Fish Protein Hydrolysates Production, Biochemical and Functional Properties

Critical Reviews in Food Science and Nutrition
ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: http://www.tandfonline.com/loi/bfsn20
Fish Protein Hydrolysates: Production,

Biochemical, and Functional Properties
Hordur G. Kristinsson & Barbara A. Rasco
To cite this article: Hordur G. Kristinsson & Barbara A. Rasco (2000) Fish Protein Hydrolysates:
Production, Biochemical, and Functional Properties, Critical Reviews in Food Science and
Nutrition, 40:1, 43-81, DOI: 10.1080/10408690091189266
To link to this article: http://dx.doi.org/10.1080/10408690091189266
Published online: 03 Jun 2010.
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Download by: [Texas A&M University Libraries] Date: 12 January 2016, At: 11:28
Critical Reviews in Food Science and Nutrition, 40(1):43–81 (2000)
Fish Protein Hydrolysates:

Production, Biochemical, and Functional
Properties
Hordur G. Kristinsson* and Barbara A. Rasco**
Institute for Food Science and Technology, The School of Fisheries, University of Washington, Seattle,
Washington 98105
Referee: Dr. George M. Pigott, President, Sea Resources Engineering Inc., 4525 105 Avenue, N.W., Kirkland, WA, 98033
Downloaded by [Texas A&M University Libraries] at 11:28 12 January 2016
* Corresponding author: Present address: Department of Food Science, University of Massachusetts at Amherst, Marine Foods
Laboratory, Marine Station, Gloucester, Massachusetts 01930; Fax: (978) 281-2618; E-mail: hordur@Foodsci.umass.edu
** Present address: Department of Food Science and Human Nutrition, Washington State University, P.O. Box 646376, Pullman,
Washington 99164
ABSTRACT: Considerable amounts of fish processing byproducts are discarded each year. By developing
enzyme technologies for protein recovery and modification, production of a broad spectrum of food ingredients
and industrial products may be possible. Hydrolyzed vegetable and milk proteins are widely used food ingredients.
There are few hydrolyzed fish protein foods with the exception of East Asian condiments and sauces. This review
describes various manufacturing techniques for fish protein hydrolysates using acid, base, endogenous enzymes,
and added bacterial or digestive proteases. The chemical and biochemical characteristics of hydrolyzed fish
proteins are discussed. In addition, functional properties of fish protein hydrolysates are described, including
solubility, water-holding capacity, emulsification, and foam-forming ability. Possible applications of fish protein
hydrolysates in food systems are provided, and comparison with other food protein hydrolysates where pertinent.
KEY WORDS: fish protein hydrolysates, fish protein, functional properties, chemical hydrolysis of fish protein,
enzymatic hydrolysis of fish protein, protein functionality, fish byproducts.
I. INTRODUCTION processes for byproduct recovery and utilization

it has to be more economically feasible than dis-
At this time there are huge amounts of pro- carding the byproducts.
tein-rich byproduct materials from seafood pro- Every year over 91 million tons of fish are
cessing plants discarded without any attempt of harvested, of which 29.5% is transformed into
recovery. At the same time many processors are fishmeal.1,2 Possibly more than 50% of the re-
no longer allowed to discard their offal directly maining fish tissue is considered to be processing
into the ocean, resulting in a very high cost of waste and not used as food.3 With a dramatically
refining the material before it is discarded. To increasing world population and a world catch of
meet the need of the seafood processing industry, fish presently on the verge of exceeding the esti-
an alternative to discarding these byproducts mated sustainable limits of the suggested 100
should be developed. Recovery and alteration of million tons/year, there is obviously an increased
the fish muscle proteins present in the byproduct need to utilize our sea resources with more intel-
material and using these as functional ingredients ligence and foresight. By applying enzyme tech-
in food systems is a very exciting and promising nology for protein recovery in fish processing, it
alternative. However, for the industry to develop may be possible to produce a broad spectrum of
Copyright© 2000, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without
the consent of the publisher is prohibited.
43
food ingredients or industrial products for a wide understand the process of protein hydrolysis it is
range of applications. This would utilize both crucial to have a good understanding of the nature
fishery byproducts or secondary raw materials of the protein substrate and the hydrolyzing agent.
and, in addition, underutilized species that would During protein hydrolysate manufacture, the pro-
otherwise be discarded. tein substrate is hydrolyzed by either a proteolytic
Enzymatic modification of proteins using enzyme or an acid or base.
selected proteolytic enzyme preparations to cleave Our diet contains a wide variety of proteins
specific peptide bonds is widely used in the food from different sources. It is generally accepted
industry.4 Hydrolysis of food proteins has a long that the relative concentration of dietary essential
history, mainly for vegetable and milk proteins; amino acids is the major factor determining the
these proteins are widely used in the food indus- nutritional value of food protein.7 Proteins de-
try. Most work on the hydrolysis of fish proteins rived from animal sources are considered to be
was conducted in the 1960s. Some fish protein nutritionally superior to those from plants be-
hydrolysate (FPH) preparations at that time were cause they contain a better balance of the dietary
quite successful.5 During the 1960s, research was essential amino acids. Of these egg and milk pro-
directed to the production of cheap nutritious proteins (casein) are frequently used as reference
tein sources for rapidly growing developing coun- proteins for evaluating protein quality. Proteins
tries, or toward animal feed production, primarily derived from meat and poultry muscle are also of
through production of fish protein concentrates very high quality and fish muscle proteins are
(FPC). Little work has been done recently on equally nutritious.8 Fish muscle contains an ex-
FPH, but some research has been directed into the cellent amino acid composition and is an excel-
potential of using powdered hydrolysates in food lent source of nutritive and easily digestible pro-
formulations. Many studies have resulted in fish teins.9,10 However, because fish is extremely
protein hydrolysates with excellent functional perishable and because chemical composition can
properties. However, taste defects, specifically vary, the utilization of fish as a basic raw food
bitterness, and process economics are still major material presents unique food processing prob-
limiting factors for FPH applications. lems.11
This review gives an overview of the differ- The muscle of different animals is very simi-
ent techniques for production of fish protein hy- lar, containing similar protein and similar amino
drolysates, past and present research on their prop- acid profiles. There are slight differences between
erties, and various methods to study the extent of fish muscle and the muscle of land animals. These
hydrolysis and product functionality. are mainly associated with the differences in
muscle structure required for swimming and buoy-
ancy. Fish are supported by a mass of water, thus
II. THE BIOCHEMICAL the muscle fibers require less structural support
CHARACTERISTICS OF FISH MUSCLE than those in land animals. Because of this, fish
PROTEIN muscle tends to have less connective tissue than
muscles from terrestrial animals, resulting in more
In foods, a protein is traditionally categorized tender texture. Also, because of the unique move-
as a fibrous or globular protein based on its ter- ment of fish, the structural arrangement of muscle
tiary conformation. Each type of food protein has fibers is quite different from terrestrial animals. A
a unique molecular structure that determines its large fraction of commercially utilized fish stocks
functional properties, in addition to a range of are cold adapted or poikilothermic, and because
environmental conditions over which it exhibits of this their muscle proteins have different bio-
such properties.6 These factors and their effect on chemical properties compared with those of en-
functionality are discussed in more detail later in dothermic animals.12 Poikilothermic characteris-
this review. tics of fish proteins make them more heat sensitive
The functional and structural properties of than mammalian muscle proteins, with a greater
food proteins thus vary tremendously, and to fully tendency to denature at elevated temperatures.
44
Fish muscle proteins from cold water species are main components of the thick filament, and thin
more prone to denaturation than those from tropi- filament, respectively. Myosin comprises 50 to
cal waters.13,14 The T-50 values (temperature re- 60% of the myofibrillar contractile proteins, and
quired for 50% denaturation) of fish muscle pro- actin only 15 to 30%.12,15 Myosin is the most
teins are also influenced by pH and were reported abundant of the single muscle proteins, making
to be 29 to 35°C at pH 7.0 and 11 to 27°C at pH up around 38% of the total, and is a large mol-
5.5.15 ecule containing two identical heavy chains
Protein composition in muscles varies by (223 kDa) and two light chains (22 and 18 kDa).
muscle type. Of the three types (striated, smooth, The molecule has two identical globular head
and cardiac muscle) of muscles, the striated regions that incorporate the light chains and a
muscles are the predominant form in fish. Striated significant fraction of the heavy chains. The tails
muscle tissue is arranged into muscle fibers that of the heavy chains form very long α-helices that
are bound together by a connective tissue to make wrap around each other18 (Figure 1). Myosin can
a fiber bundle. Fish muscle has “white” and “dark” be cleaved by proteases at two sites on the mol-
meat.15 The white meat is generally more abun- ecule, one recognized by both trypsin and chy-
dant, contains less lipids than the dark meat, and motrypsin and the other by papain. Papain cleaves
is the most widely consumed type of muscle tis- near the head region, releasing the head from the
sue. It is composed of about 18 to 23% of protein, tail. Trypsin and chymotrypsin cleave further from
depending on the species and time of harvesting. the head, dividing the molecule into two compo-
Fish proteins can be divided further into different nents called the heavy meromyosin (with the head
groups based on their solubility. About 70 to 80% region) and the light meromyosin, both with dif-
of fish muscle are structural proteins. These struc- ferent functional properties.
tural protein are soluble in cold neutral salt solu- Myosin molecules are connected via their head
tions of fairly high ionic strength. The remaining region to the polymerized actin molecules in the
20 to 30% contain sarcoplasmic proteins that are thin filaments due to the ATPase activity of the
soluble in water and dilute buffers, and a final head molecules. This complex is called actomyo-
part of the structural protein, 2 to 3%, being in- sin and is responsible for muscle contraction and
soluble connective tissue proteins.11 Recent stud- relaxation. Actomyosin plays a major role in de-
ies, however, challenge these generally accepted termining the quality of fish meat because it is
solubility data, showing that the muscle protein quite labile and easily affected during processing
components can be highly soluble at low ionic and storage. For example, during frozen storage
strenghts.16,17 the actomyosin becomes progressively less soluble
Myofibrillar proteins are the primary food and the flesh becomes increasingly tougher.19
proteins of fish, comprising 66 to 77% of the total The thin filament is a complex of actin mol-
protein in fish meat. The myofibril protein com- ecules making a double helix. Tropomyosin sits
plexes contain myosin and actin. These are the within the grooves of the thin filaments and two
FIGURE 1. Fish myosine molecule.
45
troponin molecules bind the actin filament at each the reaction and characteristics of the final prod-
helical repeat. Actin is the most prominent pro- uct.
tein of the three protein in the thin filaments, Chemical and biological methods are the most
making up about 13% of the total muscle pro- widely used for protein hydrolysis with chemical
teins. Actin occurs in two forms, G-actin, a spheri- hydrolysis used more commonly in industrial prac-
cal monomer, and F-actin, a large polymer that tices. Biological processes using added enzymes
connects to myosin. The thin filaments play a are employed more frequently, and enzyme hy-
very important role by regulating muscle contrac- drolysis holds the most promise for the future
tion. From the point of view of muscle biochem- because it results in products of high functionality
istry, thin filaments are very important, however, and nutritive value. The chemical and biological
their content is low in meat and their role with hydrolysis are discussed in more detail below,
respect to food processing has not been studied with an emphasis on hydrolysis with added en-
completely. Other contractile proteins of interest zymes. In addition, there are many potential tech-
are C-protein, α-, and β-actinin, connectin and niques for extracting protein from animal tissue.
paramyosin; however, they are of limited interest These include the use of aqueous and organic
as food proteins. With respect to protein hydroly- solvents; the conventional processes of cooking,
sis, the myofibrillar protein myosin, actin, or ac- pressing, drying, and hot oil extraction.22 The
tomyosin are subject to enzymic cleavage and are extraction of protein by means of solvent is also
the greatest focus here. worth mentioning due to its industrial and histori-
cal importance for fish protein recovery.
III. PROTEIN HYDROLYSIS

