10

Use of Amylolytic Enzymes in Brewing

N.P. Guerra, A. Torrado-Agrasar, C. López-Macías, E. Martínez-Carballo,
S. García-Falcón, J. Simal-Gándara and L.M. Pastrana-Castro Nutrition
and Bromatology Group, Department of Analytical and Food Chemistry,
Food Science and Technology Faculty, Ourense Campus, University
of Vigo, Ourense, Spain

Abstract List of Abbreviations

One of the main problems in the production of fermented alco- AA Total amylolytic activity in the mixture
holic beverages from amylaceous raw materials is the efficient AAt Total amylolytic activity (expressed as percentage
conversion of starch into fermentable sugars for Saccharomyces referred to the initial total activity) for the
cerevisiae.
-amylase, -amylase,
-glucosidase and limit dext- amylases mixture at time t in equation (10.3)
rinase are the enzymes responsible for the hydrolysis of starch AA

-Amylase activity (expressed as percentage
into maltose and glucose in beer elaboration. referred to the initial total activity) in
Two different strategies are used in traditional brewing: to equation (10.3)
favor the activity of the endogenous enzymes that are present a and b Pre-exponential parameters in equation (10.3)
in the ingredients during the malting and mashing steps (beer for each glucoamylase form present in the
in Western countries), or to use amylolytic microorganisms in a commercial enzyme.
previous step to yeast fermentation (koji in Eastern countries). DP Diastatic power
More recently, the development of technologies for the efficient E Total enzyme concentration
production of enzymes, mainly of microbial origin, has allowed EBC European Brewery Convention
the application of exogenous amylases in beer elaboration to EC Enzyme Commission
improve classical brewing by compensating enzymatic deficits EU Enzymatic units
in poor malts and by reducing the needs of malt for mashing. FAO Food and Agricultural Organization of the
But, moreover, the addition of exogenous enzymes has also United Nations
allowed the use of new starchy materials with low amylolytic GA3 Gibberellic acid
potential and the preparation of worts with adequate sugars GRAS Generally recognized as safe
composition to elaborate new kinds of beer with interesting I Inhibitor (glucose) concentration in
functional properties, as low-caloric and gluten-free beers for equation (10.2)
celiac people. ICRISAT International Crops Research Institute for the
The importance of the amylolytic potential of the amyla- Semi-Arid Tropics
ceous material, the synergic activity of the three main enzymes IoB Institute of Brewing
(
-amylase, -amylase and limit dextrinase) involved in starch IUBMB International Union of Biochemistry and
hydrolysis during mashing, and the need of a correct perforance Molecular Biology
of the malting and mashing steps to maximize the expression JECFA Joint FAO/WHO Expert Committee on
and activity of these enzymes and to minimize the losses of Food Additives
enzymatic activity due to the thermal treatments applied dur- ka and kb First order constants in equation (10.3)
ing brewing are here described, paying special attention to the for each glucoamylase form present in the
use of barley and sorghum as the most used starchy substrates commercial enzyme
and sources of amylases. Finally, examples of the application K m Operational Michaelis–Menten constant in
of exogenous enzymes in brewing for the use of new starchy equation (10.2)
materials as chestnut, and for the elaboration of new kinds of K s Operational substrate inhibition constant in
functional beers, are also included. equation (10.2)

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CH010.indd 113 1/17/2008 9:33:04 AM

