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collected (skimmed) for reuse from the surface of the

fermenting wort, whereas bottom yeasts are collected

(cropped) from the bottom of the fermenter. The

difference between lager and ales on the basis of

bottom and top cropping has become less distinct

with the advent of cylindroconical fermenters,

where the yeast sediments to the base of the vessel,

and centrifuges, where the yeast remains in suspen-

sion throughout the fermentation.

0004 There is a plethora of literature describing the gen-

etics and biochemistry of Saccharomyces cerevisiae

laboratory strains, but there is a lack of knowledge

regarding the genetics and biochemistry of industrial

Saccharomyces strains. The haploid strain that the

molecular biologist employs in the university labora-

tory as the organism of choice is usually totally un-

suitable for use in breweries. Brewing yeasts and

many other industrial yeasts have been selected over

time for those characteristics that render them un-

amenable to easy genetic manipulation in the labora-

tory. They are usually polyploid or aneuploid, lack a

mating-type characteristic, and sporulate poorly, if at

all, and the spores that do form are usually not in

fours, exhibiting a poor spore viability and rendering

tetrad analysis difficult.

0005 The objectives of wort fermentation are to consist-

ently metabolise wort constituents into ethanol and

other fermentation products in order to produce beer

with a satisfactory quality and stability. Another

objective is to produce yeast crops that can be confi-

dently repitched into subsequent brews. During the

brewing process, overall yeast performance is con-

trolled by a plethora of factors. These factors include:

0006 the yeast strains employed and their condition at

pitching and throughout fermentation;

0007 the concentration and category of assimilable

nitrogen;

0008 the concentration of ions;

0009 the fermentation temperature;

0010 the pitching (inoculation) rate;

0011 the tolerance of yeast cells to stress factors such as

osmotic pressure and ethanol;

0012 the wort gravity;

0013 the oxygen level at pitching;

0014 the wort sugar spectrum; and

0015 yeast flocculation characteristics.

These factors influence yeast performance either indi-

vidually or in combination with others and also to-

gether permit the definition of the requirements of an

acceptable brewer’s yeast strain.

0016 In order to achieve a beer of high quality, it is axiomatic

that not only must the yeast be effective in removing

the required nutrients from the growth/fermentation

medium (wort), able to tolerate the prevailing environ-

mental conditions (for example, ethanol tolerance) and

impart the desired flavour to the beer, but the micro-

organisms themselves must be effectively removed from

the wort by flocculation, centrifugation and/or filtration

after they have fulfilled their metabolic role.

0017It is worth noting that brewing is the only major

alcoholic beverage process that recycles its yeast. It

is therefore important to jealously protect the quality

of the cropped yeast because it will be used to pitch a

later fermentation and therefore will have a profound

effect on the quality of the beer resulting from it.

0018Over the years, considerable effort has been de-

voted in many research laboratories to the study of

the biochemistry and genetics of brewer’s yeast (and

industrial yeast strains in general). The objectives of

these studies have been twofold:

0019to learn more about the biochemical and genetic

makeup of brewing yeast strains; and

0020to improve the overall performance of such strains,

with particular emphasis being placed on broader

substrate utilization capabilities, increased ethanol

production, and improved tolerance to environ-

mental conditions such as temperature, high

osmotic pressure and ethanol, and finally, to under-

stand the mechanism(s) of flocculation.

When yeast is pitched into wort, it is introduced into

an extremely complex environment due to the fact

that wort is a medium consisting of simple sugars,

dextrins, amino acids, peptides, proteins, vitamins,

ions, nucleic acids, and other constituents too numer-

ous to mention. One of the major advances in

brewing science during the past 30 years or so

has been the elucidation of the mechanisms by

which the yeast cell, under normal circumstances,

utilizes in a very orderly manner the plethora of

wort nutrients.

Pathway and Regulation of Fermentation

0021Wort contains the sugars sucrose, fructose, glucose,

maltose, and maltotriose, together with dextrin ma-

terial. In the normal situation, brewing yeast strains

(ale and lager strains) are capable of utilizing sucrose,

glucose, fructose, maltose, and maltotriose in this

approximate sequence (or priority), although some

degree of overlap does occur. The majority of brewing

strains leave the maltotetraose and other dextrins

unfermented, but Saccharomyces diastaticus is able

to utilize dextrin material as a result of the secretion

of the extracellular enzyme glucoamylase. The initial

step in the utilization of any sugar by yeast is usually

either its passage intact across the cell membrane or

its hydrolysis outside the cell membrane, followed by

BEERS/Biochemistry of Fermentation 435

entry into the cell by some or all of the hydrolysis

products. Maltose and maltotriose are examples of

sugars that pass intact across the cell membrane,

whereas sucrose (and dextrin with Saccharomyces

diastaticus) is hydrolyzed by an extracellular enzyme,

and the hydrolysis products are taken up into the cell.

Maltose and maltotriose are the major sugars in

brewer’s wort, and as a consequence, a brewer’s

yeast’s ability to use these two sugars is vital and

depends upon the correct genetic complement.

