FOOD AND BEVERAGE CONSUMPTION AND HEALTH

BEER
PRODUCTION, CONSUMPTION
AND HEALTH EFFECTS

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FOOD AND BEVERAGE CONSUMPTION
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 FOOD AND BEVERAGE CONSUMPTION AND HEALTH

BEER
PRODUCTION, CONSUMPTION
AND HEALTH EFFECTS

WILLIAM H. SALAZAR
EDITOR

New York
Copyright © 2016 by Nova Science Publishers, Inc.

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 CONTENTS

Preface vii
Chapter 1 To Your Health! The Role of Beer in Ancient Medicine 1
Max Nelson
Chapter 2 Beer Production with Encapsulated Yeast Cells 27
Vesela Shopska, Rositsa Denkova and Georgi Kostov
Chapter 3 Effects of the Almond Addition and the Yeast Strain Used for
Fermentation, on the Beer Chemical Properties 101
Carlos García-Latorre, María Inmaculada Talaverano,
Juan Manuel Zapata, Oscar Santamaria and Sara Rodrigo
Chapter 4 Quantitative Analytical Methods of Organic Ingredients in the
Quality Control Process for the Production of Beer 119
Bojidarka Ivanova and Michael Spiteller
Chapter 5 Saccharomyces Spp. Role in Brewing Process and
Its Serial Repitching Impact 213
Cátia Martins, Tiago Brandão, Adelaide Almeida and
Sílvia M. Rocha
Chapter 6 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide at
Low Pressure and Quality Evaluation of the Treated Beer 257
Fumiyuki Kobayshi and Sachiko Odake
Index 275
 PREFACE

Beer is defined as a fermented alcoholic beverage made of malted cereals, water, and
yeast, and occasionally other additives, such as hops. This alcoholic beverage has been
consumed for thousands of years, when independent events revealed that some juices
fermented when left in the open air, giving as a result a completely different product. The first
chapter of this book aims to examine the role of beer in medicine from around 2000 B.C. to
A.D. 1000 in Mesopotamian, Egyptian, Greek, and Roman texts. Chapter Two presents the
possibilities of beer fermentation with encapsulated yeast cells. Chapter Three reviews the
effects of the almond addition and the yeast strain used for fermentation, on the beer chemical
properties. Chapter Four focuses on the quantitative analytical methods to organic ingredients
in the quality control process for beer production. Chapter Five studies the role of
Saccharomyces spp. in the brewing process and its serial repitching impact. Chapter Six
provides a discussion on the inactivation of beer yeast by microbubbled carbon dioxide at low
pressure and quality evaluation of the treated beer.
Chapter 1 - The aim of this chapter is to examine the role of beer in medicine from
around 2000 B.C. to A.D. 1000 in Mesopotamian, Egyptian, Greek, and Roman texts. The
focus is both on the various conceptions regarding the good or bad health effects of beer as
well as on the uses to which beer was put in ancient medical treatments. Beer, when used
medicinally, was mainly employed as a base for ingesting medicinal plants, though it was
sometimes thought to have its own inherent beneficial qualities for both internal and external
therapy. However, estimations of the salubriousness of beer, even when drunk in moderation,
as well as the importance of beer in medicine diminished gradually over time and from East
to West (from areas where beer was an everyday beverage to areas where wine was
preeminent), only to be revived in Europe in the early Middle Ages.
Chapter 2 - In recent years, the immobilization of yeast cells and their application in
brewing has been the subject of research due to their main advantages: enhanced fermentation
productivity, reduced fermentation time, easier implementation of continuous operation,
improved cell stability, facilitated cell recovery and biomass reuse.
The purpose of the present chapter is to present the possibilities of beer fermentation with
encapsulated yeast cells. The chapter begins with an overview of the application of
immobilized systems in beer production and the immobilization effect on yeast metabolism.
The data were used for the choice of a method and carriers for cell immobilization –
encapsulation in an alginate/chitosan matrix. The influence of different parameters on the
viii William H. Salazar

microcapsule formation and the effect of the fermentation conditions on the microcapsule
stability were also investigated.
Laboratory-scale beer fermentations with immobilized top- and bottom-fermenting yeast
strains were carried out. The fermentation dynamics data were used for the kinetic model
development and the description of the immobilization effect on beer flavor.
The results obtained were used for the optimization of the fermentation conditions by
applying the planned experiment methods. Furthermore, the effect of some fermentation
parameters on the main fermentation and maturation time was studied.
Chapter 3 - Beer is one of the most consumed beverages all over the world, and recently,
literature has attributed it a variety of health benefits. Among its beneficial characteristics, the
presence of polyphenols and antioxidant compounds, are nowadays widely studied. On the
other hand, nuts are known as a source of nutritious food with a potent free radical
scavenging capacities. Due to that, the addition of almonds in a craft beer brewing process
was done, using the almonds in two different level of manufacture: almond flour or grinded
almonds. Besides, two different moments for the addition were tested (in mashing or in
boiling), as well as two different yeast strains (Safale S-04 or Safbrew T-58). Influence of
variables and the interactions among them, were studied on some quality parameters such as
pH, colour, bitterness, total polyphenol index, antioxidant activity and ferric reducing ability
of plasma (FRAP). Yeast strain influenced pH and bitterness, showing T-58 a higher pH but a
lower bitterness (5.3 IBU) in comparison to S-04 (7.7 IBU). Also regarding with bitterness,
adding the almond in mashing caused the highest bitterness (8.4 IBU). The significant
interaction almond type × yeast strain × addition moment showed that the lowest amount of
polyphenols was determined when almond was added in flour format, in mashing, and
fermentation took place with S-04 yeast strain. Neither for the antioxidant activity nor for the
FRAP, significant effects were reported for any studied variable. Finally, principal component
analysis revealed that the clearest separation was caused by the yeast strain used for the
fermentation.
Chapter 4 - Although chronic alcohol abuse leads to liver diseases, hard spirits are more
harmful to human health. And thus, the great popularity of beer is associated with diet
benefits in reducing the risk heart disease; myocardial infarction and diabetes. The broad
biological antioxidant, antitoxiolytic, anticancer and antimicrobial activities are due to cereal
and hop bitter acids. Other ingredients are soluble fibers, vitamins and minerals. Isoflavones
genistein and daidzein were already implicated in cancer prevention. From an analytical
chemistry perspective, the beer exhibits a complex chemical profile of  400–600
ingredients. Their kind and concentration vary in the final beer product, and through all
brewing stages. It has complex photochemistry and intra–specification diversity based on
growing region of the plant, collecting phase and/or post–harvest treatment. The elaboration
of analytical protocols represents significant challenge, in spite of the production of beer is
from many Centuries. This chapter reflects contributions of MS to beeromics, focusing
attention to organic ingredients. The method is irreplaceable for quantitative analysis of
complex mixtures. The content deals with determination of large scale of components and
concentrations, including trace analysis. It impacts a sector of the economy, which contributes
to many interdisciplinary fields of chemistry such as analytical chemistry; food chemistry;
agricultural chemistry and so on. Analysis of microbial contamination, that may occur during
production process and which may negatively affect the final food is included. The
determination of mycotoxins, which can carry–over from contaminated grains in food–
 Preface ix

product thus causing serious health problems for humans is discussed. They exhibit a wide
range of toxic activity such as carcinogenicity and neurotoxicity, and reproductive and
developmental toxicities. Other health risk components are also shown. The chapter content is
with high relevance for “food control” in conjunction with strict legislation to regulate
presence of mycotoxins in food.
Chapter 5 - Beer is the second most consumed beverage in the world, accounting
ca. 35% of all recorded alcohol consumed in 2010. Brewing industry is extremely
competitive, highlighting the importance of constant innovation. The success of global beer
commercialization is its intrinsic quality that depends of a network of variables, namely raw
materials, yeast strain and the brewing process itself. The biochemical performance of yeasts,
usually Saccharomyces spp., is one of the parameters with significant importance in brewing
once it limits the alcoholic content of final beer and produces different metabolites with
crucial impact on beer aroma and flavor. It will be given focus to the different metabolic
pathways with significant impact on beer’s aroma profile, namely the carbohydrates and
nitrogen compounds metabolism; and also the formation of aldehydes, higher alcohols,
vicinal diketones, esters, fatty acids, sulfur and terpenic compounds. Environmental changes
can promote stress in yeasts along brewing. The stress factors will be systematized in this
book chapter, as well as the yeasts cellular effects and their biological response. Furthermore,
brewing companies tend to reused (or repitch) yeasts several times to minimize resources
costs, while maintaining quality of the final product. Also, different approaches will be
described in order to monitor yeast quality. Therefore, the impact of yeasts repitching along
brewing will be systematized, considering its associated advantages and drawbacks. In
summary, this book chapter aims to elucidate about the raw materials and the brewing
process, and also to give an overview of the Saccharomyces spp. metabolism (special
attention will be done on its impact on beer aroma profile), as well as to understand the
effects of serial repitching on yeasts’ behavior.
Chapter 6 - Inactivation of Saccharomyces pastorianus cells, beer yeast, by two-stage
system with microbubbled carbon dioxide at low pressure (MBCO2) was investigated by the
observation with scanning and transmission electron microscopies (SEM and TEM),
fluorescent analysis with propidium iodide, and measurements of leakage of nucleic acid and
protein and intracellular enzyme activation. Furthermore, yeast in unfiltered beer (UFB) was
inactivated by two-stage MBCO2 and the quality of the treated beer was evaluated. It was
observed by SEM that wrinkles in S. pastorianus cells treated by two-stage MBCO2 with a
heating coil at 50°C (MB50) and heat treatments at 50°C and 80°C (H50 and H80) were more
than those in non-treated (NT) cells. Upon observation with TEM, it suggested that MB50
had a direct effect on the intracellular substrate, although little influence on the cell
membrane, whereas H80 cells showed visible damage to cell membranes. However, it was
recognized that PI intensity in MB50 cells was great than that in NT, H50 and H80 cells, and
that the amount of nucleic acid and protein leaked from H80 cells was significantly higher
than that of NT, MB50 and H50 cells. Furthermore, the enzyme inactivation efficiency in
MB50 cells was the same as in H80 cells. These results estimated that inactivation of S.
pastorianus by two-stage MBCO2 was due to actions of MBCO2 on the cell membrane and
the intracellular substrate such as enzyme inactivation. Furthermore, 5-log reductions of yeast
in UFB could be achieved by two-stage MBCO2 with a heating coil at 50°C for 5 min, while
yeast in UFB was not completely inactivated by heat treatment at 80°C for 5 min. In sensory
evaluation of these beers, there were no significant differences on the score of flavor,
x William H. Salazar

although bitterness of two-stage MBCO2-treated beer (MBB) was significantly low on the
score of taste. In analysis of volatile compounds, dimethyl sulfide, dimethyl disulfide, β-
myrcene and styrene in MBB was less than those in UFB and heat-treated beer at 80°C for 5
min (HTB). Glucose and maltose contents in MBB and UFB were almost the same, although
those in HTB significantly decreased. Organic acids, except for acetic acid in MBB, were
nearly identical with those in UFB and HTB. Most of the free amino acids in MBB were
lower than those in UFB and HTB. Bitter units of beer increased in the order of MBB, HTB
and UFB. These results suggested that two-stage MBCO2 showed promise as a practical
technique for inactivating yeast in UFB. The inactivation efficiency of two-stage MBCO2 had
less influence of materials in beer than that of heat treatment, and two-stage MBCO2 have
little negative impact on the beer quality.
In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485-704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.

Chapter 1

TO YOUR HEALTH!
THE ROLE OF BEER IN ANCIENT MEDICINE

Max Nelson
University of Windsor, Canada

ABSTRACT
The aim of this chapter is to examine the role of beer in medicine from around 2000
B.C. to A.D. 1000 in Mesopotamian, Egyptian, Greek, and Roman texts. The focus is
both on the various conceptions regarding the good or bad health effects of beer as well
as on the uses to which beer was put in ancient medical treatments. Beer, when used
medicinally, was mainly employed as a base for ingesting medicinal plants, though it was
sometimes thought to have its own inherent beneficial qualities for both internal and
external therapy. However, estimations of the salubriousness of beer, even when drunk in
moderation, as well as the importance of beer in medicine diminished gradually over time
and from East to West (from areas where beer was an everyday beverage to areas where
wine was preeminent), only to be revived in Europe in the early Middle Ages.

INTRODUCTION
The relative healthfulness or harmfulness of alcoholic beverages, including beer, has
been a contentious issue since ancient times and remains so today. While there have been
those who encouraged moderation, others advised abstention. And while some have
prescribed beer specifically for various ailments, others have rather emphasized the ill-effects
of any beer consumption.
The aim of this chapter is to examine the role of beer (defined as any fermented, and not
distilled, beverage made from cereal) in medicine from around 2000 B.C. to A.D. 1000. The
study will range from the earliest records in Mesopotamia, the fertile land between the
Euphrates and Tigris Rivers (mainly in what is now Iraq), and Egypt, the fertile land along the
lower part of the Nile River, to later Greek and Roman and some early medieval sources. The
focus will be both on the various conceptions regarding the good or bad health effects of beer
2 To Your Health!

as well as on the uses to which beer was put in ancient medical treatments. The emphasis will
be on the surviving written sources with only the rare appeal to material evidence. The
investigation of these sources will necessarily be selective, not only in its spatial and
chronological scope (thus, for instance, Chinese and Indian texts, or beer in late medieval and
modern medicine, will not be referenced), but also in terms of mentioning only a sampling of
exemplary passages, with no pretense at completeness.1 However, the result will be a broader
look into the subject of beer in ancient medicine than has so far been published in the few
existing surveys specifically on the topic (in very brief surveys, Cornwall, 1939 and Wright-
St. Clair, 1962 cited the Ebers Papyrus as their sole ancient source on beer in medicine, while
Keller, 1958, in another very brief survey, added only the Hearst Papyrus and a tablet from
Nippur).2
Only a provisional assessment of the role of beer in ancient medicine can be attempted
because of the nature of the evidence. Much ancient medical knowledge was probably passed
down orally and never written down. Furthermore, only a small portion of what was written
down is extant, mainly through accidental preservation (such as in the case of the dozen or so
known Egyptian medical papyri) or occasionally because of the deliberate choices of a few
individuals (such as the medieval scribes who copied Galen’s works). Even the surviving
evidence is often lacunose (as is the case with broken cuneiform tablets from Mesopotamia or
fragmentary quotations from Greek authors), and some texts even remain to be published,
properly interpreted, or translated (see, for example, Nunn, 1996: 24 on the Egyptian material
and Nutton, 2013: 1-9 on the Greco-Roman material). In fact it is often very difficult or even
impossible to properly understand the sources, such as when it comes to how to translate the
terms used for various plants and other substances prescribed alongside beer in
Mesopotamian and Egyptian texts, hence often below mention will simply vaguely be made
to “ingredients” (see, for example, Böck, 2011: 696 on Mesopotamian texts and Nunn, 1996:
151 onEgyptian texts). The same can be said for the terms used for diverse types of beer, as
will be seen. Moreover the sources from different regions pose their own particular
difficulties. Thus the Mesopotamian texts for the most part offer no general explanatory or
theoretical bases for their authors’ medical understandings or physicians’ practices and the
nature of the corpora makes it difficult to determine regional variations or developments (see
the remarks in Worthington, 2009: 52-54), and so here in general they will be treated
synchronically as an undifferentiated whole.3 Issues arise in the Egyptian texts from the fact
that anonymous authors are responsible for the works and never cite any sources by name and
often also mix early with later material (see Bardinet, 1995: 33). And as for the Greek and
Roman authors, who can at least be dated relatively accurately, on the whole they have little
interest in beer, which for the most part they themselves did not consume, and they speak of it
often with contempt (see Nelson, 2005: 25-76).
So as to provide as clear a picture as possible, the material in this study will be organized
thematically, and then for each topic roughly chronologically, going first through the

1
Reference throughout will be made to translations of passages, mainly from Scurlock 2014 (for Mesopotamian
sources), Bardinet, 1995 (for Egyptian sources [in French]), and Nelson, 2001 (for Greek and Roman sources).
Jewish texts will be excluded for the sake of space, except for one which references Egyptian beer (in the
section on beer as an aperient).
2
On the other hand, wine in ancient medicine has received much scholarly attention (see, for example, the
collection of essays in Jouanna and Villard, 2002 for the ancient Greeks).
3
Scholars have attempted their own systematic analyses of Mesopotamian medicine; see, for example, Herrero,
1984 (on therapeutics) and Scurlock and Andersen, 2005 (on diagnostics).
 Max Nelson 3

Mesopotamian evidence, and then the Egyptian, Greek, and Roman evidence, and sometimes
continuing slightly later. First, ancient notions about beer as a healthful or harmful element of
the diet will be presented as well as opinions on intoxication and over-drinking. Then the uses
of beer as a vehiculum and menstruum for medicinal preparations will be examined, both for
oral ingestion and non-oral introduction. And finally some therapeutic uses of beer, both
internally and externally, will be analysed. Only occasionally will the rationale (based on
ancient opinion) or the efficacy (based on modern knowledge) of ancient beer-based
treatments will be addressed.

BEER AS A HEALTHFUL OR HARMFUL ELEMENT

OF THE DIET

By the third millennium B.C., beer (known as KAŠ in Sumerian and šikaru in Akkadian)
was one of the most popular beverages among the peoples of Mesopotamia (see, for example,
Röllig, 1970 and Stol, 1994). In ancient medical texts from the region, it is taken for granted
both that beer is a basic element of the diet and that it is generally healthful. In a diagnostic
and prognostic handbook organized in the eleventh century B.C. into forty cuneiform tablets
(of which only about half survives) by the scholar Esagil-kīn-apli of Borsippa, eating bread
and drinking beer is considered a good sign in a patient while having trouble doing so is
considered a bad sign.4 Thus for a feverish patient not to tolerate eating bread and drinking
beer is perceived as a negative symptom, (for example, DPS 4.82 and 31.46”-49” and 52”-56”
in Scurlock, 2014: 37 and 229-230, with Scurlock and Andersen, 2005: 58).5 This can even
be taken as a sign of impeding death (DPS 12.111”-112” in Scurlock, 2014: 101, with
Scurlock and Andersen, 2005: 303). Having a diminished appetite for bread and beer or
having an upset stomach after drinking beer, or else vomiting up beer, are also seen as
symptoms of gastro-intestinal problems (see the many examples in Scurlock and Andersen,
2005: 55, 121, 122, 125, 127, 128, 129, 131, 132, 135, 136, 137, 139, 144, 288, 357, and
553). In an interesting example, it is related that a patient complaining of stomach pain and
exhibiting a lack of appetite for bread and beer might not actually have any real stomach pain,
thus showing an awareness of psychosomatic illnesses (BAM 316.iv.3-4 in Scurlock and
Andersen, 2005: 373).6 Another text lists among various signs of a man suffering from
misfortune (probably depression) his diminished appetite for bread and beer (BAM 234.1-10
in Stol, 1993: 29). Yet another text mentioning not tolerating bread and beer may reference
rabies (BAM 231.i.1-18 in Scurlock and Andersen, 2005: 22-23). In general, then, a sign of
good health was an appetite for beer and a sign of bad health was a rejection of beer. The only
examples we have of Mesopotamians considering beer bad for a patient is when the beer is

4
For the handbook, abbreviated as DPS (for Diagnostic and Prognostic Series), see Scurlock, 2005: 302 and 303
and Scurlock and Andersen, 2005: 7 (with a summary of its contents at 575-677). For the rationale behind the
handbook, see Heessel, 2004.
5
Note also the further texts on fevers in Stol, 2007: 12, 17, 26-27, and 31.
6
BAM is used as the abbreviation for the texts found in Köcher, 1963-1980. In this text the cause is said to be a
personal god or goddess (see Scurlock and Andersen, 2005: 439). Compare also Stol, 1999, who does not
discuss this passage, but does speak of the notion that superhuman beings were thought to be involved in such
cases.
4 To Your Health!

contaminated (BAM 90.3’-5’ and AMT 48/2.11-14 in Scurlock and Andersen, 2005: 19 and
565),7 or else, when it is drunk in excess (as will be seen below).
In ancient Egypt, bread and beer (ḥḳt) were also the basic elements of the diet (see, for
example, Helck, 1971, Samuel, 2000: 537, Ritner, 2001: 353, and Peck, 2013: 96), and in fact
physicians were at one time paid by a government subvention which included bread and beer
(Ghalioungui 1983: 95 and Halioua and Ziskind 2005: 19). However, Egyptian medical
papyri have little to say about beer as a normal element of the diet. One papyrus from the
second century A.D. does mention throwing up sweet beer as a negative symptom which
points to deficiency in flesh and vision (Pap.Vindob. D. 6257 15.2 in Reymond, 1976: 125).8
Also in one prescription in an Egyptian gynecological work dating to about 1850 B.C. (the
Kahun Papyrus) it is proposed that a woman should not drink beer after giving birth, perhaps
since the narrowing of the uterus was thought to be connected to or aggravated by beer
consumption (6 in Bardinet, 1995: 438).9 Interestingly then, unlike the Mesopotamians,
Egyptians could conceive of times when even moderate beer drinking should be avoided.
In ancient Greek and Roman texts, beer is no longer found as a basic element of the diet
(wine being the standard everyday beverage), and it is generally considered harmful (see
Nelson, 2003, 2005, and 2014). Beer is nowhere explicitly mentioned in the corpus of
medical texts attributed to the so-called western “father of medicine” Hippocrates and his
followers and dated from the fifth and fourth centuries B.C., showing that it was not a usual
drink among ancient Greeks (Nelson, 2005: 33 and 2014: 31-33). However, at the same time
some Greek authors began to speak of beer as an inferior beverage of foreigners on various
grounds. The tragic playwright Aeschylus mentioned it as effeminate and enfeebling
(Suppliant Women 952-953 in Nelson, 2001: 284 and fr. 124 Radt in Nelson, 2001: 285,
respectively).10 Natural philosophers took an interest in it as well, with Aristotle
differentiating between wine and beer as being different sorts of substances having different
physiological effects on the drinker (fr. 7 Laurenti in Nelson, 2001: 288-289) and his student
Theophrastus considering beer as being a product which had somewhat decomposed (On the
Causes of Plants 6.11.2 in Nelson, 2001: 290). Other ancient authors also thought of beer as
barley or wheat rotted in water (Dionysius of Halicarnassus, Roman Antiquities 13.11.1 in
Nelson, 2001: 298 and Tacitus, Germany 23.1 in Nelson, 2001: 305).11
Greco-Roman medical authorities only began to discuss beer in the first century A.D., by
which time numerous peoples within the Roman Empire were beer-drinkers (Nelson, 2005:
71-74). Often the negative effects were emphasized, though sometimes beneficial uses were
in fact noted (as will be seen in the subsequent sections below). In the first century A.D., the
Hippocratic physician Rufus of Ephesus mentioned beer made with dates as being bad for the
stomach (Rufus of Ephesus, fr. 197.1 in Daremberg and Ruelle, 1879: 481). Unfortunately,
the context of this quotation has been lost, but Rufus was perhaps reacting to its use in
Egyptian medicine, as the reference to dates could suggest (see further below). Around the
same time, Aretaeus of Cappadocia stated that beer among other harsh substances, including

7
AMT is used as the abbreviation for the seventh century B.C. tablets from the library of Ashurbanipal in Nineveh
as published in Thompson, 1923. See also Worthington, 2009: 54, n. 37 on ancient advice about washing
hands when serving beer.
8
Reymond, 1976: 191 comments that beer “appears here as the main means to test the general state of health”.
9
See Strouhal, Vachala, and Vymazalová, 2014: 108 (on the text) and 180 (on the interpretation).
10
See also the comic poet Antiphanes, fr. 47 Kassel and Austin in Nelson, 2001: 287 on a weakened, beer-drinking
old woman being given a root to treat an illness.
11
See further Nelson, 2003: 105-106 and 2014: 42, n. 101.
 Max Nelson 5

Nile water, caused ulcerations on the tonsils (On the Causes and Signs of Acute Chronic
Diseases 1.9.4 in Nelson, 2001: 307).12 Again, he was probably thinking of Egyptian beer, as
the reference to Nile water suggests. Also in the first century A.D., the herbalist Dioscorides
was the first to discuss the properties of two different types of beer. The first, zūthos, a barley
beer, probably from Egypt, was said to be a diuretic, to act on the kidneys and sinews, to be
especially harmful to the membranes, and to cause flatulence, bad humours, and even
elephantiasis (On Medicinal Material 2.87 in Nelson, 2001: 303, and see also 5.32.2 in
Nelson, 2001: 304 on elephantiasis). The second, kourmi, a barley or wheat beer, probably
found among Celts, was also said to be harmful to the sinews and bad for the humours, and it
was added that it also causes headaches (On Medicinal Material 2.88 in Nelson, 2001: 304).
Aside from the diuretic nature of beer (to be returned to below) and its causing of flatulence
and headaches, most of these claims are clearly false, and presumably are not based on any
sort of careful observations but on a priori prejudice against a foreign beverage.
The second century A.D. Galen, physician to the emperor Marcus Aurelius, marked an
important stage in the ancient European medical understanding (or rather, continued
misunderstanding) of beer. He stated that it was because beer could arise out of a decomposed
substance that it was bad for the humours and it caused flatulence (On the Powers and
Mixtures of Simple Remedies 6.6.3 in Nelson, 2001: 310). Thus Galen was the first to connect
two separate notions about beer found in ancient Greek authors, and in fact made them
causally related, that beer was due to decomposition (as first found in Theophrastus) and that
it was bad for the humours and caused flatulence (as first found in Dioscorides). He further
explained precisely how it was that beer was a decomposed substance and how it was bad for
the humours, though in a very terse manner. First, he explained that decomposition was
caused by yeast, saying that yeast has putrefactive heat (On the Powers and Mixtures of
Simple Remedies 6.6.4). It is now known that any alcoholic fermentation is due to yeast
(which converts sugar into alcohol and carbon dioxide), but in antiquity, not only was there
no conception of alcohol (thus wine and beer could be distinguished as different sorts of
substances, as Aristotle had done) but not all intoxicating drinks were thought to be affected
by yeast (see Nelson, 2005: 36 and 133, n. 25 and 2011: 80, n. 18). In this case the yeast was
thought to have a deleterious effect on the cereals, since non-fermented cereal beverages or
soups could be thought of as healthful by Galen and other Roman era authors.13 Second,
Galen also spoke of beer as watery and cold, connecting it to humoural theory, in which
various comestibles were considered either cold or hot and either dry or wet (On the Powers
and Mixtures of Simple Remedies 6.6.3 in Nelson, 2001: 310). This linked beer with women,
who were considered by nature cold and wet, in contrast to men, who like wine, were thought
of as hot and dry (see Nelson, 2005: 33-34 and 72-73 and 2011: 76 and 81, n. 45 and 46).
Galen thus provided a supposedly scientific explanation for why beer was an effeminate

12
On this passage, see Jouanna, 2002: 118-120.
13
For example, Galen, On Barley Soup in Grant, 2000: 62-67 (on the health benefits of barley soup), Celsus, On
Medicine 2.18.11-12 in Nelson, 2001: 298 (on cereal drinks being more nutritious than water), and Pliny the
Elder, Natural History 23.22.37 in Nelson, 2001: 302 (on cereal drinks nourishing sinews). Arguably the last
two sources are excluding beer from the (simple) cereal drinks mentioned (Celsus’s passage was wrongly
interpreted in Nelson, 2003: 105 and 2005: 71-72).
6 To Your Health!

beverage, as Aeschylus had claimed long before, and also for why wine was better than
beer.14
Galen was highly regarded as an authority, and his ideas about beer were repeated by
later medical authors (Aetius, On Medicine 1.154 in Nelson, 2001: 329 and Paul of Aegina,
Epitome of Medicine 7.3.6 in Nelson, 2001: 334).15 Thus in the fourth century A.D. Oribasius
of Pergamum, the emperor Julian’s personal physician and a compiler of medical wisdom,
cited from Galen (Medical Compilations 14.10.10 and 15.1.6.6 in Nelson, 2001: 318-319)
and added that beer from Cyrene in northern Africa was especially prone to cause flatulence
(Medical Compilations 3.23.4 and Synopsis for Eustathius 4.22.4 in Nelson, 2001: 318).16
Oribasius also praised wine over beer, saying that barley and wheat beers were weaker than
wine (Medical Compilations 5.31.12 in Nelson, 2001: 318). Christian authors in late antiquity
generally continued to view beer as harmful. In the fifth century A.D., for instance, Cyril,
Patriarch of Alexandria, felt that beer was cold and impure and that it caused unspecified
incurable illnesses, and that it only roused more thirst when drunk (Commentary on Isaiah 2.4
in Nelson, 2001: 320, and see Theodoret, Commentary on Isaiah 6 in Nelson, 2001: 320),
while the Church Father Jerome advised against drinking beer or any intoxicant (Letter 52.11
in Nelson, 2001: 321).17
An important turning point evidently occurred in the sixth century A.D. While at that
time Venantius Fortunatus, Bishop of Poitiers, was still speaking of the impurity of beer and
it causing dropsy (or edema) (Appendix to the Poems 9.15-18 in Nelson, 2001: 327) his
contemporary Anthimus, adviser to Theuderic, King of the Franks, recommended beer as a
beneficial beverage suitable for all, which, although it was cold (in the humoural sense),
derived its usefulness from the cereal used in it, just like barley soup made in a different way
(On the Observance of Foods 15 in Nelson, 2001: 328). This was an essential step in
returning to the Mesopotamian and Egyptian conception of beer as a healthful beverage, since
while earlier authorities like Galen could admit that a preparation like non-fermented barley
soup was healthful, now the fermenting of the cereal in beer was not thought to ruin it.
After this, beer in general was normally avoided only in so far as it might lead to
intoxication. Thus in the eighth century A.D. in Ireland beer was sometimes forbidden in
some monastic communities, but at the same time in a penitential it was declared that those
who did not drink beer ordinarily had to take three sips at Easter and Christmas to ward off
illness (ee Nelson 2005: 93, with section 14 of the anonymous Old Irish penitential in
McNeill and Gamer, 1938: 158). Beer therefore came to be thought of as a general tonic. The
Irish St. Brigid was even said to miraculously make beer which cured the ill (Anonymous,
Life of St. Brigid 2.10 in Nelson, 2001: 338 and Chilienus Monachus, Life of St. Brigid 1.4-5
in Nelson, 2001: 354).

14
Galen’s near contemporary Plutarch, in Dialogue on Love 5 in Nelson, 2001: 306, further said that wine sated
one’s thirst better than beer. See also Nelson, 2003 on Greeks and Romans in general considering wine better
than beer.
15
In a treatise falsely attributed to Galen (On the Diagnosis and Therapy of Diseases in the Kidneys 7 in Nelson,
2001: 311), it is said that wine, beer, other intoxicating beverages, and even cold water harm the stomach,
liver, and sinews.
16
For the emperor Julian’s poem against beer, see Nelson, 2005: 30-31.
17
See further Nelson, 2003: 104 and 106.
 Max Nelson 7

INTOXICATION AND OVER-DRINKING

It was widely understood in ancient times that beer (among other alcoholic and narcotic
substances) could be harmful if consumed in large quantities, both in terms of adverse short-
term effects of intoxication or alcohol poisoning as well as long-term, chronic debilities.
However, while over-drinking could be thought of as a physical liability, or for that matter, a
moral or dispositional failing, there is no evidence that it was itself considered a type of
disease or pathological condition, as alcoholism is often viewed today (see, for example,
D’Arms, 1995: 316-317 and Villard, 2002: 85 for the Greek and Roman evidence).18
In Esagil-kīn-apli’s diagnostic handbook one of the negative symptoms listed is the
continual asking for beer (or wine) and being sick for ten days (DPS 7Br.13-14 in Scurlock,
2014: 62, with Scurlock and Andersen, 2005: 361). To cure excess beer consumption which
leads to headaches, amnesia, and slurred speech, one text recommends that a potion be made
of various plants ground together and left overnight in oil and beer under the star of Gula,
goddess of medicine, and then drunk before sunrise and before being kissed by anyone (BAM
59.21-28 and 575.iii.51-54 in Scurlock, 2014: 636-637).19 Interestingly, beer is used in the
cure, which shows how highly it was regarded (or how commonly and unreflectively it was
employed) in medicine. The effects of excessive beer-drinking included the same sorts of
symptoms which Mesopotamians attributed to ghost afflictions, and so in general the
pathology which would be termed alcoholism today was blamed by them on ghosts, just as
many illnesses were blamed on various superhuman beings (Scurlock and Andersen, 2005:
361-363 and 438, and see also Scurlock, 2006: 19 and 2008: 198-200).20 This may have been
at least in part because of the dehydrating aspects of alcohol poisoning and the fact that
dehydration in general was blamed on ghosts who had died of thirst or had drowned while
desperately trying to drink from a river (Scurlock and Andersen, 2005: 501-503). It was
believed that the dead had to be kept appeased with various offerings otherwise they might
harm the living in various ways and these offerings could include beer made with roasted
grain or different types of water mixed with beer and vinegar, in contrast to the ordinary beer
or wine reserved for offerings to the gods (Scurlock, 1997: 88 and 91 and, for greater detail,
see Scurlock, 2006: especially 45 and 47). Thus beer, provided as a libation, could act as a
sort of preventative treatment.21 The Mesopotamians may also have observed liver damage
from excessive alcohol consumption (without thoroughly understanding cirrhosis), as is
suggested by the fact that the goddess Ninurta was seemingly blamed for liver problems due
to over-drinking (DPS 7.B.r.10-17 in Scurlock, 2014: 62, with Scurlock and Andersen, 2005:
520-521).
The negative effects of intoxication and their treatments do not seem to appear in
Egyptian medical papyri. However, in the Instructions of Any, a text providing advice on
various subjects and dating probably to around 1500 B.C., there is a warning over
overindulgence in drinking beer because of the poor behaviour of the drunken man but also

18
The literature on alcoholism as a disease is immense and cannot be contended with here.
19
In another recipe, plants are placed rather in wine (BAM 575.iii.49-50 in Scurlock, 2014: 637). Scurlock and
Andersen, 2005: 382 take the reference to headaches, amnesia, and slurred speech to indicate addiction (rather
than mere intoxication), but this is debatable.
20
Generally on the role of superhuman beings in illnesses, see Scurlock and Andersen, 2005: 429-528, Heessel,
2007, and Attinger, 2008: 64-68.
21
Also various ingredients are mixed in beer to help against ghost-induced stomach problems in AMT 76/1.4-29 in
Scurlock, 2014: 491-492.
8 To Your Health!

because he could fall and hurt himself, presumably though his lack of balance (passage 4 in
Lichtheim, 1976: 137).
As seen above, many Greco-Roman authors spoke of the negative effects of drinking
beer, but they seem to have been thinking of it as a deleterious drink even if consumed in
moderate amounts. The only exception is Aristotle, who in a surviving quotation from his lost
work on intoxication stated that those drunk on beer fall straight on their backs and lie flat
because of its stupefying effect, contrasting this with wine which makes people heavy-headed
and fall any which way (fr. 7 Laurenti in Nelson, 2001: 288-289, with Nelson, 2005: 34-35).
A half millennium later the Roman era physician Galen similarly spoke of heavy-headedness
when drinking too much wine, and also mentioned staggering as well as restlessness during
sleep, but he made no comment concerning the effects of excessive beer consumption (On the
Coma According to Hippocrates 4 in Leibowitz, 1967: 84-85). Further Greco-Roman authors
mention various other effects stemming from drinking too much wine, from poor complexion,
disturbed vision, and headaches, to delirium tremens and liver problems, but again they leave
out beer intoxication (see the evidence collected in D’Arms, 1995: 316, n. 71 and Villard,
2002: 88-91, along with Gourevitch, 2013).

BEER AS A VEHICULUM AND MENSTRUUM FOR

THE ORAL INGESTION OF MEDICINE

A large part of ancient medicine involved phytopharmacy, or pharmacological

preparations using plants (see the general surveys of Mazars, 2002, for the Near East, and
Aboelsoud, 2010, for Egypt). Plants might be simply mixed in a liquid base or vehiculum
(also known as a vehicle, carrier, or excipient) which was immediately ingested by the
patient. However, to properly extract the active constituents of plants, some time and effort is
needed to dissolve the plant in a liquid, called the menstruum (or diluent or solvent). Today
water is the most commonly used vehiculum and menstruum because it is tasteless, non-
irritating, and pharmacologically inert. However, a solution with at least 20% alcohol strength
is necessary to ensure the sterility of the medication, in which case it can keep indefinitely.
Dissolving can be done through maceration and expression (soaking for some time, usually in
alcohol, and straining and filtering), digestion (following the steps of maceration and
expression, with the added application of gentle heat), percolation (with the solvent passing
through gradually), infusion (steeping in water, hot or cold), or decoction (steeping in boiling
water) (Hoffmann, 2003: 217-219, and see Troy, 2006: 745-747).
It has usually been emphasized that the main use of beer in ancient Mesopotamian and
Egyptian medicine was as a vehiculum or menstruum (see, for example, Keller, 1958: 153-
154, Wright-St. Clair, 1962: 513, Lucia, 1963: 155 and 164-165, Röllig, 1970: 76, Helck,
1971: 81, Darby, Ghalioungui, and Grivetti, 1977: 548, Herrero, 1984: 58, and Strouhal,
Vachala, and Vymazalová, 2014: 90). Ancient beer would not have had the benefits of either
a water solution or a 20% alcohol solution, since on the one hand it was not neutral in taste
and effect and on the other it was not high enough in alcohol to ensure sterility.22 However, it

22
The Mesopotamians and Egyptians had both weak and strong beers (as will be seen below), and although there is
no accurate method to determine what the usual alcohol content of their beers were it presumably would never
have been as high as 20% (see Nelson, 2001: 177 on the alcohol content of ancient beers). Geller, 1993: 259
 Max Nelson 9

has been pointed out that useful alkaloids from the beer could be extracted if the medicine
were left in it for a long enough period of time (Nunn, 1996: 139 and 140).
It should be noted that beer itself came to be made through infusion or decoction, with
water as a menstruum in which, in addition to ground malted cereal (normally barley), various
herbs were steeped. Beer in ancient Mesopotamia and Egypt was (at least usually) made by
crumbling baked malted loaves into water and allowing them to ferment, but in ancient
Europe beer was rather brewed, as it still is today (Nelson, 2005: 21-24).23 By the sixth
century B.C. Celts were already using hops in beer, now a ubiquitous ingredient, but various
other plants were infused in European beers in the ancient and medieval periods, such as
sweet gale (Nelson, 2014b: 16-17). Hops may have first been used as a preservative or as a
flavouring agent (adding bitterness to what might otherwise have been a rather sweet or sour
brew), but certainly, at least by the Middle Ages, hopped beer could be seen by some as a
healthful, medicinal concoction (Nelson, 2011: 77, with references).
To return to the ancient evidence, over nine hundred tablets (many fragmentary) survive
from Mesopotamia outlining the various treatments of diseases, mostly dating to the first
millennium B.C. (Scurlock, 2005: 302). Treatments relied almost completely on preparations
of plants (and also to some extent on mineral and animal products), based on generations of
trial and error (Geller, 2010: 3 and 19-21). Beer was the most common vehiculum for the
administering of plant matter (Powell 1993: 61), although others were used as well, such as
wine, vinegar, water, milk, fish brine, sesame oil, olive oil, and urine (Herrero, 1984: 57-59
and 88-92, Böck, 2009: 111 and 2011: 698, and Geller, 2010: 21). Plants were often ground
and then given to drink mixed in first quality beer, such as for urethra, gall bladder, intestine,
and other issues as attested on a Neo-Assyrian tablet copied from a vademecum originally
owned by the apprentice physician Nabū-le’ū (BAM 1.18, 24, 26-27, 30, 35, 57-58, and 67 in
Scurlock, 2014: 277, 278, and 280).24 The medical benefits of the plant seem to be the focus,
while the beer is only a means of ingesting the medicine.25 This is clear from the fact that it is
sometimes said that wine or milk can be substituted for the first quality beer.26 The beer is
evidently meant not to detract from the medicine, and so is of the first quality, though
sometimes ordinary beer is prescribed and it too can be substituted with one or more other
liquids (for example, BAM 156.21-24 in Scurlock, 2014: 332).27 Occasionally the beer is
used in combination with other vehicula, such as oil (for example, SpTU 1.14.26, 40-41, 51,

believed that modern-day bouza was fundamentally like ancient Egyptian beer, and he pointed out that the
alcohol content only reaches 4.5%. For skepticism on the modern beer being like its ancient counterpart, see
Samuel, 1996: 3. In contrast Estes, 1989: 140 (and see 29) believed that ancient Egyptian beer’s alcohol
content was between 6.2 and 8.1%, which would still make it lower than the requisite 20%.
23
The Mesopotamian method is alluded to in a hymn to the beer goddess Ninkasi from around 1800 B.C. (in Civil,
1964: 72-73, with Powell, 1994: 98-99) while a fourth century A.D. recipe outlines the Egyptian method (in
Nelson, 2001: 316-317, and see Nelson, 2005: 23-24).
24
See Herrero, 1984: 17-20 on this text. See also, for example, BAM 240.25’-28’ in Scurlock, 2014b: 110-111 (for
female bloating) and BAM 272.0’-6’ and 17’-19’ in Scurlock, 2014: 549-550 (for an erection).
25
For directly ingesting ingredients (in various liquids), see Herrero, 1984: 61-86 and Böck, 2011: 697.
26
For example, for first quality beer or wine, see BAM 1.31-34 in Scurlock, 2014: 278 and BAM 480.iv.48’-49’ in
Scurlock, 2014: 328. For first quality beer, wine, or milk, see BAM 159.i.15-20 in Scurlock, 2014: 547. See
further Herrero, 1984: 66.
27
For ordinary beer or milk, see BAM 124.i.5-7 in Scurlock, 2014: 449. For ordinary beer or wine, see BAM
159.iii.25-27 in Scurlock, 2014: 560. For ordinary beer, water, or wine, see BAM 116rev.6’-9’ in Geller, 2005:
81. For ordinary beer, milk, or wine, see BAM 396.i.19’-22’ in Scurlock, 2014: 542. For ordinary beer,
merchant’s beer, wine, or milk, see CBS 14161 in Abusch and Schwemer, 2016: 402; CBS stands for
Catalogue of the Babylonian Section, University of Pennsylvania Museum of Archaeology and Anthropology.
For old barley beer or wine, see BM 78963.48-49 in Scurlock, 2014: 478; BM stands for British Museum.
10 To Your Health!

52, 53, 62, and 63 in Scurlock, 2014: 395, 396, and 397).28 While directions for exact dosages
are uncommon in one instance ingredients are to be given in the equivalent of 0.84 litres (one
qū) of strong beer (AMT 40/1.62-63 in Herrero, 1984: 48). It could be thought that the beer
would make the medicine more palatable or simply easier to ingest.29
Often herbal ingredients were not simply placed in first quality beer but carefully
macerated in it (at room temperature) and left overnight under the stars (for example, AMT
1/3.9’ in Böck, 2009: 114, BAM 115.5’-9’ in Geller, 2005: 77, and BAM 396.i.14’-18’ and
ii.13’-15’ in Scurlock, 2014: 542).30 This ensured that the ingredients would properly dissolve
in the potion, but it also allowed for what was considered the beneficial effect of star
irradiation (see, for example, Scurlock, 2006: 21, Attinger, 2008: 19, and Böck, 2009: 112-
113). In one text the overnight maceration takes place in merchant’s beer, that is the beer sold
at a tavern (BAM 396.ii.23’-24’ and iv.3-5 in Scurlock, 2014: 543 and 545). Ingredients were
also digested in beer in a warm oven and then cooled off (see the discussion of digestion and
the sample texts provided in Böck, 2009: 114-115, with the summary at Böck, 2011: 698). On
other occasions the ingredients were doused in cold beer and then decocted by being brought
to a boil (for example, BAM 32.9’-12’ in Scurlock, 2014: 439, BAM 124.i.12-14, 17-18, and
21 and ii.2-5 and 11-18 in Scurlock, 2014: 449 and 451, and BAM 156.15-16 in Scurlock,
2014: 332).31 Sometimes the beer decoctions were also left overnight under the stars (see, for
example, AMT 97/4.19-21 in Herrero, 1984: 81 and BAM 159.i.9-11 and ii.1-5 in Scurlock,
2014: 547 and 530-531). A good example of this is from what seems to be a cure for
anaerobic lung abscess, in which nine plants are decocted in sweet wine and first quality beer,
which is left overnight, and then boiled, filtered, and cooled; after more ingredients are added
it is taken before sunrise, after which vomiting is induced and, if this is not effective, the
mixture is then poured into the patient’s anus (BAM 55.1-17 in Scurlock, 2014: 484, with
483). Aside from ordinary and first quality beers, recipes for decoctions also called for old
beer and strong beer, among others (AMT 68/2rev.18 in Herrero, 1984: 72, with other
examples, and BAM 106obv.9-10 in Scurlock, 2014: 422).
In Egyptian medicine, water (often dew) was the most common vehiculum, whereas
honey, milk, oil, wine, and beer were used as well, sometimes in combination (Nunn, 1996:
140, and see Halioua and Ziskind, 2005: 33). Occasionally the role of beer as a vehiculum is
explicitly mentioned in the medical texts. The longest surviving ancient Egyptian medical
text, the Ebers Papyrus, which was written during the reign of Amenhotep I (around 1500
B.C.), has as its first recipe a potion in which peas are mixed in beer to help against internal
pain (1 in Bardinet, 1995: 252).32 This recipe is also found in the Hearst Papyrus, a collection
of treatments dating from around the same time (53 in Bardinet, 1995: 381), but also in the
Brooklyn Papyrus from over a millennium later, in which the beverage is to be used as an

28
See also SpTU 1.14.24-25 in Scurlock, 2014: 395 where it is to be vomited; SpTU 1 is used to refer to texts in
Hunger, 1976. See also, for example, Sippar 12.20 in Heessel and Al-Rawi, 2003: 234 and BAM 1.28-29 in
Scurlock, 2014: 278. Also honey, oil, and beer are mixed together, for example, in SpTU 1.14.27-28 in
Scurlock, 2014: 395, and milk and beer are mixed together, for example, in BAM 396.i.8’-9’ in Scurlock,
2014: 541.
29
Röllig, 1970: 76 believed that beer was used by Mesopotamians internally not so much for its own therapeutic
properties but for its pleasant taste and for making medicine more palatable.
30
For maceration in general, see Böck, 2009: 111-114, with the summary at Böck, 2011: 698.
31
See the discussion of decoction in Böck, 2009: 115, with the summary at Böck, 2011: 698. Böck, 2009: 116
points out that there is no explicit evidence for infusion in Mesopotamian texts.
32
Compare Pap.Eb. 168 in Bardinet, 1995: 273 in which the ingredients are made into a flour and then drunk in
beer before bedtime.
 Max Nelson 11

emetic four days in a row for a snake bite (47.218.48 +85, 45b in Bardinet, 1995: 530). This
shows not only that there was a common store of recipes used for a long period of time but
also that treatments often concerned flushing out harmful substances, whether those thought
to cause general internal pain or else snake venom (see Bardinet, 1995: 159-160). In another
recipe in the Hearst Papyrus the ingredients are mixed in honey and then are to be swallowed
with the help of sweet beer (3 in Bardinet, 1995: 376).33 In yet another recipe, this one from
the Brugsch Papyrus (also known as the Berlin Papyrus [= Pap.Berl. 3038]) from around
1200 B.C., ingredients are made into a cake which is then consumed with sweet beer (153 in
Bardinet, 1995: 427-428). In a recipe in the Ebers Papyrus a medicinal cake is eaten either
with about 140 millilitres (10 ros) of beer or about 70 millilitres (5 ros) of wine (9 in
Bardinet, 1995: 252).34 This may be important evidence showing that the relative strengths
(based on alcohol content) of each drink is being taken into consideration, with wine being
considered twice as strong as beer. Sometimes, however, the alcohol strength does not seem
to have been an issue, as when either water or beer could be used in a recipe (Pap.Eb. 60 in
Bardinet, 1995: 259).35
Usually in the Egyptian papyri the beer is simply mentioned as one of the many
ingredients of a potion (rather than all the ingredients being said to be drunk in the beer), and
sweet beer is mentioned more often than plain beer in these cases, for example in the Ebers
Papyrus.36 There is some controversy over what this sweet beer was. Many scholars have
believed that this referred to beer made with dates (for example, Darby, Ghalioungui, and
Grivetti, 1977: 543-547, and see further Nelson, 2001: 135). However, there is little
archaeological evidence to support the contention that Egyptians often made beer with dates,
and sweet beer could perhaps instead refer to a beer made with special cereal or malt
(Samuel, 1996: 10 and 2000: 549-550). Presumably often the sweetness of the beer was used
to cover up the nasty taste of some medicinal preparations. Sometimes the ingredients are to
be macerated in sweet beer (or rarely ordinary beer) and left overnight.37 In one instance,
ingredients are ground with beer, left overnight with honey, and then mixed with a little more
beer and drunk (Pap.Eb. 59 in Bardinet, 1995: 259). Decoctions often taken place in sweet
beer as well, and again rarely in ordinary beer.38 For instance, for a woman’s stomach pain, a

33
Compare Pap.Eb. 32 and 193b in Bardinet, 1995: 255 and 278. Also similarly in Pap.Hearst 131 = Pap.Eb. 302
in Bardinet, 1995: 392 and 297.
34
For a ro being equivalent to about 14 millilitres, see Nunn, 1996: 141. For drinking down a medicinal cake with
beer, see also Pap.Eb. 13-15 in Bardinet, 1995: 253.
35
For water and beer mixed together, see Pap.Eb. 35=185 and 63 in Bardinet, 1995: 256, 259, and 275.
36
For example, for beer, see Pap.Eb. 4, 25, 251, and 586 in Bardinet, 1995: 252, 254, 290, and 334. For sweet beer,
see Pap.Eb. 6, 13, 14, 15, 34, 35, 56, 86, 130, 138, 165, 185, 238, 284bis, 300, 327, 333, and 751 in Bardinet,
1995: 252, 253, 256, 262, 268, 273, 275, 287, 294, 297, 301, 302, and 356. However, Darby, Ghalioungui, and
Grivetti (1977: 548) posit that the two types of beer were perhaps interchangeable. Aside from sweet beer and
ordinary beer, references are sometimes made to other types, not always identifiable. See, for example,
Pap.Eb. 334 and 335 in Bardinet, 1995: 302 on what is perhaps strong beer. See also, for example, Pap.Eb.
192b=195b in Bardinet, 1995: 277-278 and also Helck, 1971: 79 on Pap.Eb. 136=151 in Bardinet, 1995: 271.
Helck, 1971: 79 takes the reference in Pap.Eb. 19 in Bardinet, 1995: 253 to be to coagulated beer.
37
For sweet beer, see, for example, Pap.Eb. 21, 137=152, 187, 202b, 207, 209, 212, 297, 322, 479, 480, and 631 in
Bardinet, 1995: 254, 269, 271, 275, 280, 282, 283, 287, 297, 300, 320, and 341. Pap.Eb. 264 in Bardinet,
1995: 292 speaks of leaving the ingredients in sweet beer until they have settled. For ordinary beer, see
Pap.Eb. 210 in Bardinet, 1995: 283. For what is perhaps strong beer, see Pap.Eb. 632 in Bardinet, 1995: 341.
38
For sweet beer, see, for example Pap.Eb. 6, 24, 55, 87, 88, 89, 92, 94, 138, 186, 201b, 203d, 217, 236, 237, 278,
285, 286, 291, 293, 306, 319, 321, 326bis, 754, and 777 in Bardinet, 1995: 254, 258, 262, 263, 269, 275, 280,
284, 287, 293, 295, 298, 300, 301, 356, and 360 and Pap.Hearst 42, 43, 48, 59, 60, 64, 207, and 211 in
Bardinet, 1995: 380, 381, 382, 383, 401, and 402. For ordinary beer, see Pap.Eb. 276=281 in Bardinet, 1995:
12 To Your Health!

plant is boiled with oil and sweet beer and drunk for four days (Pap.Smith 20 in Allen, 2005:
111, with Strouhal, Vachala, and Vymazalová, 2014: 115). The Egyptians, then, seemed to
prefer sweeter concoctions than their Mesopotamian counterparts, with the inclusion of sweet
beer or honey. Occasionally, the beer mixture is not liquid but a solid mass which is chewed
and either swallowed for internal issues (Pap.Eb. 25 and 251 in Bardinet, 1995: 354 and 290)
or spat out for tooth problems, in this last case with sweet beer specifically (Pap.Eb. 745 and
748 in Bardinet, 1995: 355).
The Greek physician Antyllus from the second century A.D. mentions crushed worms (or
alternatively the unripe fruit of the sesame tree) and palm dates served in beer for good and
plentiful breast milk in women (quoted in Oribasius, Medical Compilations 34.6-7 and Aetius
of Amida, On Medicine 4.6 in Nelson, 2001: 308-309). This is a rare attestation of beer used
as a vehiculum in a Greco-Roman medical source, and is likely taken from an Egyptian
recipe. It has been shown that if Greeks adopted Egyptian recipes they would normally
replace beer with another vehiculum (Lang, 2013: 137, n. 132). Egyptians under Roman rule
apparently continued to use beer in their medicine in the same way as they had in pharaonic
times, as is evident, for instance, from a medical papyrus dating to the second century A.D.
from the libraries of the Suchos Temples in the city of Crocodilopolis in the Fayyum in which
sweet beer figures a number of times as a vehiculum (Pap.Vindob. D. 6257 6a.30, 9.9, 11.23,
13.23, 13.31, 13.33, and 13.36 in Reymond, 1976: 91, 101, 111, 119, and 121). Beer as a
vehiculum and menstruum for medicine is later attested in Old English texts of the Middle
Ages, such as in the tenth century A.D. herbal falsely attributed to Apuleius or the medical
collection known as the Lacnunga from around A.D. 1000.39

BEER AS A VEHICULUM AND MENSTRUUM FOR

THE NON-ORAL INTRODUCTION OF MEDICINE

Although beer was normally used in potions meant to be drunk, it was sometimes rather
placed in the nose, ear, urethra, vagina (in pessaries or douches), or anus (in suppositories or
enemas).
In a recipe from Uruk, ingredients mixed in first quality beer are poured in the nose
(SpTU 1.45rev.16’-17’ in Scurlock, 2014: 389). In a recipe for an ear infection various
ingredients are inserted in the ear, oil is poured over the head, and the patient is told to eat hot
things twice a day and to drink beer twice a day, with added thyme only if the right ear is at
issue (BAM 503.iii.72’-78’ in Scurlock, 2014: 385, with Scurlock and Andersen, 2005: 204).
For a genito-urinary tract problem, ingredients can be mixed in first quality beer and drunk on

293-294, as well as 312 in Bardinet, 1995: 299 with the similar recipe in Pap.Brugsch 36 in Bardinet, 1995:
414, in which sweet beer is rather called for. For decocting in the dregs of sweet beer, see Pap.Eb. 200b in
Bardinet, 1995: 280. For what may be strong beer and sweet beer together, see Pap.Eb. 284bis and 285 in
Bardinet, 1995: 294-295. For what may be strong beer alone, see Pap.Eb. 330 and 332 in Bardinet, 1995: 301
and 302. For haou-khet beer, see Pap.Eb. 593 in Bardinet, 1995: 336.
39
For ingredients placed in beer, see Pseudo-Apuleius, Herbal 2.3, 2.15, and 36.3 in De Vriend, 1984: 38, 40, and
82 and Lacnunga 57, 58, 62, 72, 73, 75, and 131 in Pollington, 2000: 201, 211, 213, and 227. For ingredients
boiled in beer, see Lacnunga 23, 54, 55, 59, 69, 124, and 139 in Pollington, 2000: 191, 201, 211, 227, and 231.
At Lacnunga 156 in Pollington, 2000: 233 mention is made of placing ingredients in beer or whatever drink is
desired.
 Max Nelson 13

an empty stomach or else blown into the urethra (BAM 396.ii.5’-12’ in Scurlock, 2014:
542).40 In one Mesopotamian recipe beer dregs are to be mixed with other ingredients and
placed in the vagina and in another various ingredients are boiled in beer and then poured into
the vagina (BAM 1.19-20 in Scurlock, 2014: 277 and BAM 240.17’-19’ and 33’-38’ in
Scurlock, 2014b: 111). In the Egyptian Kahun Papyrus it is recommended that a female
patient should sit on the dregs of sweet beer or else simply on sweet beer (17 and 27 as well
as 24, respectively, in Bardinet, 1995: 440-442). It has been suggested that such a treatment
was used for its psychological effect, in helping to calm down a patient (Strouhal, Vachala,
and Vymazalová, 2014: 145). In the Ebers Papyrus dried myrtle leaves and the dregs of
excellent beer are applied to the pelvis and pubic regions to drive away slime from the uterus
(812 in Bardinet, 1995: 447, with Strouhal, Vachala, and Vymazalová, 2014: 120). In the
same source it is recommended that to help a woman with childbirth a pessary is to be made
from various ingredients including beer (802 in Bardinet, 1995: 446) or a mixture of a type of
fruit with beer and used as a douche (805 in Bardinet, 1995: 446). In yet another prescription,
sweet beer with honey and other ingredients are used as a douche (830 in Bardinet, 1995: 449,
with Strouhal, Vachala, and Vymazalová, 2014: 122). Beer enemas are often mentioned in
Mesopotamian texts (see Böck, 2009: 117-123, with the summary at 2011: 699). In one
example, ingredients are boiled in beer and the mixture with oil and ghee is poured hot into
the patient’s anus (BAM 156.11-14 in Scurlock, 2014: 331-332.).41 Similarly, the Berlin
Papyrus and the Chester Beatty Papyrus include many recipes in which ingredients along with
sweet beer are to be placed in the anus for four days in a row.42

BEER IN INTERNAL THERAPY

As has been shown, in ancient Mesopotamia and Egypt, beer was often used simply as a
means of dissolving and ingesting the active medicinal ingredients prescribed by a physician.
However, occasionally beer was clearly used for its own particular therapeutic qualities.43 In
one Mesopotamian recipe, the use of ḫīqu beer rather than kurunnu beer ensures that the
patient will not die, and while the meaning of these terms is uncertain, it clearly shows that
different beers were thought to have different attributes (BAM 156.1-10 in Scurlock, 2014:
331).44 The positive characteristics of beer which made it useful in ancient medicine are
shown in its potential uses as an emetic (or vomit-inducer), as an aperient (or promoter of
defecation), as a diuretic (or promoter of urination), as an antitussive (or cough suppressant),
as an anthelmintic (or destroyer of parasitic worms), and as a general anaesthetic or analgesic
(or remover of sensation or pain). In most cases at least one example exists showing that beer

40
See further Geller, 2005: 43 and 77 for beer and other ingredients poured into the penis by means of a bronze
tube.
41
See also, for example, BAM 55.1-17 in Scurlock, 2014: 484 and 159.vi.1-4 and 23-33 in Scurlock, 2014: 501 as
well as the examples in Geller, 2005: 169, 171, 173, 175, 185, 193, 195, 197, 199, 201, 203, 205, 209, 213,
and 261. Powell, 1993: 61 pointed out that enemas were not simply used to wash out the lower intestinal track,
but clearly were thought to have pharmaceutical effects.
42
Pap.Brugsch after 163h, 164, 165, 168, 169, 172, 173, 174, 175, 176, and 182 in Bardinet, 1995: 430-432 and
Pap.Chest. 19, 20, 21, and 28 in Bardinet, 1995: 458-459. Compare Pap.Eb. 265 in Bardinet, 1995: 292. See
also the anal fumigation at Pap.Hearst 7 in Bardinet, 1995: 376.
43
Contrast Darby, Ghalioungui, and Grivetti, 1977: 548, who believe that in the Pap.Eb. prescriptions beer was
simply “utilized as a vehicle or base”.
44
Herrero, 1984: 57 and 59 interprets ḫīqu to mean beer mixed with water.
14 To Your Health!

on its own was thought to have had such an effect, but in other examples, in which beer is
mixed with other ingredients, it remains uncertain whether beer is used for its own properties
or simply as an inert vehiculum.

Emetic

Beer was often used for emesis, that is, causing the patient to throw up. In one
Mesopotamian text ḫīqu beer on its own could be used as an emetic, or else served with salt
or with various plants or other ingredients mixed in, for instance when a patient has a gall
bladder problem (BAM 578.i.16, i.17, and i.18-26 in Scurlock, 2014: 518-519). Presumably a
large quantity of beer would have been needed if used alone as an emetic, while in mixtures
the other ingredients might have been relied upon to induce vomiting. In a Middle Babylonian
text from Emar, two different beer-based emetics are suggested for a feverish patient,
involving different plants, including a type of juniper in one, and types of thyme, mint, and
cress in the other (Tsukimoto, 1999, lines 16-18 and 19 in Scurlock, 2014: 420).45 Another
text provides various other beer-based emetics for fever, with many ingredients, including
dates (BM 78963.1-10 in Scurlock, 2014: 476.).46
Egyptians too had beer-based emetics. In the Ebers Papyrus, for lack of appetite a purge
is made with dates served in what seems to be stale beer (189b in Bardinet, 1995: 276, with
Nunn, 1996: 90). A recipe in the Brugsch Papyrus has beer poured onto hot stones and
inhaled in a straw and then vomited up (46 in Bardinet, 1995: 414-415, with Helck, 1971:
80). In the fifth century B.C., the Greek historian Herodotus said that for three successive
days each month Egyptians for health reasons would purge themselves by taking emetics and
enemas, though he did not identify what exact preparations they used (Histories 2.77.2 in
Strassler, 2007: 150). His near contemporary, the comic playwright Aristophanes, also spoke
of Egyptians taking purgatives, and a commentator on Aristophanes pointed out that beer was
so used (Peace 1254 and fr. 276 Kassel-Austin with Didymus Chalcenterus, fr. 14, no. 55
Schmidt in Scholia to Aristophanes’s Peace 1254 in Nelson, 2001: 292, and see 381).
Emetics were not used however, simply for general health among Egyptians, but also
specifically against snake bites. In a fourth century B.C. papyrus dealing with venomous
snake bites, garlic finely crushed and drunk in either ordinary beer or sweet beer is used as an
emetic, and also used as a spray around the house to repel snakes (Pap.Brookl. 47.218.48 +
85, 41, 42b, and 57 in Bardinet, 1995: 528-529 and 535). Interestingly, the Greek medical
writer Philumenus (from sometime before the fourth century A.D.) suggested serving garlic in
beer to throw up when bitten by an asp (On Poisonous Animals and their Remedies 16.8 in
Nelson, 2001: 309, copied by Aetius of Amida, On Medicine 13.22 in Nelson, 2001: 330).
This is the sole explicit example of beer used as an emetic (as opposed to a general purgative)
in a Greek source, and it is evidently indebted to Egyptian practice, in which garlic was
considered the great anti-venom (see Bardinet, 1995: 247). Other recipes in the Egyptian
papyrus include having salt added to the garlic and beer mixture or else using willow leaves,
salt, and garlic in sweet beer (Pap.Brookl. 47.218.48 + 85, 45a and 45c for salt and 46b for
sweet beer in Bardinet, 1995: 530-531). For large vipers one can use salt and beer with the

45
For cress and beer in an emetic, see also BAM 480.i.10-12 in Scurlock, 2014: 319.
46
For other examples of beery emetics, see Herrero, 1984: 92-93.
 Max Nelson 15

“image of Horus” plant or else garlic, willow leaves, and beer with the “image of Seth” plant
(47.218.48 + 85, 65a-b in Bardinet, 1995: 537.47 In these recipes with salt or plants
specifically 25 ros of the beer are called for, that is, about 0.35 litres, thus not a very large
amount (see n. 34 above for the measure). All of these prescriptions seem strange to modern
eyes since, on the one hand, snake venom itself could lead to vomiting and, on the other hand,
vomiting would not actually help against the effects of the snake venom, because the venom
would not be present in the vomitus. It seems that it was wrongly thought that the venom
could diffuse itself throughout the entirety of the body (Sauneron, 1989: 181, with 186
generally on the use of emetics in this papyrus). In practical terms, it has been suggested that
an emetic could distract the patient’s attention from the bite and make the patient feel like
something was being done (Nunn, 1996: 189).

Aperient

Beer was sometimes thought of as an aperient (or laxative). One Mesopotamian text
seems to suggest that a patient drink a type of thyme or a type of mint mixed in beer to ensure
defecation (BAM 159.iii.7 and 9 in Scurlock, 2014: 503 and 504). Beer, among other
vehicula, was also used in aperient potions by Egyptians.48 In the Babylonian Talmud (from
the third to fifth century A.D.) Egyptian beer, which is forbidden at Passover, is described as
including safflower and salt and is said to act as a laxative for a constipated person or as an
anti-laxative for someone with diarrhoea and it is also added that it is dangerous for an invalid
or pregnant woman (Pesaḥim 42b in Freedman, 1967: n.p.).

Diuretic

Beer is certainly more diuretic than water, because of its acids and polyphenols among
other components, and this could have made it useful in ancient medicine (Bamforth 2004:
146, and see Keller, 1958: 153). This use, however, does not seem to be explicitly found in
Mesopotamian medical texts. However, in one tablet it is recommended that one drink beer,
and not water, for a stone (DPS 18.40’-41’ in Scurlock, 2014: 176). It has been suggested that
beer was successfully employed in this case in either changing the urine acidity thus causing a
stone in the urethra to dissolve or in causing relaxation of muscle spasm from the anaesthetic
effect of alcohol (Scurlock and Andersen, 2005: 106).49 Alternatively, it has been argued that
the beer was simply used to pass (and not dissolve) a kidney stone stuck in one of the ureters
(Powell, 1993: 63-65). Modern studies have at least shown that beer consumption

47
See further 43a, 45b, 49b, 59, 70, 73, 75a, 85c, and 93a in Bardinet, 1995: 529, 530, 533, 535, 538, 539, 542, and
544. Some recipes substitute wine or water for beer (46e and 46g in Bardinet, 1995: 531). In other recipes a
beer mixture is not to be thrown up (54h and 65c in Bardinet, 1995: 535 and 537). Compare the plant drunk
crushed in beer against a scorpion sting at Pap.Tur. 31 +77, 3-5 in Bardinet, 1995: 478, with Helck, 1971: 78.
48
For example, Pap.Eb. 9, 13, 14, 15, 19, 24, and 29 in Bardinet, 1995: 252, 253, 254, and 255 (and see Pap.Eb. 25
in Bardinet, 1995: 254 for a mass chewed with beer), with Nunn, 1996: 158-160.
49
See also BAM 396.ii.19’-22’ in Geller, 2005: 37 for beer and other ingredients dissolving a stone. For
constriction of the urethra various types of plants can be drunk in beer at BAM 1.21-23 in Scurlock, 2014:
277-278.
16 To Your Health!

significantly reduces the risk of developing kidney stones (see Bamforth, 2004: 146 and
Walzl, 2009: 522-523).
Beer as a diuretic is mentioned in the earliest Egyptian medical texts. In a recipe for a
urinary tract disease in the Kahun Papyrus various ingredients are served crushed and boiled
in beer, with the beer perhaps helping to purge the tract (10 in Bardinet, 1995: 439, with
Strouhal, Vachala, and Vymazalová, 2014: 150).50 In another Egyptian recipe to ensure
normal urination from a papyrus from around 1700 B.C. beer mixed with the pith of a reed is
to be drunk; presumably the diuretic effect of beer was the key to the success of this
prescription (Pap.Ramess. III.A.30-31 in Bardinet, 1995: 468, with Strouhal, Vachala, and
Vymazalová, 2014: 222, and see 112).51 In the Ebers Papyrus various ingredients (including
marsh water and fresh dates) in beer are used to ensure normal urination in a patient (271 in
Bardinet, 1995: 292-293, with Nunn, 1996: 160). As seen above, the first century A.D.
herbalist Dioscorides noted that beer was a diuretic, but evidently he viewed this as a negative
trait, whereas in fact this could be quite useful depending upon a patient’s circumstances (see
Nelson, 2003: 106).

Antitussive

Beer was often used throughout antiquity against coughs. In one Mesopotamian text salt
was given first to a coughing patient to encourage vomiting and then honey with beer to stop
the coughing (BAM 548.i.12-13 in Scurlock, 2014: 468, with 465). Hot honey beer or simply
first quality beer with cress was also given for coughing (BAM 548.i.14-16 and iv.14’-15’,
respectively, in Scurlock, 2014: 468 and 469).52 Another recipe specifies that the ingredients
be mixed in old barley beer or wine (BM 78963.48-49 in Scurlock, 2014: 478). In the Ebers
Papyrus an unidentified plant is cooked in sweet beer and drunk for four days in a row to
suppress a cough (306 in Bardinet, 1995: 298, and see 315, 319, 321, and 322 in Bardinet,
1995: 299-300, with Helck, 1971: 78). The sweetness of the beer itself or of the added honey
could have been the factor which helped to relieve coughing (Nunn, 1996: 162). In the early
fifth century A.D. the Gallic medical writer Marcellus Empiricus recommended as a remedy
for coughing salt in hot beer (either cervesa or curmi, probably meaning wheat beer or barley
beer) before sleeping for three days (On Medicaments 16.33 in Nelson, 2001: 324). In this
case, unlike with the few other late antique authors who provide beer recipes, Marcellus was
probably not indebted to earlier Egyptian practice. Instead he could in fact have been
describing an independent Celtic medical tradition (as the Celtic beer terms he uses seem to
indicate).