A. Chemical Methods for Protein
Proteolytic modification of food proteins to Hydrolysis
improve palatability and storage stability of the
available protein resources is an ancient technol- 1. Chemical Extraction: The Making of
ogy.20 Hydrolysates can be defined as proteins Fish Protein Concentrate
that are chemically or enzymatically broken down
into peptides of varying sizes.21 Protein hydroly- The extraction methods mentioned above,
sates are produced for a wide variety of uses in other than the chemical and biological hydrolysis
the food industry, including milk replacers, pro- methods, do not hydrolyze protein. They are used
tein supplements, stabilizers in beverages and fla- primarily to concentrate intact protein by the re-
vor enhancers in confectionery products. The moval of water and oil from the substrate. The
benefits of hydrolyzing food proteins to make method of solvent extraction has been frequently
functional protein ingredients and nutritional employed when producing fish protein concen-
supplements is a more recent technology, with the trate (FPC). The development of FPC was one of
first commercially available protein hydrolysates the earliest attempts to recover fish protein from
appearing only around the late 1940s. Although processing waste and to produce a protein ingre-
production is massive worldwide, the proper con- dient from underutilized species. FPC was the
trol of the process and the exact mechanism be- precursor to the field of enzyme hydrolysis of fish
hind protein hydrolysis is in most cases not fully proteins. A small but extensive research program
understood. Recent advances have given research- on the large-scale production of FPC by the Bu-
ers insight into the connection between the pro- reau of Commercial Fisheries, now the National
cess/extent of hydrolysis and the physicochemi- Marine Fisheries Service (NMFS) of the Depart-
cal mechanisms responsible for specific functional ment of the Commerce, began in 1961. The gen-
properties of the hydrolyzed protein. Recent re- eral aim of the program was to study the manufac-
search on enzyme catalysis has also aided with ture and use of FPC as a solution to global protein
the proper selection of enzyme catalysts and pro- malnutrition and as a potential economic stimulus
cessing conditions to obtain better control over to the American fishing industry.23 Solvent-ex-
46
tracted FPC (type A FPC) is produced by using first at 75°C for 90 min in isopropanol and then at
primarily isopropanol or azeotropic extraction with 75°C for 70 min with azeotropic isopropanol. The
ethylene dichloride, although other solvents such final supernatant fraction is collected, dried, milled,
as ethanol have been used successfully as well. A and screened to separate out bone particles. The
standard process presented by Sikorski and final product has a high biological value and is
Naczk24 shown in Figure 2 is to grind a whole or colorless and odorless, with less than 1% lipids.
eviscerated fish, extract it with isopropanol at a The problem with type A FPC is that it is not
low temperature (20 to 30°C) for 50 min, then readily soluble or dispersible in foods and has
collect the supernatant and extract it twice again, poor emulsification properties.9,25,26 Dubrow et al.27
FIGURE 2. A production scheme for fish protein concentrate. (Adapted from Ref. 24.)
47
reported that FPC produced at higher tempera- 2. The Chemical Hydrolysis Process
tures (50°C) compared with lower temperatures
(20°C) had significantly lower emulsifying prop- Chemical hydrolysis of proteins is achieved
erties, but both had very poor solubility. General by cleaving peptide bonds with either acid or
poor functionality, off-flavors the high cost of base. Several processes have been proposed for
production, and traces of solvent in the final prod- the acid or alkaline hydrolysis of fish.33 This has
uct made solvent extracted FPC commercially been the method of choice in the past for the
unsuccessful despite concerted efforts.3,28 Al- industry primarily because it is relatively inex-
though FPC lacks solubility, it reportedly has pensive and quite simple to conduct. There are,
good foaming properties over a wide range of pH however, many limitations to food ingredients
(pH 2 to 11), making strong, stable foams.29–31 using this method. Chemical hydrolysis tends to
Despite problems with protein functionality, sol- be a difficult process to control and almost invari-
vent extraction is the method of choice for the ably leads to products with variable chemical
abundant fatty pelagic fish species such as sar- composition and functional properties.21,38 Pro-
dine, herring, and capelin because the protein is tein hydrolysis with strong chemicals and sol-
effectively separated from the lipids, thereby re- vents is performed at extreme temperatures and
ducing stability problems normally associated with pH and generally yield products with reduced
residual oxidizable lipid. For fatty fish, isopro- nutritional qualities, poor functionality, and re-
panol was a slightly more efficient solvent than stricted to use as flavor enhancers.39,40
ethanol considering the residual amounts of lip-
ids, but absolute ethanol produced FPC of lighter
color and a neutral flavor.32 a. Acid Hydrolysis
Many studies with FPC have also been con-
ducted with solvent-extracted FPC as a substrate Acid hydrolysis of proteins is used more com-
for enzyme hydrolysis, both to defat the substrate monly than hydrolysis under alkaline conditions.
and to make it more accessible to enzymatic hy- A vast majority of hydrolyzed proteins consumed
drolysis, with excellent functional and nutritional in the U.S. are prepared by acid hydrolysis, mostly
properties.5,25,33,34,35,36 However, enzymatic hy- from inexpensive vegetable protein sources that
drolysis using FPC as a starting substrate resulted otherwise would have poor nutritive and little
in loss of some functional properties because of functional value in foods. Although the process is
excessive protein breakdown but increased nitro- harsh and hard to control, it is still the preferred
gen solubility.30 Taste and odor problems are method for hydrolyzed vegetable proteins. Hy-
generally minimized with a FPC starting mate- drolyzed vegetable protein, which are widely used
rial.33 for flavor and taste enhancement properties, re-
Recent studies with solvent-extracted FPC quire extensive acid hydrolysis.38 Applications of
have produced FPC with better protein function- hydrolyzed vegetable proteins are primarily as
ality. For example, recently Vareltzis et al.37 stud- flavoring agents in processed meat, crackers, and
ied the addition of ethanol-extracted FPC made soup mixes. Acid hydrolysis of fish protein has
from sardine to hamburger patties and found that usually involved reacting fish proteins with hy-
the overall functional properties (water binding drochloric acid, or in some cases sulfuric acid,
and cooking yield) and the penetration depth and and the proteins are completely hydrolyzed at
shear force value of the hamburger increased with high temperature, and often high pressure. The
the addition of FPC. However the hamburgers hydrolysate is then neutralized to pH 6.0 to 7.0
had a slightly unfavorable fishy flavor. Hoyle and and concentrated to either a paste or further dried.41
Merritt5 found that herring protein extracted with Because the product is hydrolyzed extensively,
ethanol in a similar manner and then hydrolyzed its primary functional property is high solubility.
with either Alcalase or papain produced a hy- Total hydrolysis of fish protein substrate can be
drolysate with a markedly reduced bitterness and achieved in 18 h at 118°C in 6N hydrochloric
less fishy odor. acid.42 In addition, following the neutralization of
48
the digest, the hydrolysate contains large amount obtained with FPC starting material, which had
of salt (NaCl), which can make the product unpal- poor solubility and dispersibility.
atable and interferes with functionality in food Several deleterious reactions occur in alka-
systems. Another drawback of acid hydrolysis is line solutions during hydrolysis. These are initi-
the destruction of tryptophan, which is an essen- ated by hydrogen abstraction from the alpha car-
tial amino acid. Orlova et al.43 proposed an acid bon of an amino acid and include racemization of
hydrolysis process of whole fish, where steam L-amino acids, which produces D-amino acids,
distillation is used to remove aromatic substances which are not absorbed by humans. Also, disul-
followed by filtration then concentration. The fide bonds are split with loss of cysteine, serine,
concentrate was used in dehydrated soup cubes and threonine via β-elimination reactions and
and as a microbial media.43 The acid hydrolysis is formations of lysinoalanine, ornithinoalanine,
also widely utilized to convert underutilized and lanthionine, and β-amino alanine can also oc-
secondary raw material from fish into fertilizer cur.31 Some of these elimination and addition re-
due to the low production cost and resulting ex- actions may lead to the formation of toxic sub-
tensive hydrolysis. stances (e.g., lysinoalanine) that are highly