 114 Beer Making, Hops and Yeast

KiC Operational competitive inhibition constant
-1,4-glucosidic bonds, and amylopectin, a branched mol-
in equation (10.2) ecule of
-1,4-glucose residues and
-1,6-glucose bonds at
KiNC Operational non-competitive inhibition con- the branching points (Figure 10.1).
stant in equation (10.2) The inability of Saccharomyces to directly utilize the
LD Limit dextrinase starch molecule as a carbon source makes necessary the
°L Degrees Lintner previous hydrolysis of the polymer into smaller sugars,
R Ratio of
-amylase/glucoamylase enzymatic preferably the monomeric (glucose) and dimeric (maltose)
units molecules. The same happens with the proteins present in
S Substrate concentration in equation (10.2) starchy materials since they must be hydrolyzed to amino
S1, S2 Synergism terms in equation (10.3) acids to be used by the yeast as nitrogen sources.
SABS South African Bureau of Standards According to that, the first steps in beer elaboration
SDU Sorghum diastatic units are directed to allow the hydrolysis of both starch and
V Initial enzymatic reaction rate in equation (10.2) proteins. Amylases, limit dextrinase (LD) and proteases are
V m Maximal initial enzymatic reaction rate in the enzymes responsible for it. The fermentability of the
equation (10.2) wort (see Figure 10.2) and the final levels of alcohol and
°WK Degrees Windisch–Kolbach remaining sugars in beer are strongly dependent on the
activity of these enzymes.
Amylases, limit dextrinase and proteases are naturally
present in the cereal grains or in the tubercles during
germination. Therefore, traditional brewing takes advan-
Introduction
tage of it stimulating the synthesis and activity of these
Brewing is one of the best examples of the important role of endogenous enzymes. Still more, the election of barley as
enzymes in the elaboration of traditional food and beverages. the most used material for brewing is not surprising con-
Briefly, beer can be defined as the alcoholic beverage elab- sidering the high content of this cereal in amylases.
orated by means of yeast fermentation with Saccharomyces Figure 10.2 shows the main stages in beer elaboration,
cerevisiae of a starch-based material. The most used material indicating those in which enzymes are implied for a correct
is barley, although there are also beers made from wheat, rice, brewing.
oats, rye, corn, sorghum, potato, cassava root or agave among During the first stage (steeping) of barley malting, the
others. Hop is added to give bitterness. Adjuncts can be also grains are soaked in water for 1–3 days at 10–15ºC to
added as additional sources of fermentable sugars and nutri- increase the humidity of the cereal. Afterwards, the steeping
ents for yeast development during the fermentation stage. water is drained away and the grain is spread out to allow
Starch is the molecule of sugar storage in plants. It germination for 4–7 days with periodical aeration. During
is the main component of cereal grains and tubercles. this phase proteases and the starch-degrading enzymes are
Starch can be defined as the mixture of two polymers of accumulated. Proteases also contribute at this step to starch
glucose: amylose, a linear molecule of D-glucose linked by degradation releasing bound -amylase from starch (Loreti

β-amylase
(endo α-1,4-bond)

Limit dextrinase
O O O O glucoamylase
N-RE ( α-1,6-bond)
O O O
O
O O O O O O Limit dextrinase
N-RE O O N-RE O O glucoamylase
O O (α-1,6-bond)
α-glucosidase O O O O O O O O
glucoamylase
N-RE N-RE
(exo α-1,4-bond) O O O O O O O
OH
α-amylase α-amylase
β-amylase (endo α-1,4-bond) (endo α-1,4-bond)
(endo α-1,4-bond) α-glucosidase
(if no α-glucosidase or glucoamylase
glucoamylase hydrolysis) (exo α-1,4-bond)

Figure 10.1 Amylopectin structure and hydrolysis points depending on the different enzymes involved in starch hydrolysis. RE, reduc-
ing end; N-RE, non-reducing end.