Brewers’ yeast may possess independent uptake

mechanisms (maltose and maltotriose permease) to

transport the two sugars across the cell membrane

into the cell. Once inside the cell, both sugars are

hydrolyzed to glucose units by the a-glucosidase

system. It is important to reemphasize that the trans-

port, hydrolysis, and fermentation of maltose are

particularly important in brewing, since maltose

usually accounts for 50–60% of the fermentable

sugar in wort. Maltose fermentation in Saccharo-

myces yeasts requires at least one of five unlinked

(each independent) MAL (maltose) loci each consist-

ing of three genes encoding the structural gene for a-

glucosidase (maltase) (MAL S), maltose permease

(MAL T) and an activator (MAL R) whose product

coordinately regulates the expression of the a-gluco-

sidase and permease genes. The expression of MAL S

and MAL T is regulated by maltose induction and

glucose repression. When glucose concentrations are

high (greater than 1% (w/v)), the MAL genes are

repressed, and only when 40–50% of the glucose

has been taken up by yeast from the wort will the

uptake of maltose and maltotriose commence. Thus,

the presence of glucose in the fermenting wort exerts

a major repressing influence on wort fermentation

rate. Using the glucose analog 2-deoxy-glucose (2-

DOG), which is not metabolized by Saccharomyces

strains, spontaneous variants of brewing strains have

been selected in which the maltose uptake is not

repressed by glucose, and as a consequence, these

variants (called derepressed) have increased wort fer-

mentation rates. Once the sugars are inside the cell,

they are converted via the glycolytic pathway into

pyruvate. To date, only derepressed ale strains have

been isolated. Repressed lager strains have not been

found.

0022 Active yeast growth involves the uptake of nitro-

gen, mainly in the form of amino acids, for the syn-

thesis of proteins and other nitrogenous compounds

of the cell. Later in the fermentation, as yeast multi-

plication stops, nitrogen uptake slows down or

ceases. In wort, the main nitrogen source for synthesis

of proteins, nucleic acids and other nitrogenous cell

components is the variety of amino acids formed from

the proteolysis of barley proteins. Brewer’s wort

contains 19 amino acids, and, as with wort sugars,

the assimilation of amino acids is ordered. Four

groups of amino acids have been identified on the

basis of assimilation patterns (Table 1). Those in

group A are utilized immediately following yeast

pitching, whereas those in group B are assimilated

more slowly. Utilization of group C amino acids com-

mences when group A types are fully assimilated.

Proline, the most plentiful amino acid in wort and

the sole group D amino acid, is utilized poorly or not

at all. Proline is usually still present in beer at 200–

300 mg l

1

. However, under aerobic conditions, pro-

line is assimilated after exhaustion of the other amino

acids, since its uptake requires the presence of mitro-

chondrial oxidase.

0023The regulation of amino acid uptake by brewers’

and related yeast strains is complex, involving car-

riers specific to certain amino acids and a general

amino acid permease of broad substrate specificity.

The utilization pattern of wort nitrogen is due to a

combination of the range of permeases present, their

specificity, and feedback inhibition effects resulting

from the composition of the yeast intracellular

amino acids.

0024The metabolism of assimilated amino nitrogen is

dependent on the phase of the fermentation and on

the total quantity provided in the wort. The majority

of amino nitrogen is ultimately utilized in protein

synthesis and, as such, is vital for yeast growth. It

would appear that amino acids are not usually in-

corporated directly into proteins but are involved in

transamination reactions, a significant proportion of

the amino acid skeletons of yeast protein being de-

rived via the catabolism of wort sugars. This explains

why the total amino content of wort is important in

determining the extent of yeast growth, the amino

acid spectrum being somewhat secondary. However,

the amino acid spectrum of wort does influence beer

flavor.

0025The yeast assimilates the wort amino acids, a trans-

aminase system removes the amino group, and the

tbl0001Table 1 Classification of amino acids according to their spread

of absorption from wort by a brewing yeast strain

(A) Fast

absorption

(B) Intermediate

absorption

(D) Little orno

absorption

Glutamic acid Valine Glycine Proline

Aspartic acid Methionine Phenylalanine

Asparagine Leucine Tyrosine

Glutamine Isoleucine Tryptophan

Serine Histidine Alanine

Threonine Ammonia

Lysine

Arginine

436 BEERS/Biochemistry of Fermentation

carbon skeleton is anabolized, creating an intracel-

lular oxo-acid pool. The oxo-acid pool generated by

the transaminases and anabolic reactions is a precur-

sor of aldehydes and higher alcohols that contribute

to beer flavor. Thus, the formation of higher alcohols

(i.e., higher in number of carbon atoms than ethanol)

is tied in with nitrogen metabolism.

0026 The main nitrogen composition of wort has far-

reaching effects on fermentation performance and

on beer flavor. Where malt is used as the principal

source of extract, the quantity and composition of

amino acids are such that these problems are not

encountered. However, care must be exercised when

using adjuncts, many of which are relatively deficient

in amino nitrogen.

0027 Wort fermentation in beer production is largely

anaerobic, but when the yeast is first pitched into

wort, some oxygen must be made available to the

yeast. Indeed, it is now evident that this is the only

point in the brewing process where oxygen is benefi-

cial. Oxygen must be excluded as far as possible from

all other parts of the process, because it will have

a negative effect on beer quality. Specifically, it

will promote beer flavor instability. The widespread

adoption of high-gravity brewing procedures has

increased our awareness of the importance of oxygen

during wort fermentation and has stimulated basic

and applied research on the mechanisms of oxygen

interactions during cell growth and the application of

this knowledge in the process.