undesirable in foods.47,48 Alkaline hydrolysis re-
action products have an inhibiting effect on pro-
b. Alkali Hydrolysis teolytic enzymes, reducing the rate of hydroly-
sis.49 Some of the possible reaction products that
The use of alkali reactants, primarily sodium may form during alkali hydrolysis are shown in
hydroxide, to hydrolyze protein often results in Figure 3.24 High collagen solubility is also ob-
poor functionality and more importantly can ad- served with alkali treatment.50
versely affect the nutritive value of the hydroly-
sate. Despite this, limited alkali treatment is used
in the food industry to recover and solubilize a B. Biochemical Methods for Fish Protein
broad range of proteins. For example, mechani- Hydrolysis
cally deboned turkey residue (MDTR) includes a
significant proportion of alkali-soluble proteins Biochemical hydrolysis to produce fish or
that can be recovered by alkali treatment and used other food protein hydrolysates is performed by
in food applications. Fonkwe and Singh44 dis- utilizing enzymes to hydrolyze peptide bonds.
cussed the use of alkali extraction to recover This can be done via proteolytic enzymes already
MDTR with an alkaline sodium chloride solution present in the fish viscera and muscle (endog-
but found it to be unsuitable due to low recovery. enous proteases), or by adding enzymes from other
Alkaline hydrolysis of fish proteins has primarily sources. To understand the process of enzymatic
used FPC as the starting substrate. During alka- hydrolysis, it is very important to understand the
line hydrolysis of fish protein, rapid cleavage to nature and activity of proteolytic enzymes.
large water-soluble polypeptides takes place, fol-
lowed by further degradation at a slower rate. 1. Proteolytic Enzymes
Alkali treatment can aid in modifying the proper-
ties of insoluble FPC.24 Tannenbaum et al.45,46 Enzymes are biochemical catalysts vital for
have studied the alkaline process for hydrolyzing living beings, because they accelerate chemical
insoluble FPC and its applications. They devel- reactions between organic constituents within the
oped a small-scale batch process that utilizes high cells that otherwise would take an extremely long
pH (12.5) and 95°C for 20 min. The product time to complete. In food science and technology
consisted of large peptides, some relatively in- enzymes are exploited to perform desired func-
soluble at the isoelectric point, but with an overall tions in processing and analysis and to facilitate
improvement in functionality with respect to the the conversions of raw materials into higher-qual-
original FPC. Use of the solubilized FPC as a ity, more desirable foodstuffs.51 Enzymes make
milk substitute gave a product far superior to that this possible because the active site of an enzyme
49
FIGURE 3. Possible chemicals that may form in the alkali-treated proteins.
is highly specific for certain substrates. Enzymes they all share a common transition state (interme-
catalyze only one specific reaction and function diate) during catalysis. To discuss enzyme kinet-
by forming a complex with the substrate whose ics in any detail would require a separate review,
transformation they catalyze. and this is not intended here. However, it is im-
Enzymes used by the food industry and in portant to know the basic steps in enzyme cataly-
food research are predominantly hydrolases, most sis and to understand the mechanism of protein
of which are carbohydrases, followed by pro- hydrolysis. Some proteases preferentially cata-
teases and lipases. Proteases are among the best lyze the hydrolysis of bonds adjacent to a particu-
characterized enzymes. Proteolytic enzyme prepa- lar amino acid residue, while others are less spe-
rations are economically the most important group cific. The catalysis by proteases occurs primarily
of enzymes, and their use is well established in as three consecutive reactions: (1) the formation
the food industry.52 Proteases are categorized ac- of the Michaelis complex between the original
cording to the specificity of the peptide bonds peptide chain and the enzyme, (2) cleavage of the
they attack (hydrolyze) and the mechanism by peptide bond to liberate one of the two peptides,
which they act.48 Four major classes of proteases and (3) a nucleophilic attack on the remains of the
are known. They are designated by the principal complex to split off the other peptide and to re-
functional group in their active site: serine, thiol, constitute the free enzyme.20,54 The hydrolysis of
carboxyl and metallo.53 Proteases are character- peptide bonds leads to an increase in the numbers
ized further by their hydrolyzing mechanism into of ionizable groups (NH3+ and COO-), with a con-
endoproteinases or exopeptidases. The endo- comitant increase in hydrophobicity and net
proteinases cleave/hydrolyze the peptide bonds charge, a decrease in molecular size of the polypep-
within protein molecules, usually at specific resi- tide chain, and an alteration of the molecular struc-
dues to produce relatively large peptides. The ture leading to the exposure of the buried hydro-
exopeptidases systematically remove amino acids phobic interior to the aqueous environment.55–58
from either the N terminus, called aminopepti- Giving the substrate the symbol S, the enzyme E
dases, or the C terminus, called carboxypepti- and the peptides in the reaction P, the overall
dases, by hydrolyzing the terminal peptide bonds. mechanism can be presented as:
In food protein hydrolysis, endoproteinases are
always used, but occasionally endoproteinases are
k k2
combined with exopeptidases to achieve a more E + S ← 1
→ ES → EP + H − P ′
k –1
complete degradation.20
Although the four classes of proteases men- k3
tioned above have different catalytic mechanisms, H
2O
→ E + OH − P + H − P ′
50
This enzyme-substrate complex may dissoci- with different activity requirements, which result
ate back to reactant substrate and free enzyme, or in end products of different molecular profiles.
to free enzyme and product molecules. In other Another complication is that the presence of cer-
words, classic Michaelis-Menten kinetics apply.20 tain digestive enzymes and their concentration
The generally accepted mechanism for proteases may be highly seasonal, gender and age specific,
indicates that the second step is the rate-determin- and can vary tremendously within a species as
ing step, thus k2 primarily determines the overall well as between species. These variations make it
reaction speed, and Km is more or less equal to the very hard to control the hydrolytic process, and
true dissociation constant. This simple mecha- direct the production of hydrolysates with spe-
nism does not, however, deal with the detailed cific molecular properties. Autolytic methods such
question of how the enzyme and substrate are as chemical methods often result in a final prod-
bound or what molecular configurations lead to uct with bad functionality. Despite these prob-
product formation. To fully understand the ca- lems, endogenous proteolytic enzymes are used
talysis, a fairly detailed explanation is in order, to produce hydrolyzed products, specifically fish
which is not the purpose of this review. sauces and fish silage.
Enzymatic hydrolysis of proteins is a complex The production of fish sauce preceded fish
process because of several peptide bonds and their silage production and is the major fermented fish
specific accessibility to enzymatic reactions.47 The product presently consumed in the world. Its pro-
specificity of enzymes is not the only factor that duction has thousands of years of tradition in
affects the peptide profile of the final product. Asia, and it is also known to have been produced
Environmental factors such as temperature and pH in Mediterranean countries in ancient times. Pres-
play an important role. Both temperature and pH ently, fish sauce is used mainly as a condiment on
can greatly affect the enzyme reaction kinetics, and rice dishes like the popular Nuoc-Nam produced
the effect of these factors is different for each in Vietnam, and the annual production in South-
enzyme. Generally, there is an optimum combina- east Asia is about 250,000 metric tons.60 The
tion of both pH and T, where an enzyme is the most production of fish sauce does not require elabo-
active. Temperature and pH extremes deactivate rate processing equipment. The substrate is usu-
the enzymes by denaturing them. ally fish from one or more pelagic species, such
as anchovies or sardines, or minced whole fish of
low commercial value. The substrate is immersed
2. Autolytic Hydrolysis in a solution containing high concentrations of
salt (20 to 40%) and at relatively high tempera-
Biochemical production of fish protein hy- tures, preferably ambient tropical temperatures.
drolysates may be carried out by employing an In the case of whole fish, the visceral proteolytic
autolytic process. An autolytic process depends enzymes start by hydrolyzing the stomach con-
on the action of the digestive enzymes of the fish tents, then work their way through the stomach
itself. There are no enzyme costs involved, and it wall, and finally reach the muscle tissue, where
is a simple operation.59 The end product of au- they join with catheptic enzymes to hydrolyze
tolytic hydrolysis is generally a fairly viscous fish muscle proteins. The pH of the fish sauce
liquid rich in free amino acids and small peptides. process is usually neutral, because no base or acid
The digestive enzymes in question are primarily is added to the reaction mixture. The acid-depen-
the serine proteases trypsin and chymotrypsin, dent proteolytic enzymes, such as pepsin and the
and the thiol protease pepsin, all major enzymes catheptic enzyme, contribute little to fish sauce
of fish viscera and digestive tract. Lysosomal production. The endogenous serine proteases very
proteases, or catheptic enzymes, present in fish slowly breakdown the fish muscle for 6 to 12
muscle cells also contribute to proteolytic break- months (Nuoc-Mam) under anaerobic conditions.
down to some extent. This slow but extensive breakdown results in a
The endogenous enzymes in autolytic hydroly- liquefied fish sauce composed predominantly of
sis are a very complex mixture of enzymes, all free amino acids, with up to 50% nitrogen recov-
51
ery. The high concentration of salt is responsible such as ambient temperature and relative amounts
for the slow hydrolysis because this slows the of the visceral organs present. The processing
activity of the proteolytic enzymes. More impor- time is much shorter than for fish sauce because
tantly, the high salt concentration and anaerobio- no salt is added.26,60 The production of fish silage
sis totally inhibits growth of spoilage microor- using lactic acid bacteria as the hydrolyzing agent
ganisms once the salt has fully penetrated the has been reported.66,67 The bacterial fermentation
tissue.61 Lower concentrations of salt, however, is initiated by mixing minced or chopped fish
result in sauces with higher yield, lower levels of with a fermentable sugar that favors the growth of
volatile acids, and better balanced composition of lactic acid bacteria, which is advantageous be-
amino acids.62 During autolysis to produce fish cause the bacteria produces acid and antimicro-
sauce, a lipid phase, an aqueous soluble phase bial factors that inhibit competing bacteria.65
that contains much protein but little lipid, and The feed applications of fish silage are prima-
insoluble sediment of protein and lipid is formed.63 rily limited to young animals due to the extensive
After the hydrolysis is completed, the liquid pro- hydrolysis of the proteins. For fish silage to be
tein hydrolysate is tapped, filtered, and bottled, incorporated successfully in animal feeds, it has
with the final product containing up to 10% free to contain the majority of the nitrogen fraction as
amino acids and low-molecular-weight peptides, intact proteins or peptides rather than as free amino
and 25% salt.26,60 Although the production of fish acids, which are less well absorbed. A shorter
sauce does not improve the nutritive value of the processing time and added commercial proteases
protein, the keeping quality is greatly increased may be useful in such instances. Also, problems
and organoleptic characteristics are generally connected with the development of bitterness in
improved.64 the hydrolyzed silage can make the product highly
The process of manufacturing fish silage is unpalatable not only for humans but also animals
different, but the product shares many character- fed feeds rich in fish silage. The utilization of fish
istics with fish sauce. The production of fish si- silage as a primary protein source in fish feeds has
lage was not started until the middle of this cen- been neither fully investigated nor commercially
tury, and it is far from being as widely employed successful; however, it is incorporated into diets
as fish sauce production. The application of fish for pigs, poultry, and mink.67
silage is primarily for animal feed production Research on fish hydrolysates made with en-
instead of food applications. The process is rapid dogenous enzymes for human food applications
and enzymes involved are very effective in pro- has been very limited. Fish sauce is almost the
ducing oil and proteins fractions that are readily only autolytically produced food of aquatic ori-
separated. The substrate is usually secondary raw gin. The main reason for this is fairly straightfor-
material from fish processing or underutilized fish ward. To produce a functional protein hydroly-
species. The substrate is mixed with strong min- sate with specific properties, a good knowledge
eral acids or organic acids such as formic acid to of the enzymes involved is crucial. Endogenous
acidify the mixture below pH 4. At this pH the enzymes in fish are a complex and highly variable
serine proteases are generally inactive, but pepsin mixture, and thus the properties of functional pro-
and the catheptic enzymes are highly active. The tein hydrolysates so prepared may vary greatly
pepsin content can be very high in the visceral under the same reaction conditions. Also, in the
portion of fish, and the enzyme is primarily re- U.S. the sale of processed fish foods containing
sponsible for fish silage production. Under acidic visceral material of any kind is prohibited by the
conditions the active endogenous enzymes par- FDA. Despite this, some work on developing fish
tially hydrolyze the fish over several weeks to silage for human consumption has been conducted.
produce a slurry containing up to 12% amino In 1972, Malcolm B. Hale and a group of scien-
acids and peptides. Usually about 80% of the tists at NMFS and the University of Maryland
protein in acid fish silage become solubilized af- conducted a comprehensive study on making func-
ter 1 week at temperatures around 23 to 30°C.65 tional FPH by enzymatic hydrolysis for human
The rate is primarily dependent on conditions consumption.33 This study involved autolysis of
52
red hake (Urophycis chuss), a relatively lean fish, native acid enzymes resulted in a product with the
and alewife (Alosa pseudoharengus), a very fatty majority of peptides of lower molecular weight
fish. The autolysis of raw hake was conducted at than the pepsin hydrolysate under the same ex-
optimal conditions for the native enzymes, 50°C perimental conditions. This indicates that protein
and pH 7.0, for 24 h where fish was 50% of the hydrolysates can be obtained through autolysis
total slurry. The resulting hydrolysate was either very efficiently at relatively mild temperatures.
spray dried or tested as a concentrate. The aver- The functional properties of the product were,
age yield of dry solids for hake was lower than for however, not investigated.
any other proteolytic enzyme employed, 10.0% The main limitation of work performed on
(dry solubles/wet fish), compared with the high of autolytic hydrolysis of food proteins is the lack of
14.3% from using Alcalase. Similarly, the chemi- research on functional properties. Studies have
cal score attained for soluble hydrolysate were shown that protein recovery can be adequate and
quite low due to low tryptophan content that is that nutritional requirements are good, but infor-
correlated with a low recovery. The protein effi- mation on functional properties of the resulting
ciency ratios for both red hake and alewife hy- hydrolysate is very important to successfully evalu-
drolysates were essentially equivalent to that of ate its use in formulated foods.
casein. Also, the inclusion of insoluble solids in
the final product resulted in very high fat content.
The results for lipid pressed alewife were similar. 3. Enzymatic Hydrolysis of Fish Muscle
However, by lowering the reaction period to 4 h Proteins with Added Enzymes
and raising the temperature to 55°C, a satisfactory
product with good nutritional value was prepared. Using added enzymes to hydrolyze food pro-
Both the red hake and alewife were only 50 to teins is a process of considerable importance used
70% soluble, requiring substantial additions of to improve or modify the physicochemical, func-
commercial enzymes, at uneconomic levels, to tional, and sensory properties of the native pro-
become fully soluble. The alewife hydrolysate tein without jeopardizing its nutritive value, and
also suffered from very fishy taste; however, the often protein absorption is improved. These en-
red hake hydrolysate had a less fishy taste and zyme-based processes occur under mild condi-
odor. Food applications of the products obtained tions over a series of stages and do not produce
by Hale and co-workers were limited and could hydrolytic degradation products via racemization
be used primarily as a protein supplement in cul- reactions observed with both acid and alkaline
tures where its taste would be acceptable and the hydrolysis.69 The process of using added enzymes
caloric value of the lipid desirable.33 instead of chemicals or endogenous enzymes of-
Shahidi et al.59 hydrolyzed ground capelin fers many advantages because it allows good con-
(Mallotus villosus) by endogenous enzymes and trol of the hydrolysis and thereby the properties of
found that it enhanced the overall extraction of the resulting products.59 Processes can be designed
the fish protein at both acid and alkaline pH, as to take advantage of substrate specificity and the
both acid and alkaline proteases are present in relative reaction rates of different enzymes under
fish muscle and viscera. The protein recovery of the reaction conditions employed. The physico-
hydrolysates produced autolytically was, however, chemical and functional properties of hydrolyzed
considerably lower compared with commercial fish proteins are discussed separately in a later
enzymes, 22.9% compared with 70.6% with section.
Alcalase. A recent study by Cui68 with chum Enzymatic hydrolysis has been employed on
salmon (Oncorhynchus keta) mince and visceral a variety of different proteins derived from live-
content showed a surprisingly extensive and rapid stock and poultry meat,39,44,47,70,71 milk,57,72–76 and
hydrolysis at an acid pH and 37°C. The hydroly- plants.77,78 Hydrolysis of fish and other aquatic
sate also showed a marked difference in the mo- foods is also being seen more frequently in the
lecular weight distribution of peptides when com- literature. Several different aquatic protein sources
pared with a commercial pepsin hydrolysis. The have been investigated for the production of func-
53
tional fish protein hydrolysates. These include several disadvantages such as (1) high cost of
Rastelliger canagurta and Barbus carnaticus using large quantities of enzymes, (2) difficulty in
both Indian tropical fishes,79,80 hake (Urophycis controlling the extent of reaction that can result in
chuss), 10,25,33 shark (Isurus oxyrinchus), 81,82 nonhomogenous products consisting of fractions
sardine (Sardina pilchardus),35,36,83,84 herring of varying molecular weight, (3) low yields, and
(Clupus harengus),5 crayfish,85 lobster (Panulirus (4) the need to inactivate enzymes by pH or heat
spp.),86 pollack (Theragra chalcogramma),87 cape- treatment at the end of the reaction, which adds to
lin (Mallotus villosus), 59 dogfish (Squalus the processing costs.91 Also, the enzymes em-
acanthias),88 chum salmon (Oncorhynchus keta),68 ployed in the process cannot be reused.20 Figure 4
Pacific whiting (Merluccius productus),89 and outlines a fairly typical process for producing fish
Atlantic salmon (Salmo salar).90 It can readily be protein hydrolysates. Each step is given a detailed
seen from the list above that the majority of the discussion below.
sources represent underutilized species or are
connected to utilization of processing wastes.
Enzymatic hydrolysis of fish protein has been a. The Substrate and Its Preparation
employed primarily as an alternative approach for
converting underutilized fish biomass, which is Lean species, or material derived from them,
commonly used in making feed or even fertilizer is the substrate of choice for enzymatic hydroly-
into edible protein products.15,88 More recently, sis as problems with lipid oxidation can be re-
fish processing waste, or, more appropriately, duced. From an economic standpoint, however,
secondary raw material, has been connected to the abundant underutilized pelagic fish would be
FPH studies. In many cases this is due to strict preferred. The small pelagics comprise 23% of
government waste regulations. Many processors the world’s catch,92 of which only 42% is used as
are no longer allowed to discard their offal di- human food. These are mostly fatty species such
rectly to sea, resulting in a very high cost of as herring, sardines, anchovies, and mackerel, and
refining the material before discarding. Second- FPH prepared from them would contain high
ary raw material is the material remaining after amounts of lipid, which would require additional
fillets are removed, and if viscera is included, this treatments such as centrifugation to remove ex-
can represent something on the order of 64% of cess fat.93 The fewer steps that are involved in the
the weight of whitefish, the protein content of this production, the more economically viable the
waste being about 10%.3 Hydrolysis of fish pro- operation becomes. If a whole fish is used, it is
tein with selected proteolytic enzymes provides eviscerated and washed, then ground in a meat
the possibility of controlling cleavage degree of grinder, usually mixed with an equal amount of
protein in the substrate. Using suitable enzyme/ water and homogenized in a blender until a vis-
substrate ratios and reaction times, this permits cous homologous mixture is achieved. In some
the production of hydrolysates with different instances a buffer solution is added to the minced
molecular structures and different functional prop- fish, for example, phosphate buffer1,88 and boric
erties that could find applications in various food acid-NaOH buffer.85 The presence of buffer salts
formulations.82 may affect the final properties of the hydroly-
The hydrolytic process and reaction condi- sates. In a study of whey protein hydrolysis,
tions differ between different substrates and en- Kuehler and Stine72 decided not to buffer the
zymes used and also depend on the properties solutions because of the influence buffer salts
desired for the hydrolysate. Most of the described might have on foaming or emulsifying properties.
processes are conducted under research condi- Processes for fatty and lean species are differ-
tions and may have limited applications in the ent. If the FPH contains more than 1% fish fat, the
industry. Commercial production of fish protein fat must either be removed by solvent extraction
hydrolysates is still limited on a worldwide basis, or stabilized by antioxidants3,60 such as butylated
but has reached a significant level in a few coun- hydroxytoluene, butylated hydroxyanisol,5 or
tries, including France, Japan, and Southeast propylgallate.10 A fish protein hydrolysate with
Asia.60 Commercial batch protein hydrolysis has high lipid content may darken. The formation of
54
FIGURE 4. A flow sheet for the enzymatic hydrolysis of fish protein to make fish protein hydrolysate
brown pigments may result from aldol condensa- reduced to 0.9 from 4.0% of raw herring. Also
tion of carbonyls produced from lipid oxidation before placing the treated substrate in the reaction
after reaction with basic groups in proteins.5 Re- vessel, chemical agents such as NaCl, sorbic acid,
searchers have developed many means of mini- or ethanol are occasionally added to the minced
mizing the lipid content in FPH. To obtain a fish to minimize bacterial degradation,24 espe-
product of a lipid content not exceeding 0.5% by cially if reaction conditions are at neutral or alka-
weight, as established by the Protein Advisory line pH. However, adding NaCl can reduce the
Groups of FAO for a fish protein hydrolysate rate of hydrolysis, increasing reaction time. Etha-
suitable for human consumption,94 Quaglia and nol can also adversely affect the reaction process
Orban36,84 defatted ground sardines by extraction in too high concentration by inhibiting protease
with isopropanol three times (solvent: substrate activity, although sorbic acid has not been found
ratio 1:1) at 46°C for 30 min, and then homog- to affect hydrolysis in concentrations up to 0.5%.24
enized the mixture with water. Hoyle and Merritt5
used an ethanol (90%) extraction directly on
minced herring at the fish/ethanol ratio of 1:2 at b. The Choice of Enzyme
70°C for 30 min, then mixed with equal volume
of water, hydrolyzed the mixture, then spray dried The water mince mixture is added to a reac-
it. Through this procedure the lipid content was tion vessel where the hydrolysis takes place. Of-
55
ten a flask, ranging from 0.5 to 3 L with a close- of enzyme(s) is usually determined by a combina-
fitting multisocket lid, has been used. The sockets tion of efficacy and economics.48
in the lid usually carry: a stirrer, driven by a The screening for a suitable enzyme in a pro-
overhead variable speed motor to ensure adequate cess or experiment is very important if the prod-
mixing of the system, a thermometer to monitor uct is to have predetermined properties. The
temperature, a pH electrode to monitor pH, and a screening process can be conducted in a variety of
“pH-stat” device, where acid or base is added to ways, and there is no standard methodology for
maintain a constant pH (Figure 5). The tempera- this selection, leaving it primarily up to the indi-
ture of the reaction vessel is controlled. After the vidual researcher what method is most appropri-
required temperature is achieved, the pH of the ate. Good examples of selection procedures are
slurry is adjusted to the desired value. It is impor- found in studies by Hale,97 Cheftel et al.,25 Arzu
tant that the mixture is well mixed and consis- et al.,98 Rebeca et al.,1 Baek and Cadwallader,85
tently stirred when the pH is added to allow for and Kristinsson and Rasco.99 In the comprehen-
uniform distribution of the added acid or base. sive study by Hale,97 the relative activities of
Processing temperature and pH is normally se- more than 20 commercially available proteolytic
lected to optimize the kinetics of the selected enzymes were measured for the hydrolysis of a
enzyme or enzyme mixture.48 A commercial pro- washed and freeze-dried fish protein substrate
tease is added in varying concentrations depend- from haddock. Preliminary tests at 1 h, 40°C, and
ing on the rate of hydrolysis needed. Given a pH 7 resulted in the plant enzyme ficin to be most
particular enzyme and a particular substrate, any active, but with papain, also a plant enzyme, hav-
hydrolysis process involves at least five indepen- ing a much higher relative ranking. When the
dent variables. These are S (protein substrate con- enzymes were tested at 24 h the picture changed,
centration: %N × 6.25), E/S (enzyme-substrate 60% digestion (the set limit) was achieved fastest
ratio in % or in activity units per kg N × 6.25), pH, by Pronase, which exhibited the greatest activity
T (temperature), and t (time).95 per unit weight. The enzymes pepsin, papain, and
A wide variety of commercial enzymes exist pancreatin were most suitable if the lowest cost
that have been used successfully to hydrolyze fish per unit of proteolytic activity was to be followed.
and other food proteins. Proteolytic enzymes from These cost estimates are less valid today due to
plants and microorganisms are most suitable to the commercial availability of bacterial enzymes.
prepare fish protein hydrolysates.26 Enzymes used Although many would prefer to use acid pro-
to hydrolyze fish protein have at least one com- teases so microbial growth could be more easily
mon characteristic: they have to be food grade, limited, they usually yield a product with low
and, if they are of microbial origin, the producing protein yield and too excessive hydrolysis for
organism has to be non-pathogenic.96 The choice food use.39,84,86 Therefore, milder enzymes at neu-
FIGURE 5. A typical enzymatic hydrolysis reaction system in the laboratory. (Adapted from Ref. 20.)
56
tral and slightly alkaline conditions have been waste:buffer ratio of 1:1 (w/v) at 60°C and pH
used more frequently in recent years. In some 9.5. The resulting hydrolysate had a high protein
cases, a high level of solubilization is desired. content with excellent nitrogen yield (up to 70%)
The acid enzyme pepsin has been most successful and an amino acid composition comparable to
in solubilizing fish protein. Liu and Pigott100 pro- fish muscle. Further, Alcalase was found to be
duced a high-quality, fluffy, water-soluble fish the most cost-effective enzyme out of five en-
protein hydrolysate by pepsin hydrolysis of rock- zyme preparations tested to hydrolyzed salmon
fish fillets. Tarky et al.101 used pepsin at 37°C and muscle proteins.99 Other new enzyme prepara-
pH 2.0 to hydrolyze the entire fish waste resulting tions have shown excellent potential for hydro-
from filleted English sole. The final product, after lyzing fish proteins to make highly functional
ultrafiltration and spray drying, was a creamy FPH, including Flavourzyme 1000L (Novo
white, nonhygroscopic, water-soluble hydrolysate Nordisk, Bagsvaerd, Denmark), Corolase 7089
with low lipid content but very poor nutritional (Rohm Enzymes; Somerset, NJ) and Corolase
value. PN-L (Rohm Enzymes, Somerset, NJ).99
Alcalase, an alkaline enzyme produced from In an extensive paper,33 Hale reported the
Bacillus licheniformis and developed by Novo effects of various processing conditions and com-
Nordisk (Bagsvaerd, Denmark) for the detergent mercially available proteolytic enzymes on yield
industry, has been proven repeatedly by many and composition of water-soluble fish protein
researchers to be one of the best enzyme used to hydrolysates. He concluded that the hydrolysis of
prepare functional FPH and other protein hydroly- raw hake (Urophycis chuss) with Alcalase at pH
sates.20,36,59,84,88,89 Shahidi et al.59 successfully used 8.5 or above gave the best balance of essential
Alcalase to optimize processing conditions to amino acids and a high yield of soluble product,
produce capelin protein hydrolysates. Alcalase- followed by pancreatin (a mixture of serine pro-
treated hydrolysates exhibited superior protein teases). One of the first studies on added enzyme
recovery (70.6%) compared with the alkaline pro- hydrolysis of fish protein was with papain, due to
tease Neutrase and papain. Alcalase-treated hy- its favorable properties of pH and temperature
drolysates also had the lowest lipid content (0.18%) optima for activity.79,80 Two fish species were
and excellent functional properties. Quaglia and used as substrate, one freshwater, Barbus
Orban36 studied the same three enzymes at opti- carnaticus, and the other marine, Rastrelliger
mal conditions on enzymatic solubilization of canagurta, and studied at 40 and 55°C, and pH 5
sardine proteins. Hydrolysates produced using and 7. Total solids and nitrogen recovery for both
Alcalase and papain were almost identical in ni- species was high, with pH 7 having the highest
trogen recovery, which increased with increasing total solids and nitrogen recovery (69.7% at 55°C
enzyme concentration (70% recovery at a en- for freshwater species) compared with pH 5, pos-
zyme/substrate ratio of 4%). Neutrase-treated sibly attributed to better hydration at pH 7.
hydrolysates at the same ratio had only over 20% To properly compare enzyme activity on the
nitrogen recovery. Hydrolysates from Alcalase same substrate, it is necessary to determine the
and papain also exhibited better functional prop- general proteolytic activity units at specific reac-
erties and high nutritional value than those from tion conditions. Unfortunately, very few research-
Neutrase. Improved nitrogen recovery of fish pro- ers have done this, and most compare enzyme
tein hydrolysates with increase of protease con- activity on a weight basis of enzymes used in the
centration has been reported elsewhere.1,79 Alcalase reaction mixture. Adding enzymes on the basis of
and Neutrase were studied further recently by weight is meaningless if relative enzyme activity
Benjakul and Morrissey89 on Pacific whiting solid is to be compared, because enzyme activity per
waste at pH 9.5, 60°C and pH 7.0, 55°C, respec- weight is different for each enzyme under experi-
tively. Alcalase had a considerably higher activ- mental conditions used. However, there exist few
ity than Neutrase and led to a more efficient hy- studies that use and compare enzymes on the
drolysis. Optimum conditions for Alcalase were basis of their proteolytic activity. Gonzalez-Tello
20 Anson Units (AU)/kg, 1 h reaction time, and et al.69,102 studied three proteases on whey protein
57
substrate by adding them to the system based on a proteolytic enzyme cleaves the peptide linkages
Anson Units (AU). Unfortunately, they did not in it. The rate at which the dye is released can be
use the three enzymes at the same AU. In addi- used to quantitatively measure the amount of pro-
tion, the activity units were obtained from the teolytic enzyme(s) activity working in a solution
manufacturer and not assayed by the researchers. by measuring the absorbance at 520 nm. The
Also, reaction conditions used in the hydrolysis enzymes were then used to hydrolyze the sub-
experiments were different from the conditions strate, all at the same activity unit according to the
used in the enzyme assays, thus making the activ- Azocoll assay, thus comparing them at the same
ity units very unreliable. activity level on the same substrate. No reports in
Benjakul and Morrissey89 studied the hydroly- the literature have taken this approach; however,
sis of Neutrase and Alcalase on Pacific whiting the use of Azocoll in fish protein hydrolysis ex-
solid wastes by adding the enzymes to the system periments has been reported by Ferreira and
based on AU units. This study has the same limi- Hultin.104
tation as the studies by Gonzalez-Tello et al.69,102
by using reaction conditions different than those