CH010.indd 114 1/17/2008 9:33:05 AM


et al., 1998) and releasing the inactive limit dextrinase
forms from their inhibitors (Longstaff and Bryce, 1993;
McCafferty et al., 2004).
Final drying (kilning) provides brown coloring and
flavor by the Maillard reaction. In the case of malt storage,
kilning also allows malt stabilization by stopping the enzy-
matic reactions and inhibiting an undesirable microbial
growth by decreasing the water activity. This malt is still
not readily fermentable by Saccharomyces cerevisiae. Since
hydrolysis must be continued in the next stage of brewing,
kilning is a critical step because an intensive heating treat-
ment can lead to enzymes deactivation.
The next step (mashing) is performed to obtain the wort.
Mashing starts by milling and mixing the malt with water
in ratios between 3:1 and 4:1. Extra carbon and nitrogen
sources can be added at this point as adjuncts. Mashing is
a critical operation in brewing. Hydrolysis of starch and
proteins must be completed at this stage by continuation
of the enzymatic hydrolysis, which had started during ger-
mination, to allow next the fermentation. This stage ends
heating at 75–80°C to inactivate the enzymes, and filter-
ing to eliminate the residual solids. The resulting wort is
thus enriched in low molecular weight compounds, mainly
maltose, glucose, and amino acids, and also in starch dex-
trins that were not hydrolyzed by the endogenous amylases.
Mashing is the last step for starch and protein hydrolysis.
Before yeast inoculation hop and adjuncts are added to
the filtered wort, which is finally boiled to inactivate resid-
ual enzymatic activities, to sterilize the medium, to coagu-
late proteins and to give flavor by improving hop extraction
and inducing Maillard reactions.
Starchy
material
Malting Gibberellic acid
steeping
germination
kilning Enzymes (glucanases)
Malt
Adjuncts Mashing Enzymes
Wort
Hops adjuncts Boiling
Yeast Fermentation Enzymes
Maturation
Clarification
clarification
Beer
Figure 10.2 Stages in beer elaboration.
CH010.indd 115
Use of Amylolytic Enzymes in Brewing 115
Besides the central role of amylases, limit dextrinase and
proteases in wort fermentability and beer composition,
there are other enzymes implied in brewing. -glucanases
are the enzymes responsible for the solubilization and
hydrolysis of the -glucans, which are major components of
the cell wall of the starchy endosperm of barley. The syn-
thesis and activity of these enzymes during germination is
essential in brewing to improve the extraction of the grain
components (mainly starch and proteins). During mashing
these enzymes are also interesting because they continue the
hydrolysis of the solubilized -glucans, which increase the
viscosity of the medium and make difficult the operations of
filtering (Georg-Kraemer et al., 2004; Kuusela et al., 2004).
Amylases in Traditional Brewing
The efficient extraction and transformation of starch into
fermentable sugars is a critical step in brewing. Hence,
amylases and limit dextrinase expression, stability and per-
formance under the conditions applied by the malting and
brewing industries are critical.
The concentration of fermentable and non-fermentable
starch-derived sugars in the wort defines the quality of
the beer in terms of alcoholic degree and caloric content.
The elaboration of high ethanol beers employs worts with
high concentrations of fermentable sugars, which can be
achieved by addition of adjuncts rich in fermentable sug-
ars and by extensive hydrolysis of the starch coming from
malt and adjuncts. On the opposite, low ethanol beers are
elaborated by limiting the activity of the enzymes during
mashing to reduce the amount of fermentable sugars.
Among the starch-derived sugars, only the monosac-
charide (glucose) and the lineal disaccharide (maltose) are
readily fermentable by Saccharomyces cerevisiae, while the

-1,6-disaccharide (isomaltose) and the lineal trisaccha-
ride (maltotriose) are only partially consumed (Yoon et al.,
2003). The lineal and branched oligosaccharides of higher
degree of polymerization are not fermentable for this yeast.
Fructose and saccharose, which are not starch sugars but
are present in many starchy materials, are also good carbon
sources for S. cerevisiae. A high degree of hydrolysis of the
starch molecule is then necessary to obtain high concentra-
tions of fermentable sugars.
The enzymatic hydrolysis of starch is carried out by the
joint action of three amylases (
-amylase, -amylase and

-glucosidase) and of limit dextrinase (Table 10.1 and
Figure 10.1).
-Amylase (EC 3.2.1.1) is an endo-act-
ing enzyme that rapidly and randomly attacks the
-1,4-
glucosidic linkages of starch and related oligosaccharides
to produce linear and branched oligosaccharides. The final
products of the exhaustive hydrolysis of starch by
-amylase
are maltose, glucose and
-limit dextrins that contain the

-1,6-glucosidic linkages of the branched molecules of
starch. The action of the
-amylase on starch produces a
1/17/2008 9:33:05 AM
CH010.indd 116
Table 10.1 Enzymes involved in starch degradation during brewing according to the IUBMB nomenclature

Trivial name Systematic name Code number Reaction Substrates Products

-Amylase, alpha-amylase, 1,4-
-D-glucan EC 3.2.1.1 Random endohydrolysis of Starch, glycogen and Oligosaccharides,
glycogenase, endoamylase, glucanohydrolase 1,4-
-D-glucosidic linkages related polysaccharides maltose, glucose
116 Beer Making, Hops and Yeast

