterobacteria at 811 days of storage. A more recent study evaluated the effectiveness of
chitosan coatings chilled storage of shrimp. The chitosan coating solution was effective in
maintaining the shrimp biochemical and sensorial quality, increasing the shelf-life in 5 days
[49]. These results indicate that chitosan, as a natural preservative can have further
application in fish and seafood products.
Algae
In recent decades, marine resources have afforded numerous unique chemical structures
with a vast array of bioactivities including antimicrobial. Within marine organisms, algae
have shown promising antimicrobial properties and many of them have the advantage of
being edible, therefore, the addition as food preservers can be much easier. In order to defend
themselves, algae produce bioactive compounds such as polysaccharides, antioxidants,
carotenoids, dietary fiber, protein, essential fatty acids, vitamins, and minerals making them
interesting candidates to be used as food supplement, source of vitamins, food additives and
antimicrobials [50-54].
Secondary metabolites of seaweeds with antimicrobial potentials include fatty acids,
halogenated compounds such as haloforms, halogenated alkanes and alkenes, alcohols,
aldehydes, hydroquinones and ketones, carbonyls, sulfur-containing heterocyclic compounds
(sulfated polysaccharides) and phlorotannins [55]. Compounds such as sterols, heterocyclic
and phenolic compounds, including quinones, flavones, flavonoids, flavonols and tannins
have exhibited some antibiotic activity with possible exploitation. Most of the compounds
responsible for the antimicrobial activity of algae are primarily phenolic and polysaccharide
components and their mechanisms of action could be cytostatic (growth inhibition of the
microorganism) or cytotoxic (death of the microorganism) [56]. The mechanism of
polyphenols toxicity against microbes may be related to inhibition of hydrolytic enzymes
(proteases and carbohydrolases) or other interactions to inactivate microbial adhesins, cell
envelope transport proteins, non-specific interactions with carbohydrates, etc. [57].
As respect to polysaccharides, their antimicrobial capacity is attributed to the presence of
glycoprotein-receptors in the cell-surface of bacteria which is capable of recognizing and
binding to the charged compounds associated with the polysaccharides molecules [58].
Recent studies suggest that the antimicrobial activity of polysaccharide molecules is related to
their molecular weight, charge density, sulfate content (in the case of sulphated
polysaccharides) and to their structural and conformational aspects.
The incorporation of algae as preservatives in food products can open opportunities for
extending food shelf-life by reducing the proliferation of spoilage and pathogenic bacteria
offering an important alternative to synthetic substances. Table 8 shown compounds isolated
from algae that evidenced antimicrobial activity against pathogenic and spoilage bacteria.
Table 8. Algae extracts tested against food pathogenic and spoilage bacteria (adapted
from Amaro et al. [59], Dominguez [53] and Gyawali et al. [9])
Main compounds /
Algae species Target bacterial species References
Extracts
Staphylococcus aureus
Listeria innocua
Bacillus subtilis
Esterococus faecallis
Ulva lactuca Semi-purified fractions [60]
Cronobacter sakazakii
Pseudomonas aeruginosa
Escherichia coli
Salmonela thyphimurium
Hemanthalia Listeria monocytogenes
Polyphenols [61]
elongata Enterococcus faecalis
Salmonella abony
Bacillus subtilis
Escherichia coli
Ulva rigida Polyphenols [62]
Listeria monocytogenes
Enterococcus faecalis
Bacillus subtilis
Sulphated Pseudomonas aeruginosa
Gracilaria ornata polysaccharide Escherichia coli [63]
Enterobacter aerogens
Salmonela choleraesuis
Salmonella typhi
Bacillus subtilis
Escherichia coli
Sargassum wightii Pseudomonas aeruginosa
and Turbinaria Polyphenols Aeromonas hydrophila [64]
ornata Proteus vulgaris
Klebsiella pneumoniae
Shigella flexneri
Main compounds /
Extracts
Salmonella enteretidis
Chaetomorpha aerea Sulphated galactan [65]
Bacillus subtilis
Micrococcus luteus
Kappaphycus alvarezii Sulphated Escherichia coli -
and polysaccharides and (12 antibiotic resistant [66]
Padina boergessenii polyphenols strains)
Vibrio parahaemolyticus
Ulva fasciata Labdane diterpenoids Vibrio alginolyticus [67]
Vibrio vulnificus
Himamthalia elongata
Laminaria saccharina Pseudomonas aeruginosa
Laminaria digitata Listeria monocytogenes
Polyphenols [68-69]
Enteromorpha Enterococcus faecalis
Spirulina sp. Salmonella abony
Palmaria palmata
Fatty acids, alkanes, Staphylococcus aureus
Haematococucs pluvialis [70]
polyphenols Escherichia coli
Bacillus subtilis
Micrococcus luteus
Odonthalia corymbifera Bromophenols [71]
Proteus vulgaris
Salmonella typhimurium
Escherichia coli
Gelidiella acerosa
Gracillaria edulis Staphylococcus aureus
Turbinaria conoides Bacillus cereus
Padina gymnospora Vibrio vulnificans
Chondrococcus hornemanni Polyphenols Salmonella typhi [72]
Hypnea pannosa Listeria monocytogenes
Dictyota dichotoma Entereococcus faecalis
Jania rubens E. coli
Haligra sp.
Sargassum sp. Unknown Escherichia coli [73]
Bacillus subtilis
Escherichia coli
Indolic compounds,
Dunaliella salina PUFAs, b-ionone, [74]
Candida albicans
neophytadiene
Aspergillus niger
Sphaerococcus Pseudomonas aeruginosa
coronopifolius Bromoditerpenes Escherichia coli [75]
Asparagopsis armata E. coli
Sphaerococcus Methanolic extracts Bacillus subtilis [76]
coronopifolius
Main compounds /
Extracts
MRSA
Listonella anguillarum
Phaeodactylum tricornutum Eicosapentaenoic acid [77; 87]
Lactococcus garvieae
Vibrio spp
Short-chain fatty acids
Escherichia coli,
Haematococcus pluvialis (butanoic acid and [70]
methyl lactate)
Unsaturated, saturated
Skeletonema costatum Vibrio spp. [78]
long chain fatty acids
Pseudomonas sp.
Aeromonas sp.
Euglena viridis Organic extracts Edwardsiella sp. [79]
Vibrio sp.
Escherichia coli
S. costatum Extra-metabolites Listeria monocytogenes [80]
Chlamydomonas reinhardtii
Chlorella spp.
Chroococcus dispersus
S. aureus spp.
Anacystis ridulans Supernadant;
Bacillus subtilis
Oscillatoria splendida Methanolic and [81]
E. coli
Phormidium sp. hexane extracts
Salmonella typhi
Nostoc muscorum
Scenedesmus obliquus
Oocystis sp.
Colpomenia sinuosa B. subtilis
Dictyota dichotoma S. aureus
Dictyota dichotoma var. Dichloromethane Enterobacter aerogenes
[82]
implexa extrats E. coli
Petalonia fascia Proteus vulgaris
Scytosiphon lomentaria Salmonella typhimurium
Chloroform/methanol
Gracilariopsis longissima Vibrio spp. [83]
extract
Phytochemicals such E. coli
as saponins, tannins, S. aureus
Chlorococcum humicola carotenoids, S. Typhiumurium [84]
flavonoids, alkaloids, P. aeruginosa
glycosides V. cholerae
Phytol, fucosterol,
neophytadiene or E. coli,
Himanthalia elongata [85]
palmitic, palmitoleic S. aureus
and oleic acids
Lipophilic compound
Asparagopsis taxiformis
(pyrrole-2-carboxylic
Laurencia ceylanica
acid, pentadecanoic Pathogenic Vibrio strains [86]
Laurencia brandenii
acid and octadecanoic
Hypnea valentiae
acid
Chlamydomonas reinhardi
Chlorophyll
Chlorella vulgaris B. subtilis [87]
derivatives
Scendesmus quadricauda
Main compounds /
Extracts
Ecklonia cava
E. kurome
S. aureus
E. stolonifera
MRSA
Eisenia aborea Phlorotannins [88]
Salmonella spp.
Eisenia bicyclis
E. coli
Ishige okamurae
Pelvetia siliquosa
Many compounds with antimicrobial activity were already identified and isolated,
however, for our knowledge, none have been applied in food industries, with the purpose of
maintaining food quality regardsing microorganism growth control. Some of the constraints
with the use of natural antimicrobial compounds are related with the method of application of
the compound since natural compounds frequently present odor and/or flavors [89].
Therefore, some strategies for the application of the natural product have to be developed.
EMERGING NATURAL ANTIMICROBIAL COMPOUNDS FOR SEAFOOD

PRESERVATION CASE OF PHENOLIC COMPOUNDS
Among all compounds isolated from natural sources, phenolic compounds exhibit an
interesting diversity of biological activities, such as antioxidant, antimicrobial, anticancer and
anti-inflammatory [90-92]. They represent one of the most diverse groups of secondary
metabolites found in edible plants being present in a wide variety of fruits, vegetables, nuts,
seeds, stems and flowers as well as tea, wine, propolis, honey and marine organisms like
algae [88, 93-95]. As referenced above (Table 8), algae are one of the major producers of
phenolic compounds and several of them have evidenced great antioxidant and antimicrobial
activities. Moreover, the brown algae are the only organisms that have capacity to produce
one specific type of phenolic compounds, the phlorotannins, which showed potential to act as
potential antimicrobial agents in the food industry [88]. Actually, between all bioactivities
evidenced, the phenolic compounds have exhibited interesting and potent antimicrobials
capacities against different spoilage and pathogenic microorganisms [96]. According with
this, the use of these compounds as antimicrobial agents in food products would provide
additional benefits, including dual-function effects of both preservation and delivery health
benefits.
More recently, the role of phenolic compounds as natural antimicrobials applied in fish
and seafood products are emerging field of study [97]. The use of tannic acid combined with
modified atmospheric packaging (MAP) exhibited a synergistic effect on the retardation of
microbial growth in striped catfish slices stored at the refrigerated temperature. This
combination comparing with control situation (kept in air without tannic acid treatment)
allowed to increase the shelf-life from 3 to 15 days, based on microbiological acceptability
limit (107 cfu/g) [98]. Other example of phenolic compounds application in seafood is ferulic
acid (FA) utilization in white shrimp (Litopenaeus vannamei) during iced storage. The shrimp
treatment with different FA solutions (1% and 2%) during 10 days, stored in ice, lead to an
inhibition of psychrophilic and mesophilic bacterial growth [99]. In addition, Nirmal and
Benjakul [100] verified that a catechin solution presents capacity to retard the growth of
psychrophilic bacteria and spoilage microorganisms including H2S-producing bacteria and
enterobacteriaceae in white shrimp (Litopenaeus vannamei) samples. In the same line, when
combined with MAP, Mentha spicata L. and Artemisia campestris extracts, which are
characterized by possessing high phenolic contents, had capacity to increase the sardine fillets
shelf-life of 10 (control) to 17 days, suggesting that these effects were mediated by the
presence of phenolic compounds [101]. In the same line, Kostaki and co-workers [102] using
thyme oil combined with MAP increased the shelf-life of sea bass from 6 to 17 days. The
antibacterial properties of thyme oil are attributed to its high phenolic content including:
carvacrol, thymol, p-cymene and -terpinene.
In fact, the phenolic compounds have evidenced great potential as natural antimicrobials
in different seafood products. They can be applied alone or combined with other processes,
resulting in the prevention of spoilage and thereby, extending the product's shelf-life.
Phenolic compounds demonstrate, therefore, great potential to be used with success, as
natural antimicrobials in food industry.
CONCLUSION
Fish and other seafood are an important part of a balanced diet and contribute to a good
nutritional status, manly due its high levels of many important nutrients that are not
commonly found in other food products. Nevertheless, the diverse nutrient composition of
seafood makes it an ideal environment for the growth and propagation of spoilage micro-
organisms and common food-borne pathogens.
Food additives are essential for preservation of ready-to-eat or minimal processed foods
in order to ensure food safety, playing an important role in preserving the freshness, taste,
appearance and texture of foods. A wide range of food grade chemicals can be added to
seafood products extending shelf-life. Nonetheless, consumers conscience of natural
products as a safer and healthier option led a demand by high quality food without chemical
additives. This situation led researchers and food processors to search for safer natural food
additives with a broad spectrum of antimicrobial activity.
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[102] Kostaki, Maria., Vasiliki, Giatrakou., Ioannis, N. Savvaidis. and Michael, G.
Kontominas. 2009. Combined Effect of Map and Thyme Essential Oil on the
Microbiological, Chemical and Sensory Attributes of Organically Aquacultured Sea
Bass (Dicentrarchus labrax) Fillets. Food Microbiology, 26(5), 475-82.
http://dx.doi.org/10.1016/ j.fm.2009. 02.008.
Chapter 17
HACCP ECONOMICS IN SEAFOOD