50
See also Pap.Eb. 784 in Bardinet, 1995: 444 on pain during urination, with Strouhal, Vachala, and Vymazalová,
2014: 150.
51
A similar recipe is found in Pap.Eb. 272bis in Bardinet, 1995: 293, with Strouhal, Vachala, and Vymazalová,
2014: 116.
52
For honey and beer, see also AMT 25/3,4.12-16 in Thompson, 1934: 6 and AMT 80/2,7.2-12 in Thompson, 1934:
7. Compare the recipe for lung problems in which, after various other remedies, the patient should drink first
quality sweet date beer (BM 78963.36-39 in Scurlock, 2014: 477).
 Max Nelson 17

Anthelmintic

Beer could also have been thought of as a useful anthelmintic (or vermifuge). In
Mesopotamian medicine, to get rid of intestinal worms, ingredients were mixed in hot beer
(BAM 159.ii.20-48 in Scurlock, 2014: 497-498). Beer of various types (including sweet beer,
thick beer, and what may be strong beer) were also used to get rid of worms in Egyptian
medicine, as attested in both the Ebers Papyrus and the Brugsch Papyrus.53 Some two
millennia later Marcellus Empiricus spoke of using beer to soak a herbal suppository to expel
intestinal worms, perhaps again relating a Celtic practice (On Medicaments 28.13 in Nelson,
2001: 325, distorted in Cassius Felix, On Medicine 72 in Nelson, 2001: 326). As late as the
sixth century A.D. Aetius of Amida recommended drinking as much as two ounces of the
juice of madwort with one ounce of beer to expel worms (On Medicine 9.37, with 1.285 and
3.156, in Nelson, 2001: 329-330). Aetius may well have been preserving a much older recipe,
perhaps ultimately Egyptian, but it is significant that he retained the beer (rather than
substituting it for wine or another liquid more common in the Byzantium of his day) since it
may show that he thought that beer in particular was efficacious against worms. Modern
anecdotal evidence also seems to confirm beer’s power as an anthelmintic.54

Anaesthetic or Analgesic

It may be assumed that often beer was used not to cure an illness, but to relieve its
symptoms, such as pain, through its anaesthetic or analgesic properties (Keller, 1958: 153,
and see Bamforth, 2004: 45). At least beer was recommended by Egyptians to relieve pain in
childbirth (Strouhal, Vachala, and Vymazalová, 2014: 103 and 173). It may also have been
used in surgeries as a sort of mild sedative.55 The fermentation of dates with beer (if indeed
that was common among Egyptians) would have likely made it a higher strength alcohol
drink and hence a more potent sedative than ordinary table beer.

BEER IN EXTERNAL THERAPY

Beer was occasionally also used externally, to wash wounds and especially as part of a
mass which was placed with a bandage or compress on sore areas or broken bones as a
cataplasm (or plaster or poultice) or even as a more solid cast. Sometimes also beer was used
in baths and shampoos, lotions and creams, and fumigations.

53
Pap.Eb. 59, 60, 63, and 71 (beer), 55, 56, 73, 76, 81, 83, and 84 (sweet beer), 65 (thick beer), 72 and 74 (what
may be strong beer), and 79 (sweet beer and what might be strong beer) in Bardinet, 1995: 258-262 and
Pap.Brugsch 4 (beer), 2 and 3 (sweet beer), and 5 (what might be strong beer) in Bardinet, 1995: 409-410. See
Helck, 1971: 78 and Nunn, 1996: 68-73.
54
For example, White, 1842: 420 recorded how he knew a man who claimed that he often voided worms
“especially after drinking rather freely of beer”.
55
Helck, 1971: 81 assumed that beer was used in Egyptian medicine for its intoxicating properties. However,
Strouhal, Vachala, and Vymazalová, 2014: 89-90 point out that there is no evidence for the use of beer or wine
in ancient Egyptian surgery. For alcohol as the main Egyptian analgesic, see also Nunn, 1996: 158.
18 To Your Health!

Wound Washes, Cataplasms, and Casts

In two Sumerian tablets from Nippur dated to around 2000 B.C., which contain some of
the earliest known treatments, mention is made of beer with other ingredients poured over
wounds (see Figure 1) (CBS 14221, 1.4, 6, 7, and 8 and 3.1 in Civil, 1960: 63 and 64 and HS
1359 in Civil, 1961: 91, with Attinger, 2008: 10).56 This has been considered a very
efficacious wound wash (Majno, 1975: 48).57 In a later text beer alone is used to wash a
wound (BAM 156.25-31 in Scurlock, 2014: 431). Occasionally in later Akkadian texts
medicinal preparations were to be rubbed onto the body or applied as bandages (Herrero,
1984: 100-103, with various examples involving beer, and Böck, 2011: 699). Beer was often
used in cataplasms placed on the head or on the eyes. For a headache various plants and other
ingredients are crushed and mixed with first quality beer and vinegar and placed in a bandage
on the head for three days (BAM 156.32-39 in Scurlock, 2014: 332). In some recipes the
ingredients are decocted in beer before being placed in the bandage for the headache (BAM
11.9-11, 19-20, and 34-35 in Scurlock, 2014: 558-559).58 In other recipes for headaches
various ingredients are placed in beer, beer dregs, or beer wort in a bandage placed on the
patient’s shaved head.59 For sore eyes a dough is made with cress, cereal, and beer dregs and
used in a bandage on the eyes (BAM 156.48-49 in Scurlock, 2014: 332-333).60
Mesopotamians also placed on limbs or feet medicated casts in which various ingredients
were boiled in beer.61

Figure 1. Sumerian cuneiform tablet from Nippur (c. 2000 B.C.) in which beer is recommended as a
wound wash. By permission of the University of Pennsylvania Museum of Archaeology and
Anthropology (CBS 14221).

56
HS stands for Hilprecht Sammlung, Jena.
57
For various other washes, see Herrero, 1984: 93-97. In Greco-Roman medicine water, vinegar, and wine were
most often used on wounds (see Nelson, 2011: 29, n. 80).
58
However, see BAM 11.28-29 in Scurlock 2014: 559, where decoction is not mentioned.
59
BAM 480.i.6, 8-9, 30-31, ii.29, 45, 50, 59, iii.10-15, 20 (beer), i.49’, 57’-58’, ii.1, iii.17-19 (beer dregs), and
i.61’, ii.21-22 (beer wort) in Scurlock, 2014: 319-324 and 425-426. See also BAM 156.32-39 in Scurlock,
2014: 332.
60
Also more fragmentarily at BAM 159.v.1-2 in Scurlock, 2014: 367. A mixture of cress and beer for the eyes is
also found at BM 54641 + 54826obv.13’ and rev. 8 in Fincke, 2009: 90 and 91.
61
BAM 124.ii.51-54 (for a broken limb) and iii.18-19 (for a foot, perhaps strained) in Scurlock, 2014: 458, and see
Scurlock and Andersen, 2005: 248.
 Max Nelson 19

The Egyptian papyri also recommend cataplasms made with different types of beer (or,
quite often, beer dregs) among other ingredients for application on wounded or achy areas of
the body.62 In one recipe a number of ingredients in sweet beer are placed as a mass on the
afflicted area (Pap.Eb. 97 in Bardinet, 1995: 264). However, unlike in the Mesopotamian
texts, beer is not at all used in Egyptian eye remedies (see Pap.Eb. 336-431 in Bardinet, 1995:
302-314). Two Greco-Roman sources preserve prescriptions similar to the Egyptian ones. In
an anonymous compilation of remedies dating to the fourth century A.D. beer dregs are to be
mixed with leaves of danewort and tied in linen to cure scrofulous tumours (Pseudo-Pliny, On
Medicine 3.6 in Nelson, 2001: 317) and in the sixth century A.D. Aetius of Amida
recommended applying crushed mustard with beer to an arrow wound (On Medicine 13.23 in
Nelson 2001: 330).
It has sometimes been thought that an antiseptic or antibacterial property of beer made
such applications effective in terms of sterilizing a wound and preventing infections.63
Certainly it would not have been the antiseptic effect of the alcohol contained in the beer
which would have made it effective, since the alcohol content of ancient beer would have
probably been relatively low (as pointed out in n. 22 above).64 However, it has been thought
that other substances in beer could have made it antiseptic (Majno, 1975: 48). Some have
claimed that tetracycline, which has been detected in ancient human remains in Egypt and
other places, could be such a substance (Nelson, Dinardo, Hochberg, and Armelagos, 2010,
with further references). It remains unclear, however, to what extent beer was deliberately
used because of any possible sterilizing effect (as pointed out in Jones-Lewis, 2016: 402). In
the Smith Papyrus (from around 1550 B.C.) which deals with treatments for external trauma
to the upper body, beer is never used to treat wounds, but other substances are found,
including honey, which is known to be an effective antibacterial agent.65

Baths and Shampoos

Among Mesopotamians patients were sometimes bathed in special liquid preparations

(Herrero, 1984: 97-98 and Böck, 2011: 699). In a Middle Babylonian text from Emar, a
feverish patient is to bathe in a mixture of incense and beer for seven days (Tsukimoto, 1999,
ll. 16-18 in Scurlock, 2014: 420). In Nabū-le’ū’s text, a patient is bathed in beer in which a
plant has been boiled (BAM 1.iii.32 in Herrero, 1984: 20.).66 In other texts from Nineveh,

62
Pap.Eb. 130 (for a wound), 200 (for back pain), 208 and 213 (for an obstruction), 605 (for knee pain), 675-676
(for stiffness), 757bis (for pain in the right side), and 807 (for pain when delivering a baby) in Bardinet, 1995:
268, 279-280, 282-283, 337, 346, 356, and 446 and Pap.Hearst 243 (for a hippopotamus bite) in Bardinet,
1995: 406. For beer dregs, see Pap.Eb. 541, 564, 568, 608, 609, 639, 662, 683, and 690 in Bardinet, 1995:
328, 332, 337-338, 342, 345, 347, and 348 and Pap.Hearst 137 in Bardinet, 1995: 393. See Helck, 1971: 81.
63
For example, Shai and Maibach, 2005: 20 believe that beer used in plasters applied to wounds and skin lesions
among Mesopotamians did have some beneficial effect as an antiseptic. Scurlock and Andersen, 2005: 16
speak generally of beer helping to eliminate bacterial contaminants when drunk. See also Walzl, 2009: 524 on
the anti-bacterial effects of beer.
64
See Ali, Dolan, Fendler, Larson, 2001 on the effectiveness of alcohol as an antisepsis in various circumstances,
making it clear that the alcohol content needed would only be possible through distillation, and not the mere
fermentation of cereals.
65
For honey, see Pap.Smith 1, 2, 3, 7, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 23, 25, 26, 27, 28, 30, 32, 34, 35, 36, 37,
38, 40, 42, 43, and 47 in Allen, 2005: 72, 73, 77, 80, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, and 105, with
Estes, 1989: 69-71, Nunn, 1996: 148 (with further references), Ritner, 2001: 354, and Allen, 2005: 42.
66
Compare AMT 22/2.1-28 in Geller, 2005: 259 and 261.
20 To Your Health!

beer is to be poured onto the patient’s feet (AMT 69/7.3-7 in Thompson, 1937: 273 and AMT
70/3.5-6 in Thompson, 1937: 278 and Herrero, 1984: 98). Mesopotamians also had medicated
shampoos, including one made by grinding various ingredients in beer (BAM 33.9-14 in
Scurlock, 2014: 430). However, there does not seem to be any Egyptian or later ancient
evidence for such beer baths and shampoos.67

Lotions and Creams

Among Mesopotamians, lotions were made from various ingredients, including beer
(Herrero, 1984: 97-98 and Böck, 2011: 699). One Babylonian text recommends adding
various ingredients into barley beer, which is then reduced and made into a lotion to be
applied on the rectum and also suggests adding various ingredients in old beer to treat lice and
itching (BM 42576 + 43546 + 43589, 1-10 and 18-21 in Geller, 2005: 265). In the Ebers
Papyrus it is suggested that beer foam with other ingredients be applied to the skin to treat
different illnesses, including in women.68 In the first century A.D., Pliny the Elder stated that
the froth of beer nourishes the skin in the faces of women (Natural History 22.82.164 in
Nelson, 2001: 302). He may here be referring to a practice of Egyptians, whom he mentions
earlier in the same passage. In any case, it has been suggested that Pliny’s claim is true and
that beer in fact is good for the skin because it contains vitamins of the B complex (B2, B5,
and B60 (Walzl, 2009: 525-526).

Fumigations

In the Brugsch Papyrus, fumigation with dregs of sweet beer is recommended for a snake
or scorpion bite (79 in Bardinet, 1995: 419).69 Mesopotamian texts also speak of fumigations,
but none seem to mention the use of beer dregs or other beer-based products (Herrero, 1984:
109-110 and Böck, 2011: 700).

CONCLUSION
From about 2000 B.C. to A.D. 500, estimations of the salubriousness of beer, even when
drunk in moderation, as well as the importance of beer in medicine diminished gradually over
time and from East to West (from areas where beer was an everyday beverage to areas where
wine was preeminent). Beer went from being a universal beverage and medicament to an
almost entirely shunned substance among mainstream physicians.

67
It is said in the Welsh laws traditionally attributed to the tenth century A.D. King Hywel the Good that the king
should receive twice a year a vat of mead large enough that he could bathe in it, or else two vats of honey beer
or four of plain beer (Dull Dyved 2.19.3-4 in Owen, 1841: 532, cited in Nelson, 2014b: 18). It is unclear,
however, if the king would actually bathe in the beverage, and whether or not that would be done for health
reasons.
68
772 (with red ochre for ear problems) and 794 (with dried feces to counteract the displacement of the placenta in
women) in Bardinet, 1995: 359 and 445. See also Pap.Eb. 548 in Bardinet, 1995: 329.
69
On the fumigations in this papyrus, see Bardinet, 1995: 216-217.
 Max Nelson 21

Mesopotamians, for whom beer was an everyday beverage, had hardly anything to say
about beer’s harmfulness, except if it was contaminated or drunk to excess, and they even
tended to blame superhuman beings on what was really alcohol abuse. They used beer as their
main base for ingesting medicinal plants, as well as macerating and decocting them, and
relied on some of beer’s own inherent qualities, for example as an emetic.
Egyptians also used beer as an everyday beverage, and tended to view beer mainly only
in a positive light, yet they did consider moderate beer drinking to be occasionally
contraindicated. They also tended to use water more often than beer to administer medication
and to rely on honey rather than beer for wound applications, but they made more use of
sweet beer in medicine than the Mesopotamians did and they also apparently considered beer
on its own to be a useful purgative.
Greek physicians from the classical period ignored beer entirely while their
contemporaries, for whom beer could be thought of as an effeminate and enfeebling foreign
beverage made of decomposed cereals, clearly disparaged its wholesomeness. Roman era
physicians in general tended to view beer as neither a healthful table beverage nor as a useful
base for medication, and in fact they believed it could actively harm various parts of the body
even if not drunk to excess. However, some medical authorities from the second century A.D.
on did provide recipes against certain illnesses and disorders which included the use of beer,
apparently transmitting prescriptions from earlier Egyptian medicine. However, at least one
text may be a record of medical practices of the Celts, who created various herb-infused
beers, including hopped beer.
Finally, after the fall of the vinocentric Roman Empire, at a time when beer was hardly
being drunk anymore in the Near East and Egypt, in Europe beer again became more
prominent as a staple of the diet and in medicine. In fact during the early Middle Ages the
Greco-Roman notions antithetical to beer were for the most part ignored or forgotten, as beer
came to be considered beneficial and to be used again to administer various types of
medication and even as a general tonic.

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In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485-704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.

Chapter 2

BEER PRODUCTION WITH ENCAPSULATED

YEAST CELLS

Vesela Shopska1, Rositsa Denkova2 and Georgi Kostov2

1
Department of Wine and Beer Technology,
University of Food Technologies, Plovdiv, Bulgaria
2
Department of Biochemistry and Molecular Biology,
University of Food Technologies, Plovdiv, Bulgaria

ABSTRACT
In recent years, the immobilization of yeast cells and their application in brewing has
been the subject of research due to their main advantages: enhanced fermentation
productivity, reduced fermentation time, easier implementation of continuous operation,
improved cell stability, facilitated cell recovery and biomass reuse.
The purpose of the present chapter is to present the possibilities of beer fermentation
with encapsulated yeast cells. The chapter begins with an overview of the application of
immobilized systems in beer production and the immobilization effect on yeast
metabolism. The data were used for the choice of a method and carriers for cell
immobilization – encapsulation in an alginate/chitosan matrix. The influence of different
parameters on the microcapsule formation and the effect of the fermentation conditions
on the microcapsule stability were also investigated.
Laboratory-scale beer fermentations with immobilized top- and bottom-fermenting
yeast strains were carried out. The fermentation dynamics data were used for the kinetic
model development and the description of the immobilization effect on beer flavor.
The results obtained were used for the optimization of the fermentation conditions by
applying the planned experiment methods. Furthermore, the effect of some fermentation
parameters on the main fermentation and maturation time was studied.

Keywords: encapsulated cells, beer fermentation, liquid core capsules, flavor formation
28 Vesela Shopska, Rositsa Denkova and Georgi Kostov

INTRODUCTION
Beer is a product that has been manufactured and consumed with a remarkable passion
for centuries. The product features unique flavor and aroma. Its moderate consumption has
beneficial effects on human health – a fact that cannot be denied even by the most fervent
critics of alcohol consumption (Kunze, 2004; Kostov 2015; Naydenova, 2014a).
Beer production is a complex process consisting of a series of operations subjected to
chemical, physical, biochemical and thermodynamic processes. Knowledge of these processes
is the foundation for the development of a high quality beverage. One of the most important
stages in beer production is alcoholic fermentation. In the fermentation process, the wort is
transformed into beer which results not only in ethanol accumulation and consumption of
fermentable sugars, but also in the accumulation of a number of by-products of the vital
activity of yeast: esters, higher alcohols, aldehydes, vicinal diketones, organic acids, lipids,
etc. They impart to the resulting beer its specific flavor known to every consumer worldwide
(Kunze, 2004; Kostov, 2015; Naydenova, 2014a).
Familiarity with the mechanism and kinetics of alcoholic fermentation in beer production
is the basis for developing new methods of fermentation. Recently, there has been growing
interest in methods for continuous fermentation of beer characterized by a number of
advantages: high degree of substrate conversion, ability for more efficient control of the
fermentation process, possibility of obtaining beer with a balanced organoleptic profile, etc.
Different approaches aimed at the shortening of the fermentation process duration are used:
bioreactor design optimization, immobilized biocatalyst introduction into beer manufacture,
and optimization of fermentation control processes. The use of immobilized cell systems both
in batch and in continuous fermentation offers the possibility of reducing the duration of beer
fermentation and maturation from one month to several days. The cell immobilization
methods ensure the retention of cells within the apparatus and high viable cell concentrations,
which is a prerequisite for shorter fermentation time. Thus the apparatus volume is reduced
and productivity increases at the expense of increased biocatalyst concentration in the
apparatus (Kostov, 2015; Naydenova, 2014a).

1. Methods and Matrices for Immobilization

Immobilized cells are physically confined or localized in a specific space, retaining their
catalytic activity and, if possible or even necessary, their viability, therefore they can be used
repeatedly and continuously (Godia et al., 1987). The immobilization process can be
temporary or irreversible (Kostov, 2007; Sinicin et al., 1991).
The application of immobilized systems in the fermentation process is characterized by
the following advantages: higher cell concentration in the working volume of the reactor
leading to increased productivity of the processes; smaller bioreactor size and, in the case of
continuous processes, decreased reaction time; easy biomass separation and regeneration,
which allows for the re-use of the same biocatalyst for an extended period of time; possibility
of application in both periodic and continuous alcoholic fermentation (Boulton and Quain,
2001; Hayes et al., 1991a; Pilkington et al., 1998). These processes have some disadvantages:
limited mass transfer because of diffusional resistances, possibility of matrix destruction at
high rates of the fermentation process and/or formation of gaseous metabolites, change in the
 Beer Production with Encapsulated Yeast Cells 29

physiological state of the immobilized culture, as well as in the growth rate and the overall
stoichiometry of the reactions in the immobilized system (Mensour et al., 1996).
According to Kourkoutas et al., 2004; Leenen et al., 1996, Margaritis and Kilonzo,
2005 and Nedovic et al., 2005, the immobilization matrix should be characterized by the
advantages presented in Table 1.
The main immobilization methods are presented on Figure 1. They can be divided into
four main groups (Kourkoutas et al., 2004).

Table 1. Features for the choice of an immobilization matrix

Feature Criterion
Solubility Low; inert
Biodegradability Low
Stability High; Good mechanical and rheological properties
Diffusion of substances High
Possible; It is necessary for the microorganisms to survive
Growth
during the immobilization procedure
Immobilization procedure Simple and feasible on an industrial scale
Adsorption of other Minimal
microorganisms
Price Low
Not to change the organoleptic profile of the resulting
Organoleptic profile
beverage

A. Cell Immobilization on Solid Carrier Surfaces (Figure 1a)

With this technique, cells attach to the surface of a solid matrix spontaneously or
as a result of external impact. Nonporous carriers which allow cell adsorption only on the
outer surface and prevent the penetration of microorganisms inside the matrix are used for
immobilization (Hayes et al., 1991a; Pilkington et al., 1998). The cell adsorption onto the
matrix is carried out through various mechanisms: formation of covalent and ionic bonds,
electrostatic interactions, Van der Waals forces, flocculation and coagulation, hydrophobic
interactions, and biospecific adsorption (Sinicin et al., 1991; Nedovic et al., 2005). Different
factors affect the absorption process: matrix type: size, shape, porosity, surface charge;
microorganism type: strain, age, surface charge; medium conditions: pH, temperature, flow
rate during immobilization, and ionic load (Kostov, 2007; Sinicin et al., 1991).
The main advantages of this type of immobilization are the facilitated mass transfer and
the carrier regeneration. The disadvantage is the lesser biomass loading and the sensitivity to
shear stresses, which increases the possibility of cell leakage in the medium, as well as the
autolysis of the cells and the change in their pH optimum (Pilkington et al., 1998; Strehaiano
et al., 2006; Krastanov, 1995).
Polyurethane and polyvinyl foam, stainless steel, ceramics, glass, wood and sawdust
cubes, etc. are used as carriers in this immobilization method (Furuta et al., 1997; Kishimoto
et al, 2002; Liu et al, 1998; Bekers et al., 2001; Willaert, 2000; Yamauchi et al., 1995;
Nedovic et al., 2005; Verbelen et al., 2010).
30 Vesela Shopska, Rositsa Denkova and Georgi Kostov

B. Immobilization by Entrapment within a Gel (Figure 1B)

Immobilization is based on entrapment of the cells in polymer matrices (e.g.,
polysaccharides, synthetic polymers and proteins) and the subsequent “gelation” of the
polymer to form a solid matrix with minimal loss of viability and maximum cell load. The
main disadvantages are the limited mechanical stability and in some cases, the inadequate
mass transfer (Boulton and Quain, 2001; Kourkoutas et al., 2004; Nedovic et al., 2005).
Collagen, carrageenan, epoxy resins, alginates, etc., in which the process of binding is carried
out by precipitation, polymerization, polycondensation and ionic bonds, are used as
immobilization matrices (Krastanov, 1995; Margaritis and Kilonzo, 2005; Naydenova, 2014a;
Kostov, 2007; Kostov, 2015).

Figure 1. Basic methods of microbial cell immobilization; (Kourkoutas et al., 2004).

C. Immobilization within a Pretreated Porous Matrix (Figure 1A)

Immobilization is achieved by attaching the microorganism cells to the inner surfaces,
flocculation and retention in pockets inside the material. In comparison with gels, porous
matrices are characterized by increased mechanical stability and higher resistance to
compression. Porous glass, ceramics, kieselguhr and others are used as matrix materials in
performing this method (Baron and Willaert, 1996; Nedovic et al., 2005; Willaert, 2000;
Verbelen et al., 2010).

D. Immobilization by Flocculation (Figure 1C)

Immobilization by flocculation is a simple method which is based on the formation of a
large numbers of cells (flocs) that may be formed naturally or artificially by the addition of
flocculating agents (Nedovic et al., 2005). Factors like pH, ionic strength, or age of the cell
culture influence the flocculation ability of the cells. The resulting preparations are either
single cells with stabilized enzyme systems, or cell aggregates (Krastanov, 1995; Sinicin et
al., 1991). The main advantages of this method are the absence of stress effects on cells and
 Beer Production with Encapsulated Yeast Cells 31

possibilities of obtaining a significant biomass concentration in the reactor (Sinicin et al.,

1991). The main disadvantages are the difficult control over the flocculation, the increased
viscosity and the high risk of cell washout (Obradovic et al., 2004; Willaert, 2000).

E. Containment between Microporous Membranes (Figure 1D)

Retention behind a membrane can be performed in 2 ways: by using pre-made
membranes or by forming a membrane around the cells (Kourkoutas et al., 2004; Willaert,
2000). The method is very “soft,” but at a great cell load, diffusion limitations (Lebeau et al.,
1998) and destruction of the membrane due to cell growth (Gryta, 2002) are possible.
Polymer membranes with proven mechanical strength are most common in the first
method. The most frequently used membranes are sheet membranes or “hollow fiber”
membranes. This method is often associated with limitations related to the formation of
gaseous metabolites, high diffusion limitations and membrane destruction (Nedovic et al.,
2005; Hayes et al., 1991a; Margaritis and Kilonzo, 2005).
In microencapsulation the membrane is formed around a natural polymer core by
chemical reactions. The porosity of this membrane depends on the coating polymer (Guisely,
1989, Nedovic and Willaert, 2004, 2005). Microencapsulation is widely applied in various
fields because of the following advantages: high cell density, high degree of retention of cell
viability and high biosynthetic rate of the desired metabolic products (Sweta Rathore et al.,
2013). It has been found that immobilized cells are very well protected against adverse
environmental conditions in all derived preparations (Annan et al., 2008; Canh Le-Tien
et al., 2004; Chávari et al., 2010; Krasaekoopt et al., 2006; Mokarram et al., 2009; Pimentel-
González et al., 2009). Examples are polyelectrolyte complexes of chitosan and alginate
formed by the binding of the negatively charged carboxyl groups of the alginate to the
positively charged amino groups of the chitosan (Murata et al., 1993; Takahashi, 1987;
Takahashi et al., 1990; Wen-tao et al., 2005). The process of forming a membrane is
influenced by different factors (Gaserod et al., 1998; Gaserod et al., 1999). The impact of
some of them will be presented in the present chapter.

2. Yeast Metabolism and Metabolic Changes during Immobilization

The immobilization of yeast cells mainly affects yeast metabolism during alcohol
fermentation.

A. Utilization of Carbohydrates, Nitrogenous Substances and Lipids, and Its Changes

as a Result of Immobilization
Wort carbohydrates are the main sources used for structural and energy exchange
by microbial cells. Around 2% of them are used for biomass synthesis and the rest
undergo fermentation. Wort carbohydrates are consumed and fermented in the following
order: monosaccharides (glucose and fructose), disaccharides (sucrose and maltose), and
trisaccharides (maltotriose). Due to catabolite repression, the consumption of disaccharides
and trisaccharides begins when the glucose concentration decreases to 0.2-0.6%. Small
amounts of maltotriose are used for glycogen and trehalose synthesis (Popova, 1992; Boulton
and Quain, 2001; Briggs et al., 2004; Kunze, 2004; Russell, 2006; Tange, 2009).
32 Vesela Shopska, Rositsa Denkova and Georgi Kostov

The amino acids assimilated by yeasts are used for synthesis of proteins and new cells.
They are metabolized in the following order (Tange, 2009):

 Group A amino acids. They are assimilated quickly: glutamine, glutamic acid,
arginine, asparagine, aspartic acid, lysine, serine and threonine;
 Group B amino acids. They are assimilated immediately after Group A amino acids:
valine, methionine, leucine, isoleucine and histidine;
 Group C amino acids. They are assimilated very slowly: glycine, phenylalanine,
tyrosine, tryptophan and alanine;
 Group D amino acids. They remain almost unutilized: proline.

Valine is required for yeast growth but it can be utilized only after Group A amino acids.
Therefore the cell begins to synthesize valine, thereby forming α-acetolactate, which is a
precursor for diacetyl synthesis. Synthesis of precursors of aldehydes, higher alcohols and
esters is also affected by the order of utilization of amino acids (Tange, 2009).
Wort lipids are directly involved in cell structures or used in other anabolic and catabolic
pathways as intermediate metabolites or regulators. Under anaerobic conditions, beer yeasts
are auxotrophic with regard to sterols and unsaturated fatty acids. Yeasts can assimilate
exogenous sterols but only under anaerobic conditions, when their synthesis in the cell is
completed (Briggs et al., 2004).
A number of authors have discussed the reasons for the changed metabolism of
immobilized cells. It is believed that the factors influencing these changes are limited mass
transfer, surface tension and osmotic pressure, reduced water activity, interactions between
cells, altered membrane permeability and composition of the nutrient medium (Melzoch et al.,
1994; Norton and D'Amore, 1994; Walsh and Malone, 1995).
The change in the cell morphology and physiology °is due not only to the interaction
between the cells and the medium, but also to the physicochemical properties of the carrier
(Rouxhet and Mozes, 1990). Comparative studies on immobilized and free cells showed
increased amounts of structural and reserve polysaccharides, modified growth rate, increased
substrate metabolism and, hence, increased product yield (Norton and D'Amore, 1994).
In immobilization in alginate gel, yeasts propagate mainly along the periphery of the core
while the quantity inside remains constant (Martynenko and Gracheva, 2003). Differences
between the size and shape of cells immobilized in alginate beads and free cells are attributed
to the limited space for yeast growth in the matrix (Melzoch et al., 1994). The slower growth
of yeasts immobilized in alginate beads leads to changes in the flavor of the final product
(Ryder and Masschelein, 1985).
Increased activity of certain enzymes is observed during immobilization, which
presupposes a faster fermentation process (Strehaiano et al., 2006). The immobilized cells
contain more DNA and fatty acids and have a different composition and structure of the cell
walls in comparison with the free cells (Brányik et al., 2008; Fumi et al., 1994). The changes
in the cell wall composition influence the membrane permeability and consequently the
absorption of sugars and amino acids (Branyik et al., 2005).
The immobilized yeasts show increased productivity, the extent of the influence being
dependent on the immobilization method (Hayes et al., 1991a; Hayes et al., 1991b; Navarro
and Durand, 1977).
 Beer Production with Encapsulated Yeast Cells 33

The immobilization process affects the pH optimum of yeast cells. A decrease in the
intracellular pH, which causes increased enzyme activity and accelerated glycolysis and
productivity of the process, is observed with some methods. The biosynthesis of reserve
carbohydrates is also accelerated under these conditions (Galazzo and Bailey, 1990). In this
situation, there are some changes in the amount of accumulated esters due to inhibition of the
acetic acid synthesis (Domeny et al., 1999; Galazzo and Bailey, 1990).
In yeast cell immobilization in alginate, the cells on the surface of the alginate beads do
not metabolize maltose because of the high glucose concentration in the outer layer of the
matrix. In contrast, the maltose intakein the cells located in the core of the alginate beads is
not inhibited (Willaert, 1999).
The immobilized yeast cells show higher resistance to ethanol than free yeast cells. The
reason is the increased amount of saturated fatty acids in immobilized cells and the protective
effect of the immobilization matrix (Hilge-Rotmann and Rehm, 1991; Norton et al., 1995;
Ciesarova et al., 1998; Shen et al., 2003).

B. Accumulation of Secondary Metabolites

By-products such as carbonyl compounds, higher alcohols, esters, organic acids and
sulfur-containing compounds which affect the beer quality are also formed during the
fermentation process (Boulton and Quain, 2001; Kunze, 2004).

Carbonyl Compounds
About 200 carbonyl compounds are found in beer. Aldehydes and vicinal diketones are
essential to beer flavor and aroma.
The majority of aldehydes are intermediates in the synthesis of higher alcohols from
α-keto acids in yeast cells, while the other part of aldehydes come directly from the
wort. Exogenous aldehydes form complexes with sulfur dioxide and the complexes formed
cannot be reduced by yeast enzymes (Briggs et al., 2004). Acetaldehyde has the highest
concentration in beer. This is of particular interest because of the fact that it is an ethanol
precursor. It is formed mainly at the beginning of the main fermentation as a result of the
pyruvate decarboxylation or ethanol oxidation by yeasts, then its synthesis slows down and its
reduction is accelerated (Popova, 1992; Kabzev and Ignatov, 2011; Russell, 2006). It forms
the "green" flavor and aroma of young beer. High temperatures, increased oxygen amount
and biomass inoculum as well as the poor physiological condition of yeasts are responsible
for the higher acetaldehyde amount formed (Geiger and Piendl, 1976; Kunze, 2004).
2,3-butanedione (diacetyl) and 2,3-pentanedione are the vicinal diketones important for
beer quality. Vicinal diketones are an indirect result of yeast metabolism. Their precursors –
α-acetohydroxy acids – are intermediate metabolites of the biosynthetic pathway of
isoleucine, leucine and valine. At the beginning of the main fermentation, part of the
intracellular α-acetohydroxy acids are released in the fermentation medium where they are
converted to diacetyl and 2,3-pentanedione by oxidative decarboxylation. This is the rate
limiting step in the formation of vicinal diketones. The formation and reduction of vicinal
diketones is affected by the following major factors: the yeast strain, the aeration, the amino
acid content in the wort, the temperature, the pH, etc. (Boulton and Quain, 2001).
The amount and type of amino acids in wort are important parameters that regulate the
synthesis of α-acetohydroxy acids. Barton and Slaughter, 1992 found that wort with a high
valine and isoleucine content inhibited the formation of vicinal diketones. Alanine, threonine,
34 Vesela Shopska, Rositsa Denkova and Georgi Kostov

glutamate, and NH4+ also inhibit the action of the α-acetohydroxy acid synthetase (Barton and
Slaughter, 1992). Increased temperature, increased biomass and faster stirring rate of the
fermentation medium accelerate the reduction of diacetyl and 2,3-pentanedione (Popova,
1992; Willaert, 2007).
In most cases, the amount of diacetyl produced by immobilized systems is higher than
that produced by free cells (Branyik et al., 2008; Kronlof et al., 1989; Ryder and Masschelein,
1985; Van De Winkel et al., 1993). This can be explained by several factors: the increased
activity of the acetohydroxy acid synthetase which leads to an increased α-acetolactate
concentration; the changes in the amino acid metabolism caused by the immobilization
technique used; the rapid utilization of amino acids by yeasts due to their rapid growth
resulting in a reduction of the valine amount and activation of the anabolic pathway of amino
acid formation; the continuous intake of Group A amino acids which inhibit the intake of
valine; the short stay in the bioreactor, which means insufficient time for the oxidative
decarboxylation of α-acetolactate to diacetyl and thus incomplete reduction of diacetyl by
yeasts (Branyik et al., 2008; Dufour and Devreux, 1986; Inoue, 1995; Kronlof et al., 1989;
Okabe et al., 1994; Shindo et al., 1994). Optimization of the α-amino nitrogen and valine
content in the wort and increase in the concentration of immobilized cells and their residence
time in the reactor are also used in order to reduce the diacetyl concentration (Branyik et al.,
2008; Pajunen et al., 2001; Petersen et al., 2004; Shindo et al., 1994).

Higher Alcohols
More than 40 higher alcohols are found in beer (Engan, 1981). The beer organoleptic
profile is influenced mainly by aliphatic alcohols: n-propanol, isobutanol, 2-methylbutanol
(amyl alcohol) and 3-methylbutanol (isoamyl alcohol), and the aromatic alcohol 2-
phenylethanol. Aliphatic alcohols enhance the alcoholic flavor and cause the warming effect
of beer, and the aromatic alcohol lends a flower aroma to beer (Meilgaard, 1974). 90% of the
higher alcohols are synthesized during the main fermentation, and the rest in the maturation
process (Tange, 2009). Higher alcohols also affect flavor because they are precursors for ester
synthesis (Boulton and Quain, 2001).
Higher alcohols are synthesized from α-keto acids, which in turn are obtained by 2
metabolic pathways (Briggs et al., 2004; Russell, 2006): a catabolite pathway for their
synthesis through deamination of wort amino acids by yeasts, and an anabolic pathway along
which they are synthesized from wort carbohydrates, i.e., they are obtained from pyruvate or
acetyl-Co A as part of the biosynthetic pathway of amino acids.
The choice of a pathway depends on the amino acid content in the wort and on the higher
alcohol type. For example, n-propanol is a product of the anabolic pathway since there is no
corresponding amino acid. The catabolic pathway is carried out when the amino acid content
is higher and the higher alcohol chain is longer (Chen, 1978). The formation of higher
alcohols depends on the yeast strain, the amino acid composition of the wort and the
fermentation conditions. Wort rich in valine, leucine and isoleucine induces the formation of
isobutanol, amyl alcohol and isoamyl alcohol. Wort poor in amino acids also leads to an
increased amount of higher alcohols due to stimulation of the catabolic pathway (Popova,
1992; Boulton and Quain, 2001; Szlavko, 1974; Sablayrolles and Ball, 1995). Increased
amounts of nutrients (amino acids, lipids and zinc), aeration, agitation and increased
temperature also lead to an increased amount of higher alcohols (Landaud et al., 2001).
 Beer Production with Encapsulated Yeast Cells 35

Different higher alcohols amount are formed as a result of fermentation with immobilized
or free cells. Reduced formation of higher alcohols due to the reduced α-amino nitrogen
consumption is observed in cell immobilization in a gel (Domeny et al., 1998; Smogrovicova
and Domeny, 1999), while in systems with a greater degree of α-amino nitrogen metabolism
(immobilization through adsorption), the amount of higher alcohols is higher and comparable
to that produced by free cells (Kronlof et al., 1989; Shen et al., 2003). The reduced production
of higher alcohols by immobilized systems is due to the limited cell growth (Willaert and
Nedovic, 2006). Generally, the yeast strain is more important than the immobilization method
(Virkajarvi and Pohjala, 2000).
The higher alcohol amount is essential for ester synthesis, hence different control
strategies are used. The stimulation of immobilized yeast growth by increasing the dissolved
oxygen concentration and the temperature leads to the formation of more fusel alcohol
quantities (Branyik et al., 2004a). High concentrations of higher alcohols can be reduced by
limiting yeast growth and / or α-amino nitrogen consumption through regulating the CO2
concentration in the medium (Shen et al., 2004).

Esters
About 100 esters are found in beer (Engan, 1981). They are characterized by low
threshold of sensation and pleasant fruity-flowery fragrance. They are divided into acetic acid
esters: ethyl acetate (fruity / solvent), isoamyl acetate (banana), and 2-phenylacetate (roses /
copper / apple); fatty acid esters with a chain length of C6-C10: ethylcaproate (apple with an
anise note) and ethylcaprylate (apple). The amount of ethyl esters is higher since ethanol has
the highest concentration in beer (Russell, 2006; Tange, 2009).
60% of the esters are formed during the exponential growth phase of yeasts (Willaert and
Nedovic, 2006). Alcohols and carboxylic acids activated by coenzyme A, as well as the
presence of the acyltransferase and ester synthetase are needed for the synthesis of esters. The
biosynthesis of esters is related to lipid biosynthesis since no esters are formed during the
formation of fatty acids and lipids. The reason is that acetyl CoA is used in the synthesis of
fatty acids and lipids, but it is also required for the synthesis of esters. Ester biosynthesis is
affected by the yeast strain, the amino acid composition of the wort and the fermentation
conditions: biomass, oxygen content, and fermentation temperature. The amino acid
composition influences indirectly the formation of higher alcohols. The ester content is
directly proportional to the amount of carbohydrates in the wort, but it must be recognized
that different carbohydrates lead to the formation of different amounts of esters, i.e., less
esters are formed from maltose than from glucose and fructose. Zn2+ stimulates the synthesis
of higher alcohols, hence the synthesis of the corresponding esters, while oxygen and lipids
inhibit ester synthesis. Increased biomass amount and increased temperature result in an
increase in the ester amount, while stirring and higher pressure inhibit their formation
(Popova, 1992; Saerens et al., 2008; Dufour et al., 2003; Younis and Stewart, 1998).
The use of immobilized yeasts in beer fermentation results in obtaining different amounts
of esters. In some cases, the use of immobilized systems leads to an increased ester amount in
comparison to the traditional fermentation with free cells (Andries et al., 1995; Kronlof et al.,
1989), but the overall trend is a reduction in the amount of esters (Branyik et al., 2008;
Verbelen et al., 2010). The limited mass transfer is the reason for the low oxygen
concentration in the immobilization matrices and, hence, the reduced cell growth. Therefore
yeasts use acetyl CoA for the synthesis of esters instead of fatty acids (Willaert and Nedovic,
36 Vesela Shopska, Rositsa Denkova and Georgi Kostov

2006). High concentrations of oxygen under continuous aeration of the wort in the reactor
lead to reduced ester synthesis (Wackerbauer et al., 2003). In immobilization by adsorption,
the amount of esters is comparable to their amount produced by free cells, whereas some
differences are observed in the entrapment in a matrix (Smogrovicova and Domeny, 1999).
The carrier-yeast strain combination is essential for the formation of esters (Virkajarvi and
Pohjala, 2000).

Organic Acids
About 110 organic acids are found in the composition of beer. A significant number of
them are formed during fermentation (acetic, pyruvic, citric, malic, etc.) and lead to a
decrease in pH. Their transition in beer is due to the lack of a mechanism for their further
oxidation. Their amount depends on the wort composition, the yeast strain, the temperature
and the degree of aeration (Meilgaard, 1975; Popova, 1992; Boulton and Quain, 2001).
There are insignificant differences in the total organic acid concentration and pH of beer
produced in a traditional way and beer produced with immobilized cells (Smogrovicova and
Domeny, 1999; Yamauchi et al., 1995).
Fatty acids that impair the sensory characteristics and foam properties of beer are formed
during fermentation. It has been found that 90% of the fatty acids in wort are palmitic,
linoleic, stearic and oleic acid; in beer, 75-80% of the acids are caprylic and capric acid. This
can be attributed to the accumulation of higher fatty acids by yeast for lipid synthesis and the
release of saturated short chain fatty acids as byproducts of this process, which, in most cases,
is due to the deterioration of the fermentation conditions. These acids are strong detergents
and disrupt the cell membrane, thereby suggesting that their secretion in the fermenting
medium is a response to stress caused by ethanol accumulation or as a result of cell autolysis
(Chen et al., 1980; Boulton and Quain, 2001; Briggs et al., 2004).

Sulfur-Containing Compounds
Sulfur-containing compounds have a direct or indirect effect on the organoleptic profile
of beer. Many of them pass straight from the wort into the beer, but some of them, i.e.,
hydrogen sulfide, sulfur dioxide, dimethyl sulfide and mercaptans, result from yeast
metabolism. When present in low concentrations, they give desirable characteristics to beer.
The hydrogen sulfide content is an important feature of top-fermented beers (Boulton and
Quain, 2001).
Hydrogen sulfide plays a significant role during post-fermentation and maturation. At the
end of the main fermentation, the H2S which was not used for the synthesis of sulfur-
containing amino acids is reused by the yeasts. At a post-fermentation and maturation
temperature of 10-12 ºC, the hydrogen sulfide excess is reduced (Willaert, 2007).

Changes in Metabolites during Beer Fermentation and Maturation

During maturation, yeasts reduce the acetaldehyde amount. Similarly to diacetyl, this
amount increases if yeast metabolism is stimulated. When using early-flocculating yeasts, the
acetaldehyde concentration is higher at the end of the maturation. During beer maturation,
yeasts secrete amino acids, phosphates, peptides, nucleic acids and the like in amounts
depending on the fermentation conditions. The duration of this stage depends on the time for
the reduction of the vicinal diketone concentration below the threshold of sensation (Willaert,
2007).
 Beer Production with Encapsulated Yeast Cells 37

The following strategies have been developed to shorten the process:

 maturation carried out at a higher temperature than that of the main fermentation
(Munroe, 2006; Willaert, 2007);
 “green” beer warming to 80-90°C and a pause for a short period of time (7-10 min)
to decarboxylate α-acetohydroxy acids. The beer is centrifuged before heating in
order to avoid autolysis of yeast cells (Willaert, 2007);
 adding the α-acetolactate decarboxylase enzyme to help the conversion of α-
acetolactate directly to acetoin. S. cerevisiae do not have that enzyme, therefore it is
isolated from a number of bacteria (Jensen, 1993; Hanneman, 2002);
 using genetically modified yeast strains, i.e., inserting the bacterial gene for α-
acetolactate decarboxylase in yeast chromosomes (Linko et al., 1993).

3. Microencapsulation of Yeast Cells: Basic Characteristics

Capsules of solid, liquid or gaseous components coated with a continuous membrane, the
yeast cells being inside the core, are formed during microencapsulation (Rathore et al., 2013;
Zuidam and Shimoni, 2010). As a result of this process, capsules having a spherical shape and
a size of between 100 and 2500 μm are obtained. They are matrix- or reservoir-type capsules,
but those with immobilized cells are actually a combination of the two types of capsules
(Rathore et al., 2013; Zuidam and Shimoni, 2010). Classical microcapsules are defined as
structures with a distinct core and a membrane. However, recently there have been capsules
whose structure is not strictly defined as a separate membrane and a core (Franjione and
Vasishtha, 1995; Gibbs et al., 1999; Kailasapathy, 2002).
Compared to the classical process of immobilization in porous matrices (alginate,
chitosan, carrageenan and others), encapsulation provides a number of advantages. The outer
membrane of the capsule can be semi-permeable and sufficiently thin so as not to hinder the
diffusion of the substrate and the products, but also to provide a targetted fermentation
process or cell protection (Jankowski et al., 1997; Kailasapathy, 2002). Last but not least, the
use of a suitable technique allows for obtaining capsules with very small dimensions, which
leads to a reduction in the diffusional resistances in the fermentation system. At the same
time, the membrane prevents the cells from contamination (Franjione and Vasishtha, 1995;
Kailasapathy, 2002).
The choice of an encapsulation method and the corresponding equipment depends on the
nature of the core and the membrane, the medium conditions and the application of the
capsules. Thus the mechanical stability of the microcapsules, the conditions of diffusion of
substances through the membrane and the core, the temperature and the pH of the medium,
etc. are important for the implementation of microcapsules in industry (Strand et al., 2004).
Microencapsulation as an immobilization method is based on the use of water-soluble
polymers. They provide mild conditions for the core formation procedures resulting in the
formation of a core with good permeability for metabolic products, and to some extent, they
protect the cells against the harmful effects of the external environment. Simultaneously, the
formed membrane protects the cells from leaking into the fermentation system, and if the
conditions for its preparation are suitable, mechanically stable capsules can be obtained.
38 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Different techniques can be used for the preparation of microcapsules with a defined shape,
dimensions and characteristics like ion polymer coating, phase inversion, surface precipitation
and the like. The easiest method to implement is the process of ion polymer coating (Strand et
al., 2004).

A. Basic Polymers Applied in the Formation of Microcapsules with Solid and

Liquid Cores
Capsule formation largely depends on the immobilization carrier. Studies have shown that
natural and/or synthetic polymers are the most commonly used matrices (Jen et al., 1996;
Rathore et al., 2013).

Alginate
Alginic acid and its salts are perhaps the most common natural polymers used to obtain
microcapsules. It is derived from brown seaweed and because of its compatibility with
microbial cells and simple gel formation, it is widely used in applied biotechnology. The
polymer is structurally composed of two sugars: D-mannuronic (M) and L-guluronic acid (G).
The M:G ratio along with their distribution determine the gelling, chemical, mechanical and
diffusional properties of the resulting gel, as well as the distribution of its charges (Melvik
and Dornish, 2004; Strand et al., 2000). The block structure of the polymer (arrangement of
M and G blocks) defines the functional characteristics of alginate and its behavior during
immobilization. Normal alginates do not possess regularly repeating units (Strand et al.,
2000).
The gelling of the alginate molecule is carried out in the presence of divalent cations. The
gelation process is described in the work of Melvik and Dornish, 2004. It depends on gel
concentration and the gelling agent type. The crosslinking in the alginate molecule is only
carried out by certain cations, in a strict affinity row that decreases as follows (Strand et al.,
2000; Melvik and Dornish, 2004):

Pb2+ > Cu2+ = Ba2+> Sr2+> Cd2+> Ca2+> Ni2+> Zn2+> Co2+

Alginates may form gels under acidic conditions. Monovalent ions and Mg2+ do not lead
to gelation (Sutherland, 1991), while ions like Ba2+ and Sr2+ form more stable gels than Ca-
alginate (Clark and Ross-Murphy, 1987).
The cores of alginate capsules are formed in two main ways: by dropping into the gelling
solution or by emulsification with an intermediate agent (Poncelet et al., 2001).

(Kappa)-Carrageenan
(Kappa)-carrageenan is a polysaccharide extracted from seaweed and composed of β-D-
galactose-4-sulfate and 3,6-anhydro-α-D-galactose (Leenen, 2001). The carrageenan gel is
formed under mild conditions (by suspension in cold water), with additional reinforcement
by some ions (K+, Ca2+, NH4+) (Imeson, 2009; Tampion and Tampion, 1987). The
operating stability of carrageenan can be improved by binding with various agents such as
glutaraldehyde, polyacrylamide and the like (Hayes et al., 1991a; Margaritis and Kilonzo,
2005).
 Beer Production with Encapsulated Yeast Cells 39

Chitosan
Chitosan is a copolymer of N-acetyl-D-glucosamine and D-glucosamine (Rinaudo, 2006;
Stolarzewicz et al., 2011). The presence of amino groups makes it one of the few cationic
polyelectrolytes in nature. The solubility of chitosan in an acidic environment and its ability
to react with the anionic polyelectrolytes impart excellent gelling properties (Dutta et al.,
2002; Krajewska, 2004). Chitosan has a number of advantages such as biocompatibility,
biodegradability, non-toxicity, physiological inertness, and affinity proteins. It is mainly
used in methods of gel incorporation by ion-formation or by chemical crosslinking with
glutaraldehyde (Fujimoto et al., 2006; Hellander et al., 2001; Liu et al., 2004; Liouni et al.,
2008; Riddle and Mooney, 2004).

Polyvinyl Alcohol (PVA)

Polyvinyl alcohol is a hydrophilic polymer whose aqueous solutions tend to gel alone
during prolonged storage. It is tasteless, odorless, and its properties depend on the number
of acetate groups. When heated, polyvinyl alcohol is softened but normally does not melt.
Its physical characteristics and specific functional applications depend on the degree of
polymerization and the degree of hydrolysis (Hassan and Peppas, 2000; Wittlich et al., 2004).

Agar / Agarose
Agar and agarose are polysaccharides extracted from red seaweeds that have the ability
to form thermally reversible gels. They are used mainly for nanoencapsulation of cells. A
disadvantage of using them is the passage of cells through the membrane after gelation (Iwata
et al., 1989; Uludag et al., 2000; Riddle and Mooney, 2004).

B. Polyelectrolyte Complexes for the Preparation of Microcapsules

The preparation of capsules is based on the formation of polyelectrolyte complexes by
the reaction between the oppositely charged polymers containing covalently attached groups
of anions (polyanions) or cations (polycations) (Park and Chang, 2000). These complexes are
in the category of physically crosslinked gels. In contrast to covalently crosslinked gels, the
number and position of the cross bonds of these complexes vary with time and temperature
(Hoffman, 2001). The formation of capsules is influenced by the following factors:

 The molecular weight of the polymers affects the formation of the rheological
characteristics of the capsules, their complex stability and the mutual distribution of
the gelation zones in the capsule formation (Gursoy et al., 1999; Thu et al., 1996).
 The inner solution must be more viscous, so that particles with spherical shape and
suitable quality of their outer surface could be created. A number of researchers have
proven that the higher the viscosity of the inner polymer is, the more spherical
particles are formed (Bartkowiak and Hunkeler, 1999; de Vos et al., 2002).
 The selected polymers should ensure the formation of complexes with high
mechanical and chemical stability. The capsule destruction can be reduced by
increasing the intensity of the polyelectrolyte reaction or by avoiding the dissolution
of the inner polymer, i.e., prevent the formation of liquid core capsules (Bartkowiak
and Hunkeler, 1999; de Vos et al., 2002).
40 Vesela Shopska, Rositsa Denkova and Georgi Kostov

 The reactivity of the ionic groups, the chain structure and the solubility of the
polyelectrolytes depend on the ionic strength and the pH of the solution. Typically,
the capsule structure is also affected by the pH of the external polymer (Bartkowiak
and Hunkeler, 1999; Brissova et al., 1998; Lacik et al., 1998; Lacik et al., 2001; Thu
et al., 1996).

Since there are different methods for the preparation of capsules, the reaction time can
very often be divided into two main steps: time for the capsule core production and time for
membrane formation (reaction time) (Lacik, 2004).

C. Effect of Microencapsulation on the Viability and Physiology of the

Immobilized Cells
Cell encapsulation may cause a variety of physical and chemical changes in the
microenvironment of the cells, which in turn can lead to changes in their physiological state
(Groboillot et al., 1994; Sun et al., 2007). They are related to the changes in the ionic charge,
the metabolites produced, the osmotic tolerance of the cells, the reduction of the water
activity and others. These changes depend to a significant degree on the microorganism types,
so they will be discussed in the relevant sections where the application of different capsules
will be examined.
In most cases, the purpose of encapsulation is to preserve cell viability against
environmental changes due to changes in the pH, the temperature and the osmotic pressure,
the accumulation of toxic cell metabolites and others. Quite naturally, loss of viability due to
the immobilization procedure will be observed (Zhao et al., 2008).
Retaining the viability of the immobilized cells is dependent on the encapsulation method
and the equipment used. The incorporation of certain ingredients such as sugars, starch and
gum acacia improves cell viability, especially when used in methods aimed at obtaining
products with vital cells intended for oral use (Desmond et al., 2002; Gardiner et al., 2002;
Kailasapathy, 2002; Lian et al., 2002; Muthukumarasamy et al., 2006).

D. Alginate and Chitosan Capsules and Their Application in Fermentation Industries

Alginate is the preferred polymer for preparing capsules with a solid or liquid core as it
can form spherical particles. A variety of polymers such as polyamino acids, chitosan,
polyamides, polyacrylates, etc. can be used as coating agents (Qi et al, 2005). Since research
is aimed at obtaining alginate and chitosan capsules, in the next few pages we will give
guidelines for their preparation and application in fermentation industries.
Usually chitosan is used as a coating agent for alginate beads in order to improve their
mechanical stability (Murata et al., 1993; Qi et al., 2005). It depends on the process of
preparing the polyelectrolyte complex (Gaserod et al., 1998; Gaserod et al., 1999). The
stability of the chitosan-alginate complex in the pH range from 2 to 5 and in the presence of
salts is substantially increased. It should be noted that if pure chitosan solution is used (in the
absence of Ca2+ ions in the chitosan solution), a relatively thin membrane is formed on the
surface of the alginate beads. Stable composition is obtained when Ca2+ ions are added to the
chitosan solution (Lacik, 2004).
Chitosan-alginate capsules may be formed for 20 min in the absence of primary gelation
of the alginate. This is done by simply spotting an alginate solution into a chitosan solution.
 Beer Production with Encapsulated Yeast Cells 41

The kinetics of membrane formation and its characteristics depend strongly on the
concentration of the components, the molecule mass of alginate and chitosan, the reaction
time, the pH and the ionic charge. However, there is no evidence of the long-term stability of
such capsules. This method of capsule formation is an excellent example of how a minor
change in the terms of encapsulation leads to the formation of unstable systems (Bartkowiak
and Hunkeler, 1999; Bartkowiak and Hunkeler, 2000).
Yan et al., 2009, examined the possibility of microencapsulation of S. cerevisiae
cells in alginate/chitosan beads. The authors found similar growth and productivity of the
immobilized cells in comparison with the free cells. Furthermore, they proved that the
concentration of the chitosan solution and its molecular weight had an impact on ethanol yield
(Yan et al., 2009).
Qi et al., 2006, investigated the metabolic change of microbial cells encapsulated in
alginate-chitosan-alginate (ACA) preparations and found that the capsules did not affect the
encapsulated culture physiology significantly. The authors concluded that by changing the
membrane parameters its permeability could be affected with respect to certain substances in
the substrate. In general, it has been found that encapsulation in this kind of carriers does not
alter substantially yeast cell metabolism as compared with the free cell processes (Qi et al.,
2006).
In the work of Qi et al., 2006, the influence of the process parameters of
microencapsulation in ACA on the cultivation of S.cerevisiae was studied. In this type of
immobilization, the capsule core is important for the process. It is the limiting factor for mass
exchange processes taking place in the system. It was found that in the case of a solid core,
the yeast cells formed small spherical aggregates evenly distributed in the solid phase. The
aggregates had a distinct boundary surface. Unlike the solid core, aggregates with accidental
shape whose boundaries were not clearly defined were formed in the liquid core. This
allowed the cells to “swim” freely in the core. It can be assumed that the aggregates in the
solid core capsules explain the poor mass transfer characteristics of the system as a whole,
which leads to deterioration in the growth characteristics compared with those of the liquid
core capsules (Qi et al., 2006).
Sun et al., 2008, examined the changes in the metabolism of various osmotically tolerant
yeast strains immobilized in ACA liquid core capsules. Studies were performed with two
yeast strains under aerobic and anaerobic conditions and a control free cell fermentation. It
was found that growth inhibition by ACA capsules was strain-specific. The authors concluded
that immobilization in ACA microcapsules affected seriously the metabolism of osmotically
tolerant yeasts and had a strong impact on their ethanol-producing properties (Sun et al.,
2008).
Almonacid et al., 2010, investigated the influence of alginate-chitosan capsules on the
main characteristics of beer, i.e., color, taste, and aroma, as well as on the physicochemical
parameters of the process: pH, density, ethanol concentration and reducing sugars. The
authors found that the capsules did not affect significantly the organoleptic and
physicochemical profile of the resulting beer (Almonacid et al., 2010).
Yoo et al., 1996, investigated lactic acid production by immobilized cells of L.casei in
liquid core capsules. It was found that the liquid core provided larger space for cell growth.
The capsules prepared with Ba-alginate had higher mechanical stability. The structure of the
capsules was stabilized after coating with chitosan (Yoo et al., 1996).
42 Vesela Shopska, Rositsa Denkova and Georgi Kostov

PURPOSE OF THE STUDY

There is no detailed information in literature on the use of alginate and chitosan capsules
in beer preparation, especially with regard to changes in the mode of fermentation.
The aim of the present study was the development and implementation of methods
of obtaining beer with yeast cells immobilized in alginate/chitosan capsules, and the
examination of the immobilization process effect on the organoleptic profile of the resulting
beer.

MATERIALS AND METHODS

1. Microorganisms

The studies in the present chapter were conducted with dry ethanol and beer yeast strains
Saccharomyces cerevisiae Actiflore BO213 (S.cerevisiae BO213), Saccharomyces cerevisiae
Safbrew S-33 (top-fermenting strain), Saccharomyces pastorianus (carlsbergensis) Saflager
W34/70 (bottom-fermenting strain) and Saccharomyces pastorianus (carlsbergensis) Saflager
S-23 (bottom-fermenting strain), stored and rehydrated according to the manufacturers’
instructions.

1.1. Fermentation Media

A. Fermentation broth (g/dm3): glucose – 118.40; (NH4)2SO4 – 2; KH2PO4 – 2.72;
MgSO4x7H2O – 0.5; yeast extract – 1.
B. Manufacture wort with extract 17.5 ± 0.5°P, diluted with sterile distilled water to the
appropriate extract according to the experimental scheme.
All culture media were sterilized at 121°C for 20 min.

2. Reagents and Chemicals

Standard substances of analytical grade were used in all experiments. Algogel 6021
sodium alginate produced by Degussa, France, and chitosan produced by Acros Organics,
Belgium, were used for the immobilization procedures.

3. Experimental Procedures

A. Immobilization of Yeast Cells

Pre-activated yeast culture was mixed with a Na-alginate 2% to 4% solution in order to
achieve a 107 cfu/cm3 concentration of active yeast cells. The mixture was homogenized and
dropped into a 2% CaCl2 solution at constant stirring. The minimum bead formation time was
30 min. Ionic polymeric coating and/or alcohol fermentation according to the accepted
experimental scheme were carried out with the preparations obtained. Crosslinking was
conducted for 30 to 120 min with a 0.2% - 2% (w/v) chitosan solution obtained in 1% (v/v)
 Beer Production with Encapsulated Yeast Cells 43

acetic acid. One part of the beads was placed for core dissolution with a 0.05 M Na-citrate
solotion. All immobilized preparations (Ca-alginate and the solid or liquid core capsules)
were kept in a saline solution in a refrigerator until use. For the optimization of the
encapsulation procedure, a 0.15-0.45% (w/v) chitosane solution obtained in 1% (v/v) acetic
acid was used. The crosslinking time varied between 48 and 132 min. The developed variants
were in accordance with the experimental scheme.

B. Alcohol Fermentation with Free and Immobilized Yeast Cells

To study the immobilization carriers, alcohol fermentation was carried out in 100 cm3
bottles equipped with fermentation stoppers and filled with 70 cm3 of sterile fermentation
broth. A certain amount of the immobilized preparation was inoculated in the bottles. The
bottles were incubated at 30°C. The process was monitored by applying the weighing method,
i.e., by weighing the bottles every 12 h. The alcohol and substrate concentrations were
determined by (Kostov et al., 2010), and the mechanical stability was determined visually by
estimating the number of destroyed capsules.
Beer alcohol fermentation was carried out in 200-500 cm3 sterile fermentation
bottles, filled with sterile wort with the corresponding extract to 75% of their geometric
volume. The bottles were equipped with fermentation stoppers. A certain amount of yeast
suspension (aiming at a viable cells concentration of 107 cfu/cm3), or 1 to 19 g of the
immobilized preparation (according to the experimental scheme), were inoculated in the
bottles. The bottles were incubated at the appropriate fermentation temperature. The process
was monitored by weighing the bottles and monitoring the changes in the extract (real or
apparent), the ethanol concentration and the concentration of the respective metabolite
groups. All laboratory beverages underwent organoleptic evaluation.

4. Methods of Analysis

A. Standard methods of the European Brewery Convention (EBC) (Analytica - EBC,

2004):

 Original, apparent and real wort extract – Methods 8.3 and 9.4;
 Final, real and apparent fermentation degree – Method 9.5;
 Alcohol concentration – Method 9.2.1;
 Wort and beer color and pH – Methods 8.5, 9.6, 8.17 and 9.35;

B. Methods for Metabolite Determination

 Determination of the acetaldehyde content of beer – the aldehyde concentration was
determined according to the bisulfite method after simple sample distillation of the
beer (Nascimento et al., 1998);
 Determination of the ester content of beer – the ester concentration was determined
by ester saponification with NaOH after simple sample distillation of the beer (The
Food Chemical Codex, 1999);
44 Vesela Shopska, Rositsa Denkova and Georgi Kostov

 Determination of the higher alcohol content of beer – the higher alcohol

concentration was determined according to the Komarovsky-Felenberg method after
simple sample distillation of the beer (Duke, 1947);
 Determination of the beer vicinal diketones – the concentration of vicinal diketones
in beer was determined spectrophotometrically at λ = 335 according to EBC Method
9.24.1 (Analytica - EBC, 2004).

C. Biomass Concentration
The biomass quantity was determined using the calculation procedure based on Balling’s
equation (Parcunev et al, 2012).

5. Determination of Fermentation Process Kinetics

The alcohol fermentation kinetics was described by the following system of differential
equations:

dX
  t  X t 
dt
dP (1)
 q t  X t 
dt
dS 1 dX 1 dP
 
dt Yx / s dt Yp/ s dt

where: X(t) – biomass concentration, g/dm3; P(t) – ethanol concentration, g/dm3; S(t) –
substrate concentration (the real wort extract), g/dm3; YP/S and YX/S – yield (economic)
coefficients; μ(t), h-1 – specific biomass growth rate; q(t), g/(g.h) – specific rate of product
(alcohol) accumulation in the medium. All these parameters were variable in time.
The models outlined in the subsequent parts of the present work were used to describe the
kinetics of alcohol fermentation with free and immobilized yeast cells.
The identification of the model parameters was performed in a Matlab 7.0 environment
using Optimization toolbox functions by minimizing the error between experimental and
model data by the least squares method, and using the 4-5 order Runge-Kutta algorithm. The
method is described in detail in (Mitev and Popova, 1995; Popova 1997; Kostov et al., 2012).

RESULTS AND DISCUSSION

1. Selection of a Method and Conditions for Encapsulation

The requirements specified in Table 1 need to be observed in order to choose the right
carrier. The immobilization method selected should allow preservation of the vital cell
activity. It should also be relatively inexpensive and easy to implement in practice.
Figure 2. Scheme of the formation of preparations of Ca-alginate and alginate/chitosan with solid and liquid core capsules.

Legend: FC – free cells; ALG – alginate beads; AC30S – AC120S – alginate/chitosan beads with solid core, received with coating time between 30 and 120
min; AC30L – AC120L – alginate/chitosan beads with liquid core, received with coating time between 30 and 120 min.
46 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Comparing different carrier types for cell immobilization is labor-intensive and requires
knowledge of a number of parameters, such as fermentation activity, stability, growth
characteristics, and others. Alginate and chitosan capsules meet the modern production
requirements: they retain high catalytic cell activity; they have certain stability and provide
optimal mass transfer characteristics. In the present study, the medium selection was carried
out on the basis of the experiment, the diagram of which is presented on Figure 2. The
experiment was conducted with the S.cerevisiae BO213 strain. The procedure included
development of 8 capsule variants with liquid or solid cores obtained from three original
alginate concentrations. Immediately after their preparation, the capsules were placed in
fermentation broth in order to study their fermentation properties (Iliev et. al., 2014). The
survey results are shown on Figure 3 to Figure 6.

а) solid core preparations

b) liquid core preparations

Figure 3. Dynamics of alcohol fermentation with encapsulated S.cerevisiae BO213 cells in a 2%

alginate solution.
 Beer Production with Encapsulated Yeast Cells 47

a) solid core preparations

b) liquid core preparations

Figure 4. Dynamics of alcohol fermentation with encapsulated S.cerevisiae BO213 cells in a 3%

alginate solution.

The fermentation dynamics of the preparations obtained from 2% alginate was not
characterized by any alteration compared with that of the free cells. Typically, the non-
crosslinked beads accumulated higher ethanol amounts compared to the free cells. The solid
core preparations accumulated a slightly higher ethanol concentration in comparison with
the free cell, but a lower ethanol concentration when compared to the non-crosslinked
preparation. The ethanol concentration in the medium decreased with the increase in the ion
polymer coating time (Figure 3). For comparison, liquid core beads accumulated a lower
ethanol concentration compared with the non-crosslinked beads and the free cells.
48 Vesela Shopska, Rositsa Denkova and Georgi Kostov

The general trend was reduction of the ethanol concentration (product yield) with the
increase in the coating time and at core dissolution. At the end of the process (about the 72nd
hour), the ethanol yield varied between 76% and 94% (Iliev et. al., 2014).
The liquid core capsules were characterized by higher mechanical stability since
the liquid core had elastic properties and adsorbed the tensions caused by the
pressurized departure of the CO2 formed during fermentation. The high fermentation activity
led to leakage of cells into the medium. Cracks began to appear in the preparations. The non-
crosslinked preparation broke first, then the destruction of the solid core capsules began. The
destruction was mostly due to the CO2 formed and the pressure created within the gel beads.
This disadvantage of the solid core capsules was compensated by core dissolution.

а) solid core preparations

b) liquid core preparations

Figure 5. Dynamics of alcohol fermentation with encapsulated S.cerevisiae BO213 cells in a 4%

alginate solution.
 Beer Production with Encapsulated Yeast Cells 49

Similar trends were observed in S.cerevisiae BO213 preparations obtained with a 3%

alginate solution. The non-crosslinked preparation accumulated higher product concentrations
in the medium. The ethanol concentration in the medium decreased with the increase in the
coating time. Additional decrease was observed with core dissolution. Similarly to the
previous variant, solid core capsules were more susceptible to fracture, and those with a liquid
core were harder to destroy due to their elastic nature (Figure 4).
Trends similar to the previous two variants were retained with preparations of
S.cerevisiae BO213 received with a 4% alginate solution (Figure 5). These preparations
showed the highest mechanical resistance due to the increased alginate concentration. A
disadvantage was the long preparation time, which could lead to culture inactivation in the
immobilization process (Iliev et. al., 2014).
The declining trend in the ethanol yield with the increase in the ion polymer coating time
and with core dissolution has a logical explanation. The longer coating time led to membrane
increase and, therefore, diffusion resistance increase. In core dissolution, the substrate access
to the central core areas was facilitated, which led to an increased process rate. At the same
time, the substrate and product diffusion in the liquid core should also be taken into account
(Kostov, 2015). However, liquid core capsules had a certain advantage – their increased
elasticity, which determined their higher mechanical stability. The ethanol yield at the end of
the fermentation process was similar for all the variants (Figure 6). However, fermentation
process dynamics must be considered in the variant choice.
The study conducted showed that there was a certain optimum zone of the parameters of
the capsule preparation process, in which maximum yield would be realized at high
mechanical stability. Such modeling can be performed by the mathematical and statistical
analysis methods using Central Composite Design (Table 2) (Kostov, 2015).

Figure 6. Ethanol yield at the 72nd hour of fermentation with S.cerevisiae BO213 for all variants.
50 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Table 2. Experiment matrix of the modeling of the ion polymer coating process of liquid
core capsules prepared with a 3% alginate solution

Coded variables Real variables Results

№ Coating Chitosan Coating Chitosan Biomass,
Yield, %
time concentration time, min concentration, % g/dm3
1 0 0 90 0,3 67,75 0,0198
2 -1 -1 60 0,2 74,38 0,0277
3 1 -1 120 0,2 67,25 0,0172
4 -1 1 60 0,4 76,76 0,0422
5 1 1 120 0,4 67,56 0,0092
6 0 0 90 0,3 69,30 0,0277
7 0 0 90 0,3 66,37 0,0092
8 -1,41421 0 48 0,3 75,13 0,0145
9 1,41421 0 132 0,3 66,92 0,0317
10 0 -1,41421 90 0,15 72,25 0,0303
11 0 1,41421 90 0,45 74,15 0,0475
12 0 0 90 0,3 68,20 0,0356

The statistical analysis of the fermentation process results is presented in Table 3. After
the insignificant coefficient removal and the adequacy testing, the following mathematical
model was proposed for the description of the ion polymer coating process:

EY  49,94  1,015* T  6,381* Ch  0,487 * T 2  1,62* Ch2 (1)

wherein: EY – ethanol yield, %; T – time; Ch – chitosan concentration (coded value).