0028 Oxygen has a profound influence on the activity of

yeasts and particularly on yeast growth. Certain yeast

enzymes only react with oxygen, and this cannot be

replaced by other hydrogen acceptors. This applies to

the oxygenases involved in the synthesis of unsatur-

ated fatty acids and sterols, which are vital compon-

ents of cell membranes. Quantitative studies on the

effect of aeration on yeast growth and fermentation

have been given little serious consideration until the

last 25 years. The traditional concept of beer fermen-

tation was that growth occurred prior to the fermen-

tation of most wort sugars and that fermentation was

carried out by nongrowing, stationary phase cells. It

is now known that yeast growth, sugar utilization,

and ethanol production are coupled phenomena. For

example, the rate of fermentation by growing, expo-

nential phase cells of an ale yeast strain is 33-fold

higher than that of nongrowing cells.

0029 For a brewery fermentation to proceed rapidly,

there must be sufficient amounts of yeast synthesized.

Inadequate growth of a brewer’s yeast culture will

result in poor attenuation, altered beer flavor, incon-

sistent fermentation times, and recovered pitching

yeasts, which are undesirable for subsequent fermen-

tations. Trace amounts of oxygen have profound

stimulatory effects on yeast fermentation and

particularly on yeast growth. Pasteur demonstrated

that oxygen was necessary for normal yeast reproduc-

tion, although excessive wort aeration caused un-

desirable flavor effects on the finished beer. Oxygen

requirements were confirmed by such early notable

brewing researchers as Adrian Brown, Horace

Brown, and Frans Windisch. Windisch concluded

that overvigorous aeration of fermenting worts led

to yeast ‘weakness,’ illustrated by increasingly slug-

gish fermentations characterized by longer lag phases,

a slower specific rate of fermentation, and/or residual

sugar remaining in the final beer. The critical import-

ance of oxygen was confirmed when, in 1954, it was

shown that, under anaerobic conditions, Saccharo-

myces yeast strains require both preformed sterols

and unsaturated fatty acids as growth factors. These

two lipids are both found in membranes and are

critical for membrane function and integrity. Both of

these lipid classes require molecular oxygen for their

biosynthesis.

0030Lipids in beer quantitatively form an almost negli-

gible component, but can influence a beer’s organo-

leptic and physiochemical properties. Malt is the

main source of unsaturated fatty acids in wort. Wort

concentrations of these acids are suboptimal and can

be growth-limiting. During fermentation, yeast can

take up free fatty acids from wort, most of which are

incorporated as structural lipids.

0031Yeast cultures synthesize fatty acids throughout

fermentation, but the ratio of the acids varies with

time. Unsaturated fatty acid (for example, palmitoleic

(C16:1) and oleic (C18:1) acids) synthesis only occurs

in the presence of dissolved oxygen. Oxygen is pre-

sent in aerated/oxygenated pitched wort for a rela-

tively short period (3–9 h), and during this period,

there is a large increase in the percentage of unsatur-

ated fatty acids. When oxygen is depleted, there is an

increase in the production of short-chain fatty acids

(C6–C12).

0032The sterol component of brewing yeast ranges from

0.05 to 0.45% of the cellular dry weight (depending

on the prevailing environmental conditions) and ac-

counts for less than 10% of the total cell lipid. Ergos-

terol is the major sterol in brewing-yeast strains and

can account for over 90% of the total sterol. The

biosynthetic pathway for sterol formation is complex.

The important fact for this chapter is that the precur-

sor sequences can be synthesized anaerobically, but

the final reaction that produces ergosterol requires

molecular oxygen. The major function of sterols in

yeast is to contribute to the structure and dynamic

state of the membranes. The primary role is to modu-

late membrane fluidity under fluctuating environ-

mental conditions. For example, ergosterol confers

BEERS/Biochemistry of Fermentation 437

increased resistance to ethanol and multiple freeze–

thaw effects. A decrease in the ergosterol level of

membranes has been directly related to a reduction

in cell viability in the presence of ethanol.

0033 Pitching yeasts are propagated under weakly aer-

ated conditions or recovered from previous fermenta-

tions. In both cases, the cells are lipid-depleted, and to

promote normal growth and attenuation, either pre-

formed lipids must be added to the wort or oxygen

must be made available for their synthesis. In

commercial brewing, only the second alternative is

feasible. Wort is cooled and aerated/oxygenated to

8–16 mg l

1

dissolved oxygen. Within a few hours of

pitching, most of this oxygen is removed from the

wort. During this time, there is intensive synthesis of

lipid (sterol and fatty acid) and a decrease in cellular

glycogen. In practice, sterol synthesis by brewing

yeasts in the presence of oxygen appears to be of

greater significance than unsaturated fatty acid

synthesis. This may be due to the contribution of

wort to the fatty acid pool. Wort does not contribute

exogenous sterol to the fermentation.

0034 Although ethanol is the major excretion product

synthesized by yeast during wort fermentation, this

primary alcohol has little impact on the flavor of the

final beer. It is the type and concentration of the many

other yeast excretion products formed during wort

fermentation that primarily determine the flavor of

the beer. The formation of these excretion products

depends on the overall metabolic balance of the yeast

culture, and there are many factors that can alter this

balance and, consequently, beer flavor. Yeast strain,

fermentation temperature, adjunct type and level,

fermenter design, wort pH, buffering capacity, wort

gravity, etc., are all influencing factors.

Flavor Formation

0035 Some volatiles are of great importance and contribute

significantly to beer flavor, whereas others are im-

portant in building background flavor. The following

groups of substances are found in beer: organic and

fatty acids, alcohols, esters, carbonyls, sulfur com-

pounds, amines, phenols, and a number of miscellan-

eous compounds. In flavor terms, the higher alcohols

(also called fusel oils) that occur in beer and many

spirits are: n-propanol, isobutanol, 2-methyl-1-buta-

nol, and 3-methyl-1-butanol. However, more than 40

other alcohols have been identified. Regulation of the

biosynthesis of higher alcohols is complex, since they

may be produced as byproducts of amino acid catab-

olism or via pyruvate derived from carbohydrate

metabolism.