used to assay the enzyme for proteolytic activity c. The Mechanism of Enzymatic Hydrolysis
and relying on enzyme units provided by the sup-
plier instead of assaying the enzymes themselves. The enzymatic hydrolysis of fish muscle pro-
Other studies on fish protein hydrolysis add en- teins is characterized by an initial rapid phase,
zymes according to AU units and also suffer from during which a large number of peptide bonds are
these same limitations.59,82 A study by Beddows hydrolyzed, after this rate of enzymatic hydroly-
and Ardeshir103 is the most carefully conducted sis decreases and reaches a stationary phase where
study in the literature with respect to using stan- no apparent hydrolysis takes place59 (Figure 6).
dardized relative enzyme activity. They assayed The shape of the hydrolysis curve has been asso-
three proteases, bromelain, ficin, and papain, by ciated with enzyme inactivation, product inhibi-
using BApNA (Benzoyl-Arg-para-Nitroanilide) to tion by hydrolysis products formed at high de-
obtain some indication of the relative proteolytic grees of hydrolysis, a low Km value for the soluble
activities of these enzymes. They then added the peptides that act as effective substrate competi-
enzymes to a system of minced Ikanbilis tors to the unhydrolyzed fish protein,1 and possi-
(Stolephorus sp.), a tropical fish, at the same ac- bly autodigestion of the enzyme.105 Shahidi et
tivity units as based on their assay. The assay, al.59 found that a high concentration of soluble
however, involved different reaction conditions fish peptides in the reaction mixture, released
than the hydrolysis experiment, which limited its during the initial phase of hydrolysis, reduced
reliability. Assaying enzymes with BApNA also both the rate of hydrolysis and the recovery of
only estimates the trypsin activity of the enzyme soluble proteins. Thus, removal of hydrolysate
preparation, not their general proteolytic activity. from the reaction mixture should enhance the
Using BapNA, therefore, gives a less accurate hydrolysis rate and the protein recovery.
and and probably underestimated value of en- By increasing the protease concentration, and
zyme activity. In a research conducted by thereby increasing the extent of hydrolysis, re-
Kristinsson and Rasco,99 where salmon muscle covery of soluble nitrogen increases,1,71,103 al-
proteins were hydrolyzed, the enzymes were as- though increasing enzyme concentrations may not
sayed under the same reaction conditions as they be cost effective. Substrate concentration has also
were used in the hydrolysis experiment. A syn- negative effects on protein recovery. Linder et
thetic protein substrate for proteolytic activity, al.47 found that more than 8% protein concentra-
Azocoll, was used to obtain a uniform level of tion in the system, regardless of enzyme concen-
proteolytic activity for all enzymes used. Azocoll tration, seemed to have an inhibiting effect on
is an insoluble cowhide preparation consisting protein recovery. Baek and Cadwallader85 reported
largely of collagen. The method is based on dye using Optimase to hydrolyze crayfish processing
release from the insoluble substrate Azocoll when byproducts the %DH increased as substrate con-
58
FIGURE 6. Hydrolysis curve for salmon muscle mince with Corolase 7089 at three different activity units (AzU =
Azocoll Units).
centration decreased to 45% (w/v), suggesting on the kinetics of protein solubilization. Their
that high %DH did not coincide with a high amount experimental data followed Michaelis-Menten
of hydrolysate. Similarly, Surowka and Fik,70 who type kinetics. Michaelis-Menten kinetics has also
measured the production of protein hydrolysate been observed with whey protein hydrolysis.69
with Neutrase from chicken heads, reported that Kristinsson and Rasco99 studied the kinetics of
hydrolysis increased as substrate concentration five different enzyme preparations during the
decreased. Ferreira and Hultin,104 using Newlase hydrolysis of salmon muscle proteins and found
A to hydrolyze cod (Gadus morhua) frames, found that the initial rate of the reaction for all enzymes
that enzyme autolysis can be reduced at higher showed a linear relationship to enzyme activity.
substrate concentrations. These experiments indicated that the initial rate
Because fish tissue is a very complex sub- constants for each enzyme tested were in the same
strate and also contains large amounts of protein- order. This confirmed previous studies by Hevia
ase inhibitors, it is impossible to explain the mecha- et al.106 and Cheftel et al.25 on menhaden fish
nisms of protein hydrolysis in detail for this protein as a substrate.
system.60 A kinetic study of the process is also A kinetic model of whey protein hydrolysis
quite complicated due to the various types of with Alcalase has been proposed, where the hy-
peptide bonds involved and their differing vulner- drolytic reaction is zero-order for substrate. The
ability to attack by enzymes during the hydrolytic enzyme denatures simultaneously via a second-
process.69 Very few studies on kinetics of fish and order reaction due to free enzymes attacking the
food protein enzymatic hydrolysis are reported. enzyme bound to the substrate.69
Sakai et al.87 conducted a kinetic study on the Moreno and Cuadrado107 hydrolyzed vegetable
hydrolysis of pollack surimi protein using a acid proteins with Alcalase and found reaction mecha-
protease derived from Aspergillus niger, deter- nism consistent with substrate inhibition and a
mining the effects of temperature, pH, initial sub- second-order deactivation with respect to the en-
strate concentration, and enzyme concentration zyme concentration. Enzyme autolysis was de-
59
pendent on the substrate concentration. Cheftel et ties of a protein hydrolysate for a given protein-
al.25 used Pronase to hydrolyze fish protein con- enzyme system.96 The degree of hydrolysis dem-
centrate and found that the rate constant decreased onstrates both theoretically and empirically that
with time, and that proteolysis did not follow four processing variables, S, E/S, T, and t, can be
first-order kinetics with respect to concentration left uncontrolled, provided that %DH is con-
of the peptide bonds. Cheftel et al.25 suspected trolled.95 From this, it is obvious that %DH is a
this to be due to the multitude of possible sub- very simple and rapid method of measuring the
strates in FPC, the number of different proteolytic extent of protein breakdown.
enzymes present, the inhibitory effects of sub- Two methods for measuring %DH have been
strate or self-digestion, as well as the different studied thoroughly and shown to be satisfactory,
specificities that are known to be present in Pro- the pH-stat technique and osmometer technique.
nase. Archer et al.108 also studied the kinetics of The pH-stat method is more commonly used and
the enzymatic hydrolysis of FPC. Their research more useful for industrial applications. The prin-
found that the enzyme is initially adsorbed to the ciple behind the pH-stat method is relatively simple
surface of the protein, with the initial rate of and is based on maintaining a constant pH during
reaction being proportional to the surface area of the reaction. By pH-stat, the %DH is calculated
substrate exposed to the aqueous phase. The over- from the volume and molarity of base or acid used
all kinetics were described by a sequence of two to maintain a constant pH. The degree of hydroly-
first-order processes, an initial, fast reaction in sis is defined as the percent ratio of the numbers
which loosely bound polypeptide chains are of peptide bonds broken (h) to the total numbers
cleaved from an insoluble protein particle, and a of bonds per unit weight (htot; meq/kg protein,
second, slower reaction in which a more com- calculated from the amino acid composition of
pacted core protein is digested.108 Langmyhr109 the substrate): %DH = (h/htot) × 100.
studied the kinetics of the hydrolytic breakdown %DH can also be expanded to:
of cod muscle with acetyltrypsin. The results in-
dicated that enzyme molecules are rapidly and B ⋅ NB
%DH = ⋅ 100
firmly bound to fish proteins and that the maxi- α ⋅ htot ⋅ MP
mum binding occurred under the optimum pH
and T conditions for the enzyme. Results also
suggested that muscle proteins are hydrolyzed in where B = base consumption in ml (or acid in
the same way and to the same extent whether they case of acid proteases), NB = normality of the base
occur separately or are integrated into intact (or acid), α = average degree of dissociation of
muscle. the –NH groups or COOH groups, MP = mass of
protein in grams (%N × 6.25). The degree of
dissociation is found by the following equation:
d. Measuring the Extent of Enzymatic
10 pH – pK
Hydrolysis α=
1 + 10 pH – pK
To follow the reaction kinetics and get a
measure for the extent of the hydrolytic degrada- This equation for %DH is valid when hydrolysis
tion, a parameter named degree of hydrolysis is conducted at a pH above the pK of the α-NH
(%DH) is employed. In principle, there are sev- group (for hydrolysis at neutral or alkaline pH).
eral control methods, but under practical indus- Under these conditions, the reaction will result in
trial conditions there are few.96 The degree of the a net release of protons (H+) when peptide bonds
hydrolysis is most commonly used to describe are cleaved and the base consumption is propor-
hydrolysis of food proteins. The advantage of tional to the number of peptide bonds split.110
using %DH as a process parameter is that %DH Above pH 6.5, the dissociation of the protonated
appears to determine unambiguously the proper- α-NH group becomes significant. Therefore, it is
60
important to know the pK values of the α-NH ∆T = K f ⋅ ω ⋅ ∆C
groups, both to determine the reaction pH and to
calculate the dissociation constant correctly. The
pK value varies significantly with temperature. where Kf is 1.86 K/mol for water, ω the osmotic
The pK values at different temperatures can be coefficient, and C the osmolality of solution. From
calculated according to Steinhardt and Beychok:111 the increase in osmolality, ∆C, %DH can be cal-
culated according to the following formula:
298 – T
pK = 7.8 + ⋅ 2400
298 ⋅ T  ∆C 1 1 
% DH =  ⋅ ⋅  ⋅ 100
 S% ⋅ fosm w htot 
where T = temperature in Kelvin.
If samples are drawn during the experiment,
it is crucial to correct for the actual value of base where ∆C = depression of freezing point mea-
consumed. Actual base consumption (B′) will be sured in milli-osmol, S% × fosm = g protein/1000
smaller than the theoretical base consumption (B) ml H20, 1/w = calibration factor for the osmom-
if this is the procedure. To correct for this, B can eter: 1.04, htot = total number of peptide bonds in
be calculated from the following formula:20 the substrate.
The fosm factor is calculated by knowing the
percent substrate dry matter, D%, present in the
reaction mixture:
1000
fosm =
100 – D%
Although the osmometer technique is a very