Model (1) indicates that the two factors had opposite effects on ethanol yield. Increasing
the time for ion polymer coating resulted in a reduction of the ethanol yield. Increasing the
chitosan concentration led to an increase in the ethanol yield. The increase in the chitosan
concentration may have improved the membrane quality, and mostly its permeability with
respect to ethanol. However, an excessive increase in the coating agent concentration resulted
in a reduction of the ethanol yield. This was associated with the negative sign before the
coefficient of Chitosan^2 (Ch2). The mathematical model determined a maximum yield of
75.21% at chitosan concentration of 0.38% and ion polymer coating time of 63 min.
On one hand, the reduced coating time indicated that this was the parameter that
influenced membrane permeability, as more permeable membranes were formed in a shorter
time. On the other hand, the expectation that the concentrated solution would reduce
membrane permeability was not justified. Perhaps, the chitosan concentration influenced the
mechanical stability of the membrane and its permeability to yeast cells.
Based on the studies carried out, the planned experiment and the statistical processing of
the experiment established the following conditions for obtaining liquid core microcapsules:
Na-alginate concentration – 3%; chitosan concentration to coat the capsules – 0.38%; ion
polymer coating time – 60 min; core dissolution time – 30 min; Na-citrate concentration –
0.05 M.
 Beer Production with Encapsulated Yeast Cells 51

Table 3. ANOVA for “Ethanol Yield"

Sum of Squares

Mean Square

Significance
reedom (Df)
Degree of f
Parameter

P- value
F-ratio
A: Time 97.57 1 97.57 61.11 0.0005 Significant
B: Chitosan Significant
3.62 1 3.62 2.27 0.01923
concentration
AA 12.62 1 12.62 7.91 0.0375 Significant
AB 1.079 1 1.079 0.68 0.4484 Insignificant
BB 39.66 1 39.66 24.84 0.0042 Significant
Block 0.000052 1 0.000052 0.00 0.9957 -
Total error 7.98329 5 1.60
Total (corr.) 155.413 11
R2 = 94.86%; R2 (за d.f.) = 90.58%; Standard error = 1.26; Absolute average error = 0.668l; Durbin-
Watson parameter = 2.851;

2. Yeast Strain Selection for Beer Preparation with Immobilized Cells

The choice of yeast strain has to be carried out under diffusion resistance conditions
because the immobilization process changes the yeast metabolism and leads to significant
changes in the beer organoleptic profile. Strain selection was carried out with the strains S.
carlsbergensis Saflager S-23, S.carlsbergensis Saflager W34/70, and S.cerevisiae Safbrew S-
33. The principal scheme of the experiment is presented in Figure 7 (Naydenova et al., 2012).

Figure 7. Scheme for yeast strain selection for beer preparation with immobilized cells.

The fermentation process dynamics is presented on Figure 8 and Figure 9. There were
differences between the three yeast strains.
During fermentation with S. carlsbergensis Saflager S-23, maximum activity was
observed between the second and the third day, then it started decreasing gradually. The
immobilized cells of the same strain possessed higher activity, which was observed about 24
hours earlier than the free cell fermentation. In free cell fermentation, a larger amount of
52 Vesela Shopska, Rositsa Denkova and Georgi Kostov

unfermented extract remained, while in immobilized cell fermentation a higher degree of

fermentation was observed at the end of the study.

a) original extract 12°P

b) original extract 17°P

Figure 8. Alcohol concentration dynamics in fermentation with free and immobilized cells.

The fermentation activity of the immobilized cells of S. carlsbergensis Saflager W / 34-

70 was similar to that of the free cells in the fermentation of wort with original extract of 12
ºP. Similar to S. carlsbergensis Saflager S-23, there was a shift of the maximum enzyme
activity and a higher real fermentation degree in fermentation with immobilized cells. In
original extract of 17 ºP, a shift of the activity peak of free cells was established compared
with that in original extract of 12 ºP due to the low osmotic tolerance of the strain. Since the
microcapsules provided partial protection from substrate inhibition (Norton and D'amore,
1994), the activity maximum of immobilized cells coincided with that of free cells.
More significant differences were observed in the behavior of the free and immobilized
cells of S. cerevisiae Safbrew S-33 that increased with increasing the wort extract. The
fermentation activity of free cells increased with the increase in the original wort extract,
which led to the assumption that this strain was osmotically tolerant. Immobilized cells
 Beer Production with Encapsulated Yeast Cells 53

generally showed a lower fermentation degree than free cells. The differences in the
physiology of growth between free and immobilized cells make this strain potentially
interesting to study (Naydenova et al., 2012).

a) original extract 12°P

b) original extract 17°P

Figure 9. Extract concentration dynamics in fermentation with free and immobilized cells.

All three tested strains accumulated relatively high biomass amounts in the medium or in
the capsules. The biomass concentration varied within the range of 4-5 g/dm3 (Naydenova et
al., 2012).
With regard to its fermentation degree, beer obtained with immobilized cells was
characterized by a deeper fermentation. For the free cell variants of fermentation of wort with
12% original extract, the real fermentation degree varied between 61-62%, and of wort with
17% original extract – in the range of 52-61%. For immobilized preparations these values
were 62-68% and 58-64% respectively. The highest fermentation degree was achieved with S.
carlsbergensis Saflager S-23 and original wort extract of 12 ºP (Naydenova et al., 2012).
An important feature of beer is its organoleptic profile. The results of the descriptive
evaluation are summarized on Figure 10. The highest scores were assigned to the beer
produced with S. cerevisiae Safbrew S-33. There were no significant differences in the
organoleptic profiles of the beers produced from wort with high or low extract. The lowest
54 Vesela Shopska, Rositsa Denkova and Georgi Kostov

tasting evaluation was received for the beverage fermented with S. carlsbergensis Saflager W
34/70.
The S. cerevisiae Safbrew S-33 and S. carlsbergensis Saflager S-23 strains had good
characteristics for beer production with immobilized cells. The beverages obtained with them
had organoleptic profiles similar to those of the beverages obtained with free cells. Both
strains had high fermentation activity, which was maintained after the immobilization
process.

Figure 10. Organoleptic profile of the beverages obtained with free and immobilized cells.

3. Comparative Study on Fermentation Conditions in Beer Preparation with

Immobilized Cells

A. Influence of the Original Wort Extract on the Beer Obtained

In most cases, fermentation processes with immobilized cells differ significantly from
those with free cells since immobilized cells grow under diffusion resistance conditions. Mass
transfer resistances influence both the primary metabolism of the immobilized culture, i.e.,
sugar absorption and ethanol accumulation, and the secondary metabolite accumulation.
Knowledge of alcohol fermentation for beer preparation in terms of diffusion resistances is
required for the development of a controlled technology. The fermentation temperature and
the original wort extract are essential to the management of the fermentation process. They
can change the immobilized yeast culture metabolism substantially along with the diffusion
resistances.
At this work stage, the fermentation process for obtaining beer using 5 different original
wort extracts – 9°P, 11°P, 13°P, 15°P and 17°P, was estimated. Parallel fermentation
processes with free and immobilized cells at a constant fermentation temperature of 15°C
were carried out. The dynamics of substrate absorption, alcohol and biomass accumulation
were compared. The dynamics of accumulation of 3 large secondary metabolite groups, i.e.,
esters, aldehydes and higher alcohols, were traced simultaneously.
 Beer Production with Encapsulated Yeast Cells 55

Development of a Kinetic Model for the Accumulation of Primary and Secondary

Metabolites in Alcohol Fermentation in Beer Manufacture
The selection of a kinetic model should be based on experimental results and the error in
their approximation with the model. In the present case, and based on the work of Ramirez
and Maciejowski, 2007 and Kostov et al., 2012, the Monod model has been chosen for the
description of the primary metabolism, as its structure is also interpolated to the model of
substrate consumption during fermentation. The reasons for the present choice will be
discussed in the presentation of the test results, but in terms of secondary metabolism, it is
necessary to make this statement now.
The primary metabolism can be described with the well-known system:

dX
  t  X t 
dt (2=1)
dP
 q t  X t 
dt
dS 1 dX 1 dP
 
dt Yx / s dt Yp/ s dt

Initially, three kinetic models were used for the description of the specific growth rate and
ethanol accumulation rate (Kostov, 2015; Pracunev et al., 2012):

Monod Model
S S (3)
  max ; q  qp max
K sx  S K sp  S

wherein: μ – specific growth rate of the yeast culture, h-1; μmax – maximum specific growth
rate of the yeast culture, h-1; qp – specific rate of ethanol accumulation, g/(g.h); qpmax –
maximum specific rate of ethanol accumulation, g/(g.h); S – substrate concentration, g/dm3;
KSX, KSP – affinity constants in Monod equation, g/dm3;

Aiba Model
S S
  max exp(K ip P )X ; q  qp max exp(K ip P )X (4)
K sx  S K sp  S

wherein: μ – specific growth rate of the yeast culture, h-1; μmax – maximum specific growth
rate of the yeast culture, h-1; qp – specific rate of ethanol accumulation, g/(g.h); qpmax –
maximum specific rate of ethanol accumulation, g/(g.h); S – substrate concentration, g/dm3;
Kip – constant of cell growth inhibition by the product; P – product concentration, g/dm3

Tiessier Model
  S    S  (5)
  max 1  exp     ; q  q p max 1  exp    
 K sx  
   K sp 

wherein: μ – specific growth rate of the yeast culture, h-1; μmax – maximum specific growth
rate of the yeast culture, h-1; qp – specific rate of ethanol accumulation, g/(g.h); qpmax –
maximum specific rate of ethanol accumulation, g/(g.h); S – substrate concentration, g/dm3;
KSX, KSP – affinity constants in Monod equation, g/dm3.
56 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Contrary to primary metabolism modeling, which has been the focus of extensive
research, the modeling of the secondary metabolism of yeast cells in alcohol fermentation is
the subject of a limited number of studies. This is due mostly to the incomplete knowledge of
cell metabolism, the relationship between metabolites and growth, etc. The proposed kinetic
model is based on the currently known data on metabolite accumulation presented in the
Introduction as well as the data contained in the works of Ramirez and Maciejowski, 2007
and Andres-Toro et al, 1998.
Higher alcohol synthesis is directly linked to yeast cell growth; the rate of synthesis
should be determined depending on the metabolic pathway, anabolic or catabolic. The latter,
however, can hardly be incorporated into the model, so the model for their synthesis can be
summarized to (Kostov, 2015; Vassilev et al., 2013):

dFA
 YFA ..X(t) (6)
dt

wherein: YFA – yield coefficient of higher alcohols per biomass unit, mg/(g.h)
Ester synthesis can also be associated directly with cell growth, especially due to the fact
that a substantial amount of esters accumulate in the exponential growth phase (Kostov, 2015;
Vassilev et al., 2013):

dE
 YE ..X(t) (7)
dt

wherein: YE – yield coefficient of esters per biomass unit, mg/(g.h);

Aldehyde synthesis is directly related to cell growth and their reduction depends on the
aldehyde concentration at the very beginning of this process (Kostov, 2015; Vassilev et al.,
2013):

dA
=YA ..X(t)  k A .A.X (8)
dt

wherein: YA – yield coefficient of aldehydes per biomass unit, mg/(g.h); kA – coefficient of

aldehyde reduction, mg/(g.h); A – aldehyde concentration, mg/dm3;

Comparative Study of Fermentation with Free and Immobilized Top-Fermenting S.

Cerevisiae Safbrew S-33 Cells at 15°C
The survey results are summarized on Figure 11 to Figure 16. In all examined variants,
fermentation was carried out normally: substrate consumption and ethanol accumulation
followed the typical trends in beer manufacture. At this temperature, the main fermentation
took about 5 days and the free cells were characterized by a faster start. The immobilized
cells quickly compensated their delay. The liquid was stirred mainly by the CO2 release.
There were no significant differences in biomass accumulation. The free cells entered the
stationary phase more rapidly, while the immobilized cells had a prolonged exponential
phase. The observed difference in the biomass change reflected on ethanol accumulation and
 Beer Production with Encapsulated Yeast Cells 57

extract consumption (Table 4 to Table 8) (Kostov, 2015; Naydenova, 2014a; Naydenona et

al., 2013b).

a) Immobilized cells

b) Free cells

Figure 11. Real extract dynamics in fermentation with S. cerevisiae Safbrew S-33 at 15°C.

The results in Table 4 to Table 8 showed that all three models described the fermentation
process kinetics very well. Contrary to the expectations, there was no strong substrate or
product inhibition as all three models yielded relatively similar values of the maximum
specific growth and product accumulation rates. The kinetic description with the Monod
model was possible and preferred since it facilitated the modeling after including metabolites
in the generalized kinetic model of the fermentation process. The only drawback of the
Monod model was that it did not describe the lag phase of the process well, but due to the
long fermentation (8-10 days) this was not a problem (Kostov, 2015; Naydenova, 2014a;
Naydenona et al., 2013b).
58 Vesela Shopska, Rositsa Denkova and Georgi Kostov

a) Immobilized cells

b) Free cells

Figure 12. Alcohol concentration dynamics during fermentation with S. cerevisiae Safbrew S-33 at
15°C.

The maximum specific growth rate determined by the Monod model was observed at 11
ºP and 13 ºP original wort extract. With the increase in the wort extract, the growth
characteristics began decreasing gradually. The yield biomass coefficient ranged from 6% to
7% (Kostov, 2015; Naydenova, 2014a; Naydenona et al., 2013b; Parcunev et al., 2015).
The data showed that immobilization had a rather negative impact on biomass growth.
This explained the observed prolonged exponential growth phase in the immobilized
preparations. The efficiency coefficient ημ began declining with increasing the wort extract.
The similar kinetics of biomass accumulation explained the late start of the processes with
immobilized cells.
Immobilization had a contradictory effect on ethanol accumulation in the fermentation
medium. The data on the efficiency coefficient ηq showed that the rates of ethanol
accumulation for free and immobilized cells were relatively close. At 15 ºP and 17 ºP wort
extract, ethanol accumulated at a slightly higher specific rate, but it was similar to that of the
free cells.
 Beer Production with Encapsulated Yeast Cells 59

Table 4. Kinetic parameters of the fermentation process with free and immobilized top-
fermenting S. cerevisiae Safbrew S-33 cells – Original extract: 9°P

EFFICIENT
PARAMETERS ERROR
COEFFICIENTS
μmax Ksx qpmax Ksp Yx/s Yp/s Kix Kip ημ ηq
d-1 g.dm-3 g.(g.d)-1 g.dm-3 - - g.dm-3 g.dm-3 - -
Monod
Free cells
0.157 200 3.39 200 0.0597 0.521 - - 0.416
1.261 0.914
Immobilized cells
0.196 226.71 3.1 216.72 0.063 0.555 - - 0.382
Tiessier
Free cells
0.181 200 1.754 100 0.086 0.444 - - 0.577
0.883 0.886
Immobilized cells
0.159 200 1.554 100 0.053 0.593 - - 0.382
Aiba
Free cells
0.165 173.2 2.321 152.11 0.085 0.532 0.231 0.427 0.725
0.794 1.877
Immobilized cells
0.131 232.1 4.357 183.12 0.088 0.572 0.133 0.674 0.621

a) Immobilized cells

b) Free cells

Figure 13. Biomass concentration dynamics during fermentation with S. cerevisiae Safbrew S-33 at
15°C.
60 Vesela Shopska, Rositsa Denkova and Georgi Kostov

The dynamics of the formation of esters, higher alcohols and aldehydes is summarized on
Figure 14 to Figure 16, and the kinetic parameters are summarized in Table 9 (Kostov, 2015;
Vassilev et al., 2013).

Table 5. Kinetic parameters of the fermentation process with free and immobilized top-
fermenting S. cerevisiae Safbrew S-33 cells – Original extract 11°P

Efficiency
Model parameters Error
coefficients
μmax Ksx qpmax Ksp Yx/s Yp/s Kix Kip ημ ηq
d-1 g.dm-3 g.(g.d)-1 g.dm-3 - - g.dm-3 g.dm-3 - -
Monod
Free cells
0.294 200 6.37 197.11 0.054 0.581 - - 0.202
0.918 0.695
Immobilized cells
0.270 226.71 4.43 226.72 0063 0.57 - - 0.18
Tiessier
Free cells
0.304 210.13 3.010 100 0.057 0.060 - - 0.321
0.651 0.729
Immobilized cells
0.198 205.7 2.195 92.11 0.0623 0.6 - - 0.116
Aiba
Free cells
0.450 62.68 5.907 100 0.063 0.65 0.086 0.035 0.131
1.564 0.789
Immobilized cells
0.704 200 4.662 200 0.125 0.56 0.066 0.021 0.733

Table 6. Kinetic parameters of the fermentation process with free and immobilized top-
fermenting S. cerevisiae Safbrew S-33 cells – Original extract 13°P

Efficiency
Model parameter Error
coefficients
μmax Ksx qpmax Ksp Yx/s Yp/s Kix Kip ημ ηq
d-1 g.dm-3 g.(g.d)-1 g.dm-3 - - g.dm-3 g.dm-3 - -
Monod
Free cells
0.395 221.214 3.297 200 0.065 0.61 - - 0.192
0.559 0.879
Immobilized cells
0.221 158.11 2.897 200 0.071 0.59 - - 0.210
Tiessier
Free cells
0.307 178.12 2.995 150 0.074 0.597 - - 0.347
0.597 0.708
Immobilized cells
0.221 199.78 2.123 150 0.075 0.61 - - 0.214
Aiba
Free cells
0.321 124.21 6.130 100 0.054 0.59 0.123 0.065 0.464
2.038 0.747
Immobilized cells
0.654 200 4.578 164 0.101 0.55 0.094 0.009 0.214

Generally, higher alcohols accumulated during the exponential growth phase (about 80%
of their total amount). Their amount in the fermentation broth increased with the increase in
the wort extract. This also affected the yield coefficient, which followed an increasing trend
with increasing the wort extract (Table 9). The immobilized cell system produced relatively
small amounts of higher alcohols due to the diffusion resistances in the system. The higher
 Beer Production with Encapsulated Yeast Cells 61

alcohol precursors – α-keto acids – were intermediate metabolites in the amino acid
biosynthesis in yeast cells, or were obtained by deamination and transamination of wort
amino acids (Briggs et al., 2004). Consequently, their accumulation occured in yeast cells,
and for the reaction to proceed, the cited substrates had to get to the yeast cells in the presence
of diffusion resistances, which was a prerequisite for reduced higher alcohol synthesis.
However, it can be summarized that their impact was relatively low and comparable higher
alcohol amounts accumulated in beer.

Table 7. Kinetic parameters of the fermentation process with free and immobilized top-
fermenting S. cerevisiae Safbrew S-33 cells – Original extract 15°P

Efficiency
Model parameters Error
coefficients
μmax Ksx qpmax Ksp Yx/s Yp/s Kix Kip ημ ηq
d-1 g.dm-3 g.(g.d)-1 g.dm-3 - - g.dm-3 g.dm-3 - -
Monod
Free cells
0,211 154,58 2,21 148,65 0,012 0,53 - - 0,196
0,597 1,200
Immobilized cells
0,126 200 2,654 167,7 0,066 0,54 - - 0,311
Tiessier
Free cells
0.407 178.12 1.995 150 0.054 0.697 - - 0.247
0.788 0.772
Immobilized cells
0.321 199.78 2.530 150 0.055 0.51 - - 0.314
Aiba
Free cells
0.321 124.21 6.130 100 0.054 0.59 0.123 0.065 0.464
2.038 0.747
Immobilized cells
0.654 200 4.578 164 0.101 0.55 0.094 0.009 0.214

Table 8. Kinetic parameters of the fermentation process with free and immobilized top-
fermenting S. cerevisiae Safbrew S-33 cells – Original extract 17°P

Efficiency
Model parameters Error
coefficients
μmax Ksx qpmax Ksp Yx/s Yp/s Kix Kip ημ ηq
d-1 g.dm-3 g.(g.d)-1 g.dm-3 - - g.dm-3 g.dm-3 - -
Monod
Free cells
0,195 155,24 1,95 126 0,01 0,52 - - 0,264
0,589 1,031
Immobilized cells
0,115 156,26 2,01 134 0,07 0,5 - - 0,367
Tiessier
Free cells
0.307 178.12 2.995 150 0.074 0.597 - - 0.347
0.597 0.708
Immobilized cells
0.221 199.78 2.123 150 0.075 0.61 - - 0.214
Aiba
Free cells
0.325 124.21 6.14 100 0.054 0.59 0.223 0.065 0.664
2.038 0.747
Immobilized cells
0.664 200 4.58 164 0.101 0.55 0.194 0.009 0.514
62 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Figure 14. Ester concentration dynamics in fermentation with free and immobilized cells of S.
cerevisiae Safbrew S-33 at 15°C.

Figure 15. Higher alcohol concentration dynamics in fermentation with free and immobilized cells of S.
cerevisiae Safbrew S-33 at 15°C.

Figure 16. Aldehyde concentration dynamics in fermentation with free and immobilized cells of S.
cerevisiae Safbrew S-33 at 15°C.

Legend: I – immobilized cells; F – free cells; 9, 11, 13, 15, 17 – original wort extract, °P.
 Beer Production with Encapsulated Yeast Cells 63

Similarly, about 60% of the esters were also accumulated in the exponential growth
phase. Depending on the original extract, the two types of systems had different behavior. At
low original wort extracts of 9 ºP and 11 ºP, the immobilized cells accumulated more esters
than free cells. With the increase in the extract concentration, the trend was reversed. The data
showed an increase in the yield coefficient with increasing the wort extract. The immobilized
preparations were characterized by reduced ester yield because most coefficients were lower
than those in free cell preparations. A decrease in the ester concentration was observed at the
end of the fermentation process because of the CO2 losses and H2CO3 losses respectively. The
most pronounced trend for the loss of this group of aromatic products was detected at low
wort extracts where fermentation ended quickly and lacked the necessary sugars for the CO2
formation to maintain the appropriate carbonic acid amounts. In actual production, this
disadvantage is avoided by increasing the pressure. The ester concentration ranged from
about 50-200 mg/dm3 at the end of the fermentation in all variants tested, which is
characteristic of beer obtained with a top-fermenting yeast strain.

Table 9. Kinetic parameters for the accumulation of secondary

metabolites in alcohol fermentation with free and immobilized
top-fermenting S. cerevisiae Safbrew S-33 cells

Original extract Fermentation type YE YFA YA KA Error

°Р mg/(g.d) -
Free 31.99 18.8 120.1 0.0345 0.462
9
Immobilized 41.75 17.36 79.81 0.046 0.346
Free 34.45 11.31 15.45 0.045 0.547
11
Immobilized 21.68 3.64 6.52 0.0678 0.247
Free 184.36 19.52 51.01 0.0456 0.101
13
Immobilized 152.43 23.44 59.82 0.081 0.246
Free 192.1 22.2 55.03 0.045 0.256
15
Immobilized 153.2 21.3 65.07 0.069 0.296
Free 175.2 26.3 66.9 0.061 0.325
17
Immobilized 126.3 24.2 71.2 0.073 0.185

Aldehyde synthesis also followed the trends already established. The general trend was
an increase in the aldehyde yield coefficient with the increase in the wort extract. Another
important feature of these variants was that the immobilized cells lacked a marked aldehyde
maximum, which can be explained by the higher cell loading in the fermentation bottles. The
reason for the lack of a clear peak was that synthesis and reduction occur at similar average
rates (determined by the biomass amount). An exception was the variant with 13 ºP wort
extract. The presence of a diffusion barrier resulted in a difference in aldehyde synthesis and
reduction. At the end of the study period, the aldehyde concentration varied between 10
mg/dm3 and 50 mg/dm3 (Kostov, 2015; Parcunev et al., 2015; Vassilev et al., 2013).

Comparative Study of Fermentation with Free and Immobilized Cells of S.

Carlsbergensis Saflager S-23 at 15°C
The results on the fermentation process dynamics are summarized on Figure 17 to Figure
22 and in Table 10 and Table 11. Only data on the kinetic characteristics from the Monod
64 Vesela Shopska, Rositsa Denkova and Georgi Kostov

model are presented since the results obtained and the analyses conducted are similar to those
of the previous strain (Naydenova, 2014; Naydenona et al., 2013a; Parcunev et al., 2015).
This process had no significant differences from the literature data. The immobilized
cells were characterized by a slow start but they caught up quickly. Interestingly, in extracts
of over 13°P, this trend was reversed, indicating low osmotic tolerance of the strain. The main
fermentation time was between 3 and 6 days and it increased with increasing the wort extract
(Kostov, 2015; Naydenova, 2014a; Naydenona et al., 2013a; Parcunev et al., 2015).
The free cell biomass entered the stationary growth phase faster, whereas a prolonged
exponential growth phase was observed with the immobilized cell biomass. The maximum
specific growth rate was observed in original wort extract of 9 ºP. With the increase in the
original wort extract, gradual reduction of the growth characteristic of the yeast population
was recorded. Ethanol accumulation also proceeded at higher specific rates.
The immobilization process favored alcohol fermentation and resulted in higher specific
rates of ethanol accumulation. The high values of the efficiency coefficients showed that the
immobilization process did not affect population development and primary metabolite
accumulation significantly (Figure 18 and Table 10).
Higher alcohol synthesis took place according to the trends established in beer
manufacture. The data showed that the free and immobilized cells accumulated similar higher
alcohol amounts, though there were certain differences in terms of the yield coefficient. This
can be explained mainly by the influence of the increased concentration of immobilized cells
in the system, hence the higher average process rate. At the end of the main fermentation (5th
– 6th day), about 85% of the total higher alcohol amount was accumulated. Their
concentration increased with the increase in the original wort extract, which was reflected to a
substantial degree in the developed mathematical model (Kostov, 2015; Parcunev et al., 2015;
Vassilev et al., 2013).
Similar kinetics of ester accumulation was observed for both fermentation systems.
During the main fermentation, around 60-70% of the total esters were accumulated. The
increase in the ester concentration was proportional to the increase in the original wort
extract. The immobilized cells produced greater ester amounts than the free cells. It was
suggested that immobilization caused inhibition of fatty acid synthesis, which led to acetyl-
CoA accumulation and together with the high ethanol levels increased ester synthesis in this
type of system was provoked (Shen et al., 2003). The high original extract led to reduced
oxygen solubility in the wort, which also provoked greater ester accumulation (Dufour et al.,
2003). It should be noted that the kinetic model did not account for the partial ester reduction
at the end of the process.
Interesting results on the ester concentration during fermentation were obtained during
the fermentation of high original extract wort (17 °P). At the beginning of the fermentation
process, about 500 mg/dm3 esters were formed, but on the 10th day their concentration was
comparable to that of the other tested variants. This problem of acetate ester overproduction
(most frequently ethyl acetate) is well known in the manufacture of high extract beer
(Naydenova, 2014a).
 Beer Production with Encapsulated Yeast Cells 65

a) Immobilized cells

b) Free cells

Figure 17. Real extract dynamics in fermentation with S. carlsbergensis Saflager S-23 at 15°C.

In contrast to esters and higher alcohols, differences between immobilized and free
cells were observed in the dynamics of aldehyde formation. The free cells produced more
aldehydes during the main fermentation, and the maximum aldehyde concentration was
observed on the second day. With the increase in the original wort extract, an increase in the
maximum aldehyde concentration was established as well. No sharp peaks were observed
during fermentation with immobilized cells. The aldehyde concentration rise to a certain
level, then remained relatively constant until the end of the process when a significant
reduction was observed. These results allowed the formulation of the hypothesis that carbonyl
compound synthesis and reduction occured at similar specific rates. This in turn explained the
differences observed between processes with free and immobilized cells. On the 10th day, the
aldehyde concentration ranged from 10 to 30 mg/dm3. The mathematical model indicated that
the process rates were relatively close in both fermentation systems (Kostov, 2015; Parcunev
et al., 2015; Vassilev et al., 2013).
66 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Table 10. Kinetic parameters of the fermentation process with

S. carlsbergensis Saflager S-23

Model parameters Efficiency coefficients Error

μmax Ksx qpmax Ksp Yx/s Yp/s ημ ηq
d-1 g.dm-3 g.(g.d)-1 g.dm-3 - - - -
9%
Free cells
0,424 150 2,042 150 0,125 0,53 0,306
0,708 1,350
Immobilized cells
0,3 57,74 2,756 166,33 0,063 0,58 0,123
11%
Free cells
0,117 150 2,03 150 0,093 0,53 0,725
2,564 2,724
Immobilized cells
0,30 204,26 5,53 226,71 0,6 0,58 0,209
13%
Free cells
0,224 150 3,839 150 0,124 0,476 0,133
1,335 1,259
Immobilized cells
0,300 221,420 4,901 226,72 0,063 0,58 0,370
15%
Free cells
0,200 150 1,956 150 0,057 0,5 0,125
1,425 1,641
Immobilized cells
0.285 225 3,21 226 0,047 0,578 0,379
17%
Free cells
0,11 150 1,547 150 0,053 0,578 0,165
2,309 1,198
Immobilized cells
0,254 223,21 1,854 150 0,051 0,594 0,412

a) Immobilized cells

Figure 18. (Continued)

 Beer Production with Encapsulated Yeast Cells 67

b) Free cells

Figure 18. Alcohol concentration dynamics in fermentation with S. carlsbergensis Saflager S-23 at
15°C.

Table 11. Kinetic parameters for secondary metabolite accumulation in alcohol

fermentation with free and immobilized bottom-fermenting S. carlsbergensis
Saflager S-23 cells

Original
Fermentation type YE YFA YA KA Error
extract
°Р mg/(g.d) -
Free 12.23 12.55 7.63 0.023 0.259
9
Immobilized 13.03 4.68 16.06 0.0205 0.345
Free 116.4 20.47 29.42 0.01 0.429
11
Immobilized 144.2 33.6 45.94 0.035 0.573
Free 61.05 13.61 21.19 0.025 0.129
13
Immobilized 31.72 8.15 11.36 0.0513 0.234
Free 73.2 36.6 22.6 0.025 0.429
15
Immobilized 36.6 10.3 13.2 0.035 0.573
Free 56.9 20.6 19.3 0.05 0.429
17
Immobilized 29.6 18.6 26.69 0.06 0.573

a) Immobilized cells

Figure 19. (Continued)

68 Vesela Shopska, Rositsa Denkova and Georgi Kostov

b) Free cells

Figure 19. Biomass concentration dynamics in fermentation with S. carlsbergensis Saflager S-23 at
15°C.

B. Effect of Fermentation Conditions on the Fermentation Process Time

The data from the experiments showed that the different factors had different effects on
the fermentation process time. Fermentation is an important field of interest for system
engineering due to its complex biological non-linear phenomena and dynamic processes
(Andres-Toro et al., 2010). Beer production with immobilized cells is generally industrialized
if the new characteristics acquired result in a more economical system, and the new
technology can be readily scaled up (Nedovic et al., 2005). Therefore, mathematical model
development is an important step towards the determination of suitable fermentation
parameters in order to achieve an optimized process (Adeyemo and Enitan, 2011). Moreover,
process optimization has been the key issue in maintaining operating conditions, increasing
product yields and ensuring product quality (Schmidt, 2005).
The aim of this working stage was to minimize the beer fermentation time while retaining
high product quality with an optimized process control strategy. Therefore, two mathematical
models for the effect of the main fermentation temperature (TMF), immobilized cell mass
(MIC) and original wort extract (OE) on the main fermentation and maturation time were
developed. The results on the optimization of the primary fermentation model were used for
beer production under laboratory conditions. Meanwhile, the system productivity was
determined and could be used for the batch fermentation transfer into a continuous mode.

Figure 20. Ester concentration dynamics in fermentation with free and immobilized S. carlsbergensis
Saflager S-23 cells at 15°C.
 Beer Production with Encapsulated Yeast Cells 69

Figure 21. Higher alcohol concentration dynamics in fermentation with free and immobilized S.
carlsbergensis Saflager S-23 cells at 15°C.

Figure 22. Aldehyde concentration dynamics in fermentation with free and immobilized S. carlsbergensis
Saflager S-23 cells at 15°C.
Legend: I – immobilized cells; F – free cells; 9, 11, 13, 15, 17 – original extract of the wort, °P.
The experiments were conducted with both strains under the immobilization conditions
defined. For the purposes of the experiment, wort with original extract content (OE) 8.5 ±
0.5°P, 10.5 ± 0.5°P, 13 ± 0.5°P, 15.5 ± 0.5°P and 17.5 ± 0.5°P was used.
Central Composite Design (CCD) of type 23 with star arm and block structure was used
for the optimization of the fermentation parameters TMF, OE and MIC. The system
productivity was calculated according to Kopsahelis et al., 2007.

Modelling of Fermentation with Immobilized S. Cerevisiae Safbrew S-33 Cells

The results of the experiment are presented in Table 12, and those of the statistical
analysis in Table 13. The dynamics of the parameters studied is summarized on Figure 23 and
Figure 24. The non-significant variables were eliminated according to their p-value at a
confidence level α = 0.95 (Table 13). The following adequate mathematical model was
obtained (10):

MF  78.7  32.3TMF  15.13OE  36.05M IC  10.44Т MF

2
 10.5OE.M IC  20,75M IC
2
(9)

The model obtained (Equation 9) showed that the main fermentation time decreased with
the increase in TMF and MIC. On the contrary, the OE increase resulted in a prolonged primary
fermentation. It is interesting to note that the model showed an increase in the main
70 Vesela Shopska, Rositsa Denkova and Georgi Kostov

fermentation time with the increase in TMF and MIC when these parameters were squared. It
can be assumed that the discrepant effects of TMF and MIC were due to the substrate and
product diffusion into and out of the beads. The model obtained (Equation 10) confirmed the
suggestion that the combined effect of the factors would be of key importance to the objective
function. Therefore, it can be summarized that the reduction of the primary fermentation time
will occur if the fermentation temperatures are high and the wort used has low original
extract. The decrease in TMF below the recommended fermentation temperature for the
selected yeast strain led to a very prolonged fermentation. The mathematical model was
optimized and the following operating conditions were determined: TMF = 17.24°C; OE =
10.24°P; MIC = 11.56 g. Under these conditions, the main fermentation time had to be 36 h. It
was found that the real main fermentation time was 12 h longer than the main fermentation
time determined by the model. This could be related to the system diffusion resistance which
hindered the substrate transfer to the cells (Naydenova et al., 2014c).
Another adequate mathematical model was developed for the effect of TMAT, OE and MIC
on the maturation time:

MatF  178.3  20.2Tmat  9.4OE  19.9M ic  28,8Т mat

2
 10.4OE 2 (10)

The model obtained (Equation 9) showed a controversial effect of TMAT on the maturation
time. First, the increase in TMAT affected the maturation time “negatively.” On the other hand,
a higher temperature led to the synthesis of more carbonyl compounds, which resulted in
prolonged maturation. The maturation time also increased with the increase in MIC.
Consequently, the maturation was affected not only by the operating conditions, but also by
the carbonyl compound concentration at the beginning of the maturation. The second
mathematical model (Equation 10) can also be optimized. However, this is not necessary
since the model structure is related to the main fermentation model (Equation 9) (Naydenova
et al., 2014c).
One of the purposes of the study was to select the batch fermentation conditions which
would be transferred into a continuous fermentation system. According to Kopsahelis et al.,
2007, one of the parameters for the comparison between batch fermentations was system
productivity, which could be estimated at various stages of fermentation and for various
parameters: ethanol, green beer, beer. The productivity depended on the fermentation time
and could be used for comparison between batch and continuous fermentation (Naydenova et
al., 2014c).
It can be summarized that the ethanol productivity increased with the increase in each of
the parameters studied (Table 12). It is interesting to note that the highest ethanol productivity
during the main fermentation was noted for variant 17 where all the factors studied were at
their upper level. When the temperature was in its star point of 19.5°C, the system
productivity was a little lower. This could be related to the OE decrease which led to the
production of less ethanol. Therefore, the ethanol productivity determined for the total
fermentation time was the highest when OE was in its star point, i.e., 17.5°P. The green beer
productivity and beer productivity depended on the ethanol productivity, the degree of
fermentation and OE. Therefore, the highest green beer productivity was recorded for variant
17, and the highest beer productivity for variant 10 (Table 12). The green beer productivity
can be used for the transfer of batch fermentation into a continuous mode. Our subsequent
 Beer Production with Encapsulated Yeast Cells 71

research showed that the system productivity in a batch mode was related to the system
productivity in a continuous mode (Naydenova et al., 2014c).

Table 12. Experimental design and results for modeling and optimization of
fermentation parameters for fermentation with S. cerevisiae Safbrew S-33

QETH, g/(dm3.h)

Q, g/(dm3.h)
№

Parameters

MatF, h
MF, h
TMF, °С OE, °P Mic, g
MF F QMF QF
coded real coded real coded real
1 -α 10.5 0 13 0 10 180 168 0.36 0.43 29.38 33.64
2 -1 12.5 -1 10.5 -1 5 144 84 0.37 0.70 19.92 34.01
3 -1 12.5 1 15.5 -1 5 192 108 0.38 0.76 40.72 86.14
4 -1 12.5 -1 10.5 1 15 96 132 0.53 0.45 28.02 21.68
5 -1 12.5 1 15.5 1 15 108 120 0.73 0.72 85.77 76.51
6 0 15 0 13 0 10 72 96 0.87 0.74 69.28 57.27
7 0 15 0 13 0 10 76 90 0.82 0.79 64.67 60.68
8 0 15 0 13 0 10 74 100 0.96 0.71 75.67 54.61
9 0 15 -α 8.5 0 10 72 84 0.54 0.54 17.92 17.64
10 0 15 +α 17.5 0 10 120 84 0.73 1.13 111.76 158.97
11 0 15 0 13 -α 1.1 228 72 0.29 0.87 23.49 68.49
12 0 15 0 13 +α 18.9 72 84 0.84 0.83 67.63 63.43
13 0 15 0 13 0 10 68 92 0.92 0.77 73.35 59.76
14 1 17.5 -1 10.5 -1 5 72 84 0.70 0.68 36.64 34.87
15 1 17.5 1 15.5 -1 5 132 84 0.57 1.00 62.99 111.51
16 1 17.5 -1 10.5 1 15 42 114 1.08 0.60 50.46 29.82
17 1 17.5 1 15.5 1 15 54 78 1.28 0.75 133.35 78.62
18 +α 19.5 0 13 0 10 54 102 1.20 0.69 107.42 51.08
TMF – main fermentation temperature; OE – original extract; Mic – immobilized cell mass; QETH –
ethanol productivity; Q – beer productivity; MF – main fermentation; MatF – maturation time; F –
for total fermentation time (F=MF+MatF); ±α=1.7885

А mathematical model was developed for the influence of the fermentation parameters on
green beer productivity (equation 12). The model showed that the increase in all tested
parameters led to an increase in the system productivity. The negative sign of the MIC2
coefficient showed indirectly that the model did not take full account of the diffusion
resistance in the system. However, the model described the experimental data with high
accuracy (R2=96.6%) and can be used for the transfer of a batch fermentation system into a
continuous mode (Naydenova et al., 2014c).

QMF  67.73  17.26TMF  24.7OE  15.02M IC  11.68OE.M IC  7.62M IC

2
(11)
72 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Table 13. ANOVA for “Main fermentation time”

Sum of Mean
Source Df F-Ratio P-Value Significance
Squares Square
A:TMF 15041.2 1 15041.2 101.17 0 Significant
B:OE 3296.19 1 3296.19 22.17 0.0022 Significant
C:MiC 18710 1 18710 125.85 0 Significant
AA 2109.95 1 2109.95 14.19 0.007 Significant
AB 18 1 18 0.12 0.7381 insignificant
AC 72 1 72 0.48 0.5089 insignificant
BB 416.955 1 416.955 2.8 0.1379 insignificant
BC 882 1 882 5.93 0.045 Significant
CC 7400.95 1 7400.95 49.78 0.0002 Significant
blocks 418.191 1 418.191 2.81 0.1374 Significant
Total error 1040.71 7 148.673
Total (corr.) 47521.8 17
R2 = 96.74%; R2 (за d.f.) = 94.96%; Standart error = 12.04; Аbsolute standart error = 7.84; Durbin-
Watson = 1.75.

a) real extract b) alcohol of beer

Figure 23. Extract and alcohol concentration dynamics in the factor space for the fermentation time.

a) aldehydes b) vicinal diketones (VDK)

Figure 24. Carbonyl compound concentration dynamics in the factor space for the fermentation time.

Modeling of Fermentation with Immobilized S. Carlsbergensis Saflager S-23 Cells

An analogical study was conducted with the bottom-fermenting S. carlsbergensis
Saflager S-23. The results are shown in Table 14 and Table 15 and on Figure 25 and Figure
26.
 Beer Production with Encapsulated Yeast Cells 73

As a result of the experiments, the effect of the examined parameters on the main
fermentation time was established. The non-significant variables were eliminated according
to their p-value at confidence level α=0.95 (Table 14) (Naydenova et al., 2014b). The
following adequate mathematical model was obtained after the removal of the non-significant
variables:

MF  176.4  103.6TMF  35.7OE  37.4M ic  54.1Т MF

2
(12)

The model obtained (equation 12) did not confirm the suggestion that the combined
effect of the factors would be of key importance to the objective function. The main
fermentation time decreased with the increase in TMF and Mic. On the contrary, the OE
increase resulted in a prolonged primary fermentation. It can be assumed that the discrepant
effect of the temperature and the original extract on the main fermentation time was due to the
mass transfer of the substrate and ethanol in the capsule. It is interesting to note that there was
a significant area in the response surface where the main fermentation time was up to 100 h.
Therefore, the optimal values of the main fermentation time were observed between the 48th
and the 72nd h. It may be noted that OE and Mic had a significant impact on the main
fermentation course for all variants at the same fermentation temperature. Mic affected the
main fermentation time but did not affect the total fermentation time. It is known that more
secondary metabolites are synthesized due to the increase in Mic, which leads to a prolonged
maturation. The low Mic led to the synthesis of low amounts of secondary metabolites, which
resulted in reduced maturation time (Naydenova et al., 2014b).
Similarly, a mathematical model was developed for the experimental data on maturation
time:

MatF  178.3  20.2Tmat  9.4OE  19.9M ic  28,8Т mat

2
 10.4OE 2 (13)

According to the model, the temperature had the most significant impact on the reduction
of maturation time. The microcapsule mass showed a “negative” effect on the objective
function. This can be easily explained by the fermentation dynamics. The increased Mic and
OE led to the synthesis of more carbonyl compounds, which resulted in increased maturation
time (Figure 25). Nevertheless, Figure 26 illustrates an interesting phenomenon: there was a
relatively small zone (between 11 and 13 °Р) wherein the increase in the extract did not lead
to an increase in the carbonyl compound concentration. This could be related to the provision
of optimal growth conditions for the immobilized cells and the diffusion resistances in the
system (Naydenova et al., 2014b).
At the beginning of the maturation, a second aldehyde peak was observed, which was
essential for the model accuracy. The first peak occurred during the main fermentation
(between the 36th h and the 96th h). The second peak was observed between the 12th h and the
24th h of maturation. The second peak was clearly defined for the higher extracts and its
height was similar to the first one. Contrariwise, the lower extract led to indistinct aldehyde
peaks. The vicinal diketone peak occurred during the main fermentation. The OE increase
resulted in the peak displacement to the later period of the main fermentation. The vicinal
diketone reduction started before the end of the main fermentation (Naydenova et al., 2014b).
74 Vesela Shopska, Rositsa Denkova and Georgi Kostov

The mathematical model was optimized and the following operating conditions were
determined: TMF = 15.7±0.5°C; OE = 9.72±1°P; MIC = 13±0.5 g. Under these conditions, the
main fermentation time had to be between 48 hours and 72 hours. It was found that the real
main fermentation time was 12 h longer than the main fermentation time determined by the
model. This could be related to the system diffusion resistance which hindered the substrate
transfer to the cells (Naydenova et al., 2014b).
In that case, the ethanol productivity of the system varied from 0.08 to 0.61 g/(dm3.d), but
for the majority of the variants it was in the 0.18-0.35 g/(dm3.d) range. The highest
productivity was observed when the main fermentation lasted for 78 hours (10.5ºP, 15ºC and
15g immobilized cells). It could be related to the fermentation time reduction at low OE, high
TMF and high MIC. On the other hand, the OE increase led to the production of more ethanol.
The green beer productivity of the system depended on the ethanol productivity, the degree of
fermentation and OE. The green beer productivity was in the range of 5.78-65.02 g/(dm3.d),
but the optimal variants had productivity of approximately 20 g/(dm3.d). The differences
between beer productivity and green beer productivity were insignificant. The beer
productivity varied between 8.26 and 69.61 g/(dm3.d) and increased with the increase in the
OE. After a similar statistical analysis of the effect of various factors on the green beer
productivity, the following mathematical model was developed (Naydenova et al., 2014b):

QP  20.33  10.73T  8.19OE  6.92M IC  4.55T .OE  6.15Т .M IC  2.47OЕ 2  2.87M IC 2 (14)

All the parameters studied affect the green beer productivity positively. The model could
be used for easy and fast calculation of productivity for any operating parameters (the data
should be pre-coded).

Comparison of the Results for S. Carlsbergensis Saflager S-23 and S. Cerevisiae

Safbrew S-33
The data obtained from the modeling of the fermentation with S. carlsbergensis Saflager
S-23 and S. cerevisiae Safbrew S-33 showed some general trends. Firstly, the amount of the
immobilized preparation used had contradictory effects on the different fermentation stages.
The immobilized preparation mass reduced the main fermentation time but increased the total
process time due to the production of more carbonyl substances. This effect can be minimized
by increasing the fermentation temperature, hence, the post-fermentation and maturation
temperature. A similar increase was related to beer deterioration. The original wort extract as
a whole provoked a fermentation time increase.
The potential productivity of the system in continuous mode was influenced by all
parameters and increased with their increase. The immobilized cell system had significant
productivity and allowed beer production in a continuous mode, the characteristics of the
resultant beer being comparable to those of commercial beers.
 Table 14. Experimental design and results on the modeling and optimization of fermentation parameters
S. carsbergensis Saflager S-23 (Naydenova et al., 2014b)

№ Parameters QETH, g/(dm3.h) Q, g/(dm3.h)

TMF, °С OE, °P Mic, g MF, h MatF, h
MF F QMF QF
coded real coded real coded real
1 -α 8.0 0 13 0 10 636 50 0.08 0.08 5.78 8.26
2 -1 10.0 -1 10.5 -1 5 288 168 0.18 0.25 9.26 9.60
3 -1 10.0 1 15.5 -1 5 324 180 0.18 0.29 15.97 27.21
4 -1 10.0 -1 10.5 1 15 192 264 0.27 0.36 13.41 14.10
5 -1 10.0 1 15.5 1 15 300 204 0.20 0.33 17.49 28.72
6 0 12.5 0 13 0 10 204 180 0.27 0.40 18.76 22.47
7 0 12.5 0 13 0 10 196 172 0.29 0.42 19.53 23.38
8 0 12.5 0 13 0 10 200 190 0.28 0.41 19.14 22.91
9 0 12.5 -α 8.5 0 10 108 156 0.39 0.54 11.51 13.41
10 0 12.5 +α 17.5 0 10 264 84 0.30 0.34 45.02 49.24
11 0 12.5 0 13 -α 1.1 252 132 0.24 0.32 18.61 19.55
12 0 12.5 0 13 +α 18.9 96 168 0.60 0.90 40.49 49.94
13 0 12.5 0 13 0 10 210 175 0.27 0.39 18.22 21.82
14 1 15.0 -1 10.5 -1 5 144 96 0.31 0.47 14.16 19.34
15 1 15.0 1 15.5 -1 5 192 96 0.35 0.50 32.65 47.82
16 1 15.0 -1 10.5 1 15 78 126 0.67 0.89 36.50 33.87
17 1 15.0 1 15.5 1 15 120 168 0.59 0.83 65.02 69.61
18 +α 17.0 0 13 0 10 120 72 0.51 0.71 40.54 42.37
TMF – temperature of main fermentation; OE – original extract; Mic – immobilized cell mass; QETH – ethanol productivity; Q – beer productivity; MF –
for main fermentation; MatF – maturation time; F – for total fermentation time (F=MF+MatF); ±α=1.7885.
76 Vesela Shopska, Rositsa Denkova and Georgi Kostov

Table 15. ANOVA for “Main fermentation time” and “Maturation time”
(Naydenova et al., 2014b)

Sum of
Source DF Mean Square F-ratio P-value Significance
Squares
“Main fermentation time”
A: TMF 154598.21 1 154598 49.25 0.0002 significant
B: OE 18351.3 1 18351.3 5.85 0.0462 significant
C: Mic 20104.8 1 20104.8 6.40 0.0392 significant
AA 35716.9 1 35716.9 11.38 0.0119 significant
AB 378.125 1 378.125 0.12 0.7387 insignificant
AC 36.125 1 36.125 0.01 0.9176 insignificant
BB 1961.46 1 1961.46 0.62 0.4552 insignificant
BC 561.125 1 561.125 0.18 0.6851 insignificant
CC 3465.47 1 3465.47 1.10 0.3283 insignificant
blocks 4565.37 1 4565.37 1.45 0.2670 insignificant
Total error 21974.7 7 3139.24
Total (corr.) 274597. 17
R2 = 91.99%; Standard error = 5.063; Absolute average error = 2.832; Durbin-Watson = 3.18;

“Maturation time”
A: TMF 5866.31 1 5866.31 2.80 0.1380 significant
B: OE 1261.83 1 1261.83 0.60 0.4629 significant
C: Mic 5696.13 1 5696.13 2.72 0.1430 significant
AA 12883.0 1 12883.0 6.16 0.0421 significant
AB 1012.5 1 1012.5 0.48 0.5091 insignificant
AC 40.5 1 40.5 0.02 0.8933 insignificant
BB 1748.82 1 1748.82 0.84 0.3910 significant
BC 112.5 1 112.5 0.05 0.8233 insignificant
CC 28.8288 1 28.8288 0.01 0.9099 insignificant
blocks 6092.72 1 6092.72 2.91 0.1317 significant
Total error 14648.7 7 2092.67
Total (corr.) 48580.9 17
R2 = 89.95%; Standard error = 5.75; Absolute average error = 5.67; Durbin-Watson = 2.82;

CONCLUSION
The high cell load in yeast cell immobilization in matrices / carriers of different nature
was the basis for the reduction of the bioreactor working volume and the fermentation time.
This led to high volumetric productivity of the system and reduced capital expenditure.
Simultaneously, immobilized cell systems provided opportunities for continuous fermentation
processes with simple equipment and at reduced microbial contamination risk. All these
advantages determine the significance of immobilized cell introduction into beer
manufacture.
The present study was focused on the development of technological solutions for
obtaining beer with immobilized yeast cells in liquid core capsules. The yeast strains S.
cerevisiae Safbrew S-33 and S. carlbergenisis Saflager S-23 were selected because they did
not lose their activity in the course of immobilization, which allowed their application as
 Beer Production with Encapsulated Yeast Cells 77

immobilized cell preparations. An accelerated beer method for obtaining a beverage with an
interesting organoleptic profile can be developed using immobilized preparations of the
selected strains.

a) real extract b) alcohol of beer

Figure 25. Extract and alcohol concentration dynamics in the factor space for the fermentation time;
(Naydenova et al., 2014b).

Along with the selection of a starter culture for beer production, the present study was
aimed at developing an immobilization procedure that would lead to obtaining mechanically
stable capsules, which would retain the maximum activity of the yeast culture. Thus, a
fundamentally new approach for simultaneous assessment of both the stability of the capsules
and their fermentation activity was carried out. As a result, an immobilization procedure for
obtaining liquid core capsules of alginates crosslinked with chitosan was developed. The data
indicated that the membrane formation time and the forming solution concentration had
significant influence on the mechanical stability and the gel formulation activity. These data
were consistent with the results reported by Lacik, 2004; Thu et al, 1996; Lacik et al., 1998;
Brissova et al., 1998; Lacik et al., 2001.

a) aldehydes b) vicinal diketones (VDK)

Figure 26. Carbonyl compound concentration dynamics in the factor space for the fermentation time;
(Naydenova et al., 2014b).

The immobilization process affected primary and secondary yeast metabolism to varying
degrees. This effect depended on the immobilization method and carrier, and the cultivation
conditions. The results obtained in the present work indicated that the yeast culture retained
its activity after immobilization. This provided grounds for the concusion that the primary
yeast metabolism did not affect the immobilization process materially; the only difference
was observed with the slow start of the process with immobilized cells. The lag was within 12
hours, which could be attributed to the presence of diffusion resistances. It was compensated
78 Vesela Shopska, Rositsa Denkova and Georgi Kostov

quickly and the two beer types obtained by the two fermentation types did not differ
substantially in terms of their basic physicochemical parameters at the end of the
corresponding fermentation process. The results obtained from the fermentation processes
conducted showed that immobilization had a more pronounced effect on the formation of
different groups of secondary metabolites.
Regardless of the type of fermentation, with free or immobilized cells, up to 80% of the
higher alcohols were synthesized during the main fermentation. The higher alcohol content
accumulated in the immobilized cell system was about 10-15% lower in comparison with
similar free cell processes. The experimental data were in accordance with the results cited by
Willaert and Nedovic, 2006; Ryder and Masschelein, 1985; Smogrovicova and Domeny,
1999; Mensour et al., 1997; Curin et al., 1987; Cop et al., 1989, showing that this decrease
was due to the limited cell growth, which was also confirmed by the results on the
fermentation process kinetics. The immobilization process affected the balance of the higher
alcohols produced: depending on the selected immobilization method and carrier, various
amounts of the different higher alcohol groups were synthesized in the various systems.
The experimental results showed contradictory effects on ester synthesis. The
predominant ester amount (about 60%) was formed during the main fermentation. The
immobilization process affected ester formation substantially. This had its explanation in
strain specificity. Another interesting result was the heightened ester formation at high
original wort extracts. These data were consistent with the observations of Peddie, 1990;
Nykanen and Nykanen, 1977; Engan and Aubert, 1977; Gee and Ramirez, 1994. Similarly to
the higher alcohol trend, the immobilization process affected the balance between the ester
groups produced.
Little attention is paid in literature to aldehyde production and reduction. But it should be
taken into account that in the post-fermentation and maturation processes aldehydes are
reduced to certain limits imposed by the relevant standards of the brewing industry.
A number of experiments demonstrated that vicinal diketone synthesis and reduction
were strongly dependent on the fermentation conditions chosen. The increase in the original
wort extract, the fermentation temperature and the immobilized preparation amount provoked
increased synthesis of this group of metabolites, but also led to more rapid carbonyl
compound reduction. In literature, data on this subject are contradictory but a large number of
authors note that immobilized cells produce greater amounts of diacetyl, which is reduced
faster (Virkajarvi and Kronlof, 1998; Virkajarvi and Pohjala, 2000; Kronlof et al., 1989;
Andersen et al., 1999; Onaka et al., 1985).
All of these observations served as the basis of the planned experiment conducted, which
aimed at the sеlection of fermentation process conditions for the two yeast strains (S.
carlsbergensis Saflager S-23 and S. cerevisiae Safbrew S-33) in order to ensure its
completion within 120 hours. It can be pointed out that, depending on the fermentation mode
calculated for the corresponding strain, the main fermentation process took place within 24-
72 hours. Along with the process intensification, the resulting beer was comparable in its
organoleptic profile and quality to commercial beers.
 Beer Production with Encapsulated Yeast Cells 79

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BIOGRAPHICAL SKETCHES
Name: Vesela Nevelinova Shopska

Affiliation: University of Food Technologies, Department of wine and brewing

technology
 Beer Production with Encapsulated Yeast Cells 89

Education:

 2014 - University of Food Technologies - PhD – Technology of alcoholic and non-

alcoholic beverages;
 2006 - University of Food Technologies – Master Degree – Wine and brewing
technology;

Address: 26 Maritza blvd, 4002 Plovdiv, Bulgaria

Research and Professional Experience:

2015 Assistant professor, University of Food Technologies

2012-2013 Specialist of bioengineering and technological processes management,
Institute of System Engineering and Robotics at Bulgarian Academy of Science

Research experience in the area of: beer fermentation; immobilized cells; beer
microbiology; ethanol production

Professional Appointments:

2015 Assistant professor, University of Food Technologies

2012-2013 Specialist of bioengineering and technological processes management,
Institute of System Engineering and Robotics at Bulgarian Academy of Science

Publications Last 3 Years:

1. Naydenova V., Kostov, G., Popova, Z. (2012). Comparative study of brewing yeast
strains for beer production with immobilized cells, Proceedings of 6th Central
European Congress on Food, 23-26 May 2012, Novi sad, Serbia, 1012-1017.
2. Kostov G., Yoncheva, T., Spassov, H., Koprinkova-Hristova, P., Stoyanov, N.,
Naydenova, V. (2012). Neural network models for quality assessment of red wines
Preprints of IFAC Workshop on Dynamics and Control in Agriculture and Food
Processing, 13-16 June 2012, Plovdiv, Bulgaria, 113-118.
3. Parcunev I, Naydenova, V., Kostov, G., Yanakiev, Y., Popova, Z., Kaneva, M.,
Ignatov, I. (2012). Modeling of alcohol fermentation in brewing - some practical
approaches, Proceedings of 26th European Conference on Modelling and
Simulation, 29 May – 1 June 2012, Koblenz, Germany, 434-440.
4. Naydenova V., Vassilev, S., Kaneva, M., Kostov, G. (2013). Encapsulation of
brewing yeast in alginate/chitosan matrix: comparative study of beer fermentation
with immobilized and free cells, Bulgarian Journal of Agricultural Science, 19
(2):123–127, IF=0.19.
5. Kostov G., Pircheva, D., Naydenova, V., Iliev, V., Lubenova, V., Ignatova, M.
(2013). Kinetics investigation of bio-ethanol production with free and immobilized
cells, Comptes rendus de l’ academie bulgare des sciences, 66 (10):1463-1472,
IF=0.21.
90 Vesela Shopska, Rositsa Denkova and Georgi Kostov

6. Popova S., Kostov, G.. Ignatova, M.. Lubenova, V., Naydenova, V., Pircheva, D.,
Nakov, L., Angelov, M., Mihailova, I. (2013). State and kinetic parameters
estimation of bio-ethanol production with immobilized cells, Proceedings of 2nd
International Conference on Applied and Computational Mathematics (ICACM '13),
14-16 May 2013, Athens, Greece, 51-57.
7. Vassilev S., Naydenova, V., Badova, M., Iliev, V., Kaneva, M., Kostov, G., Popova,
S. (2013). Modeling of alcohol fermentation in brewing – comparative assessment
of flavor profile of beers produced with free and immobilized cells, Proceedings of
27th European Conference on Modelling and Simulation, 27-31 May 2013, Alesund,
Norway, 415-421.
8. Naydenova V., Badova, M., Vassilev, S., Iliev, V., Kaneva, M., Kostov, G. (2014).
Encapsulation of brewing yeast in alginate/chitosan matrix: lab-scale optimization
of lager beer fermentation, Biotechnology and Biotechnological Equipment, 28 (2):
277-284 IF=0.622.
9. Iliev V., Naydenova, V., Kostov, G. (2014).Bioreactors with light-beads fluidized
bed: the voidage function and its expression, Acta Universitatis Cibiniensis, Series
E: Food Technology (AUCFT), 18 (2), 35-42.
10. Iliev V., Naydenova, V., Kostov, G. (2014). Study on the application of the
chitosan-alginate capsules and alginate-chitosan-alginate capsules in ethanol
fermentation, Proceeding of II International Congress “Food Technology, Quality
and Safety” FOODTECH 2014, 28-30 October 2014, Novi Sad, 494-499.
11. Iliev S., Popova, S., Mitrouchev, P., Naydenova, V., Iliev, V., Kostov, G. (2014).
Adaptive control of bio-ethanol production with immobilized cells, Proceedings of
International Conference “AGRI-FOOD SCIENCES, PROCESSES AND
TECHNOLOGIES” AGRI-FOOD 2014, 14-15 May 2014 Sibiu, Romania, 203-213.
12. Iliev V., Shopska, V., Kostov, G. (2014). Bioreactors with light-beads fluidized
bed: fluidization on aqueous ethanol solutions, Scientific works of University of
Food Technologies, VOLUME LXI, 210-216.
13. Kopanarova G., Naydenova, V., Iliev, V., Kostov, G. (2014). Immobilization of
yeast cells in polyvinyl alcohol for ethanol production. I. Preparation of PVA
particles by freeze-thawing method, Proceedings of International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD
2014, 14-15 May 2014 Sibiu, Romania, 214-222.
14. Naydenova V., Iliev, V., Kaneva, M., Kostov, G., Koprinkova-Hristova, P., Popova,
S. (2014). Modeling of alcohol fermentation in brewing – carbonyl compounds
synthesis and reduction, Proceedings of 28th European Conference on Modelling
and Simulation, 27-30 May 2014, Brescia, Italy, 279-284.
15. Naydenova, V., Iliev, V., Kostov, G. (2014). Lab-scale optimization of beer
fermentation with immobilized top-fermenting yeast, Proceeding of II International
Congress “Food Technology, Quality and Safety” FOODTECH 2014, 28-30 October
2014, Novi Sad,45-49.
16. Vassilev S., Shopska, V., Iliev, V., Kaneva, M., Kostov, G., Popova, S. (2015).
Modeling of alcohol fermentation in brewing – integrated approach for control of
continuous alcohol fermentation, Proceedings of 29th European Conference on
Modelling and Simulation, 26-29 May, Albena, Bulgaria, 286-291.
 Beer Production with Encapsulated Yeast Cells 91

17. Kostov, G., Lyubenova, V., Shopska, V., Petelkov, I., Ivanov, K., Iliev, V.,
Denkova, R., Ignatova, M. (2015).Software sensors for monitoring the biomass
concentration and the kinetics of continuous beer fermentation with immobilized
cells, Comptes rendus de l’Academie bulgare des Sciences, 68(11):1439-1448,
IF=0.284.
18. Denkova, R., Goranov, B., Denkova, Z., Kostov, G. (2015). Determination of the
kinetic parameters of batch fermentation of Lactobacillus plantarum X2 with
probiotic potential isolated from spontaneously fermented sourdough, Ukrainian
journal of food science, 4 (1): 59-66.
19. Goranov, B., Shopska, V., Denkova, R., Kostov, G. (2015). Kinetics of batch
fermentation in the cultivation of a probiotic strain Lactobacillus delbrueckii ssp.
bulgaricus B1, Acta Universitatis Cibiniensis, Series E: Food Technology (AUCFT),
19 (1): 63-74.
20. Denkova R., Goranov, B., Shopska, V., Denkova, Z., Kostov, G. (2015).
Determination of the kinetic parameters of batch fermentation of Lactobacillus
plantarum strains with probiotic potential, Journal of food and packaging science,
technique and technologies, 6: 52-57.

Name: Rositsa Stefanova Denkova

Affiliation: Department of “Biochemistry and molecular biology,” University of Food

Technologies, Plovdiv, Bulgaria.

Education: PhD in Biotechnology (Technology of bioactive substances), Master Degree

in “Industrial biotechnology,” Bachelor Degree in “Biotechnology.” All degrees were
received from Sofia University “St. Kliment Ohridski,” Sofia, Bulgaria.