0036 Esters are important flavor components that

impart flowery and fruit-like flavors and aromas to

beers, wines, and spirits. Their presence is desirable at

appropriate organoleptic concentrations, but failure

to properly control fermentation can result in un-

acceptable beer ester levels. Organolepiticaly import-

ant esters include ethyl acetate, isoamyl acetate,

isobutyl acetate, ethyl caproate, and 2-phenylethyl

acetate. In total, over 90 distinct esters have been

detected in beer.

0037Some 200 carbonyl compounds are reported to

contribute to the flavor of beer and other alcoholic

beverages. Those influencing beer flavor, produced as

a result of yeast metabolism during fermentation,

are various aldehydes and vicinal diketones, notably

diacetyl. Also, carbonyl compounds exert a signifi-

cant influence on the flavor stability of beer. Excessive

concentrations of carbonyl compounds are known to

cause stale flavor in beer. The effects of aldehydes on

flavor stability are reported as grassy notes (propanol,

2-methyl butanol pentanol) and a papery taste (trans-

2-nonenal, furfural).

0038Quantitatively, acetaldehyde is the most important

aldehyde. This is produced via the decarboxylation of

pyruvate and is an intermediate in the formation

of ethanol. It may be present in beer at concentrations

above its flavor threshold (approx. 10 mg l

1

), at

which it imparts an undesirable ‘grassy’ or ‘green

apple’ character. Acetaldehyde accumulates during

the period of active growth. Levels usually decline in

the stationary phases of growth late in fermentation.

As with higher alcohols and esters, the extent

of acetaldehyde accumulation is determined by the

yeast strain and the fermentation conditions.

Although the yeast strain is of primary importance,

elevated wort oxygen concentration, pitching rate,

and temperature all favor acetaldehyde accumula-

tion. In addition, the premature separation of yeast

from fermented wort does not allow the reutilization

of excreted acetaldehyde associated with the later

stages of fermentation.

0039Other important flavor-active carbonyls, whose

presence in beer is determined in the fermentation

stage, are the vicinal diketones, diacetyl (2,3-butane-

dione) and 2,3-pentanedione. Both compounds

impart a ‘butterscotch’ flavor and aroma to beer.

Quantitatively, diacetyl is the most important, since

its flavor threshold is approx. 0.1 mg l

1

and

is 10-fold lower than that of 2,3-pentanedione. The

organoleptic properties of vicinal diketones con-

tribute to the overall palate and aroma of some ales,

but in most lagers, they impart an undesirable

character. A critical aspect of the management of

larger fermentations and subsequent maturation is

to ensure that the mature beer contains concentra-

tions of vicinal diketones lower than their flavor

threshold.

438 BEERS/Biochemistry of Fermentation

0040 Diacetyl and 2,3-pentanediones arise in beer as

byproducts of the pathways leading to the formation

of valine and isoleucine. The a-acetohydroxy acids,

which are intermediates in these biosyntheses, are

in part excreted into the fermenting wort. Here,

they undergo spontaneous oxidative decarboxyla-

tion, giving rise to vicinal diketones. Further metab-

olism is dependent on yeast dehydrogenases. Diacetyl

is reduced to acetoin and, ultimately, 2,3-butanediol,

and 2,3-pentanedione to its corresponding diol. The

flavor threshold concentrations of these diols are

relatively high, and therefore the final reductive

stages of vicinal diketone metabolism are critical in

order to obtain a beer with acceptable organoleptic

properties.

0041 The diacetyl concentration peak occurs towards

the end of the period of active growth. The reduction

of diacetyl takes place in the later stages of fermenta-

tion when active growth has ceased. In terms of prac-

tical fermentation management, the need to achieve a

desired diacetyl specification may be the factor that

determines when the beer may be moved to the con-

ditioning phase, filtered or centrifuged (depending on

the processing procedures). Thus, diacetyl metabol-

ism is an important determinant of overall vessel

residence time, which clearly affects the efficiency of

plant utilization.

0042 Sulfur compounds make a significant contribution

to the flavor of beer. Although small amounts of

sulfur compounds can be acceptable or even desirable

in beer, in excess, they give rise to unpleasant off-

flavors, and special measures such as purging with

or prolonged maturation times are necessary to

remove them. Many of the sulfur compounds present

in beer are not directly associated with fermentation

but are derived from the raw materials employed.

However, the concentrations of hydrogen sulfide

(rotten egg aroma) and sulfur dioxide (burnt match

aroma) are dependent on yeast activity. Failure to

manage fermentation properly can result in unaccept-

ably high levels of these compounds occurring in the

finished beer.

0043 The concentration of hydrogen sulfide and sulfur

dioxide formed during fermentation are primarily

determined by the yeast strain used, although the

wort composition and the fermentation conditions

are major factors, particularly where levels are abnor-

mally high. Both compounds arise as byproducts of

the synthesis of the sulfur-containing amino acids

cysteine and methionine from sulfate. Their synthesis

is influenced by wort composition in that the yeast

will preferentially assimilate sulfur-containing amino

acids. It is only when wort is depleted in such

amino acids that the biosynthetic route comes into

operation.