Taka-amylase A and oligosaccharides with (low amounts),

three or more 1,4-
-linked
-limit dextrin
D-glucose units

-Amylase, beta-amylase, 1,4--D-glucan EC 3.2.1.2 Successive hydrolysis of Starch, glycogen and -maltose, -limit
saccharogen amylase, maltohydrolase 1,4-
-D-glucosidic linkages to remove related polysaccharides dextrin
glycogenase -maltose units from the non-reducing and oligosaccharides
ends of the chains

-Glucosidase, maltase,
-D-glucoside EC 3.2.1.20 Successive hydrolysis of terminal Polysaccharides
-D-glucose
glucoinvertase, glucosidoinvertase, glucohydrolase non-reducing 1,4-linked
-D-glucose Oligosaccharides (more
maltase-glucoamylase,
-glucoside residues with release of
-D-glucose rapidly)
hydrolase, glucosidosucrase,

-Glucopyranosidase
Glucoamylase, 1,4--D-glucan EC 3.2.1.3 Successive hydrolysis of terminal Polysaccharides (more -D-glucose
amyloglucosidase, glucose glucohydrolase non-reducing 1,4-linked
-D-glucose rapidly) Oligosaccharides
amylase, exo-1,4-
-glucosidase, residues with release of -D-glucose
-amylase, acid maltase, Hydrolysis of terminal 1,6-
-D-
Y-1,4-glucan glucohydrolase, glucosidic linkages when the next
lysosomal
-glucosidase bond in the sequence is 1,4
Limit dextrinase, R-enzyme, Dextrin
-1,6- EC 3.2.1.142 Hydrolysis of 1,6-
-D-glucosidic
- and -limit dextrins of Oligosaccharides,
amylopectin-1,6-glucosidase glucanohydrolase linkages amylopectin (complete maltose (as the
reaction), amylopectin smallest sugar
(incomplete reaction) released)

1/17/2008 9:33:05 AM

fast reduction of the viscosity of the medium. For that rea-
son
-amylase is also called liquefying amylase. -amylase
(EC 3.2.1.2) is an exo-acting enzyme that cleaves -maltose
from the non-reducing ends of the lineal chains, but does
not hydrolyze the
-1,6-glucosidic linkages. Consequently,
the final products of starch hydrolysis by -amylase are
-maltose and -limit dextrins.
-glucosidase (EC 3.2.1.20)
is an exo-acting enzyme that cleaves non-reducing
-1,4-
linkages liberating glucose. This amylase participates in
starch hydrolysis mainly during the early stages of starch
degradation in germinating barley seeds (Sun and Henson,
1991). -Amylase and
-glucosidase are also called saccharify-
ing amylases. Limit dextrinase (EC 3.2.1.142) is an endo-act-
ing enzyme that removes the
-1,6-linkages in
- and -limit
dextrins to allow their further hydrolysis by -amylase.
The activity of mainly
-amylase, -amylase and limit
dextrinase is collectively called “diastatic power” (DP). In
the brewing industry, DP is a key parameter of malting
quality since it is an estimate of the capacity of the malt
to degrade starch into fermentable sugars (Delcour and
Verschaeve, 1987). Methods for estimating the diastatic
activity of malt are generally based on its ability to generate
reducing sugars. The main units and criteria used to measure
the DP of a malted cereal are:
●
Degrees Lintner (°L), defined by the JECFA (the Joint
FAO/WHO Expert Committee on Food Additives)
and the IoB (Institute of Brewing) as follows: “A malt
has a diastatic power of 100°L if 0.1 ml of a clear 5%
infusion of the malt, acting on 100 ml of a 2% starch
solution at 20°C for 1 h, produces sufficient reducing
sugars to reduce completely 5 ml of Fehling’s solution.”
For a complete description of the method see at http://
www.fao.org/ag/agn/jecfa-additives/specs/Monograph1/
Additive-270.pdfhttp://www.fao.org/ag/agn/jecfa-addi-
tives/specs/Monograph1/Additive-270.pdf. The DP is
around 35–40 for standard barley malts, but it can be as
high as 100–125 for lager malts, and over 160 for some
high protein North American malts. The latter have far
more enzymatic power than they require to hydrolyze the
starch from the malt. Therefore, they enable the brewer
to use these malts as an amylases source in the case of
unmalted starch adjuncts addition (The BREWER Inter-
national, 2002, p. 29).
●
degrees Windisch–Kolbach (°WK), used by the EBC
(European Brewery Convention), which can be con-
verted to Lintner units as follows:
 WK 16
DP L 
3.5
●
Sorghum diastatic units (SDU), used by the SABS
(South African Bureau of Standards) especially sorghum
and not easily comparable to ºL and ºWK (EtokAkpan,
2004).
CH010.indd 117
Use of Amylolytic Enzymes in Brewing 117
Next, the importance of every stage during brewing to
enhance the activity of the enzymes and to obtain an ade-
quate wort for beer elaboration is briefly described.
Malting: Germination and Kilning The first step of ger-
mination is critical in the brewing process if no exogenous
enzymes will be added. At this stage, all the enzymes must
be expressed and activated to start the process of starch sol-
ubilization and hydrolysis.
-Amylase,
-glucosidase and
bound limit dextrinase are synthesized de novo during seed
germination, while -amylase (Ziegler, 1999) and free limit
dextrinase are activated by endogenous proteases. As well
as the effect of genotype on enzymes expression, the main
factors that affect both expression and activation of these
enzymes during germination are temperature, water/solid
ratio, oxygen availability and concentration of gibberellic
acid (GA3) – the hormone that stimulates the synthesis of