Address: 4002, 26 “Maristza” blvd, Plovdiv, Bulgaria

Research and Professional Experience: isolation and identification of microorganisms,

selection of microorganisms for development of starters, examination of probiotic properties,
development of starters for functional foods and beverages

Professional Appointments:

2014 – Present, Assistant professor, Department of “Biochemistry and molecular

biology,” University of Food Technologies

Publications Last 3 Years:

1. R. Denkova, V. Yanakieva, Z. Denkova, D. Blazheva (2013). Antimicrobial activity

of probiotic lactobacilli, bifidobacteria and propionic acid bacteria, isolated from
different sources. In: Microbial pathogens and strategies for combating them:
92 Vesela Shopska, Rositsa Denkova and Georgi Kostov

science, technology and education (A. Méndez-Vilas, Ed.). Formatex 2013, 857 -
864.
A. Slavov, Z. Denkova, R. Denkova, Z. Urshev (2013). Morphological characteristics
and genetic identification of seven bacterial strains for wastewater treatment. Journal
of Food and Packaging Science Technique and Technologies, Issue II, №2, 2013, 16
- 20. ISSN 1314-7773.
2. R. Denkova, V. Yanakieva, Z. Denkova, P. Mancheva (2013). Antimicrobial activity
of a probiotic strain Lactobacillus acidophilus A2 of human origin against pathogens.
Journal of Food and Packaging Science Technique and Technologies, Issue II, №2,
2013, 26 - 30. ISSN 1314-7773.
3. R. Denkova, V. Yanakieva, Z. Denkova, P. Hristozova (2013). Inhibitory activity of
the probiotic strain Bifidobacterium bifidum Bif. 4 of human origin against
pathogens. Journal of georgievaFood and Packaging Science Technique and
Technologies, Issue II, №2, 2013, 21 - 25. ISSN 1314-7773.
4. N. Valcheva-Jekova, Z Denkova, R. Denkova (2013). Physicochemical and
microbiological characteristics of spring waters in Haskovo. Journal of Food and
Packaging Science Technique and Technologies, Issue II, №2, 2013, 21 - 25. ISSN
1314-7773.
5. V. Lyubenova, G. Kostov, R. Denkova, D. Pircheva, M. Ignatova, M. Angelov
(2013). Adaptive Control of Continuous Fermentation with Immobilized Yeasts
Saccharomyces Cerevisiae BO 213. Recent Advances in Industrial and
Manufacturing Technologies. 35 – 40. ISBN: 978-1-61804-186-9.
6. Kostov G., R. Denkova, D. Pircheva, V. Lubenova, L. Nakov, M. Ignatova, M.
Angelov (2013). Kinetics investigation of bio-ethanol production with free and
immobilized cells. Comptes rendus de l academie bulgare des sciences 66 (10),
1463-1472.
7. R. Denkova, V. Yanakieva, Z. Denkova, V. Nikolova, V. Radeva (2013). In vitro
inhibitory activity of Bifidobacterium and Lactobacillus strains against Candida
albicans. Bulgarian Journal of Veterinary Medicine (2013), 16, No 3, 186−197.
ISSN 1311-1477 (print); ISSN 1313-3543 (online).
8. R. Denkova, Z. Denkova, V. Yanakieva, S. Ilieva (2013). Antimicrobial activity of
the probiotic strain Lactobacillus delbrueckii ssp. bulgaricus GB of human origin
against pathogens, Food and Environment Safety, Volume XII, Issue 2 – 2013, p.
123 – 129. ISSN 2068 – 6609.
9. R. Denkova, V. Yanakieva, Z. Denkova, L. Georgieva (2013). Enzymatic profile of
Lactobacillus brevis strains isolated from different sources. Научни трудове на
Русенския Университет - 2013, Scientific Works of the University of Ruse,
Proceedings v. 52, s. 10.2, 61 - 65. ISSN 1311-3321.
10. R. Denkova, Z. Denkova, V. Yanakieva, V. Radeva, Y. Tumbarski (2013).
Antimicrobial activity of lactobacilli of human origin against Pseudomonas
aeruginosa. Научни трудове на Русенския Университет - 2013, Scientific Works
of the University of Ruse, Proceedings v. 52, s. 10.2, 11 - 15. ISSN 1311-3321.
11. Z. Denkova, V. Yanakieva, R. Denkova, I. Dobrev, S. Kozludzhova (2013).
Examining the possibilities for application of pea milk in obtaining fermented
probiotic foods. Научни трудове на Русенския Университет - 2013, Scientific
 Beer Production with Encapsulated Yeast Cells 93

Works of the University of Ruse, Proceedings v. 52, s. 10.2, 31 - 35. ISSN 1311-
3321.
12. R. Cholakov, R. Denkova, V. Yanakieva, Z. Denkova, Z. Urshev, S. Syuleyman
(2013). Molecular-genetic and biochemical characterization of Saccharomyces
cerevisiae strain 36-6G. Научни трудове на Русенския Университет - 2013,
Scientific Works of the University of Ruse, Proceedings v. 52, s. 10.2, 83 - 87. ISSN
1311-3321.
13. B. Goranov, Z. Denkova, G. Kostov, R. Denkova (2013). Possibilities for the
biological acidification of mash in the production of wort: Kinetics of lactic acid
production in a free cell culture of Lactobacillus delbrueckii ssp. bulgaricus M3.
Научни трудове на Русенския Университет - 2013, Scientific Works of the
University of Ruse, Proceedings v. 52, s. 10.2, 36 - 40. ISSN 1311-3321.
14. Y. Tumbarski, Z. Denkova, V. Yanakieva, I. Dobrev, R. Denkova (2013). Role of
the noroviruses as substantial foodborne pathogens: A review. Научни трудове на
Русенския Университет - 2013, Scientific Works of the University of Ruse,
Proceedings v. 52, s. 10.2, 70 - 74. ISSN 1311-3321.
15. R. Denkova, V. Yanakieva, Z. Denkova (2013). Enzymatic profile of strains of
Lactobacillus fermentum isolated from different sources. Селскостопанска
Академия Институт за Изследване и Развитие на Храните. Международна
научно-практическа конференция “Храни, технологии и здраве,” 2013, Сборник
доклади / Agricultural Academy Food Research and Development Institute.
International Scientific-Practical Conference “Food, Technologies and Health,” 2013
Proceedings Book. ISBN 978-954-24-0229-9.
16. Z. Denkova, D. Dimitrov, R. Nikolova, R. Denkova, I.Gavrailov, D. Dimbareva
(2013). Biological preservation of cosmetic creams with probiotic bacteria.
Agricultural Academy Food Research and Development Institute. International
Scientific-Practical Conference “Food, Technologies and Health,” 2013 Proceedings
Book. ISBN 978-954-24-0229-9.
17. R. Cholakov, Z. Denkova, Z. Urshev, V. Yanakieva, R. Denkova (2013).
Biochemical and molecular-genetic characterization of Weisella confusa strain BP15,
isolated from fermented cereal beverage. Agricultural Academy Food Research and
Development Institute. International Scientific-Practical Conference “Food,
Technologies and Health,” 2013 Proceedings Book. ISBN 978-954-24-0229-9.
18. M. Yordanova, S. Ilieva, B. Goranov, Y. Evstatieva, K. Milanova, K. Bozhanchev,
R. Denkova, D. Nikolova (2014). Optimization of nutrient medium composition by
mathematical modeling for production of Rhizopus arrhizus KB-2 lipase. Bulgarian
Journal of Agricultural Science 20 (1): 87–92. ISSN 1310-0351.
19. R. Denkova, Z. Denkova, V. Yanakieva, L. Georgieva (2013). Highly active freeze-
dried probiotic concentrates with long shelf life. Food and Environment Safety -
Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava,
Volume, Issue III – 2013, (in press). ISSN 2068 – 6609.
20. R. Denkova, S. Ilieva, Z. Denkova, L. Georgieva, A. Krastanov (2013).
Examination of the technological properties of newly isolated strains of the genus
Lactobacillus and possibilities for their application in the composition of starters.
Biotechnology and biotechnological equipment, 28 (3): 487 – 494, DOI: 10.1080/
13102818.2014.918701.
94 Vesela Shopska, Rositsa Denkova and Georgi Kostov

21. Z. Denkova, I. Dobrev, R. Denkova, V. Yanakieva, S. Kozludzhova (2014). Pea

probiotic foods and beverages during storage. Journal of Food and Packaging
Science, Technique and Technologies №3, 69 - 73. ISSN 1314-7773.
22. R. Denkova, L. Georgieva, Z. Denkova (2014). Freeze-dried sourdough starters.
Journal of Food and Packaging Science, Technique and Technologies №3, 74 - 80.
ISSN 1314-7773.
23. R. Denkova, L. Georgieva, Z. Denkova, V. Yanakieva, S. Ilieva (2014). Highly
active probiotic concentrates with long shelf life obtained using cryoprotectants of
plant origin. Journal of Food and Packaging Science Technique and Technologies
№4, 2014, 66 – 70. ISSN 1314-7773.
24. Denkova Z., I. Murgov, R. Denkova, R. Enikova (2014). Evaluation of the
toxicological safety of a freeze-dried probiotic preparation. Food and Environment
Safety, Volume XIII, Issue 2 – 2014, 109 - 115. ISSN 2068 – 6609.
25. Denkova R., H. Strinska, Z. Denkova, G. Dobrev, D. Todorov, K. Mladenova, S,
Shishkov (2014). Study of the adhesion of Lactobacillus plantarum strains with
probiotic properties to MDCK. Food and Environment Safety, Volume XIII, Issue 3
– 2014, 214 - 217. ISSN 2068 – 6609.
26. Dobrev I., R. Denkova, Z. Denkova, E. Filipov, V. Yanakieva (2014). Non-
traditional yogurt from cow's milk enriched with pea protein. Journal of Food and
Packaging Science, Technique and Technologies №5, 2014:38 - 44. ISSN 1314-
7773.
27. N. Vulcheva-Zhekova, R. Denkova, Z. Denkova, R. Nikolova (2014).
Characterization of a strain isolated from a thermal healing spring in Stara Zagora
Mineral Baths. Bulgarian Journal of Veterinary Medicine 17 (1): 115 – 116. ISSN
1311-1477.
28. D. Teneva, Z. Denkova, R. Denkova, T. Atanasova, N. Nenov (2014). Antimicrobial
activity of essential oils and extracts from black pepper, cumin, coriander and
cardamom. Bulgarian Journal of Veterinary Medicine 17 (1): 113 – 114. ISSN 1311-
1477.
29. Dobrev, Z. Denkova, V. Yanakieva, E. Filipov, R. Denkova, B. Goranov (2014).
Non-traditional pea yogurts. Bulgarian Journal of Veterinary Medicine 17 (1): 111 –
112. ISSN 1311-1477.
30. R. Denkova, B. Goranov, Z. Denkova, G. Kostov, I. Georgieva (2014).
Determination of the kinetic parameters of batch fermentation of Lactobacillus
paracasei RN5 with probiotic potential. Scientific Works of the University of Ruse -
2014, Proceedings, Volume 53, book 10.2 Biotechnologies and food technologies, 9 –
13. ISSN: 1311-3321.
31. R. Denkova, H. Strinska, Z. Denkova, G. Dobrev, D. Todorov, K. Mladenova, S.
Shishkov (2014). Study of the adhesion of Lactobacillus acidophilus strains with
probiotic properties to MDCK. Scientific Works of the University of Ruse - 2014,
Proceedings, Volume 53, book 10.2 Biotechnologies and food technologies, 22 – 26.
ISSN: 1311-3321.
32. R. Denkova, V. Vlaseva, E. Filipov, Z. Denkova, I. Dobrev, B. Goranov, P.
Merdzhanov (2014). Obtaining chocolate masses with probiotic lactobacilli strains.
Scientific Works of the University of Ruse - 2014, Proceedings, Volume 53, book 10.2
Biotechnologies and food technologies, 55 – 58. ISSN: 1311-3321.
 Beer Production with Encapsulated Yeast Cells 95

33. D. Teneva, R. Denkova, Z. Denkova (2014). Selection and biochemical

characterization of lactic acid bacteria strains isolated from salad dressings.
Scientific Works of the University of Ruse - 2014, Proceedings, Volume 53, book 10.2
Biotechnologies and food technologies, 32 – 35. ISSN: 1311-3321.
34. D. Teneva, Z. Denkova, R. Denkova, R. Cholakov, B. Goranov (2014).
Characteristics of lactic acid bacteria strains isolated from salad dressing. Scientific
Works of the University of Ruse - 2014, Proceedings, Volume 53, book 10.2
Biotechnologies and food technologies, 51 – 54. ISSN: 1311-3321.
35. R. Denkova, S. Ilieva, Z. Denkova, L. Georgieva, M. Yordanova, D. Nikolova, Y.
Evstatieva (2014). Production of wheat bread without preservatives using sourdough
starters. Biotechnology and Biotechnological Equipment 28 (5), 889-898. DOI:
10.1080/13102818.2014.965057.
36. R. Cholakov, R. Denkova, D. Teneva, V. Yanakieva, I. Dobrev, Z.
Denkova, Z. Urshev (2014). Molecular-genetic and biochemical characterization of
Saccharomyces cerevisiae strain 25-G, isolated from fermented cereal beverage.
Food and Environment Safety, Issue 4 – 2014: 360 - 364. ISSN 2068 – 6609.
37. R. Denkova, B. Goranov, V. Shopska, Z. Denkova, G. Kostov (2015).
Determination of the kinetic parameters of batch fermentation of Lactobacillus
plantarum strains with probiotic potential. Journal of Food and Packaging Science,
Technique and Technologies №6, 2015:52 - 57. ISSN 1314-7773.
38. D. Teneva, R. Denkova, B. Goranov, Z. Denkova, P. Popova (2015). Antimicrobial
activity of Lactobacillus plantarum strains against pathogens. Journal of Food and
Packaging Science, Technique and Technologies №6, 2015: 117 – 123. ISSN 1314-
7773.
39. D. Teneva, R. Denkova, B. Goranov, Z. Denkova, P. Popova (2015). Probiotic
properties of Lactobacillus plantarum strains isolated from salad dressings. Journal
of Food and Packaging Science, Technique and Technologies №6, 2015: 58 - 63.
ISSN 1314-7773.
40. R. Denkova, B. Goranov, Z. Denkova, V. Shopska, G. Kostov (2015).
Determination of the kinetic parameters of batch fermentation of Lactobacillus
plantarum X2 with probiotic potential isolated fron spontaneously fermented
sourdough. Ukrainian Food Journal 4 (1): 59 – 66. ISSN 2313-5891 (Online); ISSN
2304-974X (Print).
41. B. Goranov, R. Denkova, D. Teneva, Z. Denkova, P. Popova (2015). Molecular-
genetic identification of Lactobacillus strains, isolated from homemade yoghurt.
Ukrainian Food Journal 4 (1):67 – 76. ISSN 2313-5891 (Online); ISSN 2304-974X
(Print).
42. B. Goranov, V. Shopska, R. Denkova, G. Kostov (2015). Kinetics of batch
fermentation in the culturing of a probiotic strain Lactobacillus delbrueckii ssp.
bulgaricus B1. Acta Universitatis Cibiniensis, Series E: Food Technology (AUCFT),
XIX(1): 61 – 72.
43. Teneva D., Z. Denkova, R. Denkova, T. Atanasova, N. Nenov (2015). Antimicrobial
activity of essential oils and extracts from black pepper, cumin, coriander and
cardamom. Bulgarian Journal of Veterinary Medicine ONLINEFIRST. DOI:
10.15547/bjvm.881. ISSN 1311-1477.
96 Vesela Shopska, Rositsa Denkova and Georgi Kostov

44. Denkova R., G. Dobrev, B. Goranov, D. Teneva, Z. Denkova, V. Shopska, S. Fortier

(2015). Obtaining probiotic yogurts with phytosterol esters. Scientific Works of the
University of Food Technologies (in press).
45. Teneva D., R. Denkova, B. Goranov, Z. Denkova (2015). Probiotic properties of
Lactobacillus delbrueckii ssp. bulgaricus strains isolated from salad dressings.
Scientific Works of the University of Food Technologies (in press).
46. Teneva D., R. Denkova, B. Goranov, Z. Denkova (2015). Biochemical and
molecular-genetic identification of Lactobacillus strains, isolated from salad
dressings. Научни трудове на Русенския Университет - 2015 / Scientific Works of
the University of Ruse – 2015, 54 (10.2): 50 - 56. ISSN 1311-3321.
47. Teneva D., B. Goranov, R. Denkova, Z. Denkova (2015). Antimicrobial activity of
Lactobacillus delbrueckii ssp. bulgaricus strains against Candida albicans NBIMCC
74. Научни трудове на Русенския Университет - 2015 / Scientific Works of the
University of Ruse – 2015, 54 (10.2): 18 - 23. ISSN 1311-3321.
48. Denkova R., B. Goranov, G. Dobrev, Z. Denkova, G. Kostov, P. Boyanova, D.
Teneva, S. Fortier (2015). Rheological properties of probiotic yoghurts with
phytosterol esters. Ukrainian Food Journal 4 (3): 440 - 452. ISSN 2313-5891
(Online); ISSN 2304-974X (Print).
49. Kostov G., V. Lyubenova, V. Shopska, I. Petelkov, K. Ivanov, V. Iliev, R. Denkova,
M. Ignatova (2015). Software sensors for monitoring the biomass concentration and
the kinetics of continuous beer fermentation with immobilized cells. Comptes rendus
de l’Academie bulgare des Sciences, 68 (11): 1439-1448. ISSN 1310–1331 (Print) /
ISSN 2367–5535 (Online). IF=0,284.

Name: Georgi Atanasov Kostov

Affiliation: University of Food Technologies, department “Technology of wine and

brewing”

Education:

 2015 - Doctor of Sciences – Food technology, University of Food technologies,

Plovdiv;
 2007 - PhD – Machines and apparatus for food and flavor industry; University of
Food technologies, Plovdiv;
 2006 – Master degree - Machine engineering, University of Food technologies,
Plovdiv;
 2003 – Bachelor degree - Machine engineering, University of Food technologies,
Plovdiv;

Address: 4002, 26 “Maristza” blvd, Plovdiv, Bulgaria

Research and Professional Experience: bioreactors; fermentation technologies;

immobilized cells;
 Beer Production with Encapsulated Yeast Cells 97

Professional Appointments:

 System engineer, Institute of System engineering and robotics, Bulgarian Academy

of Sciences, 2011-2014;
 Associated professor, University of Food Technologies, department “Technology of
wine and brewing,” 2011 and present;
 Scientific secretary of Technological faculty, University of Food Technologies,
2011-2015; Secretary of Academic council, University of Food Technologies, 2014-
2015;

Publications Last 3 Years:

1. Goranov B., D. Blazheva, G. Kostov, Z. Denkova, Y. Germanova, Lactic acid

fermentation with encapsulated Lactobacillus casei ssp. rhamnosus ATCC 11979
(NBIMCC 1013) in alginate/chitosan matrices, Bulgarian Journal of Agricultural
Science, 19 (2) 2013, 101–104, IF=0.19.
2. Kostov G., M.Angelov, Z.Denkova, P.Koprinkova-Hristova, P.Pandzarov,
Characteristics of yeast strains for ethanol fermentation for the purpose of biofuels
and food industries, Food Science and Technology, Харчовая наука i технологiя,
3, 2010, Одеса, 29-32.
3. Kopanarova G., V. Naydenova, V.Iliev, G. Kostov, Immobilization of yeast cells in
polyvinyl alcohol for ethanol production. I. Preparation of PVA particles by freeze-
thawing method, Proceedings of the International Conference “AGRI-FOOD
SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014, Sibiu,
Romania, 2014, 214-222.
4. Parcunev I, V. Naydenova, Kostov G., Y. Yanakiev, Z. Popova, M. Kaneva, I.
Ignatov, Modeling of alcohol fermentation in brewing - some practical approaches,
ECMS 2012, Proceedings 26th European Conference on Modelling and Simulation
©ECMS Klaus G. Troitzsch, Michael Möhring, Ulf Lotzmann (Editors), 2012, DOI:
10.7148/2012-0434-0440434-440, 434-440.
5. Vassilev S., V. Naydenova, M. Badova, V. Iliev, M. Kaneva, G. Kostov, S. Popova,
Modeling of alcohol fermentation in brewing – comparative assessment of flavor
profile of beers produced with free and immobilized cells, Proceedings of 27th
European Conference on Modelling and Simulation © Webjørn Rekdalsbakken,
Robin T. Bye, Houxiang Zhang (Editors), ISBN: 978-0-9564944-6-7, 415-421.
6. Naydenova V., S. Vassilev, M. Kaneva, G. Kostov, Encapsulation of brewing yeast
in alginate/chitosan matrix: comparative study of beer fermentation with
immobilized and free cells, Bulgarian Journal of Agricultural Science, 19 (2) 2013,
123–127, IF=0.19.
7. Naydenova V., M. Badova, S. Vassilev, V. Iliev, M. Kaneva, G. Kostov,
Encapsulation of brewing yeast in alginate/chitosan matrix: lab-scale optimization
of lager beer fermentation, Biotechnology and Biotechnological Equipment, 28, 2,
277-284, DOI: 10.1080/13102818.2014.910373, IF=0.622.
8. Naydenova V., V. Iliev, M. Kaneva, G. Kostov, P. Koprinkova-Hristova, S. Popova,
Modeling of alcohol fermentation in brewing – carbonyl compounds synthesis and
98 Vesela Shopska, Rositsa Denkova and Georgi Kostov

reduction, Proceedings 28th European Conference on Modelling and Simulation

©ECMS Flaminio Squazzoni, Fabio Baronio, Claudia Archetti, Marco Castellani
(Editors), ISBN: 978-0-9564944-8-1, 2014, 279-284.
9. Kostov G., D. Pircheva, V. Naydenova, V. Iliev, V.Lubenova, M. Ignatova. Kinetics
investigation of bio-ethanol production with free and immobilized cells, Comptes
rendus de l’ academie bulgare des sciences, ISSN 1310-1331, vol. 66, (10), 1463-
1472, IF=0.21.
10. Iliev V., V.Naydenova, G.Kostov, Study on the application of the chitosan-alginate
capsules and alginate-chitosan-alginate capsules in ethanol fermentation,
PROCEEDINGS of II International Congress “Food Technology, Quality and
Safety” FOODTECH 2014, Novi Sad, ISBN 978-86-7994-043-8,494-499.
11. Lyubenova V., St. Dudin, G. Kostov, M. Ignatova, P. Mitrouchev, M. Angelov, New
technology for ethanol production, Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD
2014, Sibiu, Romania, 2014, 129-139.
12. Nedyalkov, P., M. Kaneva, St. Kemilev, G. Kostov, K. Micheva, P. Poneva,
Influence of the temperature and the duration of extraction on the composition and
the properties of extracts from Alchemilla Mollis, Научни трудове на УХТ, 62,
2015, 198-203.
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In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485-704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.

Chapter 3

EFFECTS OF THE ALMOND ADDITION AND

THE YEAST STRAIN USED FOR FERMENTATION,
ON THE BEER CHEMICAL PROPERTIES

Carlos García-Latorre1, María Inmaculada Talaverano2,

Juan Manuel Zapata3, Oscar Santamaria1 and Sara Rodrigo1,
1
Agricultural Engineering School, University of Extremadura,
Badajoz, Spain
2
CICYTEX-Technological Institute of Food and Agriculture-INTAEX
(Government of Extremadura) Badajoz, Spain
3
Cervecería Artesana Natural Extremeña S.L., Pol. Industrial El Nevero
Calle Dieciocho, Badajoz, Spain

ABSTRACT
Beer is one of the most consumed beverages all over the world, and recently,
literature has attributed it a variety of health benefits. Among its beneficial
characteristics, the presence of polyphenols and antioxidant compounds, are nowadays
widely studied. On the other hand, nuts are known as a source of nutritious food with a
potent free radical scavenging capacities. Due to that, the addition of almonds in a craft
beer brewing process was done, using the almonds in two different level of manufacture:
almond flour or grinded almonds. Besides, two different moments for the addition were
tested (in mashing or in boiling), as well as two different yeast strains (Safale S-04 or
Safbrew T-58). Influence of variables and the interactions among them, were studied
on some quality parameters such as pH, colour, bitterness, total polyphenol index,
antioxidant activity and ferric reducing ability of plasma (FRAP). Yeast strain influenced
pH and bitterness, showing T-58 a higher pH but a lower bitterness (5.3 IBU) in
comparison to S-04 (7.7 IBU). Also regarding with bitterness, adding the almond in
mashing caused the highest bitterness (8.4 IBU). The significant interaction almond type
× yeast strain × addition moment showed that the lowest amount of polyphenols was
determined when almond was added in flour format, in mashing, and fermentation took


Tel: +34 924 289 300; Fax: +34 924 286 201. E-mail: saramrodrigo@gmail.com.
102 Carlos García-Latorre, María Inmaculada Talaverano, Juan Manuel Zapata et al.

place with S-04 yeast strain. Neither for the antioxidant activity nor for the FRAP,
significant effects were reported for any studied variable. Finally, principal component
analysis revealed that the clearest separation was caused by the yeast strain used for the
fermentation.

Keywords: beer, almond, phenols, antioxidant activity, nutrients

INTRODUCTION
Beer History and Present

Beer is defined as a fermented alcoholic beverage made of malted cereals, water, hops,
and yeast. This alcoholic beverage is elaborated and consumed for thousands of years, when
independent events revealed that some juices fermented when leaving in the open air, giving
as a result a completely different product (Hough, 1990).
The fact of drinking alcoholic beverages is different from culture to culture, and it is
evolving over the time. Thus, for several cultures alcohol drinking is completely forbidden for
religious reasons while for some others, only through getting drunk, the contact with the Gods
is possible. Nowadays, the perception of drinking alcoholic beverages has totally changed in
The West: from being a mere support in social relations to emerge as an important component
in the diet, especially wine and beer, both beverages with low alcohol content. In this way,
numerous studies have focused in the last decades on wine and beer beneficial effects in
human health.
Regarding with that, wine market drew on the health beneficial of this product many
years ago, focusing them on the presence of polyphenols, antioxidants, procyanidins, etc.
However, brewers are now pointing out the presence of similar compounds in beer, in
addition to the hop derivate products, which have been shown important beneficial effects in
human health.

Beer and Health

It is assumed that a moderate beer consumption contributes significantly to energy intake

due to its ethanol content (7 kcal mL-1 FW), protein (4 kcal mL-1) and essential amino acids
(between 5 - 10 mg 100 g-1) and carbohydrate (3.75 kcal mL-1) which includes starch
partially degraded in a non-fermentable form (Bamforth, 2002; Bamforth, 2004). Beer also
contains a range of antioxidants, polyphenols, phenolic compounds, folates, carbohydrates,
soluble fibre, vitamins and minerals (Mayer et al., 2001; Bamforth, 2002; Romeo et al., 2008;
Rodrigo et al., 2014). Among the vitamins, it should be pointed out the vitamin B (B1, B2, PP
and B12), coming from malt and yeast (Polak et al., 2013).
On the other hand, the low alcohol content of beer also presents beneficial effects in
human health, and it is established that a moderate ethanol intake helps in the prevention of
coronary diseases and ischemic stroke risk (Klatsky, 2010; Movva and Figueredo, 2013), in
the improvement of the immune response (Romeo et al., 2008) or in the reduction in
atherosclerosis developement (Hashimoto et al., 2001). Moderate alcohol consumption is
 Effects of the Almond Addition and the Yeast Strain Used for Fermentation … 103

defined as an alcohol intake of 10-12 mL d-1 for women and 20-24 mL d-1 for men according
to Díaz et al., (2002), which is equivalent to 1 - 3 drinks d-1 for studies carried out in the UK
by Rimm et al., (1996). In the same line, Romeo et al., (2008) connected moderate beer
consumption with positive effects on good cholesterol, increasing the HDL proportion in
blood; this fact reinforces the preventive nature of beer regarding cardiovascular diseases.
As written above, beer is also an important source of minerals, whose concentration
varies between countries and beer styles (Rodrigo et al., 2015). Thus, in the UK, the Food
Standards Agency periodically publish Food Composition tables, where 18 of the entries
correspond to beer, and for which, concentrations of Ca, Cl, Cu, Fe, I, K, Mg, Mn, Na, P, Se
and Zn are reported. Furthermore, beer contains an interesting amount of another beneficial
components such as folic acid (Mayer et al., 2001; Bamforth, 2002), whose deficiency allows
the development of certain types of cancers or Alzheimer (Aceituno-Medina et al., 2015), and
also isohumulones, compounds derivate from hop exhibiting beneficial effects in diabetes and
obesity, due to its capacity to stimulate alpha peroxisome proliferator-activated receptors,
which positively influence the lipid metabolism (Obara et al., 2009). However, one of the
most important beneficial effects of beer is directly linked to the presence of phenolic
compounds and their bioavailability. These important benefits are supported by the protecting
role of both, polyphenols and flavonoids, against cardiovascular diseases (Mohamed and
Darbar, 2010). These compounds come from both, malt (70% – 80%) and hop (20% – 30%)
(Inns et al., 2007; Qingming et al., 2010; Quifer-Rada et al., 2015), and their importance lies
on their capacity of increasing antioxidant activity in blood plasma (Fumi et al., 2011).
According to the literature, the main phenolic compounds in beer are the hydroxybenzoic
acids, the cinnamic acids (i.e., ferulic acid) and the flavonoids (Gerhäuser, 2005).

Beer Supply and Craft Beer

All these considerations are boosting the beer consumption all around the world and as a
result, in the past decade the world beer supply increased from 22.72 to 26.10 kg capita-1 year-
1. This fact is partly due to the emergent beer market and consumption in Africa and Asia,

where the beer supply increased in the past ten years from 8 to 11 and from 9 to 15 kg capita-1
year-1, respectively in both continents; in Europe, America and Oceania, the beer supply
remains more or less stable over the time, showing supplies of around 70, 60 and 70 kg
capita-1 year-1 respectively (FAO, 2016).
However, the consumption of beer is changing in different countries, and these changes
are reflecting the importance that the consumers are now giving to both, beer composition and
brewing process. Thus, in the 1970s strongly arose the craft beer concept, in response to the
big industrial processes, popping up consumers who were interested in taking a higher quality
product, even when this implied an increase in the price (Berkhout et al., 2014). The
development of microbreweries leads us to have 8 microbreweries in 1980 to find 1800 in
2014, according to Brewers Association (2015).
Even when these winds of change started in the USA, is in 1971 with the foundation of
Campaing for Real Ale (CAMRA) in England, when the craft beer movement broke in as a
response to the low variety in the European beer market (CAMRA, 2015). Thus, craft beer is
defined as a beer produced in microbreweries following traditional processes and having
distinctive flavours and aromas (Scarpa, 1996). Due to their own characteristics, these
104 Carlos García-Latorre, María Inmaculada Talaverano, Juan Manuel Zapata et al.

microbreweries produce up to 1000 hl of beer per year (Sturm et al., 2012; Van de Walle,
2014), and they make of innovation their own hallmark (Acanfora et al., 2009; CAMRA,
2015; Mascia et al., 2015).

May We Create a Healthier Beer?: Yeast Type Importance and Almond as

Antioxidants Promoter

Even when the German Beer Purity Law written in 1516 established that only water, malt
and hop were allowed ingredients to use in beer brewing, after yeast discovery they were
included as the fourth legal ingredient.
The importance of yeast, unicellular fungi generally belonging to Saccharomyces genus,
lies in their capacity to ferment sugar and turn it into ethanol and other secondary metabolites,
such as CO2 or a number of aromatic compounds (Hellborg and Piskur, 2009). The relevance
of this ingredient is such that the most usual beer classification it is usually done based on the
yeast behaviour during fermentation. Thus, beers are divided in ales (S. cerevisiae) and lagers
(S. carlsbergensis) (Blanco et al., 2015); while formers use high fermentation yeasts, working
in the upper part of the tank, last are low fermentation and develop their activity in the bottom
of the tank, requiring lower temperatures. This main difference implies a higher aromas and
flavours concentration in ale beers, due to a higher ester concentration (Hiralal et al., 2014),
presenting lager beers softer flavours.
However, it is necessary to take into account that even when it is possible to classify
yeasts in these two big groups, strain variability inside both groups is almost unlimited. This
high diversity greatly influences the concentration of organic acids, esters, aldehydes or
diacetyl, during fermentation or maturation of beer, which enormously affects organic
properties of the final product (Heggart et al., 2000; Hellborg and Piskur, 2009; Pires et al.,
2014). Therefore, it is extremely important the selection of yeasts that are going to be used in
the beer fermentation to increase the beer flavor (Carrau et al., 2014).
The yeasts market shows a higher availability of specific strains (mostly ale type) which
are mainly classified according to the aromas that they endow to the beer. Thus, S-04 endows
neutral aromas, while K-97 endows fruity aromas and T-58 spiced ones.
But, are only malt, water, hop and yeasts the ingredients that should be use in beer
brewing? The answer is a big no. As written above, in the last decades there has been a strong
increase in the attention that the beer consumers pay to the craft beer, and the possibility that
a microbrewery offers to create new products with better organoleptic characteristics, most of
times creating new recipes including new ingredients or natural additives.
This way, for the present work the addition of almond to the beer brewing was proposed,
due to its high nutritional value. Almond is an important source of vitamin E (alpha-
tocopherol) (Jambazian et al., 2005) or phytosterols and monounsaturated fatty acids
(Damasceno et al., 2011), mainly oleic acid (Yada et al., 2011). Thus, Hyson et al., (2002)
stated that the intake of almonds can lower plasma triacylglycerols and total and low-density
lipoprotein (LDL) cholesterol which implies an increase in the levels of high-density
lipoprotein (HDL) cholesterol. Similarly, another studies pointed out that the almond
consumption can reduce coronary heart diseases risk, due not only to the lipid profile, but to
the presence of phenolic compounds showing a high antioxidant activity that could prevent or
delay the oxidation of fatty acids (Wijeratne et al., 2006; Monagas et al., 2008; Vinson and
 Effects of the Almond Addition and the Yeast Strain Used for Fermentation … 105

Cai, 2012). Moreover, it is known the high amount of polyphenols, mainly present in the skin,
are easily absorbed during digestion processes (Mandalari et al., 2010) and those
polyphenolic compounds act synergistically with vitamin C and E enhancing the antioxidant
protection of the LDL (Esfahlan et al., 2010; Jamshed and Gilani, 2014). All these
biologically active compounds such as flavonoids, phenolic compounds, tannins, vitamin E,
etc. (Rao, 2012; Xie et al., 2012) present in the almond have a number of secondary health
beneficial effects among which anti-inflammatory, anticarcinogenic and hepatoprotective
effects, can be highlighted (de Pascual-Teresa et al., 2010; Truong et al., 2014; Wijeratne et
al., 2006; Xie et al., 2012). Therefore, it is expected that the almond addition to the beer
brewing could increase the healthy properties of this (already healthy) beverage, and thus
showing positive results on consumer’s health.
Thereby, the present chapter’s aim is to increase the knowledge about the possible
changes in the beer characteristics after brewing it adding almond and fermenting with
different yeast strains.

MATERIAL AND METHODS

Ingredients and Brewing Conditions

Only five ingredients were used for the beer brewing: barley malt (Pilsen and Biscuit),
tap water, hops (pellets of cv. Perle), yeast - two Saccharomyces cerevisiae strains, S-04
(Safale) and T-58 (Saltbrut)- and almond. Almond was used in two different forms (grinded
and flour) and was added in two different moments. Barley malts were used in 9:1 proportion,
Pilsen:Biscuit, and both were milled through a Roppi 1100 mill trying to get 1/3 of flour, 1/3
of broken grains and 1/3 of whole grains.
Mashing was carried out at 67ºC in a Speidel Braumeister 50 L designed for small scale
brewing, adding 5 L water per malt kilogram. After 60 minutes at 67 ºC, eight aliquots were
taken. Each aliquot contained 1 L wort and 35 g malt.
Experimental design consisted of a main treatment of moment of almond addition, in
mashing and in early boiling; a secondary treatment of almond form (grinded and flour); and
a tertiary treatment depending on the yeast strain used for fermentation.
The first moment for almond addition took place when the temperature of 78ºC was
achieved and kept during 15 more minutes, and this process was carried out in a heating bath.
In every aliquot hops was added for the last 15 minutes of mashing in a rate of 5 g L-1.
After mashing, filtration took place and wort of every sample was boiled for 75 minutes
at 105ºC in an autoclave (Autester ST DRY PV IlI, Selecta). Previously, almond addition
took place in the corresponding aliquots. The amount of almond was always the same: 20 g
per 1 L wort. Once the samples were boiled, a quick cooling was carried out in an ice bath
and wort temperature before yeasts inoculation was 25ºC – 30ºC; then inoculation was done
after yeast hydration with 100 ml sterile water at 25 ºC, using a rate of 0.23 g L-1.
Fermentation took place at room temperature 20ºC – 22ºC during four days and then,
before bottling sugar addition at a rate of 7 g L-1 was done for second fermentation (22ºC –
26ºC) in bottle. Each sample was re-fermented separated in three 33 cl brown glass bottles
during six weeks and each bottle was considered as a replicate.
106 Carlos García-Latorre, María Inmaculada Talaverano, Juan Manuel Zapata et al.

Beer Analysis

Beer samples (120 mL) were degassed by stirring 15 – 25 minutes in a covered

beaker to avoid alcohol volatilization and then filtered thanks to filter paper whatman 11 um.
Alcohol content was determined according to the official method described in Spanish
regulation BOE 23/10/85, where 100 g of degassed sample was distilled by using a
distillation unit K-350/K-355 (Buchi) to get 85 – 90 mL of extract which was completed up to
100 g with distilled water. Prepared sample was cooled down until 20ºC and then density
was measured with a triple scale hydrometer. Conversion into alcohol content was done
thanks to the conversion tables described in the regulation. Also using degassed sample pH
was determined in 20 mL beer with a Crisom pHmeter. Meanwhile, beer colour was analyzed
with the two variants that Seaton and Cantrell (1993) proposed in their method. Thus, both
EBC and IOB Recommended Method Wavelength were done, determining the absorbance by
spectrophotometer (B13627 Unicam Helios Alpha UV-VIS) at 430 nm and 530 nm,
respectively. For this determination, degassed beer was used and the colour was determined
in duplicate multiplying absorbance value by 12.7, according to the method.
Bitterness in the beer was measured in IBUs (International Bitterness Units) by following
the method described by Howard (1968). An aliquot of the degassed beer (10 mL) was
pipetted into a flask where later 1 mL of hydrochloric acid 3 N and 20 mL of isooctane were
poured. The flask was stoppered and shaken vigorously for 2 minutes using a Vibromatic 384
shaker (Selecta) and one more minute in a vortex. After that, the isooctane layer was
transferred to a 1-cm cell in which the optical density is determined at 275 nm in the
spectrometer, using isooctane as reference. Bitterness values in IBU were obtained in
duplicate multiplying the absorbance values by 50.
Total polyphenols content (TP) was analysed according to the Folin-Ciocalteau method
(Singleton y Rossi, 1965), using gallic acid as a standard and expressing the results as gallic
acid equivalent (GAE) in milligrams per litre of beer. Samples of 0.5 mL (two replicates)
obtained adding 0.1 mL beer and 0.4 mL distilled water, were introduced into test tubes and
then 25 mL of distilled water, 2.5 mL of Folin-Ciocalteu reagent and 10 mL of anhydrous
Na2CO3 20%, were added. Distilled water was added up to 50 mL and shaken in vortex prior
to their storage in darkness for 30 minutes. After that, absorbance was measured at 750 nm.
To obtain the results, a pattern curve was done with different concentration of gallic acid as
standards (0, 20, 40, 50, 100, 150 and 500 mg L-1).
The antioxidant activity was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) as
a free radical, following the method described by Von Gadow et al., (1997). A concentration
of DPPH 0.6 mM was prepared and then a root solution methanol:DPPH in a rate 4.5:1 was
measured in spectrophotometer at 515 nm having methanol as stardard. Absorbance of the
root solution was 1.25. For the analysis of every sample three absorbance values were done:
one blank sample created with 150 µL + 2850 µL of root solution, one control sample created
with 150 µL sample and 2850 µL methanol and the sample to analyse created with 150 µL
sample and 2850 µL root solution. Once prepared, every sample was stored in darkness for 24
hours and absorbance was measured at 515 nm. Antioxidant activity was determined applying
the following equation:
Finally, the ferric reducing ability of plasma (FRAP) was calculated by following Pulido
et al., (2000) method, where a Trolox standard curve (0.8; 0.6; 0.4; 0.3; 0.2; 0.1 and 0.025
mM) was created. Each point of the curve was obtained by measuring the absorbance at 515
 Effects of the Almond Addition and the Yeast Strain Used for Fermentation … 107

nm of a solution done from 150 µL of the Trolox standard with 2850 µL of root solution used
in the antioxidant activity. The solution was stored in darkness for 24 hours before the
measurement. Calculation of FRAP expressed in mM of Trolox was done thanks to the
equation: % antioxidant activity = (absorbance of sample × absorbance control) × 100/
absorbance blank

Statistical Analysis

Alcohol content, pH, colour, bitterness, TP, AA and FRAP were subjected to a 3-way
ANOVA, including ‘moment of addition’ (mashing or boiling), ‘almond form’ (grinded or
flour, ‘yeast type’ (S-04 and T-58) and their interactions in the model. When significant
differences were found in ANOVA, means were compared using Fisher’s protected least
significant difference (LSD) test at p ≤ 0.05. Pearson correlation tests were performed
between the analyzed parameters. All these analyses were performed with the Statistix v. 8.10
package. Principal component analysis (PCA) was conducted on the eight studied parameters
for each treatment, and these analyses were performed with the XLStat (Addisoft, USA) ‘add-
on’ for Microsoft Excel.

RESULTS AND DISCUSSION

Alcohol content in all the samples was exactly the same: 4.5%. This fact was expected
due to the relationship between the alcohol content and the final density of the beer after first
fermentation (1011 g L-1 in all the treatments). The only difference to remark resided in the
density initial decrease, faster in beers fermenting with T-58 (data not shown). This fact could
be explained by a better adaptation to the conditions of this strain what means an earlier
conversion of sugars in alcohol. The obtained alcohol content is in the range of usual alcohol
content for craft beers that Strong and England (2015) recorded between 4.5% and 6%.

Table 1. Summary of the three-way ANOVA for pH, EBC colour, IOB colour and
bitterness in the analyzed beers

DF pH EBC IOB Bitterness

MS MS MS MS
Moment of addition
(Moment) 1 3.40×10-4 15.67 0.37 83.81*
Almond form (Form) 1 1.50×10-3 48.74*** 15.75** 24.30
Yeast strain (Strain) 1 4.18×10-1*** 5.48 2.73 33.73*
Moment * Form 1 2.20×10-3 0.02 0.03 43.61
Moment * Strain 1 4.00×10-3 5.78 1.54 83.81**
Form * Strain 1 1.84×10-3 24.59** 12.19** 51.48**
Moment * Form * Strain 1 2.00×10-4 0.01 0.07 25.94*
MS: Mean Square.
, and *** significance at p ≤ 0.05, 0.01 and 0.001 respectively following the ANOVA.
* **
108 Carlos García-Latorre, María Inmaculada Talaverano, Juan Manuel Zapata et al.

The three-way ANOVA performed to evaluate the effect of the almond addition (moment
and form) and the yeast strain used for fermentation, on the pH, colour and bitterness showed
a significance of the strain for the pH, of the almond form and the interaction almond form ×
yeast strain for colour and for the bitterness both main treatments, moment of addition and
strain, and all the interactions including strain (Table 1). For this reason, the multiple
comparison test was carried out with the data of the different treatments.
The different amount of CO2 and/or organic acids that each yeast strain can excrete
during the fermentation process could explain the significant differences found in the pH data
in this study. However, another hypothesis can be given to explain the yeast strain influence
on beer pH; Coote and Kirsop (1976) observed different behaviour of pH in beer depending
on the contact yeast-wort. Thus, while S-04 strain flocculates quite fast, T-58 hardly
precipitates, which means a longer contact between the yeast and the wort’s sugars, favouring
a higher pH (Table 2).
According to the literature, best results regarding to the quality are found in S-04 yeast
strain (Table 2), due to that a pH below 4.4 contribute to have a more stabile foam and better
organoleptic characteristics in the beer (Bible, 2007). Both pH values, for S-04 and T-58
yeast strains, being below 4.5, promoted a correct antimicrobial activity (Wunderlich and
Back, 2009). Moreover, it is necessary to highlight that the pH values for both strains are in
the normal range of the pH for ales beer (4-4.5) according to DeLange (2013).
Regarding to the colour (both IOB and EBC, highly correlated; r = 0.95), ANOVA
analysis showed significant differences for almond form and the interaction almond form ×
yeast strain (Table 1). In Table 2, colour values are presented and it can be observed that the
beers of the experiments were light-coloured and golden. The main reason for obtaining
golden beers is the use of Pilsen malt (one of the lightest malt in the market) in a high
percentage (Shellhammer, 2009); in this study, 90% of Pilsen malt was used as base malt.

Table 2. Effect of the almond addition (moment and form) and yeast strain on beer pH,
EBC colour, IOB colour and bitterness mean (with standard error) in
the analyzed beers

pH EBC IOB Bitterness

Moment of addition
(Moment)
Late mashing 4.31 ± 0.05 9.44 ± 0.75 4-03 ± 0.51 8.39 ± 1.61a
Early boiling 4.30 ± 0.04 7.82 ± 0.66 3.78 ± 0.41 4.65 ± 0.68b
Almond form (Form)
Grinded 4.30 ± 0.04 10.06 ± 0.70a 4.72 ± 0.44a 7.52 ± 1.75
Flour 4.31 ± 0.05 7.20 ± 0.52b 3.10 ± 0.34b 5.51 ± 0.65
Yeast strain (Strain)
S-04 4.17 ± 0.01b 9.11 ± 0.95 4.25 ± 0.56 7.70 ± 1.77a
T-58 4.44 ± 0.02a 8.15 ± 0.43 3.57 ± 0.31 5.33 ± 0.55b
Mean 4.31 ± 0.04 8.63 ± 0.74 3.91 ± 0.45 6.52 ± 1.33
Different low case letters means significant differences (p < 0,05) according to LSD tests.
 Effects of the Almond Addition and the Yeast Strain Used for Fermentation … 109

Figure 1. EBC (left) and IOB (right) colour as influenced by the interaction almond form × yeast strain.
Vertical bars on the left corner represent LSD (p < 0.05); the one located more to the left, for the same
level of almond form and the other for different level of almond form.

Figure 2. Bitterness (IBUs) as influenced by the interaction almond form × moment of addition × yeast
strain. Vertical bars on the left corner represent LSD (p < 0.05); the one located more to the left, for the
same level of moment of addition and almond form, the one in the middle for the same level of moment
of addition and the one located in the right for different level of moment of addition.

The influence of the almond form in the colour could be explained by the almond peel:
while almond was grinded with peel, the almond flour came from peeled raw material. Thus,
some pigments such as β-carotens and some polyphenols present in the almond peel (Yada et
al., 2011) could have favoured darker colours in the beer when grinded almond was added.
However, significant differences in colour regarding to the almond form were only observed
when S-04 yeast strain was used for fermentation, presenting homogeneous values the colour
of the beers fermented with T-58, no matter the almond form added in the brewing process.
This fact could be explained by the higher concentration of polyphenols in the beer when the
grinded almond was added that could have prompted more frequent oxidation process, which
can cause higher polyphenol-protein interaction. Knowing that this interaction is strongly
110 Carlos García-Latorre, María Inmaculada Talaverano, Juan Manuel Zapata et al.

influenced by yeast strain (Shellhammer, 2009), higher turbidity in the beer at the end of the
process due to the presence of proteins, can present higher absorbance values.
Statistical analysis for bitterness results pointed out that both main treatments, moment of
addition and yeast strain showed significance, as well as all the interaction including yeast
strain (Table 1). The most remarkable thing to highlight is that all the values were relatively
low regarding to the more usual data of bitterness in ales, which according to Strong and
England (2015) are in the range from 15 to 40. This low bitterness could be attributable to the
variety of hops used, Perle in pellets, whose alpha-acids level is relatively low.

Table 3. Summary of the three-way ANOVA for total polyphenol content (TP), the
antioxidant activity (AA) and the ferric reducing ability of plasma (FRAP) in the
analyzed beers

DF TP AA FRAP
MS MS MS
Moment of addition (Moment) 1 80.67 3.18 4.17×10-4
Almond form (Form) 1 210.04 50.20 1.84×10-3
Yeast strain (Strain) 1 198.38** 5.41 4.17×10-6
Moment * Form 1 0.17 0.14 3.75×10-5
Moment * Strain 1 80.67* 1.05 3.75×10-5
Form * Strain 1 18.38 1.06 9.38×10-4
Moment * Form * Strain 1 54.00* 17.02 5.04×10-4
MS: Mean Square.
, and *** significance at p ≤ 0.05, 0.01 and 0.001 respectively following the ANOVA.
* **

Likewise, the fact of adding almond in mashing moment could explain the higher
bitterness level obtained (Table 2) due the release of amygdalin, a glycoside (Lee et al., 2013)
that could set free all the bitter compounds when diastases work, taking place this work only
under 70ºC (Palmer, 2006).
With reference to the yeast strain influence on bitterness, Popescu et al., (2013) stated
that alpha-acids molecules can bind yeast cell wall, being discarded with the dead yeast at the
end of the fermentation process by filtrating or decanting. Again, the different flocculation
level of the two yeast strains used could explain the significant differences found in the data
after ANOVA study; while S-04 highly precipitates from the beginning, T-58 remains in
suspension in the wort for a longer time (Fermentis, 2012a; Fermentis, 2012b), which
prompted a higher contact between yeast cells and the alpha-acids molecules, discarding more
bitter compounds in clarification moment.
Regarding to the results of the significant interaction almond form × moment of addition
× yeast strain (Figure 2), the only remarkable thing is the fact of having a significantly higher
value of bitterness when yeast strain S-04 was used, what reinforces once more all the stated
above.
Results from the three-ways ANOVA done for the total polyphenol content (TP), the
antioxidant activity (AA) and the ferric reducing ability of plasma (FRAP) are presented in
Table 3. Yeast strain and the interactions moment of addition × yeast strain and almond form
× moment of addition × yeast strain, significantly affected TP results.
 Effects of the Almond Addition and the Yeast Strain Used for Fermentation … 111

Even though the TP data obtained in this study are similar to the ones found by Saura-
Calixto and Goñi (2004) in their beers, another studies in the literature (ElKaoutit et al., 2008;
Estruch et al., 2010) reported considerably lower TP values for this beverage. The higher TP
content found in the beers brewed in this experiment could be related to the high phenols
concentration present in the almond (Bartolomé et al., 2010; Mandalari et al., 2010), whose
influence could be independent of the moment of addition. This increase in the TP content is
really positive, due to the antioxidant capacity and good sensorial properties that the phenols
give to the beer (Vanbeneden et al., 2008; Fumi et al., 2011).
The differences found in TP content regarding to the yeast strain could be due to the
different secondary metabolites that these organisms can excrete, such as pyruvic acid or
acetaldehyde, which usually react with polyphenols, especially pigments. Thus, it is possible
to find new compounds favouring colour stability, but this fact reduces the polyphenols
concentration (Fulcrand et al., 1998; Caridi et al., 2004). The hypothesis here is that yeast
strain S-04, showing a lower TP content, produces the type of metabolites cited above, or
produces them in bigger amount.

Table 4. Effect of the almond addition (moment and form) and yeast strain on beer total
polyphenols content (TP), antioxidant activity (AA) and ferric reducing ability of
plasma (FRAP) mean (with standard error) in the analyzed beers

TP AA FRAP
(mg GAE L-1) (%) (mM Trolox)
Moment of addition (Moment)
Late mashing 483.90 ± 20.00 8.19 ± 0.87 0.80 ± 0.01
Early boiling 520.60 ± 16.70 7.46 ± 0.64 0.80 ± 0.01
Almond form (Form)
Grinded 472.70 ± 17.60 9.27 ± 0.64 0.79 ± 0.01
Flour 531.80 ± 16.40 6.38 ± 0.64 0.81 ± 0.01
Yeast strain (Strain)
S-04 473.50 ± 21.00b 8.30 ± 0.69 0.80 ± 0.01
T-58 531.00 ± 12.30a 7.35 ± 0.83 0.80 ± 0.01
Mean 502.30 ± 18.80 7.83 ± 0.76 0.80 ± 0.01
Different low case letters means significant differences (p < 0.05) according to LSD tests.

As written above, the interaction almond form × moment of addition × yeast strain
revealed statistical significance for TP data. However, as shown in Figure 3, the most
remarkable difference was the one caused by the lower value of TP in beers when the S-04
yeast strain was used for fermentation, fact explained previously in this section.
However, even when there are no important significant differences among the rest of the
samples, it can be glimpsed a tendency for a higher TP contents in beers brewed with almond
in grinded form; maybe promoted, again, by the polyphenols presented in the almond peel
(Yada et al., 2011). Finally, it is important to highlight, in this triple interaction, the positive
effect in the analyzed parameter when T-58 yeast strain was used combined with the grinded
almond, showing this combination the highest value of TP (Figure 3).
The analysis of variance (ANOVA) done for the antioxidant activity and the ferric
reducing ability of plasma (FRAP) data did not show any effect of the different treatments,
112 Carlos García-Latorre, María Inmaculada Talaverano, Juan Manuel Zapata et al.

neither their interactions. Despite the close relationship that usually polyphenols content and
antioxidant activity show (Guido et al., 2005; Saura-Calixto and Goñi, 2006; Zhao et al.,
2010), Caridi et al., (2004) stated in their study that not always a higher amount of TP means
higher AA, due to the fact that some phenolic compounds not always have antioxidant
capacity. According to Verhagen (2010), only the low molecular weight polyphenols own
antioxidant capacity, not showing such effect the high molecular weight polyphenols. In the
same way, some phenolic compounds can act as pro-oxidants in media with a high metal ions
concentration (25 – 100 µM) (Rice-Evans et al., 1996). The high metal content, such as iron
and zinc, present in the almond (Yada et al., 2011) could have favoured this situation,
increasing the metal concentration in beer wort.

Figure 3. Total polyphenols content (TP) as influenced by the interaction almond form × moment of
addition × yeast strain. Vertical bars on the left corner represent LSD (p < 0.05); the one located more
to the left, for the same level of moment of addition and almond form, the one in the middle for the
same level of moment of addition and the one located in the right for different level of moment of
addition.

PCA was applied to evaluate trends in the data. In the PCA, the two principal
components (PC1 and PC2) explained 83.45% of the total variance (56.65% and 26.60%,
respectively). In Figure 4 it can be seen that EBC and IOB colour, as well as TP and FRAP,
are located at positive values of PC1, and AA at negative values in this PC. In the same way,
pH is located at positive values of PC2 and bitterness in negative ones. The PCA shows a
clear separation among the beers fermented with the two yeasts strains; while T-58 beers are
located at the positive values of PC2, S-04 beer are located at the negative ones. In the same
figure (Figure 4), it can be seen how beers fermented with T-58 yeast strain show higher
values of pH and TP, and lower bitterness, comparing with the results of the beers fermented
with S-04 strain. According to the PCA, the beer colour was significantly affected by the
almond form, being more intense the colour when grinded almond was used as additive.
Thus, beers brewed with grinded almond added in both moments, mashing or boiling, and
fermented with S-04 yeast strain, showed more intense colour. Moreover, adding almond in
mashing meant higher bitterness and FRAP values.
 Effects of the Almond Addition and the Yeast Strain Used for Fermentation … 113

Figure 4. Correlation between loadings and factors on beer parameters in the Principal Components
Analysis (PCA). In the figure beer codes are the following: MG04 (mashing – grinded – S-04), MG58
(mashing – grinded – T-58), BG04 (boiling – grinded – S-04), BG58 (boiling – grinded – T-58), MF04
(mashing – flour – S-04), MF58 (mashing – flour – T-58), BF04 (boiling – flour – S-04), BF58 (boiling
– flour – T-58).

CONCLUSION
The findings presented in this chapter suggested that the addition of almond during
the brewing process can slightly vary the chemical and organoleptic characteristics of the
beer. Thus, adding the almond during mashing increases the bitterness, while the addition of
grinded almond causes intensification in the beer colour. On the other hand, yeast strain used
for fermentation modifies not only the total polyphenols content, but also the type of phenolic
compounds found in the final product. However, neither antioxidant activity nor ferric
reducing ability of plasma are affected by almond addition or yeast strain.

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In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485-704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.

Chapter 4

QUANTITATIVE ANALYTICAL METHODS

OF ORGANIC INGREDIENTS IN THE
QUALITY CONTROL PROCESS FOR THE
PRODUCTION OF BEER

Bojidarka Ivanova1* and Michael Spiteller

Lehrstuhl für Analytische Chemie, Institut für Umweltforschung,
Fakultät für Chemie und Chemische Biologie, Universität Dortmund,
Dortmund, Nordrhein-Westfalen, Deutschland

ABSTRACT
Although chronic alcohol abuse leads to liver diseases, hard spirits are more harmful
to human health. And thus, the great popularity of beer is associated with diet benefits in
reducing the risk heart disease; myocardial infarction and diabetes. The broad biological
antioxidant, antitoxiolytic, anticancer and antimicrobial activities are due to cereal and
hop bitter acids. Other ingredients are soluble fibers, vitamins and minerals. Isoflavones
genistein and daidzein were already implicated in cancer prevention. From an analytical
chemistry perspective, the beer exhibits a complex chemical profile of  400–600
ingredients. Their kind and concentration vary in the final beer product, and through all
brewing stages. It has complex photochemistry and intra–specification diversity based on
growing region of the plant, collecting phase and/or post–harvest treatment. The
elaboration of analytical protocols represents significant challenge, in spite of the
production of beer is from many Centuries. This chapter reflects contributions of MS to
beeromics, focusing attention to organic ingredients. The method is irreplaceable for
quantitative analysis of complex mixtures. The content deals with determination of large
scale of components and concentrations, including trace analysis. It impacts a sector of
the economy, which contributes to many interdisciplinary fields of chemistry such as
analytical chemistry; food chemistry; agricultural chemistry and so on. Analysis of
microbial contamination, that may occur during production process and which may
negatively affect the final food is included. The determination of mycotoxins, which can

*
Corresponding author: Tel.:+ 49 231 755 4089, Fax. + 49 231 755 40 84; E-mail: B.Ivanova@infu.uni-
dortmund.de; B.Ivanova@web.de.
120 Bojidarka Ivanova and Michael Spiteller

carry–over from contaminated grains in food–product thus causing serious health

problems for humans is discussed. They exhibit a wide range of toxic activity such as
carcinogenicity and neurotoxicity, and reproductive and developmental toxicities. Other
health risk components are also shown. The chapter content is with high relevance for
“food control” in conjunction with strict legislation to regulate presence of mycotoxins in
food.

Keywords: quantitative analysis, beer, analytical chemistry, analytical instrumentation, mass

spectrometry

ABBREVIATIONS AND ACRONYMS

ACIT Array capillary in–tube
APCI Atmospheric pressure chemical ionization (mass spectrometry)
BD Between–day–analysis
CE 1Capillary electrophoresis
2Collision energy
CE
CID Collision–induced dissociation
CXP Cell exit potential
CZE Capillary zone electrophoresis
DESI Desorption electrospray ionization (mass spectrometry)
DIESI Direct–injection electrospray ionization (mass spectrometry)
DLLME Dispersive liquid–liquid micro–extraction
1Degrees of polymerization
DP
2Declustering potential
DP
DSME Dispersive suspended micro-extraction
ECD 1Electron capture detection (mass spectrometry)
ECD 2Electrochemical detector
ED Electrochemical detection
EESI Extractive electrospray ionization (mass spectrometry)
EI Electron impact (mass spectrometry)
ELSD Evaporative light scattering detection
EP Entrance potential
ESI Electrospray ionization (mass spectrometry)
FAB Fast atom bombardement
FACE 1Fluorescence assisted carbohydrate electrophoresis
FACE 2Fluorophore–assisted carbohydrate electrophoresis
FIE Flow injection electrospray–ionization (mass spectrometry)
FP Focusing potential
FPLC Fast protein liquid chromatography
Fs Fluorescence (detection, spectroscopy)
FT–ICR Fourier transform ion cyclotron resonance (mass spectrometry)
FWHM Full width at half maximum
GC Gas chromatography
HPAEC High–performance anion exchange chromatography
 Quantitative Analytical Methods of Organic Ingredients … 121

HPLC High performance liquid chromatography

HPLC DAD–MS – High-performance liquid chromatography
with diode–array detection
HR/AM High–resolution accurate–mass
HRE Reflux extraction
HS Head–space (dynamic)
ITEX In–tube extraction
LAESI Laser ablation electrospray ionization
LMW Low molecular weight
LOD Limits of detection (concentration)
LOQ Limits of quantitation (concentration)
LPME Liquid phase micro–extraction
LTQ Linear ion trap quadrupole
MAE Microwave–assisted extraction
MALDI Matrix–assisted laser desorption/ionization
(mass spectrometry)
MEEKC Micro-emulsion electro–kinetic chromatography
MEKC Micellar electrokinetic chromatography
MOS Maltooligosaccharides
MPLC Medium–pressure liquid chromatography
MRM Multiple reaction monitoring
MS Mass spectrometry
MS–USAEME Manual shaking–enhanced
ultrasound–assisted emulsion cation micro–extraction
Nano–DESI Nanospray desorption electrospray ionization
(mass spectrometry)
NIMS Nanostructure–initiator mass spectrometry
PAD Pulsed amperometric detection
PDA Photodiode array (detection)
PSD Post source decay (mass spectrometric fragmentation)
QMSD Quadrupole mass detection
QqQ Triple quadrupole
Q–TOF Quadrupole–time–of–flight
QuEChERS Quick easy cheap effective rugged safe
r.a. Relative abundance
RSD Relative standard deviation
SD Standard deviation
SDME Single drop micro–extraction
SDS–PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SFE Supercritical fluid extraction
SIM Single ion monitoring
SIMS Secondary ion mass spectrometry
SFO Solidification of floating organic drop
SPDE Solid phase dynamic extraction
SPE Solid phase extraction (conventional)
SPME Solid–phase micro–extraction
122 Bojidarka Ivanova and Michael Spiteller

TC Temperature controlled
TOF Time–of–flight (analyzer)
UEA Ultrasound–assisted extraction
UHPLC Ultra–high performance liquid chromatography
UPLC Ultra performance liquid chromatography
UV Ultraviolet (detection, spectroscopy)
WD Within–day–analysis

INTRODUCTION
The production of beer and brewing are the oldest biotechnological processes [1]. The
ethanol toxicity and the chronic alcohol abuse are important factors for liver related diseases.
Epidemiological studies, however, were shown that hard spirits for human health are more
harmful than fermented alcoholic beverages [2].
According to ‘German Beer Purity Law’ with Bavarian Reinheitsgebot (Since 1516),
there are four ingredients in beer (water, hops, barley, and yeasts (Saccharomyces
cerevisiae)) [3, 4]. The complex chemical content of compounds with high chemical, thermal
and photochemical instability; and complex photochemistry, determine the “analytical
chemistry” of beer as a challenging research task. In addition to still, not well understood
biochemical reactions and mechanisms of brewing. There are numerous contributions in
brewing technology and quantitative protocols. The quantitative analysis is of prime
importance for “quality control” in connection with brewing industry. The beer quantification
initially comprises an extraction phase. Solid–liquid extraction techniques are soaking and
Soxhlet; supercritical fluid extraction and distillation [5–10]. SFE and DSME are used. To
methods of solvent extraction such as UAE and MAE are those for quantitation of organics.
They are often coupled with QuEChERS. Along with conventional liquid–liquid extraction,
which is widely used in the analytical practice reflux extraction method, DLLME, LPME,
ITEX and in–needle extraction approaches are utilized as well. The in–tube techniques
include in–capillary extraction. The solid–phase extraction methods SPME and SPDE are
high capacity enrichment techniques. The extracted analytes are injected into GC– or LC–
instruments. The second state of beeromics is, thereby, a chromatographic separation. The
GC– method yields reliable analytical information mainly on thermally stable volatile
analytes. A substantial amount of organic analytes in beer exhibit low thermal stability and
hence they are not accessible to analytical quantification. The LC– method is low selective
and sensitive at low concentrations of analytes in presence of complex matrices of naturally
occurring compounds. To ensure reliable analytical information for a large scale of analyte
concentrations of a multi-component analyte mixture of chemical, photochemically and
thermally unstable substances there are improvements and optimizations of numerous
analytical methods together with methodological instrumental developments related to sample
pretreatment steps and detection methods. They include HPLC, UPLC, UHPLC, MPLC and
the like. Under separation process mainly HPLC and GC are used as instrumental approaches
to the determination of carbohydrates in beer to high and low volatile matter; alcohol content
( [0.5–10] % w/w); phenyl flavonoids; simple phenolic compounds; vitamins; amino–acids;
hop bitter acids (concentration range  [17–55] ppm, iso–α–acids)) and more [11–23]. Other
 Quantitative Analytical Methods of Organic Ingredients … 123

separation techniques based on electrophoresis as capillary electrophoresis; micellar

electrokinetic and microemulsion electrokinetic chromatography were implemented routinely
for such as purposes [11].
The detection of analytes is third stage of beeromics. The determination of carbohydrates
in beer is performed measuring the refractive index as a detection methods for the HPLC–
separation phase [24].
Mass spectrometry is irreplaceable method for environmental analytical chemistry;
agricultural sciences and food industries [11–23]. The main reasons are: (i) its instrumental
flexibility allowing coupling with various separation methods; (ii) ability to on–line and off–
line monitoring of chemical components; (iii) large amount of ionization approaches for the
simultaneous analysis of numerous analytes in complex mixtures (up to 26,000 components);
(iv) ability to operate on a large scale of concentrations of analytes. A great feature of
some instrumental MS methods, consist of its ability to operate without “extraction” and
“separation” steps of the sample treatment.
The EI–MS as a hard ionization approach was used to metabolomics [25–27]. The GC–
EI–MS approach shows disadvantages towards the analytical uptime  24–48 h daily [21].
GC–based metabolomics dependents on the volatility of the sample. If MS– can be used as a
detection method, where the intensity of analyte signals depends on ionization efficiency,
then volatility and ionization are somehow opposed. Because of non–charged volatile
analytes not efficiently ionized. In addition, as part of an operation of about 200 samples,
GC–column matrix undergo gradual significant change analytical properties, thus increasing
in non-reproducibility of the data. Improving the chromatographic separation step was
performed in hybrid MS–based methods such as HPLC and UPLC, but have technical
constraints. They are too slow and expensive for the purpose of routine application.
The soft ionization ESI–MS/MS is commonly used for analysis in solution, allowing
detection of a large scale polar and non–polar compounds. The ESI causes for formation of
various anionic, cationic species and/or adducts depending on experimental conditions. The
lack of extensive MS fragmentation of analytes due to soft ionization character provides for a
high reproducibility of MS profile at certain experimental conditions, thus allowing molecular
fingerprint, qualitative analysis and quantification of unknown compounds in mixtures [26,
27].
The DIESI–MS or FIE–MS (‘flow injection’ or ‘flow infusion’) methods may
produce large analytical information at low cost. And thus they have a great potential for
routine implementation in analytical practice [25, 26]. Other methodological instrumental
elaborations have led discovery of 'ambient' methods such as DESI for direct analysis of
solids without sample preparation and separation steps. The contribution of random and
systematic errors of chromatographic measurements and different samples pretreatments is
reduced. The MALDI, SIMS, LAESI, NIMS and nano–DESI allow 2D and 3D MS imaging
[27]. This technique is especially prominent for environmental analysis, including analysis of
food. Moreover, imaging with tandem MS/MS approach and multiple reaction monitoring can
identify macromolecules and low molecular weight analytes in complex multi–component
mixtures. The flexible MS instrumentation, allows experimental design based on specific
features of analytes and matrices. The instrumental design includes improvements in
resolution, accuracy, sensitivity and robustness through various analyzers such as single (or
hybrid) quadrupole instruments; TOF analyzers; Ion trap; Orbitrap analyzers and FT–ICR–
MS. Single quadrupole ion trap detectors provide robust and rapid scanning in a large m/z
124 Bojidarka Ivanova and Michael Spiteller

range, despite variability in resolution. The ion trap instruments with ion storage permit
tandem MSn operation, which contributes to structural analysis. The TOF instruments have
theoretically high resolution at m/z 500. A performance about 10 000 FWHM with ground
fault expected  5–10 ppm. The next generation instruments with ultra–high resolution and
accuracy using Orbitrap analyzers or FT–ICT, show resolution 240 000 and 1 600 000 with
18 T magnets. The precision is sub–ppm errors [25–27]. In particular, imaging, MALDI MS
has a number of advantages [28–34], yielding a high spatial resolution of the size of the laser
beam. Achievements in this regard have raster step size 2.5 m of 1 m laser spot diameter
[28–34]. The spacial feature is undercellular dimensions, so that MS–based visualization of
cellular fibers, granular and Purkinje cells and so on. This capability corresponds to high-
resolution optical microscopy. The instrumental features of MALDI–MS with Orbitrap
analyzer and imaging technology make this method of choice for analytical environmental
chemistry of multicomponent analysis of complex analyte mixtures with wide range
molecular weights of LMW analytes to (bio)macromolecules. We highlight herein, the ability
of MALDI–MS to detect large concentration ranges of analytes of trace and ultra–trace
amounts to attomol and zmol ranges; and analysis of macro–components. It can be utilized to
study complex concentration dynamics, yielding to exact thermodynamics and kinetics of
processes in food matrix. The more recent employment of MALDI FTICR imaging MS for
proteomics in bacterial micro-colonies of Staphylococcus aureus shows a significant mass
resolution ~75 000 (m/z 5000) and accuracy < 5 ppm. The molecular weights of proteins to ~
12kDa have been identified by LC–MS/MS [33]. The method performances are much
lower then typical quantities of MALDI–Orbitrap and MALDI (ESI)–ICR–MS methods,
due to high complexity of the biological tissues. The methods provide accurate analytical
information of complex food samples as phases, including volatile analytes in gas–phase;
homogeneous and heterogeneous compositions with gradients distribution of ingredients in
bulk and surface. MALDI MS enables direct testing; off–line and on–line screening and
imaging in all stages of beer production. This method was developed as a quantitative method
for clinical diagnostics [28–34]. The Orbitrap mass analyzer yields meaningful analytical
information on each complex analyte mixture. Both ESI– and MALDI–MS are used for
carbohydrate analysis in beer, with LC–, GC–, CE–, and FACE– based analysis protocols.
ELSD is regarded as semi–universal mass detector for carbohydrate quantification in beer
[28]. The technique is, on the determination of non-volatile analyte by light scattering after
nebulization and evaporation of a solvent phase. Other approach is HPAEC–PAD. It achieves
good resolution of these analytes, however, there is not to obtain structural information. Or, it
operates with standards for quantification at each analyte. The large scale organic ingredients
in beer, however, difficult implementation of standards for each analyte. Often carbohydrate
isomers are distinguished using gel electrophoresis by FACE [35]. The concentration LODs,
however, are relatively high. For carbohydrates in beer were obtained LODs  [100–313] M
[35]. The protocols for proteomics are mainly based on 1D SDS–PAGE, HPLC and FPLC,
respectively [36]. The same is valid for analytical protocols for phenolic compounds. The
high–resolution MS measurements provide a reliable information on unknown analytes in
complex food mixture at high accuracy and precision. Tandem MSn operation mode produces
crucial information about the molecular structure of the analyte [37]. The developments in
analytical techniques for food control involve MS reaction monitoring mode with individual
tuning of analytes [11–23]. They are introduced by a direct infusion into a MS instrument via
 Quantitative Analytical Methods of Organic Ingredients … 125

syringe pump. Appropriate MS transitions are selected together with a series analyte
dependent parameters (e.g., DP and CE). ECHO technique is followed by injection of
reference and unknown samples. If retention times close, analyte components are equally
affected by matrix components [20, 38].

1. BEER PROTEOMICS, PEPTIDOMICS AND ANALYSIS

OF AMINO ACIDS

1.1. Proteomics – A General Overview

The protein content in beer is ~ 500 mg.L-1 [39]. The amino acid masses are  [5–100]
Da. The proteins in beer derived from cereal grains (Table 1).

Table 1. Representative proteins from barley, cereal and yeast

(MALDI–TOF– or ESI–MS data)

Accession (NCBI/other) Protein name Refs.

Yeast proteins
gi|171455, gi|6321968 Enolase 1 and 2 [39]
gi|6322409, gi|219564301 Glyceraldehyde-3-phosphate dehydrogenase 1, 2 [39]
gi|1708241 Hydroxymethylglutaryl-CoA synthase [39]
gi|124702, gi|124703 Invertase 1 and 2 [39]
gi|6321718, gi|6323964 Probable family 17 glucosidase SCW4 and SCW10 [39]
gi|1723734, gi|731298 Probable glycosidase CRH1 and transporter SEO1 [39]
gi|347595651, gi|968906 Protein EGT2, NCA3 [39]
gi|125609, gi|6325103 Pyruvate kinase and Saccharopepsin [39]
gi|1723755, gi|97218363 Uncharacterized protein YGR237C, YOR020W-A [43]
Barley proteins
gi|1310677, gi|75282567 Serpin-Z4, Z7 [39–42]
Lipid transfer proteins [39]
gi|47168353, gi|6492243 Lipid transfer proteins [39, 40]
gi|75251746, gi|128377 Non-specific lipid-transfer proteins [39, 42]
Trypsin/α-amylase inhibitors [39]
gi|123970, gi|2506771 -Amylase inhibitor BDAI-1 and BMAI-1 [39, 40, 42]
Cereal proteins
gi|29839419 13S globulin seed storage protein 3 [39]
gi|7209265, gi|215398472 -Gliadin and Globulin 3B [39]
gi|209972037, gi|10638433 -Gliadin [39]

Proteins in grain and barley influence foam stability of beer. Quantitative protocols were
reported [40, 43–61]. Serpin-Z4 and lipid transfer protein 1 are known allergens causing for
urticaria and/or Baker's asthma. Coeliac disease is a non-IgEmediated autoimmune disorder
associated with “gluten” (see next Section 1.2). It causes for chronic gastrointestinal [39].
126 Bojidarka Ivanova and Michael Spiteller

1.2. Analysis of Prolamins and Glutenins in Beer

The term “gluten” refers to a class of so-called proteins prolamins and glutenins [62–69].
Their consumption leads to autoimmune reaction affecting by Coeliac disease about 1% of
people worldwide. It is intolerance to 5% of the population worldwide [62]. For this reason,
we have chosen analytical MS–based developments for quantitative determination of gluten
in beer to discuss as a separate subsection. The MALDI–TOF–MS and LC–ESI–MS methods
were used to determin peptide sequences (Tables 2 and 3).

Table 2. The (HPLC)–ESI–, (SDS–PAGE) MALDI–MS/MS analysis of peptide

sequences of proteins (confidence level ≥ 95%) of 60 commercially available beers

Peptide name MS [m/z] Refs. Peptide name MS [m/z] Refs.