Flocculation

0044The flocculation property or, conversely, lack of floc-

culation of a particular yeast culture is one of the

major factors when considering important character-

istics during brewing and other ethanol fermenta-

tions. Unfortunately, some degree of confusion has

arisen by the use of the term flocculation in the scien-

tific literature to describe different phenomena in

yeast cell behavior. Specifically, flocculation, as it

applies to brewer’s yeast is ‘ the phenomenon wherein

yeast cells adhere in clumps and either sediment from

the medium in which they are suspended or rise to the

medium’s surface.’ This definition excludes other

forms of aggregation, particularly that of ‘clumpy-

growth’ and ‘chain formation.’ This nonsegregation

of daughter and mother cells during growth has

sometimes erroneously been referred to as floccula-

tion. The term ‘nonflocculation’ therefore applies

to the lack of cell aggregation and, consequently, a

much slower separation of (dispersed) yeast cells

from the liquid medium. Flocculation usually occurs

in the absence of cell division, but not always, during

late logarithmic and stationery growth phase and

only under rather circumscribed environmental con-

ditions involving specific yeast cell surface compon-

ents (proteins and carbohydrate components) and an

interaction of calcium ions. Although yeast separ-

ation often occurs by sedimentation, it may also be

by flotation because of cell aggregates entrapping

bubbles of CO

as in the case of ‘top-cropping’ ale

brewing yeast strains.

0045Individual strains of brewer’s yeast differ consider-

ably in flocculating power. At one extreme, there are

highly nonflocculent, often referred to as powdery,

strains. At the other extreme, there are floccu-

lent strains. The latter tend to separate early from

suspension in fermenting wort, giving an underatte-

nuated, sweeter, and less fully fermented beer. Beers

of this nature, because of the presence of fermentable

sugars, are liable to biological instability. By contrast,

poorly flocculent (nonflocculent or powdery) yeasts

produce a dry, fully fermented, more biologically

stable beer in which clarification is slow, leading to

filtration difficulties and the possible acquisition of

yeasty off-flavors. The disadvantages presented by

the two types of yeast strain are especially relevant

to more traditional fermentation systems where the

fermentation process is dependent upon the sedimen-

tation characteristics of the yeast.

0046Contemporary brewing technology has largely re-

versed this situation where yeast sedimentation

characteristics are now fitted into the fermenter

design. The efficiency, economy, and speed of batch

fermentations have been improved by the use of

BEERS/Biochemistry of Fermentation 439

cylindroconical fermentation vessels and centrifuges

(which are often, but not always, employed in

tandem). There is no doubt that differences in the

flocculation characteristics of various yeast cultures

are primarily a manifestation of the culture’s cell wall

structure. Several mechanisms for flocculation have

been proposed. One hypothesis is that anionic groups

of cell wall components are linked by Ca

2þ

ions. In all

likelihood, these anionic groups are proteins. Another

hypothesis implicates mannoproteins specific to floc-

culent cultures acting in a lectin-like manner to cross-

link cells; here, Ca

2þ

ions act as ligands to promote

flocculence by conformational changes. Most people

working in the field agree that the latter hypothesis is

the most credible. In addition to flocculation, there is

the phenomenon of coflocculation. Coflocculation is

defined as the phenomenon where two strains are

nonflocculent alone, but flocculent when mixed to-

gether. To date, coflocculation has only been observed

with ale strains, and there are no reports of cofloccu-

lation between two lager strains of yeast. There is a

third flocculation reaction that has been described

where the yeast strain has the ability to aggregate

and cosediment with contaminating bacteria in the

culture. Again, this phenomenon appears to be con-

fined to ale yeast strains, and cosedimentation of

lager yeast with bacteria has not been observed.

0047 As described above, flocculation requires the pres-

ence of surface protein and mannan receptors. If these

are not available or are masked, blocked, inhibited, or

denatured, flocculation cannot occur. The onset of

flocculation is an aspect of the subject where there is

great commercial interest but about which relatively

little is known. As previously discussed, the ideal

brewing strain remains in suspension as fermenting

single cells until the end of fermentation when the

sugars in the wort are depleted, and only then does

it rapidly flocculate out of suspension. What signals

the onset of activation or relief from inhibition? This

is still an unanswered question that is currently being

studied by a number of research laboratories.

0048 Yeast flocculation is genetically controlled, and

research on this aspect of the phenomenon dates

from the early 1950s. However, because of the poly-

ploid/aneuploid nature of brewing yeast strains,

most, but not all, of the research on flocculation

genetics has been conducted on haploid/diploid gen-

etically defined laboratory strains. Numerous genes

have been reported to directly influence the floccu-

lent phenotype in Saccharomyces spp. Four dominant

flocculation genes have been identified: FLO1 (whose

alleles are FLO2, FLO4, FLO8), FLO5, FLO9,and

FLO10, as well as semidominant gene, flo3, and two

recessive genes, flo6 and flo7. In addition, mutations

in several genes, including the regulatory genes,

TUP1 and SSN6, have been found to cause floccula-

tion or ‘flaky’ growth in nonflocculent strains. In

total, at least 33 genes have been reported to be

involved in flocculation or cell aggregation. Although

the role of many of these genes is far from under-

stood, FLO1 and other FLO genes have

been successfully cloned into brewing strains and

the flocculation phenotype expressed.

See also: Alcohol: Properties and Determination; Beers:

History and Types; Raw Materials; Chemistry of Brewing;

Yeasts

Further Reading

Reed G and Nagodawithana TW (1991) Yeast Technolo-

gy.New York: Van Nostrand Reinhold.