good method to determine the degree of hydroly-
sis, it is being less employed frequently. The de-
gree of hydrolysis is now being more commonly
determined by using either the trichloroacetic acid
where B′1, B′2, etc. are the actual base consumption (TCA) method or the trinitrobenzenesulfonic acid
at the drawing of samples number 1, 2, etc. The (TNBS) method. Both of these methods are very
value n is the sample number and m is the sample useful when working within the pH 3 to 7 range,
size (ml). The pH-stat method, however, has some where the pH-stat is unusable. Several variations
limitations. Use of pH-stat as a means of control of these methods exist. The most commonly used
is only practical outside the approximate pH in- TCA method is based on determining the ap-
terval 3 to 7.96 This has to be taken into consider- proximate degree of hydrolysis (%DH) of protein
ation when deciding on pH values for the hy- hydrolysates by the ratio of percent 10% TCA
drolysis reaction. Within this pH range, process soluble nitrogen in the hydrolysate compared to
control may depend on other methods. Therefore, total amount of protein in sample. This is gener-
the pH-stat method for %DH determination is ally done by removing aliquots at selected inter-
useful mainly under alkaline conditions. vals and mixing with 20% TCA to create 10%
The other commonly used method to measure TCA-soluble and TCA-insoluble fractions. These
hydrolysis, the osmometer technique, is more mixtures are then centrifuged and the supernatant
universally applicable. This technique measures analyzed for nitrogen. The degree of hydrolysis is
the freezing point of samples drawn at regular thus calculated from the following equation:
intervals during hydrolysis to construct a hydroly-
sis curve. For this method, the freezing point
depression (∆T) is measured and converted to %DH = (10% TCA-soluble N in sample/Total N
milli-osmol (∆C) as follows:95 in sample) × 100
61
This method has been used successfully in fish5 been reported to need at least 90°C for 30 min to
and other food protein hydrolysis.44,70 The TNBS be fully inactivated.5 Terminating the reaction by
method is based on the concentration of primary thermal means is undesirable114 because of the
amino groups in the hydrolysate. It is a spectro- effects of heat denaturation on the protein that
photometric assay of the chromophore formed by leads to exposure of hydrophobic residues and
the reaction of TNBS with liberated amino groups subsequently protein aggregation. Diniz and Mar-
(420 nm), at slightly alkaline conditions.112 The tin88 suggest that this form of heating can have the
degree of hydrolysis can then be calculated as advantage of being very effective in the separa-
presented by Baek and Cadwallader, 85 and tion of oil from the fish protein substrate, al-
Benjakul and Morrissey,89 for crayfish hydrolysis though Webster et al.39 found that some protein-
and whiting hydrolysis, respectively: fat interaction occurred at elevated temperatures
that prevented their separation, when using bo-
Lt – L0 vine lungs as the protein substrate.
% DH = ⋅ 100
Lmax – L0 The temperatures at which various proteins
denature and unfold vary enormously. Because