PROCESSING PLANTS
Aurora Zugarramurdi1,2,*, Mara Amelia Parin1

and Hctor Mateo Lupin3
1
College of Engineering, National University of Mar del Plata, Mar del Plata, Argentina
2
Scientific Research Commission of Buenos Aires Province (CIC),
Buenos Aires, Argentina
3
Southern Regional Centre, National Institute of Industrial Technology (INTI),
Former FAO (UN) Senior Officer, San Martin, Argentina
ABSTRACT
The economics related to HACCP implementation in actual seafood plants is
analysed as a function of quality cost changes. To evaluate the effectiveness of the
HACCP-based system implemented in fish processing plants, a mathematical quality cost
model developed for the food industry is applied to fish salting plants and compared with
experimental values obtained from industry.
Experimental results obtained from Argentinean anchovy (Engraulis anchoita)
salting plants are presented and compared with those calculated from the analytical
model. The proportion of variance explained by the model was 0.929 for total quality cost
(TQC), showing high performance for the model. For the salted anchovy plants analysed,
it can be observed that when the product quality level increases from fairly good to very
good, the controllable costs were increased from 4 to 67% of TQC, while the costs of
failure were decreased from 96 to 33% of TQC. When the product quality level improves
from good to very good, TQC decreases 50%. The evaluation of the quality cost and its
relationship to the level of product quality constitutes a useful tool to make decisions
intended to implement systems of quality assurance.
Keywords: HACCP, economics, quality, TQM, fish processing
*
Corresponding author: Aurora Zugarramurdi. College of Engineering, National University of Mar del Plata,
Argentina. Email: auroraz@mdp.edu.ar.
304 Aurora Zugarramurdi, Mara Amelia Parin and Hctor Mateo Lupin
INTRODUCTION
There is global concern in the implementation of the HACCP system by the food
industry, especially for high-risk foods such as meat, poultry or fishery products [1, 2]. The
HACCP system is based on a scientific, systematic, rational, multi-disciplinary, and cost-
effective approach controlling safety problems [3, 4, 5].
Many fish processing plant managers feel that quality programs decrease plant
productivity, thereby making such programs costly (i.e., luxury). This may be true for an
initial implementation period, but it is not indefinitely true [6]. A study of HACCP adoption
in the seafood industry shows that firms are adopting HACCP in order to meet customer as
well as legal requirements and to gain improvements in operating efficiency [7]. Also, in the
agricultural sector, it has been shown that an increase of the quality does not necessarily mean
higher costs [8].
The cost-benefit analysis is a tool to study the economics of HACCP. Few studies have
addressed the costs and benefits of HACCP implementation in the fish business. Some of the
findings demonstrate that a HACCP-based system implementation reduces failure costs,
improves quality and allows for better knowledge of production planning and control,
showing that in the first years of HACCP implementation, when failure costs are over 80% of
TQC, each dollar expended in controllable costs returns more than two dollars in failure
savings [9]. As a consequence, it is difficult to evaluate the extent to which costs and benefits
to businesses act as an incentive/disincentive to the further adoption of HACCP within the
food sector [10, 11].
Previous studies on the estimates of the costs of HACCP programmes have identified
benefits at both the industry and firm levels for seafood [12]. Some studies could also be
found on the quality costs for different industries and how to use them to improve company
performance, cost reduction and competitiveness [13, 14].
To evaluate the effectiveness of a HACCP-based system, a realistic estimate of quality
costs is essential. Quality costing activities can be carried out to gain top management
commitment, direct improving efforts, and, above all, estimate the benefits of quality
improvement [3, 15].
Quality costing is the first step for preparing a case for a Total Quality Management
(TQM) initiative. Moreover, a realistic estimation of quality costs is an essential element of
any TQM planning. However, only a minority of organizations uses formal quality costing
methods because quality costs are hard to measure.
The aim of this chapter is to assess the economic feasibility of a HACCP-based system
through the analysis of quality cost changes, to apply a previously developed mathematical
model [16] for the calculation of the quality costs associated with a specific quality level in
food processing plants and to compared the results from literature on quality costs models
applied to fish processing industry.
DETERMINATION OF TOTAL QUALITY COSTS (TQC)

Among several methods that can be used to collect, categorize and measure quality costs
[17], the PAF (Prevention, Appraisal and Failure) method was the proposed approach to
HACCP Economics in Seafood Processing Plants 305
quantify the economic impact on actual plants, both before and after the introduction of
HACCP. The PAF model is a well-known model used to analyse quality costs [16, 17, 18,
19], and, as such, it has been standardized for its application [20]. The extension of the PAF
technique to the analysis of HACCP economics relies on three basic assumptions. The first
one is that safety is an essential element of quality and as such usually understood by the fish
industry [21, 22]. The second is that the economic input/output of management decisions
taken at plant level to implement HACCP will play, in plant economy, a role equivalent or
similar to the decisions taken for quality reasons. The third assumption is that such inputs/
outputs and decisions could be specifically recorded, disaggregated from general records, or
studied and assessed or measured from current plant operation.
In general, HACCP implementation will be echoed in quality improvement. This is
because HACCP entails knowing the system in detail, and once the system is known, this
knowledge can be very often put into practice to enhance the economic performance of the
plant [12, 19]. The opposite is not always necessarily true. For instance, efforts in enhancing
quality aspects like appearance or packaging will not necessarily improve the safety level.
The developed quality cost model [16] identifies three cost categories for total quality
costs: Prevention costs, Appraisal costs and Failure costs. This methodology has also been
used by other authors for different food processing plants [14, 23].
The costs of conformance or controllable costs are comprised of costs associated with
prevention and appraisal. Non-conformance costs also called resulting costs are made up of
internal and external failure costs.
Prevention costs are those associated with the design, implementation and maintenance of
the HACCP system. They encompass the costs of assembling and operating the HACCP team
(e.g., for drafting the HACCP and Hygiene plans), HACCP personnel training (and re-
training), and preventive maintenance. These costs are usually planned and incurred before
the actual operation. A survey of the Turkish seafood industry showed that inadequacy of
employee education is the major difficulty, regarding to maintain hygiene and sanitation.
Employing permanent workers is needed to deal with this difficulty as suggested by Mol [24].
Appraisal costs are those related to the evaluation of incoming raw materials, processes,
intermediate and final products and services to ensure they conform to HACCP and Hygiene
plans in the first place, and to HACCP-based regulations required for different markets/
countries. The costs of operating and maintaining a monitoring and control system at all
critical control points (CCPs) fall within this category; also verification and internal HACCP
audit costs. However, they exclude re-work or re-inspection costs following failure.
Failure costs are usually divided into internal and external failure costs [15, 25, 26].
Internal failure costs are those associated with inadequate compliance with HACCP and
Hygiene plans, identified before ownership transfer from the industry to customer/consumer.
Generally speaking, they include scrap, rework, re-test, re-inspection, modification and
downtime costs among others. Particularly, corrective actions costs are typical internal failure
costs.
External failure costs are those related to the inadequate compliance with HACCP and
Hygiene plans, identified after ownership transfer of products from the industry to the
customer/consumer.
The model consists of two controllable costs and resulting sub models. This model
assumes that quality costs can be calculated from ten different components (Table 1). Each
component of quality cost is calculated using quality, market and production parameters
(Table 2) and equations are shown in Table 3.
The total safety/quality costs can be expressed by Eq. (11) that represents the total safety/
quality cost per unit of product as follows:
TQC(s) = SCP(s) + SCA + SCF (s) (11)
where: TQC(s) = total safety/quality costs per unit of product, SCP(s) = summation of all
prevention costs per unit of product, SCA(s) = summation of all appraisal costs per unit of
product, SCF(s) = summation of all failure costs per unit of product.
Table 1. List of categories and elements of quality costs
Controllable Costs Resulting Costs

Prevention Costs Internal Failure Costs
P1. Design, development and implementation of a F1. Scraps, reprocessing or spoilage
quality assurance plan
P2. Quality training programs for suppliers and F2. Low labour productivity and low
production personnel process yield
P3. Hygiene and sanitation of the plant F3. Inefficient use of plant capacity
P4. Preventive maintenance and additional
supervision
Appraisal Costs External Failure Costs
A1. Receipt and control of incoming material F4. Claims, rejected and recalled products
A2. Sampling and laboratory analysis
A3. In-process inspection
Table 2. Quality, market and production parameters
Quality Qm = raw material quality (dimensionless parameter)

parameters Qp = product quality (dimensionless parameter)
YQm* = yield for optimum quality level Qm* (kg product/kg raw material)
XQm* = productivity for optimum quality level Qm* (kg product/h-worker)
Nscp = number of sanitation control points
Nccp = number of critical control points
Market PQm = purchase price of raw material (US$/kg)
parameters PQp = selling price for quality level Qp (US$/kg)
PQp* = selling price for optimum quality level Qp* (US$/kg)
Production d = number of working days per year
parameters K = daily production (kg product/day)
Ri = raw material sampling inspection rate (kg raw material/h)
S = average labour rate for trained workers (US$/h)
IF = total fixed investment (US$)
XQm = productivity for quality level Qm (kg product/h-worker)
YQm = yield for quality level Qm (kg product/kg raw material)
L = total labour cost (US$/kg product)
Table 3. Relationships used to estimate quality cost components
Type of cost Relationship Eq. no.

Prevention costs
P1. Design, development and CP1 = P1 IF/d K (1)
implementation of a quality assurance
plan
P2. Quality training programs for CP2 = P2 L Qp (2)
suppliers and production personnel
P3. Hygiene and sanitation of the plant CP3 = (P3 L [1 + 0.01 Nscp] Qp)/z (3)
P4. Preventive maintenance and CP4 = [P4 IF/(d K) + P5 L] Qp (4)
additional supervision
Appraisal Costs
A1. Receipt and control of incoming CA1 = (A1 S/YQm Ri) Qp (5)
material
A2. Sampling and laboratory analysis CA2 = A2 L Qp (6)
A3. In-process inspection CA3 = A3 Nccp L Qp (7)
Internal Failure Costs
2
F1. Scraps, reprocessing or spoilage CF1 = (PQp* - PQp) (1-Qp) (8)
F2. Low labour productivity and low CF2 = S (1/XQm - 1/XQm*) + PQm (1/YQm - (9)
process yield 1/YQm*)
F3. Inefficient usage of plant capacity CF3 = PQp (YQm* - YQm)/YQm* (10)
The level(s) corresponds to safety when compliance (with the reference HACCP-based
system) is achieved. When regulatory compliance is attained, s becomes the quality level,
assessed as pertaining to the specific product.
MODEL VALIDATION: FISH SALTING PLANTS

The quality cost model (QCM) hereby exposed was applied to manual salting processing
plants located in Mar del Plata city, Argentina. These plants elaborate salted and ripened
anchovy in drums for export and already have proper Good Manufacturing Practices (GMP)
and Sanitation Standard Operating Procedures (SSOPs). These regulations must be fulfilled
prior to implement a HACCP-based system. Technical and economical data for the evaluation
of quality costs were elaborated from previous works and from experience in actual fish
salting plants [1, 27-30]. Experimental results obtained in Argentinean anchovy (Engraulis
anchoita) salting plants are presented and compared with those calculated from the analytical
model. Other food industries could also apply a similar approach to evaluate the advantages
of being able to support the safety and quality of their products. The evaluation of the quality
cost and its relationship to the level of product quality constitutes a useful tool to make
decisions intended to implement systems of quality assurance.
Raw material. Anchovies were caught on the Argentine platform in the South-western
Atlantic Ocean from 37.4S to 38.8S during the spring, when they approach the coast in
schools for spawning. All the catches occurred from September-December (anchovy season)
when the air temperature is 10-25C and the water temperature is 13-16C. Weight range for
individual fish was 29.5-36.3 g, length was 14-15.5 cm, moisture was 75.6-77.8%, and fat
was 3.6-5.2%. Usually these plants operate during anchovy season with fresh raw material for
about 50 working days per year.
Process. Fish are transported to the salting plant immediately after landing and are
immersed for one day in saturated brine, then manually beheaded and partially gutted. Part of
the viscera is removed, leaving the pyloric caeca and gonads (reproductive organs), to provide
the pyloric caeca enzymes essential for the formation of the correct ripe flavour. Washed
anchovies are packed in 250 kg drums, in layers with food grade dry salt [1, 31]. A round
piece of plastic is placed on the top layer of salt and pressed (pressure range 120-130 g/cm2).
Drums are stored for ripening at least for three months until their sale.
Quality cost model. To calculate the quality costs associated with a specific quality level,
a relationship between the raw material and final product quality is needed. Chemical,
physical, and sensory parameters of fresh anchovies (raw material) and salted and maturated
anchovies quality were jointly analysed in a dimensionless basis [30]. From the evaluation of
sensory assessment and belly bursts of fresh anchovy, different intervals are defined in Table
4 as a function of raw material quality.
The parameters were normalised in a range from 0 to 1, where the value 1 corresponds to
the optimum quality. A general linear equation was obtained for the normalised experimental
raw material quality and final product quality values of fatty fish and Eq. (12) was found (R2
= 0.972, Students t test, P < 0.01):
Qp = 0.938 Qm + 0.107 (12)
Quality cost components. To calculate quality cost components the equations shown in
Table 3 were used. Total quality costs per unit of product (TQC) were calculated by the
summation of Eqs. (1) to (10). It should be noted that internal failure costs were evaluated as
direct company losses. External failures costs were not considered because of a lack of
availability data from fishery industry managers or owners. Due to the commercial
implications, it is very difficult to find values of this type of cost [1].
Controllable cost. Each component of the controllable costs is affected by coefficients
that depend on quality level. The values of these coefficients are shown in Table 5 and
explained below.
Prevention costs. Fish processing plants already complying with GMP-SSOPs only need
as much as one per cent of IF for planning and implementing the quality system so as to reach
a very good quality [32]. One per cent was used for the P1 value corresponding to optimum
quality. In this case study, plant capacities felt within the 10-30 raw material t per day range.
A fish salting plant of 15 t/day capacity has a IF of approximately US$ 300 000 [1].
The cost of training and P2 coefficient descend from 0.5 to 0.1 when the level of product
quality is decreased from extra quality to good [4].
In the fishery industry, the denominator of Eq. (3) was fixed at 0.842 [33]. It was
determined that cleaning expenses have a cost structure where overall labour comprises the
bulk of costs (average 84.2%), detergents and disinfectants accounting for only a minor part
(average 10.6%) and cleaning equipment for even less (average 5.2%). Hygiene labour costs
in fish processing plants were found to be up to 15 per cent of the total labour costs. In
general, the SSOPs plans have 7 sanitation control points [34].
Table 4. Raw material quality levels for fish salting plants
Quality level Qm 100

Poor 29
Fairly good 30-49
Good 50-69
Very good 70-89
Extra quality 90
Table 5. Coefficients for fish salting plants as a function of quality level
Quality level P1 P2 P3 P4 P5 A1 A2 A3
Extra 0.01 0.5 0.15 0.002 0.05 1 0.04 0.02
Very good 0.005 0.3 0.08 0.0015 0.04 0.5 0.03 0.01
Good 0.003 0.1 0.04 0.001 0.02 0.3 0.01 0.005
Fairly good 0 0 0.01 0.0005 0.01 0.1 0 0
Poor 0 0 0.01 0 0
Coefficient P4 was determined between 2 and 10 per cent of maintenance costs.