D Hordein
gi|1167498 879.5097 {[M+H]+} [66] B1-F2 1377.8601 {[M+H]+} [62, 70]
gi|1167498 1172.6078 {[M+H]+} [66] B3-hordein 1249.7639 {[M+H]+} [70]
gi|1167498 1398.7826 {[M+H]+} [66] B1-hordein 1080.5495 {[M+H]+} [70]
gi|1167498 1729.9203 {[M+H]+} [66] B-hordein 857.4152 {[M+H]+} [70]
gi|1167498 1816.9456 {[M+H]+} [66] B3-hordein 1604.9843 {[M+H]+} [70]
gi|1167498 2054.1543 {[M+H]+} [66] B1-hordein 1049.5661 {[M+H]+} [70]
gi|1167498 2313.2151 {[M+H]+} [66] B1-hordein 1163.5879 {[M+H]+} [70]
gi|1167498 2928.6033 {[M+H]+} [66] B1-hordein 1290.6515 {[M+H]+} [70]
gi|1167498 4192.0498 {[M+H]+} [66] (gi|82548225) 1227.7014 {[M+H]+} [66]
gi|82548225 1372.6578 {[M+H]+} [66] B3-hordein 921.4833 {[M+H]+} [70]
gi|82548225 1834.0010 {[M+H]+} [66] B3-hordein 1144.6775 {[M+H]+} [70]
B3-hordein 1628.8599 {[M+H]+} [70] -Hordein
gi|123459 920.5563 {[M+H]+} [66] gi|123464 1047.5946 {[M+H]+} [66]
gi|123459 1213.7061 {[M+H]+} [66] gi|123464 1133.5771 {[M+H]+} [66]
gi|123459 1487.6241 {[M+H]+} [66] gi|123464 1725.8370 {[M+H]+} [66]
gi|123459 1501.7865 {[M+H]+} [66] gi|123464 2687.8840 {[M+H]+} [66]
gi|123459 1650.8342 {[M+H]+} [66] C Hordein
gi|123459 1666.8291 {[M+H]+} [66] gi|167016 1425.8262 {[M+H]+} [66]
Peptide name MS [m/z] Refs. Peptide name MS [m/z] Refs.
gi|123459 1834.0010 {[M+H]+} [66] gi|167016 2131.0924 {[M+H]+} [66]
C Hordein
gi|167016 2156.1287 {[M+H]+} [66] gi|442524 2103.7651 {[M+H]+} [66]
gi|167016 2794.4128 {[M+H]+} [66] gi|442524 2156.1287 {[M+H]+} [66]
gi|442524 1171.6066 {[M+H]+} [66] gi|442524 2466.2600 {[M+H]+} [66]
gi|442524 1425.7820 {[M+H]+} [66] gi|442524 2794.4128 {[M+H]+} [66]
gi|442524 1989.9334 {[M+H]+} [66]

1.3. Beer Peptidomics and Analysis of Amino Acids

The MS was used to determine amino acids and oligpopeptides in beer [70–78]. The
chemical substitution of peptide chains was performed. As a tagging reagent 2-(9-carbazole)-
ethyl chloroformate was used (Figure 1). The homo-glycyl-di– to hexapeptides were
examined (Table 4). Fmol concentration ranges were proven. The LMW peptide analysis is
shown in Table 5.
 Quantitative Analytical Methods of Organic Ingredients … 127

Table 3. MALDI–TOF–MS or LC–MS/MS analysis of hordeins

Accession (NCBI/other) Protein name Refs

gi|326501830, gi|338817618 Avenin-like protein A3 and A4 [41]
gi|110832715, gi|123459*, gi|51556918 B-hordein [39, 41]
gi|18929, gi|224385, gi|224386
gi|75220900, gi|123458, gi|809031 B1-hordein [39, 41]
gi|122220131, gi|671537 B3- and D-hordein [39]
gi|75220910, gi|123461, gi|75102504 C-hordein [39]
gi|226755, gi|1708280*, gi|34329257 -Hordein-1 and -Hordein-3 [39]
gi|20210 Glutelin [39]
gi|75250230, gi|164457873 HMW glutenin subunit x and y [39]
gi|329745049, gi|17425212 LMW glutenin subunit [39]

Table 4. The ESI–, UHPLC (or LC)– Q–TOFMS, and 2D GC–MS/MS analysis of amino
acids in beer; tR – Retention time [mins]; Concentration LODs; r –
Correlation coefficient

Analyte MS tR [mins] r LODs [units] Refs.

[m/z]
Arginine 0.12 0.9995 12.9 fmol [71, 72]
Asparagine 366.11 0.11 0.9996 4.3 fmol; 70.1 g.mL-1; [71, 72, 79]
Serine 339.10 0.14 0.9996 4.3 fmol; 72.9 g.mL-1 [71, 72, 79]
Glutamine 380.12 0.10 0.9996 17.6 fmol; 65.0 g.mL-1 [71, 72, 79]
Threonine 353.11 0.16 0.9997 17.5 fmol; 75.1 g.mL-1 [71, 72, 79]
Glycine 309.09 0.13 0.9997 13.5 fmol; 65.5 g.mL-1 [71, 72, 79]
Alanine 323.10 0.11 0.9998 24.8 fmol; 63.0 g.mL-1 [71, 72, 79]
0.9992 0.01 mg.L-1 [76]
Proline 349.12 0.15 0.9998 33.2 fmol; 72.7 g.mL-1 [71, 72, 79]
0.9973 0.05 mg.L-1 [76]
Methionine 383.10 0.08 0.9997 39.8 fmol; 68.3 g.mL-1 [71, 72, 79]
Valine 351.13 0.08 0.9996 19.2 fmol; 85.9 g.mL-1 [71, 72, 79]
0.9985 0.05 mg.L-1 [76]
Phenylalanine 399.13 0.08 0.9996 16.7 fmol; 88.8 g.mL-1 [71, 72, 79]
Tryptophan 438.14 0.08 0.9997 35.5 fmol; 83.7 g.mL-1 [71, 72, 79]
Isoleucine 365.15 0.06 0.9996 18.6 fmol; 86.9 g.mL-1 [71, 72, 79]
0.9993 0.01 mg.L-1 [76]
Leucine 365.15 0.06 0.9999 16.4 fmol; 84.1 g.mL-1 [71, 72, 79]
Cysteine 355.07 0.07 0.9999 126 fmol; 64.0 g.mL-1 [71, 72, 79]
Histidine 622.17 0.07 0.9993 18.6 fmol; 69.0 g.mL-1 [71, 72]
Ornithine 599.19 0.06 0.9999 5.7 fmol; 100.4 g.mL-1 [71, 72]
Lysine 613.21 0.06 0.9999 4.0 fmol; 106.1 g.mL-1 [71, 72, 79]
Tyrosine 648.18 0.06 0.9997 5.3 fmol; 101.2 g.mL-1 [71, 72, 79]
Citrulline 392.13 80.9 g.mL-1 [71, 72]
Norleucine 365.15 79.6 g.mL-1 [71, 72]
-Aminobutyric acid 0.9999 0.05 mg.L-1 [76]
128 Bojidarka Ivanova and Michael Spiteller

Figure 1. Chemical substitution of amino acids or peptides [71].

Table 5. The ESI–, UHPLC– (or LC)–Q–TOFMS quantitative analysis of peptides in

beer; tR – Retention time [mins]; Concentration LODs

Analyte/Sequence MS [m/z] tR LODs Refs.

(Gly)2 303.1 10.71 38.8 fmol; 0.8 nM [71, 73]
(Gly)3, (Gly)4 36.8, 48.5 fmol [71]
(Gly)5, (Gly)6 65.4, 64.3 fmol [71]
Peptide 428.164, 291.172 3.3, 7.8 [74]
Peptide 455.240, 864.788 8.2, 10.3 [74]
Peptide 287.068, 749.111 10.5, 14.1 [74]
-Glu-Val-Gly (kokumi) 175.1, 229.2 0.09–0.18 mg.L-1 [75]
nsLTP 1 (H. vulgare)
VPYT, ISPDID 479.2538, 659.3261 [70]
GIHNLNLN 447.7458 [70]
GIHNLNLNNAAS 619.3251 [70]
GIHNLNLNNAASIPSK 831.9859 [70]
nsLTP 2 (H. vulgare)
DTLNLCGIPVPHC 748.3888 [70]
α-Amylase/trypsin inhibitor CMd [70]
FPTNLLG 761.4248 [70]
LLVAPGQCNLATIHNVR 626.0268 [70]
LVAPGQCNLATIHNVR 588.3351 [70]
AAAATDCSPGVAFPTNLLGHCR 762.7199 [70]
AATDCSPGVAFPTNLLGHCR 715.3589 [70]
DYVLQQTCAVFTPGSK 907.4689 [70]
α-Amylase inhibitor BDAI-1 [70]
LLVAGVPALCNVPIPNEAAGTR 744.7604 [70]
α-Amylase/trypsin inhibitor CMa [70]
MGLPSNPLE 957.4798 [70]

To volatile N-containing ingredients biogenic and other amines are assigned (Subsection
11.1); amino acid ethyl esters (Table 4); different pyrazines; pyridines; pyrroles; or thiazoles
[78]. To diketopiperazines belong cyclic dypeptides (Figure 2). They are objects of quality
control [78]. Electron impact MS data show m/z values (Relative intensity, %): 154 (96), 125
(12), 86 (26), 70 (100), 55 (12), 43 (17), 41 (23), 30 (13) (cyclo(Leu-Pro)); 168 (35), 140
(10), 125 (34), 97 (33), 70 (100), 55 (17), 44 (90), 41 (49), 28 (51) (cyclo(Ala-Pro); 228 (32),
 Quantitative Analytical Methods of Organic Ingredients … 129

167 (44), 154 (99), 139 (32), 70 (100), 61 (28), 56 (21), 41 (32), 28 (26) (cyclo(Met-Pro));
244 (32), 153 (51), 125 (100), 91 (52), 70 (88), 43 (8), 41 (27), 28 (34) (Cyclo(Phe-Pro) and
m/z 194 (24, M), 166 (5), 138 (7), 124 (5), 110 (7), 96 (13), 70 (100), 55 (10), 41 (37), 28 (17)
(cyclo(Pro-Pro), respectively.

Figure 2. Diketopiperazines in beer [78].

2. BEER LIPIDOMICS AND ANALYSIS OF FREE FATTY ACIDS

Lipidomics and analysis of free fatty acids is an important analysis in beeromics
due to effect of lipids on yeast metabolism and quality of beer [78]. These ingredients are
in the brewing as different forms, like simple lipids of free fatty acids; mono–, di– and
triacylglycerols; complex lipids and lipids which are bound. Lipopolysaccharides of
pathogenic microorganisms in beer are also assigned. Lipidomics was performed, determining
pathogenic agents [80–85]. This section is relevant to subsection 11.4. The anaerobic species
Pectinatus cerevisiiphilus and Pectinatus frisingensis belong to bacteria of genus Pectinatus.
They are spoilage microorganisms in packaged beer. The MS methods using desorption–
ionization approaches were employed determining location and type of lipids in this bacterial
strain [81]. The ESI–MS/MS protocols were developed for lipidomics of Pectinatus
bacterium [83, 84]. The structure of lipid A is depicted in Figure 3. It was an anomeric
glycosidic phosphate residues found.
130 Bojidarka Ivanova and Michael Spiteller

Figure 3. Chemical diagram of lipid A from bacteria genus Pectinatus in beer [81–84].

Figure 4. (Continued)
 Quantitative Analytical Methods of Organic Ingredients … 131

Figure 4. The ESI–Orbitrap–MS/MS fragmentation of plasmenyl-triacyl-cardiolipin [84].

Lipidomics of Megasphaera and Pectinatus species in spoiled beer using ESI–MS/MS

was reported [82]. HR/ESI–MS/MS by Orbitrap analyzer was used to identify cardiolipins of
different species of Pectinatus bacterium [84]. The fragmentation pattern of plasmenyl-
triacyl-cardiolipin is shown (Figure 4).
The free fatty acids with carbon chain lengths C6–C18 and their ethyl esters are
components in beer [86–95]. The concentration ranges are  (1–22.8) mg.L-1 (C6); [1.8–14.7]
mg.L-1 (C8); [0.1–5.2] mg.L-1 (C10) and [0.05–0.007] mg.L-1 (C2) (free fatty acids), and 
[0.07–0.37] (C6) and [0.08–0.60] mg.L-1 (C8) (fatty acids ethyl esters) [96]. GC–MS
quantitation protocols were reported [86–92].

3. CARBOHYDRATE QUANTITATION, DETERMINATION OF MONO –

AND OLIGOSACCHARIDES AND PENTOSANS

Carbohydrates are main components in beer [24, 35, 93–125]. These are assigned glucose
(content  [0.5–5] g.L-1), fructose ( [0.5–2] g.L-1); maltose ( [0.5–15] g.L-1); maltotriose
( [0.5–10] g.L-1) as monosaccharides ( [0.5–5] g.L-1) and glucose oligosaccharides. The
saccharides are linked to beer testing; diet and physical properties. The MALDI–MS and
quantum chemistry, using density functional theory of D-fructose were used to study the
fragmentation mechanisms [126]. This analyte stabilizes – and –pyranose, and – and –
fructose isomers (Figures 5 and 6). The reactions depend on deprotonation side in molecule of
monosaccharide.
132 Bojidarka Ivanova and Michael Spiteller

Figure 5. Chemical diagrams of –D–fructopyranose and –D–fructofuranose [24, 35, 93–132].

Figure 6. (Continued)
 Quantitative Analytical Methods of Organic Ingredients … 133

Figure 6. Molecular dynamic simulation of fragmentation processes of monosaccharide -D-fructose;

Fragmentations of – and –anomers [126].

The synthetic substitution of carbohydrate scaffold permits selective determination of

each conformers. The 1–naphthylamine was used to functionalize the compound. The
quantitative analysis by HPLC–Fs (and MS) and FACE methods shows concentration
LODs 1.2 M. Concentration LODs 110 M by FACE were reported (Table 6) [35]. The
fragmentation patterns are: ribose, m/z 278.5 (r.a. 45%), 260.4 (12), 242.4 (13), 170.5 (61),
168.6 (26), 156.4 (100), 144.3 (91), 143.3 (86), 129.3 (54); Xylose, 278.5 (28), 260.4 (16),
242.4 (11), 170.5 (44), 168.6 (21), 156.4 (100), 144.3 (69), 143.3 (71), 129.3 (47); Glucose,
308.4 (51), 290.5 (31), 272.5 (7), 170.6 (36), 168.4 (13), 156.3 (100), 144.3 (49), 143.3 (37),
129.3 (30); maltose, 470.6 (35), 452.6 (2), 308.5 (100), 290.6 (13), 156.4 (6), 144.3 (4);
Isomaltose, 470.6 (36), 452.6 (1), 308.5 (100), 290.6 (11), 156.4 (4), 144.3 (2); Maltotriose,
654.7 (21), 632.7 (100), 614.8 (1), 470.5 (2), 308.5 (75), 290.6 (3); Maltotetraose, 816.9 (21),
794.8 (100), 470.5 (2), 308.5 (9), 144.4 (2); Maltopentaose, 978.8 (41), 956.9 (100), 632.5
(9), 470.7 (4), 308.6 (17), 290.3 (2); Maltohexaose, 1119.0 (100), 794.9 (51), 632.7 (43),
470.5 (26), 308.5 (89); and maltoheptaose, 1281.1 (73), 995.9 (14), 956.8 (64), 794.8 (100),
65246 (39), 632.7 (87), 470.6 (27), 308.5 (99), respectively [35]. The fragmentation pattern of
maltotetraose under ESI(–)–CID–MS conditions is shown in Figure 7. The FAB–MS
fragmentation of matohexaose is depicted in Figure 8.
The quantitative MS analysis of oligosaccharides was based on peak at m/z 991 of
{[M+H]+} ion, observed under FAB–MS experimental conditions (Figure 9). The increasing
in concentration of analyte leads to disappearance of matrix signals. Fragmentation patterns
134 Bojidarka Ivanova and Michael Spiteller

of carbohydrate were detected [131]. At a fixed concentration of matrix component (glycerol,

4 g), a liner relationship between MS ion intensities of analyte normalized against matrix
MS peak intensity at m/z 553 as a function of amount of analyte was found (Figure 9). As in
MALDI–MS, the matrix components in FAB–MS effect behavior of analyte signals. The use
of glycerol matrix with diethanolamine causes of the shown in Figure 10 relationship between
normalized intensities of carbohydrate and amount of the analyte.

Figure 7. ESI(–)–CID–MS fragmentation of maltotetraose [130].

Figure 8. FAB–MS fragmentation of maltohexaose [131].

 Quantitative Analytical Methods of Organic Ingredients … 135

Figure 9. Normalized MS intensities of malohexaose signals (matrix signal at m/z 553, glycerol, 4g)
versus the amount of the carbohydrate [131].

Figure 10. FAB(+)–MS spectrometric data of malohexaose signals (matrix signal at m/z 526,
diethanolamine, 4g) versus the amount of the carbohydrate [131].

Main fragmentation pathways of matooligosaccharides under FAB(+)–MS and FAB(–)–

MS experimental conditions (Figure 11, DP  [3, 7] (n  [1, 5])).
136 Bojidarka Ivanova and Michael Spiteller

Figure 11. Fragmentation pathways of maltoligosaccharides under FAB(+)–MS and FAB(–)–MS

operation modes; DP  [3, 7] (n  [1, 5]) [131].

Table 6. Carbohydrate analysis of beer in bottles using chemically substitution by 1–

naphthylamine [35]; HPLC–Fs and MRM–MS (ESI(–)) parameters; FACE data; DP
[V]; CE [V]; CXP [V]; Concentration LODs [g.L-1]; tR – Retention time [mins]

Analyte MRM–MS MS (r.a., %) DP CE CXP HPLC FACE tR

LODs [g.L-1]
Ribose 278.3156.2 300.4,{[M+Na]+}(27) 31 30 27 0.04 - 64.8
Arabinose 300.4{[M+Na]+} (25) 0.08 - 60.8
Glucose 308.3156.2 330.5{[M+Na]+} (28) 75 32 27 0.03 0.1 54.5
Maltose 470.4308.3 492.6{[M+Na]+} (19) 71 27 20 0.07 0.2 49.1
Maltotriose 632.5308.3 670.8 {[M+Na]+} (1) 72 35 18 0.7 1.0 44.7
Isomaltose 492.6{[M+Na]+} (23) 0.9 1.4 43.2
Maltotetraose 794.5308.3 832.8 {[M+K]+} (2) 100 45 17 5.1 4.3 39.7
Maltopentaose 956.6308.4 994.8 {[M+K]+} (3) 108 56 17 2.6 6.0 36.0
Maltohexaose 1118.7308.4 1141.0{[M+Na]+(60) 125 70 16 1.7 2.6 32.7
Maltoheptaose 1280.9308.5 1303.0{[M+Na]+(31) 93 81 17 1.0 2.4 30.9
 Quantitative Analytical Methods of Organic Ingredients … 137

Maltooligosaccharides with different degree of polymerization in beer were determined

by MALDI(+) and MALDI(–) MS [103] (Table 7).
On this subsection we analyze β-glucans in beer. These are carbohydrate polymers, which
contain β–glycosidic bonds, which occur in the beer a result of degradation processes of grain
and yeast cell walls [133].

4. DETERMINATION OF OFF–FLAVORS IN BEER

Esters and diketones are also associated with quality of beer. They can cause a negative
effect on fermentation process. These analytes are objects of online and offline quantification
[134–154]. The normally detected in beer off–flavors are isoamyl acetate, ethyl acetate, ethyl
hexanoate and 2,3–butanedione, respectively [134]. The concentration ranges are 1400,
15.000, 200 and 40 ng.mL-1, respectively. A HS–SPME–GC–MS based quantification
was reported [134]. Dynamic HS–SPME analysis with GC–qMSD in beer were performed
[138] (Table 8). The main part analytical protocols based on GC–MS involves determination
of total volatile fraction of beer. They often include quantification of terpenes as well [140].

Table 7. The MALDI(+) and MALDI(–) MS analysis of maltooligosaccharides with

different degree of polymerization in beer; Fragmentation patterns [103]

DP {[M+Na]+} {[M+K]+} {[M-H]-} {[M-R1-H]-} [M-R2-H2O-H]- [M-R1-R2-H2O-H]-

3 527.1582 543.1322 503.1617 383.1195 455.1406 335.0984
4 689.2111 705.1850 665.2146 545.1723 617.1934 497.1512
5 851.2639 867.2378 827.2674 707.2251 779.2463 659.2040
6 1013.3167 1029.2906 989.3202 869.2779 941.2991 821.2568
7 1175.3695 1191.3435 1151.3730 1031.3308 1031.3308 1103.3519
8 1337.4223 1353.3963 1313.4258 1193.3836 1265.4047 1145.3625
9 1499.4752 1515.4491 1475.4787 1355.4364 1427.4575 1307.4153
10 1661.5280 1677.5019 1637.5315 1517.4892 1589.5104 1469.4681
11 1823.5808 1839.5547 1799.5843 1679.5420 1751.5632 1631.5209
12 1985.6336 2001.6076 1961.6371 1841.5949 1913.6160 1793.5737
13 2147.6864 2163.6604 2123.6899 2003.6477 2075.6688 1955.6266
14 2309.7393 2325.7132 2285.7428 2165.7005 2237.7216 2117.6794
15 2471.7921 2487.7660 2447.7956 2327.7533 2399.7745 2279.7322
16 2633.8449 2649.8188 2609.8484 2489.8061 2561.8273 2441.7850
17 2795.8977 2811.8717 2771.9012 2651.8590 2723.8801 2603.8378
18 2957.9505 2973.9245 2933.9540 2813.9118 2885.9329 2765.8907
19 3120.0034 3135.9773 3096.0069 2975.9646 3047.9857 2927.9435
20 3282.0562 3298.0301 3258.0597 3138.0174 3210.0386 3089.9963
21 3444.1090 3460.0829 3420.1125 3300.0702 3372.0914 3252.0491
22 3606.1618 3622.1358 3582.1653 3462.1231 3534.1442 3414.1019
23 3768.2146 3784.1886 3744.2181 3624.1759 3696.1970 3576.1548
24 3930.2675 3946.2414 3906.2710 3786.2287 3858.2498 3738.2076
25 4092.3203 4108.2942 4068.3238 3948.2815 4020.3027 3900.2604
26 4254.3731 4270.3470 4230.3766 4110.3343 4182.3555 4062.3132
27 4416.4259 4432.3999 4392.4294 4272.3872 4344.4083 4224.3660
28 4578.4787 4594.4527 4554.4822 4434.4400 4506.4611 4386.4189
29 4740.5316 4756.5055 4716.5351 4596.4928 4668.5139 4548.4717
30 4902.5844 4918.5583 4878.5879 4758.5456 4830.5668 4710.5245
138 Bojidarka Ivanova and Michael Spiteller

Table 8. Selected volatile and semi–volatile components in beer determined by MS–

based methods; tR – Retention time [mins];
Concentration LODs

Analyte tR [mins] LODs Method Refs.

Ethyl butanoate 4.521 ng.L-1–mg.L-1 GC–qMSD [140]
2-Methyl-1-propanol 6.368 ng.L-1–mg.L-1 GC–qMSD [140]
8.62 8.3 ppm GC–MS/FID [142]
Isoamyl acetate 6.907 ng.L-1–mg.L-1 GC–qMSD/GC–MS [134, 138, 139]
56.4 112.6 ng.L-1 HS–SPME–GC–MS [140]
6079.05 g.L-1 HS–SPME–GC–MS [140]
16.76 1.7 ppm GC–MS/FID [142]
ESI–MS/MS [143]
Ethyl hexanoate 12.104 ng.L-1–mg.L-1 GC–qMSD/GC–MS [134, 138]
56.4 9298.4 ng.L-1 HS–SPME–GC–MS [148]
Styrene 13.19 ng.L-1–mg.L-1 GC–qMSD [138]
1-Methyl-3-(1-methylethyl)- 13.768 ng.L-1–mg.L-1 GC–qMSD [138]
benzene
Hexyl isobutanoate 24.427 ng.L-1– mg.L-1 GC–qMSD [138]
Furfural 27.948 ng.L-1– mg.L-1 GC–qMSD [138]
(Z)-3-Hexenyl hexanoate 42.679 ng.L-1– mg.L-1 GC–qMSD [138]
4,6-Di(1,1dimethylethyl)-2- 61.177 ng.L-1– mg.L-1 GC–qMSD [138]
methylphenol
2.3-Butanedione 41.2 ng.L-1 HS–SPME–GC–MS [140]

5. ANALYSIS OF TERPENES IN BEER

Terpenes derived mainly of hops essential oil (Figure 12) and contribute to sensory
characteristics of beer [155–167]. They are chemically instable and participate in oxidation
processes. Typical terpenes in beer are classified as terpenes hydrocarbons, epoxydes and
alcohols, respectively [155]. The monoterpenoids participate in biotransformation reactions
in presence of filamentous fungi. As far as these substances are in contact with yeasts
during beer production, such reactions may occur [157]. GC–MS analysis was shown that
species Saccharomyces cerevisiae and Kluyveromyces lactis may reduce terpene geraniol to
citronellol [157]. Kluyveromyces lactis and Torulaspora delbrueckii may transform geraniol
to nerol (Figure 13).
The chemical isomerisation and degradation of hop ingredients in beer; and processes of
photochemical transformations; oxidation and/or reduction may occur. Specifically, –
ahumulene (g) and –caryophyllene were widely discussed [168]. The chemical diagrams of
respective products of interaction are depicted in Figure 14.
 Quantitative Analytical Methods of Organic Ingredients … 139

Figure 12. Chemical diagrams of hops terpenes [154]; Chemical names: β–myrcene (a); limonene (b);
terpinolene (c); p–cymene (d); α–pinene (e); β–pinene (f); α–humulene (g); β–caryophyllene (h); trans–
β–farnesene (i); 1,4–cineole (j); β–citronellol (3,7–dimethyl–6–octen–1–ol) (k); nerol (3,7–dimethyl–
cis–2,6–octadien–1–ol) (l); geraniol (3,7–dimethyl–trans–2,6–octadien–1–ol) (m); linalool (3,7–
dimethyl–1,6–octadien–3–ol) (n); α–terpineol (1–p–menthen–8–ol) (o); and nerolidol (p), respectively.

Figure 13. Biochemical transformation of geraniol and nerol, catalyzed by species Saccharomyces
cerevisiae, Kluyveromyces lactis and Torulaspora delbrueckii [157].
140 Bojidarka Ivanova and Michael Spiteller

Figure 14. (Continued)

 Quantitative Analytical Methods of Organic Ingredients … 141

Figure 14. Chemical transformations of –ahumulene (g) and –caryophyllene [168].

6. QUANTITATIVE ANALYSIS OF PHENOLIC COMPOUNDS IN BEER BY

MASS SPECTROMETRIC BASED METHODS
6.1. Analysis of Monophenolic Ingredients

There are numerous phenolic compounds in beer [3, 4]. About 30% are from hops, while
another 70–80% – from malt. The main phenolic compounds in dried hops ( 14.4%) are
phenolic acids; cathechins; flavonoids; chalcones; and proanthrocyanidins, respectively. The
flavonoids kaempferol and quercetin; hop bitter acids xanthohumol, it iso–derivative
isoxanthohumol and 8–prenylnaringenin; simple phenolic compounds tyrosol and ferulic acid
(Figure 15) show anti–inflammatory and anti–oxidative activities; and an anti–carcinogenic
one. This biological activity causes for their application in dermatology against skin
cancer [3, 4]. The MPLC–UV and HPLC–MS/MS were used studying monophenolic
components in tannin and resin extracts; hard and soft resins (isolated as insoluble fraction
in n-hexane but, which is soluble in ether and cold methanol (first set resins) and a
sample set resins soluble in saturated hydrocarbons, having low-boiling points such as n-
hexane); and hop pellets (Figure 15) [169–197]. The reports show content < 0.01 mol.
(100g)-1 of 1-methoxy-4-prenylphloroglucinol in soft resin and extracts and; 1022
mol.(100g)-1 for hard resin and 68.38 mol.(100g)-1 for tannin extract [169]. Total content
substituted cis/trans–p–coumaric acids and their methyl and ethyl esters is < 0.01 mol.
(100g)-1 in soft resins and extracts; and 6158 mol.(100g)-1 in hard resin. The analysis of 1–
O–β–D–(2–methylpropanoyl) phloroglucinol glucopyranoside, 1–O–β–D–(2–methylbutyryl)
phloroglucinol glucopyranoside, phloroisovalerophenon–3,5–di–C–β–D–glucopyranoside and
trans–N–feruloyltyramine has shown contents  [3.85–4280], [1.31–800.6], (0.01–1038] and
(0.001–2999] mol.(100g)-1 in different matrixes. The quantities of phenolic acids in beer
vary (Figure 15): p–hydroxybenzoic acid ( [0.017–16.84] mg.L-1), gallic acid ( [0.015–
3.5] mg.L-1), vanillic acid ( [0.01–1.79] mg.L-1), syringic acid ( [0.017–0.68] mg.L-1),
gentisic acid ( [0–1.5] mg.L-1), p–coumaric acid ( [0.06–1.67] mg.L-1), caffeic acid (
[0.01–0.98] mg.L-1), ferulic acid ( [0.15–14.2] mg.L-1), and sinapic acid ( [0.056–1.04]
142 Bojidarka Ivanova and Michael Spiteller

mg.L-1), respectively [174]. The MS–based quantitative protocols are shown in Table 9. The
stable isotope dilution analysis was reported [196].

Figure 15. Chemical diagram of monophenolic compounds [169, 170, 195–197].

 Quantitative Analytical Methods of Organic Ingredients … 143

Table 9. The MS quantitative analysis of monophenolic compounds; tR –

Retention time [mins]; MS data [m/z]; Concentration LODs [g.g-1]; **–
Stable isotope labeled compound

Analyte tR MS [m/z] LODs MS/MS–method Refs

Gallic acid 1.16 169.0142 ESI-LTQ-Orbitrap [171]
Caffeic acid 4.6 179.0349 ESI-LTQ-Orbitrap; [171, 196]
184** ESI-TSQ
Caffeic acid-O-hexoside I 1.55 341.0877 ESI-LTQ-Orbitrap [171]
Caffeic acid-O-hexoside II 2.23 341.0877 ESI-LTQ-Orbitrap [171]
Ferulic acid* 7.03 193.0506 ESI-LTQ-Orbitrap [171, 196]
198** ESI-TSQ
Vanillic acid 4.5 167.0349 ESI-LTQ-Orbitrap [171, 196]
175** ESI-TSQ
p-Coumaric acid 5.94 163.0400 ESI-LTQ-Orbitrap [171]
168** ESI-TSQ
4-Hydroxybenzoic acid 3.54 137.0241 ESI-LTQ-Orbitrap [171, 196]
8-8’-Disinapic acid 401.0357.0 2.30±0.10 UPLC-ESI/MRM [195]
8-5’-Diferulic acid 385.1341.2 0.05±0.01 UPLC-ESI/MRM [195]
8-O-4’-diferulic acid 385.1193.1 2.78±0.26 UPLC-ESI/MRM [195]
8-3’-Dicoumaric acid 325.1281.2 0.15±0.01 UPLC-ESI/MRM [195]

The oxidation reactions of coumaric, ferulic and sinapic acids in vitro yield to dimers
(Table 9). These dimers, e.g., 8-8’-disinapic, 8-5’-diferulic, 8-O-4’-diferulic and 8-3’-
dicoumaric acids respectively were quantified by UHPLC–MS/MS MRM/MS in light beer
[195]. Single oxidation reactions and mechanism of formation of dimeris are depicted in
Figure 16.

Figure 16. (Continued)

144 Bojidarka Ivanova and Michael Spiteller

Figure 16. Dimerization reactions with participation of hydroxycinnamates [195].

6.2. Determination of Monomeric Phenolic Flavonoids

The metabolomics of flavonoids [171–190], is an issue of enormous efforts,

because of they exhibit important biological antioxidant, antiproliferative, anti-inflammatory,
anticarcinogenic and antiviral activities. Thye are found in plants, fruits, nuts, vegetables,
flowers, seeds, and bark [198–211]. Series review articles are presented [198–211]. In this
subsection the attention is focused only on MS contributions to analysis of flavonoids in beer.
These types chemicals rang from phenolic acids (subsection 6.1) to simple flavanoids,
anthocyanidins, and tannins. The term “oligomeric” is connected with phenolic flavonoids
(dimeric, trimeric) to heptameric structures (n  [2, 7], Figure 26, subsection 6.3). The term
 Quantitative Analytical Methods of Organic Ingredients … 145

“polymeric” (tannins) phenolic flavonoids denotes molecular structures with n  [2, 20].
The two types condensed products of polyhydroxy flavan–3–ol units such as catechin,
gallaocatechin and their epimers (Figure 17) are discussed in the next subsection 6.3.

Figure 17. Flavanol molecular scaffold in hops [37].

Online LC–APCI (+)–MS/MS and offline ESI(+)–MS/MS studies of catechin,

epicatechin, and gallocatechin were reported [37, 211–212]. The fragmentation pathways are
shown in Figure 18. Epicatechin shows {[M+H]+} ion at m/z 291 and fragment species at m/z
165 (MS/MS product ions m/z 126), 139 (152), 169 (122), 151 (140), and 147 (144),
respectively. Gallocatechin exhibits characteristic MS peaks at m/z 307 {[M+H]+} and
product MS/MS ions at m/z 181 (m/z 126), 139 (168), 169 (138), 151 (156), and 163 (144),
respectively [39].

Figure 18. Fragmentation pathways of monomeric catechin [39, 179].

146 Bojidarka Ivanova and Michael Spiteller

Figure 19. Nomenclature of fragmentation ions of di–substituted flavonoid glycosides [38]; Chemical
diagram of a flavonoid scaffold (in black); R1,R2– substituents such as –CH3, –OCH3, glycoside.

The MS methodological development for proteomics (approaches (i)–(iv),

“Introduction”) are applicable to flavins [38, 201, 203, 208, 209]. Series flavins were
examined with approach (iv). Under ESI(–)/CID experimental conditions fragmentation
pathways of flavonoids were studied, using {[M-2H]-} anion radical. It is obtained as a result
of redox process in presence of FeII/FeIII ions (Figure 19).
Isoflavones in beer genistein and daidzein (Figure 20) were implicated in cancer
prevention [213]. The quantitative GC–MS and HPLC–DAD–MS protocols were reported
[213, 214] (Tables 10 and 11). This experimental scheme is a common one to many
quantitative protocols based on MS [215–225]. Xanthohumol (Figure 20) with prenylated
chalcone scaffold [208, 209] is main phenolic compound in hop extracts and beer. The
HPLC–MS/MS determination was reported [208, 209, 214]. The APCI–MS data were shown
MS peak at m/z 354. APCI(+)–MS spectra exhibit peaks at m/z 371 {[M+NH4]+}, 355
{[M+H]+}, and 299 {[M+H–C4H8]+}; MS/MS analysis (m/z 355) shows fragment ions at m/z
299 {[M+H–C4H8]+} (r.a. 100%), 235 {[M+H–C8H8O]+} and 179 {[M+H–C4H8–C8H8O]+}.
The APCI(-)–MS spectra were shown peak at m/z 353 {[M–H]-} (100%). The MS/MS data
show peaks at m/z 233 {[M–H–C8H8O]-} and 119 {[M–H–C8H8O–C8H2O]-} [214]. The
experimental parameters are: direct infusion at a flow rate 100 mL.min-1; pressure of
nebulizing gas 15 psi; flow rate 4 L.min-1 and T = 300 and 350oC of APCI heater. The second
experimental scheme of HPLC/MS analysis is at flow rate 1 mL.min-1, pressure 70 psi, flow
rate of drying gas 5 L.min-1 and T = 350 and 450oC [215]. Same experimental schemes were
used for analysis of hop bitter acids [215] (subsection 7.1). The determination of other
polyphenolic flavonoids in beer (Figure 20) was performed using fragment molecular ions:
isoxanthohumol, m/z 371 {[M+NH4]+}, 355 {[M+H]+} and 299 {[M+H–C4H8]+} (r.a. 100%).
The MS/MS analysis (m/z 355) shows peaks at m/z 299 {[M+H–C4H8]+} (100%), 235
{[M+H–C8H8O]+}, and 179 {[M+H–C4H8–C8H8O]+}, respectively. Negative–ion APCI(-)–
MS spectra reveal peak at m/z 353 {[M–H]-} (100%). Its MS/MS analysis reveals peaks at
m/z 233 {[M–H–C8H8O]-}. The 8–prenylnaringenin is characterized with following ions
under APCI(+)–MS experiments, i.e., peak at m/z 341 {[M+H]+} (100%) and 285 {[M+H–
C4H8]+}. The MS/MS fragmentation data (m/z 341) of show peak at m/z 285 {[M+H–C4H8]+}
(100%). The MS/MS analysis of m/z 285 shows peaks at m/z 165 {[M+H–C4H8–C8H8O]+}.
 Quantitative Analytical Methods of Organic Ingredients … 147

APCI(-)–MS spectra exhibit peaks at m/z 339 {[M–H]-} (100%) and MS/MS fragments at m/z
219 {[M–H–C8H8O]-}; desmethylxanthohumolreveals MS peaks (APCI(+)–MS) at m/z 341
{[M+H]+} and 285 {[M+H–C4H8]+}. The MS/MS (m/z 341) shows peaks at m/z 285 {[M+H–
C4H8]+}, 221 {[M+H–C8H8O]+} and 165 {[M+H–C4H8–C8H8O]+}, respectively. The MS/MS
data reveal peaks at m/z 165 {[M+H–C4H8–C8H8O]+} (100%). APCI(-)–MS spectra show
peak at m/z 339 {[M–H]-}. The MS/MS results are peaks at m/z 295 {[M–H–C2H3O]} and
219 {[M–H–C8H8O]-} [215].

Figure 20. (Continued)

148 Bojidarka Ivanova and Michael Spiteller

Figure 20. Chemical diagrams of phenolic flavonoids in beer [213, 215, 217, 226, 227].
 Quantitative Analytical Methods of Organic Ingredients … 149

Table 10. Quantitative MS analysis of flavonoids; Linearity range [g.(mL)-1];

Correlation coefficient r2, LODs [g.g-1]; Recovery % (RSD, %); k – and tR –
Retention factor and time [mins]

Analyte Linearity range r2 LOD Recovery k Refs.

Isoxanthohumol 2.1–166.0 0.9989 0.8 1.95 [16, 214,225]
0.02 97.6 (0.91) [214]
0.9989 0.012 93 [228]
8-prenylnaringenin 3.8–152.0 0.9984 1.0 3.11 [214, 225]
6-prenylnaringenin 2.2–177.0 0.9999 0.8 4.18 [214, 225]
Xanthohumol 1.3–81.0 0.9997 0.3 [214]
0.02 93.7 (3.75) 5.46 [214]
0.9999 0.008 90 [228]
Cohumulone 0.02 90.5 (2.74) 9.13 [214, 229]
Humulone 0.02 89.0 (3.00) 10.2 [214]
Adhumulone 0.05 102 (3.02) 10.4 [214]
Colupulone 0.02 96.4 (4.36) 13.1 [214, 229]
Lupulone 0.03 82.5 (3.28) 14.3 [214, 229]
Adlupulone 0.03 104 (1.03) 14.6 [214]

Table 11. Quantitative analysis of phenyl flavonoids;

Accuracy and precision of HPLC–MS–MS/MRM–MS methods;
tR – Retention time [mins]

Analyte tR Analysis Refs.

One hop extract One beer sample All beer samples
BD WD BD WD BD WD
Xanthohumol 14.18 4.7 4.1 6.9 4.7 1.5 7.4 [230]
Isoxanthohumol 9.13 15.8 5.5 2.6 3.6 12.8 10.8 [230]
8–Prenylnaringenin 11.63 12.6 7.4 6.0 5.9 8.2 8.8 [230]
Desmethylxanthohumol 12.45 6.5 5.0 - - - - [230]
6–Prenylnaringenin 13.27 5.6 5.3 2.1 4.7 3.5 8.9 [230]
6–Geranylnaringenin 16.82 13.0 11.4 8.0 6.2 4.8 12.1 [230]

The HPLC–APCI(+)–MS/MS analysis of polyphenolic flavonoids and chalcones in

hop plant extract (Figure. 20) was reported [217]. The fragmentation scheme of xanthohumol
E is depicted in Figure 21. The data are: xanthogalenol, m/z 355 ({[M+H]+}), daughter
ion m/z 179 (r.a. 100%); 4'–O–methylxanthohumol (369 ({[M+H]+}), 193 (100), 163 (13));
4',6'–di–O–methylchalconaringenin (301 ({[M+H]+}), 181 (100), 166 (11), 147 (10));
desmethylxanthohumol (341 ({[M+H]+}), 183 (58), 165 (100)); 3'-geranylchalconaringenin
(409 ({[M+H]+}), 285 (7), 183 (22), 165 (100)); 3',5'–diprenylchalconaringenin (409
({[M+H]+}), 297 (14), 233 (49), 195 (47), 177 (100), 121 (15)); 5'–prenylxanthohumol (423
({[M+H]+}), 247 (19), 191 (100)); xanthohumol B (371 ({[M+H]+}), 251 (45), 233 (100),
191 (24), 179 (93), 147 (30), 109 (10)); xanthohumol C (353 ({[M+H]+}), 233 (100), 191
(19), 109 (10)); xanthohumol D (371 ({[M+H]+}), 251 (6), 233 (89), 191 (26), 179 (100), 109
(13)); isoxanthohumol (355 ({[M+H]+}), 179 (100)); 7–O–methyl–6–prenylnaringenin (355
({[M+H]+}), 179 (100), 149 (12), 121 (6)); 7–O–methyl–8–prenylnaringenin (355
({[M+H]+}) 179 (100), 149 (10), 121 (5)); 5,7–di–O–methyl–8–prenylnaringenin (369
150 Bojidarka Ivanova and Michael Spiteller

({[M+H]+}), 193 (100), 163 (21)); 5,7–di–O–methylnaringenin (181 (100), 166 (8), 147 (12),
119 (8)); 6–prenylnaringenin (341 ({[M+H]+}), 183 (18), 165 (100)), 8–prenylnaringenin
(341 ({[M+H]+}), 183 (21), 165 (100)); 6,8–diprenylnaringenin (409 ({[M+H]+}), 297 (19),
233 (44), 177 (100), 121 (18)); 6–geranylnaringenin (285 (20), 183 (17), 165 (100)), and 8–
geranylnaringenin (409 ({[M+H]+}), 285 (10), 183 (19), 165 (100)), respectively.
The APCI–CID–MS method determins with a high degree of accuracy structural isomers
of isoxanthogalenols 7–O–methyl–6–prenylnaringenin and 7–O–methyl–8–prenylnaringenin
(Figure 20). The MS peaks are at m/z 179, 149 and 121, respectively. However, due to
different energies of ions of both phenyl flavonoids from thermodynamic point intensities of
maxima of MS are fragmenting ions clearly distinguishable (Figure 22). The high
reprobusibility of data allows quantitative and structural of flavonoids mixtures.
Other advantages of tandem MS/MS methods for quantitative and structural analysis of
structurally similar molecular scaffolds of phenyl flavonoids in beer were demonstrated,
studying 6–prenylnaringenin (Figure 20) by APCI–CID–MS (Figure 23) [215–225]. The
main process is a formation of {[M–H]-} anions, producing [A1–H]- and [Bl–H]- fragments
(Figure 23). Under positive operation mode, the main fragmentation pathway is cleavage of
isoprenoid fragment. Under negative operation mode (APCI(–)–CID–MS experiments) this
process is unfavorable, due to nucleophilic nature of isoprenoid functional group. The
cleavage of isoprenoid group is a charge-dependent process.

Figure 21. APCI(+)–CID MS fragmentation of xanthohumol E [217].

 Quantitative Analytical Methods of Organic Ingredients … 151

Figure 22. Fragmentation patterns of 7–O–methyl–6–prenylnaringenin and 7–O–methyl–8–

prenylnaringenin [217] under APCI (+)–CID–MS experiments.

Figure 23. (Continued)

152 Bojidarka Ivanova and Michael Spiteller

Figure 23. (Continued)

 Quantitative Analytical Methods of Organic Ingredients … 153

Figure 23. The APCI((+) and (–))–CID–MS fragmentation schemes of chalcones and phenyl flavanones
[218, 228]; Fragmentation pathway of cyclic product of xanthohumol to isoxanthohumol under basic
experimental conditions [242]; MS data of 8-prenylnaringenin [243].

A MS–based metabolomics of 8-prenylnaringenin by catalyzed activity of human liver

microsomes was reported [243] (Figure 24). The human exposure is mainly via beer
consumption, and other hops extract as food supplements. The intake of high doses may have
potential toxic effect on people through different metabolic products. They exhibit different
154 Bojidarka Ivanova and Michael Spiteller

pharmacological activities, including estrogenic one. The MS data according [243] are: M4
(8-prenylnaringenin aldehyde), m/z 353.1011 {[M-H]-} (fragment ions 353.10 (relative
abundance 40%); 247.06 (10); 233.04 (100); 207.07 (10); 119.05 (50); M5 (5,4’-dihydroxy-
2”,2”-dimethylpyrano-[7,8:6”,5”] flavanone), 337.1048 {[M-H]-}, (337.11 (20); 217.05 (85);
149.06 (14); 119.05 (100); 93.04 (22)); M6 (8-prenylapigenin): 337.1048 {[M-H]-} (337.11
(100); 293.04 (10); 282.05 (15); 281.04 (21); 268.04 (9); 219.06 (3); 117.03 (5)); M7 (5,7,4’-
trihydroxy-8-(2-hydroxy-3-methylbut-3-enyl) flavanone), 355.116 {[M-H]-} (355.12 (100);
337.11 (10); 285 (5); 284 (5); 283 (5); 235.05 (96); 217.05 (50); 165.03 (30); 119.05 (95)); and
M8 (3-hydroxy-8-prenylnaringenin), 355.117 {[M-H]-} (355.12 (20); 193.09 (100); 161.01
(11); 125.03 (11); 124.02 (21)), respectively.

Figure 24. Metabolic pathways (A and B) of 8-prenylnaringenin [243].

 Quantitative Analytical Methods of Organic Ingredients … 155

From an analytical point of view, quantitative analysis of phenyl flavonoids is difficult,

due to their great chemical instability, yielding cyclic products under acidic experimental
conditions [243–245] (Figures 23 and 25). Under basic experimental conditions, a cyclization
of xanthohumol occurs via a mechanism of Michael’s addition [245, 246] (Figure 25). The
optimization of experimental conditions and a quantitative protocol based on SPE–LC–
MS/MS (MRM–MS) methods were reported [244]. The quantitative determination was
performed using MS peaks at m/z 353  119 (isoxanthohumol, DP = –60 V, FP = –200 V,
CE = –35 V, EP = –11 V and time 600 ms). Under alkali experimental conditions, a low
reliability of data was found. The confidence levels are  84–87%. Excellent correlation
coefficients were achieved at pH = 5 (r2 within [1–0.98]). Concentration LODs is 0.03 g.L-1.
The recovery is 97.1 ± 0.03%.

Figure 25. Acidic and alkali catalyzed cyclization of phenyl flavonoids [244–246].

A MS protocol (Table 10) for beeromics includes determination of retention factor

(equation (1)) and recovery (equation (2)) [215–225]. The tR and t0 (Eq. (1)) denote retention
times of components and solvents used. In equation (2) S.A is spiked amount, while O.A. is
original amount.
156 Bojidarka Ivanova and Michael Spiteller

Eq. (1)

Eq. (2)

The content [mg.g-1] of hops and flavonoids in solid phase extracts is obtained by
equation (3), where As is peak area; Ai is peak area of internal standard; a,b – slope and
intercept of calibration curve; Ci is concentration of internal standard; V is volume of extract;
f is dilution factor; and Ws is sample weight [214]:

Eq. (3)

6.3. Analysis of Procyanidins and Prodelphinidins

In subsection 6.2 monomeric forms of phenyl flavonoids in beer were shown. Here, there
is discussed MS analysis of procyanidin and prodelphinidin dimers and oligomers (Figure
26), which derivate by monomeric phenyl flavonoids. Approximately 30% of ingredients are
from hops plant [243].

Figure 26. (Continued)

 Quantitative Analytical Methods of Organic Ingredients … 157

Figure 26. Chemical diagrams of procyanidin and prodelphinidin dimers and oligomers; B–type dimeric (top
figure) C4–C8 bonded proanthocyanidins (procyanidin, B3 (R1 = H, R2 = OH, R3 = H, R4 = OH, R5 = R6 = H);
prodelphinidins, B3 (R1 = H, R2 = OH, R3 = H, R4 = R5 = OH, R6 = H), B9 (R1 = OH, R2 = H, R3 = H, R4 = R5
= OH, R6 = H)); A–type dimeric prodelphinidin (bottom figure) ent–epigallocatechin–(4–8, 2–O–7)–
catechin and A–type dimeric prodelphinidin (bottom figure) ent-epigallocatechin–(4–6, 2–O–7)–catechin
[37, 247–249].
The HPLC–ESI–MS/MS analysis of these ingredients includes chemical substitution
(thiolysis), yielding to mixture of enantiomers (Figure 27). The synthetic conditions are:
treatment of extract of polymeric substances with phenyl–methanethiol (T = 20oC, t = 10 h,
40 L sample object, 40 mL CH3OH containing 3.3% HCl (v/v), and 80 L toluene––thiol
(5% v/v in CH3OH). For pale malt sample and hop, there was taken 5 mg, mixed with 400 L
CH3OH containing 3.3% HCl (v/v) and 800 L toluene––thiol [247]. The RP–HPLC–ESI(–
)–MS/MS analysis of substituted derivatives of Sephadex LH20 extracts from two type lager
beers was shown concentration proportion of 3,4–– and –gallocatechin benzylthioether in
pure fraction 6% dimers and 73% trimers. The oligomeric concentration is  (45–47) %. The
total concentration of proanthocyanidins (mg.L-1 of beer) is  8–13.9. The MS peaks at m/z
289 and 305 of substituted derivatives were used for quantitative analysis.
The ESI–MS/MS study of B–type dimeric proanthocyanidins was reported [37] (Figure
28). Both diastereoisomers have shown different retention times.
158 Bojidarka Ivanova and Michael Spiteller

Figure 27. Thioacidolysis of trimeric (+)–catechin [247]; Chemical diagrams of the products (+)–
catechin (gray), 3,4––catechin benzylthioether and 3,4––catechin benzylthioether.

Figure 23. (Continued)

 Quantitative Analytical Methods of Organic Ingredients … 159

Figure 28. (Continued)

160 Bojidarka Ivanova and Michael Spiteller

Figure 28. MS fragment roads of A– and B–types dimers and trimers [37].

Figure 29. Fragment processes of tannins under MALDI–PSD analysis [250].

 Quantitative Analytical Methods of Organic Ingredients … 161

The proanthocyanidins in beer originate  (70–80) % from, barley and  (20–30) % of

hops [249]. The health benefits of tannins were widely discussed. They are associated with
decreasing in risk of coronary artery disease; cancer; and ulcers. Tannins, however, exhibit
significant variation as molecular skeleton and thus different biological and physiological
functions occur. This must address the risk assessment for human health and nutrient positive
features fast, accurate and precise quantitative protocols [248]. The degree of polymerization
and quantification of proanthocyanidin oligomers were obtained by MALDI–TOF–MS, ESI–
MS and FAB–MS methods [250–253]. The study of specific sequences of individual
molecular chains, highlighting peptide ones based on MALDI–MS was extended to PSD
fragment pathways [250, 251]. There are the first data in the literature on MS fragmentation
of tannins. The MS patterns and mechanism of MS fragment ion formation are shown in
Figure 29.

7. THE METHODS OF MASS SPECTROMETRY IN THE QUANTITATION

OF HOP BITTER ACIDS

7.1. Analysis of –Acids (Humulones)

Hop bitter acids (Figure 20) are important ingredients in beer with health benefits for
humans [254–280]. They exhibit great chemical diversity; structural similarity; thermal and
chemical instability, and complex photochemistry [168, 214, 226–229, 244, 281–369]. Their
complex quantitative analysis is complicated by the lack of reference standards available at
each analyte as well. Early works on low resolution MS were reported [370]. The fragment
schemes are depicted in Figure 30. The keto–enolic forms were studied (Figures 31 and 32).

Figure 30. (Continued)

162 Bojidarka Ivanova and Michael Spiteller

Figure 30. Fragment patterns of –acids and their tautomers [370].

Figure 31. Tautomerism in hop bitter acids [370].

Despite enormous efforts in improving chromatographic separation carriage for

selectivity and sensitivity, and implementation of MS based method for detection, its
linearity and matrix effect require application of internal standards for accurate quantitative
determination [215–225,371,372]. As promising strategy ECHO–technique for quantification,
using non–labeled standards of hop bitter acids was highlighted [372] (Table 12). Amounts
added are  [0.02–5.56] mol.L-1. The recoveries are  [98–111] %. The concentration
LODs/LOQs, achieved by APCI (–)(or (+))–MS/MS for – and –acids are 0.01/0.1. While
the confidence level is r2 = 0.9996 [215].

Figure 32. Chemical diagrams of –, and –acids [215, 281]; Nomenclature: ()–(prefix)acids.
 Quantitative Analytical Methods of Organic Ingredients … 163

The CE–ESI–MS based quantitative protocol was developed, studying oxidation

reactions of hop bitter acids (Figure 33) [11]. These processes yield to double acyloin
residue, which is main molecular scaffold of abeo-isohumulones. They were considered by
isohumulones as derived and were obtained via an oxidation process of humulone, however
[11]. The reproducibility is 3.6% [11].

Table 12. The MS analysis of –acids by APCI–and

HPLC–ESI–MS/MS/ECHO–MS methods

Analyte [M-H]- [M+H]+ Fragment pattern (r.a.) Refs.

Humulon 363 307 {[M+H-C4H8]+}; 295 {[M+H- [215, 281]
C5H8]+}; 239 {[M+H-C5H8-C4H8]+};
MS/MS (239): 221 {[M+H-C5H8-C4H8-
H2O]+}
361 292 {[M-H-C5H9]-}; MS/MS (361): 343 [215, 281]
{[M-H-H2O]-}; 292 {[M-H-C5H8]-};
MS/MS (292): 274 {[M-H-C5H9-H2O]-};
249 {[M-H-C5H9-C3H7]-}
361.2020 361 (100), 292 (98), 249 (31), 221 (31); [372]
Cohumulone 347.1864 347 (100), 278 (94), 234 (35), 207 (29) [372]
Adhumulone 361.2023 292 (98), 249 (31), 221 (31); [372]

Figure 33. Oxidation process of humulinone [11].

The MS data of oxidation and auto-oxidation products of – and –acids,

during storage process of hops were reported [373]. The cis/trans–humulinic acids,
tricyclodehydroisohumulone, and Ashurst’s analyte in the oxidized hops products were found
(Figure 34).
164 Bojidarka Ivanova and Michael Spiteller

Figure 34. Chemical diagrams of tricyclodehydroisohumulone and Ashurst’s analytes [373–388].

Figure 35. Mechanism of auto-oxidation of humulone [384] (R = CH(CH3)2, CH2CH(CH3)2, and

CH(CH3)CH2CH3).

The auto-oxidation of humilone during storage of beer was investigated by UFLC–

MS/MS and single crystal X–ray diffraction [384]. The acyloin rearrangement (Figure 35)
causes for isolation of tricycloperoxyisohumulone A. It MS peak of {[M–H]-} anion is at m/z
319.1543. The crystal structure of tricycloperoxyisohumulone A in it enolic form is depicted in
Figure 36. The compound crystallizes in a noncetrosymmetric orthorhombic space system and
group P212121. Strong intra-molecular hydrogen bond OH…O with bond length r(O…O) =
2.531 Å occurs. The 2–fold screw chains are formed via moderate intermolecular OH…O=C
hydrogen bonds (r(O…O) = 2.878 Å). The quantitative data including between–day–analysis
are shown in Figure 37. This auto-oxidation product is minor component (4 mol.g-1).
Despite the fact that the concentration of the analyte increases rapidly in the first 5 weeks, it is
stable for a 40 week period (Figure 37).
 Quantitative Analytical Methods of Organic Ingredients … 165

Figure 36. Crystal structure and chemical diagram of tricycloperoxyisohumulone A [384]; PLUTON
diagram; hydrogen bond network;View along a – axis.

Figure 37. Between–day–analysis of auto-oxidation of humulone in beer during storage; C –

Concentration of analytes [mol.g-1] [384].
166 Bojidarka Ivanova and Michael Spiteller

7.2. Analysis of Iso––Acids (Isohumulones)

Chemical diagrams of iso–a–acids are depicted in Figure 38.

Figure 38. Chemical diagrams of iso––acids, tetrahydro–iso––acids and dihydro–iso––acids;

Corresponding cis– and trans–isomers of iso––acids [215, 216]; Nomenclature: Trans(cis)–
tetrahydro(dihydro)–iso––(prefix)acids.

The APCI–MS analysis (subsection 6.2 [215]) of iso––acids in beer shows fragment
ions: iso–cohumulon, APCI(+)–MS data: m/z 349 {[M+H]+} (r.a. 100%), 331 {[M+H-
H2O]+}; 313 {[M+H-2H2O]+}; and 281 {[M+H-C5H8]+}, respectively. MS/MS of m/z 349
yields to MS peaks at m/z 331 {[M+H-H2O]+} (100); 313 {[M+H-2H2O]+}; 281 {[M+H-
C5H8]+}. The tandem MS/MS operation mode of MS peak at m/z 331 yields to peaks at m/z
313 {[M+H-2H2O]+} (100), 295 {[M+H-3H2O]+}, 275 {[M+H-H2O-C4H8]+}. The APCI(–)–
MS spectra show peaks at m/z 347 {[M-H]-} (100), and 251 {[M-H-C6H8O]-}, respectively.
MS/MS analysis of peak at m/z 347 causes for fragment ions exhibiting peaks at m/z 329
{[M-H-H2O]-}; m/z 278 {[M-H-C5H9]-}; m/z 251 {[M-H-C6H8O]-} (100%). The
 Quantitative Analytical Methods of Organic Ingredients … 167

corresponding MS/MS data (m/z 251) are: m/z 233 {[M-H-C6H8O-H2O]-}; m/z 207 {[M-H-
C6H8O-C2H3O]-}. The results of iso–n–humulon and iso–adhumulon under APCI(+)–MS
conditions are peaks at m/z 363 {[M+H]+} (100), m/z 345 {[M+H-H2O]+}; m/z 327 {[M+H-
2H2O]+}; m/z 295 {[M+H-C5H8]+}. The MS/MS spectra (m/z 363) exhibit peaks at m/z 345
{[M+H-H2O]+}; m/z 327 {[M+H-2H2O]+} (100%); m/z 307 {[M+H-C4H8]+}; m/z 295
{[M+H-C5H8]+}. MS/MS data of m/z 345 are peaks at m/z 327 {[M+H-2H2O]+} (100%); m/z
289 {[M+H-H2O-C4H8]+}; m/z 277 {[M+H-H2O-C5H8]+}; MS/MS (m/z 327): m/z 309
{[M+H-3H2O]+} (100%). The APCI(–)–MS data are peaks at m/z 361 {[M-H]-} (100%); m/z
265 {[M-H-C6H8O]-}; MS/MS (m/z 361): m/z 343 {[M-H-H2O]-}; m/z 317 {[M-H-C2H3O]-};
m/z 265 {[M-H-C6H8O]-}. MS/MS (m/z 265): m/z 247 {[M-H-C6H8O-H2O]-} [215]. The
concentration LODs/LOQs are 0.006/0.02 (xanthohumol), 0.01/0.02 (isoxanthohumol), and
0.006/0.03 (8–prenylnaringenin), respectively. The level of confidence is r2  [0.9999–1]
[215]. The APCI(–)–MS data about cis–tetrahydro–iso–cohumulone are {[M–H]–} ion (m/z
351), {[M-H2O-H]-} (m/z 333) and {[M-H2O-C6H10O-H]-} (m/z 235), respectively [216].
Cis–dihydro–iso–cohumulone shows different fragment pathway as result of different type of
substituents. Mass spectrometric peaks at m/z 349 {[M-H]-}, 251 {[M-C6H10O-H]-} and 233
{[M-C6H10O-H2O-H]-} were found [216]. The HPLC–ESI–MS/MS/ECHO–MS methods
were used for quantitative analysis of iso––acids in beer [372]. The data show: trans–
isocohumulone, LC–TOF–MS, m/z 347.1871; MS/MS (–30 V): m/z (%) 347 (10), 329 (10),
278 (15), 251 (80), 235 (25), 233 (35), 209 (30), 207 (25), 182 (100), 181 (10); cis–
isocohumulone, m/z 347.1877, MS/MS: m/z 347 (15), 329 (25), 278 (10), 251 (70), 235 (10),
233 (50), 209 (35), 207 (30), 182 (100), 181 (30); trans–isohumulone, LC–MS (ESI–) m/z
(%) 361 (100) {[M-H]-}; MS/MS: m/z 361 (15), 343 (10), 292 (10), 265 (100), 247 (25), 235
(25), 223 (25), 221 (20), 196 (80), 195 (20); cis–isohumulone, m/z 361.2037, LC–MS (ESI–)
m/z 361 (100) {[M-H]-}; MS/MS: m/z 361 (25), 343 (25), 292 (15), 265 (90), 247 (35), 235
(15), 223 (30), 221 (30), 196 (100), 195 (20); trans–isoadhumulone, m/z 361.2019; MS/MS:
m/z (%) 361 (15), 343 (15), 292 (10), 265 (75), 247 (20), 235 (15), 223 (25), 221 (25), 196
(100), 195 (15); cis–isoadhumulone, m/z 361.2007, MS/MS: m/z 361 (25), 343 (25), 292 (15),
265 (95), 247 (45), 235 (10), 223 (25), 221 (25), 196 (100), 195 (20); xanthohumol, m/z
353.1394; MS/MS: m/z 353 (80), 233 (100), 119 (50); isoxanthohumol, m/z 353.1404; MS/MS:
m/z (%) 353 (80), 233 (100), 119 (50), respectively.
The oxidation products of iso––acids of hops during storage were studied [373]. The
reaction pathways yielding to 4’-hydroxy-allohumulinones from corresponding humulinones
are shown in Figure 39. The 4’-hydroxy-allocohumulinone and 4’-hydroxy-allo-n-
humulinone have shown negative {[M–H]-} anions at m/z 379.1754 and 393.1912, respectively.
The chemical transformations of hops, including cyclic reactions, rearrangement and
oxidation as a result of wort boiling were discussed [386]. The keto–enol tautomerizm of –
acids results to iso––acids (next subsection). In parallel, there is a chemical transformation
during brewing process and beer aging, leading to different molecular scaffolds of hop
acids. Lingering and harsh testing substances such as tricyclohumols, tricyclohumenes,
isotricyclohumenes, tetracyclohumols and epitetracyclohumols were obtained. They are main
products of chemical transformation of iso––acids (Figure 40). The LC–(ESI)–TOF–MS
analysis shows concentration ranges in stored and fresh beer as listed in Table 13.
168 Bojidarka Ivanova and Michael Spiteller

Figure 39. Obtaining of 4’-hydroxy-allohumulinones from corresponding humulinones [373]; R =

CH(CH3)2, CH2CH(CH3)2 and CH(CH3)CH2CH3.

Table 13. Concentration ranges (C, [mol.L-1]) (SD) of transformation products of iso–
–acids in stored and fresh beer, and hydrolytic cleavage along with LC–ESI(–)–TOF–
MS and MRM optimized parameters for quantitative analysis; DP [V]; CE [V]; CXP
[V] [385, 386]

Analyte C [mol.L-1] (SD) Mass transition DP CE CXP

Tricyclocohumol 1.00 (±0.00) 365.3165.0 –105 –48 –9
Tricyclohumol 1.60 (±0.10) 379.3179.0 –105 –48 –9
Tricycloadhumol – 379.3179.0 –105 –48 –9
Tricyclocohumene 0.30 (±0.03) 347.3165.0 –60 –52 –7
Tricyclohumene 0.36 (±0.03) 361.2179.0 –60 –52 –7
Tricycloadhumene – 361.2179.0 –60 –52 –7
Isotricyclocohumene 0.28 (±0.04) 347.3165.0 –60 –52 –7
Isotricyclohumene 0.44 (±0.03) 361.2179.0 –60 –52 –7
Isotricycloadhumene 0.12 (±0.03) 361.2179.0 –60 –52 –7
Tetracyclocohumol 0.42 (±0.02) 365.3193.1 –120 –46 –11
Tetracyclohumol 0.65 (±0.03) 379.3207.1 –120 –46 –11
Tetracycloadhumol 0.14 (±0.01) 379.3207.1 –120 –46 –11
Epitetracyclocohumol 0.08 (±0.02) 365.3193.1 –120 –46 –11
Epitetracyclohumol 0.36 (±0.04) 379.3207.1 –120 –46 –11
Epitetracycloadhumol – 379.3207.1 –120 –46 –11
Tricyclocohumulactol – 339.2193.0 –90 –38 –15
Tricyclohumulactol – 353.2207.0 –90 –38 –15
Tricyclocadhumulactol – 353.2207.0 –90 –38 –15
Scorpiocohumol – 363.3164.9 –90 –46 –13
Scorpiohumol – 377.3178.9 –90 –46 –13
Scorpioadhumol – 377.3178.9 –90 –46 –13
Trans–humulinic acid – 265.1154.9 –75 –30 –24
Hydroxy–transalloisohumulone – 377.1195.2 –90 –41 –16
 Quantitative Analytical Methods of Organic Ingredients … 169

HPLC–MS/MS–MRM analysis of 18O–labeling compounds was performed in order to

study the reaction pathways yielding to tricyclohumulactols and scorpiohumols from trans–
iso––acids (Figure 41, Table 13) [386].
The molecular transformation of trans–iso––acids as a result of hydrolytic cleavage,
yielding to 3-methylbutyric and 4-methyl-3-pentenoic acids was discussed [385]. The
HR/MRM–MS method was used (Table 13). The mechanism of the reactions is depicted in
Figure 42. The concentration ranges detected are  [35–86] mmol.mol-1.

Figure 40. Transformation reactions of iso––acids yielding to tricyclohumols, tricyclohumenes,

isotricyclohumenes, tetracyclohumols and epitetracyclohumols [386]; R = CH(CH 3)2, CH2CH(CH3)2,
and CH(CH3)CH2CH3.

Figure 41. Chemical transformation of trans–iso––acids to tricyclohumulactols and scorpiohumols

[229, 338, 386]; R = CH(CH3)2, CH2CH(CH3)2, and CH(CH3)CH2CH3, respectively.
170 Bojidarka Ivanova and Michael Spiteller

Figure 42. Mechanism of β-dicarbonyl cleavage as a result of hydrolysis of trans-isohumulone, yielding

to 3-methylbutyric and 4-methyl-3-pentenoic acids [385].

The oxidation process of trans–isohumulone in beer upon UV–irradiation was studied by

GC–MS [388]. A set volatile short chain organics of amount 75±8% were found (Figure 43).

Figure 43. Photochemistry of trans–isohumulone in beer upon UV–irradiation [388].

7.3. Analysis of –Acids (Lupulones)

Early works devoted to low resolution MS analysis of transformation products of –acids

as a result of extraction of hops components under boiling at pH = 5.5, which is part of
brewing technology, provided comprehensive structural analysis of those ingredients [389].
The main fragment pathways are shown in Figure 44.
 Quantitative Analytical Methods of Organic Ingredients … 171

Figure 44. Main fragmentation pathways of transformation products of –acids as a result of boiling at
pH = 3 in aquatic medium [389].
172 Bojidarka Ivanova and Michael Spiteller

A HR/AM–LC–MS/MS analysis of –acids (Figure 31) was reported [229]. The MS

parameters are shown in Table 14. The HPLC–MS/MS by ECHO and MRM techniques were
used. Quantitative and structural data about –acids are shown in Figure 45 and Table 14
[371]. The quantitative determination was performed without sample treatments of the
commercial beer. The low LODs and LOQs  [0.8–3.0] nmol.L-1 and [1.3–5.0] nmol.L-1 were
achieved. The analytical quantity of cohulupone is 121.1 nmol.L-1 (LOD) [371]. The products
of chemical transformation of colupulone as a result of wort boiling are shown in Figure 46.

Figure 45. Transformation products of –acids during wort boiling [229, 371].
 Quantitative Analytical Methods of Organic Ingredients … 173

The ESI–IT–MS/MS was used studying interactions of –acids with 1–hydroxyethyl

radical as an effort to understand the mechanism of oxidative decomposition yielding to loss
of antimicrobial activity of those acids towards Gram–positive bacteria such as Lactobacillus
spp., Streptococcus spp., Staphylococcus spp., Micrococcus spp., and Bacillus spp.,
respectively [383]. The reaction kinetics (Figure 47) (rate constant obtained k2 = 3.1.107
L.mol-1.s-1) was reported. The competitive kinetic approach was employed [390, 391]. The
thermodynamics was also reported. A free Gibbs energy value G = 106 kJ.mol-1 was
obtained [383].

Table 14. MS parameters of –acids using HR/MS/MS analysis

(HR/AM–LC–MS/MS, ESI–TOF–MS/MS/MS–ECHO, MRM);
Daughter ions (r.a. %); Molecular anion {[M-H]-} (r.a. %)

Analyte [M-H]- Fragmentation pattern (r.a. %); Ionic fragment Refs.

/[M+H]+
Colupulone 399.3 194.06,219.07,275.13,287.13 [229, 371]
399 287(100),399(75),330(30) [371]
401 345 {[M+H-C4H8]+} [215, 281]
399 355 {[M-H-C2H3O]-}; 330 {[M-H-C5H9]-}; [215, 281]
287 {[M-H-C5H9-C3H7]-}
399.2547 287(100),399(75),330 (30); [372]
Cohulupone 317.2 184.04,205.05,220.11,233.08, 248.10 [229, 371]
Hulupinic acid 263.1 1165.05,181.05,193.05,209.04, 263.13 [229]
Norticyclocolupone 357.2 219.17, 287.17 [229]
Dehydrotricyclocolupone 397.2 189.13, 232.04, 259.21, 264.06, 327.19 [229]
Hydroxytricyclocolupone 415.3 277.22, 345.21 [229, 371]
Hydroperoxytricyclocolupone 431.2 111.05,233.08,287.13,341.17 [229]
Epoxycohulupone 333.2 165.06,181.05,193.05,209.05, 263.13 [229]
Lupulone 413.3 208.07,233.08,289.15,301.15 [229, 371]
415 359 {[M+H-C4H8]+}; [215]
291 {[M+H-C4H8-C5H8]+};
MS/MS (291):
235 {[M+H-2C4H8-C5H8]+}
413 369 {[M-H-C2H3O]-} [215, 371]
Hulupone 331.2 125.06,194.06,198.05, [229]
219.07,234.13,247.10,262.12
Norticyclolupone 371.2 217.18, 287.17 [229]
Dehydrotricyclolupone 411.3 189.13,246.09, 259.21, 287.94, 327.20 [229]
Hydroxytricyclolupone 429.3 277.22, 345.21 [229]
Hydroperoxytricyclolupone 445.3 125.06,247.10,301.15,355.19 [229]
Epoxyhulupone 347.2 151.00,165.06,181.05,193.05, 209.05, 263.13 [229]
Adlupilone 413 301 (44), 233 (11) [371, 372]
174 Bojidarka Ivanova and Michael Spiteller

Figure 46. Reactions of chemical transformation of colupulone [371].