-amylase and other hydrolases.

-Amylase synthesis is synergically improved with
the increase of temperature from 20–25ºC to 30–35ºC
and the addition of exogenous GA3 (Singh et al., 1988).
-Amylase, which is synthesized during the development
of the grain and accumulated in an inactive starch-bound
form, is released from starch and activated by a proteolytic
process. Anoxic conditions during germination prevent
the production of the endoprotease involved in
-amylase
activation. Oxygen deficit also inhibits
-amylase synthe-
sis (Hanson and Jacobsen, 1984; Loreti et al., 1998). Limit
dextrinase in barley is partially synthesized following ger-
mination (Hardie, 1975) under the influence of GA3
(Lee and Pyler, 1984) and the hydration conditions.
Continuous humectation during germination produces
higher levels of limit dextrinase than addition of water at the
beginning (Pratt et al., 1978; Longstaff and Bryce, 1993;
Stenholm, 1997). Nevertheless, limit dextrinase activity is
usually low in malted cereals, making up less than 20% of
the potential total activity (Longstaff and Bryce, 1991) and
leading to high concentrations of non-fermentable limit
dextrins. This low activity is due to the major presence of
LD as inactive forms of the enzyme bound to endogenous
proteinaceous inhibitors (MacGregor et al., 2000). LD
activation probably occurs by proteolysis of these inhibitors
by endogenous thiol proteases (Longstaff and Bryce, 1993)
and/or specific reduction of inhibitors by thioredoxin (Cho
et al., 1999). In agreement with it, anaerobically germinated
grain, following a period of normal malting, produced
grains containing a limit dextrinase activity constituting
over 80% of the potential total limit dextrinase activity
(McCafferty et al., 2004) due to the application of reduc-
ing conditions that could improve the activity of cysteine
proteases (McCafferty et al., 2000). In consequence, con-
sidering the high levels of limit dextrinase inhibitors usu-
ally present in malt, it does not seem suitable to improve
the levels of this enzyme in the wort by selecting cereal lines
with a high potential for limit dextrinase synthesis. It will be
1/17/2008 9:33:05 AM
 118 Beer Making, Hops and Yeast
more effective to optimize those malting and mashing con-
ditions that promote the release of the free enzyme from the
inhibitors-bound enzyme (MacGregor et al., 1999).
In spite of enzymes synthesis and activation during germi-
nation, at this stage starch is only attacked in a small extent
since starch in the grain is in a crystalline non-soluble form
that shows resistance to the enzymatic hydrolysis.
-Amylase
and
-glucosidase are the only enzymes able to partially
attack native (not boiled) starch (Sun and Henson, 1990).
Consequently, the enzymes present in the germinated grain
must retain their activity during the heating treatment of
kilning to be active in the next step of mashing. Kilning is
then a critical operation in brewing because excessive tem-
peratures will lead to dramatic losses of the amylolytic abil-
ity of the malt. The thermostability of the synthesized and
activated enzymes during germination is also of great impor-
tance for correct mashing. It depends on the enzyme, its ori-
gin and even on the variety of the starchy material (Evans
et al., 2003). Under a typical kilning regimen, approximately
30% of the -amylase activity is irreversibly deactivated
(Evans et al., 1997).
-Amylase,
-glucosidase and limit
dextrinase are also negatively affected, although the inhibi-
tor-bound forms of this last enzyme are more resistant to
thermal degradation (Sissons et al., 1995).
Mashing At this stage, starch hydrolysis must be com-
pleted to produce wort with an adequate composition of
fermentable sugars. The use of malt with high DP will
be the first requirement if no exogenous enzymes will be
added. Nevertheless, some considerations must be made at
this respect.
The DP of a malted starchy material depends on the total
and relative amounts of enzymes present in the malt. In the
case of barley, -amylase has been significantly correlated
to DP (Gibson et al., 1995; Clarke et al., 1998), what is in
agreement with the presence of maltose in barley wort as the