Maintenance cost for fish salting plants is estimated at 2 per cent of IF. The maximum for
additional supervision cost was a 5 per cent of the direct labour costs for optimum quality,
and consequently coefficient P5 was estimated as shown in Table 5 [1].
Appraisal costs. In order to apply QCM to fish salting plants, the sampling plan used was
designed according to regulations proposed by the Canadian Quality Management Program
[35] for fish processing plants at standard quality level. The sample size is determined
according to the number of fish in the lot that is related to the weight of the fish. Parameter
A1 indicates the inspection activities performed in the actual plan in connection with the
activities that should be done to reach optimum quality, in which case can be as high as 1.
In Argentina, a typical requirement for inspection activity is about 20 kg raw materials per
hour (R). In the fish processing sector average labour rate is around 4 US$/h.
Sampling and laboratory costs in fish processing plants can rise up to 4% of labour cost
for maximum product quality [1].
The number of process critical control points (Nccp) in fish salting plants is usually 4
(reception of raw material, brining, drum filling and maturating) [34, 36, 37]. Labelling and
packing are not taken into account as critical control points when fraud is not considered as a
HACCP factor.
Resultant cost. Failure cost. Process yield and labour productivity for salted anchovy
have been analysed in previous works [30, 38] as a function of raw material or product
quality.
The linear relationship between filleting yield (Y) and product quality for anchovy
processing found by Zugarramurdi [30] was used; and is shown in Eq. (13) (R2 = 0.95,
Students t test, P < 0.01):
YQm = 89.95 0.413 Qp (13)

Eq. (14) shows the linear relationship between labour productivity or workers training
level (X) and product quality found by Zugarramurdi [30] (R2 = 0.876, Students t test, P <
0.01).
XQm = 102.97 0.848 Qp (14)
For salted anchovy, yield and productivity increase 18% and 30%, respectively when the
quality level of the product is increased from good (Qp = 0.60) to very good (Qp = 0.80). In
factories using poor raw material quality, it can be observed inefficient usage of plant
capacity, due to the decrease in the overall yield of the whole process. Also, in the meat and
dairy industry, it has been shown that the implementation of a HACCP system led to the
improvement of the efficiency of the production process [39].
Raw material price ranged from 0.3 to 0.5US$/kg while selling price ranged from 1.5 to
2.1 US$/kg as a function of quality. Then Eqs. (12), (13) and (14) were used to estimate
internal failure costs due to losses or decreases in sale price (less quality) and inefficiencies in
labour and raw material.
Total quality costs. Regression analysis has been applied to the controllable costs, failure
costs, and total quality costs per unit of product for different levels of quality resulting from
the application of the QCM. Regression equations for the model were obtained fitting
polynomial curves to the model values by least squares estimations and were plotted in Figure
1.
In order to validate the model, controllable costs, failure costs and total quality costs in
actual salted anchovy processing plants were also collected. The points in Figure 1
correspond to experimental values. To evaluate how this model fitted the experimental values,
the proportion of variance accounted for in the dependent variable by the model was
computed. The proportion of the variance explained was 0.940 (for controllable costs), 0.951
(for failure costs) and 0.929 (for total quality costs). This proves the high performance of the
model described. In fish salting plants, after the HACCP-based system was applied and
complied, not only costs were reduced but also there were additional benefits such as increase
in product quality, selling price and improve usage of plant capacity. It has also been shown
for other food processing plants like the Turkish Seafood Processing Firms already
mentioned, that implementation of food safety systems improves quality, increase the sales,
and decrease total safety/quality costs [24].
For salted anchovy plants analysed, it can be observed in Figure 1, that when product
quality level increases from fairly good to very good, controllable costs increase from 4 to
67% of TQC and failure costs decrease from 96 to 33% of TQC. It can also be observed that
when product quality level improves from good to very good, TQC decreases 50%. At the
same time, there is a zone with lower values around very good level of product quality.
Without the recognition of the existence of failure costs, the optimisation of quality costs
is impossible. In those cases, the usual actions of the companies are the reduction of
prevention and appraisal costs that lead to lower quality levels.
The company could decide an increase in the prevention and appraisal costs above the
minimum point based on considerations such as volume of sales, safety, prestige (brand
image) and company reputation.
Figure 1. Total quality costs, failure costs and controllable costs as a function of product quality for
salted anchovy processing plants.
CONCLUSION
It has been shown that the model developed by Zugarramurdi et al. [16] can be
successfully applied for the estimation of quality costs as a function of product quality level
with a very good regression coefficient for total quality costs, showing that the model
explains the 92.9% of the experimental values from actual fish salting processing plants.
It is also shown that due to the poor quality of inputs, failure costs are over 95% of the
total quality costs, while for a very good quality level, failure costs descend below 20% of
total quality costs.
This study demonstrated that after the HACCP-based system was applied and complied,
not only costs were reduced but also there were additional benefits such as increase in product
quality, selling price and improve usage of plant capacity.
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[33] Dunsmore, David G., Tomlinson, Paul, and Robert J. Ashley. 1983. Cleaning practices
in the Australian seafood processing Industry. Food Technology in Australia 35 (12):
566-70.
[34] SENASA. 1996. Manual de procedimientos. Aplicacin del Sistema HACCP. Anlisis
de Riesgos y Puntos crticos de control (Procedures manual. HACCP implementation.
Hazard Analysis and Critical Control Points). Buenos Aires: Artegrfica Impresos.
[35] Fisheries and Oceans Canada. 1989. QMP specifications. Sampling plan and acceptance
numbers for the inspection of fish/shellfish. Quality Management Program. Submission
Guide. Canada.
[36] National Marine Fisheries Service. 1990. HACCP Regulatory Model Raw Fish. Model
Seafood Surveillance Project. Pascagoula: Office of Trade and Industry Services.
[37] Yeannes, Mara I. 1991. HACCP in anchovy salting and marinating. Paper presented at
Expert Consultation on Quality Assurance in the Fish Industry. FAO/DANIDA,
Lyngby, Denmark, August 26 - September 6.
[38] Gadaleta, Liliana, Parin, Mara A., Lupin, Hctor M., and Aurora Zugarramurdi. 2003.
Calidad de la materia prima y su influencia sobre la calidad y costos de produccin en
productos pesqueros (Quality of raw materials and their influence on the quality and
production costs in fishery products). Revista de Tecnologa e Higiene de los
alimentos (Journal of Technology and Food Hygiene). Alimentaria 343: 41-47.
[39] Romano, Donato, Cavicchi, Alessio, Rocchi, Benedetto, and Gianlucca Stefani. 2004.
Costs and Benefits of Compliance for HACCP Regulation in the Italian Meat and
Dairy Sector. Paper presented at the EAAE 84th Seminar, Zeist, The Netherlands,
February 8-11.
Chapter 18
HYGIENE AND SANITATION APPLICATIONS

IN SEAFOOD INDUSTRY
Abdullah Diler 1,2,* and Frantiek Vcha3

1
Suleyman Demirel University, Fisheries Faculty,
2
zmir Katip elebi University, Fisheries Faculty,
Fishing and Processing Technology Department, zmir, Turkey
3
Faculty of Agriculture, Department of Agricultural Products Quality,
University of South Bohemia, esk Budjovice, Czech Republic
ABSTRACT
Food hygiene applications are necessary practices in terms of maintaining the quality
and safety of food products, particularly in aquatic foods. Implementation of good
hygiene practices (GHP), good manufacturing practices (GMP), as well as hazard
analysis and critical control point (HACCP) in seafood plants is one part of hygiene
applications. In this chapter, hygiene application in seafood plants is compiled in detail
based on a from-farm-to-fork approach. Regarding this, basic operations of seafood
processing steps, hygienic and sanitary measures of seafood processing, hygiene
monitoring steps for both the application itself and personnel carrying it out, sanitation
steps from harvesting to retail level and finally basic control and measurement steps were
compiled and reviewed in detail.
Keywords: seafood hygiene, sanitation, safety application methods, HACCP, GHP, GMP,
operation measurements
INTRODUCTION
The term sanitation comes from the Latin word sanitas, which means health.
*
Corresponding author: E-mail: abdullahdiler@sdu.edu.tr, abdullah.diler@ikc.edu.tr.
316 Abdullah Diler and Frantiek Vcha
In this context, the purpose of sanitation applications is to remove visible dirt from the
used equipment and reduce the level of microorganisms to a safe consumption level (e.g.,
depending on the type of microorganism, some bacteria such as Salmonella sp. should
completely be removed) [1]. Hygiene and sanitation applications are one of the most
important steps in the seafood industry in terms of assuring the quality and safety of the
fishery products. Processing starts just after harvesting or capture, and during processing (on
a boat or in a facility) the workers have to pay more attention to the perishable foods (e.g
seafood and fishery products). Due to their nutritional composition and several factors (i.e
pH, water content etc.) seafood tends to spoil faster and is more susceptible to contamination
or re-contamination during handling than other products [8].
To maintain the quality and safety of seafood during all stages of processing (Table 1),
factors such as temperature fluctuation, improper handling, and inadequate hygiene of both
workers and equipment have to be taken into account and controlled intensely [2].
The applications of hygiene and sanitation involve the use of chemical agents and heat
treatment together with an effective Hazard Analysis and Critical Control Points (HACCP)
implementation.
The seafood industry contains a broad range of products (fish, crustacean, mollusks) and
each product has their own hazards (biological, chemical or physical), so a HACCP plan
should be facility specific [3].
In this chapter the hygiene and sanitation applications, HACCP plans and requirements
and definitions in HACCP implementations and control measures from farm to the retail level
was investigated and reviewed.
1. Receipt
Marketable live freshwater fishes originating from quality living conditions, free of
contaminants (such as PCBs, heavy metals and a variety of other degradation products) are
used for processing.
The delivery of fish shall be accompanied with a certificate, issued by the relevant body
of veterinary inspection, characterizing the delivery in terms of any occurrence of diseases
and parasitic infections, applied drugs/preparations and any other ways of treatment. The
document shall provide a statement that the delivery of concern is suitable for processing.
Neither dead nor apparently sick fish shall be accepted for further processing. Just
asphyxiated fishes are acceptable for processing in the cases of the trout, tilapia, great
maraena and peled whitefish.
Other asphyxiated fishes may only be taken into processing subject under the approval of
the relevant body of veterinary hygienic service obtained in advance.
Live marketable fishes are delivered in suitable containers filled with quality water
identical with water used for the pisciculture of the fish species in question and containing
sufficient amount of oxygen. The fish shall not suffer from any mechanical damage during
unloading.
Dead fish are not transported in water. In case that such transport is made for a longer
distance and during the season of air temperatures in excess of 5C, the fish are mixed with
flaked or crushed ice and placed in suitable transport containers provided with drains for
water produced by ice thawing.
Hygiene and Sanitation Applications in Seafood Industry 317
Table 1. Basic operational sequence in seafood processing
2. Slaughtering
In the places of their regular processing fish can be stunned, with subsequent bleeding,
using impulse current, carbon dioxide gas (CO2) or other suitable gas subject to a specific
regulation. Various technological systems are employed. In general, strict safety regulations
and operational rules shall be regulated due to specific features of equipment design.
3. Descaling
On descaling fish scales are removed using a cold pressure water jet or other mechanical
means. Due care should be taken to prevent fish bodies from any damage or injury in the
course of descaling, as such a lesion can act as a potential source of meat contamination
during subsequent fish processing stages. Damaged and impaired fish are eliminated from
further processing [5].
Scales need not be removed with the trout, tilapia, great maraena, tench and some other
fish species.
4. Evisceration
In terms of operational hygiene evisceration should be considered as an extraordinarily

important process. On the opening of the abdominal cavity there is a risk of bowel incision
that would result in the consequent contamination of the product with undesirable micro-
organisms [10]. After processing particular fish lots and all the processing equipment and
working tools shall be thoroughly cleaned. Fish may be eviscerated either by the manual or
mechanical way. The operation starts with an incision led longitudinally from the anus up to
the head, using either a knife or a power cutter. After removing the viscera, the gall bladder
should be removed. Edible parts are placed in a separate container. The head is removed
using a curved cut made in front of the gill bone. Dorsal, tail, anal and pectoral fins are cut
off. Subject to customers requirement, both the head and fins may be included in the edible
parts.
5. Cutting into Portions
Subject to customers requirements and processing plant conditions, the final