 Quantitative Analytical Methods of Organic Ingredients … 175

Figure 47. Reaction interactions of lupulones and hydroxyethyl radicals [383]; R = CH(CH3)2,
CH2CH(CH3)2, and CH(CH3)CH2CH3, respectively.

8. ANALYSIS OF THIOL – CONTAINING INGREDIENTS IN BEER

The volatile S–containing ingredients (Figure 48) contribut to the aroma of many foods
and beverages [382–407]. They exhibit a low odor thresholds and sensory impact at trace
concentration levels. The quantitative procedure includes extraction step, consisting of: to a
500 mL extract of hops or beer was added 2.5 mnol 4-methoxy-2-methylbutane-2-thiol as
176 Bojidarka Ivanova and Michael Spiteller

internal standard [394]. The analysis by HPLC–HR–ESI–MS results to concentration LODs

 0.0001–68.2 ng.L-1 (Table 15) [392–407].

Figure 48. Chemical diagrams of volatile S–containing ingredients in beer [392–407]: 3-mercapto-
propan-1-ol (a); 3-mercapto-2-methyl-butan-1-ol (b); 3-mercaptohexan-1-ol (c); 3-mercapto-butan-1-ol
(d); 3-mercapto-pentan-1-ol (e); 3-mercapto-heptan-1-ol (f); 3-mercapto-octan-1-ol (g); 3-mercapto-
nonan-1-ol (h); 3-mercapto-2-methyl-propan-1-ol (i); 2-mercaptomethyl-butan-1-ol (j); 2-
mercaptomethyl-hexan-1-ol (k); 3-mercapto-2-methyl-pentan-1-ol (l); 3-mercapto-3-methylbutan-1-ol
(m); 4-mercapto-4-methylpentan-2-one (n); 5-mercapto-hexan-3-one (o); 4-mercapto-butan-2-one (p);
4-mercapto-5-methyl-hexan-2-one (q); 4-mercapto-pentan-2-one (r); 4-mercapto-3-methyl-pentan-2-
one (s); 4-mercapto-nonan-2-one (t); 4-mercapto-butan-2-ol (u); 1-mercapto-pentan-3-ol (v); 5-
mercapto-6-methyl-heptan-3-ol (w); 4-mercapto-pentan-2-ol (x); 5-mercapto-hexan-3-ol (y); 5-
mercapto-decan-3-ol (z); 2-amino-3-mercapto-3-methyl-butyric acid (zwitterion) (aa); 2-mercapto-
ethyl-ammonium cation (ab); 3-sulfanyl-4-methylpentan-1-ol (ac); 3-sulfanyl-4-methylpentyl acetate
(ad); 2-methyl-furan-3-thiol (ae); 4-methoxy-2-methyl-butane-2-thiol (af); furan-2-yl-methanethiol
(ag); 3-methyl-but-2-ene-1-thiol (ah); 1-phenyl-ethanethiol (ai); hexane-1-thiol (aj); hexanoic acid 3-
mercapto-hexyl ester (ak) and acetic acid 3-mercapto-hexyl ester (al), respectively.
 Quantitative Analytical Methods of Organic Ingredients … 177

Table 15. Quantitative analysis of thiols in beer; Concentration LODs [ng.L-1]; MS data
and fragmentation patterns; Analyte labeling is given in Figure 48

Analyte LOQ MS fragmentation, m/z (r.a. %) Refs.

(a) 0.2 57 [100]; 41 [94]; 58 [82]; 45 [59]; 47 [59] [397]
(b) 0.01 41 [100]; 60 [93]; 45 [89]; 61 [86]; 71 [61] [397]
(c) 0.004 55 (100), 41 (72), 57 (64), 61 (46), 67 (30) [394, 397,
405]
(d) 0.1 57 [100]; 55 [86]; 41 [86]; 72 [74]; 43 [74] [397]
(e) 0.001 41 [100]; 57 [70]; 55 [47]; 61 [43]; 69 [43] [397]
(i) 0.07 41 [100]; 47 [67]; 57 [62]; 55 [49]; 72 [42] [397]
(m) 0.01 41 (100), 69 (73), 71 (35), 86 (24), 43 (24) [397, 405]
(n) 0.05 43 (100), 132 (26), 75 (21), 55 (19), 99 (120) [392, 397,
405]
0.20 99.0797 {[MH-CH3CH2O(CO)CH=CHSH]+}
27 101.0941 {[MH-CH3CH2O(CO)CH=CHSH]+} [392]
(x) 0.0001 56 [100]; 45 [96]; 55[71]; 61 [67]; 100 [58] [397]
(v) 0.005 47 [100]; 57 [99]; 59 [94]; 45 [79]; 41 [71] [396, 397]
(aa) 297 [400]
(ac) 68.2 134 {[M]+}, 100 {[M-H2S]+}; 91 {[M-CH(CH3)2]+}; 73 {[M- [394]
CH(CH3)2-H2O]+}; 61 {[M-CH3CH2CH2-CH2OH]+}.
(ad) 1.8 176 {[M]+}, 116 {[M-CH3COOH]+}; 101 {[M-CH3COOH- [394]
CH3]+}; 73 {[M-(CH3)2CHCH(SH)CH2]+}
(al) 0.19 143.1056 {[MH-CH3CH2O(CO)CH=CHSH]+} [394]

The MS study of light-struck flavor shows a photo-oxidation process, involving

penicillamine and cysteamine [400]. Non–volatile analyte was not detected by ESI(–)–MS. In
contrast, ESI(+)–MS detects trace concentrations of photo-oxidation products of both
substances. The MS peaks at m/z 297 and m/z 153 were obtained. The reaction
mechanisms are shown in Figure 49. The MS analysis of 4-sulfanyl-4-methylpentan-2-one,
3-sulfanylhexan-1-ol and 3-sulfanylhexyl acetate was discussed [392] (Table 15).
The concentration LODs  [0.19–27] ng.L-1 were achieved. Formation of {[MH–CH3
CH2O(CO)CH=CHSH]+} cations via cleavage mechanism occurs (Figure 50).

Figure 49. Photo-oxidation mechanism of penicillamine and cysteamine [400].

178 Bojidarka Ivanova and Michael Spiteller

Figure 50. The GC–Q–TOF–MS/SCD fragmentation of 4-sulfanyl-4-methylpentan-2-one [392].

The chemical reactivity of 1-hydroxyethyl radical towards thiols in a beer was studied [406, 407]. The
model interaction with glutathione is depicted in Figure 51.

Figure 51. Mechanism of interaction of glutathione with 1-hydroxyethyl radical [406, 407].

9. ANALYSIS OF VITAMINS IN BEER

Despite simplicity of vitamins as molecular structures and their fundamental importance
as food additives to food industry, little effort was devoted to MS protocols for their
determining in food [408–423]. Ascorbic acid is added in concentration range  [30–50]
mg.L-1 to many beer products, due to its large antioxidant activity to reduce content of
molecular oxygen [419]. Vitamin C is not naturally occurring ingredient in beer. The
sensitivity of vitamin C to oxidation makes its quantitative analysis difficult. It requires rapid
and sensitive quantification in an inert atmosphere. The routine method for analysis are HPLC
with electrochemical detection [419, 420]. Vitamin C and vitamins riboflavin (vitamin B2,
m/z 377.2 {[M+H]+}; 399.1 {[M+Na]+} [408–423]), niacin (vitamin B3), pyridoxine (vitamin
B6), D-pantothenic acid, folic acid, biotin (vitamin B12, vitamin H, concentration ranges
 Quantitative Analytical Methods of Organic Ingredients … 179

(1.99–0.076) g.mL-1), thiamine (vitamin B1) [408–423] were determined in beer. The
concentration ranges are g.mL. Despite scarce reports in MS–based protocols for
quantification of vitamins in beer, there are elaborations of selective determination by
MALDI–MS [424]. The Ag–nanoparticle supported desorption–ionization approach for
analysis of riboflavin was performed in concentrations 1.3 nmol.(0.5L) [424].

10. OTHER INGREDIENTS IN BEER

There are more components at micro and trace concentrations in beer, like
benzeneacetonitrile--(-D-blucopyranosyloxyne-), succinic anhydride, benzoil acid, 2,4,6-
trimethyl-2,4,6-trimethylphenyl ester, benzenepentanoic acd, d-oxo-, 2-phenyl-hex-5-en-
3-ol, naphthalene, 2-ethenyl, 2,6-octadiene,2,6-dimethyle, 2-butenoic acid-3-methyl-2-
methylene-3-butenyl ester, propanoic acid, 2-methyl-3-methylbutyl ester; 4H-phyran-
4-one, 5-hydroxy-2-(hydroxymethyl)-; alanine, N-methyl-N-allyloxycarbonyl,pentyl ester;
1-cyclohexene-1-carboxaldehyde,-4-(1-methylethyl)-; -D-mannofuranoside-; 2-cyclohexen-
1-one-4-carboxylic acid, 4-(3,7-dimethyl-2,6-octadien-1-yl)-3-methyl-, and ethyl ester;
chromone,5-hydroxy-6,7,8-trimethoxy-2,3-dimethyl-, respectively. Their MS quantification
was shown [288, 289]. The early work dealing with structural analysis of hops material by
MS includes molecular scaffold of naturally occurring hop substances with structurally
similar molecules [347–369]. The EI data of 2,2-dimethyl-5,7-dihydroxychroma (Figure 52)
show MS peak at m/z 139 of tropylium or benzyl ions. This ion, having a high stability yields
to relative intensity of 30% of the total ion current. Chromone, 2,2,8,8-tetramethyl-
3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chromen-5-ol, 1-(5-hydroxy-2,2,8,8-tetramethyl-3,4,
7,8-tetrahydro-2H,6H-pyrano[3,2-g]chromen-10-yl)-2-methyl-propan-1-one and 8-acetyl-7-
hydroxy-2,2-dimethyl-6,6-bis-(3-methyl-but-2-enyl)-2,3,4,6-tetrahydro-chromen-5-one were
also studied [347].

Figure 52. (Continued)

180 Bojidarka Ivanova and Michael Spiteller

Figure 52. Electron impact fragment patterns of 2,2-dimethyl-5,7-dihydroxychroma, chromone, 2,2,8,8-

tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chromen-5-ol, 1-(5-hydroxy-2,2,8,8-tetramethyl-
3,4,7,8-tetrahydro-2H,6H-pyrano[3,2-g]chromen-10-yl)-2-methyl-propan-1-one and 8-acetyl-7-
hydroxy-2,2-dimethyl-6,6-bis-(3-methyl-but-2-enyl)-2,3,4,6-tetrahydro-chromen-5-one, respectively
[347].

In this subsection the MS methods for determination of phytoalexins as benzoxazinoids in beer are
discussed (Figure 53). The concentration LODs are  [35–70] mg.L-1 [15].
 Quantitative Analytical Methods of Organic Ingredients … 181

Figure 53. Chemical diagrams of benzoxazinoids [15].

The HRGC–MS analysis of argpyrimidine (N-(5-hydroxy-4,6-dimethylpyrimidine-2-

yl)-L-ornithine) (Figure 54) was reported [114]. This chemically substituted amino acid is a
new arginine-containing structure in beer, that originated by chemical conversion of maltose
(Figure 54).

Figure 54. Mechanism of chemical conversion of maltose [114].

11. QUANTITATIVE ANALYSIS OF HEALTH RISK INGREDIENTS IN

BEER BY MASS SPECTROMETRIC BASED METHODS
11.1. Analysis of Biogenic Amines in Beer

Biogenic amines are metabolites of plants, animal and microorganisms and are frequently
found in foods such as wine and beer [424–426]. They are classified as a quality marker,
indicating degree of degradation and fermentation processes. Their high concentration in
foods may cause health disorders in humans such as cold sweat, headaches, hypo– or
hypertension, renal intoxication and many others [424]. Risk to human health from
consumption of beer is reported on some consumers. The distribution of biogenic amines in
beer depends on raw material, processes of brewing technology and hygiene. The following
amines are most important for food analysis, i.e., histamine, cadaverine, putrescine, tyramine,
182 Bojidarka Ivanova and Michael Spiteller

tryptamine, agmatine, β-phenylethylamine, spermidine, spermine, and spermidine,

respectively (Figure 55).

Figure 55. Chemical diagrams of biogenic amines in beer.

11.2. Analysis of Mycotoxins in Beer

Mycotoxins are class of naturally occurring toxins; secondary metabolites of fungi,

causing for acute toxic, mutagenic, carcinogenic, teratogenic and estrogenic effects on
humans [427, 428]. The beer produced as a cereal product is prepared by brewing of barley
and wheat. And so, these grains with mycotoxin contamination associated as well. This
subsection is devoted to MS analysis of natural toxins in beer [429–479]. Mycotoxin levels
can in unrefined grain vary by fungal growth during malting. They may contaminate final
food product. Despite this fact, the assessment of risk to human health includes determination
of these contaminants during whole production stages. Brewing grains are contaminated
primary by Fusarium mycotoxins (species F. graminearum, F. crookellense, and F.
acuminatum) deoxynivalenol and zearalenone, leading to serious health problems in humans
[431]. Despite, mycotoxins derived from first raw material, and diluted in brewing,
were reduced concentration level in the final beer product one order of magnitude [429–454].
In addition, about 60% of zearalenone in spent grains detected. It chemical conversion
to -zearalenol, -zearalenol, -zearalanol, and -zearalanol occurs (Figure 56). The
deoxynivalenol based mycotoxins are associated with deoxynivalenol-3-glucoside,
deoxynivalenol-3-di-, deoxynivalenol-3-tri-, and deoxynivalenol-3-tetraglucoside [452]
(Figure 56).
 Quantitative Analytical Methods of Organic Ingredients … 183

Figure 56. (Continued)

184 Bojidarka Ivanova and Michael Spiteller

Figure 56. Chemical conversion of zearalenone to main metabolites during fermentation [431];
Chemical diagrams of mycotoxins in beer [429–454, 480].

For this reason within the frame of European Union communities there are strict
legislations about maximum concentration levels of deoxynivalenol and zearalenone in
cereals and corresponding cereal–based foods, thus preventing mycotoxicoses in humans.
These amounts are for deoxynivalenol 1250 mg.kg-1 (raw cereals) and 750 mg.kg-1 for
cereals, including malt [481–484]. While corresponding amounts for zearalenone are 100 and
75 mg.kg-1. However, there is no maximum permitted levels in beer food shown [481–484].
Another group of mycotoxins, objects of strict regulation in food are ergot alkaloids and
Alternaria toxins [481–484]. In the direction of the first family alkaloids, which are mainly
 Quantitative Analytical Methods of Organic Ingredients … 185

obtained from Claviceps purpurea, it is known that may cause ergotism in humans associated
with abdominal pain or vomiting, and neurological signs. Nevertheless, that there are ergot
alkaloid based pharmaceutics for treatment of neurological diseases; migraine and/or
Parkinson diseases. Alternaria toxins have been associated with esophageal cancer in humans.
Amongst most toxic under the species is tenuazonic acid; a metabolite by Phomasorghina and
Pyricularia oryzae (Alternaria spp.) (Figure 56). According to legislation directives of
European Union maximal admissible levels of Alternaria toxins in foods are not yet defined.
The maximum admissible level of 0.5 g.kg-1 for ergot sclerotia in certain cereals by
Commission Regulation (EU) is more recently legislated (2015/1940, 10.2015) [483].
Ochratoxin A (Figure 56) is a secondary metabolite from Aspergillus and Penicillium fungi
(P. Verrucosum, A. ochraceus, A. carbonarius and A. niger) [432]. It can infect wheat and
barley, so contaminates final food products, including beer. This mycotoxin causes of
nephrotoxic, teratogenic, hepatotoxic and immunotoxic effects along with liver tumors in rats.
The guidelines of the European Union regulations maximum residual content of ochratoxin A
legislate values 5 mg.kg-1 (raw grain) and 3 mg.kg-1 (treated grain, including malt) [481–484].
There are not shown explicitly levels in beer–product. The determination is made of this
mycotoxin in malt [429–454]. Weekly intake concentration level of ochratoxin A according
to World Health Organization is 100 ng.kg-1 body weight [481–484]. The level was obtained
by potentially carcinogenic effect of ochratoxin A for humans. The maximum permissible
levels in corn for aflatoxin is 4 g.kg-1. For aflatoxin B1 the level discussed is 2 g.kg-1 [481–
484]. The mycotoxins beauvericin and enniatins A, A1, B and B1 have large a scale in
biological activity, including toxicity to human cells such as fibroblast foetal lung cells
(MRC–5) and hepatocellular carcinoma cells (Hep G2) [480]. The IC50 lies in range g.kg-1.
The determination of these mycotoxins in foods frequently includes enzyme immunoassay.
The application to beer food was resulted to concentration LODs for ergometrine (0.06 g.L-
1), zearalenone (0.14 g.L-1), deoxynivalenol (2.1 g.L-1) and alternariol (0.18 g.L-1),

respectively [429]. The LC–ESI–MS/MS quantification of zearalenone and it main

metabolites during fermentation was reported [431]. The MS quantitative and qualitative
characteristics are shown in Table 16. Limit of quantitation LOQ 2 ng.g-1 (ppb) was achieved
for zearalenone. This result highlights the great advantages of MS–based methods for
quantitative analysis of mycotoxins in food. Between–day change of chemical structure of
initial mycotoxin was monitored. The data are depicted in Figures 57 and 58. The online
SPE–LC–ESI–MS/MS protocol for quantitative analysis of ochratoxin A in beer was reported
(Table 16) [432]. Concentration LOD is 0.1 ng.mL-1. The MS fragment pattern is shown in
Figure 57. Table 16 summarizes quantitative MS data of mycotoxins in beer [429–454]. The
MALDI–MS based protocol for quantification of deoxynivalenol and nivalenol in beer was
developed [448]. The concentration range is  [600–20 000] g.kg-1. The MALDI
optimization sample pretreatment procedure includes testing of various matrixes, in order to
obtain an efficient ionization of the analyte. The protocol was based on matrixes such as
sodium azide or hydrazine hydrate. In contrast, the use of graphite as a matrix leads to a low
ionization efficiency, thus adversely affecting method performance [448]. The great
advantages of matrixes of organic salts [485–487] for quantification of beer components is
that efficient ionization of analytes gives rise to low detection limits. For deoxynivalenol in
beer the values are 2 ng or 8.5.10-13 mol [448].
186 Bojidarka Ivanova and Michael Spiteller

Figure 57. The ESI(+/–)–MS fragment pattern of ochratoxin A [447].

Table 16. The (SPE–HPLC; QuEChERS – HPLC; SPE–UHPLC,

SPE–LC; or DSME–LC) ESI–(QqQ or Orbitrap)–MS/MS/(MRM–MS) analysis of
mycotoxins obtained during beer fermentation and final beer product; Concentration
LODs and LOQs

Analyte Product ion (m/z) (Collision LODs Refs.

energy, eV)
Zearalenone 131 (28) 2 ng.g-1 (LOQ) [431, 451]
319.1547 (20) {[M+H]+} 32 ng.mL-1 [433]
317.1 {[M-H]-} 4 g.kg-1 [434]
0.44 g kg-1 [435]
-zearalenol 174 (22) 2 ng.g-1 (LOQ) [431]
 319.2 {[M-H]-} 10 g.kg-1 [434]
-zearalanol 277 (22) 2 ng.g-1 (LOQ) [431]
 321.1702 (20) {[M+H]+} 12 ng.mL-1 [433]
-zearalenol 275 (18) 2 ng.g-1 (LOQ) [431]
 319.2 {[M-H]-} 10 g.kg-1 [434]
-zearalanol 277 (22) 2 ng.g-1 (LOQ) [431]
Ochratoxin A 404 {[M+H]+} ng.mL-1; 0.02 g.L-1 [432, 438, 440, 446, 447, 479]
404.0903 5 ng.mL-1 [433, 436]
Aflatoxin B1 313.0712 (35) {[M+H]+} 5 ng.mL-1 [433, 436, 438, 444, 450, 451]
Aflatoxin B2 315.0868 (35) {[M+H]+} 4 ng.mL-1 [433, 436, 438, 444, 450, 451]
Aflatoxin G1 329.0662 (29) {[M+H]+} 5 ng.mL-1 [433, 436, 438, 444, 450]
Aflatoxin G2 331.0816 (32) {[M+H]+} 4 ng.mL-1 [433, 436, 438, 444, 450]
Sterigmatocystin 325.0711 (35) {[M+H]+} 7 ng.mL-1 [433, 436, 444, 453]
0.75-50 µg kg-1
Nivalenol 313.1289 (18) {[M+H]+} 50 ng.mL-1 [433, 436, 437, 451]
Deoxynivalenol 297.1337, [M+H]+ (20) 20 ng.mL-1; [434, 436, 437, 442, 444]
0.05 g.L-1 [450, 451]
59.1 (38) 40 g.kg-1 [434]
Fumonisin B1 722.3962 (54) {[M+H]+} 105 ng.mL-1 [433, 436, 438, 444, 450, 451]
Fumonisin B2 706.4017 (25) {[M+H]+} 95 ng.mL-1 [433, 436, 438, 444, 450, 451]
Fumonisin B3 706.4016 (25) {[M+H]+} 95 ng.mL-1 [433, 450, 451]
 Quantitative Analytical Methods of Organic Ingredients … 187

Beauvericin 801.4437 4 ng.mL-1; 0.8 g.kg-1 [433, 453]

(50) {[M+NH4]+}
Tenuazonic acid 2 g.kg-1 [441, 480]
Enniatin A 1.2 g.kg-1 [480]
Enniatin A1 0.4 g.kg-1 [480]
Enniatin B 0.8 g.kg-1 [480]
Enniatin B1 1.2 g.kg-1 [480]
Ergometrine 0.02±0.003 g.L-1 [429]

Figure 58. The LC–ESI–MS/MS between–day monitoring of chemical transformation of zearalenone to

main metabolites during fermentation [431].

Figure 59. Chemical substitution of tenuazonic acid [441].

188 Bojidarka Ivanova and Michael Spiteller

The quantitative analysis of tenuazonic acid in beer [441] was performed via chemical
substitution (Figure 59). The ESI–MS/MS (MRM–MS) analysis uses fragment pathway m/z
378140. The method validation was based on analysis of 43 beers of different style and
concentration range 8–500 g.kg-1 of mycotoxins (Table 16).
The ESI–MS/MS analysis of beauvericin and ennamin A1 was reported [488, 489]. The
ion–trap CAD spectrum of the first analyte reveals a peak of {[M+Na]+} adduct. It produces
exclusively bn* ions. The analysis of {[M+H]+} cation shows formation of cn and bn ions. The
first set ions are more abundant [489].

11.3. Analysis of Other Human Health Risk Ingredients in Beer

An ESI–MS based quantitative protocol for analysis of trans–2–nonenal, representing

off–flavor aldehyde in beer was reported [490]. This cytotoxic and genotoxic ingredient is
obtained as product of lipid oxidation [490].

11.4. Determination of Contamination by Microbial Communities

The beer as a foodstuffs matrix is a hostile environment for growth of microorganisms

comparing with other food matrices. The central reasons are anaerobic conditions with low
oxygen content > 0.1 ppm; acidic character (pH  [3.8–4.8]); ethanol content < 10%; and
CO2 amount (ca. 0.5% w/w) [491, 492]. The antimicrobial activity of hops contribute to a
high sterility of the beer–food. The grow of pathogens in beer as a function of pH is shown in
Figure 60 [495]. To so–called “bad” bacteria in brewing are assigned [491–504]: (a) lactic
acid bacteria (Lactobacillales). It can adapt to hops and to spoiling the beer product. There
are two common spoilage bacteria Lactobacillus brevis and Pediococcus damnosus; (b) acetic
acid bacteria (10 species known). To the brewing industry is only awarded Gluconobacter
(Gluconobacter oxydans); (c) Enterobacteriaceae. Among known species the following ones
are connected with the beer production, i.e., Rahnella aquatilis, Obesumbacterium proteus,
and Citrobacter freundii; (d) Zymomonas. The isolated species from beer is Zymomonas
mobilis (Achromobacter anaerobium); (e) Pectinatus spp. In beer is found bacterium P.
cerevisiiphilus; (f) Megasphaera spp. To beer industry is associated M. cerevisiae; and (g)
miscellaneous [491]. The pathogens shown as (a) (Lactobacillus (L.)) are responsible for
majority of incidents with spoilage of beer [491–504]. The quantification of pathogens is
among the main concerns of "food control" and assessing risk to human health through
consumption of contaminated beer. The MALDI–TOF–MS study of Lactobacillus (L.) brevis
strains in beer was reported [492]. The ESI–MS/MS determination of proteins fragments was
performed. The acyl carrier protein sequence of Lactobacillus (L.) brevis (ATCC 367 ACP)
and protein residues were identified by LC ESI MS/MS. Series possible protein candidates
with molecular weight 9435 Da ± 600 ppm are listed in Table 17.
 Quantitative Analytical Methods of Organic Ingredients … 189

Figure 60. Growth pathogens in 444 beers vs. pH [495].

Table 17. Possible protein candidates with molecular weight 9435 Da ± 600 ppm in beer;
LC–ESI MS/MS and MALDI–TOF analysis

Strains Accession number/Gene sequence Refs.

Lactobacillus brevis gi|545616974,gi|116098938,gi|544228539, [492, 493, 495]
gi|472406752, gi|116333591,
gi|499986959
buchneri KF910134, AM087681
parabuchneri KF910135 [295]
harbinensis HQ419067
perolens KF910115, KF910136, KF910131
Gluconobacter albidus AB178392, AB178405, AB178431 [494]
cerevisiae HG424633, HG329625, HG329624 [494]
cerinus X80775, KF910106, KF910111,
KF910094
japonicus AB178410, HG329573
kanchanaburiensis AB459530
nephelii AB540148, KF700364
oxydans X73820
thailandicus AB128050, FN391803
uchimurae AB193244
wancherniae AB511060
Acetobacter aceti AB680674 [494]
malorum KF537504
pasteurianus KF910100, KF910095 [495]
Pediococcus acidilactici AM749814 [495]
damnosus KF910126 [495]
190 Bojidarka Ivanova and Michael Spiteller

11.5. Analysis of Pesticides in Beer

A wide range of pesticides in agricultural practice are used against insects, molds and
fungi. They can occur in raw material, and can be transmitted in the final food product [505–
526]. So, the quantitative analysis of pesticides in beer is of importance. It ensures a safety for
human health consumption. Despite pesticides are not naturally occurring “ingredients” in
beer, but are considered as agrochemicals, contaminating the food, in this subsection are
shown contributions in implementation of precise protocols for quantitative analysis of
pesticides in beer, based on mass spectrometric methods [504–526]. Analysis pesticides in
beer samples by UHPLC–MS/MS was reported [505]. The concentration LODs was  0.01–
5.61 gL-1 (level of confidence r2 > 0.95). The fiber liquid phase microextraction used (Table
18).

Table 18. The MS quantitative analysis of pesticides in beer [506–526]; tR – Retention

time [mins]; MS transitions (m/z) (Collision energy [eV]); Concentration (C)

Analyte tR MS Recovery C; LODs Refs.

Simazine 3.32–3.43 202.1132.1 (18) 93.9 (6.5) 25 g.L-1 [505]
13.03 186 (58.2) 173 (34.3) 5.ng.ml-1 [510]
Desethyl 3.53–3.67 202.3146.1 (16) 111.6 (12.7) 25 g.L-1 [505]
terbuthylazine
Carbaryl 3.54–3.68 202.1  145.1 (10) 75.9 (4.6) 25 g.L-1
17.94 115 (51.6)  116 (35.5) 25.ng.ml-1 [510]
Monolinuron 3.63–3.77 215.1  126.1 (18) 73.0 (11.5) 25 g.L-1 [505]
Chlorotoluron 3.79–3.94 213.2  72.1 (15) 91.7 (13.4) 25 g.L-1
Metobromuron 3.83–3.96 259.1  170.0 (20) 109.0 (8.3) 25 g.L-1
Atrazine 3.88–4.03 216.1  174.1 (18) 95.1 (8.8) 25 g.L-1
13.21 215 (59.2)  202 (34.0) 5.ng.ml-1
Metazachlor 3.89–4.06 278.3  134.0 (20) 80.9 (9.1) 25 g.L-1
Lenacil 3.92–4.10 235.2  153.0 (15) 75.3 (11.5) 25 g.L-1
Fensulfothion 3.94–4.11 309.1  281.2 (15) 76.2 (10.2) 25 g.L-1
Isoproturon 3.97–4.13 207.2  72.1 (30) 103.8 (8.6) 25 g.L-1
Diuron 4.05–4.20 233.1  72.0 (25) 98.0 (15.2) 25 g.L-1
Azaconazole 4.08–4.23 300.1  159.0 (23) 90.0 (11.0) 25 g.L-1
Iodosulfuron 4.08–4.28 530.4  163.2 (16) 88.2 (12.3) 25 g.L-1
methyl
Bensulfuron methyl 4.18–4.30 411.3  149.2 (18) 76.1 (7.6) 25 g.L-1
Diethofencarb 4.30–4.45 268.3  226.3 (10) 103.8 (11.7) 25 g.L-1
Sebuthylazine 4.33–4.51 230.4  174.1 (18) 98.2 (4.9) 25 g.L-1
Propazine 4.36–4.53 230.4  188.2 (16) 85.7 (12.3) 25 g.L-1
Linuron 4.37–4.54 249.1  160.0 (18) 74.4 (11.9) 25 g.L-1
Spiroxamine 4.38–4.62 298.3  144.2 (20) 106.7 (11.8) 25 g.L-1
Methiocarb 4.44–4.59 226.1  169.2 (10) 83.2 (12.6) 25 g.L-1
Terbuthylazine 4.46–4.60 230.2  174.1 (15) 109.4 (6.8) 25 g.L-1
Paclobutrazol 4.51–4.67 294.2  70.0 (25) 108.5 (6.1) 25 g.L-1
Flutalonil 4.52–4.66 324.4  242.3 (25) 96.9 (8.9) 25 g.L-1
Promecarb 4.53–4.68 208.2  151.2 (9) 79.0 (6.9) 25 g.L-1
 Quantitative Analytical Methods of Organic Ingredients … 191

Analyte tR MS Recovery C; LODs Refs.

Prometryn 4.54–4.69 242.0  157.9 (17) 104.2 (13.7) 25 g.L-1
17.34 184 (70.8)  226 (55.3) 5.ng.ml-1 [510]
Propyzamide 4.61–4.72 256.2  190.0 (16) 97.6 (11.2) 25 g.L-1 [505]
Triazophos 4.66–4.80 314.2  162.1 (18) 82.6 (4.7) 25 g.L-1
Tepraloxidim 4.69–4.85 342.4  250.3 (12) 109.7 (5.9) 25 g.L-1
Iprovalicarb 4.70–4.82 321.4  119.1 (15) 106.3 (8.5) 25 g.L-1
Triadimenol 4.73–4.86 296.2  70.0 (12) 105.5 (6.0) 25 g.L-1
Fenhexamide 4.78–4.90 302.2  97.1 (25) 87.9 (5.1) 25 g.L-1
Epoxiconazole 4.82–4.95 330.2  121.1 (20) 102.4 (6.9) 25 g.L-1
Tebutam 4.88–4.98 234.3  90.9 (18) 107.7 (8.0) 25 g.L-1
Metolachlor 4.91–5.02 284.3  252.4 (15) 82.9 (12.5) 25 g.L-1
Fenbuconazol 4.91–5.02 337.3  70.1 (25) 108.2 (12.9) 25 g.L-1 [505]
32.20 198 (60.2)  125 (32.8) 25.ng.ml-1 [510]
Diflubenzuron 4.97–5.07 311.1  158.1 (12) 101.2 (7.3) 25 g.L-1 [505]
Cyprodinil 4.97–5.09 226.0  108.1 (20) 109.5 (9.9) 25 g.L-1
Thiazopyr 5.05–5.16 397.2  377.2 (28) 102.3 (13.6) 25 g.L-1
Furmecyclox 5.13–5.26 252.5  170.2 (13) 106.2 (10.0) 25 g.L-1
Spinosad 5.14–5.47 732.7  142.2 (30) 95.9 (7.2) 25 g.L-1
Bitertanol 5.29–5.39 338.3  269.3 (10) 102.7 (6.2) 25 g.L-1
Pencycuron 5.40–5.45 329.3  125.1 (19) 103.5 (12.7) 25 g.L-1
Trifloxystrobin 5.43–5.51 409.3  186.2 (18) 110.6 (10.8) 25 g.L-1
Triflumizole 5.51–5.59 346.2  73.1 (17) 71.4 (10.3) 25 g.L-1
Clethodim 5.53–5.62 360.3  164.1 (20) 92.3 (4.0) 25 g.L-1
Cycloxydim 5.53–5.62 326.3  280.4 (13) 80.3 (4.5) 25 g.L-1
Fluazifop buthyl 5.60–5.67 384.3  282.3 (20) 85.6 (11.5) 25 g.L-1
Sethoxydim 5.68–5.76 328.5  178.1 (18) 84.4 (5.5) 25 g.L-1
Hexythiazox 5.87–5.95 353.3  228.1 (15) 103.8 (10.2) 25 g.L-1
Fenazaquin 6.26–6.37 307.3  161.2 (16) 82.0 (12.6) 25 g.L-1
Trifluralin 32.51 264 (80.9)  290 (14.4) 6.4 ng.g-1 [506]
Fenarimol 21.41 107(79.9)  219(50.1) 10.7 ng.g-1
6.12 139 (100), 295 (18) 5.10-4 ppm
30.41 219 (68.1)  251 (59.3) 5.ng.ml-1
Malathion 21.87 173(93.7)  93 (93.2) 28.7 ng.g-1
18.78 127 (77.7)  125 (86.5) 5.ng.ml-1
Propoconazole I 28.91 259(97.1)  69 (65.6) 17.7 ng.g-1
26.91 259 (94.6)  69 (68.4) 5.ng.ml-1
Naurimol 29.59 139(76.1)  235 (72.3) 6.8 ng.g-1
Chlorfenapyr 3.22 59 (100), 247 (8) 5.10-4 ppm [509]
Quinoxyfen 4.13 237 (100), 272 (37) 5.10-4 ppm
Tebuconazole 4.52 83 (42%), 125 (96) 5.10-4 ppm
27.47 250 (100.7) 70 (42.2) 10.ng.ml-1
Pyridaben 6.70 147 (100), 309 (9) 5.10-4 ppm
Z-dimethomorph 8.94 165 (30), 301 (100) 5.10-4 ppm
E-dimethomorph 9.29 165 (30), 301 (100) 5.10-4 ppm
192 Bojidarka Ivanova and Michael Spiteller

CONCLUSION
Beer is a complex food matrix of an analytical chemistry point of view. It contains CO2,
ethanol, inorganic salts and  (400–600) organic ingredients. The chemical ingredients vary
due to chemical, thermal and photochemical instability, thus determining a complex
photochemistry of the beer. The development of precise analytical quantitative protocols
provide an important research challenge. The methods of MS have irreplaceable place due to
their great instrumental functions. Among various MS methods, ESI and MALDI methods
greatly expand the analytical application of MS for thermally instable analytes and these
with high polarity and masses. A wide range analytes from LMW to macromolecules may
be examined. The MS sample preparation techniques are applicable to homogeneous and
heterogeneous samples in solid– and semi–liquid states. Among the most prominent
contribution of MALDI–MS method to beeromics is its ability to in vitro and in vivo
screening an dimaging of microorganisms, that spoil the beer. Our chapter reflects recent
developments in analytical chemistry of food, focusing the attention on beeromics. The
chapter summarizes, outstanding contributions of MS–based analytical protocols to the
“Quality control” of food, including assessment of risk to human health from contaminating
agents in foods.

ACKNOWLEDGMENTS
The authors thank Deutsche Forschungsgemeinschaft (DFG) and NRW–EU–Ziel2–
Programmes for high resolution mass spectrometric equipment and research funding; Central
instrumental laboratories for structural analysis at Dortmund University of Technology
(Federal State Nordrhein–Westfalia, Germany); analytical and computational laboratory
clusters at Institute of Environmental Research at the same University. Competing interests:
Michael Spiteller received research funding (255/21–1, 255/22–1, DFG); Bojidarka Ivanova
received research funding (255/22–1, DFG).
This chapter was carefully carried out. Nevertheless, authors and publisher do not warrant
the information therein to be free of errors.

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BIOGRAPHICAL SKETCHES
B. Ivanova has received MSc degree in “Chemical physics and theoretical chemistry”
(1997) and PhD one in “Inorganic and analytical chemistry” (2001) at Sofia University “St.
Kliment Ohridski” (Bulgaria). In 2006 she achieved a Habilitation of “Analytical chemistry”
at the same Institution. Ivanova has been “associate professor” of analytical chemistry, and
 Quantitative Analytical Methods of Organic Ingredients … 211

“Head of the group” of “Molecular Spectroscopy and Structural Analysis” (2006–2010).

She occupied research position at the Institute of Environmental Research (INFU) of the
Faculty of Chemistry and Chemical Biology at the Dortmund University of Technology
(Germany) (2010–2011). At the same Institution she hold research positions under projects
(2006,2010) sponsored by Deutscher Akademischer Austausch Dienst and Deutsche
Forschungsgemeinschaft (Germany). She has been visiting researcher at Ruhr–University
Bochum (Germany) under project initiatives sponsored by Alexander von Humboldt Stiftung
(Germany) (2003,2007–2009). Ivanova is author and co-author of more than 220 scientific
contributions, including books, textbooks and distance–learning e Textbooks, focusing her
interest on molecular spectroscopy and structural analysis, inorganic and metal-organic
chemistry of d– and f–elements, organometallic synthesis and catalysis. Ivanova has been
awarded with Rectors’ Council award “Best young scientist" (Sofia University “St. Kliment
Ohridski,” 2003) and “Pitagor Award” for “Best young scientist in whole area of sciences” of
Bulgarian Ministry of Education (2009).

Michael Spiteller finished his study (1976) and earned a doctoral degree (1979) in
Chemistry at Georg–August University (Göttingen, Germany). Habilitation he achieved at
Institute for Soil Science and Forest Hygiene (Göttingen) in 1985. After that, he lead
“Research Group” at the Institute for Metabolism and Environmental Fate (Bayer Corp.,
Germany) for 8 years. He has appointed as professor “extraordinarius” at Georg–August
University in 1990 and for “full” professor at University of Kassel (Germany). Now, Spiteller
is professor and “Head” of the Institute of Environmental Research of the Faculty of
Chemistry and Chemical Biology at the Dortmund University of Technology (Germany). In
the same Institution, Spiteller holds “Research Group Leader Position” of “Environmental
Analytical Chemistry” under the “focus” programme of Deutsche Forschungsgemeinschaft.
He is member of numerous expert commissions at German Federal Offices and Councils. He
serves also as member of editorial advisory boards of international specialized Journals
such as “Fresenius Environmental Bulletin,” “Advances in Food Science,” “Pflanzenschutz
Nachrichten” (Bayer), “Journal of Serbian Chemical Society” and the fourth. His work has
led to over 500 scientific papers and presentations, review articles and monographs. He has
been honoured member of Serbian Chemical Society and Institute of Organic Chemistry at
the Bulgarian Academy of Sciences. He has been honoured with doctor title “honoris causa”
from the University of Novi Sad (Serbia, 2006) and from the University of Sofia “St. Kliment
Ohridski” (Bulgaria, 2009). Spiteller’s scientific interests encompass environmental analytical
chemistry, mass spectrometric methodology and, natural products chemistry.
In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485-704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.

Chapter 5

SACCHAROMYCES SPP. ROLE IN BREWING PROCESS

AND ITS SERIAL REPITCHING IMPACT

Cátia Martins1,2, Tiago Brandão3,4,

Adelaide Almeida2 and Sílvia M. Rocha1,
1
Departamento de Química and QOPNA,
Universidade de Aveiro, Aveiro, Portugal
2
Departamento de Biologia and CESAM,
Universidade de Aveiro, Aveiro, Portugal
3
Unicer Bebidas, SA, Leça do Balio, Portugal
4
President of the European Brewing Convention, russels, Belgium

ABSTRACT
Beer is the second most consumed beverage in the world, accounting ca. 35%
of all recorded alcohol consumed in 2010. Brewing industry is extremely competitive,
highlighting the importance of constant innovation. The success of global beer
commercialization is its intrinsic quality that depends of a network of variables, namely
raw materials, yeast strain and the brewing process itself. The biochemical performance
of yeasts, usually Saccharomyces spp., is one of the parameters with significant
importance in brewing once it limits the alcoholic content of final beer and produces
different metabolites with crucial impact on beer aroma and flavor. It will be given focus
to the different metabolic pathways with significant impact on beer’s aroma profile,
namely the carbohydrates and nitrogen compounds metabolism; and also the formation of
aldehydes, higher alcohols, vicinal diketones, esters, fatty acids, sulfur and terpenic
compounds. Environmental changes can promote stress in yeasts along brewing. The
stress factors will be systematized in this book chapter, as well as the yeasts cellular
effects and their biological response. Furthermore, brewing companies tend to reused (or
repitch) yeasts several times to minimize resources costs, while maintaining quality of the
final product. Also, different approaches will be described in order to monitor yeast
quality. Therefore, the impact of yeasts repitching along brewing will be systematized,
considering its associated advantages and drawbacks. In summary, this book chapter aims


Corresponding Author address; Email: smrocha@ua.pt.
214 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

to elucidate about the raw materials and the brewing process, and also to give an
overview of the Saccharomyces spp. metabolism (special attention will be done on its
impact on beer aroma profile), as well as to understand the effects of serial repitching on
yeasts’ behavior.

Keywords: Saccharomyces spp., metabolism, repitching, beer, raw materials, brewing

process

BEER GENERAL CHEMICAL COMPOSITION AND CONSUMPTION

The name of beer comes from the Latin word “bibere” that means to drink. Historically,
beer was started to be produced by the summarians in Mesopotamia (now Iraq) from more
than 6000 years ago. In this region, the oldest surviving beer recipe was found in cuneiform
(clay tablets) that refers it in a hymn to Ninkasi, the patron goddess of brewing. In that period,
it is known that the beer was made by women bakers, from barley via bread. Then, a new
civilization appeared in Mesopotamia, around 1770 BC, the Babylonians, which had beer in
their diet, made from barley or emmer (Triticum dicoccum, wheat’s ancient form), and they
developed more than twenty different beers, ruled by Hammurabi code. This brewing
tradition had spread from Mesopotamian region to Egypt, where beer was the primary
beverage for Pharaoh and all peasants (fact recorded in several pictures and sculptures), and
was also considered as a product with medical and religious purposes. Egyptians’ brewing
techniques were passed to Greeks and Romans (around 500 BC), but beer was not well
accepted by the privileged classes that preferred to drink wine. In the Middle Age, European
monasteries had an important contribution in the development of modern beer, namely in the
introduction of hops flowers to flavor and preservation of beers (rule written in 822 by abbot
Adalhard in a monastery in Corbie, northern France) (Barth, 2013). At this time, beer was
used as commodity for commerce, payment and taxes, being disseminated in Europe. From
16th century, beer became a global beverage due to the universal expansion of the brewing
techniques. The Industrial Revolution (18th century) had a huge impact on the brewing
industry once the beer production at an artisanal manufacture was converted into an industrial
scale. Several scientific advances were recorded, such as the development of hydrometers and
thermometers, saccharometer, and artificial refrigeration, which allowed a greater knowledge
about the final product and better process control. Moreover, between 1855 and 1876, Louis
Pasteur discovered the pasteurization methods to prevent the development of undesirable
microorganisms. Also, Emil Christian Hansen had an important contribute to brewing
science, once he isolated and classified the yeasts as bottom-fermenting lager yeasts
(Saccharomyces uvarum, or also called Saccharomyces carlsbergensis) and top-fermenting
ale yeasts (Saccharomyces cerevisiae). His work contributed to the beer quality, and also the
flavor standardization.
In the 21st century, the great developments of the brewing industry allow that consumers
have hundreds of different beers available on markets, being the actual trends concerning
about the quality and stability control of beer, as well as the developing microbrewing
industries (in Europe, there was an increase of 16%, from 2013 to 2014) and the development
of innovative products (e.g., non-alcoholic and flavored-mixed beers) to attract consumers.
 Saccharomyces spp. Role in Brewing Process … 215

The latest statistical results (Figure 1) report that the beer global consumption and,
consequently, its production, has been stagnating either considering the world, as well as the
European Union (EU) plus 3 countries (Norway, Switzerland and Turkey). This stagnation of
the beer consumption was not only induced by the actual economic context but there are other
major factors that are correlated such as: lower per capita consumption; higher taxes for beer
in bars, restaurants in some countries, which leads to the consumption of beer at consumer’s
house; and the consumers buy less beer premium brands. Only Asia and Africa markets have
a contrary trend, where an slight increase of beer consumption have occurred, for instance
representing attractive markets for Europe’ exportation that increased 15% since 2008 until
2014.

414500 195000

404500 190000

Worlwide data (x103 kL)

394500 185000
EU data (x103 hL)

EU production
384500 180000
EU consumption

374500 175000 Worldwide production

Worldwide consumption
364500 170000

354500 165000
2009 2010 2011 2012 2013 2014
Year

Figure 1. Beer production and consumption in the European Union plus 4 countries (Croatia, Norway,
Switzerland and Turkey) and in world, between 2009 and 2014 (Kirin Beer University Report, 2015;
Walle, 2015).

Beer is a fermented beverage that is produced from several raw materials: water, hops or
other hop products (e.g., pellets or hop oil), malted grains (normally barley), and yeast. Some
other cereals can be combined with barley malt (e.g., wheat malt, unmalted cereal adjuncts)
contributing for other starch- and/or sugar-containing raw materials. Usually the final product
contains: ethanol, compounds from hops, high concentration of carbon dioxide (CO2, around
0.5% p/p), low pH value (between 3.8 and 4.7) and low concentration of oxygen (< 0.1 ppm)
(Dragone et al., 2008). Figure 2 represents the average chemical composition of a lager beer,
which main component is water (ca. 90 – 94%). The major yeasts fermentation products are
ethanol and carbon dioxide. Yeasts, as well as other raw materials (derived from malt and
hops that survive wort boiling), are responsible for most of the volatile compounds formation
(responsible for the bouquet or aroma of beer), which then that can be found in beer, namely
higher alcohols (10– 300 mg/L), organic acids (50 – 250 mg/L), esters (25 – 40 mg/L),
aldehydes (30 – 40 mg/L), vicinal diketones, sulfur compounds (1 – 10 mg/L), and terpenic
compounds (Briggs et al., 2004; Buiatti, 2009). Despite their lower amount (ca. 0.5%),
volatile components have great impact on beer flavor, as well as their interaction with non-
volatile components, thus contributing for the achievement of the aroma characteristics of
216 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

each beer style. There are also non-volatile components present in beer (ca. 4%), such as
inorganic salts, sugars, amino acids, nucleotides, polyphenols, vitamins, lipids, and hop
resins. Additionally, the majority of the carbohydrates, present in wort, are metabolized by
yeasts, being only detected the residues that yeasts can not metabolize (e.g., D-ribose, L-
arabinose, or D-xylose) or those that come from ‘primings’ addiction, that are other sugar
sources without being barley (Briggs et al., 2004; Boulton, 2013). Thus, hundreds of different
compounds were already identified in beer, which belong to different classes, being their
concentration dependent of the different raw materials that are used, the different brewing
steps applied by each brewing company, as well of the yeast strain (Boulton, 2013).

Volatiles 0.5 %
Higher alcohols
Volatile organic acids
Extract 4 % Esters
Ethanol 3-5 % Aldehydes
Vicinal diketones
Sulfur compounds
Water Terpenic compounds

90-94% CO2 0.5 %

Carbohydrates (80 – 85 %)
Proteins (4.5 – 5.2 %)
Minerals (3 – 4 %)
Bitter substances (2 – 3 %)
Organic acids (0.7 – 1.0 %)

Figure 2. Average chemical composition of a lager beer based on data collected from (Vanderhaegen et
al., 2006; Eßlinger, 2009; Eßlinger and Narziß, 2012).

1. BREWING PROCESS
This topic is divided in two sections, the first one will comprise the four raw materials
used in beer production, and then, the complexity of the brewing process will be explained
through the description of the different steps, being each one clarified from a scientific
point of view. The brewing process comprises several steps, represented in Figure 3, which
can be sectioned in three different and independent zones within a company: brewhouse;
fermentation, and storage cellar; and filtration and filling. Furthermore, it is also indicated in
which step of the process the different raw materials can be added.
 Saccharomyces spp. Role in Brewing Process … 217

Wort

Figure 3. Brewing process workflow, in which is indicated the steps where raw materials are added.

1.1. Raw Materials

In order to produce beer, there are four main ingredients: malted grains, hops, water, and
yeast. A brief description of each raw material will be presented, as well as some key
concepts that are important to understand the brewing process.

Malted Grains
The cereal grain that is more often used is barley (Hordeum vulgare), due to the easier
regulation and control of the germination process of these grains comparing with others; it has
a favorable price; and allows the creation of innovative beer styles through special coloring
and flavoring (O’ Rourke, 1999). The barley seeds are the raw material used in the production
of malted barley (starch’ source and enzymes required for starch break down) (Barth, 2013).
The main carbohydrates present in barley are the α-glucans, namely amylose and amylopectin
of starch (55 – 57%). Moreover, barley proteins (9 – 10.5%) are important for beer’ stability,
foam and taste, and yeast nutrition; while lipids are only partially used on malting process.
218 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

Another important constituents are phosphates (ca. 0.3%), minerals (2.5 – 3.5%), vitamins
(ca. 0.5 × 10-3%), and phenolic substances (ca. 0.2%) (Eßlinger and Narziß, 2012). Mixtures
of malted and unmalted barley can be used, nevertheless the portion of unmalted grains
should not exceed 50%, because it can promise the activity of the endogenous hydrolases,
present in malt, during the mashing process (Meilgaard, 1976; Eßlinger, 2009).
Other cereal grains (e.g., wheat, maize, rice, sorghum) can be added as adjuncts, in order
to have different sources of extract (fermentable sugars). They can be unprocessed raw
cereals or semi-purified extracts. Regarding the solid cereal adjuncts (‘mash tun’ adjuncts), it
is required some prior processing (e.g., milling and mashing) for starch release and/or is
required the use of protein-rich barley malts with very high enzyme contents (amylases)
(Briggs et al., 2004; Eßlinger and Narziß, 2012). In the case of liquid extracts (soluble sugars
or syrups), they may be added during boiling wort (‘copper’-or ‘kettle’-adjuncts), increasing
and adjusting the wort fermentability that is required for high-gravity brewing; or they can be
added post-fermentation (called ‘primings’) for flavoring and/or coloring the finished beer
(usually that undergo a secondary conditioning process) (Briggs et al., 2004).
Partially unmalted wheat (Triticum aestivum) can be added to the mash, being its
composition similar to barley: contains 65% of starch and other carbohydrates, 12 – 14% of
proteins, and 1.7% of fat. Maize or corn (Zea mays L.) is used as a malt replacement (requires
prior starch purification through dry and wet milling), with extract yields of 87 – 91% (after
removing the oil-rich embryo), 8.5 – 9% of proteins, and residual fat content (< 1%)
(Eßlinger, 2009; Eßlinger and Narziß, 2012). Rice (Oryza sativa L.) requires a pre-processing
to be used as adjunct, and it is composed by extract 93 – 95%, protein 8 – 9%, and fat 0.5 –
0.7%. Sorghum (Sorghum vulgare) can be used as a malt additive, however it has only
regional importance in Africa (Eßlinger and Narziß, 2012).
In the case of some cereals such as sorghum, rice, or maize, it is required higher
temperatures (60 – 90°C) along mashing, comparing with barley (50 – 70°C), thus requiring a
separate treatment in a cereal cooker (Boulton, 2013).

Water
Another important raw material is water, which content varies between 90 – 94% in beer.
As it is the main component of beer, its chemical and biological composition requires high
quality control, namely be ‘aesthetic’ (clear, colorless, neutral in odor and taste), without
presence of pathogens and heavy metals (namely iron and manganese), and should not be
corrosive (Briggs et al., 2004; Eßlinger and Narziß, 2012). Water contributes mainly with the
inorganic constituents present in beer, having impact on the palability of the finished beer,
once the ionic composition of the water varies with the geographical area (Boulton, 2013).
The major cations are the potassium, sodium, calcium, and magnesium; while the major
anions are chloride, sulphate, nitrate, and phosphate. There are many ions that are essential
for yeast growth, even at trace amount (e.g., zinc, copper or iron); while in case of other
constituents, their larger amounts may be toxic to yeasts (e.g., nitrate), being their content
limited by law (Briggs et al., 2004).

Hops
Hops are the dried cones of the female part of the plant Humulus lupulus. They can be
added as flowers or by the form of pellets/hop extract, depending on the wanted amount of
resin and essential oils. They are widely used as beer raw material due to several reasons,
 Saccharomyces spp. Role in Brewing Process … 219

namely they give a bitter taste and specific aroma, they improve and stabilize the beer foam,
and also increase the biological shelf-life through their antibacterial properties (especially
against Gram-negative, but also against Gram-positive bacteria) (Eßlinger, 2009). The hops
bitter components are α-acids (humulones, 3 – 17%), β-acids (lupulones, 2 – 7%) and the
oxidation products of bitter acids, namely, the soft and hard resins. The α-acids are the most
important components because of their high bitterness potential (Eßlinger, 2009; Eßlinger and
Narziß, 2012). In conventional brewing, hops are added to the sweet wort and are boiled
during one hour and half to two hours, in which the α-acids are isomerized into the iso-α-
acids, and the essential oil components (up to 3%) are vaporized. Nevertheless, a portion of
choice ‘aroma’ hops can be added later (‘kettle-hop aroma’), in order to replace the loss of
some volatile compounds, mainly in lager beers. Moreover, dry hopping technique can be
applied, which corresponds to the introduction of dry hops in beer, either in casks or
conditioning tanks, thus increasing the ‘dry-hop aroma’ (mainly applied in ale beers
production) (Briggs et al., 2004).
Hop essential oil is present in the lupulin glands, being responsible for the characteristic
pleasant aroma of hops, and composed by a complex mixture of more than 300 compounds.
Each individual variety has specific compositions and concentrations of components.
Nevertheless, hydrocarbons are the major fraction (50 ± 80%), being the major components
the monoterpene myrcene (17 – 37%) and the sesquiterpenes β-caryophyllene and
humulene (Briggs et al., 2004; Eßlinger, 2009). The ripeness of the cones can be reflected
in the myrcene percentage, while humulene/caryophyllene ratio is considered a varietal
characteristic. The hydrocarbons components are more volatile and then more easily lost
during wort boiling when comparing with the oxygenated fraction, which are more probable
to be found in beer. The oxygenated fraction have intense odors and aromas, and can
contribute with different aromas to beer (e.g., citrus, floral, spicy), being linalool the major
impact compound for hop aromatic beers (Boulton, 2013).
Hops flavor is conditioned by several parameters, such as growing temperature, soil,
moisture, and other climate issues. As small craft brewers cannot have a big hop inventory,
their product cannot be so consistent between batches (Barth, 2013).

Yeast
Brewing yeasts are unicellular fungi, that reproduce vegetatively by multilateral budding
or fission, and usually they belong to the Saccharomycetaceae family and Saccharomyces
genus (Eßlinger and Narziß, 2012). They are able to ferment either in anaerobic or semi-
anaerobic conditions, and are also able to ferment one or more sugars (e.g., glucose, fructose)
(Barnett, 1992).
There are two types of Saccharomyces yeasts involved in beer fermentation: top-
fermenting (or ale) and bottom-fermenting (or lager) yeasts, which vary according to their
fermentative ability, rate of sugar utilization, tolerance to temperature, flocculation
characteristics and volatiles’ profile (Eßlinger, 2009). While ale yeasts are usually classified
as S. cerevisiae; lager yeasts’ classification is still a controversy, but S. pastorianus, S.
carlsbergensis and S. uvarum are classified as lager yeasts. There are other yeasts species,
namely some of the genus Brettanomyces, that are commonly used in a Belgian ale style
called lambic (Barth, 2013).
220 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

1.2. Brewing Process Steps

The first step on beer production (Figure 3) is the conversion of grains or seeds of
different plants (barley is the most commonly used) into malt through a malting process,
which is usually performed by an independent company. Malting involves three main steps:
steeping, germination, and kilning. Firstly, the grains are immersed in water (steeping), being
allowed their germination under controlled temperature and humidity. It is promoted the
activation of some enzymes (amylolytic, proteolytic, and cellulytic) present in barley, leading
to the partial degradation of the starch (present in the grains’ endosperm). The germination is
stopped by heating (kilning), forming the green malt in its finished form (water content of
malt of ca. 4%), which then can be stored in silos (Eßlinger and Narziß, 2012). Furthermore,
Maillard reactions can also be promoted in the kilning step, leading to formation of
darker malts. These malts can be employed to contribute with different and specific
aromatic/coloring compounds to beers, although enzymes might not resist the applied high
temperatures.
The brewing process itself starts at the brewhouse, in which the wort is produced through
several main steps, namely milling, mashing, filtration, wort boiling, and wort clarification.
The malt grains and other solid adjuncts are broken down and reduced into small particles,
through a mill, obtaining thick flour called grist. The milling conditions should be adapted in
order to obtain the best yield of the obtaining extract during mashing.
Grist (in combination with other solid adjuncts or enzymes) is added to hot water (step
called mashing) at controlled time (or periods of time), temperature (or range of temperatures)
and pH, in order to promote the most efficient hydrolysis of starch and other biomolecules,
e.g., proteins and lipids. At high temperatures (around 65ºC), the starch granules start to
gelatinize, allowing an easier access to amylase enzymes (- and -amylase), which initiates
the starch degradation (breaking of the -1,4 glycosidic bonds) into fermentable sugars.
Moreover, some protein degradation can occur due to some remaining peptidase activity
(most of these enzymes which have high concentrations in malt before kilning, are denatured
in the kilning step). Mashing can take from 2 to 4 hours, and finish at approximately 75ºC.
The variation of time, pH and temperature, as well as the presence of coenzymes, cofactors,
substrate concentration or presence of activators and inhibitors, will allow the activation and
inactivation of enzymes, in a sequential manner along mashing step. Sweet wort is obtained
after mashing, being composed mainly by fermentable sugars (e.g., glucose, maltose,
maltotriose, and sucrose) and non-fermentable sugars (such as small branched dextrins), and
the sum of these sugars corresponds to the wort extract, that is usually measured in degrees
Plato (ºP): sugar content expressed in grams of sugar per 100 grams of wort (Huuskonen et
al., 2010).
Very high gravity (VHG) fermentations are a current brewing trend that allows to obtain
beer with 14 – 16% (v/v) of ethanol content instead of 4 – 5% in low or normal gravity
fermentations. The main difference is related with the amount of dissolved solids in wort, in
which high amount of fermentable sugars are available (18 ºP or more, instead of 11 – 12 ºP).
VHG fermentations have several advantages such as: considerable reducing amount of water
need, increased productivity (more alcohol content), lower energy needs, less capital loss,
among others; thus increasing the overall brewery productivity. However, the high osmo-
stress conditions that yeasts are submitted in VHG fermentations, compromise their
 Saccharomyces spp. Role in Brewing Process … 221

fermenting ability and viability, as recently review by Puligundla et al., 2011. This stress can
lead to an unbalanced flavor composition to beer (mainly overproduction of acetate esters)
(Anderson and Kirsop, 1974; Saerens et al., 2008; Yu et al., 2012) due to changes the
expression of several genes associated to the metabolic pathways as observed by Nam et al.,
2009. The increase of fermentation temperature can lead to higher fermentation rate, but can
promote higher stress to yeasts. Nevertheless, the efficiency of the fermentation can be
increased if the oxygen conditions were improved, without interfering with the physiological
conditions of yeast and quality of beer, namely using preoxygenation of yeast combined with
aeration as Verbelen et al., 2009c suggests.
Sweet wort can be filtrated to separate an insoluble part (spent grains – currently sold for
animal feed) and the soluble part (wort), usually by the use of a lauter tun. This step takes 2 –
3 hours and occurs at 75 – 80ºC. Furthermore, wort is vigorously boiled around one hour and
half to two hours, in a vessel called kettle, in which hops are added. The boiling process leads
to loss of most of the hop oil volatile components (Simpson, 1993; Kishimoto et al., 2005),
which can lead to the addition of ‘aroma’ hops in the end of boiling, the ‘kettle-hop aroma.’
The boiling process is performed in order to allow: the solubilisation and transformation of
the hops substances, namely the extraction of -acids and their isomerization into iso--acids
that contribute for the bitter taste (Schönberger and Kostelecky, 2011); elimination of
unpleasant volatile compounds (mainly derived from malt; e.g., decomposition of S-
methylmethionine into dimethyl sulfide, that has a cooked vegetable flavor (Barth, 2013));
sterilization of the wort; precipitation of proteins with high molecular weight (trub) and the
formation of complexes between proteins and polyphenols (important in beer haze); fixation
of the final concentration of wort (original extract); denaturation of the exogenous enzymes
and malt, for guarantee unforeseen alterations along the following brewing steps. At this step,
Maillard reactions are also promoted due to the applied high temperatures, which lead to
melanoidins formation, which contributes to wort color. Moreover, liquid adjuncts can be also
added at this phase. Subsequently, a decanter (gravity’s action) or whirlpool (centripetal
force) is used to separate the wort from trub and hops components that did not solubilize.
Wort is then cooled until 7 – 22ºC (depending on the fermentation temperature) and airy in
sterile conditions. Thus, wort is a rich and complex source of nutrients for yeasts, being
mainly composed by carbohydrates (ca. 90 – 92% of the total solids) (Barth, 2013). The
nitrogen-compounds represent ca. 5% of the total solids, from which 85% are represented by
free amino acids, peptides, and proteins. In fact, the wort amino acids and small peptides
come from malts and adjuncts (that contain nitrogen) and their content is expressed as free
amino nitrogen (FAN), representing the main source of assimilable nitrogen by yeasts (Briggs
et al., 2004; Boulton, 2013).
Prior to fermentation, it is needed to perform yeast propagation, whereas yeasts are
transferred consequently into larger wort volumes, until is obtained large amount of yeasts
needed to pitch (pitching rate). Cold oxygenated wort is inoculated with yeasts (pitching)
during its transfer into the fermenter. The fermentation takes place in closed vessels, usually
cylindroconical vessels, being the selection of yeast strain dependent of the required beer
type: ale or lager. Typical ale fermentation occurs at 16 – 25ºC, while lager fermentation is
usually performed at 7 – 14ºC, requiring more days to be completed. Normally, yeasts tend to
reproduce between four- and six fold, during fermentation. Throughout this step, yeasts
uptake amino acids and sugars from the wort, which metabolize for reproduction and
production of energy and two main products, ethanol and CO2 (Figure 4). Moreover, several
222 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

other biochemical reactions are mediated by yeasts, which lead to the release of hundreds of
chemical compounds that not only can contribute to beer flavor, but also to other beer
desirable characteristics. The different metabolic pathways associated to yeasts fermentation
will be further explained in detail.

Figure 4. Summary of the main biochemical reactions mediated by yeasts in the brewing fermentation
(adapted from (Lewis and Young, 1995)).

The subsequent step is the beer maturation, where green beer is transformed into
its finished form. It is enabled the adjustment of the beer carbonation, once the remaining
yeasts perform a secondary fermentation; moreover, some off-flavors components, like
vicinal diketones (VDK’s) diacetyl (2,3-butanedione) and 2,3-pentanedione, are converted
enzymatically by yeasts, reducing their content until it is below the flavor threshold.
Furthermore, after yeasts had converted all the available sugars and other nutrients, there is a
reduction of the convention currents (promoted by CO2 formation), the metabolism rate starts
to decrease, and yeast cells tend to adhere and form flocs – flocculation process. This inherent
characteristic of yeasts is extremely important once if it happens too late, it will interfere in
the filtration; but if it occurs too earlier, beer will have high sugars concentration, low ethanol
concentration, and presence of off-flavors (Verstrepen et al., 2003; Lodolo et al., 2008). After
flocculation process is completed, yeasts are removed from the fermenter (usually as
concentrated slurry), being transferred to a collection vessel, in which they are maintained at
4ºC, until be reused again (repitching).
Clarification is performed by the beer filtration, in order to bright it, once consumers tend
to consider bright beer as a quality parameter. There is the removal of particles down to about
0.5 μm. Nowadays, kieselguhr (light soil consisting of siliceous diatom remains) is the most
common filter. Clarification can be combined with stabilization, where beer is allowed to
stabilize at -1 – 2ºC (during 1 – 3 days), in order to promote the precipitation of proteins and
polypeptide-polyphenols complexes, thus inhibiting the haze formation and colloidal
instability. There are some stabilizing agents that can be added: to adsorb polyphenols (e.g.,
 Saccharomyces spp. Role in Brewing Process … 223

polyvinylpolypyrrolidone – PVPP) or proteins (e.g., silica gel); to degrade proteins and

polypeptides (proteases, for instance papain); or to precipitate proteins (with tannic acid).
The last step is filling where the beer can be packed in different containers: bottles (glass
or plastic), cans, kegs, casks, or bulk tanks. Some important factors should be considered for a
suitable package, especially the barrier properties in order to prevent the CO2 loss and
entrance of oxygen that could induce beer staling (Vanderhaegen et al., 2006). The biological
stabilization can be done at low temperatures by filtration, or high temperatures through
pasteurization (flash or tunnel).

2. SACCHAROMYCES SPP. ROLE IN THE BREWING PROCESS

This second topic is divided in four sections, the first one will comprise the
characterization of the two main yeasts types used in the brewing process (lager and ale), as
well as aspects related to their taxonomy; then the yeasts metabolic pathways associated to
the volatile compounds formation will be presented in the second sub-section; the third part
will describe the different stress factors that yeasts suffer during brewing process and their
biological response, and also the description of the different parameters (viability and vitality)
that can be used to monitor yeasts quality; and the fourth part will comprise the impact of
serial repitching on yeasts.

2.1. Saccharomyces spp. Used in the Brewing Process and Their Taxonomy

Yeasts that are used in the brewing process are mostly members of the genus
Saccharomyces, being their taxonomy classification described in Figure 5. However, some
difficulties have emerged for determination of the current taxonomy of Saccharomyces
species, once initially brewers classified yeasts according to their brewing properties (e.g.,
flocculation process). Currently, the development of molecular tools allows a more accurate
determination, and new classifications have occurred. Thus, in this sub-section, it is intended
to illustrate the progress of the yeasts taxonomy over the years, as well as to describe the
differences between the two main types of yeasts used in the brewing process: ale and lager.
Saccharomyces cerevisiae was firstly named as top (ale) fermenting brewing yeast in
1883, when Emil Hansen from Carlsberg brewery was able to characterize Saccharomyces
pure cultures, describing its unique and reproducible industrial fermentations skills.
In 1908, Hansen called the bottom (lager) brewing yeast as Saccharomyces
carlsbergensis (Barnett, 1992; Lodolo et al., 2008; Saerens et al., 2010), which sometimes is
used as a synonym for S. pastorianus. Martini and Martini, 1987 reported the existence of
three different species of Saccharomyces based on the DNA sequences: S. cerevisiae and S.
bayanus only had 22% of their genomes in common; S. pastorianus shared 52 and 72% of the
genome with S. cerevisiae and S. bayanus, respectively, which led to the possibility of S.
pastorianus to be a natural hybrid of S. cerevisiae and S. bayanus. Rainieri et al., 2006,
through polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP)
analysis confirmed that S. pastorianus yeasts contain components of S. cerevisiae-like
genome and non-S. cerevisiae portions of genome derived from S. bayanus and/or S. uvarum.
224 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

Only Libkind et al., 2011 finally isolated (association with Nothofagus trees in Patagonia) and
identified S. eubayanus as the non-S. cerevisiae parent of S. pastorianus. The genome of S.
eubayanus was almost completed sequenced by Baker et al., 2015. These authors verified that
these yeasts have experienced increased rates of evolution since their hybridization with S.
cerevisiae yeasts (formation of domesticated alloploid hybrids), and also some alterations on
the expression of certain genes associated to metabolism.

Genus Saccharomyces

Family Saccharomycetaceae

Order Saccharomycetales

Class Saccharomycetes

Sub-phylum Saccharomycotina

Phylum Ascomycota

Kingdom Fungi
Domain Eukarya

Figure 5. Taxonomic classification of Saccharomyces species.