Smart K and Boulton C (2000) Brewing Yeast Fermentation

Performance. New York: Blackwell Science Inc.

Stewart G G and Russell L (1986) One hundred years of

yeast research and development in the brewing industry.

Journal of the Institute of Brewing 92: 537–558.

Chemistry of Brewing

C W Bamforth, University of California, Davis, CA,

USA

Introduction

0001The word ‘brewing’ is used in at least two contexts in

beer making. Strictly speaking, the most accurate

usage of the term is to describe the process by which

is produced the feedstock, wort, that will be fed to the

yeast. Thus it is customary for those within the indus-

try to talk of ‘brewing and fermentation.’ Frequently,

however, the word brewing is used to describe the

entirety of the operation by which malted barley

(and other sources of sugar) and hops are converted

to beer. For the purposes of a description of the chem-

istry of brewing I am taking the latter definition, but

we must not ignore the prior process of malting (the

controlled germination of barley) because the conver-

sion of barley into wort involves this stage also. It is

very difficult to divorce a discussion of the mashing

stage of brewing from malting, for in reality they are

but successive stages in the enzymic conversion of

barley into wort. Figure 1 summarizes the structure

of barley and the distribution of the key polymers

within the starchy endosperm, whilst Table 1 sum-

marizes the key process stages of malting and

brewing. I will approach this somewhat complex

440 BEERS/Chemistry of Brewing

issue by focusing on the chemistry of the finished

product and highlighting how the balance of compon-

ents in beer is determined by the various stages occur-

ring in the maltings and brewery.

The Chemistry of Beer

0002 The properties of beer result from the presence of a

diversity of chemical compounds, some large (macro-

molecules) and some small. For the majority of beers

these materials are derived in their entirety from the

raw materials or are generated through the metabol-

ism of brewing yeast, Saccharomyces cerevisiae. Most

brewers are reluctant to introduce additives at any

stage of the process and will also place stringent

specifications on the water, grist, and hop materials

in respect of what may or may not be used in the

processes of their suppliers. Some brewers will use

propylene glycol alginate to protect foam on

the product from the deleterious impact of lipophilic

materials entering the beer at point of sale, for

example, fats from food or detergent in inadequately

rinsed glasses. Sulfur dioxide or ascorbic acid is some-

times (but increasingly sparingly) employed to protect

beer from staling. (See Yeasts.)

Ethanol

0003The equation at the heart of the production of all

alcoholic beverages is:

!

Yeast

OH þ 2CO

In the case of the majority of beers the sugar is derived

during mashing of malted barley. In fact, most of the

sugar derived in conventional mashing operations is

maltose, with lesser quantities of glucose, malto-

triose, sucrose, fructose, and dextrins.

0004The majority of beers worldwide contain between 3.5

and 5.5% alcohol by volume (ABV).

0005Ethanol impacts on the quality of beer in several

ways. It contributes directly to flavor, with a recog-

nizable warming characteristic absent from nonalco-

holic or low-alcohol products. It also affects the

contribution to aroma of other volatiles by influen-

cing their partitioning between the beer and its head

space.

0006Ethanol lowers surface tension, thereby promoting

bubble formation. Conversely, it competes with other

surface-active molecules for sites in the bubble wall,

leading to a lessening of foam stability. (See Alcohol:

Properties and Determination.)

fig0001 Figure 1 The structure of barley and of one of the cells of the starchy endosperm. A, embryo; B, starchy endosperm; C, aleurone;

D, hull; E, micropyle; F, cell wall (75% b-glucan; 20% arabinoxylan; 5% protein; traces ferulic and acetic acids); G, protein; H, large

starch granule; I, small starch granule.

BEERS/Chemistry of Brewing 441

Carbon Dioxide

0007 A typical brewery fermentation will produce some

150 g CO

per hectoliter for every degree Plato (1

degree Plato is basically equivalent to a 1% sugar

solution). Most of this CO

sweeps out and is lost

during fermentation. Many packaged beers will con-

tain 500–550 g CO

1

and extra gas needs to be

introduced to the product prior to packaging. At

1 atm pressure (as CO

) and 0



C, a beer will dissolve

no more than 200 g hl

1

. Achievement of the

very high levels of CO

demands the pressurizing of

beer. None the less, when a beer container is opened,

the gas usually stays in solution: the beer is ‘supersat-

urated’ with CO

. When fobbing occurs spontan-

eously when a can or bottle is dispensed, it is called

‘gushing.’ In the absence of agitation as a cause, the

most likely reason for the phenomenon is the presence

of low-molecular-weight, highly hydrophobic pep-

tides contributed by infection of barley, notably

from Fusarium.

0008CO

affords the ‘sparkle’ to beer, through its reac-

tion with the pain receptor mechanism of the trigem-

inal nerve. Apart from this influence on mouth feel,

establishes the extent of foam formation during

tbl0001 Table 1 The key events in malting and brewing

Processstage Treatments Events

Steeping of barley Water added, separated by air rests, to raise water

content of embryo and endosperm (target in

whole grain is usually 44–46%); takes up to 48 h

at 14–18



Water enters through micropyle. Distribution

through embryo, aleurone, and starchy

endosperm critical to enable modification.