fish muscle proteins are adapted to function at
where Lt = the amount of a specific liberated lower temperatures, they may not be particularly
amino acid at time t, L0 = the amount of the heat stable. Denaturation is usually undesirable
specific amino acid in original substrate (blank), because it results in altered physicochemical prop-
Lmax = the maximum amount of the specific amino erties, particularly a loss in protein solubility and
acid in the substrate obtained after hydrolysis. functionality.31 Spinelli et al.34 are among few
New methods and modifications of previous researchers that have recognized the adverse ef-
methods for determining the degree of hydrolysis fect elevated temperatures may have on fish pro-
are being developed. In the special case of fish, a tein hydrolysates. To both terminate the enzy-
simple method for monitoring the enzymatic hy- matic reaction and quantitatively recover the
drolysis of fish protein was developed by Ukeda protein fractions, they reacted enzymatically hy-
et al.113 The degree of hydrolysis was monitored drolyzed muscle proteins from rockfish with 5%
by an amino group determination method with sodium hexametaphosphate (HP), following slight
gluteraldehyde (GA). The method is based on the acidification to form an insoluble protein-phos-
consumption of dissolved oxygen during the re- phate complex. By isoelectric precipitation, they
action between gluteraldehyde and the liberated recovered up to 90% of the protein. In addition,
amino groups of the protein substrate. The results the complex could be washed free of occluded
by the GA method were in agreement with the nonprotein nitrogen components with no loss of
TNBS method previously described, with a corre- protein nitrogen.
lation coefficient of r = 0.992. Chemical inactivation would be to either lower
or raise the pH of the slurry to a point where the
e. Termination of Enzymatic Reaction enzyme deactivates. Some enzymes are more sen-
sitive to pH changes than they are to temperature
When a desired %DH is attained, it is neces- changes. Alcalase is a relatively thermostable
sary to terminate the enzymatic reaction. This is enzyme, but it is very sensitive to acid pH. Com-
very important as otherwise the enzymes would plete inactivation of Alcalase therefore is obtained
remain active in the substrate and further hydro- by lowering the pH to 4.0.20,59 Neutralizing the
lyze the protein and peptides. Deactivation of slurry to pH 7.0 would inactivate pepsin and most
enzymes is achieved either by chemical or ther- other acid proteases. Extremes of pH can also,
mal means. Usually the slurry of hydrolysate and like elevated temperatures, have detrimental ef-
enzymes are transferred to a heat bath, where the fects on protein and peptides. Many proteins un-
enzymes are deactivated by exposing them to fold at pH values less than about 5 or greater than
temperatures ranging from 75 to 100°C for 5 to 10. Unfolding at such extremes of pH usually
30 min, depending on the type of enzyme. For occurs because the folded protein (or oligopeptide)
example, papain is very heat tolerant, and has has groups buried in nonionized form that can
62
ionize only after unfolding.52 Stabilizing salt- fraction. Other separation methods for fish pro-
bridges between ionizing groups can also be dis- tein hydrolysates have been reported such as suc-
rupted by extreme pH values. The termination of tion filtration of the sludge82 and filtering the
the enzymatic reaction thus is a formidable ob- slurry by passing it through a 2-mm mesh screen.86
stacle in any FPH operation. The choice of inac- The removal of colored and odiferous matter has
tivation method should be carefully made and also been reported by treating the first soluble
based up on what the enzyme under study is fraction obtained by centrifugation with 1% w/v
sensitive to, whether it is heat or pH. In some charcoal at 55°C and 30 min before the next
cases a combination of elevating temperatures separation step.59
and lowering pH have been used.82 In a commercial operation, the final soluble
fraction is generally spray dried to convert the
hydrolysate to a powdered form, which can be
f. Protein Hydrolysate Concentration incorporated into food formulations. The insoluble
fraction or the sludge precipitated during cen-
Commonly, the slurry is desludged by centrifugation may be used as animal feed. Spray
trifugation, which results in several fractions (Fig- drying of the soluble fraction is one of the most
ure 7): sludge in the bottom, aqueous layer in the energy consuming and expensive steps in the pro-
middle, lipid-protein fraction between aqueous duction of protein hydrolysates. In industrial pro-
layer and sludge, aqueous and oil layers and oil duction a compromise must be made between the
layer on the top.68 A single centrifugation step can hydrolysate yield and the amount of water that
eliminate the vast majority of the lipids present. must be removed to obtain a dry product.60 A
The oil layer overlying the aqueous layer is then suspension of equal amounts of water and fish
removed and the soluble fraction collected. Be- substrate appears to be convenient from an eco-
cause lipid in the final hydrolysate is a major nomic point of view. Rebeca et al.1 performed a
concern for FPH, it is important to remove it. study where eviscerated mullet (Mugil cephalus)
Lipid residues in FPH must be lower than 0.5% to was enzymatically hydrolyzed without the addi-
prevent alteration of the lipid fraction during stor- tion of water, to lower the cost of spray drying.
age.34 More than one centrifugation step is often Interestingly, the experiment resulted in higher
required to separate the soluble proteins from the dry matter content, with high protein recovery, of
lipids and insoluble solids. The second centrifu- the soluble fraction and the cost of drying was
gation step would be performed only on the soluble reduced. Generally, in the laboratory, hydroly-
CLEAR OIL
LIGHT LIPID-PROTEIN
LIPID-PROTEIN SMEAR
AQUEOUS
HEAVY LIPID-PROTEIN
SLUDGE
FIGURE 7. Different fractions obtained when recovering soluble fish protein hydroysates.
63
sates are neutralized and freeze dried. By neutral- functional properties. The functional properties
ization the final product can have a fairly high salt of fish protein hydrolysates are important, par-
content, which is undesirable (HCl + NaOH → ticularly if they are used as ingredients in food
H2O + NaCl). This can be mostly avoided if the products.60 Enzymatic hydrolysis of fish proteins
soluble fraction is passed through an ion exchange generates a mixture of free amino acids, di-, tri-,
column before freeze-drying. Dialysis to desalt and oligopeptides, increases the number of polar
the soluble fraction can also be a useful process. groups and the solubility of the hydrolysate, and
Increasingly, ultrafiltration membranes have therefore modifies functional characteristics of
been introduced into the production process for the proteins, improving their functional quality
hydrolysates. The hydrolysate after enzyme inac- and bioavailability. The choice of substrate and
tivation is filtered directly through a membrane proteases employed and the degree to which the
with a specific molecular weight cut-off value. protein is hydrolyzed affect the physicochemical
The ultrafiltration process can contain more than properties of the resulting hydrolysates.105 En-
one filter, yielding several molecular weight frac- zyme specificity is important to peptide function-
tions depending on the product desired. A major ality because it strongly influences the molecular
advantage for such a process is the control of size and hydrophobicity of the hydrolysate.117
molecular size of selected peptides so that a uni- Thus, the peptides obtained have different mo-
form product is possible,76 as the molecular size lecular profiles, and the surface energy of the
of the hydrolyzed proteins is a key factor in deter- hydrolysate is different, depending on the en-
mining functional properties of hydrolysates.115 zyme used; these variations have a bearing on the
The method has, however, not found its way into functionality of the mixture.114,118 The more nar-
fish protein hydrolysate production. The reason row the specificity, the are the larger peptides
probably being that in order to successfully use produced; the broader the specificity, the smaller
the ultrafiltration, the sample has to be very pure are the peptides generated. As the range of enzy-
and free of lipids. It is, however, possible that the matic activities within commercial preparations
method could be applied to highly purified and is increased, the hydrolysate becomes more com-
defatted FPH powders. The method has found to plex.4 The chain length of peptides or breaking of
be very useful in soy,115 whey,75,116 and casein linkage is also dependent on the extent of hy-
protein hydrolysis.76 In the case of whey protein, drolysis; conditions of hydrolysis; concentration
large-molecular-weight fractions are primarily of enzyme and the type of protein to be hydro-
responsible for allergic reactions. However, small lyzed.41 Due to the complex peptide profile, it is
peptides and free amino acids are less well ab- often useful to calculate the average peptide chain
sorbed by humans. Therefore, protein hydrolysis length (PCL) introduced by Adler-Nissen and
should not be more extensive than the minimum Olsen119 to express the composition of the protein
required to eliminate allergic responses. By ultra- hydrolysate. PCL is calculated from the degree of
filtration therefore it is possible to exclude both hydrolysis in the following way:
the too large and too small peptides and collect
100
the molecular weight in between by passing the PCL =
hydrolysate solution through two filters, the first % DH
with a higher cut-off value than the second.116 This approximation is acceptable in nearly all
Applying this technique to FPH would be an inter- practical cases.20 The relationship between PCL
esting task to undertake. and %DH is shown in Figure 8.
Manipulating the reaction conditions during
IV. PHYSICOCHEMICAL AND enzymatic hydrolysis of food proteins produces
FUNCTIONAL PROPERTIES OF FISH hydrolysates with different solubility and emulsi-
PROTEIN HYDROLYSATES fying characteristics, foaming properties, or taste
characteristics.4 The control of the enzymatic re-
As previously mentioned, one of the major action is very important, as previously discussed.
advantages and goals of enzymatically hydrolyz- Uncontrolled or prolonged hydrolysis of fish pro-
ing fish proteins is to modify and improve their teins may result in the formation of highly soluble
64
FIGURE 8. The relationship between average peptide chain length (PCL) and degrees of hydrolysis (%DH).
peptides, completely lacking the functional prop- such as emulsification and foaming, are affected
erties of the native proteins. This may promote by solubility,121 and therefore it is an excellent
the formation of undesirable bitter peptides. By indicator of the protein hydrolysate functionality,
controlled hydrolysis it is possible to eliminate and its potential (and limitations of) applica-
these drawbacks and to obtain hydrolysates with tions.31,58 Hydrophobic and ionic interactions are
different physicochemical and functional proper- the major factors that influence the solubility char-
ties, in some cases even better than the originals.36 acteristics of proteins. Hydrophobic interactions
The physical and chemical properties that govern promote protein-protein interactions and result in
protein functionality include size, shape, amino decreased solubility, whereas ionic interactions
acid composition and sequence, net charge and promote protein-water interactions and result in
distribution of charges, hydrophobicity/hydrophi- increased solubility. Ionic residues on the surface
licity ratio, peptide structures, molecular flexibil- of peptides and proteins introduce electrostatic
ity/rigidity, and the ability to interact/react with repulsion between protein molecules and repul-
other components.120 Functionality of food pro- sion between hydration shells around ionic groups,
teins has been defined as: “those functional and both major contributors to increased solubility of
chemical properties which affect the behavior of proteins. Solubility of protein and protein hy-
proteins in food systems during processing, stor- drolysates is generally measured by employing
age, preparation and consumption”.31 The follow- the nitrogen solubility index (NSI), a standard-
ing sections discuss the main functional proper- ized method developed by AOCS and later modi-
ties that fish protein hydrolysates exhibit, that is, fied by Morr et al.122 The NSI is determined by
solubility, water holding, emulsifying, foaming suspending a protein hydrolysate sample in water
and sensory properties, and research aimed to- then stirring and centrifuging the mixture. The
ward evaluating these properties for various FPH supernatant is then analyzed for nitrogen content
preparations. by the Kjeldahl procedure, and the NSI calculated
as a percentage of the soluble nitrogen to the
A. Solubility percentage of total nitrogen in the sample.20
Intact fish myofibrillar proteins have the prob-
Solubility is probably the most important of lem of the lack of solubility in water over a wide
protein and protein hydrolysate functional prop- range of pH,13,123 and enzymatic hydrolysis is very
erties. Many of the other functional properties, important in increasing the solubility of these
65
proteins. The effects of enzymatic hydrolysis on pared with the neutral and acid ones. Gel chroma-
fish protein are straightforward. Enzymatic break- tography showed that hydrolysates prepared with
down of the protein involves a major structural the alkaline proteases had a lower average mo-
change in that the protein is gradually cleaved lecular weight. The increased hydrophilicity of
into smaller peptide units, and as the degree of these preparations could be due to their larger
enzymatic hydrolysis increases the solubility of charge to size ratios compared with the longer
fish proteins increases. The enhanced solubility proteins such as those found in nonhydrolyzed
of the hydrolysates is due to their smaller molecu- proteins from a similar source.44 Interestingly,
lar size compared with the intact protein, and the pepsin had lower solubilized protein ratio than all
newly exposed ionizable amino and carboxyl of the alkaline proteases, but pepsin is generally
groups of the amino acids, that increase the hy- considered one of the best enzymes to solubilize
drolysate hydrophilicity.58,117 To effectively bind fish protein.35,36,68,97,100 Quaglia and Orban35,36 also
to water molecules, the peptides have to have the studied the properties of hydrolysates produced
ability to form hydrogen bonds between its hy- from sardine and concluded that Alcalase and
drophilic polar amino acid side groups and the papain, at optimum pH and temperature, both
water molecules. Hydrolysis exposes some of the gave hydrolysates characterized by high solubil-
hydrophobic groups to the surface, but at the ity. Hydrolysates made with Alcalase at higher
same time converts even more hydrophobic groups degrees of hydrolysis showed a decrease in high-
to hydrophilic groups by generating two end car- molecular-weight fractions, and increased solu-
bonyl and amino groups. Thus, the smaller pep- bility.35 This connection between %DH and solu-
tides from myofibrillar protein hydrolysis have bility is also reported for other food protein.124 A
proportionally more polar residues, with the in- significant increase in solubility was observed
creased ability to form hydrogen bonds with wa- with an increase in %DH from 2.5 to 15% for an
ter. This increases protein solubility to that of the enzymatically hydrolyzed single cell microbial
intact protein. In addition, ion-dipole interactions protein (“Pruteen”), from less than 10% solubility
between water and simple ions such as Na+ and to over 90%.124 Similar results were observed
Cl- are also important in the interactions between with soy protein hydrolysates from 1 to 8.3%
the polar or charged groups on biomolecules and DH.96 Yu and Fazidah125 hydrolyzed Aristichthys
water.18 Biomolecules tend to be very soluble at noblis, a Chinese freshwater fish, with protease P
favorable NaCl conditions. The increased solubil- “Amano” 3, and reported excellent solubility at
ity is partly through the formation of sodium salts 15% DH after a 3-h digestion.
of carboxyl groups of proteins, COONa.13 This is Hoyle and Merritt5 found that enzymatically
true for fish myofibrillar proteins, but the hydro- hydrolyzed ethanol-extracted herring FPH had
phobicity of globular proteins, such as milk pro- highest solubility compared with nonextracted
teins, may increase with exposure of apolar amino enzymatically hydrolyzed FPH. This finding sug-
acid residues after hydrolysis.117 Although in- gests that the lower lipid content of ethanol-ex-
creased solubility has a positive relationship to tracted herring FPH may have resulted in less
the extent of hydrolysis, care has to be taken that competitive water binding of proteins compared
the substrate is not too extensively hydrolyzed. A with hydrolysates with higher lipid contents. Vieira
very high degree of hydrolysis may lead to high et al.86 studied the functional properties of hy-
solubility, but this can have very negative effects drolysates from lobster processing wastes and
on the rest of the functional properties. To main- found these to be highly soluble, pepsin yielding
tain or improve functionality, generally low de- a fraction with higher solubility than papain or a
grees of hydrolysis are necessary.4 fungal protease. The highest nitrogen solubility
Sugiyama et al.84 studied enzymatic hydroly- index (NSI) for these hydrolysates was found at
sis with several alkaline, neutral, and acid pro- extremes of pH. The solubilities of all the prod-
teases on defatted sardine meal. The result showed ucts were low at pH 5, approximately correspond-
that all the alkaline proteases had higher ability to ing to the isoelectric point of protein, at which it