The most recent definition of the Saccharomyces sensu stricto group includes seven
different species: S. cerevisiae, S. paradoxus (encompassing S. cariocanus), S. mikatae, S.
kudriavzevii, S. arboricolus, S. eubayanus, and S. uvarum (Borneman and Pretorius, 2015). S.
cerevisiae is used to produce wine, bread, ale beer and sake; S. eubayanus that can produce
wine and cider; while S. pastorianus is involved in lager beer fermentation (Rainieri et al.,
2003).
Two distinct lineages can be associated to lager yeasts:

I. Group I: Saaz. They are taxonomically classified as S. carlsbergensis, they have

slightly greater capacity to grow at low temperatures and relatively poor fermentation
performance (lower maltose and maltotriose utilization, comparing with Frohberg
strains) (Wendland, 2014). They have retained proportionally more DNA derived
from the S. eubayanus parent (Dunn and Sherlock, 2008).
II. Group II: Frohberg. They are taxonomically classified as S. pastorianus. Their
fermentation performance is slightly better than group I, thus they are more
predominant in modern industrial-scale brewing (Gibson et al., 2013). They have
retained proportionally more DNA from the S. cerevisiae parent (Dunn and Sherlock,
2008).
 Saccharomyces spp. Role in Brewing Process … 225

Nevertheless, both lineages are alloploid hybrids from S. cerevisiae  S. eubayanus,

which produce different esters concentrations, thus contributing with different impact on beer
aroma (Dunn and Sherlock, 2008; Gibson et al., 2013; Wendland, 2014). For instance, these
different features can be achieved through yeasts domestication. Hybridization studies have
been vastly explored in order to produce yeasts with different fermentation performances,
being able to be used in high-gravity brewing and serial repitching (Steensels et al., 2014a,
2014b; Baker et al., 2015; Krogerus et al., 2015).
Brewing yeasts are divided in two types: ale and lager. According to their flocculation
pattern, ale yeasts (Figure 6A) are classified as top-fermenting, while lager yeast (Figure 6B)
are bottom-fermenting. Ale yeasts show flotation, trapping the CO2 bubbles, and going to the
top of the vessel; while lager yeasts clump together, sedimenting on the vessels’ bottom
(Speers et al., 1992; Verstrepen et al., 2003). This purely natural property of yeast cells to
flocculate allows the brewing industry to have an cost-effective, environment-friendly, and
simple method to take apart yeast cells, at the end of fermentation, from green beer
(Verstrepen et al., 2003). Nevertheless, in modern brewery industries, the vessels type
(cylindroconical) and volume allow that ale yeasts also sediment on the bottom at the end of
fermentation (Soares, 2010; Vidgren and Londesborough, 2011). There are two possible
reasons: one is related to greater hydrostatic pressure in vessels that limits the CO2 bubbles
size and also promote more turbulence, which leads the liberation of CO2 bubbles from flocs;
other is related with a possible selection of ale mutants in order to have a more suitable
cropping of yeasts (Vidgren and Londesborough, 2011).

A B

Yeasts

Yeasts

Figure 6. Flocculation pattern of ale (A) and lager (B) yeasts. Ale yeasts were initially top-fermenting
but in modern breweries they are bottom-fermenting such as lager yeasts.

Flocculation occurs in the presence of calcium ions that ensure that the lectin-like
proteins (flocculins that are only present in the flocculent strains) have the correct
conformation for the connection with the α-mannan residues (associated with mannoproteins)
of the cell wall of the neighbor cell. The flocculins synthesis seems to be associated with the
presence of FLO genes, being the yeasts’ flocculation dependent of several factors, namely
the presence of cations (e.g., calcium), pH (the optimum value is between 3.0 – 5.0),
temperature, oxygen, sugars (competition with the flocculation receptors, so their promote the
226 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

reversible dispersion of flocs), ethanol, chronological age (older cells tend to be more
flocculent), cell density (it is required a minimum threshold), and mechanical agitation
(promotes the physical proximity between yeasts cells) (Soares, 2010).
Besides the difference at the flocculation behavior, ale yeasts (e.g., S. cerevisiae) are
genetically more diverse and require high temperatures to ferment (between 16 – 25ºC), while
lager yeasts (e.g., S. pastorianus) are more conserved and need low fermentation temperatures
(between 7 – 14ºC) (Saerens et al., 2010).
The phenotypic characteristic of the two yeast types can be distinguished based on
their colony morphology due to the formation of light green colonies, being ale yeasts
characterized by an undulating appearance, while lager yeasts have smooth surface (Powell
and Diacetis, 2007), when grown in a specific growth media – Wallerstein Laboratory
Nutrient Broth, WLN. The budding process is different between the yeast types, being
possible to be observed under a microscope: ale yeasts tend to be stuck together, even when a
new bud occurs; while lager yeasts tend to separate after a budding process (Eßlinger, 2009).
Yeasts can also be differentiated by fermentation characteristics, namely melibiose
(disaccharide that can be present in wort) is only metabolized by lager yeasts, once ale yeasts
do not have the enzyme α-galactosidase (melibiase) (Lodolo et al., 2008; Eßlinger, 2009).
Moreover, the yeast strain has a huge impact on the final product, as Tosi et al., 2009 showed
through the comparison of fermentation performance of two strains (S. cerevisiae and S.
uvarum). They observed different patterns according to the strain used, namely the content of
compounds with direct impact on aroma, such as ethanol, ethyl acetate, and 2-phenylethanol.
Ale beers tend to be more fruity and estery, whereas lager beers do not have such a complex
aroma, which is partly sulfurous (Eßlinger, 2009).
Besides the common phenotypical approach, other attempts have been used to
distinguish the yeast strain, namely: spectroscopic methodology (pyrolysis mass
spectrometry and Fourier transform infrared spectroscopy) developed by Timmins et al.,
1998; genetic approach through PCR-RFLP (homologous genes) or amplified fragment
length polymorphism (AFLP, genetic fingerprinting) used by Pope et al., 2007; metabolic
footprinting through direct injection mass spectrometry and gas chromatography time-of-
flight mass spectrometry applied by Pope et al., 2007, complemented with multivariate
analysis (PCA – Principal Components Analysis and CVA – Canonical Variants Analysis).
American Society of Brewing Chemists recommends the use of PCR of inter-delta regions of
chromosomal DNA, for genetic fingerprinting, which allows strains identification and
mutants’ detection (Boulton, 2013).

2.2. Saccharomyces spp. Metabolism

Yeasts metabolism is conditioned not only by the raw materials that are used, but also by
their initial concentration and the initial oxygen content. This section intends to explain the
importance of these parameters that can influence the yeasts metabolism. Moreover, a special
attention will be given to the metabolic pathways involved in the formation of volatile
compounds that will have further impact on beer flavor.
Saccharomyces spp. are added to oxygenated wort during its fermenter transfer, at a
certain concentration (pitching rate). Yeasts pitching rate corresponds to the amount of viable
cells per wort volume, being usual to use 10 millions of viable cells per milliliter in a 10 ºP
 Saccharomyces spp. Role in Brewing Process … 227

wort. This parameter should be monitored and revised according to the wort gravity and the
amount of viable cells (> 95%).
At the beginning, the presence of oxygen promotes the yeasts growth (cell size and
number). It is important to satisfy the yeasts requirements during fermentation through the
presence of enough dissolved oxygen in the pitched wort, in order to allow their adaptation
from aerobiosis to anaerobiosis. Moreover, oxygen is also important for yeasts survival
along repitching, once they suffer some starvation after the end of previous fermentation and
storage steps (Boulton, 2013). Indeed, new yeast cell biosynthesis needs the production of
lipids (main cell membrane component), that requires the presence of oxygen. Oxygen
promotes the formation of unsaturated molecules (like unsaturated fatty acids – UFA) and
sterols, both from acetyl coenzyme A (CoA) molecule (formed through carbohydrate
metabolism or from wort fatty acids) (Verbelen et al., 2009c).

Table 1. Major yeast volatile metabolites present in beer, their flavor threshold and
concentration in beer, as well as their aroma descriptor (Kobayashi et al., 2008; Russell
et al., 2009; Boulton, 2013)

Flavor threshold Typical concentrations

Metabolites Aroma descriptor
(mg/L) in beers (mg/L)
Aldehydes
Acetaldehyde 25 2 – 20 Sweat, pungent
Higher alcohols
Ethanol 14000 25000 – 50000 Alcohol
n-Propanol 600 5 – 50 Alcohol
Iso-butanol 100 5 – 100 Alcohol
Alcohol, banana, sweet,
2-Methylbutanol 50 – 70 10 – 130
aromatic
Alcohol, banana, medicinal,
3-Methylbutanol 50 – 65 25 – 180
aromatic
Phenyl ethanol 40 5 – 102 Roses, sweet, fragrant
Esters
Ethyl acetate 25 – 30 5 – 35 Fruity, solvent-like
Ethyl hexanoate 0.20 0.05 – 0.20 Apple
Ethyl octanoate 0.90 0.04 – 0.50 Aniseed
Isoamyl acetate 1 0.30 – 4.00 Pear drops, banana
Isobutyl acetate 0.40 – 1.60 0.10 – 0.30 Fruity
Phenyl ethyl acetate 3.50 0.10 – 0.80 Rose or floral
Vicinal diketones
2,3-Butanedione 0.070 – 0.15 0.008 – 0.600 Butterscotch or toffee
2,3-Pentanedione 0.9 0.008 – 0.600 Honey
Sulfur compounds
Hydrogen sulfide 0.004 – 0.005 0.001 – 0.002 Rotten eggs
Dimethyl sulfide 0.050 0.010 – 0.150 Asparagus, corn, molasses
3-Methylthio-1- Cauliflower, cabbage,
1.2 0.002 – 0.050
propanol potatoes
228 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

At anaerobic stage, yeasts metabolize the fermentable carbohydrates present in the wort.
Through the nutrients available (mainly carbohydrates and nitrogenous compounds), yeasts
convert them principally into ethanol and CO2; they also produce energy (adenosine
triphosphate – ATP), and use it to generate new cell components or new biomass, as well as
the synthesis and secretion of hundreds of metabolic products into wort. If these metabolites
are volatile, they can give characteristic flavor and aromas to beer (Figure 4) (Lewis and
Young, 1995), being the major yeasts volatile metabolites presented in Table 1.

2.2.1. Metabolic Pathways

Carbohydrates Metabolism
Maltose is the main sugar present in wort, around 50 – 55% of the total amount of
carbohydrates, and its uptake depends first by its transport into yeast cell by maltose
permease; then occurs its hydrolysis, into two molecules of glucose, by maltase (-
glucosidase) (Figure 7). Nevertheless, yeasts prefer to uptake firstly monosaccharides, such as
glucose and fructose, followed by the increase of the carbohydrates complexity, namely
disaccharides (maltose), and then trisaccharides (maltotriose) (Lodolo et al., 2008). Dextrins
are not metabolized by yeasts, so they will remain in beer, contributing for its body and
mouth feel.
Brewing yeasts have ability to grow either at aerobic and anaerobic conditions, being
capable of a fully oxidative respiratory growth. Nevertheless, relatively high amount of
fermentable carbohydrates can switch off the genes responsible for this respiratory oxidative
phosphorylation, leading to the carbohydrates catabolism via glycolysis. This phenomena is
called catabolite repression or Crabtree effect (Boulton, 2013). The high carbohydrates
concentration present in wort promote the metabolization of glucose residues by yeasts
(Figure 7), through the glycolytic pathway to produce pyruvate that, in anaerobic conditions,
is converted into ethanol and CO2, via acetaldehyde. This produced ethanol allows yeasts
to have an advantage in natural environments due to its inhibitory effects on many
microorganisms. The CO2 is usually recovered from the fermenters for beer carbonation
(Lewis and Young, 1995).

Aldehydes Formation
Yeasts can remove carbonyl compounds from wort (e.g., 2-methylbutanal, 3-
methylbutanal, and 3-methylpropionaldehyde), that are not desired in beer due to their
“worty” aroma. They can also lead to the production of other carbonyl compounds that are
intermediates in higher alcohols formation.
Acetaldehyde is formed from pyruvate (by pyruvate decarboxylase action, Figure 7),
which content can be accumulated in mid- to late fermentation, which leads to its increase
until concentrations above the flavor threshold (25 mg/L) (Table 1). Nevertheless,
acetaldehyde concentration can decrease during cool conditioning by yeasts. Some factors
contribute for high levels of acetaldehyde in beer, namely elevated: fermentation
temperatures, wort oxygenation or pitching rates. Acetaldehyde accumulation in beer is not
desired once it contributes with green apple and grassy flavors to beer (Boulton, 2013).
 Saccharomyces spp. Role in Brewing Process … 229

Fructose
Glucose
CO2

transporter
Invertase
Sucrose Glucose + Fructose

Hexose
Ethanol

Maltose
Fructose
Glucose

Glycolysis Ethanol
CO2
Maltose

Pyruvate Acetaldehyde
Maltotriose

Maltose Acetate

Maltotriose Pyruvate

TCA Acetyl-CoA Acetyl-CoA

Figure 7. Carbohydrates metabolism of yeasts, via aerobic and anaerobic conditions. Maltose,
maltotriose, sucrose, fructose and glucose are the main carbohydrates present in wort.

Nitrogen Compounds Metabolism

Besides the metabolism of wort carbohydrates, also nitrogen compounds (main sources in
wort: amino acids, ammonium ion, and some di- and tripeptides) are metabolized by yeasts.
Oligosaccharides and proteins are not metabolized by yeasts, so they will remain in beer, and
can contribute for haze and head formation (Boulton, 2013). Wort-free amino nitrogen (FAN)
level and composition have a great impact on the content of several yeasts metabolites,
namely: higher alcohols, esters, vicinal diketones, and hydrogen sulfide, most of them with
impact on beer flavor. The uptake of nitrogen compounds is dependent of the yeast strain,
being performed by general (GAP) or specific amino acids permeases.
Like fermentable carbohydrates, yeasts tend to metabolize amino acids according with
the following order groups: group A – arginine, asparagine, aspartate, glutamate, glutamine,
lysine, serine and threonine; group B – histidine, isoleucine, leucine, methionine and valine;
group C – alanine, glycine, phenylalanine, tyrosine, tryptophan, and ammonia (only absorbed
after total consumption of amino acids from group A); and group D – proline (Lodolo et al.,
2008). Moreover, Procopio et al., 2013 evidenced that the amino acid uptake by yeasts is
correlated with beer flavor profile, where leucine, isoleucine, valine, glutamine, cysteine, and
proline were the amino acids that had more impact on the synthesis of aroma-active
compounds in fermentations performed by S. pastorianus.

Higher Alcohols Formation

The amino acids from wort can be incorporated directly to protein production, or they can
be metabolized leading to formation of higher/fusel alcohols. Higher alcohols formation can
230 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

occur through the amino acids catabolism, “Ehrlich pathway,” or from the amino acid
synthesis “Genevois pathway” (where α-keto acids are formed in carbohydrates metabolism)
(Eßlinger, 2009). Basically, in the Ehrlich pathway (Figure 8), the amino group from the
amino acid may be transferred by an enzyme called transaminase (transamination) leading to
the formation of an oxo-acid (α-keto acid) product, that then suffers a decarboxylation by
decarboxylase enzyme, producing CO2 and an aldehyde. This aldehyde is then reduced
(action of alcohol dehydrogenases) to a higher/fusel alcohol (Lewis and Young, 1995; Pronk
et al., 1996; Pires et al., 2014). Around 90% of the higher alcohols have been synthetized at
the end of the primary fermentation (Eßlinger, 2009).

Transamination Reduction
Decarboxilation

CO2
Amino acid α-Keto acid Fusel aldehyde Higher alcohol

Figure 8. Ehrlich pathway for higher alcohols formation by yeasts.

Higher aliphatic and aromatic alcohols are important flavor compounds once they are the
most abundant volatile components present in beer. Nevertheless, their specific impact on
beer aroma is not always noticeable, once these compounds are present, in some cases, at
concentrations lower than their flavor threshold (Table 1).
Higher fermentation temperatures lead to higher fermentation rates with increased
concentration of higher alcohols, as observed by Landaud et al., 2001. Moreover, higher
pitching rates and oxygen levels in wort, as well as high level of FAN in wort, contribute for
higher alcohols concentration in beer; but the main key factor is the yeast strain (ale strains
tend to produce higher alcohols concentration than lager yeasts) (Boulton, 2013).

Vicinal Diketones Formation

Vicinal diketones (VDK’s) production (Figure 9) is directly correlated with amino acid
metabolism, once their precursors, -acetolactate and -acetohydroxybutyrate, are
intermediates of the biosynthesis of valine and isoleucine, respectively (Lewis and Young,
1995; Lodolo et al., 2008). These intermediates (-acetolactate and -acetohydroxybutyrate)
are released outside the cell, where they suffer a spontaneous oxidative decarboxylation,
leading to the formation of diacetyl and 2,3-pentanedione, respectively. Yeasts can uptake
again these compounds, reducing them into 2,3-butanediol and 2,3-pentanediol, which
threshold is higher, not contributing for beer flavor (Eßlinger, 2009).
VDK’s have low flavor thresholds, namely 0.07 – 0.15 mg/L for diacetyl and 0.9 mg/L
for 2,3-pentanedione (Table 1). Due to their intense butterscotch or toffee aromas, these
compounds are considered defect in pilsner-type lager beer; while in some ales and stouts,
they can be desirable compounds if present in concentrations above their threshold.
The amino acids concentration in wort is the main key factor for diacetyl formation,
namely valine, once it inhibits the α-acetolactate formation (Boulton, 2013). Krogerus and
Gibson, 2013 showed that modifications in the amino acid profile of wort (namely valine
and other branched-chain amino acids) can be a successful way to reduce the diacetyl
concentration along fermentation.
 Saccharomyces spp. Role in Brewing Process … 231

Fructose Acetoin
Glucose
2,3-Butanediol

transporter
Invertase
Sucrose Glucose + Fructose

Hexose
Maltose Diacetyl
Fructose
Glucose CO2

Spontaneous
2,3-Butanediol
Glycolysis

Maltose Acetoin Diacetyl

Pyruvate α-Acetolactate α -Acetolactate
Maltotriose

Maltose Isoleucine Valine

Maltotriose
Threonine α-Acetohydroxybutyrate
2,3-Pentanediol 2,3-Pentanediol

2,3-Pentanedione

Threonine

Spontaneous
α-Acetohydroxybutyrate 2,3-Pentanedione

CO2

Figure 9. Metabolic pathway of vicinal diketones (diacetyl and 2,3-pentanedione) formation by yeasts.
Maltose, maltotriose, sucrose, fructose and glucose are the main carbohydrates present in wort.

Esters Formation
Esters are the most important aroma compounds produced by yeasts, once they have low
thresholds (µg to mg/L, Table 1). Nevertheless, their concentration is lower comparing with
other yeast metabolites. The formation of an ester molecule involves the reaction between an
acetyl CoA molecule (derived from pyruvate or from direct reaction between CoA and
acetate, through acetyl-CoA synthase activity) with an alcohol molecule. These reactions
occur during the vigorous stage of the primary fermentation (Boulton, 2013; Pires et al.,
2014).
Beer esters can be divided in two groups: acetate esters (e.g., ethyl acetate, isoamyl
acetate, and phenyl ethyl acetate), where there is a reaction between an acyl-CoA and ethanol
or higher alcohol; and ethyl esters (e.g., ethyl hexanoate, ethyl octanoate and ethyl
decanoate), where first occurs a reaction between CoA and medium-chain-length fatty acid,
and then with an alcohol (by action of an alcohol acyl transferase). The main key factor that
promotes higher esters concentration is the increase of temperature.
Ester compounds contribute with floral and fruity flavors in beer (Table 1), being already
identified around 100 esters in beer (Lodolo et al., 2008; Pires et al., 2014). However, higher
ester concentrations can lead to bitter taste instead of fruity taste, contributing with negative
flavors to beer, so brewers need to have special attention to the optimum conditions for a
proper balanced ester profile in beer (Pires et al., 2014). The most frequent esters present in
beer are described in Table 1 (Lewis and Young, 1995).
232 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

Short-Chain Fatty and Organic Acids Formation

The short-chain fatty acids (e.g., butanoic, hexanoic, and octanoic acids), formed during
lipid metabolism, can be released in the beer, contributing for yeasty flavors in beer and
inhibiting the formation of beer foam; while long-chain fatty acids (e.g., palmitic, linoleic,
stearic, and oleic) are uptake from wort by yeasts (Briggs et al., 2004; Lodolo et al., 2008;
Eßlinger, 2009).
Organic acids (e.g., pyruvate, citrate, malate, acetate and succinate) can also be produced
from pyruvate or from the repressed tricarboxylic acid cycle. They are responsible for the pH
decrease during fermentation and also sour flavors (Lodolo et al., 2008).

Sulfur Compounds Formation

Yeasts can also produce sulfur compounds, being hydrogen sulfide (H2S) and sulfur
dioxide (SO2) the main ones. Volatile sulfur compounds are usually not desired in beers, due
to their strong vegetable aromas. The formation of these compounds is related with the
metabolism of sulfur-containing amino acids (e.g., cysteine and methionine) and sulfate ions
from wort. Moreover, some dimethylsulfide (DMS) can be produced by yeasts that have
dimethyl sulphoxide (DMSO) reductase activity, contributing with vegetables or celery
aromas to beer (Eßlinger, 2009; Boulton, 2013).
During primary fermentation, firstly exogenous sulfur-containing amino acids are
used for sulfur compounds formation, while sulphate uptake is depressed and the S-
adenosylmethionine pool is small. S-adenosylmethionine is an important intracellular
metabolite that monitors the formation of S-containing metabolites: when its concentration is
low, occurs the uptake of sulphate (by specific permeases), which is reduced by a specific
NAD+ linked reductase, into sulphite and sulphide (used in the biosynthesis of S-containing
metabolites); the excess of S-adenosylmethionine leads to inhibition of genes expression
related with sulphate uptake and its utilization. Then, the formed SO2 is used for the amino
acids synthesis. The decline of the metabolic rate in mid- to late fermentation promotes the
excretion of SO2 into extracellular medium; an accumulation of S-adenosylmethionine pool
can occur, and consequently of sulphite; and then there is a stagnation of sulfur compounds
production. Furthermore, sulphite can bind to aldehydes and form adducts, being important
during beer staling due to its possible reaction with staling precursors, namely (E)-2-nonenal.
Thus, aldehydes may persist in beer, once they are not available for enzymatic reactions
(Boulton, 2013).
Duan et al., 2004 reported that there were increased levels of sulfur compounds when
wort had higher concentration of cysteine. Despite their biosynthesis pathway is closed
linked, Duan et al., 2004 verified that the environmental conditions can constrain differently
their rate of formation.

Terpenic Compounds Formation

Terpenic compounds mainly derive from the raw materials, particularly the hops.
Nevertheless, yeasts seem to have ability to produce these compounds that can contribute
with pleasant flavors to beer.
 Saccharomyces spp. Role in Brewing Process … 233

Figure 10. Terpenic compounds formation through mevalonate pathway, based on KEGG (Kanehisa et
al., 2016).

Terpenic compounds can be de novo synthesized through mevalonate pathway, in yeasts

cytosol (Figure 10)(Carrau et al., 2005; Khor and Uzir, 2011; Rodriguez et al., 2014).
Additionally, they can suffer bioconversion by yeasts (King and Dickinson, 2003; Takoi et
al., 2010; Gamero et al., 2011), in fact King and Dickinson, 2000 showed the S. cerevisiae
ability of geraniol and nerol biotransformation into linalool and α-terpineol.
Metabolic engineering of Saccharomyces spp. have also been performed so that yeasts
can produce higher concentrations of terpenic compounds (Hu and Lu, 2015). Basically,
genes of certain enzymes, namely geraniol synthase (Pardo et al., 2015), linalool synthase
(Amiri et al., 2016), or farnesyl diphosphate synthase (Erg20) (Fischer et al., 2011; Ignea et
al., 2015), are cloned from a vegetable source and introduced in a plasmid from Escherichia
coli, which is expressed in a Saccharomyces spp. This approach was successful in the
application of wine production (Gamero et al., 2011; Pardo et al., 2015).

2.2.2. Yeast Stress Factors in Brewing Process

Yeast cells are exposed to severe environmental changes along the brewing process,
namely during yeast propagation, fermentation, and yeast storage. The different stress factors
that yeasts can suffer along the brewing process are presented in Table 2, as well as the
cellular effects and respective yeast response.
At the first period of fermentation, temperature shock, hyper osmotic solution and
oxidative stress are experienced by yeasts, in aerobic conditions. Then, there is an
anaerobiosis period, in which hydrostatic pressure inside fermenters increases, as well as the
amount of acetaldehyde and ethanol. Internal acidification and starvation are the main
parameters that the yeasts are exposed (Bleoanca and Bahrim, 2013).
 Table 2. Yeast stress factors along the brewing process (Lodolo et al., 2008; Bleoanca and Bahrim, 2013)
Stress Cellular effect
 Proteins, lipids and DNA are the cellular
components more affected;
 Lipid peroxidation – decreased membrane
fluidity, inactivation of membrane receptors and
enzymes;
 Protein oxidative damage – formation of
Oxidative
hydrogen peroxide, protein aggregation or
fragmentation, changes in electrical charges;
 DNA – mitochondrial DNA is more prone; if
the damage is not repaired, can occur punctual
mutations, intrachromosomial recombination, or
crossing- over, deletions or insertions.
 Hydrogen bonds and hydrophobic interaction
are broken;
 Denaturation of proteins and nucleic acids;
 Under high temperatures – death, atypical
budding, appearance of respiratory-deficient,
Thermal
lower pH, increased fluidity of plasma membrane;
 Under lower temperatures – shrinkage of cells,
destruction of vacuolar membranes, membrane
integrity is compromised because of a transitory
gel-like phase (fatty acids/sterols).
 Glycogen consumption and variation of the
trehalose content;
Mechanical
 Reduction of viability and vitality;
(physical or
shear stress)  pH increase in slurry;
 Leakage of intracellular proteases.
 Contact with hypertonic medium leads to
plasmolisis;
 Adaptation - restructuring of the actinic
cytoskeleton, temporary arrest of life cycle and
Osmotic
metabolisms’ reprogramming; also increase of the
amount of glycerol, elimination of toxic ions;
 Contact with hypoosmotic medium leads to
turgescence.
Yeast response
 Non- enzymatic antioxidant
mechanisms: ROS binding by
glutathione, polyamines,
erithroascorbic acid,
metallothioneines,
flavohemoglobines;
 Enzymatic antioxidant
mechanisms: catalase, superoxide
dismutase, glutathione- peroxidase,
glutathione-reductase,
thioredoxinperoxidase, and
peroxiredoxins.
 Increase synthesis of the Heat
Shock Proteins (HSP);
 Accumulation of protecting
compounds (trehalose and glycerol)
or enzymes- catalase, mithocondrial
superoxide- dismutase;
 Presence of poliamines
(spermine and spermidine) that
improve the membrane integrity.
 Release of cell wall enzymes
(invertase and melibiase) and cell
wall polysaccharides (mannan and
glucan) in the slurry supernatant;
 Haze generation in beer and
impaired flocculation performance.
 Induction of Stress Responsive
Elements (STRE).
Brewing steps affected and main effects
 Yeast propagation (increased catalase
activity and elevated concentrations of
glycogen and trehalose);
 Fermentation (activation of a oxidative
stress regulator responsible for the
activation of several genes to respond to
several stress conditions);
 Yeast storage (different consumption
of trehalose according to the storage
temperature).
 Yeast propagation (higher temperatures
comparing with the rest of the process);
 Yeast storage (low temperatures used;
and depending on the period of time,
different storage temperatures should be
applied).
 Whenever yeasts are moved within
brewery: natural process (convection
current within the fermentation vessel) or
artificial process (e.g., pumping,
centrifugation).
 Fermentation (mainly hyperosmotic
stress);
 The new brewing trend of Very High
Gravity Brewing promotes this type of
stress on yeasts.
Stress Cellular effect
 Similar effects as thermal and oxidative stress;
 Cellular membranes could be an effect of the
lipids’ high sensitivity to high pressure, once
affects the membrane fluidity;
High pressure
 Vacuoles suffer acidification due to proton
dissociations;
 DNA conformation changes (from double-
helix into zig-zag-ed structure).
 Increase of membrane fluidity and
permeability;
 Deficient transport system of essential
compounds (amino acids and glucose);
 Growth, cellular metabolic rate, and viability
Ethanol reduction;
 Repression of cellular pathways (e.g., protein
biosynthesis, cell growth, RNA metabolism);
 Activation of cellular pathways, such as cell
integrity pathway and ergosterol synthesis.
 Starvation for “natural” nutrients (carbon,
phosphate, nitrogen, or sulfate) – low death rates;
 Starvation of amino acids or other metabolites
in auxotrophic mutants – rapid loss of viability;
Nutritional  Inhibition of ribosomal RNA synthesis;
 Variations on trehalose and glycogen
concentration;
 Autophagy – membrane transport catabolic
phenomenon.
Yeast response
 Homeoviscous adaptation –
increase of the membrane fatty acid
unsaturation (e.g., cholesterol);
 Accumulation of trehalose.
 Appearance of vacuolar
acidification;
 Reduction of saturated fatty
acids (e.g., palmitic acid), and
increase of unsaturated fatty acids
(e.g., oleic acid);
 Biosynthesis of squalen and
ergosterol;
 High concentration of trehalose;
 Increased of the activity of
superoxidismutase.
 Interruption of fermentation if
assimilable wort nitrogen is below
150 mg/L;
 Zinc depletion leads to
“sluggish” fermentations;
 Impaired flocculation capacities
(changes on cellular surface and
malfunction of genes responsible
for intracellular adhesion).
Brewing steps affected and main effects
 Fermentation – mainly gaseous
pressure due to the formation of CO2 by
yeasts.
 Fermentation and storage –
accumulation and persistence of ethanol
leads to danger on the yeast cell;
 The new brewing trend of Very High
Gravity Brewing promotes this type of
stress on yeasts due to higher ethanol
contents produced;
 Yeast storage – combination with
higher temperatures, decreases the yeast
viability.
 Yeast storage – if yeasts are stored for
longer periods, there is an induction of
delayed flocculation which leads to
problems in yeast separation, and beer
quality is compromised.
236 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

If yeast cells are exposed to toxic levels of intracellular reactive oxygen species (ROS),
disturbance in yeasts performance may occur, and therefore, lead to a cellular damage
response, process called oxidative stress. ROS are produced by mitochondria in the normal
aerobic metabolism of yeasts. Gibson et al., 2008 showed that aging may cause damage in
mitochondrial DNA, thus more tendency for yeast cells to become respiratory-deficient
yeasts. Indeed, respiratory-deficient yeasts are the most frequent mutations along the brewing
process, where they have a loss of the respiratory function possible due to mitochondrial
DNA damage caused by oxidative stress (Gibson et al., 2008), especially if the yeasts are
serial repitched.
Measurement of glycogen, trehalose and sterol levels may also be used to predict if
yeasts are under stress and if they are properly stored (Winkler et al., 1991; Jenkins et al.,
2003b; Cheong et al., 2007). This information allows performing an adequate pitching of
yeast when they are reused, letting to have increased fermentation performance. Glycogen is
an intracellular carbohydrate that reflects the nutritional status of the cell, and allows to
indicate if there is an efficient storage and yeast handling. Glycogen is required for sterol
synthesis (Jenkins et al., 2003b), while trehalose is produced to confer stability to plasma
membrane; both compounds are synthesized when cells are under stress. Trehalose is also
used during starvation periods, being regulated by stress-responsive elements (STRE)
(Jenkins et al., 2003b). The 2,3,5-triphenyltetrazolium chloride (TTC) overlay technique is
most used methodology to verify the existence of respiratory-deficient yeasts (Jenkins et al.,
2003b; Gibson et al., 2008).

2.3. Parameters of Yeasts Quality Control

The physiological and healthy state of yeasts influences their performance, being
extremely important to manage their quality, not only along the brewing process, but also in
serial repitching. In fact, the improvement of quality assurance procedures allows the
production of more consistent beer products, namely their quality, flavor, alcohol content, etc.
Yeasts quality can be evaluated through their viability and vitality, being in this sub-section
presented the different methodologies used and the reference values established for some
methodologies are presented in Table 4.
Viability determines the number of living cells, providing the cells ability to grow and
reproduce when they are pitched into wort (Briggs et al., 2004). The pitching rate
conventionally used in brewing fermentations is around of 5 - 20×106 viable cells/mL (Erten
et al., 2007; Verbelen et al., 2009a). A healthy culture is considered when it has  95% of
viable cells (Table 4), and it is inadvisable to use cultures, especially for repitching, with
viabilities lower than 85% (Lewis and Young, 1995).
One possible approach to measure yeasts viability is the cells staining with a vital dye
(e.g., methylene blue, methylene violet, or 1-anilino-8-naphthalenesulphonic acid (MgANS)).
Methylene blue is the most used stain test in brewing industry, in which the dead cells are
stained in blue once they cannot exclude the dye, while viable cells uptake the dye, they will
reduce it through cellular dehydrogenase activities, turning it colorless. The yeasts
measurement is performed in a haemocytometer counting chamber, using a simple light
microscopy. Nevertheless, it is time consuming and has user-dependent variations. Other
main problem of this procedure is correlated with the reliability for viabilities below 90%
 Saccharomyces spp. Role in Brewing Process … 237

(Briggs et al., 2004). This method is recognized as international method by the Institute of
Brewing, European Brewing Convention, and the American Society of Brewing Chemists.
Another possible staining test is with methylene violet, which action is similar with
methylene blue (Smart et al., 1999; Maskell et al., 2003). The fluorescent dye MgANS test
consists in the penetration of the dye into the nonviable cells and binds to the cytoplasmatic
proteins to produce yellow/green fluorescence. The necessity of having a fluorescence
microscopy led to exclude this method to be implement in brewery industries (McCaig,
1990). Flow cytometry can also help brewers to measure the yeasts viability once it measures
the number of yeasts and DNA content, it also gives information about cell cycle (Novak et
al., 2007). Novak et al., 2007 reported the use of flow cytometry to monitor the propagation
of yeasts under anaerobic and aerobic conditions, using acriflavine (ACR) and fluorescein
isothiocyanate (FITC) as graduating fluorescent dye solutions; Kobayashi et al., 2007 also
used this approach but with different fluorescent dyes: dehydrorhodamine 123 (DHR) and
bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). In traditional microbiology, the
viability of yeast cells is quantified through the colony counting by plating serial dilutions
(Jenkins et al., 2003a; Maskell et al., 2003). The viable cell concentration is expressed
through the number of colony forming units (CFU) (Briggs et al., 2004). This technique can
also be applied to detect contaminations by other microorganisms, by selecting the proper
culture medium and incubation temperature (Lewis and Young, 1995). Particle counter can
also be used to monitor the population growth and the cell size, as showed by Albertin et al.,
2011. More recently, Laverty et al., 2013 developed an automated method that allows the
quantification of yeast budding percentages and yeasts viability, through the use of an image
cytometry method. This automated method employs a dual-fluorescent nucleic acid dye that
specifically penetrates into stain live cells, thus allowing the distinction of the morphological
characteristics of budding yeasts. Saldi et al., 2014 applied this image cytometry method,
compared its efficiency with several others, and it showed to be a good alternative to the
current methodologies, not only to monitoring yeast viability, as well as their vitality.

Table 4. Sum up of the reference values for some methodologies used to control yeasts
quality (viability and vitality)

Yeasts’ quality
Methodologies Reference value Reference
control parameters
Vital dye staining
Flow cytometry or (Lewis and
Viability  95% of viable cells (healthy culture)
particle counter Young, 1995)
Colony counting
 Between 2.4-2.7 (highly active with
good fermentation potential);
Acidification (Gabriel et
 Below 2 (reduced metabolic activity);
power (AP) al., 2008)
 Below 1.8 (low metabolic
competence).
Intracellular pH  High vitality: pH 6.2 – 7.2; (Weigert et
Vitality
(ICP)  Stressed populations: pH < 5.2. al., 2009)
 Industrial brewer’s yeasts: 40 – 90%;
 Extremely flocculent laboratory yeasts:
Flocculation (Verstrepen
90 – 100%;
capacity et al., 2003)
 Nonflocculent laboratory yeasts: 0 –
15%.
238 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

The fermentation process can be highly affected by yeasts because they may be viable,
yet they may not be actively proliferating. Indeed, yeasts vitality has been described for
several purposes such as the measurement of activity after the transfer from a nutrient-poor to
a nutrient-rich media, the fermentation performance, and the yeasts behavior in the presence
of physiological stress (Smart et al., 1999). The available tests for vitality are based in one
specific feature of yeast metabolism. These tests have different degrees of relevance for
brewing industry, so there is not only one recommended vitality method until now.
Flow cytometry has been used to monitor yeasts vitality, through the determination of the
physiological state of single cells during propagation or to set the vitality before fermentation
(the optimal time for pitching propagated yeast cultures is different for each yeast strain,
being the amount of yeast cells in phase G2/M determines the optimal conditions for pitching
(Novak et al., 2007)). With this technique is possible to establish the population distribution
within the cell cycle, once the signal obtained is proportional to the amount of yeast cells in
each phase (Hutter, 2002; Kobayashi et al., 2007; Lodolo and Cantrell, 2007; Novak et al.,
2007). It also allows to distinguish the yeast type, once the DNA profile achieved with flow
cytometry has 1/2 and 3 or more peaks for lager (diploid DNA) and ale (polyploid or
aneuploid DNA) yeasts, respectively (Hutter, 2002).
Acidification power (AP) test is a fast and simple method that allows the determination of
yeast vitality through their ability to perform a successful fermentation. This method
promotes the metabolic activity of cells through a glucose-induced medium acidification,
which leads to the excretion of acidity compounds (production of CO2 and organic acids,
H+/K+ exchange), lowering the pH of the medium (Sigler et al., 2006; Gabriel et al., 2008).
Gabriel et al., 2008 established that AP values from 2.4-2.7 means that yeast was highly
active with good fermentation potential, AP values below 2 was registered when yeasts had
reduced metabolic activity, and AP values below 1.8 when yeast showed low metabolic
competence (Table 4).
The intracellular pH (ICP) is another parameter that allows the yeasts vitality monitoring.
The majority of the yeast enzymes have an optimal activity pH in a neutral range. During
fermentation or propagation, the production of CO2 and organic acids lowers the extracellular
medium pH, which leads to the protons’ pumping by yeasts, in order to maintain the
extracellular pH. Higher proton extrusion rate corresponds to higher yeasts metabolic activity,
thus higher ICP indicates higher yeasts vitality. ICP was recently monitored by flow
cytometry, in which Weigert et al., 2009 were able to differentiate between exponential and
stationary phases along yeasts propagations (bottom-fermenting S. cerevisiae strain W34/70),
as well as to detect less vital yeast subpopulations. ICP varied between 6.2 and 7.2 for highly
active yeasts, while stressed populations had ICP lower than 5.2 (Table 4).
Yeasts sedimentation is one of the most important measurable parameter of industrial
strains, which can be measured through yeasts flocculation capacity according to several
parameters: bond strength, morphology, extent of sedimentation and rate of sedimentation
(Verstrepen et al., 2003). The flocculation capacity, based on degree of sedimentation, counts
the free cells in a flocculating culture, and compares them to the total cell number (before
flocculation or after deflocculation). Helms sedimentation test (Jenkins et al., 2003b; Strauss
et al., 2003) is the most used method in brewing (also the method recommended by ASBC)
(Verstrepen et al., 2003) and also the modified Stratford flocculation test (Novak et al., 2007).
It is estimated that extremely flocculent extremely laboratory strains have around 90 – 100%
 Saccharomyces spp. Role in Brewing Process … 239

flocculation, most industrial brewer’s yeasts have 40 – 90%, toward the end of fermentation,
and nonflocculent laboratory yeast strains have 0 – 15% (Table 4) (Verstrepen et al., 2003).

2.4. Saccharomyces spp. Serial Repitching

Each brewery has its own yeast culture line, which requires the propagation before the
first fermentation. Thus, yeasts are pitched, cropped, and then repitched in consecutive
fermentations, in order to have more efficient processes. Between the reuses, yeasts are stored
with a high concentration cell suspension (slurry) or maintained in extended stationary phase.
The yeasts performance along serial repitching can be affected by several parameters, which
will be described in this sub-section (namely yeasts age and population selection). Moreover,
it is intended to list the research papers that mention the application of yeasts serial
repitching, and to understand the impact of this brewing procedure in yeasts performance.
The number of yeast reuses is dependent of some parameters, such as: microbial stability,
yeast apparent robustness, final product quality, and the company policy. The number of
reuses can vary between 7 and 20 times, or sometimes longer, especially for top-cropping
yeasts, once they have lower contamination with trub and other non-yeast solids. However,
the number of yeasts repitching may be compromised due to the possible stress that yeasts
can suffer, thus the number of repitching should be reduced to 5 – 10 times (Powell et al.,
2003). Moreover, the application of high-gravity brewing practices affects yeasts repitching
once it is limited by yeasts’ capacity to tolerate higher ethanol levels and to attenuate very
concentrated worts. In some cases, it is possible to adapt some parameters, such as the
composition of malts and the ionic composition of wort (adjust or supplement metal ions,
e.g., Mg2+) for less yeasts stress and consequently less problems along repitching (Boulton,
2013).
Yeasts age can be measured chronologically by the number of times that yeasts are
recycled along repitching. In order to maintain the yeasts quality, it is required to reduce their
metabolism through decrease of the storage temperature (Eßlinger, 2009). Before a new
fermentation, it is needed to determine the yeasts viability, to adjust the amount of pitching
yeast (Powell et al., 2000). Aeration and short-term storage are essential for the good
physiological conditions of yeasts (Eßlinger and Narziß, 2012).
Lager beers represent around 90% of the beer market, so most of the research has been
focused on lager yeasts (Saerens et al., 2010). After the fermentation, where the brewing
yeasts typically divide 4 – 6 times (Boulton, 1991), lager yeasts are allowed to settle on the
cylindroconical vessels. The yeast sedimentation seems to occur within an age gradient as
reported by Hodgson et al., 1997, where older yeasts are in the bottom of the cone and
younger yeasts are more frequently found on the cones’ top. These results confirmed the
hypothesis formed by Barker and Smart, 1996 that already mentioned that older cells tended
to flocculate earlier, while younger cells had the opposite behavior. The formation of bud
scars in the surface of yeast cells are correlated with the replication lifespan (range from 10 -
30 divisions) of yeasts and can be used as an indicator of cell age – budding index. This
analysis can be done by confocal microscopy as reviewed by Powell et al., 2000, where virgin
cells tend to have smooth surfaces, with practically no protruding structural components;
while older cells have rough surface, with more wrinkles, leading to more tendency to
flocculate, supporting previous reports (Barker and Smart, 1996; Hodgson et al., 1997).
240 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

The selection of the yeast population has impact on the fermentation performance: if the
population contains higher older cells concentration, the cells will have slower division rates,
reducing the size of the inoculum, and then slowing the yeasts growth and high lag phase.
Moreover, Barker and Smart, 1996 reported an increased tendency of older “mother” cells to
retain “daughter” cells, influencing the flocculation process. If the population has mainly
newly budded virgin cells, it will need more time to reach the critical size needed for the first
division. So, it is important to compromise both types of cells in order to not have
inappropriate fermentative characteristics, which may lead to fermentation inconsistency,
poor flavor development, and reduced flocculation performance (Hodgson et al., 1997;
Powell et al., 2003). Powell et al., 2004 suggested that the yeast strain and the dimensions of
the fermentation vessel influence the localization of aged cells within the cone, that cannot
follow the previous reports; but still the bottom yeasts had lower fermentation capacities.
These authors also mentioned that yeast crop is composed by a diverse range of cells with
different phenotypes, and not by a single homogenous mass, which can promote some
variability on fermentation performance. Thus, the population of yeast to repitch should be
with young to middle aged cells, once they are the most active portion (fast growth and yeast
biomass production) (Hodgson et al., 1997; Powell et al., 2000). Bühligen et al., 2014 also
verified an age gradient along yeasts sedimentation, in which the older and younger cells
fractions had the higher dead cells concentration, and should not be used along serial
repitching. Nowadays, in the brewing process, after the sedimentation, the first fraction of
cropped yeast is discarded as waste, because it contains older and dead cells among protein
debris (trub). The rest of the yeasts (middle-aged and virgin cells) are transferred to a yeast
collection vessel, until be used into a new fermentation (Powell et al., 2000, 2003).
Several studies (Smart and Whisker, 1996; Jenkins et al., 2003a; Lodolo et al., 2008)
reported the deterioration of yeasts along serial repitching and cropping due to: hygiene issues
(cross-contamination with others microorganisms such as wild yeast or bacteria); selection of
yeasts with certain characteristics that affect the performance such as age, cell size, increased
flocculation capacities; and selection of yeasts with certain characteristics related with
quality, for instance respiratory-deficient yeasts, which physiological changes can be caused
by stress and genetic changes.
In order to understand the impact of serial repitching on yeasts (Table 5), different
parameters have been studied, namely their: viability, vitality (through the analysis of
flocculation capacity, acidification power test, the glycogen and trehalose contents, and petite
mutations), fermentation performance (by analysis of volatile compounds, sugars and amino
acids/proteins uptake and degradation, lipids analysis), and genetic modifications.
As previously mentioned, the yeasts taxonomy suffered several modifications along time,
especially through the development of molecular tools that allowed a more consistent yeasts
classification. This problem reflects on the yeasts strain characterization along Table 5, which
shows the diversity of yeasts names and some incorrect taxonomy, for instance Miller et al.,
2013 refer that S. cerevisiae is a synonymous of S. pastorianus. Thus, it is difficult to verify
the impact of serial repitching in each yeast strain due to this duality of criteria used in yeasts
taxonomy.
 Table 5. Sum up of the different studies implied with yeasts serial repitching, regarding the following studied factors: yeast
strain, tested parameters, repitching times and the main results/conclusions

Repitching time and wort

Yeast strain Parameters Main results / conclusions Ref.
conditions
 Viability  First 10 fermentations: increase of viability and vitality until
 30 successive
(methylene blue); 100%, being stable until 24 fermentations; (Smart and
Ale fermentations (lab scale)
 Flocculation  After 24 fermentations: flocculation and viability became Whisker, 1996)
 Malt wort (10-12 ºP)
(Helm's test). highly variable and decreased in overall magnitude.
 Shaking flasks fermentation:
- No effects on yeasts viability along serial repitching;
- At 5th fermentation in a 20 ºP, yeasts were not able to
 Viability
attenuate properly, being the ethanol amount decreasing along
(methylene blue);
serial repitching;
 Vitality
 5 - Decrease of yeasts vitality along serial repitching;
(acidification (Cunningham and
S. cerevisiae  Wort  Static fermentation:
power test); Stewart, 2000)
(12 and 20 ºP) - Viability decreases in static fermentations;
 Sugar analysis
- Decrease of yeasts vitality along serial repitching;
(HPLC);
- No effects on yeasts fermentation performance;
 Ethanol content.
 Oxygen stimulates yeasts growth, allowing them to tolerate
stress conditions (e.g., acid treatments, high-gravity
fermentations).
 Substantial increase in isoamyl acetate (up to 2.25 fold), and
 Volatile esters  7
S. cerevisiae ethyl acetate content (from 25 to 32 ppm) along repitching; (Quilter et al.,
and alcohols (HS-(lab scale)
5610  Isoamyl acetate content increased steadily up until 5th 2003)
GC-FID).  ~12 ºP
fermentation, and then began to decrease.
 Acidification  AP value decreased with repitching, having higher SD along (Gabriel et al.,
S. cerevisiae 95 4
power test. repitching. 2008)
 Phosphatidylcholine (PtdCho) and
Phosphatidylethanolamine (PtdEtn) content altered along
repitching, but mostly in 1st generation, and then yeasts
 Lipid analysis
S. cerevisiae gradually accommodated to unfavorable fermentation (Jurešic et al.,
(phospholipids and 3
(lager) conditions; 2009)
fatty acids).
 Yeasts are capable to adapt to stress conditions partially by
modifying the PtdCho/PtdEtn ratio and the unsaturation degree
of their fatty acids.
 Table 5. (Continued)

Repitching time and wort

Yeast strain Parameters Main results / conclusions Ref.
conditions
 Proportion of neutral lipid classes did not vary significantly
along repitching (high intrinsic stress resistance);
S. cerevisiae var.  Triacylglycerols together with steryl esters and squalene
(Rupčić and
carlsbergensis  Neutral lipids. 3 constituted the major part of neutral lipids (70%);
Jurešič, 2010)
(lager)  Increased ergosterol content and an increased
unsaturated/saturated (UFAs/SFAs) ratio (major changes: shift
from 16:0 to 16:1, but less from 18:0 to 18:1 species).
 Specific growth rate and maximum optical density
 Ethanol and
 8 decreased, otherwise there was an increase in isoamyl alcohol
isoamyl alcohol
S. carlsbergensis (lab scale) and ethanol content along repitching; (Kobayashi et al.,
content (GC);
BH092  Yeast extract-dextrose  Gradually increase of intensities of fluorescence dyes along 2007)
 Cell staining and
medium repitching, indicating higher exposition to reactive oxygen
flow cytometry.
species.
 Viability
 Viability decreased as generation number increased for all
(methylene blue,
strains (reduction not statistically significant, between 80 –
MgANS, plate
Lager: SCB1,  5 (SCB2), 100%);
counts); (Jenkins et al.,
SCB2, SCB3,  8 (SCB4)  AP declined with increased generation number to a greater
 Acidification 2003a)
SCB4  10 (SCB1 and SCB3) extent;
Power Test;
 Overall increase in glycogen and intracellular trehalose
 Glycogen and
levels.
trehalose content.
 Appearance of 2% petite mutations only in 5th fermentation,
and then 7% in the 6th fermentation;
 As the fermentation progresses, the ability of yeasts to
 Petite mutation;
reduce diacetyl decreases;
 Flocculation; (Jenkins et al.,
Lager SCB3 8  Low VDK uptake of propagation slurries due to the
 VDKs 2003b)
requirement for enzymes’ induction as a result of acetolactate
determination.
generation and beer diacetyl challenge;
 Slight decrease on flocculation, but remained unchanged in
4 to 6 fermentations (around 80%).
 Repitching time and wort
Yeast strain Parameters Main results / conclusions Ref.
conditions

 No accumulative increase in petite mutation after six

 1
 Petite mutations; repitchings;
Lager yeast  3 (Lawrence et al.,
 mtDNA RFLP  Majority of petites isolated from yeasts slurries exhibited
CB11  9 2012)
analysis. identical mtDNA restriction digest patterns;
 10
 Disruption of mitochondrial DNA may not occur randomly.

 Viability  Different flavor profiles (esters and higher alcohols) were

(methylene blue); exhibited in beer from generation 0 and generation 4;
Diamond lager  5
 Flocculation;  No statistical changes in flocculation along repitching;
yeast (Lallemand, (lab scale) (Powell and
 Petite mutations;  Despite the lower viability of G0 dried yeasts, repitched
Inc.) (wet and  Lager-style wort (12.5 Fischborn, 2010)
dried)  Volatile cultures consistently contained high proportion of live cells;
ºP)
compounds (GC-  No phenotypic or genetic variants accumulated over time
FID). (no petite mutations).
 No significant differences in the rate of sugars assimilation;
 2  Repitching had impact on the utilization of nitrogenous
S. cerevisiae  Sugar analysis
(lab scale) compounds by yeasts, in wort;
(lager brewing (HPLC); (Miller et al.,
 Wort 80:20 (malt: high  Increased intracellular storage of amino acids may occur as
yeast, syn. S.  Amino acids 2013)
maltose syrup adjunct) a result of repitching, and may progressively be reduced the
pastorianus) analysis.
 14.7 ºP uptake of particular amino acids from wort on subsequent
fermentation.
 Six to eight generations do not cause a significant increase
 Viability (flow in cell age (preoxygenated wort);
cytometry);  No trend was observed in glycogen concentration after
 Volatile analysis repitching, while for trehalose concentration there was a
S. cerevisiae  8
(GC-FID); decreased trend (adaptation of yeasts to the fermentation and (Verbelen et al.,
(pastorianus) (lab scale)
 Glycogen and storage related stresses); 2009b)
CMBSPV09  15 ºP
trehalose content;  More stress in non-preoxygenated yeasts;
 Total fatty acids  Both higher alcohols and esters are not influenced by
of yeasts. repitching;

 Diacetyl content increased drastically after two repitchings

(possibly due to increased fermentation speed starting from 3rd
generation);
 Decrease of unsaturation of fatty acids along repitching.
 Table 5. (Continued)

Repitching time and wort

Yeast strain Parameters Main results / conclusions Ref.
conditions
 AP decreased (being up to 10%) with increased wort
osmolarity and yeasts generation, however values were always
 Acidification
very high;
power test;  3
 Acetaldehyde level in green beer was slightly above or (Sigler et al.,
S. pastorianus 95  Volatile analysis
(lab scale)
within the flavor threshold; 2009)
(GC-ECD and GC-  12, 16 and 20 ºP
 Levels of esters ethyl acetate were within the usual limits or
FID).
slightly above them, and had small variations; same trend for
higher alcohols.
 2
S. pastorianus  Acidification  Small drop in AP value was observed with repitching, (Matoulková and
(lab scale)
RIBM 95 power. nevertheless AP remained high throughout. K. Sigler, 2011)
 Malt wort (12 ºP)
 Signs for advanced aging could not be detected after 20
 RT-qPCR;
repitchings;
 Microarrays
 Expression of flocculation genes remained stable during all
(expression of  20
S. pastorianus serial repitchings; (Bühligen et al.,
genes for aging, (4 different cycles)
HEBRU  Higher wort aeration led to increased expression of stress 2013)
flocculation,  12 ºP
response genes, especially connected to ROS defense;
metabolism and
 Higher amounts of oxygen in the wort led to lower levels of
stress).
isoamyl acetate in beer.
 Supplementation of oxygen in worts allow the rejuvenation
of yeasts cells and prevent cell aging;
 Bud scar
 No loss of flocculation during serial repitching;
counting and size
 No significant changes were observed for genes associated
measurement;  6 and 17 (industrial
S. pastorianus to stress; (Bühligen et al.,
 Viability; scale)
HEBRU  Gradient of age observed for yeasts sedimentation, being the 2014)
 mRNA analysis;  12, 12.5 and 14 ºP
older cells the first to flocculate;
 Expression of
 Older and younger cells fractions contained the higher dead
specific genes.
cells concentration, and should not be used to repitch;
 No increase of replicative age cells during serial repitching.
 Viability  No detectable changes in yeasts fermentation ability;
(methylene blue);  No changes in viability of yeasts after repitching;
(Kordialik-
S. pastorianus  Glycogen and  10 (10 ºP)  Drop in the glycogen level during fermentation of 15°P wort Bogacka and
308 and B4 trehalose content;  8 (15 ºP) was observed earlier than 10°P wort – higher osmotic stress Diowksz, 2013)
 Volatile analysis and ethanol concentration generated when higher gravity wort
(GC-FID). was fermented;
 Repitching time and wort
Yeast strain Parameters Main results / conclusions Ref.
conditions
 No significant variation in beer flavor produced along
repitching; no difference in the ‘higher alcohols to esters’ ratio
was noticed either.
 Ethanol
 Extract consumption and ethanol production was consistent
determination;
along serial repitching;
 Metal ions
S. pastorianus  Serial repitching does not affect the initial and overall (Deželak et al.,
analysis; 11 (lab scale)
TUM 34/70 uptake of zinc ions by yeasts; 2015a)
 Fermentable
 After the 6th fermentation, there was a slight increase of
carbohydrates
glucose utilization.
analysis.
 Asn, Ser, Val, Phe, Ile, Leu, and Lys assimilation profiles
S. pastorianus  Amino acids were less affected by serial repitching; (Deželak et al.,
11 (lab scale)
TUM 34/70 analysis.  Gln, Gly+His, Arg, Ala, and Trp assimilation profiles were 2015b)
more affected by serial repitching.
 Yeast karyotype;  Only 3 middle-molecular weight chromosomes had
S. pastorianus (Deželak et al.,
 Protein 11 (lab scale) significant and consistent size changes;
TUM 34/70 2014)
profiling.  Total protein synthesis declines along serial repitching.
 No obvious or little pattern that influences methanol and
acetaldehyde formation during serial repitching, respectively;
 Volatile  Low influence of serial repitching in higher alcohols
S. pastorianus (Deželak et al.,
compounds (GC- 11 (lab scale) formation (2- and 3-methylbutanol, 2-phenylethanol, 1-
TUM 34/70 2015c)
FID). propanol, and isobutanol);
 Higher rate of esters formation (ethyl acetate, isoamyl
acetate, and 2-phenylethyl acetate) from 1st to 4th repitching.
 Flocculation;  Some changes in macromorphological characteristics of ale
 RAPD  98 generations for the strain, however there were no genomic variations or changes in
fingerprinting ( ale strain terms of fermentation characteristics;
BridgePort ale (Powell and
regions);  135 generations for the  No significant differences were registered for fermentation
and lager strains Diacetis, 2007)
 RFLP lager strain analysis, flocculation assessment and genetic fingerprinting of
fingerprinting (Ty  13.5 ºP nuclear DNA, between fresh lager yeasts and after 135
elements). generations.
246 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

Taking into account the serial repitching of S. cerevisiae (ale yeast), only two parameters
can be compared: viability and vitality (AP test). Smart and Whisker, 1996 registered
decrease of yeasts viability after 24 fermentations at lab scale, but in most of the cases not
registered viabilities lower than 80%. Cunningham and Stewart, 2000 only verified decrease
on yeasts viability when there was no wort oxygenation. S. cerevisiae vitality was monitored
by AP test which revealed that there is a decrease of the yeasts acidification power along
serial repitching, nevertheless the values were superior to 2.2 (Cunningham and Stewart,
2000; Gabriel et al., 2008), showing that yeasts were not critically affected by the handling
during the brewing process.
Regarding lager yeasts’ (in the broadest sense) serial repitching, it is only possible to
compare yeasts vitality through the analysis of petite mutations. Jenkins et al., 2003b only
registered the presence of petite mutants after the 5th (2%) and 6th repitching (7%), while
Lawrence et al., 2012 verified the presence of petite mutants in different serial repitchings,
but concentrations always lower than 1%. These lower values of petite mutants’ percentage
were obtained, once warm cropping techniques were used, causing less stress in yeasts, which
lead to less formation of petite mutants.
Several studies report the serial repitching effects of S. pastorianus strains, covering a
wide range of studied parameters. Regarding yeasts viability, there were registered no
significant changes along repitching, even using high-gravity worts (8 to 10 times) (Verbelen
et al., 2009b; Kordialik-Bogacka and Diowksz, 2013). Oxygen dissolved in wort proved to be
important for yeasts growth, and consequently for their viability. Yeasts use oxygen to
produce unsaturated fatty acids and sterols, which are used for the plasmatic membrane
structure and its integrity. Thus, there is an efficient biomass production that can tolerate
different stress conditions (e.g., acid treatments, high-gravity fermentations) which yeasts can
be exposed along serial repitching, and prevent cell aging (Verbelen et al., 2009b; Bühligen et
al., 2014).
As observed for S. cerevisiae strains, there is a decrease of the S. pastorianus
acidification power along serial repitching, however yeasts were not critically affected by the
handling. The flocculation capacity is another parameter that reflects yeasts vitality, and S.
pastorianus serial repitching showed no changes in the flocculation genes expression, until 20
repitchings (Bühligen et al., 2013, 2014).
Sugars uptake by yeasts seem to not have significant interference along serial repitching
(Miller et al., 2013; Deželak et al., 2015a). Regarding amino acids uptake by S. pastorianus
strain, Deželak et al., 2015b verified different trends of assimilation of the amino acids along
11 serial repitchings. Miller et al., 2013 also observed reduced uptake of several amino acids
by S. pastorianus strain, only in 2 serial repitchings, due to an increase of amino acids
intracellular storage. The total protein synthesis in S. pastorianus strain also declined during
11 serial repitchings, as registered by Deželak et al., 2014.
Kordialik-Bogacka and Diowksz, 2013 verified an increase of trehalose content in S.
pastorianus strain until 10 serial repitchings, while the glycogen content decreased in older
cultures. Regarding glycogen, Kordialik-Bogacka and Diowksz, 2013 showed a greater
decrease when using a high-gravity wort (15 ºP) that promotes higher osmotic stress to yeasts.
However, Verbelen et al., 2009b registered no trend in glycogen concentration and a decrease
of trehalose content along 8 serial repitchings, which results suggest that yeasts adapt to
fermentation and storage related stress cycles. These results obtained by Verbelen et al.,
2009b and Kordialik-Bogacka and Diowksz, 2013, are contradictory maybe due to strain
 Saccharomyces spp. Role in Brewing Process … 247

variability, fermentation conditions (one work was performed at laboratory conditions and
other at industrial scale), and wort composition.
In terms of yeast volatile producing compounds (higher alcohols and esters), Verbelen et
al., 2009b and Kordialik-Bogacka and Diowksz, 2013 showed that their formation by S.
pastorianus strains was not influenced until 10 serial repitching, while Sigler et al., 2009
observed slight variations, but only repitched S. pastorianus strain 3 times. On the other hand,
Deželak et al., 2015c verified little influence on higher alcohols formation (2- and 3-
methylbutanol, 2-phenylethanol, 1-propanol, and isobutanol) during 11 serial repitchings of S.
pastorianus, but higher rates of esters formation (ethyl acetate, isoamyl acetate, and 2-
phenylethyl acetate) from 1st to 4th repitching.
DNA techniques such as random amplified polymorphic DNA – polymerase chain
reaction (RAPD-PCR) and RFLP were also used to understand the genetic changes during
long term serial repitching by Powell and Diacetis, 2007. Either for ale, as well as for lager
yeasts, Powell and Diacetis, 2007 did not have significant differences along the 98 and 135
generations, respectively, in terms of genetic fingerprinting. These results showed that with
absence of selective pressures, yeasts can be resistant to genetic changes, being that
phenomenon related to the yeast strain used. Bühligen et al., 2013 also verified the same
trend: with more than 20 repitchings of S. pastorianus, there was not observed an advanced
aging through mRNA (messenger ribonucleic acid) expression; as well as no significant
changes in the expression of genes associated to aging, stress, storage compounds metabolism
and cell cycle through the analysis by low-density microarrays (Bühligen et al., 2014).
However, no studies to detect phenotypic or metabolic alterations of yeast cells were done by
the authors. Yeast karyotype was performed by Deželak et al., 2014, and only three middle-
molecular weight chromosomes had significant changes along S. pastorianus serial
repitching.

CONCLUDING REMARKS AND FUTURE TRENDS

Beer aroma profile is dependent of a network of several effects, from which raw
materials and the yeast strain have a key role. In fact, Saccharomyces spp. are the most
commonly yeasts used in brewing, and through their complex metabolic pathways, different
metabolites can be formed, namely volatiles which contribute for the beer sensorial
characteristics. Moreover, serial repitching showed to have slight impact, even when applied
in high-gravity fermentations, in different studied parameters, namely viability, vitality,
oxidative stress, genetic modifications or volatile compounds formation. The presence of
oxygen revealed to be one important parameter in yeasts serial repitching once it is essential
for yeasts growth. Nevertheless, there is still required to have a deep knowledge about the
metabolic pathways correlated with the formation of compounds that will have impact on
beer sensorial characteristics.
The competitive environment that brewing companies are involved, promotes their
constant innovation, either for the development of new products or for the maximization
resources and reduction of costs. Indeed, the production of different beer styles is a current
brewing trend, once consumers wish to have different sensorial experiments. Furthermore, the
use of by-products from others industries, as adjuncts, is an increasingly common practice,
248 Cátia Martins, Tiago Brandão, Adelaide Almeida et al.

either for the development of new products, but also because of their cheap price. However, it
is required to investigate more about the yeasts performance in these conditions, mainly due
to initial high carbohydrates content. High-gravity fermentations are another current brewing
trend, which require yeasts selection or genetic modifications in order to be able to tolerate
higher ethanol contents. Yeasts genetic modifications are a possibility for the achievement of
interesting beer products.
In conclusion, the metabolic complexity of yeasts reveals a great potential to be
exploited, nevertheless they do not reach the goal by themselves, being necessary the
combination with different raw materials to lead to the constant innovation in this sector, and
consequently to the development of new products.

ACKNOWLEDGMENTS
Thanks are due to FCT/MEC for the financial support to the QOPNA research Unit (FCT
UID/QUI/00062/2013) and CESAM (project PEst-C/MAR/LA0017/2013), through national
founds and where applicable co-financed by the FEDER, within the PT2020 Partnership
Agreement. C. Martins thanks the FCT/MEC for the PhD grant (SFRH/BD/77988/20112010)
through the program POPH/FSE.

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In: Beer: Production, Consumption and Health Effects ISBN: 978-1-63485-704-8
Editor: William H. Salazar © 2016 Nova Science Publishers, Inc.

Chapter 6

INACTIVATION OF BEER YEAST BY

MICROBUBBLED CARBON DIOXIDE
AT LOW PRESSURE AND QUALITY EVALUATION
OF THE TREATED BEER

Fumiyuki Kobayshi and Sachiko Odake

Faculty of Applied Life Science, Nippon Veterinary and Life Science University,
Kyonanntyo, Musashino, Tokyo, 180-8602 Japan

ABSTRACT
Inactivation of Saccharomyces pastorianus cells, beer yeast, by two-stage system
with microbubbled carbon dioxide at low pressure (MBCO2) was investigated by the
observation with scanning and transmission electron microscopies (SEM and TEM),
fluorescent analysis with propidium iodide, and measurements of leakage of nucleic acid
and protein and intracellular enzyme activation. Furthermore, yeast in unfiltered beer
(UFB) was inactivated by two-stage MBCO2 and the quality of the treated beer was
evaluated. It was observed by SEM that wrinkles in S. pastorianus cells treated by two-
stage MBCO2 with a heating coil at 50°C (MB50) and heat treatments at 50°C and 80°C
(H50 and H80) were more than those in non-treated (NT) cells. Upon observation with
TEM, it suggested that MB50 had a direct effect on the intracellular substrate, although
little influence on the cell membrane, whereas H80 cells showed visible damage to cell
membranes. However, it was recognized that PI intensity in MB50 cells was great than
that in NT, H50 and H80 cells, and that the amount of nucleic acid and protein leaked
from H80 cells was significantly higher than that of NT, MB50 and H50 cells.
Furthermore, the enzyme inactivation efficiency in MB50 cells was the same as in H80
cells. These results estimated that inactivation of S. pastorianus by two-stage MBCO2
was due to actions of MBCO2 on the cell membrane and the intracellular substrate such
as enzyme inactivation. Furthermore, 5-log reductions of yeast in UFB could be achieved
by two-stage MBCO2 with a heating coil at 50°C for 5 min, while yeast in UFB was not
completely inactivated by heat treatment at 80°C for 5 min. In sensory evaluation of these


Corresponding author: F. Kobayashi. Faculty of Applied Life Science, Nippon Veterinary and Life Science
University, 1-7-1, Kyonanntyo, Musashino, Tokyo, 180-8602. E-mail: fkoba@nvlu.ac.jp.
258 Fumiyuki Kobayshi and Sachiko Odake

beers, there were no significant differences on the score of flavor, although bitterness of
two-stage MBCO2-treated beer (MBB) was significantly low on the score of taste. In
analysis of volatile compounds, dimethyl sulfide, dimethyl disulfide, β-myrcene and
styrene in MBB was less than those in UFB and heat-treated beer at 80°C for 5 min
(HTB). Glucose and maltose contents in MBB and UFB were almost the same, although
those in HTB significantly decreased. Organic acids, except for acetic acid in MBB, were
nearly identical with those in UFB and HTB. Most of the free amino acids in MBB were
lower than those in UFB and HTB. Bitter units of beer increased in the order of MBB,
HTB and UFB. These results suggested that two-stage MBCO2 showed promise as a
practical technique for inactivating yeast in UFB. The inactivation efficiency of two-stage
MBCO2 had less influence of materials in beer than that of heat treatment, and two-stage
MBCO2 have little negative impact on the beer quality.

INTRODUCTION
Pressurized carbon dioxide (CO2) processes that include supercritical (SC) CO2 have
been widely studied as a new food pasteurization method because heating may cause
undesirable changes in the taste and flavor of foods (Damar and Balaban, 2006; Garcia-
Gonzalez et al., 2007a; Spilimbergo and Bertucco, 2003). However, the SCCO2 process is
prohibitively expensive from a practical perspective, because it requires maintaining high-
pressure conditions (10-30 MPa) in order to inactivate microorganisms effectively.
Furthermore, SCCO2 may also result in a loss of food flavor because it is used as an
extracting solvent for organic compounds. Therefore, we recently developed a pasteurization
technique that uses CO2 microbubbles (MBCO2) at a pressure lower than those required for
SCCO2 and reported that Escherichia coli, Saccharomyces cerevisiae and Lactobacillus
fructivorans could be effectively inactivated by the MBCO2 (Kobayashi et al., 2009, 2010,
2012a, b). In addition, to improve the inactivation efficiency of MBCO2, the MBCO2
equipment was devised from a batch system to a two-stage system in which MBCO2 is
generated and mixed into a sample solution at a lower temperature before the sample solution
is heated to a moderate temperature (two-stage MBCO2). It was then reported that the quality
of sake in which L. fructivorans and some enzymes were inactivated by the two-stage
MBCO2 retained good taste and flavor on sensory evaluation and measurement of volatile
compounds (Kobayashi et al., 2013). However, the mechanisms of action of two-stage
MBCO2 on microbial cells are still not clear.
Beer yeast (S. pastorianus NBRC11024) was used in the study. Beer is alcohol beverage
produced most in the world and in Japan about 5,600,000 kL of beer have been consumed in
2011 (Kirin Holdings Co., Ltd., 2012). Beer yeast and some bacteria present in fermented
beer cause the quality to deterioration (Miyamoto, 2001), so it is treated by pasteurization at
70°C or by filtration. However, changes in the taste and flavor of pasteurized beer are induced
by heating. At some beer companies in Japan, preservation and distribution of unpasteurized
beer is permitted at room temperature after removing solid materials such as substances
bound to proteins and polyphenols, hop resins, yeasts and some bacteria in fermented beer by
filtration (Inui, 2001). In recent years, unfiltered beer (UFB) containing yeast has become
popular in Japan due to its mellow taste and rich flavor. In addition to its unique taste and
flavor, the UFB also contains some nutrients, including amino acids, vitamins and minerals
(Arimura, 2000; Sogawa, 1990). However, it is not widely distributed due to its short shelf-
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 259

life. Therefore, it is considered that UFB would be more widely distributed if the yeast could
be inactivated without heating.
In this study, to investigate the effect of two-stage MBCO2 to the membranes and
interiors of S. pastorianus cells, observation with scanning and transmission electronic
microscopy and fluorescence analysis with propidium iodide (PI) were performed (Kobayashi
et al., 2014). Furthermore, the amounts of protein and nucleic acid leaked from S. pastorianus
cells were estimated by a spectrophotometer and intracellular enzyme activities were
measured with the APIZYM kit. In addition, yeast in UFB was inactivated by two-stage
MBCO2, and the quality of its treated beer was determined by the sensory evaluation and the
measurement of the components (Kobayashi and Odake, 2015).