major fermentable sugar (typically 55%) (Kunze, 1996)
and with the presence of -amylase as the enzyme that con-
tributes in a greater extent to the total amylolytic activity
in barley malt. Nevertheless, the levels and composition of
fermentable sugars not only depend on -amylase, but also
on the synergistic interaction between all the enzymes that
are involved in starch degradation in such a way that, for
the same content in -amylase, the differences in
-amylase
and limit dextrinase activities will be responsible for the dif-
ferences in the levels of non-fermentable sugars in the wort
(MacGregor et al., 1999). In effect, in spite of the impor-
tance of -amylase to generate maltose, the activity of this
enzyme is positively affected by
-amylase, which improves
synergically the activity of the -amylase providing it oli-
gosaccharides of adequate molecular weight for this saccha-
rifying enzyme. On the other hand, limit dextrinase is the
only enzyme capable of hydrolyzing
-1,6-linkages. Since
high levels of limit dextrins, which can constitute as much
as 25% of total carbohydrates, often remain in the wort as
CH010.indd 118
non-fermentable sugars (Enevoldsen and Schmidt, 1974),
wort fermentability also depends strongly on the activity
of the free limit dextrinase (Stenholm and Home, 1999).
Nevertheless, this enzyme cannot attack starch granules
without the previous action of
-amylase on starch (Maeda
et al., 1978). On the other hand, high levels of maltose,
produced by the intensive action of -amylase, can inhibit
the activity of the free limit dextrinase during the mashing
stage of brewing (MacGregor et al., 2002). In conclusion,
optimal starch hydrolysis will require an adequate equilib-
rium between
- and -amylase and free limit dextrinase,
as reflects the empirical model obtained by MacGregor
et al. (1999) including the main effects of
- and -amy-
lase, as well as the interactions between these enzymes and
the free limit dextrinase.
The relative ratio and total amounts and activity of
each enzyme in malt depends first of all on the genotypic
characteristics of the starchy material, the variety, the cul-
tivar and the malting conditions, as commented before.
Nevertheless, the activity of the enzymes during mashing
is strongly affected by the conditions of pH, which must
be in the range 5–6 – as usual in the wort – by the presence
of Ca2, which is necessary for the activity and stability
of the
-amylase, and mainly by the temperature and
time of operation.
During mashing, temperature must reach at least 60ºC
to ensure starch gelatinization that makes it adequate for
the enzymatic hydrolysis (Slack and Wainwright, 1980).
This increase of temperature affects the activity of every
enzyme in a different extent depending on their specific
activities at each temperature and on their resistant to ther-
mal denaturation, these effects being stronger as the time
of operation increases. This way, an adequate amount and
ratio of the enzymes could be suboptimal if the conditions
of mashing are particularly inadequate for one of them.
In general terms,
-amylase is the most resistant enzyme
to heat inactivation and the amylase with higher optimal
temperature (around 70ºC). -Amylase and limit dextri-
nase show maximum enzymatic activities at 60–62.5ºC
(Stenholm and Home, 1999), but while limit dextrinase {AQ1}
retains most of its activity at these temperatures, espe-
cially under conditions of high gravity mashing (Stenholm,
1997), -amylase suffers an important inactivation. This
is in agreement with the apparent excess of -amylase
needed in malt compared to in vitro assays, what could be
explained by the need of higher amounts of this enzyme to
counterpart the loss of activity due to heat inactivation dur-
ing mashing (MacGregor et al., 1999). In this sense, there
is already some evidence that improving the heat stability
of -amylase may be more beneficial than selecting barleys
with higher -amylase levels to increase the levels of fer-
mentable sugars in the wort (MacGregor et al., 1999).
Mashing can be performed isothermally at approximately
65°C (infusion mash) or by using a ramped temperature
profile from approximately 45°C to 70°C (temperature
1/17/2008 9:33:06 AM