Synthesis of hormones (including

gibberellins) by embryo, hydration of

substrate (starchy endosperm)

Germination of barley Controlled sprouting (modification) of grain –

typically 4–5 days at 16–20



Synthesis of enzymes by aleurone (triggered by

hormones) and migration into starchy

endosperm; sequential degradation of cell

walls, some protein, small starch granules,

and pitting of large granules

Kilning Heating of grain via increasing temperature regime

(50–220



C) for desired properties: enzyme

survival, removal of moisture for stabilization,

removal of raw flavors, development of malty

flavors and color

Enzyme survival greater with low-temperature

start to kilning and lower final curing

temperature. Increased heating of malts of

increased modification (i.e., higher sugar and

amino acid levels) gives increasingly

complex flavors and colors via Maillard

reactions

Malt storage 3–4 weeks’ ambient storage, otherwise wort

separation problems occur later

Unknown

Mashing Extraction of milled malt at temperatures between

40 and 75



C; typically < 1–2 h. Separation of wort

from spent grains by lautering or mash filtration

with sparging to recover extract fully. Spent

grains go to cattle feed

Enzymolysis continued, especially of starch

after gelatinization at > 62



Use of adjuncts Solid adjuncts used in brewhouse, taking

advantage of malt enzymes; rice and corn need

‘cooking’ at up to 100



C, < 1 h (liquid sugars are

products of acid and enzyme action in sugar

factory and added at boiling stage)

Cereals with higher starch gelatinization

temperatures than for barley need

precooking before combining with main mash

Boiling 1–2 h at 100



C, before clarification (< 1 h) and

cooling

To sterilize, extract hops, concentrate, and kill

all residual enzymes. Precipitation of

insoluble materials as trub, enhancing final

product stability

Fermentation Wort pitched with yeast and fermented for 3–14

days at 6–25



Fermentation of glucose, maltose, sucrose,

maltotriose to alcohol; enzymic production of

various flavorsome compounds (alcohols,

esters, fatty acids, sulfur-containing

compounds, etc.) Synthesis and removal of

diacetyl as an offshoot of amino acid

production

Cold conditioning and filtration 1



C for 3 days; possibly stabilization (silica

hydrogels, tannic acid or papain to remove

protein; PVPP to bind polyphenol); then filtration

Precipitation, sedimentation, and removal of

solids

PVPP, polyvinylpolypyrrolidone.

442 BEERS/Chemistry of Brewing

dispense and of course impacts heavily the delivery of

volatiles into the head space of beers and therefore the

aroma.

Other Gases in Beer

0009 Oxygen has major negative effects on beer via its

oxidation of various components, leading to card-

board and other stale notes and to the formation of

haze. Brewers therefore strive to minimize the oxygen

level in beer (preferably to 0.1 mg l

1

or ideally even

less) by avoiding ingress into the product downstream

of the fermenter (yeast is a powerful oxygen scaven-

ger and many brewers feel that it is only after the

removal of yeast that oxygen is a concern).

0010 The stale papery notes are due to a series of unsat-

urated carbonyl compounds, which are usually held

to include trans-2-nonenal, though this has been no

means fully substantiated. Several components of

beer can degrade to such carbonyl substances

during oxidation, including the ‘higher’ alcohols, the

iso-a-acids, and the unsaturated fatty acids.

0011 Nitrogen has long been introduced to beer to pro-

mote foam stability. The foams produced on beers

containing N

tend to have populations of much

smaller bubbles. These foams are more resistant to

collapse and the beer displays a much smoother

mouth feel. Typical levels of N

are 20–50 mg l

1

which are three orders of magnitude lower than

those of CO

Water

0012 The vast majority of beers contain between 90 and

95% water: apart from ethanol and carbon dioxide,

the remaining constituents usually amount to less

than 1% of the product. The water used for brewing

must have no taints or hazardous components and

water is often treated coming into the brewery to

adsorb any contaminants by charcoal filtration and

ultrafiltration. The ionic composition of water is also

significant (although the grist also contributes salts).

High levels of calcium, e.g., 200–350 mg l

1

, are

claimed to be desirable in the production of ales.

Much lower levels of calcium are traditionally associ-

ated with the brewing of lagers, for example, the

water in pilsen (the home of the classic lager-style)

contains less than 10 mg l

1

calcium. A scientific

justification for this is not entirely proven, but may

relate to the role of calcium in promoting the surface

behavior of the top-fermenting ale yeasts (calcium

promotes the flocculation of yeast). Brewers may

add salts or remove them (e.g., by reverse osmosis)

to adjust the ionic composition of the product.

0013 Two key impacts of calcium are on pH and the

levels of oxalic acid surviving into beer. Calcium

reacts with phosphate to lower the pH to the appro-

priate level for mashing:

3Ca

2þ

þ 2HPO

2

! Ca

ðÞ

þ2H

By precipitating out malt-derived oxalic acid in the

brewhouse, calcium eliminates a material that, if sur-

viving into beer, causes problems such as haze, gush-

ing, and the blocking of dispense pipes.

Carbohydrates

0014Although most of the sugars found in wort are con-

verted to ethanol by yeast, those containing four or

more glucoses (i.e., maltotetraose and bigger, and

known as dextrins) are not fermented. In order to

sweeten the finished beer, some brewers may contrive

to leave a proportion of fermentable sugar uncon-

verted, or may add ‘priming’ sugar.