arrangement of fish for delivery may be negotiated between the supplier and the customer. If
the fish is halved, a cut is made along the backbone from the head up to fishs tail. With the
carp, the portions are made either by transversal cuts of either the whole fish (into
horseshoes) or the halved fish (into portions).
In filleting the muscular tissues of the body are separated from the backbone and ribs.
The operation is often supplemented with the destruction of muscular bones.
6. Washing
Washing is another very important operation that presents a risk of mass contamination
of the product. Drinking water is used for the purpose of the quality meeting the requirements
of drinking water standards. At present, drum washers are employed for fish washing. The
machines are operated in a discontinuous mode, one batch containing about 150 kg of fish.
The washing cycle takes about 2 or 3 minutes for one batch. New washing water is used for
each batch of processed fish. In order to cool down the batch quickly and efficiently, the use
of crushed ice addition is recommended. The final fish product quality significantly depends
on the process of washing. Todays trends in washing technology are characterized by the
application of water spraying (in shower systems) on the raw material processed. Fish are
then left to drip.
7. Cooling and Storage
In order to prevent any microbial growth in the product, the latter shall be cooled
immediately after washing. The quick cooling of the material with ice water, i.e., water mixed
with flaked or crushed ice is needed.
This way the product can be cooled down to the pith and any microbial growth can be
eliminated significantly. Cooling by air would be much less efficient. Product intended for
sale should be cooled down to a temperature between -1C to +5C. Cooled products are
stored usually in cold stores, where a stable temperature is maintained within the range of -
1C to +5C.
8. Packing and Labelling
The product shall be packed in a packing safe for human health. The application of
vacuum packing will generally extend the shelf life of products. Product description and
storage instructions shall comprise an integral part of product packing and related
implementary regulations [18].
9. Transportation
Fish are sensitive products that should be distributed without delay. Therefore, such
products shall be dispatched immediately after raw fish have been processed. Fish should be
transported employing thermal insulated or refrigerated means of transport at the
temperatures in the range of -1C to +5C. The temperature conditions set out for fish product
storage shall be adhered to package [18].
10. Hygienic and Sanitary Measures in Fish Processing
The measures shall involve the sanitation of areas, buildings and all equipment, the
hygiene and monitoring of personnel health condition as well as the operational hygiene in
fish processing steps. The requirements of the sanitary steps and applications are shown in
Table 2.
Table 2. Measures in the Sanitary Rules of the Premises
Any areas adhering to the operational areas of the fish processing plant shall be the
subject of consistent maintenance and kept clean and free of dust. Proper water draining
shall be provided.
Operational buildings shall be kept in proper technical condition and cleanliness. Any
defect having a potential impact on operational hygiene and product safety for human
health shall be removed preferentially and in due time.
Rooms where fish are treated shall be cleaned on a regular basis. Their floors shall have
a suitable gradient towards draining gullies and must not be slippery. The areas shall be
equipped with efficient means to secure them from any access of rodents and insect.
Storage areas shall be kept clean and stored products shall be properly stowed and well
arranged.
Each area shall be provided with a readily accessible first aid kit comprising bandage
material and means necessary for basic disinfection.
Other rooms used for auxiliary and support activities shall be kept clean and in such
condition that will not put in question both the health of personnel and product safety
for human health.
Heating, illumination and ventilation shall meet the requirements of occupational
hygiene. Ventilation equipment shall be secured against any access of rodents and
insect. Production equipment, including vessels, containers, tools and accessories shall
be properly maintained, operable, clean and kept in faultless condition to eliminate any
negative effects on personnel health and product safety for human health. Operational
benches should be made of readily washable material.
All coatings shall be smooth, undamaged and resistant to commonly used disinfection
agents. All equipment, tools and jigs shall be made of materials that have no
detrimental effect on product composition, taste and appearance.
11. Occupational Hygiene and Operational Personnel Health Monitoring
The personnel of operational areas shall follow strictly the rules of personal hygiene,
including their working garments and personal protection equipment. The personnel shall
wash their hands properly before commencing work, especially after performing a dirty
operation or using the lavatory. Any toilet care (combing, e.g.) is forbidden at work.
Similarly, the prohibition of smoking and eating in processing and storage areas shall be
strictly observed [9].
At work, the worker shall wear a specified working garment, a suitable hair cover and
prescribed protection equipment. Every worker is obliged to keep, with utmost care, his/her
workplace, equipment and accessories in consistent cleanliness and order during and after
finishing the working shift. Any employee who works with or handles raw materials and
products or even enters in operational areas are obliged:
a) to pass the prescribed medical examination before entering the job;

b) to possess his/her valid health card;
c) in case that there is a suspicion the worker or his/her family member has been
diseased by an infection, to notify the shop manager of this fact without any delay;
d) to notify his/her attending physician on his/her employment in food industry.
The processing shop management shall keep records on the medical examinations and
health cards of its employees. No person can be admitted to the immediate product processing
or handling, failing to submit his/her valid health card. All employees shall be acquainted
with the set of hygienic and sanitary measures and due records shall be kept of such
hygienic/sanitary training. No access to processing areas shall be permitted to unauthorized
persons. The principles of occupational hygiene and product-specific technological
procedures shall be strictly observed in fish processing, storage and transport. Every
employee shall notify the responsible shop manager of any suspect feature shown by the
product the employee may note during processing, packing, storage or dispatch. Such suspect
features can include, e.g., any indication of unfitness for human consumption or deviation
from product usual appearance, colour, odour or other typical property. Production
machinery, jigs, tools, vessels, containers, crates and similar equipment and accessories used
in processing shops shall be consistently kept in proper cleanliness [4, 7]. Every day after use
they have to be thoroughly cleared of residues and carefully washes with warm water
containing suitable cleaning and disinfecting agent. The final rinse should be made with hot
water fit for human consumption. After the shift end the workstation should be cleaned.
Benches employed for fish processing should be washed with hot water, degreased and
disinfected. Floors in operational and auxiliary rooms are washed with hot water containing
suitable cleaning and disinfecting agent. The entire processing area should be thoroughly
cleaned on the weekly basis, while the cleaning of the whole processing plant premises is
made two times a year [13].
12. Sanitation
Sanitation in seafood processing application and plants is the main and possibly the
important part of maintain the quality and safety. The beginning point of sanitation is
harvesting and accepting seafood (i.e fish, mollusk, crustacean) to the facility [6]. Sanitation
operations in fish from harvesting to retail will be assessed in this section.
12.1. Accepting and Keeping Live Fish in Tanks

Live marketable fish is accepted and transferred in tanks according to the conditions laid
down in national standard. In particular, the fish health condition is to be monitored.
Disputable cases shall be referred to the relevant body of veterinary supervision for decision.
Storage tanks shall be disinfected once a year, at least, using burnt slaked lime [14].
12.2. Fish Processing

Working benches, belts, machines and tools shall be kept clean all the time. Cold water
spray should be used for removing any larger contamination during the shift. After the shift
end coarse dirt should be removed and surfaces scoured with warm water (50C - 60C). On
the following morning, before commencing ones work, benches and tools should be rinsed
with warm water. Thorough cleaning is carried out once a week and consists in the careful
removal of dirt and degreasing of surfaces, a hot water rinse and disinfection. Dirty floors are
rinsed with cold water in operation and should be sprayed with cold water after the shift end.
Once a week the floors are degreased, rinsed with water and disinfected. After the period of
disinfection has elapsed, the floor should be scoured with warm water [15]. Walls - tiling
should be washed with warm water after the shift end. Once a week, walls are degreased,
scoured with water and disinfected. Disinfecting solution is then removed by a warm water
scour. Draining gullies - to be disinfected once a week using chlorinated lime. Working
garments shall be changed once a week, or even more often in case of heavier contamination.
Rubber aprons should be rinsed with warm water after the shift and disinfected/rinsed once a
week. Washrooms and lavatories - floors, wall tiling and washbasins shall be washed daily
with hot water and detergent.
Disinfection should be made once a week. Changing rooms should have separate
compartments for working garments and plain clothes. Floors and lockers should be washed
every day using water with detergent addition and disinfected once a week. Working areas,
including the areas designed for storing production waste - to be cleaned once a week and
disinfected (using chlorinated lime) once a month. General cleaning including disinfection is
made two times a year. Cold stores are scoured with cold water once a week and their full
cleaning is made each month. The latter comprises degreasing, rinsing with warm water,
disinfection and final scouring with warm water. Freezing stores shall be thoroughly cleaned
once a year after defrosting (to be carried out at the time suitable in respect to the specific
requirement of store operation). The cleaning consists in degreasing, scouring with warm
water and disinfection. Finally, disinfecting agent is scoured away with warm water [16]
(Table 3).
Means of transport shall be washed with cold water immediately after unloading cooled
fish.
In general, they are cleaned once a week by degreasing, scouring with warm water,
disinfection and final warm water scour. A separate room shall be reserved for the storage of
cleaning and disinfecting agents. Similarly, the vessels and implements used for cleaning and
disinfection shall be stored separately from those used for fish processing. Approved
disinfection agents only may be used for disinfection [17].
13. Term and Definitions explanation
Within the HACCP System certain specific terms are used, which are defined as follows:
HACCP is a new approach to the control of food hygiene. In the literature it is

described as the Hazard Analysis Critical Control Points System. The name describes
the two most important features of the whole system, i.e., the analysis of the hazard
that the health or hygienic safety of a certain food product or meal might be impaired
and the identification of the critical control points in the production, processing,
preservation, storage, transport, distribution, cooking or any other method of its
preparation for consumption.
Table 3. Operation steps of control measurements in seafood
Control Hazard Criteria Preventive Inspection Inspection Corrective

Operation Point no. Measures Method Frequency Measures
1. Receipt CP 1 biological fish diseases, to accept only each new lot delivery each not to accept,
parasitic sound fish shall be notes, delivery for processing,
infestation, accompanied accompanied veterinary any fish lacking
mould-caused with a with certificates the veterinary
diseases veterinary veterinarians certificate
certificate judgement
(delivery notes)
CP 2 chemical PCBs, toxic according to location records of according to such fish shall
substances, the monitoring monitoring analyses the cycle of be eliminated
oil products, of the fish (prevention of pisciculture from processing
heavy metals location of contamination) and pond
origin location
CP 2 physical metal objects, visual inspection of linked to the on a running foreign matter
gravel, stones, inspection of each delivery delivery basis removal
wood each delivery notes
(due care
during
loading)
2. Slaughtering CP 2 physical animal torture correct inspection of equipment daily checks adjustment of
functioning of equipment efficiency of operation equipment
technological operation monitoring intensity o technical
equipment to parameters
preset
parameters

3. Descaling CP 2 physical damaged and provision of inspection of equipment on the adjustment of
broken fish correct equipment function running equipment
bodies functioning of operation and monitoring basis technical
technological observation of functions,
equipment, technological separation of
observation of times subject to damaged fish
descaling time external bodies from
conditions
4. Evisceration CP 2 biological meat cutting the responsible work visual on the provision of
contamination intestine on the production inspection on running thorough fish
by the through shall line, working processing basis in body cleaning
contents of be avoided bench scouring processing and washing
intestine
CP 2 physical bowels thorough responsible work visual on the provision of
residues left removal of on the production inspection running proper cleaning
in the fish bowels line basis in completion
body processing
5. Cutting into CP 2 physical incorrectly responsible sharp working visual, on on the to take care of
Portions made cuts, work in tools processing running correct cutting
torn muscular processing basis into portions, to
tissues use working
tools sharp
enough
Washing CP 2 biological water water to take due care to follow on random repeated
contaminated exchange to be of water quality processing basis (spot washing after
by micro- provided in and its exchange procedures, checks) in water
organisms quality with each batch water the course of exchange
meeting the analysis processing,
requirements to standard
of the standard requirements
for drinking
water
CP 2 physical higher water to observe the to observe the measurement spot checks, adjustment of
temperatures maximum recommended by 3 times a water
promoting permissible water thermometers shift, at least temperature
faster water temperature and fast initial
microbial temperature of material
growth 10 C cooling
7. Cooling and CP 1 bological propagation of to observe the to observe the micro- micro- repeated
Storage undesirable limit values prescribed water biological biological micro-
micro- for microbial temperatures; sampling and sampling biological
organisms counts correct analysis on and analysis examination
(delayed functioning of the random basis on random
cooling or cooling basis, once
non- equipment in three
observance of months, at
cooling least
temperatures)

CP 1 physical insufficient the proper stowing of temperature 2 times in to provide for
cooling temperature initial materials measurement the shift the correct
temperature, must not to prevent its in cooled functioning
damaged exceed 5C spaces by a
product deformation recording of cooling

exterior due to (bends or device or equipment, to
unsuitable raw unsuitable thermometer eliminate any
material overlapping of impaired
stowing worked parts), to initial
provide for its materials from
uniform stowing, further
observance of processing
prescribed
temperatures
8. Packing and CP 2 biological microbial adherence to to provide inspection of to eliminate