programed mash). The time of operation is usually between
1 and 3 h. According to all that, an accurately selection
of the mashing conditions of temperature and time of
operation must be carefully done depending on malt and
adjuncts composition (if added) and on the cereal enzymes
profile to improve the activity of the enzymes that con-
trol the extent of starch hydrolysis (Brandam et al., 2003),
according to the desired final characteristics of the beer,
and to optimize extraction from malt. This way, when
it is necessary to reduce the amount of non-fermentable
sugars in the wort to obtain beers with high levels of alco-
hol or a low-caloric content, different mashing temperature
profiles can be assayed to minimize limit dextrinase and
-amylase thermal losses by avoiding long periods of high
temperature. On the opposite, in the case of low ethanol
beers low levels of fermentable sugars are needed in the
wort and, consequently, mashing at high temperature can
be a good strategy to reduce the activity of -amylase and
limit dextrinase.
Consequently with all of it, it is suggested to add to the
conventional malt quality DP criterion the description of
the individual enzymatic activities, mainly for
-amylase,
-amylase and free limit dextrinase, to make easier to brew-
ers the choice of the kind of malt and the mashing condi-
tions that will allow the elaboration of good quality and
special beers (Evans et al., 2003).
Koji
Koji can be considered the substitute of malt in the Eastern
World. Koji elaboration consists on the solid-state culture
of molds on seeds to produce hydrolytic enzymes, includ-
ing amylases and proteases. The koji thus obtained can
be used as source of enzymes for the hydrolysis of starchy
materials as a previous step in the manufacture of a variety
of Oriental fermented foods as sake (the traditional alco-
holic beverage of Japan made from rice), soy sauce or sufu
(soybean cheese). There are different kinds of koji depend-
ing on the material used (mainly rice and soybean, but also
barley), the mold inoculated (mainly Aspergillus oryzae, but
also Aspergillus flavus and species belonging to Zygomycetous
group) and the final use of the koji. In all cases, microor-
ganisms growth is not only a source of enzymes, but also
of vitamins and flavors that give the particular organoleptic
character to the final food or beverage.
The Role of Amylases in Modern
Brewing
Nowadays, the development of the enzyme technology
has allowed optimizing the enzymatic steps of brewing to
secure an adequate and constant quality of the wort (see at
The BREWER International (2002, p. 17) a “first enzy-
matic aid kit” to solve different problems that can appear
CH010.indd 119
Use of Amylolytic Enzymes in Brewing 119
during all the brewing process). As well as the optimiza-
tion of temperature and time conditions during the enzy-
matic steps, the addition of exogenous enzymes, especially
thermostable amylases, has been introduced in brewing to
compensate enzymatic deficits in case of incomplete germi-
nation or in case of materials with low enzyme production.
This has allowed extending the number of starchy materials
suitable for beer elaboration, offering the possibility to use
local cultivars for brewing and to elaborate new beers with
special tastes and nutritional characteristics, as gluten-free
beers for celiac people made from gluten-free starchy mate-
rials with low DP, as sorghum (Maccagnan et al., 1999).
The use of exogenous enzymes has also allowed elabo-
rating beer for diabetics (Curin et al., 1988) or diet (low
caloric) beers (Annemueller and Schober, 1999) with
a caloric content reduction between 15% and 50%.
Considering that the caloric content in beer can be calcu-
lated approximately as follows (Lewis and Young, 1995):
Calories in 10 cl  4 % (w/v) solids 7
% (w/v) alcohol (10.1)
and considering that in a typical beer residual dextrins
account for 75% of the solids, diet beers are elaborated by
reducing the remaining non-fermentable dextrins with the
addition of exogenous enzymes, mainly glucoamylase.
Substitution of the malting step by addition of all the nec-
essary amylolytic enzymes to avoid the synthesis of -gluca-
nases during germination is also described as a strategy to
elaborate functional beers, mainly from oats or barley, with
beneficial coronary effects due to the high content of soluble
glucans in these cereals (Triantafylloy, 2000) (Table 10.2).
The most used amylases in beer elaboration are