0015Most of the starch survives malting, because it is

relatively resistant to enzymatic hydrolysis when in

the form of large granules. When starch is gelatinized

by heating, however, its constituents (amylose and

amylopectin) become accessible. Mashing, therefore,

incorporates a stage, typically at around 65



C, to

allow for the gelatinization of malt starch. The starch

in some other cereals has higher gelatinization tem-

peratures, e.g., rice and corn starches gelatinize over

the range 70–80



C and so are cooked separately

from the main mash and then mixed with the malt

mash to be degraded by the amylases from malt. (See

Carbohydrates: Classification and Properties.)

0016Starch hydrolysis starts with a-amylase, a highly

heat-resistant enzyme abundantly present in malt. It

is endo-acting, catalyzing the hydrolysis of a-1!4

bonds within the starch, releasing dextrins. From

amylose the enzyme release linear dextrins, and

from amylopectin it produces dextrins with side

chains. (See Starch: Structure, Properties, and Deter-

mination.)

0017b-Amylase is an exoenzyme, which removes mal-

tose units from the nonreducing ends of starch and

dextrin molecules. This enzyme has sufficient stabil-

ity at 65



C to achieve most of the conversion of

starch and dextrins of which it is capable but

if mashing is carried out at somewhat higher

temperatures, e.g., 72



C, then b-amylase is rapidly

inactivated and the ensuing wort contains a high

dextrin level and low content of fermentable sugars.

Such high-temperature mashing can be used in the

production of low-fermentability worts which will

give low-alcohol beers.

0018b-Amylase cannot pass the a1!6 branch points in

the dextrins formed from amylopectin. Limit dextri-

nase carries out this function but is generally present

in fairly low concentrations because it is developed

BEERS/Chemistry of Brewing 443

very late during barley germination and because it is

inactivated by association with another protein. Fur-

thermore, limit dextrinase is relatively heat-labile. It

is because of the limited action of limit dextrinase that

20–25% of the starch remains as dextrins in most

conventionally mashed beers.

0019 In so-called ‘light’ or ‘lite’ beers all of the starch is

converted to ethanol. One way of achieving this is to

add heat-stable glucoamylase or pullulanase from

microbial sources to the mash or to the fermenter,

although those brewers seeking to avoid any add-

itions may modify their brewhouse and fermenter

operations so as to take maximum opportunity of

endogenous enzymes.

0020 The major component of the barley cell walls is

a linear b-glucan comprising approximately 67%

b1!4 links with 33% b1!3 bonds that are for the

most part every third or fourth linkage. The key

enzyme hydrolyzing this molecule is an extremely

heat-sensitive endo-b-glucanase, which is destroyed

within 5 min of mashing at 65



C. Accordingly, one

of the main purposes of malting is to remove the cell

walls, in large part through the action of this enzyme.

In practice some cell wall material (at the distal end of

the barley) will survive and if it is not properly

degraded it can afford increased viscosity to the

wort and cause sluggish wort separation in the brew-

house and retarded beer filtration. Some brewers

mash-in at low temperatures such as 40–50



Cto

allow b-glucanase to act, before raising the tempera-

ture to that needed for starch gelatinization. Alterna-

tively, heat-stable b-glucanases from bacteria (e.g.,

Bacillus subtilis) or fungi (e.g., Trichoderma, Penicil-

lium) may be used.

Proteins, Polypeptides, and Amino Acids

0021 Amphipathic polypeptides in beer stabilize the

bubbles in foam. Their hydrophobic regions ‘drive’

them into the surfaces produced during foaming and

are largely responsible for their interaction with other

hydrophobic molecules, notably the iso-a-acids. The

matrix formed counters the force of surface tension

that seeks to minimize increased surface area gener-

ated in foaming. Some of the protein in beer can react

with polyphenols to form hazes.

0022There is no advantage in having significant levels of

residual amino acids in beer; rather they are a risk in

so far as they represent assimilable nitrogen sources

for spoilage microorganisms. However, it is import-

ant that wort contains the correct balance of amino

acids to support yeast growth and fermentation. Suf-

ficient proteolysis must occur during malting and

mashing to yield these amino acids and to remove

haze-potentiating proteins, whilst leaving ample

foam-positive polypeptide. (See Protein: Chemistry.)

0023The native proteins of barley undergo considerable

degradation and denaturation in malting and

brewing. During germination of barley, endopro-

teinases develop and attack the heart of substrate

proteins to release polypeptides and peptides. An

exoacting enzyme, carboxypeptidase, acts during

malting and mashing by splitting off amino acids

successively from the carboxyl-terminus of the pep-

tides produced by the endoproteinases.

Lipids

0024Barley comprises some 3% by weight lipid, most of it

in the embryo and aleurone. Very little survives into

beer, because it is removed by adsorption on insoluble

matrices (e.g., spent grain, trub) during the process.

Lipids are severely detrimental to beer foam, disrupt-

ing the network of proteins and iso-a-acids in the

bubble wall. As observed earlier, the unsaturated

fatty acid component of lipids is suspected to be at

least one precursor of the stale flavors (e.g., ‘card-

board’) that develop in beer. (See Fats: Classification.)

Flavors from Hops

0025Hops afford the bitterness (from the hop resins) and

aroma (from the essential oils) to beer.

0026The most important resins are the a-acids (Figure 2),

accounting for 2–15% of the dry weight of the hop,

Isomeri-

zation

trans cis

α-Acids Iso-α-acids

fig0002 Figure 2 The isomerization of a-acids. R ¼ –CH

CH(CH

)

in humulone and isohumulone. R ¼ –CH(CH

)

in cohumulone and

isocohumulone. R ¼ –CH(CH

)CH

in adhumulone and isoadhumulone.

444 BEERS/Chemistry of Brewing