Labelling contamination the limit packing storage stores contaminated
values of in a clean, dry and damaged
microbial place, packing
counts unobjectionable
in terms of
hygiene
CP 2 chemical use of to employ reference to on each not to use
packing approved the delivery of unapproved
harmful to materials only certificate new packing packing
human health issued by the
packing
manufacturer
CP 2 physical missing or labelling to provide the visual each suspension of

inadequate subject to proper product inspection processed lot product
product implementary description dispatch and
description, regulations on (comprising any completion of
damaged food labelling prescribed marks its labelling
packing and labelling)
9. CP 2 biological development to observe the to maintain the monitoring on the repair of the
Transport of undesirable storage recommended the functions running cooling
micro- temperatures temperature of the basis equipment of
organisms of 1C to 5C mode transport the vehicle
vehicle
cooling
equipment
CP 2 physical product prevention of visual on the to provide for
deformation undesirable inspection running product proper
and external product bends basis stowing, to
damage due to and deformation, remove
improper to provide for its deformed
stowing proper stowing products.
The English word control expresses both the inspection carried out by monitoring
certain parameters at a critical point and the control performed by implementing
measures to meet the requirements of good hygienic and of technological practice
providing product hygienic and health safety [19].
Hazard comprises those biological, chemical and physical factors and conditions
or certain situations able to put food hygienic and health safety in jeopardy.
Therefore, such hazard consists particularly in an infection or contamination and in
the survival and growth of pathogenic bacteria. The term hazard relates also to the
production of bacterial metabolites - toxins, enzymes and biogenic amines. Other
pathogens falling under the term of hazard include moulds, myco-toxins, viruses,
parasites, bio-toxins produced by plants, fungi and animals, chemicals, radio-
nuclides and foreign matter in food. Other factors that can affect food quality should
be deemed a potential hazard, too.
Also the conditions favourable for microbial growth should be counted among
hazards, such as the thick layers of meat that slow down the process of cooling
making undesirable microbial growth possible [12].
Weight is the expression of hazard quantification as to its consequences for human
health or product quality.
Risk is defined as the estimated probability that a hazard will take effect.
Hazard analysis means the process of data collection and interpretation.
It involves the aggregate results of the evaluation of all the operations included in the
production, processing, preservation, storage, transport distribution, cooking or any
other way of preparation and consumption of products. Raw materials, ingredients
and products are analysed in order:
1. to identify hazardous raw/initial materials and food in terms of any potential

presence of alimentary pathogens and toxic substances, or, possibly, saprophytic
micro-organisms and other factors responsible for food spoilage;
2. to find, whether raw materials or food are able to support microbial growth;
3. to identify the possible sources of hazard and points of contamination of or
access to the food chain;
4. to determine the probability that micro-organisms will survive or propagate in
food in the course of its production, processing, preservation, storage, transport
distribution, cooking or any other way of preparation;
5. to evaluate the weight and risks of hazards related to product harmfulness to
human health or to its impaired quality.
Critical control point (CP) means the process operation, point or area that are
subject to systematic inspection (monitoring) and where preventive/protective
measures are applied on order to eliminate the hazard in question, to control or
reduce it to an acceptable level.
Criterion (the critical limit) means the boundary separating the range of
acceptable values from that of unacceptable ones. Criteria are subject to
measurement or monitoring in the critical control points. It is left on the
responsibility of producers and other operators to set their own criteria (target
values), stricter than generally accepted to secure the correct function of CPs.
Monitoring means the systematic observation, recording, measurement, etc., of the
pre-set criteria in the selected critical control points in order to determine, whether
the CB of concern is under control.
Protection means that the conditions of the process operation at a CB are
controlled to the extent providing the operational mode in which the process runs
according to prescribed instructions and meets the set criteria.
Corrective measure means a measure that may be taken to eliminate a hazard and
or reduce the impact of its consequences to an acceptable level.
Corrective action means an intervention to be carried out in response to the
indication of a deviation from correct functioning or a non-compliance with the set
criteria revealed by the monitoring applied to a CP.
Intervention involves an action or actions to be taken in order to bring the
operational parameters of concerned control points back in the acceptable range.
Validation or verification means the use of determination methods (other than
those applied in monitoring the criteria) in order to find whether the practical
implementation of HACCP complies with the elaborated plan. It is made also if the
plan requires re-evaluation or should be modified.
A facilitys plan of action should contain the general layout of the processing plant,
including the description of particular activities, flow diagram of raw/initial materials and
products, list of approved product range(s), and other relevant characteristics. Water can be a
primary source of contamination in plants and periodically analysed by means of quality.
Additionally, the water distribution system should include the employed water supply and
allow for traceability, as well as a detailed description of the water supply and method(s) of
water treatment employed. If the factorys own water source is used, the employed dosage of
chemicals should be given; chlorination should be indicated, how the presence of chlorine is
determined on the daily basis, points of water withdrawal in the plant should be indicated,
described and numbered, and any responsible persons name should be given.
14. Disinfection and Disinfestation
If provided on a contractual basis, the contractor and related service contracts shall be
given. For the disinfection the following shall be provided [11]:
Sanitation order/rules of the premises (see enclosure)

Disinfectant types employed in each room shall be given, including their
concentration(s) and application method(s)
Records kept of both the inventory of agents and their consumption
Application frequency as well as the list of works carried out in regular cleanings (the
daily, weekly, monthly or annual basis)
Persons performing the disinfection and relevant responsibilities
Persons handling the goods and raw materials
Means of transport cleaning and disinfecting system, including relevant

responsibilities and documents of the consumption of chemical for
cleaning/decontamination.
15. Disinfestation
General layout of the premises; the points where baits are laid should be indicated
and numbered.
Employed agent(s)
Records kept of both the inventory of agents and their consumption
Evaluation of deratisation efficiency.
16. Education and Training
Frequency of courses signed by the trainees should be recorded and kept in separate
folders for each employee.
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consumption, Official Journal of the European Union, L226, 25.06.2004: 83127.
[16] EC (European Commission). 2004b. Corrigendum to Regulation (EC) No 852/2004 of
the European Parliament and of the Council of 29 April 2004 on the hygiene of
foodstuffs, Official Journal of the European Union, L226 25.06.2004: 321.
[17] EC (European Commission). 2004c. Corrigendum to Regulation (EC) No 853/2004 of
the European Parliament and of the Council of 29 April 2004 laying down specific
hygiene rules for food of animal origin, Official Journal of the European Union, L226
25.06.2004: 2282.
[18] FAO. 2006. Traceability and labelling in fish trade. [online]. Committee on Fisheries,
Sub-Committee on Fish Trade, Tenth Session, Santiago de Compostela, Spain.
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[19] ILSI (International Life Sciences Institute). 1997. A simple guide to understanding and
applying the Hazard Analysis Critical Control Point concept. 2nd edition. Monograph
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[20] FAO. 2014. Assessment and management of seafood safety and quality Current
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agrculture organzaton of the unted natons Rome.
Chapter 19
BASICS OF SEAFOOD QUALITY INDICES
Frantiek Vcha1, and Abdullah Diler2,3

1
Faculty of Agriculture, Department of Agricultural Product Quality,
University of South Bohemia, Czech Republic
2
3
zmir Katip elebi University, Fisheries Faculty,
Fishing and Processing Technology Department, zmir, Turkey
ABSTRACT
Quality deterioration is the main problem in seafood and primarily caused by
microorganisms and enzymatic reactions. Several methods are available to determine the
quality changes of seafood during storage. Sensory methods that are non-destructive are
widely used and could be conducted either instrumentally or based on the sensory
methods (quality index method (QIM), Tory scale etc.). Biogenic amines on the other
hand could be used to estimate both the quality and safety of seafood. In this chapter, the
usage of instrumental methods for shelf life decisions and the importance of lipids, fatty
acids and biogenic amines existing in seafood as quality indicators of seafood are
reviewed.
Keywords: instrumental methods, quality, biogenic amines, lipids
INTRODUCTION
Instrumental Methods
Texture can be regarded as a manifestation of the rheological properties of a food.

Texture of fish muscle may be measured by sensory and instrumental procedures. Destructive
Corresponding author: Faculty of Agriculture, Department of Agricultural Product Quality, University of South
Bohemia, Czech Republic, Email: fvacha@zf.jcu.cz.
334 Frantiek Vcha and Abdullah Diler
instrumental methods such as texture profile analysis, Kramer test and puncture test are
important and effective in food texture assessment [1]. Textural quality is primarily a sensory
attribute that is quantified in foods by several instrumental methods. According to Bourne and
Szczesniak (2003) [2] and Cardoso et al. (2009) [3], a texture profile analysis (TPA) resulting
in a forcegenerated time curve is used to quantify a number of textural parameters that
correlate well with results from sensory evaluation.
Myofibrillar proteins and collagen, which constitute 70% to 80% of total protein content,
control the structure and the specific rheological properties of fish muscle [4]. Postmortem
textural changes are caused directly or indirectly by physicochemical changes in myofibrillar
proteins and changes in extracellular structure such as loss of fiber compaction and increase
of extracellular space between fibers [5]. Muscle texture is also affected by other parameters
such as fish rearing practices, seasonality and methods of capture, handling and processing [6,
7]. The main quality parameters for fresh fish are fat, color, texture and freshness.
Double compression makes it possible to perform TPA from a plot of forcetime curves
[8]. Other terms used to describe texture are firmness, stiffness and yield point [9]. When
using instrumental methods, such measurements are limited by the instrumental behavior of
materials in terms of stress, strain and time effects. Many attempts have been made to
correlate physical measurements with sensory evaluation of texture (Botta 1991).
Reproducibility of texture measurements is affected by the sampling technique because of the
heterogeneity of the fillets, this is mentioned, especially, for freshwater fish species such as
common carp, tench, perch and some others [10]. Therefore, it is difficult to find a
representative average sample in fish and measurements of textural properties may depend on
the location within the fillet. Some authors recommend raw fish to be tested in the form of a
fillet or a part of a fillet [11].
Texture profile analyses of fish flesh tends to commercial use where the data can be used
in several forms of product development, as to what can be incorporated into new fish
products and also has a substantial effect on the desirable storage period of frozen fish,
maintained in proper conditions, during their shelf life as mentioned in [8]. Besides fish
farming, the results can be used in different methods of fish processing into value added
products. In general, commercial exploitation of fish is directed to simply marketing fish
products as a favorite food.
Lipids
Dietary n-3 polyunsaturated fatty acids (PUFA), namely eicosapentaenoic acid (EPA -
(20:5n-3) and docosahexaenoic acid (DHA-22:6n-3), are essential in human nutrition for
prevention of cardiovascular disease, and for countering certain visual and mental disorders.
Recommendations on the required amounts of the fatty acids DHA and EPA have existed
since 1994. The minimum recommended daily intake 200 mg of EPA and DHA has not been
saturated in Western diet. The intake of both the acids has been derived, more or less
exclusively, from fish and fish products. In contrast to marine fish, the natural diet of many
freshwater fish is not rich in DHA, but is rich in linoleic acid and -linolenic acid, and, to a
lesser extent, in EPA. Many freshwater fish, therefore, readily convert C18 PUFA to their C20
and C22 homologues. The fatty acid composition of freshwater fish species is affected by
several factors and tabular values for the individual species can thus be less easily
Basics of Seafood Quality Indices 335
available. Commonly, oleic, palmitic, palmitoleic and linoleic acids, EPA and DHA are the
major acids in fish flesh. Tailoring the fatty acid composition of farmed fish by controlling
their dietary lipids is possible, while it seems that water temperature per se does not fully
control the PUFA content. Unsaturated fatty acids, namely EPA and DHA, seem to be stable
under the usual culinary treatments. Thus, lipids of freshwater fish are a most valuable source
of the essential PUFA [12].
Fish Fatty Acid Composition
In general fish are the most important food source for n-3 highly unsaturated fatty acids,
namely carbon chain length C20 with 5 double bonds and C22 with 6 double bonds, (n-3 highly
unsaturated fatty acids - HUFA) in the human diet. The importance of n-3 fatty acids,
especially n-3 HUFA in human health, has been reviewed in a number of publications [13-
17], with a large amount of evidence on the positive health effects of fish consumption. Even
when it is valuable for health reasons, locally produced freshwater fish is generally not
recognized as the best source of these fatty acids and their role in the human food chain is
frequently underestimated [18].
Marine fish are commonly divided into oily (pelagic) and non-oily (demersal) species
[19, 20]. Oil reserves of the former group, embracing herring (Clupea harengus), mackerel
(Scomber scombrus), capelin (Mallotus villosus), sprats, sardines, pilchards and anchovies,
are stored chiefly in fillets at a level of 20% or more of the wet weight of the fish. Oil
reserves of the latter group are stored in the fish liver, not in the remaining flesh. In cod,
haddock and whiting, oil comprises over 50% of liver wet weight. In so-called northern (or
high-latitude) marine fish, body oil content is the predominant source of potential metabolic
energy for the fish including palmitic acid, oleic acid, gadoleic acid, cetoleic acid, EPA and
DHA. Marine fish so far studied probably have lost the ability to convert C18 PUFA to their
C20 and C22 homologues. Their diet consists chiefly of high PUFA level, including HUFA.
Marine zooplankton retain the n-3 PUFA ingested in their phytoplankton diet and, moreover,
can produce and store long-chain monounsaturated gadoleic acid and cetoleic acid that are not
present in the phytoplankton. Plankton composition varies widely with location and season.
Marine fish have a much higher requirement for dietary n-3 PUFA than higher terrestrial
vertebrates - including man.
Common carp (Cyprinus carpio), one of the most important farmed freshwater fish
species in Central Europe and Asia, is a well established, cultured species with a well-known
production cycle and is widely consumed as a traditional food. Carp are traditionally reared in
earth ponds on a natural food based diet, with cereal supplementation to increase production.
As cereals are rich in carbohydrates and have very low levels of n-3 fatty acids, farmed carp
flesh generally contains a high level of oleic acid and a low level of favorable n-3 HUFA
[21]. The n-3 HUFA content in pond-produced, carp flesh content is influenced by several
factors, of which diet is the most important [22]. Carp are omnivores and their natural food
consists mainly of plankton, zooplankton, zoobenthos and detritus [23]. Plankton [24, 25] and
benthos [26, 27] naturally contain high levels of n-3 fatty acids (FA), including EPA and
DHA. There is a common misconception that farmed fish in general are not as rich in n-3
PUFA as wild fish. These dissimilarities are very likely caused by the immense differences in
fat quality noted in the natural food available to wild fish v. the pellets given to cultured fish.
Proper pond management, resulting in a suitable structure of plankton and benthic