EQUIPMENT AND PROCEDURE FOR TWO-STAGE MBCO2

Schematic diagram of the experimental equipment for two-stage MBCO2 was shown in
Figure 1. This equipment was constructed by Izumi Kosyo Co. Ltd. (Chigasaki, Japan) based
on our design, and was used as follows: 10 L sample solution (physiological saline solution
containing 5% ethanol and 1.0 × 106 CFU/mL S. pastorianus, or UFB) was injected into the
mixing vessel (300 mm diameter × 220 mm height; volume 15 L) from valve A and was
cooled to 10°C. The valve A was then closed and gaseous CO2 was fed into the headspace of
the mixing vessel to reach the treatment pressure by opening valve B and C. MBCO2 was
generated by sending the mixture of sample solution and gaseous CO2 introduced at 2 L/min
from valve D to an ejector-type MB generator (Aura-tec Co. Ltd., Kurume, Japan) using a
circulating pump (0.75 kW motor pump, Teikoku Electric Mfg. Co. Ltd., Tatsuno, Japan) at
15 L/min until the dissolved CO2 concentration in the sample solution reached saturation
at 10°C and the treatment pressure (5 min). The bubble size of MBCO2 measured in our
previous study (Kobayashi et al., 2013) was 37 m. Valve E was opened and the CO2-
saturated sample solution was continuously pumped using a metering pump (Nihon Seimitsu
Kagaku Co., Ltd., Tokyo, Japan) into a heating coil (5 mm dia. × 5100 mm length, volume
100 mL). The exposure time in the heating coil was set by adjusting the flow rate of the
metering pump. The treated sample was collected from the back pressure valve B or A
mounted at the exit of the heating coil or the cooling coil (set at -5°C and 4 MPa),
respectively.

INACTIVATION OF S. PASTORIANUS AND BEER YEAST

The number of surviving S. pastorianus cells after treatment by two-stage MBCO2
decreased dramatically after 1 min of exposure time at all of the tested temperatures of the
heating coil (Figure 2). A 6-log reduction in the S. pastorianus population occurred at 45°C
and 50°C for 1 min, 40°C for 3 min, and 35°C for 5 min, respectively. On the other hand,
little inactivation of S. pastorianus was induced by heat treatment alone at 35°C and 40°C. In
addition, the number of surviving S. pastorianus cells after heat treatment alone at 45°C and
50°C decreased approximately 1.5-log after 1 min of exposure time, and slowly to 5 min.
Heat treatment alone at 80°C showed the same inactivation effect as two-stage MBCO2 at
260 Fumiyuki Kobayshi and Sachiko Odake

45°C and 50°C. Therefore, it was recognized that the inactivation efficiency of the two-stage
MBCO2 on S. pastorianus was significantly higher than that of heat treatment alone and
increased with increasing temperature in the heating coil. It was noted that increased
diffusivity of CO2 and fluidity of the cell membrane are induced by higher temperatures upon
pasteurization with pressurized CO2 (Haas et al., 1989). In addition, the number of surviving
yeast in UFB dramatically decreased at 1 min as with S. pastorianus suspension and then the
decrease became slow (Figure 3), although a 6-log reduction of S. pastorianus population
was achieved by both treatments at 1 min in Figure 2. It was reported that inactivation
efficiency of pressurized CO2 was decreased by sucrose, NaCl, gelatin and glycerol at high
concentration (Garcia-Gonzalez et al., 2009) and that lowering of inactivation efficiency of
MBCO2 on L. fructivorans was caused by using buffer containing phosphoric and/or citric
acids (Kobayashi et al., 2012b). In addition, the type of yeast was considered to contribute to
the lowering of inactivation efficiency, since yeast in UFB was wild species and differed from
S. pastorianus.

Figure 1. Schematic diagram of the equipment of two-stage method with low-pressurized carbon
dioxide microbubbles.
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 261

The two-stage MBCO2 treatment was performed under the following conditions: Temperature and
pressure of the mixing vessel were set at 5°C and 2 MPa, respectively; the temperature and
pressure of the heating coil were set at 35°C, 40°C, 45°C or 50°C and 4 MPa, respectively. Heat
treatment alone was performed under the following conditions: The temperature was set at 35°C-
80°C, respectively.

Figure 2. Inactivation of S. pastorianus by two-stage MBCO2 and heat treatment alone.

MORPHOLOGICAL OBSERVATION, FLUORESCENCE STAINING,

LEAKAGE OF NUCLEIC ACID AND PROTEIN, AND
ENZYMATIC ACTIVITIES IN S. PASTORIANUS CELLS
In scanning electron microscopy (SEM) of S. pastorianus cells after the two-stage
MBCO2 with the heating coil at 50°C (MB50) and heat treatment alone at 50°C and 80°C
262 Fumiyuki Kobayshi and Sachiko Odake

(H50 and H80) (Figure 4), more wrinkles were observed on the surface of S. pastorianus cells
following the MB50, H50 and H80 treatments than on those with non-treatment (NT). The
damage to the cell membrane and budding sites were confirmed in SEM images of MB50 and
H80 cells, although no effect of H50 on the cell membrane in S. pastorianus cells was
observed. However, the membrane damage in S. pastorianus cells of H80 was great than that
of MB50. Therefore, the permeability of the cell membrane was suggested to be increased in
S. pastorianus cells of MB50 and H80. This result showed a similar pattern as previous
reports that wrinkles and damages to the membrane of S. cerevisiae cells and E. coli O157:H7
cells caused by pressurized CO2 were observed by SEM (Guo et al., 2011; Hong and Pyun,
1999; Kim et al., 2007).
In transmission electron microscopy (TEM) of NT, MB50, H50, and H80 cells (Figure
5), cell organelles could be observed at NT and H50 cells, while cellular structure was not
maintained at MB50 and H50 cells. In addition, obvious rupture of cell membranes and/or
walls was recognized in H80 cells. Also, expansion of the periplasm was confirmed in H50
cells. MB50 was suggested to have a direct effect on the intracellular substrate with affecting
on cell membranes and/or walls less than H80.
Also, it suggested that H50 increased the permeability of cell membrane because it had
an effect on the periplasm in the S. pastorianus cells. Substrate in H80 cells could not be
confirmed, although a slight remnant of membrane structure in the cells was observed. These
were estimated to be organelles with double membrane structures such as the nuclear
membrane and mitochondria, although they could not be identified as such because heat
denaturation of almost all intracellular organelles was induced by higher temperature.

Two-stage MBCO2 treatment was performed under the following conditions: temperature and pressure
in the mixing vessel were set to 5°C and 2 MPa, respectively; temperature, pressure and exposure
time in the heating coil were set to 50°C and 4 MPa, respectively; and temperature, pressure and
exposure time in the cooling coil were set to -5°C and 4 MPa, respectively. Heat treatment was
performed at 80°C. Different letters indicate significant differences in each exposure time by t-test
(p < 0.01).

Figure 3. Inactivation of yeast in unfiltered beer by two-stage MBCO2 and heat treatment alone.
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 263

NT, Non-treatment; MB50, two-stage MBCO2 treatment with the mixing vessel at 5°C and 2 MPa and
the heating coil at 50°C and 4 MPa for 5 min; H50, heat treatment alone at 50°C for 5 min; H80,
heat treatment alone at 80°C for 5 min.

Figure 4. Scanning electron microscopy images of S. pastorianus cells treated with two-stage MBCO2
and heat treatment alone.

NT, MB50, H50 and H80 was the same as in Figure 4.

Figure 5. Transmission electron microscopy images of S. pastorianus cells treated with two-stage
MBCO2 and heat treatment alone.
264 Fumiyuki Kobayshi and Sachiko Odake

The PI intensity of MB50 cells was the highest (Figure 6). The PI intensity of H50 and
H80 cells was not correlated with inactivation efficiency and was almost the same.
Furthermore, the PI intensity of MB50 cells, which showed the same degree of inactivation as
H80, was significant higher than that of H80 cells. H80 is suggested to leak nucleic acid from
S. pastorianus cells and/or to induce DNA denaturation, since PI intercalates in the double
bond of intracellular nucleic acid (Garcia-Gonzalez et al., 2007b). This result was also
consistent with the previous reports that the damage to cell membrane caused in the first stage
of pressurized CO2 was not directly attributable to microbial inactivation (Bertoloni et al.,
2006; Spilimbergo et al., 2009). Therefore, the permeability to cell membrane of S.
pastorianus was altered by two-stage MBCO2.
The amounts of nucleic acid and protein leaked from MB50 cells were almost the same
as those from H50 cells, while the inactivation efficiency differed substantially between
MB50 and H50 (Figure 7). Therefore, it was considered that the leakage of nucleic acid and
protein from S. pastorianus cells was caused by the heat treatment rather than the two-stage
MBCO2. The amounts of nucleic acid and protein leaked from H80 cells was the highest,
fully half as much again as that from MB50 cells, which nevertheless had the same
inactivation effect as H80. Thus, it was estimated that heat treatment at higher temperature
caused the damage to cell membrane and consequently allowed leakage of intracellular
substrate. On the other hand, two-stage MBCO2 was suggested to have less effect on the cell
membrane than heat treatment alone as shown in Figure 5 and to affect the cell interior
directly. Therefore, the damage of cell membrane was considered to have an impact on the
inactivation by pressurized CO2, although this result supported the agreement that the injury
of cell membrane would not be the primary cause of cell death (Wu et al., 2007).

NT, MB50, H50 and H80 was the same as in Figure 4. Different letters indicated significant differences
at the Tukey-Kramer method (P < 0.05).

Figure 6. PI intensity in S. pastorianus cells treated with two-stage MBCO2 and heat treatment alone.
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 265

Table 1. Enzyme activities in S. pastorianus cells treated with two-stage MBCO2 and
heat alone measured with an APIZYM kit

Enzymes NT MB50 H50 H80

Esterase 1 0 0 0
Esterase lipase 1 0 1 0
Leucine arylamidase 5 0 3 0
Valine arylamidase 1 0 1 0
Acid phosphatase 5 0 5 0
Naphthol-AS-BI-phosphohydrolase 2 1 1 1
α-Mannosidase 2 0 1 0
NT, MB50, H50 and H80 was the same as in Figure 4. The data presented were the means of the results
of triplicate experiments.

NT, MB50, H50 and H80 was the same as in Figure 4. Different letters indicated significant differences
at the Tukey-Kramer method (P < 0.05).

Figure 7. The quantities of nucleic acid and protein leaked from S. pastorianus cells treated with two-
stage MBCO2 and heat treatment alone.

In enzymatic activities in NT, MB50, H50, and H80 cells measured with an APIZYM kit
(Table 1), which permits monitoring of 20 different constitutive enzyme activities from a
complex sample that has not been purified (Ballestra et al., 1996), seven types of enzyme -
esterase, esterase lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-
AS-BI-phosphohydrolase and α-mannosidase - showed the activities in NT cells. However,
enzymes except for naphthol-AS-BI-phosphohydrolase in MB50 and H80 cells and only
esterase in H50 cells were completely inactivated. MB50 had a high effect on enzyme
inactivation, equivalent to H80 despite the same temperature as H50. In this study, it was
suggested that the decrease of enzyme activity in S. pastorianus cells by two-stage MBCO2
was caused by the lowering of intracellular pH by the penetration of CO2 into the cells
because residual activity of enzymes in MB50 and H80 cells was the same despite the release
of protein from MB50 cells less than H80. Therefore, inactivation of enzymes in MB50 cells
may be one of the mechanisms on microbial inactivation by two-stage MBCO2.
266 Fumiyuki Kobayshi and Sachiko Odake

UFB, unfiltered beer; MBB, beer treated by two-stage MBCO2 with the mixing vessel at 5°C and 2
MPa and the heating coil at 50°C and 4 MPa for 5 min; HTB, beer heat-treated at 80°C for 5 min.
Sensory evaluation was performed by 26 panelists (age, 21~50 years; 14 men and 12 women). The
panelists evaluated the orthonasal, retronasal and total aromas, sweetness, sourness, bitterness and
total taste of the beers on a five-point scale (4, very good; 3, good; 2, normal; 1, bad and 0, very
bad). Different letters indicate significant differences in each items by the Tukey-Kramer method
(p < 0.01).

Figure 8. Sensory evaluation of unfiltered, heat-treated and two-stage MBCO2-treated beers.

QUALITY OF BEER TREATED WITH MBCO2

In sensory evaluation of UFB, heat-treated beer at 80°C (HTB) and beer treated with two-
stage MBCO2 containing a heating coil at 50°C (MBB) (Figure 8), there were no significant
differences at all of the tested items in the evaluation of aroma, although orthonasal aroma
score of MBB was more than that of UFB and HTB, and retronasal aroma tended to become
weaker. Also, orthonasal and retronasal aroma scores of HTB were almost the same as those
of UFB, and total aroma score of UFB and MBB was better than that of HTB. There were no
significant differences at the items except for bitterness in the evaluation of taste. Bitterness
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 267

score of MBB was significantly less than that of UFB and HTB, and sourness and sweetness
scores were slightly stronger and weaker, respectively. Sourness score of HTB was slightly
lower than that of UFB. Almost panelists evaluated that MBB was most quaffable. Since
bubble number and size, bubble growth rate and ascending bubble velocity due to the
modification of the CO2 environment can affect the sensory properties of beverages (Descoins
et al., 2006), fine bubbles such as MB may be an effect on the taste of MBB. On the other
hand, it indicated that taste and flavor derived from heat treatment of high temperature was
induced, because it was evaluated to feel flavor like soy sauce, miso and cheese from HTB.
Since phenomena associated with the Mailard reaction such as appearance of off-flavor and
increase of chromaticity by heating beer was reported (Ohata and Kamata, 1983), volatile
compounds, free amino acids, organic acids and bitter units in each beer were measured.
In volatile compounds in UFB, HTB and MBB (Table 2), β-myrcene significantly
decreased, and dimethyl sulfide, dimethyl disulfide and styrene in MBB tended to be less than
those in UFB, while volatile compounds in HTB was almost the same as those in UFB. β-
Myrcene having green odor is a hop-derived aroma compound of poor hydrophilic property
and dimethyl sulfide is of great important compound as beer aroma derived from malt and
gives mellowness to beer aroma at adequate dose, although sulph-compound is generally off-
flavor and give immature or rotten odor at excess dose (Kashiwada, 2001). So, MBB
preferred on sensory evaluation may be due to decrease of dimethyl sulfide. In addition, ethyl
hexanoate showing banana-like aroma is main compound of beer and sake, and the loss from
beer and sake by pressurized CO2 treatment have been reported (Ishikawa et al., 1995; Dagan
and Balaban, 2006; Tanimoto et al., 2008). However, decrease of ethyl hexanoate in MBB
was not recognized in this study. Also, components derived from the Mailard reaction in HTB
could not be confirmed because of small threshold value (Okumura, 1991).

Table 2. Volatile compounds in unfiltered, heat-treated and

two-stage MB-CO2-treated beers

KIz Compoundsy UFB HTB MBB

725 dimethyl sulfide 9.6 ± 3.9x aw 8.5 ± 4.1 a 4.7 ± 4.1 a
902 ethyl acetate 10311.1 ± 2493.7 a 10004.4 ± 3483.2 a 12428.8 ± a
1397.6
911 2-methyl-butanal 90.0 ± 21.4 a 98.1 ± 39.2 a 111.3 ± 4.8 a
916 3-methyl-butanal 138.7 ± 26.5 a 143.4 ± 32.4 a 210.3 ± 16.9 a
955 ethyl propanoate 183.7 ± 13.7 a 223.9 ± 17.0 a 250.5 ± 19.8 a
968 ethyl 2-methyl-propanoate 105.1 ± 23.4 a 94.3 ± 3.0 a 97.2 ± 3.5 a
974 n-propyl acetate 211.1 ± 61.6 a 203.3 ± 69.1 a 230.1 ± 11.7 a
1006 methyl isobutyl ketone 93.6 ± 22.1 a 91.7 ± 28.8 a 105.6 ± 2.9 a
1013 2-methylpropyl acetate 682.2 ± 181.8 a 632.6 ± 228.8 a 658.4 ± 11.2 a
1038 ethyl butanoate 395.1 ± 101.1 a 391.6 ± 129.3 a 424.1 ± 19.9 a
1056 1-propanol 1109.6 ± 457.6 a 1026.6 ± 442.5 a 1211.9 ± 93.4 a
1073 dimethyl disulfide 46.3 ± 13.4 a 41.9 ± 18.4 a 30.1 ± 1.5 a
1130 2-methyl-1-propanol 4562.8 ± 1442.2 a 4472.2 ± 1497.5 a 5488.2 ± 174.0 a
1134 isoamyl acetate 2511.3 ± 798.7 a 2397.6 ± 1155.2 a 3072.5 ± 1226.6 a
1157 β-myrcene 105.0 ± 0.6 b 86.7 ± 11.3 b 55.3 ± 2.4 a
1167 1-butanol 47.7 ± 19.6 a 44.0 ± 15.7 a 49.1 ± 8.9 a
1240 isoamyl alcohol + 10875.3 ± 3544.9 a 11113.1 ± 3687.4 a 14354.2 ± 809.5 a
2-methyl-1-butanol
268 Fumiyuki Kobayshi and Sachiko Odake

Table 2. (Continued)

KIz Compoundsy UFB HTB MBB

1241 ethyl hexanoate 302.9 ± 59.1 a 281.8 ± 13.4 a 287.6 ± 19.0 a
1256 styrene 719.8 ± 112.7 a 543.0 ± 183.3 a 315.3 ± 37.3 a
1278 hexyl acetate 51.1 ± 13.4 a 58.0 ± 17.2 a 50.8 ± 3.6 a
1323 4-methyl-1-pentanol 49.9 ± 17.9 a 45.0 ± 7.4 a 54.1 ± 7.1 a
KIz Compoundsy UFB HTB MBB
1361 1-hexanol 23.3 ± 7.2 a 21.8 ± 6.2 a 26.1 ± 2.0 a
1373 hexahydro-1,1-dimethyl-4- 13.2 ± 5.5 a 13.0 ± 4.4 a 13.9 ± 0.8 a
methylene-4H-
cyclopenta[c]furan
1378 heptyl acetate 8.0 ± 2.8 a 7.8 ± 3.3 a 9.1 ± 0.4 a
1411 3-methyl-1-hexanol 9.7 ± 5.3 a 9.8 ± 4.3 a 11.0 ± 3.6 a
1424 ethyl octanoate 364.6 ± 140.1 a 389.0 ± 164.3 a 397.8 ± 74.4 a
1436 Heptanol 14.4 ± 7.1 a 14.8 ± 5.3 a 16.3 ± 1.2 a
1486 3,7-dimethyl-1,6-octadien-3-ol 29.5 ± 19.5 a 35.7 ± 17.4 a 56.5 ± 11.6 a
1493 1-octanol 9.7 ± 4.1 a 10.3 ± 4.1 a 10.4 ± 0.9 a
1610 caryophyllene 11.7 ± 0.8 a 9.6 ± 0.1 a 12.4 ± 0.8 a
1632 ethyl decanoate 47.1 ± 1.8 a 49.9 ± 10.9 a 51.7 ± 1.8 a
1644 α-caryophyllene 29.2 ± 6.0 a 23.2 ± 1.9 a 30.8 ± 2.1 a
1828 2-phenylethyl acetate 4.2 ± 1.0 a 3.8 ± 1.5 a 4.9 ± 1.1 a
1859 ethyl dodecanoate 1.8 ± 0.1 a 1.5 ± 1.1 a 1.8 ± 0.0 a
1998 phenylethyl alcohol 1.5 ± 0.7 a 1.7 ± 0.1 a 2.8 ± 0.7 a
UFB, HTB and MBB was the same as in Figure 8.
z
Kovats index on a DW-WAX column.
y
Volatile compounds identified by GC-MS.
x
g/L.
w
Different letters indicate significant differences in each compounds by the Tukey-Kramer method (p <
0.01).

Since UFB was used in the study, the contents of organic acid, free amino acids and sugar
were higher than those reported previously (Brewing Society of Japan, 1999). There were
no significant differences in organic acid contents except for acetic acid in measured beers
(Table 3). Acetic acid contents in UFB, HTB and MBB were 421, 379 and 383 mg/L,
respectively, and that in UFB was significantly higher than that in HTB and MBB. It was
considered that the result was the cause for the lowering of sourness in HTB and MBB
showed on sensory evaluation. It has been noted that acetic acid in beer is produced by
metabolism of yeast during fermentation and is susceptible to producing process than other
organic acids (Brewing Society of Japan, 1999). Free amino acids have little direct influence
on beer taste because of content less than threshold level (Brewing Society of Japan, 1999),
and contribute to beer taste by causing the Mailard reaction with sugars (Kunitake, 1976).
However, free amino acid contents in UFB were higher than those reported previously
(Brewing Society of Japan, 1999) and may contributed to beer taste, since UFB was used in
the study. Total amount of free amino acids was highest at UFB and in order to HTB, MBB
(Table 4). The result may be related to lowering of bitterness showed on sensory evaluation.
The difference of free amino acid contents in beer was probably due to decompose peptides
by residual yeast in UFB, decompose proteins and/or peptides by excess heat on HTB and
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 269

leak free amino acids from inactivated yeast (Guo et al., 2011), although it is not clear yet.
Glucose and maltose contents were highest at UFB and in order of MBB, HTB (Figure 9). It
was considered that a decrease of glucose and maltose in HTB was caused by the Mailard
reaction. Because it was noted that the Mailard reaction enhanced with increasing temperature
(Ohata and Kamata, 1983), it was suggested that glucose and maltose contents in HTB at
80°C was less than those in MBB at 50°C. Furthermore, the bitter unit of MBB was
significantly lower than that of UFB and HTB (Figure 10). Bitter substance of beer is iso-α
acid such as isohumulone (Brewers Convention of Japan, 1997). The result was considered to
account for the lowering of bitterness of MBB on sensory evaluation. A decrease of bitterness
of MBB may be due to be absorbed and concentrated to MB generated by two-stage MBCO2,
because isohumulone in beer is absorbed to surfaces of CO2-bubbles with forming protein and
reached in high concentration (Hashimoto, 1980).

Table 3. Main organic acids in unfiltered, heat-treated and

two-stage MBCO2-treated beers

Organic acid UFB HTB MBB

z y
succinic acid 1513 ± 123.6 a 1531 ± 205.9 a 1423 ± 99.4 a
acetic acid 421 ± 6.7 b 379 ± 13.5 a 383 ± 11.8 a
lactic acid 456 ± 45.3 a 430 ± 36.0 a 448 ± 32.3 a
pyruvic acid 218 ± 4.3 a 209 ± 7.1 a 218 ± 7.7 a
malic acid 207 ± 4.3 a 198 ± 8.8 a 193 ± 0.7 a
citric acid 59 ± 3.8 a 56 ± 3.8 a 57 ± 6.5 a
UFB, HTB and MBB was the same as in Figure 8.
z
mg/L.
y
Different letters indicate significant differences in each row by the Tukey-Kramer method (p < 0.01).

UFB, HTB and MBB was the same as in Figure 8. Different letters indicate significant differences in
each component by the Tukey-Kramer method (p < 0.01).

Figure 9. Glucose and maltose contents in unfiltered, heat-treated and two-stage MBCO2-treated beers.
270 Fumiyuki Kobayshi and Sachiko Odake

UFB, HTB and MBB was the same as in Figure 8. Different letters indicate significant differences by
the Tukey-Kramer method (p < 0.01).

Figure 10. Bitter unit (BU) of unfiltered, heat-treated and two-stage MBCO2-treated beers.

CONCLUSION
The present results suggest that two-stage MBCO2 have a direct influence on the interior
and membrane of S. pastorianus cells. It is considered that leakage of intracellular substrate
was not a major factor in the inactivation of S. pastorianus by two-stage MBCO2, because
leakage of nucleic acid and protein from MB50 cells was less than that from H80 cells.
However, the inactivation efficiency of enzymes in MB50 cells was the same as that in H80
cells. Therefore, it was estimated that inactivation of S. pastorianus by two-stage MBCO2
resulted in intracellular changes such as lowering of intracellular pH and enzyme inactivation
caused by CO2 passing through cell membranes and penetrated into the cells and the changes
of membrane permeability by CO2 contacted to or penetrated into cell membrane. However,
further research is needed since enzyme inactivation was only confirmed concerning the
effect of two-stage MBCO2 on the cell interior.
In addition, to study the possibility of two-stage MBCO2 as a pasteurization technique of
beer, yeast in UFB was inactivated by the two-stage MBCO2, and sensory evaluation and
analysis of components of the treated beer were performed. Five-log reductions could be
achieved by two-stage MBCO2 with a heating coil at 50°C for 5 min. It was suggested that a
few decreases of volatile compounds in beer by two-stage MBCO2 had little effects on
sensory evaluation of beer, because there was no significant difference on the evaluation of
total aroma. There was no significant difference among UFB, MBB and HTB on analysis of
volatile compounds, although it was recognized that HTB contained heat-derived taste and
flavor on the sensory evaluation. Also, bitterness and sourness of MBB was lower than those
of UFB and HTB. The reason was considered due to the lowering of free amino acids showed
bitterness, glucose and maltose contents, and acetic acid. Therefore, it was suggested that
two-stage MBCO2 was promising as pasteurizing technique for inactivating yeast in UFB.
 Inactivation of Beer Yeast by Microbubbled Carbon Dioxide … 271

Table 4. Amino acids in unfiltered, heat-treated and two-stage MB-CO2-treated beers

Amino acids UFB HTB MBB

P-Ser 382.9 ± 0.6z ay 438.6 ± 1.3 c 394.3 ± 2.8 b
Tau 867.6 ± 10.9 a 852.1 ± 1.8 a 819.6 ± 26.7 a
PEA 1212.2 ± 12.9 b 1170.6 ± 10.1 b 1100.0 ± 19.9 a
Asp 262.5 ± 53.3 ab 300.4 ± 5.4 b 186.3 ± 2.1 a
Thr 100.1 ± 1.0 a 102.4 ± 0.4 a 101.4 ± 2.1 a
Ser 164.7 ± 3.2 b 149.5 ± 1.6 a 149.1 ± 3.9 a
Asn 119.1 ± 2.1 a 117.6 ± 2.5 a 112.8 ± 1.1 a
Glu 260.4 ± 4.0 b 228.3 ± 0.4 a 226.2 ± 2.7 a
Gln 308.2 ± 1.2 c 274.9 ± 1.0 b 267.2 ± 2.0 a
Gly 226.6 ± 1.5 b 212.4 ± 1.9 a 204.8 ± 4.2 a
Ala 393.3 ± 3.2 b 310.4 ± 1.5 a 308.3 ± 11.1 a
Val 215.8 ± 1.5 b 204.8 ± 0.4 a 202.8 ± 2.6 a
Cys 8.4 ± 0.8 a 7.6 ± 0.5 a 6.6 ± 0.4 a
Met 56.2 ± 2.5 a 56.2 ± 0.5 a 54.7 ± 0.3 a
Cysta 10.3 ± 0.5 b 8.2 ± 0.3 a 6.7 ± 0.5 a
Ile 135.5 ± 0.2 b 130.0 ± 0.7 a 130.7 ± 1.9 a
Leu 266.2 ± 1.0 b 250.7 ± 1.6 a 251.8 ± 3.0 a
Tyr 163.5 ± 0.9 b 154.7 ± 1.1 a 152.8 ± 1.9 a
b-Ala 234.7 ± 3.5 b 231.0 ± 2.4 ab 219.6 ± 5.2 a
Phe 131.3 ± 0.6 b 124.9 ± 0.5 a 125.2 ± 1.1 a
b-ABA 194.9 ± 28.3 a 195.6 ± 19.0 a 206.2 ± 16.3 a
GABA 219.4 ± 1.4 b 215.2 ± 1.1 b 204.3 ± 1.8 a
MEA 263.8 ± 4.5 ab 271.9 ± 2.2 b 257.6 ± 3.8 a
NH3 158.5 ± 4.0 a 176.5 ± 2.5 a 171.9 ± 10.0 a
Orn 20.7 ± 0.6 a 14.8 ± 0.5 a 15.9 ± 3.3 a
His 35.2 ± 5.2 a 35.5 ± 2.0 a 32.7 ± 0.5 a
Lys 175.9 ± 4.2 a 165.1 ± 4.6 a 157.3 ± 10.7 a
Arg 149.7 ± 1.5 b 140.6 ± 2.1 ab 132.2 ± 4.2 a
Pro 1652.3 ± 3.0 b 1657.4 ± 4.6 b 1581.0 ± 7.9 a
Total 8389.9 ± 75.5 c 8198.0 ± 0.6 b 7780.2 ± 70.6 a
UFB, HTB and MBB was the same as in Figure 8.
z
mol/L.
y
Different letters indicate significant differences in each row by the Tukey-Kramer method (p < 0.01).

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 INDEX

aldehydes, 28, 32, 33, 54, 56, 60, 65, 72, 77, 78, 104,
A 213, 215, 227, 228, 232
algorithm, 44
acetaldehyde, 33, 36, 43, 81, 111, 228, 233, 245
alkaloids, 9, 184
acetic acid, 33, 35, 43, 176, 188, 258, 268, 269, 270
allergens, 125
acid, 34, 35, 36, 38, 39, 43, 63, 64, 82, 91, 97, 103,
almonds, 101, 104, 114, 116, 117, 118
106, 111, 115, 117, 127, 141, 143, 155, 168, 173,
alpha-tocopherol, 104
176, 178, 179, 185, 187, 188, 223, 229, 230, 231,
amines, 128, 181, 182
234, 235, 236, 238, 241, 246, 257, 264, 265, 268,
amino acid, 31, 32, 33, 34, 35, 36, 61, 80, 102, 125,
269
126, 127, 128, 181, 216, 221, 229, 230, 232, 235,
active compound, 229
240, 243, 246, 258, 267, 268, 270
adaptation, 107, 227, 235, 243
ammonium, 176, 229
additives, 104
amnesia, 7
adenosine, 228
amylase, 125, 220
adenosine triphosphate, 228
anaesthetic, 13, 15, 17
adhesion, 94, 235
analgesic, 13, 17
adjuncts, 215, 218, 220, 221, 247, 252
aneuploid, 238
adjustment, 222
ANOVA, 51, 72, 76, 107, 108, 110, 111
adsorption, 29, 35, 36
anthelmintic, 13, 17
aesthetic, 218
antibacterial, 19, 81, 219
aflatoxin, 185
antioxidant, 101, 102, 103, 104, 106, 107, 110, 111,
Africa, 103, 215, 218
113, 115, 116, 117, 118, 119, 144, 178, 234
aggregation, 234
antiseptic, 19
agmatine, 182
aperient, 2, 13, 15
agricultural chemistry, 119
aqueous solutions, 39
alanine, 32, 179, 229
arginine, 32, 181, 229
alcohol abuse, 119, 122
aromatic compounds, 104
alcohol consumption, 28, 102, 114, 117
Asia, 103, 215
alcohol content, 8, 11, 19, 44, 78, 102, 106, 107, 122,
aspartate, 229
220, 236, 242
aspartic acid, 32
alcohol poisoning, 7
assessment, 77, 81, 86, 87, 89, 90, 97, 99, 182, 192,
alcohol production, 88
245
alcohol strength, 8, 11
assimilation, 243, 245, 246
alcoholic beverage, 1, 86, 89, 102, 122
asthma, 125
alcoholism, 7, 22, 23
atherosclerosis, 102
alcohols, 28, 32, 33, 34, 35, 54, 56, 60, 65, 78, 83,
ATP, 228
87, 116, 138, 213, 215, 227, 228, 229, 230, 241,
attachment, 79, 86
243, 244, 245, 247
276 Index

autolysis, 29, 36, 37, 80

C

B Ca2+, 38, 40
cabbage, 227
bacteria, 37, 82, 83, 84, 91, 93, 95, 129, 130, 173, CAD, 188
188, 219, 240, 258, 273 calcium, 218, 225
bacterial strains, 92 calibration, 156
beer filtration, 222, 272 cancer, 115, 119, 146, 161
beer flavor, 27, 33, 104, 215, 222, 226, 229, 230, 245 candidates, 188, 189
beer maturation, 36, 81, 222 capillary, 120, 122
Belgium, 42, 213 capital expenditure, 76
beneficial effects, 28, 102, 103, 105 capsule, 37, 39, 40, 41, 46, 49, 73, 79, 81, 83
benefits, 101, 103, 119, 161 carbohydrates, 31, 33, 34, 35, 102, 120, 122, 123,
benzene, 138 124, 133, 134, 135, 137, 213, 216, 217, 218, 221,
beverages, 43, 54, 83, 86, 89, 91, 94, 101, 102, 122, 227, 228, 229, 230, 231, 236, 245, 248
175, 267 carbon dioxide, 215, 257, 258, 260, 271, 272, 273,
bioactive agents, 80 274
bioavailability, 103 carboxyl, 31
biocompatibility, 39 carboxylic acid, 35, 179
bioconversion, 83, 233, 250 carcinogenicity, 120
biodegradability, 39 cardiovascular disease, 103
biological activity, 141, 185 cardiovascular risk, 114
biologically active compounds, 105 case study, 116, 117
biomass, 27, 28, 29, 31, 33, 34, 35, 44, 53, 54, 56, catabolism, 228, 230
58, 63, 64, 82, 91, 96, 99, 228, 240, 246 catalysis, 211
biomass growth, 44, 58 catalytic activity, 28
biomedical applications, 82 cataplasms, 18, 19
biomolecules, 220 cation, 121, 176, 188
biopolymer, 80 cell culture, 30, 93
biosensors, 114 cell cycle, 237, 238, 247
biosynthesis, 33, 35, 61, 79, 227, 230, 232, 235 cell death, 264
biotechnology, 38, 83, 84, 85, 86, 87, 91 cell membranes, 257, 262, 270
biotin, 178 cell metabolism, 41, 56
blood, 103, 117 cell organelles, 262
body fat, 116 cell size, 227, 237, 240
body weight, 185 Central Europe, 89
BOE, 106 cereals, 5, 19, 21, 102, 184, 215, 218
boiling, 8, 101, 105, 107, 108, 111, 112, 113, 141, CH3COOH, 177
167, 170, 171, 172, 215, 218, 219, 220, 221 cheese, 81, 267
bonds, 29, 30, 39, 137, 220, 234 chemical, 28, 31, 38, 39, 40, 113, 114, 119, 120, 122,
bottom-fermenting lager yeasts, 214 123, 126, 138, 155, 157, 161, 165, 167, 172, 174,
boundary surface, 41 178, 181, 182, 185, 187, 188, 192, 215, 216, 218,
brevis, 92, 188, 189 222
brewing process, 79, 80, 101, 103, 109, 113, 115, chemical composition of a lager beer, 215, 216
117, 167, 213, 214, 216, 217, 220, 223, 233, 234, chemical reactions, 31, 178
236, 240, 246 chemical stability, 39
budding, 219, 226, 234, 237, 239, 262 chemicals, 144
Bulgaria, 27, 89, 90, 91, 96, 210, 211 chemopreventive agents, 115
by-products, 28, 115, 247 chitin, 83
 Index 277

chitosan, 27, 31, 37, 39, 40, 41, 42, 45, 46, 50, 51, coughing, 16
77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 97, covering, 246
98 Creams, 17, 20, 93
cholesterol, 103, 104, 235 Croatia, 215
chromatography, 120, 121, 123, 226 crop, 240
CID, 120, 133, 134, 146, 150, 151, 153 crystal structure, 164
cirrhosis, 7 cultivation, 41, 77, 84, 86, 88, 91, 99
civilization, 214 cultivation conditions, 77
clarification, 110, 220, 222 culture, 29, 41, 42, 49, 54, 55, 77, 81, 82, 85, 86, 88,
classes, 214, 216, 242 99, 102, 236, 237, 238, 239
classification, 104, 219, 223, 224, 240 culture media, 42
cleavage, 150, 168, 169, 170, 177 culture medium, 237
climate, 219 cycles, 244, 246
clusters, 192 cysteine, 229, 232
CO2, 35, 48, 56, 63, 86, 104, 108, 188, 192, 215, cytometry, 237, 238, 242, 243
221, 222, 223, 225, 228, 230, 235, 238, 258, 259, cytoskeleton, 234
260, 262, 264, 265, 267, 269, 270, 271, 272, 273
coenzyme, 35, 227
color, 41, 43, 115, 117, 221
D
colour stability, 111
dates, 4, 11, 12, 14, 16, 17
combined effect, 70, 73
death rate, 235
commerce, 214
decay, 121
commercial, 74, 78, 117, 118, 172
decomposition, 173, 221
commodity, 214
deficiency, 103
communities, 184
degradation, 137, 138, 181, 220, 240
compatibility, 38
degrees Plato, 220
competition, 225
Degussa, 42
complexity, 124, 216, 228, 248
dehydration, 7
composition, 32, 34, 35, 36, 40, 80, 82, 93, 98, 103,
delirium tremens, 8
118, 215, 216, 218, 221, 229, 239, 247
denaturation, 221, 262, 264
compounds, 33, 36, 70, 73, 90, 97, 101, 102, 103,
density functional theory, 131
105, 110, 111, 112, 117, 122, 123, 141, 142, 143,
depression, 3
169, 213, 215, 219, 220, 221, 222, 223, 226, 227,
derivatives, 117, 157
228, 229, 230, 231, 232, 233, 234, 235, 236, 238,
dermatology, 141
240, 243, 245, 247, 258, 267, 268, 270
desorption, 121, 129, 179
compression, 30
destruction, 28, 31, 39, 48, 234
conditioning, 218, 219, 228
detection, 120, 121, 122, 123, 162, 178, 185, 226,
constitutive enzyme, 265
244
consumers, 103, 104, 181, 214, 215, 222, 247
detergents, 36
consumption, 28, 31, 35, 55, 56, 102, 103, 104, 116,
deviation, 121
117, 126, 153, 181, 188, 190, 215, 229, 234, 245
diabetes, 82, 103, 119
containers, 223
diet, 102, 115, 117, 119, 131, 214
contamination, 37, 76, 119, 182, 239, 240
differential equations, 44
cooling, 105, 259, 262
diffraction, 164
copolymer, 39
diffusion, 31, 37, 49, 51, 54, 60, 63, 70, 71, 73, 74,
copper, 35, 218
77
coronary artery disease, 161
diffusivity, 260
coronary heart disease, 104, 115, 117
digestion, 105
correlation, 107, 155
diploid, 238
cosmetic, 93
directives, 185
cost, 123, 225
discrimination, 114
278 Index

diseases, 102, 122, 185 environmental change, 40, 233

disorder, 125 environmental conditions, 31, 232
dispersion, 226 enzyme, 30, 32, 33, 37, 52, 83, 185, 217, 218, 220,
displacement, 73 221, 226, 230, 233, 234, 238, 242, 257, 258, 259,
dissociation, 120 265, 270, 272, 273
dissolved oxygen, 35, 227 epoxy resins, 30
distillation, 43, 44, 81, 106, 122 equipment, 37, 40, 76, 82, 93, 192, 258, 259, 260
distilled water, 42, 106 erosion, 84
distribution, 38, 39, 117, 124, 181, 238, 258 ESI, 116, 120, 123, 124, 125, 126, 127, 128, 129,
disturbed vision, 8 131, 133, 134, 136, 138, 143, 145, 146, 157, 161,
diuretic, 5, 13, 15, 16 163, 167, 168, 173, 176, 177, 185, 186, 187, 188,
diversity, 104, 114, 119, 161, 240 189, 192
DNA, 32, 194, 223, 224, 226, 234, 235, 236, 237, esophageal cancer, 185
238, 245, 247, 264 esters, 28, 32, 33, 34, 35, 43, 54, 56, 60, 63, 64, 65,
DOI, 86, 93, 95, 97, 98, 195, 210 78, 80, 83, 88, 96, 104, 115, 116, 128, 131, 137,
domestication, 225 141, 176, 179, 213, 215, 221, 231, 225, 227, 229,
dregs, 12, 13, 18, 19, 20 231, 241, 242, 243, 244, 245, 247, 251, 252
dressings, 95, 96 ethanol, 28, 33, 35, 36, 41, 42, 43, 44, 47, 48, 49, 50,
dropsy, 6 51, 54, 55, 56, 58, 64, 70, 71, 73, 74, 75, 79, 80,
drying, 83, 88, 146 81, 82, 83, 85, 88, 89, 90, 92, 97, 98, 99, 102,
duality, 240 104, 122, 188, 192, 215, 220, 221, 222, 226, 227,
dyes, 237, 242 228, 231, 233, 235, 239, 241, 242, 244, 245, 248,
dyslipidemia, 116 259
ethyl acetate, 35, 64, 137, 226, 227, 231, 241, 244,
245, 247, 267
E Europe, 103, 114, 214, 215
European Union, 184, 215
E. coli, 262
evaporation, 124
ear infection, 12
evidence, 41, 114
edema, 6
evolution, 224
education, 92
excretion, 232, 238
Egypt, 214
experimental condition, 123, 133, 135, 146, 153, 155
Ehrlich pathway, 230
experimental design, 123
elaboration, 119
exposure, 79, 259, 262
electrodes, 114
external environment, 37
electron, 116, 257, 263
extract, 8, 42, 43, 44, 52, 53, 54, 57, 58, 59, 60, 61,
electron microscopy, 263
62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75,
electron paramagnetic resonance, 116
77, 78, 94, 95, 98, 106, 116, 141, 146, 149, 153,
electrophoresis, 120, 121, 123, 124
156, 157, 175, 218, 220, 221, 242, 245
elephantiasis, 5
extraction, 98, 120, 121, 122, 123, 170, 175, 221
emetic, 11, 13, 14, 21
employment, 124
emulsions, 85 F
enantiomers, 157
encapsulation, 27, 37, 40, 41, 43, 83, 87, 113 fat, 218
endosperm, 220 fatty acids, 32, 35, 36, 80, 104, 129, 131, 213, 227,
enemas, 12, 13, 14 232, 234, 235, 241, 243, 246
energy, 31, 102, 120, 186, 190, 220, 221, 228 fermentable carbohydrates, 228, 229
engineering, 68, 96, 97, 98, 233 fever, 14
England, 103, 107, 110, 117 fiber, 31, 113, 119, 124, 190
entrapment, 30, 36, 79 filling, 216, 223
environment, 39, 44, 86, 188, 225, 228, 247, 267 filtration, 105, 216, 220, 222, 223, 258, 272
 Index 279

financial, 248 Galen, 2, 5, 6, 8, 22

financial support, 248 gall bladder problem, 14
Finland, 79, 87, 88 garlic, 14
fission, 219 gastro-intestinal problems, 3
fixation, 221 GC-FID, 241, 243, 244, 245
flatulence, 5, 6 gel, 32, 35, 38, 39, 48, 77, 82, 84, 87, 121, 124, 223,
flavonoids, 103, 105, 117, 122, 141, 144, 146, 148, 234
149, 150, 155, 156 gel formation, 38
flavor, 27, 28, 32, 33, 34, 79, 80, 86, 87, 88, 90, 96, gelation, 30, 38, 39, 40
97, 104, 114, 116, 117, 177, 188, 213, 214, 215, genes, 221, 224, 225, 228, 232, 233, 234, 235, 244,
219, 221, 222, 226, 227, 228, 229, 230, 236, 240, 246, 247
243, 244, 245, 258, 267, 270 Genevois pathway, 230
flexibility, 123 genito-urinary tract problem, 12
flight, 121, 122, 226 genome, 223
flocculation, 29, 30, 110, 222, 223, 225, 226, 234, genus, 93, 104, 129, 130, 219, 223
235, 238, 240, 241, 242, 243, 244, 245, 246 Germany, 89, 192, 211
flour, 101, 105, 107, 109, 113, 220 germination, 217, 220
flowers, 144, 214, 218 Gibbs energy, 173
fluid, 121, 122 glucoamylase, 81
fluid extract, 121, 122 glucose, 31, 33, 35, 42, 87, 131, 219, 220, 228, 229,
fluidized bed, 90, 99 231, 235, 238, 245, 269, 270
fluorescence, 237, 242, 259 glucoside, 182
folate, 116 glutamate, 34, 229
folic acid, 103, 113, 178 glutamic acid, 32
food, 79, 81, 82, 88, 91, 94, 95, 96, 97, 99, 101, 113, glutamine, 32, 229
119, 123, 124, 153, 178, 181, 182, 184, 188, 190, glutathione, 178, 234
192, 258, 272 glycerol, 134, 135, 234, 260
food additive, 178 glycine, 32, 229
food industry, 81, 178 glycogen, 31, 234, 235, 236, 240, 242, 243, 244, 246
food products, 88, 185 glycolysis, 33, 228
force, 221 glycolytic pathway, 228
formation, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, glycoside, 110, 146
39, 40, 41, 42, 45, 60, 63, 65, 77, 78, 79, 80, 81, graphite, 185
83, 86, 87, 88, 123, 143, 150, 161, 188, 213, 215, gravity, 218, 220, 221, 225, 227, 239, 241, 244, 246,
220, 221, 222, 223, 224, 226, 227, 228, 229, 230, 247, 248
231, 232, 233, 234, 235, 239, 245, 246, 247, 273 Greece, 90
fragments, 147, 150, 188 Greeks, 214
France, 42, 214 growth, 29, 31, 32, 34, 35, 41, 46, 53, 55, 56, 57, 58,
freezing, 81 60, 63, 64, 73, 78, 81, 83, 86, 88, 182, 188, 218,
fructose, 31, 35, 131, 133, 219, 228, 229, 231 226, 227, 228, 235, 240, 241, 242, 246, 247, 267
fruits, 144 growth rate, 29, 32, 55, 242, 267
FTICR, 124 guidelines, 40, 185
Fumigations, 17, 20
functional food, 91
funding, 192
H
fungi, 104, 138, 182, 185, 190, 219
harmful effects, 37
furan, 176, 268
haze, 221, 222, 229
headache, 5, 7, 8, 18, 24, 181
G healing, 94
health, 101, 102, 105, 114, 116, 117, 120, 161, 181,
GABA, 271 182
280 Index

health problems, 120, 182 in vivo, 116, 192

heart disease, 119 India, 114
heavy metals, 218 indirect effect, 36
height, 73, 259 induction, 235, 242
hepatocellular carcinoma, 185 industrial processing, 116
hexane, 141, 176 industry, 37, 40, 78, 82, 96, 97, 122, 123, 188, 213,
High pressure, 235, 272 214, 225, 236, 237, 238, 247
higher alcohols, 28, 32, 33, 34, 35, 54, 56, 60, 65, 78, infrared spectroscopy, 226
87, 116, 213, 215, 227, 228, 229, 230, 243, 244, ingredients, 40, 88, 104, 105, 119, 122, 124, 128,
245, 247 129, 138, 156, 157, 161, 170, 175, 176, 179, 190,
Hippocrates, 4, 8, 21, 25 192, 217, 272
histamine, 181 inhibition, 33, 41, 52, 55, 57, 64, 125, 128, 232
histidine, 32, 229 inoculation, 105
homocysteine, 116 inoculum, 33, 240
homologous genes, 226 insects, 190
honey, 10, 11, 13, 16, 19, 20, 21, 227 integrity, 234, 235, 246
hops, 9, 102, 105, 110, 122, 138, 139, 141, 145, 153, interference, 246
156, 161, 163, 167, 170, 175, 179, 188, 214, 215, internal pain, 10
217, 218, 219, 221, 232, 253 intoxication, 3, 6, 7, 8, 181
hormone, 82 ionization, 120, 121, 123, 129, 179, 185
human, 28, 92, 102, 114, 119, 122, 153, 161, 181, ions, 38, 40, 145, 146, 150, 154, 166, 173, 179, 188,
182, 185, 188, 190, 192 218, 225, 232, 234, 245
human exposure, 153 Iraq, 214
human health, 28, 102, 119, 122, 161, 181, 182, 188, iron, 112, 218
190, 192 irradiation, 170
humidity, 220 isolation, 91, 164
humour, 5 isoleucine, 32, 33, 34, 229, 230
hybrid, 123, 223 isomerization, 221
hybridization, 224 isomers, 124, 131, 150, 166
hydrazine, 185 isotope, 142, 143
hydrocarbons, 138, 219 Israel, 209
hydrogels, 81, 82 issues, 219, 240
hydrogen, 36, 164, 165, 229, 232, 234 Italy, 90
hydrogen bonds, 164 itching, 20, 226
hydrogen peroxide, 234
hydrogen sulfide, 36, 229, 232
hydrolysis, 39, 170, 220, 228
J
hygiene, 181, 240
Japan, 257, 258, 259, 268, 271, 272, 273
hyperglycemia, 116
hypertension, 181
hypothesis, 65, 108, 111, 239 K

K+, 38, 238

I kaempferol, 141
karyotype, 245, 247
identification, 44, 86, 91, 92, 95, 96, 226
kidney stone, 15
immobilization, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37,
kidneys, 5, 6
38, 40, 41, 42, 43, 44, 46, 49, 51, 54, 58, 64, 69,
kinetic model, 27, 55, 56, 57, 64
76, 77, 78, 80, 82, 83, 84, 85, 86, 87, 88
kinetic parameters, 60, 90, 91, 94, 95, 98, 99
immune response, 102
kinetics, 28, 41, 44, 57, 58, 64, 78, 81, 86, 91, 96, 99,
improvements, 122, 123
124, 173, 272
in vitro, 116, 143, 192
 Index 281

mashing, 101, 105, 107, 108, 110, 111, 112, 113,

L 218, 220
mass, 28, 29, 30, 32, 35, 41, 46, 68, 71, 73, 74, 75,
labeling, 169, 177
116, 120, 121, 124, 190, 192, 209, 211, 226, 240
lack of balance, 8
mass spectrometry, 116, 120, 121, 209, 226
lactic acid, 41, 82, 88, 93, 95, 188, 269
materials, 30, 83, 87, 88, 214, 215, 216, 258
Lactobacillus, 80, 84, 85, 88, 91, 92, 93, 94, 95, 96,
matrix, 27, 28, 29, 30, 32, 33, 36, 37, 50, 84, 85, 87,
97, 99, 173, 188, 189, 258, 272, 273
89, 90, 97, 123, 124, 125, 133, 135, 141, 162,
LDL, 104, 115
185, 188, 192
lead, 34, 35, 36, 38, 40, 49, 73, 77, 211, 221, 222,
matter, 109, 122
228, 230, 231, 236, 240, 246, 248
maturation process, 34, 78
leakage, 29, 48, 257, 264, 270
maximum specific growth rate, 55, 58, 64
learning, 211
measurement, 80, 107, 123, 124, 236, 238, 244, 257,
legislation, 120, 185
258, 259, 272
leucine, 32, 33, 34, 229, 265
mechanical, 29, 30, 31, 37, 38, 39, 40, 41, 43, 48, 49,
liberation, 225
50, 77, 79, 80, 83, 98, 226, 234
light, 90, 99, 108, 120, 124, 143, 177, 222, 226, 236
mechanical properties, 80, 83
lipid metabolism, 103, 232
media, 112, 226, 238
lipid oxidation, 188
medical, 214
lipids, 28, 32, 34, 35, 114, 115, 116, 129, 216, 217,
Mediterranean, 117
220, 227, 234, 235, 240, 242
medium composition, 93
liquid chromatography, 120, 121, 122
melt, 39
liquid phase, 190
membrane permeability, 32, 50, 270
Listeria monocytogenes, 272
membranes, 31, 234, 235, 259, 262
Lithuania, 205
menstruum, 3, 8, 9, 12
liver, 7, 8, 119, 122, 153, 185
Mesopotamia, 214
long-chain fatty acids, 232
messenger ribonucleic acid, 247
low temperatures, 223, 224, 234
metabolic, 31, 41, 87, 154, 228, 231, 233, 248, 250,
low-density lipoprotein, 104
252, 254
LSD, 107, 108, 109, 111, 112
metabolic pathways, 34, 154, 213, 221, 222, 223,
Luo, 203
226, 247
lysine, 32, 229
metabolism, 27, 28, 31, 32, 33, 34, 35, 36, 40, 41,
51, 54, 55, 56, 57, 61, 63, 73, 77, 78, 84, 86, 104,
M 111, 114, 129, 181, 182, 184, 185, 187, 213, 214,
222, 224, 226, 227, 228, 229, 230, 231, 232, 235,
macromolecules, 123, 192 236, 238, 239, 244, 247, 268
magnesium, 218 metal ions, 112, 239
magnets, 124 methanol, 106, 141, 245
magnitude, 182, 241 methodology, 211, 226, 236
Maillard reaction, 220, 221 methylene blue, 236, 241, 242, 243, 244
majority, 33, 74, 188, 216, 238 mevalonate pathway, 233
MALDI, 121, 123, 125, 126, 127, 131, 134, 137, Mg2+, 38, 239
160, 161, 179, 185, 188, 189, 192, 209 microbial cells, 31, 38, 41, 83, 85, 86, 87, 258
malt extract, 116 microemulsion, 123
malted grains, 215, 217 micronutrients, 118
malting, 80, 83, 115, 182, 217, 220 microorganisms, 29, 30, 40, 81, 86, 91, 129, 181,
maltose, 31, 33, 35, 88, 131, 133, 181, 220, 224, 228, 188, 192, 214, 228, 237, 240, 258, 272
243, 258, 269, 270 microscopy, 236, 239, 259
management, 54, 89 microsomes, 153
manganese, 218 microspheres, 79, 81
microstructure, 88
282 Index

milligrams, 106 nutrient, 32, 34, 93, 102, 161, 221, 222, 228, 235,
milling, 218, 220 238, 258
minerals, 102, 103, 119, 218, 258 nutrition, 114, 217
Ministry of Education, 211 Nutritional, 104, 114, 115, 117, 235, 236
mitochondria, 236, 262 nutritional status, 236
mitochondrial DNA, 234, 236, 243
mixing, 259, 261, 262, 263, 266
models, 44, 57, 68, 89, 99
O
modifications, 230, 240, 247, 248
obesity, 103
moisture, 219
Oceania, 103
molasses, 227
off-flavors, 222
molds, 190
oil, 115, 138, 215, 218, 219, 221
molecular biology, 91
oleic acid, 36, 104, 235
molecular oxygen, 178
oligomers, 156, 157, 161
molecular structure, 124, 145, 178
olive oil, 114
molecular weight, 39, 41, 112, 121, 123, 188, 189,
operations, 28
221, 245, 247
opportunities, 76
molecules, 110, 179, 227, 228
optical density, 106, 242
monosaccharide, 131, 133
optical microscopy, 124
monoterpenoids, 138
optimization, 27, 28, 43, 68, 69, 71, 75, 85, 90, 97,
monounsaturated fatty acids, 104
98, 99, 155, 185
morphology, 32, 226, 238
organ, 211
mRNA, 244, 247
organelles, 262
mtDNA, 243
organic acids, 28, 33, 36, 104, 108, 215, 232, 238,
multivariate analysis, 226
258, 267, 268, 269
mutations, 234, 236, 240, 242, 243, 246
organic compounds, 258
mycotoxins, 119, 182, 184, 186, 188
ornithine, 181
myocardial infarction, 119
Osmotic, 32, 40, 52, 64, 87, 233, 234, 244, 246, 253
osmotic pressure, 32, 40
N osmotic stress, 87, 244, 246
overlay, 236
NaCl, 260 overproduction, 64, 221
NAD, 232 oxidation, 33, 34, 36, 104, 109, 115, 116, 138, 141,
naphthalene, 179 143, 163, 164, 165, 167, 170, 177, 178, 219
natural polymers, 38 oxidative, 33, 34, 116, 141, 173, 228, 230, 233, 234,
Netherlands, 88 235, 236, 247
neural network, 86 oxygen, 33, 35, 64, 188, 215, 221, 223, 225, 226,
neurological disease, 185 227, 230, 244, 246, 247
neurotoxicity, 120
neutral, 104, 218, 238, 242
neutral lipids, 242
P
next generation, 124
pain, 185
niacin, 178
pancreas, 82
nitrogen, 34, 35, 213, 221, 229, 235
pantothenic acid, 178
nitrogen compounds, 213, 229
parallel, 167
Nitrogen Compounds Metabolism, 213, 229
pasteurization, 214, 223, 258, 260, 270, 273
Norway, 90, 215
pathogens, 91, 92, 93, 95, 188, 189, 218
nuclear membrane, 262
pathways, 32, 33, 34, 56, 81, 116, 135, 136, 145,
nucleic acid, 36, 234, 237, 257, 259, 264, 265, 270
146, 150, 153, 154, 161, 167, 169, 170, 171, 188,
nucleotides, 216
228, 230, 231, 232, 233, 235, 247
 Index 283

PCR, 223, 226, 247 polyphenols, 15, 101, 102, 103, 105, 106, 109, 111,
peptidase, 220 112, 113, 114, 115, 116, 118, 216, 221, 222, 258
peptides, 36, 126, 128, 161, 221, 268 polyploid, 238
permeability, 37, 41, 50, 79, 80, 81, 235, 262, 264 polysaccharides, 30, 32, 38, 39, 81, 87, 234
permeable membrane, 50 polyvinyl alcohol, 39, 90, 97
permit, 124 poor complexion, 8
peroxidation, 234 population, 64, 116, 126, 237, 238, 239, 240, 259
pessary, 13 population growth, 237
pH, 29, 30, 33, 36, 37, 40, 41, 43, 101, 106, 107, porosity, 29, 31
108, 112, 114, 155, 170, 171, 188, 189, 215, 220, Portugal, 87, 213
225, 232, 234, 237, 238, 254, 265, 270 potassium, 218
pharmaceutics, 185 precipitation, 30, 38, 221, 222
phase inversion, 38 preparation, 37, 39, 40, 42, 43, 46, 47, 48, 49, 51, 54,
phenolic compounds, 102, 103, 104, 112, 113, 122, 74, 78, 94, 123, 192
124, 141 preservation, 44, 93, 214, 258, 274
phenotypes, 114, 240 prevention, 102, 119, 146
phenylalanine, 32, 229 Principal Components Analysis (PCA), 102, 107,
phosphate, 36, 125, 129, 218, 235 112, 113, 226
phospholipids, 241 principles, 114
phosphorylation, 228 probiotic, 79, 80, 81, 82, 83, 84, 91, 92, 93, 94, 95,
photochemical transformations, 138 96, 99
physical characteristics, 39 process control, 68, 214
physical properties, 81, 131 process duration, 28
physicochemical properties, 32 project, 211, 248
physics, 210 proline, 32, 229
physiology, 32, 41, 53, 86 propagation, 221, 233, 234, 237, 238, 239, 242
phytopharmacy, 8 protection, 37, 52, 105
phytosterols, 104 protein sequence, 188
pitching, 221, 226, 228, 230, 236, 238, 239, 249, protein synthesis, 245, 246
250, 254 proteins, 30, 32, 39, 110, 124, 125, 126, 188, 217,
pitching rate, 221, 226, 228, 230, 236 218, 220, 221, 222, 225, 229, 234, 237, 240, 258,
plants, 144, 181, 220 268
plasma membrane, 234, 236 proteomics, 124, 146
plasmid, 233 protons, 238
Plato, 220 Pseudomonas aeruginosa, 92
PLS, 252 publishing, 79
pola purgative, 14, 21
polarity, 123, 192 purification, 218
policy, 239 PVA, 39, 81, 90, 97
polyacrylamide, 38, 121 P-value, 76
polyamides, 40 pyridoxine, 178
polyamines, 234 pyrolysis, 226
polycondensation, 30
polyelectrolyte complex, 31, 39, 40
polymer, 30, 31, 38, 39, 40, 47, 49, 50
Q
polymerase, 223, 247
quality assurance, 236
polymerase chain reaction, 223, 247
quality control, 122, 128, 218, 237
polymerization, 30, 39, 120, 137, 161
quantification, 122, 123, 124, 137, 161, 162, 178,
polymers, 39, 40, 137
179, 185, 188, 237
polymorphism, 226
quantum chemistry, 131
polypeptide, 222, 223
quercetin, 141
284 Index

R S

radicals, 175 S. cerevisiae, 37, 41, 52, 53, 54, 56, 57, 58, 59, 60,
raw materials, 213, 214, 215, 216, 217, 226, 232, 61, 62, 63, 69, 71, 74, 76, 78, 104, 219, 223, 224,
247, 248 225, 226, 233, 238, 240, 241, 242, 243, 246, 252,
reaction mechanism, 177 262
reaction time, 28, 40, 41 S. pastorianus, 219, 223, 224, 226, 229, 240, 243,
reactions, 29, 122, 131, 138, 143, 144, 163, 167, 169, 244, 245, 246, 247, 251, 257, 258, 259, 261, 262,
222, 231, 232 263, 264, 265, 270
reactive oxygen, 236, 242 Saccharomyces sensu stricto group, 224
reactivity, 40 safety, 94, 190
receptors, 103, 225, 234 salt, 14, 15, 16, 38, 40, 185, 192, 216
recombination, 234 saturated fatty acids, 33, 235
recommendations, iv saturated hydrocarbons, 141
recovery, 27, 155 saturation, 259
red wine, 89, 99 sawdust, 29
reducing sugars, 41 scanning electron microscopy, 261
refractive index, 123 science, 79, 91, 92, 99, 214
regeneration, 28, 29 scientific papers, 211
regulations, 185 scrofulous tumours, 19
reinforcement, 38 SDS, 121, 124, 126
relevance, 104, 114, 120, 238 secondary metabolism, 55, 56
reliability, 155, 236 secrete, 36
repitching, 213, 214, 222, 227, 236, 239, 241, 242, secretion, 36, 82, 228
243, 244, 245, 246, 247, 249, 250, 251, 252, 253, sediment, 225
254 sedimentation, 238, 239, 240, 244
replication, 239 seed, 125
repression, 31, 228 selectivity, 162
reproduction, 221 selenium, 117
requirements, 44, 46, 227, 242 sensation, 35, 36
research funding, 192 sensitivity, 29, 123, 162, 178, 235
researchers, 39 sensors, 91, 96, 99
residues, 129, 163, 188, 216, 225, 228 Serbia, 89, 211
resins, 141, 216, 219, 258 serial repitching, 214, 223, 225, 236, 239, 240, 241,
resistance, 30, 33, 49, 51, 54, 70, 71, 74, 79, 242 244, 245, 246, 247
resolution, 116, 121, 123, 124, 161, 170, 192 serine, 32, 229
resources, 213, 247 Shampoos, 17, 19, 20
respiratory-deficient yeasts, 236, 240 shape, 29, 32, 37, 38, 39, 41
response, 36, 73, 87, 103, 213, 223, 233, 234, 235, shear, 29, 234
236 shelf life, 93, 94
restaurants, 215 shock, 233
restriction fragment length polymorphis, 223 short-chain fatty acids, 232
restructuring, 234 showing, 78, 101, 103, 104, 111, 112, 246, 267
Rhizopus, 93 signals, 123, 133, 135
riboflavin, 178 signs, 185
ribose, 133, 216 silica, 223
ribosomal RNA, 235 simulation, 133
RNA, 235 sinews, 5, 6
robotics, 97 skeleton, 161
Romania, 90, 97, 98 skin, 19, 20, 25, 105, 141
room temperature, 105, 258, 273 skin cancer, 141
 Index 285

slurred speech, 7 substrate, 28, 32, 37, 41, 43, 44, 49, 52, 54, 55, 56,
smoothness, 83 57, 61, 70, 73, 74, 220, 257, 262, 264, 270
snake bite, 11, 14 sucrose, 31, 220, 229, 231, 260
snake or scorpion bite, 20 sulfate, 38, 121, 232, 235
snake venom, 11, 15 sulfur, 33, 36, 213, 215, 227, 232
social relations, 102 Sun, 40, 41, 83, 87, 88, 194, 201, 204, 209, 210
sodium, 42, 87, 185, 218 surface tension, 32
solid matrix, 29, 30 survival, 79, 80, 85, 227
solid phase, 41, 156 sustainable energy, 117
solubility, 39, 40, 64 sweat, 181
solution, 38, 39, 40, 41, 42, 46, 47, 48, 49, 50, 77, sweet beer, 4, 11, 12, 13, 14, 16, 17, 19, 20, 21
81, 106, 107, 123, 233, 258, 259 sweet gale, 9
solvents, 155 sweet wine, 10
sore eyes, 18 Switzerland, 209, 215
Spain, 101 synthesis, 31, 32, 33, 34, 35, 36, 56, 61, 63, 64, 65,
speciation, 117 70, 73, 78, 80, 90, 97, 211, 225, 228, 229, 230,
species, 123, 129, 131, 138, 139, 145, 182, 185, 188, 232, 234, 235, 236
219, 223, 224, 236, 242, 260 synthetic polymers, 30, 38
spectroscopy, 120, 122, 211
stability, 27, 30, 37, 38, 39, 40, 41, 43, 46, 48, 49,
50, 77, 81, 125, 179, 214, 217, 236, 239
T
stabilization, 222, 223
tanks, 219, 223
stale beer, 14
tannins, 105, 144, 160, 161
standard deviation, 121
taxes, 214, 215
standard error, 108, 111
taxonomy, 223, 240
standardization, 214
techniques, 38, 122, 124, 172, 192, 214, 246, 247
starch, 40, 82, 102, 215, 217, 218, 220
technologies, 83, 88, 91, 94, 95, 96, 99
starch granules, 220
technology, 54, 68, 81, 88, 89, 92, 96, 98, 122, 124,
starvation, 227, 233, 236
170, 181, 272
state, 29, 40, 122, 192, 209, 236, 238
temperature, 29, 33, 34, 35, 36, 37, 39, 40, 43, 54,
statistical processing, 50
56, 68, 70, 71, 73, 74, 75, 78, 80, 83, 86, 98, 105,
statistics, 114, 117
219, 220, 221, 225, 231, 233, 234, 237, 239, 258,
steel, 29
260, 261, 262, 264, 265, 267, 269, 273, 274
sterile, 42, 43, 105, 221
tensions, 48
sterility, 8, 188
terpenes, 137, 138, 139
sterilizing, 19
terpenic compounds, 213, 215, 232, 233
sterols, 32, 227, 234, 246
testing, 50, 124, 131, 167, 185
stimulation, 34, 35
tetracycline, 19, 24
stoichiometry, 29
Thermal, 94, 122, 161, 192, 234, 235, 273
stomach pain, 3, 11
thermal stability, 122
storage, 39, 83, 94, 106, 124, 125, 163, 164, 165,
thermodynamics, 124, 173
167, 216, 227, 233, 234, 235, 236, 239, 243, 246,
thick beer, 17
247
threonine, 32, 33, 229
stress, 30, 36, 213, 220, 223, 233, 234, 235, 236,
threshold level, 268
238, 239, 240, 241, 242, 243, 244, 246, 247
toluene, 157
stroke, 102
tooth problems, 12
strong beer, 8, 10, 11, 12, 17
top-fermenting ale yeasts, 214
structure, 32, 37, 38, 40, 41, 55, 69, 70, 129, 165,
toxic effect, 153
181, 185, 235, 246, 262
toxicity, 39, 122, 185
styrene, 258, 267, 268
transformation, 139, 167, 168, 169, 170, 171, 172,
substitution, 126, 128, 133, 136, 157, 187, 188
174, 187, 221
286 Index

transformation product, 168, 170, 171 vitamin B3, 178

transformations, 141, 167 vitamin B6, 178
transmission, 257, 259, 262 vitamin C, 105, 178
transmission electron microscopy (TEM), 257, 262 vitamin E, 104
transplantation, 80 vitamins, 102, 119, 122, 178, 216, 218, 258
transport, 228, 235 volatile compounds, 215, 219, 221, 223, 226, 240,
treatment, 92, 105, 107, 115, 119, 123, 157, 185, 243, 245, 247, 258, 267, 268, 270
218, 257, 259, 261, 262, 263, 264, 265, 267, 272, volatility, 123
273 volatilization, 106
trial, 117 vomiting, 185
tricarboxylic acid, 232
tricarboxylic acid cycle, 232
W
trypsin, 128
tryptophan, 32, 229
water, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 18, 21, 32,
tumors, 185 37, 38, 40, 42, 102, 104, 105, 106, 122, 215, 217,
Turkey, 215 218, 220
tyramine, 181 water-soluble polymers, 37
tyrosine, 32, 127, 229 wine, 1, 2, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 20, 23,
27, 88, 89, 96, 97, 102, 114, 115, 116, 117, 181,
U 214, 224, 233, 249, 252, 254
wood, 29
ulcerations, 5 workflow, 217
ultrasound, 121 World Health Organization (WHO), 185, 209
United States (USA), 103, 107, 195, 248 worldwide, 28, 126
urinary tract disease, 16 wort, 18, 28, 31, 32, 33, 34, 35, 36, 42, 43, 44, 52,
urticaria, 125 53, 54, 58, 60, 62, 63, 64, 65, 68, 69, 70, 74, 78,
80, 85, 87, 88, 93, 105, 108, 110, 112, 117, 167,
172, 215, 218, 219, 220, 221, 226, 227, 228, 229,
V 230, 231, 232, 235, 236, 239, 241, 242, 243, 244,
245,246, 248, 249, 251, 252, 253
validation, 188
Wound Washes, 18
valine, 32, 33, 34, 85, 229, 230, 265
variables, 50, 69, 73, 101, 213
variations, 236, 244, 245, 247 X
vegetables, 144, 232
vehiculum, 3, 8, 9, 10, 12, 14 xenotransplantation, 82
velocity, 267
Very high gravity (VHG) fermentations, 220, 252
Y
vessels, 221, 225, 239
viability, 28, 30, 31, 40, 115, 221, 223, 234, 235, yeast propagation, 221, 233, 234
236, 237, 239, 240, 241, 242, 243, 244, 246, 247, yeast volatile metabolites, 227
252, 253 Yeasts metabolism, 226
vicinal diketones, 28, 33, 44, 72, 77, 213, 215, 222,
yeasts taxonomy, 223, 240
227, 229, 230, 231
yield, 32, 41, 44, 48, 49, 50, 56, 58, 60, 63, 64, 143,
viscosity, 31, 39 163, 220
vitality, 115, 223, 234, 236, 237, 238, 240, 241, 246,
247, 250, 251, 252, 253, 254
vitamin B1, 178 Z
vitamin B12, 178
vitamin B2, 178 zinc, 34, 112, 218, 245