produce highly soluble protein hydrolysates com- precipitates. Shahidi et al.59 also found that cape-
66
lin protein hydrolysates (CPH) prepared by dif- peptone production.129 In the U.S., the main appli-
ferent enzymes had different solubility profiles at cation of FPH is not for foods but as weanling
different pH conditions. However, in contrast to feed for piglets, and increasingly as a pellet coat-
the findings of Vieira et al.,86 Shahidi et al.59 ing for pet food.60
found lower solubility at pH 7 to 8, and with close
to 100% solubility for Alcalase at pH 5. The
lowest solubility value at pH 7 to 8 was, however, B. Water-Holding Capacity
84%, thus CPH had excellent solubility over the
range of pH 2 to 11. Hydrolyzed salmon muscle Water-holding capacity refers to the ability
proteins exhibited excellent solubility at 5 to 15% of the protein to imbibe water and retain it against
DH, between 92 to 100%, with over 96% solubil- gravitational force within a protein matrix, such
ity in the pH range of 2 to 11.90 as protein gels or beef and fish muscle, and it is
The solubility of enzymatically hydrolyzed positively correlated with water-binding capac-
fish protein is generally much higher than for ity.120 Water-holding capacity of proteins added
chemically produced fish protein concentrate to muscle tissue is of great importance to the food
(FPC). A FPC type-B air-dried fish powder had industry because retaining water in a food system
extremely poor solubility at a wide range of NaCl often improves texture. The functional properties
molarity and pH,126 and alkaline hydrolysis of red of proteins in a food system depend in part on the
hake exhibited lower than 30% solubility at pH 1 water-protein interaction, and the final outcome
to 7.45,46 greatly depends on how well the protein binds
Pure fish myofibrillar protein are very in- and holds water in a food system. However, a
soluble close to their isoelectric point; therefore, successful specific application of a functional
high solubility of FPH over a wide range of pH is protein ingredient or additive for enhancing a
a very useful property for many food applica- single specific functional property may not be
tions, including beverage applications. The good transferable to other food systems. Fish protein
solubility and good nutritive value of enzymati- hydrolysates are highly hygroscopic, and this has
cally hydrolyzed fish protein hydrolysates also to be considered when producing them. Proper
makes them well suited to produce milk replacers packaging and low relative humidity of air during
for weanling animals.1,10,124 Presently, this is be- processing is an important consideration. The
ing done in Japan and France. In Japan, one com- presence of polar groups such as COOH and NH2
pany is making “bio-fish flour” by enzymatic that increase during enzymatic hydrolysis have a
digestion of sardines that is used in feeds as a substantial effect on the amount of adsorbed wa-
milk replacer for calves and piglets.60,126 Menha- ter and moisture sorption isotherm for these ma-
den hydrolysate produced by pancreatine was terials. The recommended maximum water con-
found to be an excellent milk replacer with high tent of FPH for storage is 0.075 g/g at less than
PER value with the process cost half as much as 15% RH.130
dried skimmed milk.127 Soluble FPH is an excel- Fish protein hydrolysates have excellent wa-
lent amino acid source for supplementing cereal ter-holding capacity, and thus useful properties
proteins10 and to be used in bakery products, soups, for certain food formulations. Kristinsson90 made
and infant formulas.1 Pasta enriched up to 3.5% of salmon FPH with several enzymes to 5, 10, and
fish protein hydrolysate increased the protein 15% DH and added these to minced salmon pat-
content by 5%, with a dramatic increase in the ties at 1.5%. Water loss after freezing (48 days)
dietary essential amino acids lysine (37.5%), va- and thawing was reduced to 1% compared with
line (31%), and threonine (18%).128 Soluble FPH 3% for the control. In this study there was no
from extensive hydrolysis is an excellent source connection observed between %DH and water
of nitrogen for microbial growth. Extensive hy- loss, FPH made using Alcalase had the best wa-
drolysis results in a product of free amino acids ter-holding properties in salmon mince patties,
and low-molecular-weight peptides, and there- but all FPH exhibited better water-holding prop-
fore has found to be very promising for microbial erties than egg albumin and soy protein concen-
67
trate. Onodenalore and Shahidi82 increased cook- loops in the apolar oil phase, while the polar
ing yield of comminuted pork by 2.4 to 9.3% by segments extend into the aqueous phase. Desir-
adding 0.5 to 3.0% of a shark protein hydrolysate. able surface active proteins and protein hydroly-
Cooking yield increased with increasing addition, sates have three major attributes: (1) ability to
and the hydrolysates obtained via an Alcalase- rapidly absorb to an interface, (2) ability to rap-
assisted process from the washed myofibrillar idly unfold and reorient at an interface, and (3) an
shark proteins were more effective than hydroly- ability, once at the interface, to interact with the
sates made from untreated shark muscles. Shahidi neighboring molecules and form a strong cohe-
et al.59 found similar results with capelin protein sive, viscoelastic film that can withstand thermal
hydrolysates (CPH) when added at a 3% level to and mechanical motions.120,132
communited pork, CPH increased cooking yield Emulsifying capacity and emulsifying stabil-
of approximately 4%. At even lower addition lev- ity are two methods generally used to measure the
els there was a large reduction in the amount of ability of protein hydrolysates to form and stabi-
drip loss, indicating that CPH has strong water- lize emulsions. Emulsifying capacity is usually
binding capacity. In addition, CPH at 0.5 to 3.0% defined as the volume of oil (ml) that can be
level inhibited the formation of TBARS by 17.7 emulsified by the protein hydrolysate (g), before
to 60.4%, suggesting that CPH may have antioxi- phase inversion or collapse of emulsion occurs.31
dant properties, perhaps due to chelation effects. Because FPH produces emulsions of low viscos-
Hatate et al.131 also found that sardine myofibril ity, the most accurate way to measure this is to
protein hydrolysates exhibited antioxidant activ- use an oil titration method,133 and measure the
ity. More importantly, hydrolysates appeared to electrical resistance during oil titration. A sudden
act synergistically with several commercial anti- increase in resistance is observed when the maxi-
oxidants. These antioxidant effects are highly mum emulsifying capacity is reached. Emulsion
dependent on the amino acid composition and stability refers to the ability of an emulsion to
molecular size of the hydrolysate peptides. resist changes in its properties over time134 and
Fish protein concentrate (FPC) produced from can be determined simply using gravitational
sardine with an ethanol process has also been methods. Measurement involves blending the
studied in a meat model system with respect to hydrolysate with oil and water, centrifuging, and
water holding. FPC was added to a hamburger- measuring total volume of emulsion, its stability
type product made from beef at 20, 40, and 60% expressed as the difference between total volume
addition, and increased percentage of FPC in the of an emulsion and the aqueous volume to total
formulations significantly improved the cooking volume.135,136
yields of the products.37 The emulsifying properties of hydrolyzed
protein are improved by carefully controlling the
extent of hydrolysis. Extensive hydrolysis results
C. Emulsifying Properties in a drastic loss of emulsifying properties.58 Al-
though small peptides are highly stable and dif-
The emulsifying properties of FPH are di- fuse rapidly and adsorb at the interface, they are
rectly connected to their surface properties, or less efficient in reducing the interfacial tension
how effectively the hydrolysate lowers the inter- because they cannot unfold and reorient at the
facial tension between the hydrophobic and hy- interface, like proteins with higher molecular
drophilic components in food. Proteins adsorb to weight.137 Solubility seems to play an important
the surface of freshly formed oil droplets during role in emulsification because rapid migration to
homogenization and form a protective membrane and adsorption at the interface are critical.138
that prevents droplets from coalescing.6 Hydroly- However, complete solubility is not an absolute
sates are surface active and promote oil-in-water requirement. Solubility and emulsifying proper-
emulsions because they have hydrophilic and hy- ties have been found to correlate up to 25% pro-
drophobic functional groups and are water tein solubility.120 Casein hydrolyzed with pancre-
soluble.121 Hydrolysates orient their hydrophobic atin showed a linear decrease in emulsifying
68
activity with increase in %DH. Casein hydroly- Various emulsification properties have been
sate at 67% DH consisted solely of amino acids, found for hydrolyzed seafood protein products.
di-, and tripeptides with drastically reduced emul- Hydrolyzed processing wastes from lobster using
sifying activity.57 Smaller and smaller peptides papain and an undefined fungal protease had a
are formed as the enzymatic hydrolysis progresses rather poor capacity for oil emulsification.86 Also,
and this impacts emulsifying properties.138 Pro- relatively poor emulsifying capacity and stability
tease specificity also plays a key role in the emul- were reported for enzymatically produced capelin
sifying properties of protein hydrolysates because protein hydrolysate59 and shark protein hydroly-
this strongly influences the molecular size and sates;82 however, neither of these studied defined
hydrophobicity of the resulting peptides. Kuhler the %DH of the products. Enzymatically hydro-
and Stine72 found that whey protein hydrolyzed lyzed myofibrillar proteins from rockfish fillets
with Prolase yield large-molecular-weight pep- showed an increase in emulsifying capacity and
tides with excellent emulsifying stability and ac- stability with hydrolysis compared to the intact
tivity. In contrast, Pronase, which has a broad protein.34,123 Emulsifying capacity reached a point
specificity, produced much smaller peptides and at which there was no change with %DH.34 Hy-
yielded hydrolysates with very poor emulsifica- drolysates from rockfish muscle made with bro-
tion properties. Hence, a careful choice of en- melain had relatively poor emulsifying capacity
zymes and low degrees of hydrolysis are recom- and yielded unstable emulsions.136 Removing lip-
mended if good emulsifying properties are desired. ids from rockfish protein hydrolysates resulted in
There is a relationship between %DH and a dramatic loss in emulsifying capacity; the loss
emulsifying properties for fish protein hydroly- increased with increasing extraction temperature.34
sates. Enzymatic hydrolysis had a negative influ- Fish protein derivatives with good emulsify-
ence on the capacity to form and stabilize emul- ing properties can also be prepared by acetyla-
sions as degree of hydrolysis increased for sardine tion.60 Acylating agents such as acetic or succinic
protein hydrolysates83 and salmon protein hydroly- anhydrides can react with the amino groups of
sates.90 Different molecular weight distributions proteins in an alkaline environment, forming amide
of the hydrolysates show that a higher content of groups.24,142 The acetyl residues participate in the
high-molecular-weight protein fractions plays an formation of additional hydrogen bonds or else
important role in stabilizing emulsions. Hydroly- can contribute to hydrophobic adherences, thus
sates with lower degrees of hydrolysis have higher increasing emulsifying properties. Groninger and
surface hydrophobicity and sardine hydrolysates Miller136,142 are among researchers that reported
at low %DH have better emulsifying capacity an increase in emulsifying properties of fish pro-
than commercial sodium caseinate.83 Cui68 ob- tein by acetylation. Miller and Groninger136 found
tained similar results for enzymatically hydro- that emulsifying properties of FPH prepared by
lyzed chum salmon muscle. bromelain increased until 43 to 59% of the free
Hydrophobicity plays an important positive amino groups were acetylated, with further acety-
role in determining emulsifying properties. Kato lation having no effect.
and Nakai139 reported that effective hydrophobic- Emulsifying properties of fish protein con-
ity determined fluorometrically showed signifi- centrates have also been reported.27 Fish protein
cant correlation with interfacial tension and emul- concentrate produced by isopropanol extraction
sifying activity of a wide variety of proteins had a decreased emulsifying capacity with in-
studied. Li-Chan et al.140 also found that surface creased solvent extraction. This loss of emulsifi-
hydrophobicity predicted the emulsifying proper- cation capacity was believed to be tied to a loss in
ties of meat proteins. A positive correlation be- solubility.
tween surface activity and peptide length has been The emulsifying ability of other food pro-
found,73 and it has since been generally accepted teins, primarily milk protein, has been studied
that a peptide should have a minimum length of extensively. Enzymatically hydrolyzed whey pro-
>20 residues to possess good emulsifying and tein had excellent emulsifying capacity and sta-
interfacial properties.141 bility when made with the protease Prolase (~3.0 g
69
oil/mg protein).72 Pepsin and Pronase hydrolyzed surfactant lowering the surface and interfacial
whey protein yielded emulsions of less capacity tension of the liquid.31 The amphiphilic nature of
and stability, both decreasing with time of hy- proteins makes this possible; the hydrophobic
drolysis and %DH.72 The degree of hydrolysis portion of the protein extends into the air and the
was, however, not specified. Each of these en- hydrophilic portion into the aqueous phase.
zymes have different substrate specificities and Townsend and Nakai143 showed that total hydro-
action patterns. Pepsin and Pronase hydrolysates phobicity, or the hydrophobicity of exposed or
had higher %DH values than Prolase hydroly- unfolded protein, have a significant correlation to
sates. Enzymatic hydrolysis progressively de- foaming formation. The surface hydrophobicity,
creased the emulsifying activity index of casein, which is important for emulsification, does not
with a more than twofold reduction after hydroly- correlate with foam formation.
sis to a %DH of 58%, with an additional small Many different methods have been developed
decrease at 67% DH.57 A drop in emulsifying to measure foaming properties of proteins and
capacity was highly correlated with a drop in protein hydrolysates. Most, if not all, of these
hydrophobicity, and reduced molecular size, and methods have the drawback of poor experimental
thus in agreement with our findings.90 Trypsin reproducibility, thus making it hard to compare
hydrolysis was found to greatly improve the emul- findings between laboratories. Foaming proper-
sifying properties of casein and whey protein and ties are usually expressed as foam formation and
was highly influenced by pH.138 foam stability. Phillips et al.144 developed a method
Chicken head hydrolysates70,71 and turkey where the ability of protein to form foams is
waste hydrolysates44 have also been tested for described as overrun. Overrun is the percentage
emulsifying capacity, and both had poor emulsi- of excess volume produced by whipping a protein
fication ability. A significant drop in emulsifying containing liquid compared with the initial vol-
properties as hydrolysis progressed was observed ume of the liquid. Foam stability is measured by
when alpha-chymotrypsin was used to hydrolyze whipping the protein solution and measuring the
peanut protein fractions, reaching a stationary time required to decrease half of the volume.
phase after a certain time.78 Foam stability is a highly empirical method, but
There are many different factors that may is simple to perform and has found useful appli-
account for the difference observed between hy- cations with FPH.
drolysates in both the ability to form an emulsion Very few studies have been performed on
and emulsion stability. Peptides molecular char- foaming properties of fish protein hydrolysates.
acteristics and peptide chain length are the major Shahidi et al.59 and Onodenalore and Shahidi82
reason for the different emulsification ability of studied the foam formation, or whippability, and
hydrolysates. Environmental conditions such as foam stability of shark and capelin protein hy-
pH, ionic strength, temperature, etc. also have an drolysates, respectively. In both processes Alcalase
effect on the emulsification properties. Also, some was the protease used for hydrolysis. The capelin
synergistic effects of peptides on emulsifying protein hydrolysate had good whippability, 90%,
properties have been noted.117 In short, the fore- at a 12% DH. Shark protein hydrolysates from
going indicates that peptide behavior is complex shark meat had higher whippability, 106%, and
and not easy to explain. hydrolysates from washed shark myofibrillar pro-
teins had whippability of 130%. Capelin hydroly-
sates had higher foam stability after 30 s that
D. Foaming Properties dropped significantly compared with hydrolyzed
shark myofibrillar protein. Hydrolyzed whey pro-
The chemistry underlying foaming properties tein isolates prepared using Alcalase had excel-
of protein and protein hydrolysates have many lent foaming formation and stability at a 3% DH.145
things in common with emulsifying properties. The foaming ability for whey was much higher
Both rely on the surface properties of protein. than for shark and capelin protein hydrolysates.
Food foams consist of air droplets dispersed in There is a connection between the degree of
and enveloped by a liquid containing a soluble hydrolysis and foaming properties. Kuehler and
70
Stine72 concluded that whey proteins hydrolyzed drolysates are mixed with a specified amount of