communities, is highly important when managing carp fatty acid composition in the resulting
food products.
Important freshwater species within world aquaculture are silver carp
(Hypophthalmichthys molitrix), common carp, grass carp (Ctenopharyngodon idella), bighead
carp (Aristichthys nobilis), rainbow trout (Oncorhynchus mykiss), tilapias and catfish
(Ictalurus punctatus). China has been the world's leading producer with carp being the major
fish reared.
Compared to marine fish, freshwater fish contain comparable levels of oleic acid,
palmitic acid and palmoleic acid, while higher levels of linoleic acid and -linolenic acid
(ALA) and also substantial concentrations of EPA and DHA. The ratio of total n-3 to n-6
fatty acids is lower for freshwater fish, usually ranging between 0.5 and 4.0, due to the higher
proportion of n-6 PUFA, mainly of linoleic acid. The lipid pattern is markedly influenced by
the natural food of the fish. Freshwater algae, crustacea, and aquatic larvae of insects are
generally rich in linoleic acid [12].
Fish soft roe (ova, eggs) and hard roe (spermatozoa) are also consumed. Great differences
in total lipid content, between 0.4 - 1.1 and 6.3% of fresh weight are reported for soft roe of
common carp from Central Europe [28] and Australia [29], respectively. Total lipid content in
soft roe ranged between 0.5 and 3.2% of fresh weight in winter and summer respectively. The
order of major fatty acid content is similar to that in hard roe [28]. Lipid content and
composition in hard and soft roes of rainbow trout is greatly influenced by the essential fatty
acids available from the fish diet [30].
Biogenic Amines in Seafood
Biogenic amines (BAs) such as putrescine (PUT), cadaverine (CAD), spermidine

(SPD) spermine (SPM), histamine (HIM), tyramine (TYM), tryptamine (TRM) an
phenylethylamine (PEA) are widely distributed in proteinaceous foods. BAs are formed by
decarboxylation of amino acids as a result of metabolic processes. In stored fish flesh, amines
are generated by the action of spoilage bacteria decarboxylases. There are two main reasons
for amine determination in food quality assessment. The first is their potential toxicity, the
second is the possibility to use them as decomposition markers.
The high content of proteins in the fish meat represents a risk of rapid formation of BAs.
Amines are found at very low levels in fresh fish, and their accumulation is associated with
bacterial spoilage [31]. The key factors contributing to the presence and accumulation of BAs
are the availability of free amino acids, pH level, water activity and temperature. The
optimum pH of amino acid decarboxylases is acidic, ranging from 2.5 to 6.5 [32]. The quality
of the raw material, time duration and temperature of storage are critical to the accumulation
of BAs [33]. HIM is the amine, most frequently studied in fish, due to its toxicity and to its
rapid formation in many fish species. HIM can be produced readily by bacterial
decarboxylases mainly in scombroid fish, belonging to both Scombridae and
Scomberesocidae families [34], that have relatively high free histidine levels in their muscles
when alive [31]. Bacteria, present in the water environment or introduced during fish
handling, produce histidine decarboxylase, which converts histidine to HIM, a process which
is accelerated if fish are not kept chilled or frozen [35]. The importance of microbial
contamination for BAs formation is evident. Gutted hake (Merluccius merluccius) stored in
ice contained much higher content of BAs and showed lower sensory scores compared to
ungutted fish [36]. HIM is more toxic in the presence of other BAs, viz. PUT and CAD, since
these diamines may inhibit the in vivo mechanisms of HIM detoxification, mainly by the
action of diamine oxidase.
Biogenic amines can serve as indicators of the decomposition of fish. PUT, CAD, HIM
and TYM are most often discussed in this context [37]. CAD may be regarded as the specific
spoilage marker for hake (Merluccius merluccius) stored at chilling temperatures, however,
for this kind of preservation the sum of PUT, CAD HIM and TYM content seems to be a
better indicator of spoilage [38]. The levels of CAD usually increase later than that of PUT,
but the final concentrations of CAD are generally higher. These trends are observed not only
for marine, but also for fresh water species [39-41]. For rainbow trout PUT, TYM, SPD and
SPM content is recommended as the best quality indicator of spoilage [42].
Importance of Biogenic Amines in Fish
In foods of all kinds BAs are primarily produced by microbial decarboxylation of amino
acids or by enzymes present in the raw material [43]. Aromatic amines, HIM and TYM, are
vasoactive. The effects of TYM on healthy individuals are usually limited to headache or
migraine [44]. HIM is associated with "scombroid poisoning" - nausea, hypo-tension,
flushing, urticaria [45]. Enzyme action destroying aromatic amines in the digestive tract can
be inhibited by medications - antidepressants, tuberculostatics, mucolytics [46] or by
coincident ingestion of PUT and CAD. The toxic levels of aromatic amines to humans are
still uncertain. HIM levels above 500 - 1000 mg/kg and TYM levels above 100 - 800 mg/kg
are considered potentially dangerous to human health [47]. Oral toxicity levels for PUT and
CAD have been a subject of ongoing discussions. A large study of dietary exposure
assessment focusing on polyamines determined the tolerable level of CAD in fish as 510
mg/kg [48]. People having deficient natural mechanisms for detoxifying BAs for genetic
reasons or through inhibition due to the intake of anti-depression medicines are more
susceptible to BAs poisoning [46].
HIM, TYM, PUT and CAD are significant both in the safety and in quality determination
of fish as a human food. These four BAs are used as indicators of spoilage of sardines [37] or
pike-perch (Sander lucioperca) [49]. Fresh water species have been studied to a much smaller
extent compared to marine fish. PUT and TYM are proposed to be good quality evaluation
indices for crucian carp (Carassius auratus) [50]. As BAs are formed mainly by the action of
bacteria, suppression of their proliferation is crucial in BAs regulation. Some other authors
recommend using PUT, CAD and TYM as indicators, due to low content of SPD and SPM in
carp [51].
Different fish species might reach different BA levels at the sensory rejection time.
Ozogul et al. (2006) [52] found, that samples of European eel (Anguilla anguilla) at the
sensory rejection level contained 4688 mg PUT/kg. For red mullet (Mullus barbatus) PUT
content exceeding 10 mg/kg was associated with poor quality samples [53]. Similar values of
1314 mg/kg was found by Chytiri et al. (2004) [42] for rainbow trout. Supposing that 15 mg
PUT/kg represents a critical concentration, Kek et al. 2002 [54] tried to find the respective
critical times (days), when the given concentration had been reached.
As the kinetic curves of PUT formation usually show a smooth increase, they can be
described by regression equations [38, 40]. By solving these equations for the proposed
critical concentration (15 mg/kg), we can obtain estimates of the respective critical storage
time in days). The critical content of PUT at level 15 mg/kg is reached in 1115 days in
filleted flesh kept at 3C and in 610 days in fish flesh which has been minced. At 15C the
differences between fillets and mince are not so pronounced, but a higher storage temperature
shortened this time to 23 days.
The content of PUT seems to be a good quality indicator for common carp, rainbow trout
and perch (Perca fluviatilis) [purpose, CAD can be also used as an alternative measure,
because both of these amines demonstrate the best correlation with sensory levels. Histamine
and TYM are formed preferentially in samples at 15C and thus their occurrence may signal
improper temperature conditions during storage. The CAD, HIM and TYM sometimes
showed a slower rate of formation compared to sensory signals. According to the calculated
number of critical days for the PUT content, it was found that fish fillets and fish mince of
common carp, rainbow trout and perch do not significantly differ in their susceptibility to
spoilage. The mincing action accelerated the spoilage, especially in samples stored at 15C.
Fish fillets and fish mince stored at 15C signaled decay in chemical and in organoleptic
indicators in 2 days, fish mince stored at 3C in about 8 days while fish fillets stored at 3C
showed spoilage after about 14 days. Samples of fish flesh and fish mince did not contain
toxicologically significant concentrations of HIM or TYM at the later time. PUT, CAD and
SPD levels increased throughout the storage period of European eel flesh especially in
samples stored without ice. The levels present at the time the samples were rejected at the
sensory level contained 5090 mg/kg of PUT and 3090 mg/kg of CAD [52]. For rainbow
trout PUT, TYM, SPD and SPM are recommended as quality indicators [55].
CONCLUSION
There are numerous methods to determine the quality of seafood under particular
processing, transport and storage conditions. Instrumental methods are steadily gaining
importance in determining the sensory quality of seafood. The main problem with
instrumental methods is the low correlation between results from instrumentally examined
samples and samples that are analyzed by assessors. This study concludes that a combination
of sensory and instrumental methods is the best form of decision making on the shelf life of
seafood. Biogenic amines on the other hand are widely used as the K values have high
correlation with quality indices (i.e., sensory values). However, there is still a need to validate
the methods (i.e., sensory and biogenic amine content) to determine the quality indices of
seafood under different conditions.
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ABOUT THE EDITORS
smail Yksel Gen, Research Assistant, MSc and PhD candidate

Fishing and Processing Technology Department
Email: ismailgenc@sdu.edu.tr, ismailygenc@gmail.com
smail Yksel Gen is a Ph.D candidate in the Department of fishing and Processing
Technology at the University of Suleyman Demirel where he has been faculty member since
2011.
smail Yksel completed his M.Sc. at Suleyman Demirel University and he has been in
Portugal for the research of his M.Sc. thesis. He has been in Denmark for his Ph.D thesis at
Denmark Technical University (DTU) with a national grant that was provided by The
Scientific and Technological Research Council of Turkey (TUBITAK). His research interests
involve predictive modeling, shelf life prediction of seafood and interaction models to
determine both the quality and safety of fishery products under different conditions. He has
collaborated national and international projects on determination of quality and safety of
seafood products, new product development and estimating the current situation of some
fishery products to take precautions on quality and safety. He is still a member of scientific
associations such as ISEKI-Food and International Association for Food Protection (IAFP).
He has currently authored and co-authored seven peer-reviewed publications and attended
more than twenty peer-reviewed national and international conferences.
Dr. Eduardo Esteves, Professor

Departamento de Engenharia Alimentar, Instituto Superior de Engenharia,
Universidade do Algarve and CCMAR Centre of Marine Sciences
Email: eesteves@ualg.pt
Eduardo Esteves worked on fish biology and ecology for ca. 10 years (BSc with honors
in Marine Biology and Fisheries, Universidade do Algarve (UAlg), MSc in Ecology from the
Universidade de Coimbra (UC), and PhD in Population Ecology from UAlg). In the last 10
years, Eduardo Esteves changed the focus of his research interests to seafood quality thus
making his primary interests coincide with his current appointment as Professor Adjunto in
344 About the Editors
the Universidade do Algarve (South Portugal), wherein he teaches undergrad (BEng) and
grad (MSc) courses in Sensory Analysis (Sensometrics), Seafood Quality/Processing and
Transformation of Aquatic Products, Applied Statistics and Experimental Design, and
Statistical Process Control, and has supervised several undergrad and MSc students' final
internships and thesis. Eduardo represents the university in the technical committee on
seafood (CT-25) of the Instituto Portugus da Qualidade. As a member of the CCMAR
Centre of Marine Sciences, his current research interests deal with the study and statistical
modelling of sensory, physical-chemical and microbiological changes in fish and seafood
products, both high-valued species such meagre, shrimp or clams as well as under-utilized
species like mackerels or catsharks.
Dr. Abdullah Diler, Professor

Department of Fishing and Processing Technology, Faculty of Fisheries,
University of Suleyman Demirel, Isparta and Department of Fishing and Processing
Technology, Faculty of Fisheries, University of Katip elebi, zmir
Email: abdullahdiler@sdu.edu.tr
Abdullah Diler had his B.Sc. in Veterinary Science, Ankara University and Ph.D in
Aquaculture Engineering from Suleyman Demirel University. His research interests are
consisting of seafood quality and safety, welfare in fish, hygiene, seafood processing
technology, food chemistry, food microbiology and seafood borne diseases. More than 10
years he teaches undergraduate (B.Sc) and graduate (M.Sc. and Ph.D) courses in accordance
with his research area. He supervised more than 10 M.Sc. and ca. 3 Ph.D. thesis. He is
currently a member of several scientific commissions namely, Veterinary food hygienists
council, Veterinary chamber of Isparta and foundation of Turkish veterinary society. He
authored and co-authored more than 30 peer-reviewed articles and had place as keynote
speaker nearly 10 scientific symposiums.
INDEX
A C
active packaging, 178, 185, 186, 194, 198, 199, 201, calibration, 48, 49, 55, 61, 66, 67, 70, 79
260, 262 Cambaridae, 100
Agricultural Research Center (ARS), 230 carassius carassius, 100, 106, 118, 283
Alaska Pollock, 71, 81, 82, 83 carp, 53, 54, 64, 74, 100, 101, 102, 103, 104, 105,
amines, 41, 42, 45, 58, 63, 102, 103, 111, 114, 116, 106, 112, 113, 115, 116, 117, 118, 198, 266, 267,
125, 200, 242, 333, 336, 337, 338, 340, 341 268, 277, 282, 283, 295, 318, 335, 336, 337, 338,
Amur catfish, 100 340, 341, 342
antibody-based methods, 247 catfish, 74, 83, 100, 101, 110, 124, 125, 277, 281,
appraisal costs, 305, 306, 309, 310 291, 294, 301, 331, 336
Arrhenius, 225 catla, 100, 105, 106, 117
Asian swamp eel, 100, 111 cepstrum, 76
aspect ratio, 72 CFU, 14, 111, 183, 249, 250, 251, 252, 253, 268,
astaxanthin, 51, 52, 66, 68, 79, 80 291
Atlantic salmon, 51, 52, 62, 67, 68, 71, 74, 78, 79, channa argus, 100, 110, 124, 267, 272
80, 81, 83, 99, 100, 107, 119, 120, 121, 122, 145, channel, 24, 88, 100, 110, 124
149, 197, 198, 203, 205, 266, 339, 340 channidae, 100
authentication, 51, 98, 139, 140, 141, 145, 146, 148 chemical, vii, 6, 7, 8, 9, 11, 14, 15, 21, 32, 40, 41,
autolytic, vii, 14, 40, 41, 42, 102, 140, 176 42, 47, 48, 49, 50, 52, 55, 57, 60, 61, 62, 63, 68,
89, 94, 99, 102, 103, 104, 105, 106, 109, 110,
111, 112, 113, 114, 115, 117, 118, 119, 122, 123,
B 124, 125, 127, 133, 135, 139, 140, 141, 143, 144,
145, 146, 147, 161, 179, 180, 192, 193, 194, 197,
bacteria-bacteria interactions, 241
199, 204, 205, 209, 210, 211, 214, 215, 259, 263,
Bighead carp, 53, 100, 105, 116, 117, 266, 336
273, 275, 276, 277, 279, 287, 292, 299, 300, 301,
biogenic, vii, 9, 11, 19, 32, 41, 53, 58, 63, 102, 103,
308, 316, 323, 327, 328, 330, 338, 341, 342
106, 111, 114, 116, 117, 118, 119, 123, 124, 125,
Chinese mitten crab, 55, 100, 111, 125
140, 153, 226, 242, 328, 333, 336, 337, 338, 340,
chuatsi, 100, 111, 114, 125
341, 342
cichlidae, 100, 106
biogenic amines, vii, 9, 11, 32, 41, 53, 58, 102, 103,
circularity, 72
106, 114, 116, 119, 123, 124, 140, 153, 227, 328,
cirrhinus mrigala, 100, 105, 117
333, 336, 337, 338, 340, 341, 342
clams, 8, 71, 81, 149, 184
black carp, 100, 106, 118
cod, 3, 6, 29, 41, 43, 50, 52, 74, 76, 81, 83, 91, 97,
brochothrix thermosphacta, 224, 242
121, 143, 148, 153, 261, 265, 271, 277, 330, 335,
339
color, 6, 14, 32, 34, 35, 36, 37, 38, 39, 43, 45, 47, 51,
53, 58, 60, 65, 66, 67, 68, 69, 73, 74, 76, 77, 78,
346 Index
79, 80, 81, 83, 84, 85, 90, 102, 104, 108, 109, fat content, 52, 68, 80, 108, 129, 235, 244, 282
129, 131, 176, 192, 197, 199, 200, 202, 263, 275, finfish, 7, 52, 53, 100, 101, 183, 216, 249
334, 339 fish roes, 67, 80
color change index, 67 fish salting, 303, 307, 308, 309, 310, 311
color retention index, 66 Fish Salting, 303, 307, 308, 309, 310, 311
color standard, 67 fish sorting system, 76, 84
color temperature, 66, 67 fish trade, 152, 154, 156, 162, 166, 167, 168, 169,
colorimeter, 7, 49, 51, 66, 67 170, 330, 331
combase, 224, 228, 229 food borne pathogens, 224, 281, 341
common carp, 99, 100, 104, 113, 115, 116, 334, 335, food hygiene, 157, 161, 165, 315, 322, 330
336, 338, 339, 340 Food MicroModel, 224
consumption, vii, 1, 4, 5, 6, 7, 8, 10, 13, 15, 16, 17, food safety systems, 151, 154, 160, 209, 210, 310
18, 20, 24, 25, 33, 34, 40, 41, 42, 46, 49, 51, 88, food spoilage and safety predictor (FSSP), 224, 225,
102, 106, 127, 128, 129, 139, 140, 142, 146, 147, 227, 232, 233
152, 154, 159, 169, 170, 180, 192, 193, 194, 199, food-borne hazards, 209, 211
202, 209, 210, 211, 216, 217, 218, 219, 220, 224, freshness, 1, 6, 14, 15, 21, 23, 29, 31, 32, 33, 34, 35,
227, 229,243, 247, 282, 316, 321, 322, 328, 329, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 49, 50,
330, 331, 335 51, 52, 53, 55, 56, 57, 58, 59, 63, 70, 77, 79, 84,
control measurements, 323 92, 99, 102, 105, 106, 109, 111, 112, 113, 118,
correlation, 31, 50, 51, 52, 53, 54, 55, 62, 63, 72, 73, 121, 125, 128, 139, 140, 179, 195, 241, 292, 334,
74, 80, 82, 107, 109, 117, 129, 338 341, 342
crab, 16, 18, 55, 64, 76, 84, 100, 111, 125, 127, 128, freshwater crayfish, 100, 101, 112, 133
130, 131, 135, 136, 184, 249, 250, 255, 265 fuzzy logic, 74
crawfish, 74, 83
crayfish, 97, 100, 112, 125, 127, 128, 132, 133, 135,
136, 137, 184 G
crucian carp, 100, 106, 118, 283, 337, 342
gender predictor, 71
crustaceans, v, 3, 6, 14, 16, 20, 33, 100, 101, 127,
generic growth, 227
128, 130, 133, 140, 184, 198, 277, 286
genospecies, 17
ctenopharyngodon idella, 53, 100, 102, 112, 113,
geographic origin, 139, 140, 141, 143, 144, 145, 148
336
geometric morphometrics, 71, 81
cyprinidae, 100, 102, 112
good hygiene practices (GHP), 7, 161, 315
cyprinus carpio, 54, 64, 100, 104, 113, 115, 116,
good manufacturing practices (GMP), 7, 10, 161,
335, 339, 340, 341
163, 307, 308, 315
grass carp, 53, 100, 102, 112, 113, 114, 336
D green-shelled mussels, 73
grey mullet, 74
defects, 65, 66, 73, 81, 196 gurnard, 76, 84
E H
Eastern Regional Research Center (ERRC), 230 halotolerant, 17, 28

edible films, 109, 123, 191, 192, 196, 198, 202, 262, hazard analysis and critical control point (HACCP),
263 vi, 7, 10, 26, 33, 157, 158, 159, 160, 161, 162,
eriocheir sinensis, 55, 100, 111, 125 163, 165, 240, 303, 304, 305, 307, 309, 310, 311,
euclidian distance, 72 312, 313, 314, 315, 316, 322, 329, 330
experimental designs, 240 heavy metals, vii, 9, 139, 140, 141, 142, 143, 145,
exponential and square-root, 225 146, 147, 153, 211, 220, 316, 323
herring, 3, 33, 34, 37, 40, 43, 50, 58, 73, 74, 79, 83,
226, 265, 271, 277, 335
F hypophthalmichthys molitrix, 53, 63, 100, 103, 112,
114, 115, 267, 282, 336
failure costs, 304, 305, 306, 308, 310, 311
Index 347
hypophthalmichthys nobilis, 100, 105, 113, 117 MicroHibro, 230

model, 46, 48, 49, 51, 72, 73, 74, 76, 93, 96, 105,
106, 109, 158, 211, 216, 217, 218, 219, 226, 227,
I 229, 230, 231, 232, 233, 234, 235, 236, 237, 239,
240, 242, 243, 244, 272, 303, 304, 305, 307, 308,
ictaluridae, 100
310, 311, 312, 313, 314
Ictalurus puntactus, 100, 110
modified atmosphere, 6, 10, 37, 104, 116, 122, 123,
illuminant, 66
125, 133, 176, 179, 180, 182, 189, 191, 192, 194,
image analysis, 32, 45, 65, 66, 67, 68, 70, 71, 72, 73,
195, 196, 199, 224, 241, 242, 261, 262, 263, 267,
74, 77, 78, 79, 80, 81, 82, 83, 84, 85
269, 330
instrumental, v, 6, 9, 21, 31, 32, 40, 44, 49, 50, 51,
monopterus albus, 100, 111, 125
53, 55, 56, 60, 62, 63, 93, 121, 132, 136, 333,
mrigal, 100, 105, 117
334, 338, 339
mrigal carp, 100
intelligent packaging, 191, 192, 194, 198, 199, 202
multivariate, 31, 46, 48, 61, 71, 145, 330
interaction models, 241
mylopharyngodon piceus, 100, 106, 118
international trade, 7, 156, 168, 169, 170, 171, 173,
191, 209, 210, 211
N
L National Science and Technology Support Program,
94
Labeo rohita, 100, 105, 117, 118
neural network, 46, 72, 75, 77, 84, 90
laser triangulation, 73
nile tilapia, 100, 106, 118, 119
liquid smoke, 78, 85
northern snakehead, 100, 110, 124
listeria monocytogenes, 10, 11, 15, 17, 20, 22, 26,
nucleic acid-based methods, 247, 253
29, 113, 124, 128, 129, 140, 177, 178, 182, 185,
216, 219, 220, 224, 225, 241, 242, 243, 244, 245,
247, 248, 255, 256, 257, 271, 272, 277, 281, 282, O
288, 289, 290, 294, 295, 299, 331
lobster, 19, 127, 128, 131, 132, 134, 136, 203 oncorhynchus mykiss, 82, 85, 100, 109, 114, 116,
122, 123, 124, 198, 267, 282, 283, 336, 340, 342
oreochromis niloticus, 10, 52, 100, 106, 118, 119,
M 296
oysters, 8, 15, 20, 24, 26, 50, 52, 68, 71, 73, 80, 82,
machine vision, 40, 45, 58, 65, 67, 70, 71, 72, 74, 75,
92, 183, 184, 188, 211, 216, 217, 218, 219, 220,
77, 78, 79, 80, 82, 83, 84, 85
242, 249, 250, 255, 256, 277
mahi mahi, 78
mandarin fish, 100, 111, 125
MAP, 47, 90, 301 P
mathematical models, 224
megalobrama amblycephala, 100, 106, 118, 271 packaging, vi, vii, 6, 8, 85, 104, 109, 112, 122, 123,
mesophilic, 15, 17, 18, 20, 26, 102, 176, 227, 265, 125, 130, 131, 133, 156, 158, 162, 168, 176, 178,
266, 283, 284, 291 179, 180, 182, 184, 185, 186, 187, 189, 191, 192,
metabolic pathways, 145 193, 194, 195, 196, 198, 199, 200, 201, 202, 203,
microbial, vii, 6, 10, 14, 18, 21, 22, 23, 25, 28, 29, 204, 206, 207, 224, 241, 260, 261, 262, 263, 265,
32, 40, 43, 45, 52, 53, 55, 87, 102, 104, 105, 109, 267, 269, 270, 271, 280, 287, 291, 295, 301, 305,
117, 122, 123, 124, 127, 128, 129, 130, 131, 132, 330
134, 135, 136, 149, 176, 177, 178, 179, 184, 185, packed, 6, 10, 52, 62, 104, 106, 108, 110, 115, 116,
191, 192, 193, 194, 197, 198, 199, 202, 203, 204, 119, 121, 124, 130, 131, 133, 135, 178, 184, 195,
205, 210, 220, 226, 227, 229, 241, 242, 243, 244, 199, 200, 204, 241, 242, 266, 295, 301, 308, 319,
254, 259, 261, 263, 264, 270, 272, 277, 286, 287, 341, 342
291, 293, 294, 295, 297, 298, 319, 325, 326, 328, pandemic, 16, 24
336, 337, 341 pangasianodon hypophthalmus, 100, 110, 111, 124
microbial responses viewer, 229 pangasidae, 100
microbial spoilage models, 227 pangasius, 100, 101, 110, 111, 124, 125
348 Index
Pathogen Modeling Program (PMP), 224, 230, 232,