to a limited degree had a increased foaming abil- excess fat for a particular time and then centri-
ity but reduced foam stability. This was attributed fuged at a low centrifugal force and the fat ad-
to more air being incorporated into solution of sorption expressed as the millilter of fat bound by
smaller peptides, but the smaller polypeptides do 1 g protein hydrolysate59,90 for FPH. The mecha-
not have the strength required to give a stable nism of fat absorption, as assessed by this method,
foam. This was seen in the initial 30 min of is attributed mostly to physical entrapment of the
hydrolysis with further hydrolysis resulting in oil, and thus the higher bulk density of the protein
peptides that lack the ability to stabilize the air the more fat absorption.31 Fat-binding capacity of
cells in the foam. Weak foams are commonly proteins correlates with surface hydrophobicity.
observed when food proteins are hydrolyzed. Fat absorption of salmon protein hydroly-
However, the advantage of using hydrolyzed pro- sates produced using different enzymes to 5, 10,
teins as foaming agents is their insensitivity to and 15% DH was studied by Kristinsson.90 The
change in pH. The pH of the dispersing medium FPH at all %DH exhibited excellent fat absorp-
markedly affects foaming, particularly foam station, greater than both egg albumin and soy pro-
bility, with foaming properties being highest close tein concentrate. The hydrolysates at 5% DH had
to the isoelectric point of the protein.146 Fish pro- significantly higher fat absorption (5.98 to 7.07 ml
tein hydrolysates and especially fish protein con- oil/g FPH) than 10% DH (3.22 to5.12 ml oil/g
centrates (FPC) have the unusual property of hav- FPH) and 15% DH (2.86 to 3.86 ml oil/g FPH)
ing good foaming properties, and of making strong, due to the larger peptide sizes. Only two other
stable foams over a wide pH range.29-31 The foam publications dealing with fat absorption for FPH
formation of FPC is provided by the soluble pro- could be found, for capelin protein hydrolysates59
teins, only 1% of total FPC, while the remaining and shark protein hydrolysates.82 Both studies fail
denatured proteins act as foam stabilizers.30 to define the units used to express fat absorption,
Foaming properties of fish protein hydroly- and thus it is impossible to compare them to other
sates can be improved by acylation. Groninger studies.90
and Miller147 succinylated hydrolysates produced Other protein hydrolysates have been studied
by hydrolyzing rockfish fillets with bromelain. with respect to fat absorption. Casein hydroly-
These foams were significantly more stable com- sates with excellent oil-holding capacity, made
pared with foams from modified soy protein and with papain, were dissociated by SDS, indicating
egg white; however, they had smaller foam vol- that hydrophobic interactions were primarily re-
ume. The succinylated protein foams were more sponsible. The substrate specificity of enzymes
stable over a wide range of pH (3 to 9) compared also seemed to play a major role in this.114 This
with the other protein. The succinylated FPH was was further confirmed by Kristinsson,90 who
incorporated into a dessert topping, a soufflé, and showed that different enzymes used to hydrolyze
both a chilled and a frozen dessert, and the prod- salmon muscle protein had different fat absorp-
ucts were as acceptable to a taste panel as similar tion ability. The capacity of a hydrolysate to ab-
foods containing no fish protein. Ostrander et sorb fat/oil is an important attribute that not only
al.148 incorporated succinylated FPH, as prepared influences the taste of the product, but it is also an
by Groninger and Miller,147 into whipped gelatin important functional characteristic that is required
desserts. The succinylated FPH formed a stable especially for the meat and confectionery indus-
foam with smooth texture, highly acceptable with try. Fish protein hydrolysates therefore could very
respect to flavor and mouthfeel. well be used in such applications.
E. Fat Absoption F. Sensory Properties
Various methods for the measurement of fat Although enzymatic hydrolysis of proteins
absorption have been described. Commonly, hy- develops desirable functional properties, it has
71
the disadvantage of generating bitterness. This is sates, particularly exopeptidases that split off
a common problem with fish protein hydroly- hydrophobic amino acids from bitter peptides.
sates, and the major reason for its slow accep- However, for an enzyme preparation to be effec-
tance as a food ingredient. The mechanism of tive for hydrolyzing protein, both exopeptidases
bitterness is not very clear, but it is widely ac- and endoproteinases are required. Many studies
cepted that hydrophobic amino acids of peptides have shown that proteolytic preparations contain-
are a major factor. In fact, the hydrophobicity of ing exopeptidases and endoproteinases produce
peptides can be measured, and it has been found less bitter peptides than single proteases.152-155
that most bitter peptides have values over 1400 Trials have shown that by the additional action of
kcal/mol, whereas nonbitter peptides have values exopeptidases in proteolytic processes, it is pos-
below 1300 kcal/mol. This principle is, however, sible to move the bitterness point up to higher
only valid for molecular weights below 6000 D.149 degree of hydrolysis.156
Peptides with a molecular weight from 1000 to Despite the problem with bitterness of fish
6000 D and with hydrophobic characteristics have protein hydrolysates, little research has been con-
shown most likely to be bitter.102 Hydrolysis of ducted. While FPH is highly nutritious and safe
protein results in exposing buried hydrophobic and functional properties are good, the sensory
peptides, which will be readily able to interact properties are extremely important for the suc-
with the taste buds resulting in detection of bitter cessful adaptation and acceptance by the food
taste. An extensive hydrolysis to free amino ac- industry and the consumer. Yu and Fazidah125
ids, however, decreases the bitterness of these found a connection between the degree of hy-
bitter peptides because hydrophobic peptides are drolysis and the intensity of bitterness when hy-
far more bitter compared with a mixture of free drolyzing Aristichthys nobilis with a commer-
amino acids. Free amino acids, are however, un- cial protease. As the %DH increased so did the
desirable from a functional standpoint. Strict con- bitterness, and after a 5-h hydrolysis samples
trol of any hydrolysis experiment and termination were described as “very bitter”. Interestingly, no
at low %DH values therefore is desirable to pre- bitterness was observed until after 4 h of hy-
vent the development of a bitter taste and the drolysis. Papain-hydrolyzed herring had higher
retention of functional properties. bitterness scores than Alcalase-hydrolyzed her-
Common features found in the structure of ring.5 Alcalase hydrolyzed samples at higher
bitter peptides are, for example, N, or C, terminal degree of hydrolysis were less bitter than pa-
basic amino acid residues, C, or N, terminal hy- pain-hydrolyzed samples at the same %DH. The
drophobic peptide fragments and peptide chain greater hydrophobic specificity of Alcalase com-
folding due to proline residues within the peptide pared with papain could result in a reduced bitter-
structure.150 Bitterness is due to particular arrange- ness. Enzymatically hydrolyzed ethanol-extracted
ments of certain chemical groups in the peptides. herring, with either Alcalase or papain, had very
Two functional groups are necessary to produce low bitterness, suggesting that reduced bitter-
bitterness, such as a pair of hydrophobic groups ness could be due in part to lower lipid content.
or a hydrophobic or a basic group.151 The sensa- Lalasidis et al.157 hydrolyzed cod filleting offal
tion of bitterness may also be exerted by chemical with Alcalase, which produced a product with a
compounds having a hydrophobic region and one bitter taste. This bitter taste was eliminated when
hydrophilic group spaced 0.3 nm apart.95 Because the hydrolysate was treated further with
enzymes have different preferences for amino pancreatine, which is rich in exopeptidase activ-
acids, choosing the most appropriate enzyme ity.
preparation for hydrolysis can control the bitter- Fish protein hydrolysates have been in-
ness. Enzymes with a high preference for hydro- corporated into food systems to evaluate their
phobic amino acids such as Alcalase are often acceptability, and sometimes with satisfactory
preferred and frequently yield products of low results. 147,148 Proteins from Oreochromis
bitterness.20,96 The use of exopeptidases, as op- mossambicus, a freshwater fish, were hydrolyzed
posed to endoproteinases, can also be helpful in with Alcalase to produce a soluble hydrolysate
overcoming the bitterness in fish protein hydroly- that was incorporated into crackers.158 Sensory
72
evaluation showed the fried crackers with up to The plastein reaction is among few that have
10% addition of FPH were highly acceptable. been studied with respect to fish protein hydroly-
Vieira et al.86 hydrolyzed lobster processing sates. Figure 9166 outlines the steps involved in the
waste for 5 h with several commercial enzymes plastein reaction. Heck164 investigated the plas-
and found no difference in taste between the hy- tein reaction in FPH from peptic hydrolysis and
drolysates and a control sample, and detected no found that it reduced the bitterness but with an
bitterness. This is perhaps due to the extensive accompanied loss of FPH solubility. Some suc-
hydrolysis. The hydrolysate possessed a distinct, cess using the plastein reaction has been reported
pleasant smell, and it was proposed for use in with fish waste167 and sardines.168
various formulated food products such as flavor Plastein can be defined as a protein substrate
enhancers. Is has long been known that proteases that may be prepared by reversing hydrolysis with
can be used for recovery of flavor.159 Baek and a protease such as pepsin or papain in a concen-
Cadwallader85 attempted to fully utilize crayfish trated protein hydrolysate.60,169 The plasteins are a
processing byproducts as flavorings by hydrolyz- mixture of peptides of varied composition and
ing them with Optimase at optimal conditions. molecular weight between 1000 and 500.000
They found that it is possible that the hydroly- D.24,166 Three factors are necessary for completion
sates could serve as feedstocks for the production of the plastein reaction: (1) a low-molecular-weight
of value-added seafood flavor extracts. Fujimaki protein hydrolysate as a substrate, (2) a high sub-
et al.160 reported that a fish protein concentrate strate concentration, and (3) a reaction environ-
treated with Pronase had a flavor potentiating ment of pH 4 to 6 regardless of the enzyme in-
activity like that of monosodium glutamate (MSG) volved.164 During plastein synthesis, a high
but was accompanied by an unfavorable bitter concentration of hydrolysate (30 to 50%) is incu-
flavor. A low molecular acidic peptide fraction bated with an enzyme resulting in a condensation
contributed significantly to this MSG-like flavor. of the peptides. As new polypeptides are formed,
The acidic peptides isolated later from the same they aggregate via hydrophobic associations. The
fish protein hydrolysate showed that at least four final product has a low lipid content and special
dipeptides (Glu-Asp, Glu-Glu, Glu-Ser and Thr- hydrophobic functional properties.31,41,60 Tanimoto
Ser) and five tripeptides (Asp-Glu-Ser, Glu-Asp- et al.170 have studied the mechanism of plastein
Glu, Glu-Gln-Glu, Glu-Gly-Ser, and Ser-Glu-Glu) synthesis with α-chymotrypsin and found that it
had a flavor quantitatively resembling that of proceeds in two stages. The first stage is charac-
MSG, but with weaker intensities.161 Hevia and terized by a reaction of the peptide with the hy-
Olcott162 analyzed basic tripeptides that were re- droxyl group of Ser 195 that produces a peptidyl-
sponsible for bitter taste in fish protein hydroly- α-chymotrypsin complex that then undergoes
sates. The tripeptides contained asparagine and aminolysis as a result of a nucleophilic attack of
lysine as the second and C-terminal residues, re- a second peptide. The plastein reaction has also
spectively, with the N-terminal residue leucine or been found useful for protein recovery from ex-
glycine. tensively autolytically produced fish silage.171 It
Many techniques have been suggested to re- is strongly recommended that this process be stud-
duce or mask bitterness in hydrolysates, but few ied further for fish protein hydrolysis.
of them applied to FPH. These include treating
hydrolysates with activated carbon that partly
removes bitter peptides with absorption,59,163 ex- V. FUTURE DEVELOPMENTS AND
tracting bitter peptides with solvents,163 applica- POTENTIAL APPLICATIONS
tion of further enzyme treatment with exopepti-
dases,152 and by using the so-called plastein The use of FPH as a functional food ingredi-
reaction.164 Chakrabarti165 reported success in ent still has a long way to go until it becomes
debittering fish protein hydrolysate using ethyl economically feasible and accepted by industry
alcohol. Higher concentrations of alcohol low- and consumers. However, there is a wonderful
ered the bitter fraction in the hydrolysate. opportunity for this to happen, due to regulations
73
FIGURE 9. The plastein reaction. (Adapted from Ref. 166.)
of processing waste and the abundance of small FPH from extensive hydrolysis is an excellent
pelagic underutilized species that will be the fron- source of nitrogen for maintaining the growth of
tiers of fisheries in this century. These factors different microorganisms. Previously, it was noted
should stimulate the conversion of low value fish that extensive hydrolysis of fish protein results in
materials into more valuable and palatable prod- a product containing free amino acids and low-
ucts. Other applications outside the food industry molecular-weight peptides, which is very promis-
may also be a feasible option for FPH. Soluble ing for microbial peptone production.129
74
The beneficial chemical composition of FPH Another little-studied property of FPH is its
and FPC has also led to using these materials as antioxidant potential. Studies have found power-
fertilizer with good results. The FPH might be too ful antioxidant activity in intact fish protein131 and
expensive for this purpose, as added enzymes are in hydrolysate.59 These effects are probably highly
used, and the product is usually dried. However, dependent on the amino acid composition and
Ferreira and Hultin104 developed an enzymatic molecular size of the FPH peptides and deserve
process using the acid protease Newlase A to further study.
extensively hydrolyze cod frames. This process is
currently being used. The use of FPC as fertilizer VI. CONCLUSION
is more common, either from chemical hydrolysis
or autolysis. A successful process using second- There have been several attempts to make
ary raw material from processing plants (frames functional fish protein hydrolysates with enzymes,
and viscera) and using the acid endogenous en- some successful and some not. Most of these
zymes to extensively hydrolyze the material is studies are rather crude, perhaps due to the ap-
being operated in Washington State. The final plied nature of the field, and have many short-
product is sold in bulk to local farmers, mainly comings, specifically failing to compare enzymes
cranberry growers, and with superior results com- at the same activity level, controlling the %DH
pared with other commercially available fertiliz- properly and characterizing the chemical and func-
ers.172 The mechanism behind the effect of the tional properties of the final products. There is
FPH and FPC to stimulate growth and develop- potential for these products to be produced and
ment better than synthetic fertilizers need to be sold as functional food ingredients, but at present
studied further. Novel applications taking advan- other applications such as plant nutrients, fertiliz-
tage of plant growth-stimulating effect of FPH ers, and animal feeds might be more feasible.
and FPC could possibly be developed. An exten- Standardized procedures to examine the functional
sive research program supervised by Dr. Kalidas properties are needed, as well as more studies on
Shetty at the University of Massachusetts at using endogenous enzymes to make functional
Amherst examines the effect of FPH on plants. In FPH. With more basic research on the molecular
one study the effects of using FPH to stimulate level the future of FPH could be bright, especially
somatic embryogenesis in Anise (Pimpinella in light of the environmental problems facing
anisum) when compared with proline, a known fisheries. It is time for FPH research to take a
stimulator, was examined.173 The conclusion was new, fresher, and more scientific direction, be-
that FPH could well become a proline and amino cause the field has been more or less stagnant for
acid substitute in plant tissue culture applications. the last 20 years or so.
The positive effects of FPH due to proline and its
precursor glutamate on plant growth was con-
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Critical Reviews in Food Science and Nutrition

ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: http://www.tandfonline.com/loi/bfsn20

Fish Protein Hydrolysates: Production,

Hordur G. Kristinsson & Barbara A. Rasco

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Fish Protein Hydrolysates:

I. INTRODUCTION processes for byproduct recovery and utilization

FIGURE 1. Fish myosine molecule.

III. PROTEIN HYDROLYSIS

tensive hydrolysis. stances (e.g., lysinoalanine) that are highly

by using reaction conditions different than those

Although the osmometer technique is a very

denature and unfold vary enormously. Because

E. Fat Absoption F. Sensory Properties

FIGURE 9. The plastein reaction. (Adapted from Ref. 166.)

tions, J. Food Sci. Technol., 33(1), 19, 1996. 125, 1982.

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