235, 236, 237, 244
Q
PCA, 46, 49, 51, 52, 54, 55, 68
Q value, 53, 102, 105
percichthyidae, 100
quality costs, 304, 305, 306, 307, 308, 310, 311, 313
phage-based detection systems, 247
Quality Index Method (QIM), 32, 33, 35, 36, 37, 38,
photobacterium phosphoreum, 109, 224, 261, 270
39, 40, 49, 50, 52, 53, 54, 57, 58, 333
physical, 15, 21, 47, 50, 52, 54, 61, 62, 64, 70, 71,
quality indicators, 139, 141, 333, 338
78, 81, 89, 99, 102, 103, 112, 113, 114, 116, 118,
119, 127, 135, 158, 161, 177, 180, 191, 193, 194,
202, 203, 205, 210, 216, 220, 308, 316, 323, 324, R
325, 326, 327, 328, 334, 339
physical parameters, 70 rainbow trout, 50, 68, 72, 78, 80, 99, 109, 114, 116,
pink salmon, 73, 77, 83 122, 123, 124, 198, 220, 263, 267, 282, 283, 336,
pixels, 67, 69, 72 337, 338, 340, 341, 342
PLSR, 46, 51, 52 Rainbow Trout, 61, 82, 85, 109, 206, 271, 294,
polarized light, 69, 77 295, 340
post mortem, 99, 102, 103, 104, 105, 106, 108, 112, rapid detection methods, 23, 247, 248, 251, 253
128, 131 regulatory frameworks, 151, 154, 161, 167
predictive microbiology, 223, 224, 232, 240, 241, relative rate of spoilage, 225
243, 244, 245 rigor development, 72, 107, 119
prevention costs, 305, 306, 307, 308 rigor mortis, 41, 67, 80, 81, 103, 105, 107, 108, 109,
private standards, 151, 152, 166, 167, 168, 169, 170, 115, 120, 121, 202
173 risk assessment, vii, 8, 10, 11, 156, 159, 160, 161,
Procambarus clarkia 209, 210, 211, 214, 215, 216, 217, 218, 219, 220,
Procambarus clarkii, 92, 100, 112, 125, 133, 136, 223, 224, 229, 237, 240, 241, 243, 245
137 risk ranger, 224, 229, 232
processing, v, vi, vii, 1, 4, 5, 6, 7, 8, 9, 10, 18, 20, 21, rock sole, 75, 84
22, 23, 26, 29, 46, 47, 51, 55, 57, 58, 62, 63, 65, rohu, 100, 105, 117, 118
68, 71, 73, 74, 75, 76, 78, 79, 82, 84, 85, 91, 95,
96, 115, 116, 121, 124, 127, 128, 129, 130, 131,
132, 133, 134, 135, 136, 144, 145, 152, 157, 158, S
160, 163, 164, 172, 178, 183, 184, 185, 186, 188,
189, 192, 193, 194, 203, 211, 218, 220, 223, 235, safety, v, vi, vii, 1, 6, 7, 9, 10, 11, 21, 22, 23, 26, 27,
237, 238, 245, 247, 248, 250, 255, 256, 259, 260, 29, 47, 49, 56, 57, 58, 59, 60, 61, 87, 88, 93, 97,
266, 270, 273, 276, 295, 303, 304, 305, 307, 308, 105, 111, 113, 134, 140, 143, 146, 151, 152, 153,
309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
320, 321, 322, 323, 324, 325, 326, 328, 329, 330, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,
331, 333, 334, 338, 339 175, 176, 177, 178, 179, 182, 184, 185, 186, 188,
production, 1, 2, 3, 4, 5, 6, 10, 13, 17, 18, 21, 52, 62, 191, 192, 194, 197, 199, 202, 203, 204, 205, 210,
88, 91, 92, 97, 99, 100, 101, 103, 107, 110, 111, 219, 220, 221, 223, 224, 225, 229, 235, 243, 244,
112, 124, 128, 129, 140, 141, 144, 145, 152, 153, 248, 253, 259, 264, 268, 275, 276, 277, 279, 292,
158, 159, 162, 164, 167, 169, 176, 180, 181, 185, 293, 300, 304, 305, 306, 307, 310, 312, 315, 316,
196, 205, 209, 210, 216, 217, 218, 221, 230, 245, 318, 320, 321, 322, 328, 333, 337, 342
248, 253, 279, 299, 304, 306, 307, 310, 312, 313, salmo salar, 51, 52, 57, 63, 80, 91, 100, 107, 119,
314, 320, 321, 322, 324, 328, 335 120, 121, 122, 149, 197, 198, 203, 205, 265, 266,
pseudomonads, 128, 195, 196, 224, 232, 233, 235, 339, 340
240, 242, 244 salmon, 6, 10, 11, 28, 51, 52, 62, 66, 67, 68, 71, 72,
psychro, 17 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 99, 100,
psychrophilic, 14, 18, 28, 268, 283, 291 107, 108, 119, 120, 121, 122, 145, 147, 149, 154,
psychrotrophic, 14, 102, 109, 176, 180, 188, 227, 178, 189, 196, 197, 198, 203, 204, 205, 220, 237,
244, 265, 266, 283, 284 238, 239, 242, 244, 245, 250, 253, 256, 265, 266,
public health, 8, 15, 21, 140, 156, 159, 161, 209, 271,277, 281, 286, 294, 339, 340
210, 216, 219, 235, 247, 259, 294 salmonidae, 100, 107
Index 349
Sanitary and Phytosanitary Measures (SPS) spoilage, 6, 8, 9, 10, 13, 14, 21, 24, 28, 29, 32, 42,
Agreement, 155 43, 46, 47, 51, 52, 53, 59, 93, 102, 109, 110, 122,
sanitation, vi, 157, 159, 182, 219, 305, 306, 307, 128, 129, 130, 131, 132, 134, 140, 144, 145, 176,
308, 315, 316, 319, 321, 329, 330 177, 178, 179, 180, 182, 183, 184, 185, 186, 187,
sanitation applications, 316 188, 191, 192, 193, 195, 196, 200, 207, 223, 225,
seabass, 71, 81, 196, 204 226, 227, 228, 229, 232, 235, 240, 241, 242, 244,
seabream, 71, 77, 196 259, 261, 262, 264, 268, 269, 270, 275, 276, 277,
seafood packaging, 191, 192, 194, 199, 202 279, 282, 283, 288, 291, 292, 300, 306, 307, 328,
seafood plants, 303, 315 336, 337, 338, 342
seafood safety, 9, 10, 24, 26, 88, 161, 169, 170, 172, stable isotopes, 139, 140, 141, 143, 144, 145, 146,
185, 186, 189, 191, 209, 219, 312, 331 148, 149
seafood safety and quality, 10, 24, 26, 161, 169, 170, statistical, 31, 48, 55, 56, 90, 216, 218, 238, 239, 240
191, 331 stunning, 102, 104, 106, 107, 108, 109, 116, 119,
sensory analysis, 15, 31, 32, 40, 44, 50, 55 120, 122
sensory changes, 56, 123, 127, 140, 341 subjective, 7, 33, 46, 56, 66, 215
septicemia, 15, 16 superchilling, 102, 109, 121, 122
shape, 17, 19, 35, 37, 39, 44, 45, 47, 65, 66, 71, 73, swamp eel, 100, 111
74, 75, 76, 81, 82, 83, 184 synbranchidae, 100
shelf life, 1, 8, 10, 31, 32, 33, 43, 102, 103, 104, 105,
106, 107, 108, 109, 110, 112, 114, 115, 122, 123,
130, 175, 176, 177, 178, 179, 180, 182, 183, 185, T
189, 196, 199, 200, 226, 233, 241, 265, 266, 267,
tertiary modeling, 223, 224, 240
276, 319, 333, 334, 338
thermal inactivation, 223, 229, 235, 236, 240, 241,
shellfish, 7, 8, 16, 19, 20, 29, 32, 41, 58, 78, 80, 87,
244
92, 93, 97, 128, 130, 132, 133, 134, 157, 170,
tilapia, 10, 52, 62, 74, 78, 83, 85, 100, 106, 118, 119,
177, 179, 183, 184, 188, 195, 210, 211, 219, 249,
266, 277, 287, 296, 316, 318
254, 256, 277, 286, 293, 314, 340, 341
top view, 73
Shewanella putrefaciens, 14, 176, 224, 261, 266, 270
trace elements, 139, 140, 141, 142, 143, 145, 146,
shine, 34, 66, 69, 78
147
shrimp, 17, 18, 19, 22, 25, 29, 53, 54, 63, 66, 68, 71,
traceability, 60, 61, 92, 139, 140, 141, 144, 149, 158,
72, 80, 81, 82, 97, 98, 127, 128, 129, 130, 134,
160, 167, 173, 240, 329, 331
135, 154, 163, 167, 172, 182, 183, 184, 188, 189,
trout, 74, 100, 109, 122, 123, 124, 189, 198, 263,
196, 198, 204, 205, 206, 250, 256, 267, 268, 273,
266, 267, 277, 282, 283, 316, 318, 338, 341, 342
286, 287, 291, 296, 300, 301
turbot, 6, 54, 64, 74, 83
side view, 72, 73
turn angle, 72, 82
Siluridae, 100
typhoid fever, 19
silurus asotus, 100
silver carp, 53, 100, 102, 103, 112, 114, 115, 198,
267, 268, 282, 336, 340 U
silver salmon, 73
siniperca, 100, 111, 114, 125, 311 uniformity ratio, 72, 82
siniperca chuatsi, 100, 111, 114, 125 United States Department of Agriculture (USDA),
size, 6, 45, 47, 63, 65, 66, 71, 72, 74, 75, 76, 77, 81, 230, 244
105, 117, 125, 153, 184, 195, 309
sizing, 66
slaughter, 6, 99, 102, 104, 106, 107, 108, 109, 112, V
113, 116, 119, 120, 122, 123
slaughtering, 99, 104, 107, 109, 122, 159, 317, 323 vacuum, 52, 57, 63, 104, 108, 109, 112, 116, 121,
smoking, 5, 78, 192, 238, 320 123, 124, 130, 131, 133, 178, 180, 192, 194, 195,
snapper, 52, 57, 63, 76, 84 199, 204, 242, 265, 271, 277, 279, 295, 301, 319,
software applications, 223, 240 341, 342
sorting, 65, 66, 75, 80, 81, 83 varunidae, 100
specific spoilage organisms, 7, 176, 224, 232, 261
350 Index
vibrio parahaemolyticus, 15, 20, 24, 25, 128, 177,

182, 184, 189, 216, 217, 219, 220, 224, 242, 248,
W
254, 255, 256, 257, 277, 289
whiting, 10, 34, 43, 50, 77, 84, 196, 204, 243, 335
visible defects, 73
wuchang bream, 100, 106
visual attributes, 65, 66, 67, 74, 84
visual texture, 65, 66
Y
Yan Zhang, v, 87, 272

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FOOD SCIENCE AND TECHNOLOGY

QUALITY AND SAFETY MAINTENANCE

Additional books in this series can be found on Novas website

Additional e-books in this series can be found on Novas website

QUALITY AND SAFETY MAINTENANCE

SMAIL YKSEL GEN

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Names: Gen, smail Yksel, editor. | Esteves, Eduardo, editor. | Diler,

Published by Nova Science Publishers, Inc. New York

Chapter 10 Elimination and Control of Pathogens by Novel

GENERAL INTRODUCTION TO SEAFOOD QUALITY

Eduardo Esteves1,*, Abdullah Diler2 and smail Yksel Gen2

Keywords: seafood production and consumption, quality and safety

SEAFOOD PRODUCTION AND UTILIZATION

Adapted from [2].

Adapted from [3].

Fish and Seafood Products Utilization

Adapted from [1].

Social and Economical Importance

Adapted from [14].

Occurrence of chemical hazards in seafood is generally due to improper conditions of the

[6] Alasalvar, C., F. Shahidi, K. Miyashita, and U. Wanasundara, 2011. Handbook of

[23] Pouillot, R., G. Veronique, M. L. Delignette-Muller, A. Mahe, and M. Cornu. 2009.

MICROBIOLOGY OF FISH AND FISH PRODUCTS

Regine Helena Silva dos Fernandes Vieira1,2,3,*,

Keywords: seafood, bacteria, legislations, human pathogens

BACTERIAL DECOMPOSITION IN FISH

BACTERIA THAT MAY CAUSE HARM TO THE CONSUMER

Group 1: Bacteria Normally Present in Fish Habitats

Group 2: Bacteria Generally Present in the Environment

Listeria monocytogenes is a mobile, Gram-positive bacterium which grows at 37oC but at

Group 3: Bacteria that Usually Have Man and Other

OTHER CONTAMINANT BACTERIA IN FISH AND FISHERY PRODUCTS

RISK CLASSIFICATION IN A DIETARY FISH CONSUMPTION

BACTERIA WITH SPECIFIC RESTRICTIONS

Table 1 presents a comparison of the bacteria used as a standard to assess the

DETECTION METHODS FOR PATHOGENS

RELATING SENSORY AND INSTRUMENTAL

Keywords: sensory analysis, instrumental methods, multivariate statistical techniques,

METHODS TO ASSESS SEAFOOD FRESHNESS AND QUALITY

US Seafood Inspection Program

Quality Index Method

Attribute Description Score

Quality parameter Description Score

Quality parameters Attributes Score

Quality parameters Description Score

Parameters Description Score

Physicochemical and Instrumental Methods

Table 7. Torry scoresheet for freshness evaluation of cooked herring

Score Odor Flavor Texture

Odor Flavor Score

QI = (histamine + putrescine + cadaverine)/(1 + spermidine + spermine)

HPLC is commonly carried out to determine BA concentrations because of its sensitivity,

Total Volatile Basic Nitrogen

Torrymeter, Fischtester, Freshtester

Machine Vision System

Electronic Nose and Tongue

Multi-sensor data fusion and the Artificial Quality Index

RELATIONSHIPS BETWEEN INSTRUMENTAL AND SENSORY TRAITS

Successes in Well-Known, Recognized Species

of spectroscopic techniques (Vis/NIR spectroscopy) are listed, e.g., detection of astaxanthin

Applications Pertaining to Less-Know, Emerging Species

S = [(LP - LNP)2 + (aP - aNP)2 + (bP - bNP)2]1/2 (1)