-amylase,
-glucosidase and glucoamylase (see Table
10.1), although the use of these two last enzymes can
affect negatively the fermentation of mashing worts with
high concentrations of maltose since the presence of high
concentrations of glucose as a result of the action of these
amylases could inhibit maltose consumption in some
yeast strains (Stewart et al., 1979; Phaweni et al., 1993).
-Amylase is also added, especially in case of those materi-
als, as sorghum, lacking this enzyme.
The addition of limit dextrinase is not suitable since the
inhibitors present in the malt will complex and deactivate
part of the added enzyme (Stenholm, 1997). Pullulanase
(EC 3.2.1.41) can substitute limit dextrinase in brewing
(Enevoldsen, 1970; Odibo and Obi, 1989) since this enzyme
is not inhibited as limit dextrinase. However, pullulanase is
commonly used in the production of syrup adjuncts, but
it is not approved for use in brewing in many countries
(MacGregor et al., 1999). Exogenous hydrolysis of
-1,6-
linkages can be performed by glucoamylase, which offers
the advantage over
-glucosidase of also hydrolyzing
-1,6-
glucosidic bonds when the next bond is
-1,4. Care must
be taken in the use of thermostable glucoamylases (many
1/17/2008 9:33:06 AM
CH010.indd 120
Table 10.2 List of patents including the use of exogenous enzymes in brewing for different purposes

Patent number Title

General brewing; starch sources for brewing
US2005095315 Process for the production of alcoholic beverages using maltseed
DD99179 Highly fermented beer manufacture with barley processing
ZA9803237 Process for brewing beer
WO2002074895 Improved fermentation process
CN1594525 Method for manufacturing barley extracts for beer production
DE2153151 Starch degradation for the manufacture of ethanol beverages
ZA2003009381 A method of producing a fermentable wort
120 Beer Making, Hops and Yeast

RU2190012 Method for manufacture of beer Arsenal’noe Temnoe 4

FR2203875 Manufacturing beer
DE2352906 Enzymes in beer wort manufacture
ZA7004735 Brewers’ wort for making beer
NL7713669 Brewer’s wort
SU379615 Beer
US3066026 Wort treatment with hectorite and enzyme
CN1616636 Preparation of a potato syrup for special use in beer
ZA6900044 Production of beer
WO9805788 Improved process for the production of alcoholic beverages using maltseed
DD109400 Beer wort after substitution of traditional raw materials
JP2004024151 Beer-like alcoholic beverages brewing from wheat starch
Enzymatic preparations
DE10027915 Methods for isolation from malted grain of enzymes and enzyme mixtures and their application in beer and food production
DE10241647 Preparation of enzymes and culture media components from malted grain for use in beverage and food industries
WO9742839 Enzyme granulate for use in food technology
US6031155 Isolation and characterization of barley endoxylanase gene and products and their use to enhance arabinoxylan degradation in brewing processes
FR2676456 Thermostable variants of Bacillus amyloliquefaciens
-amylase and their preparation and use
Special new beers with nutritional benefits
CN1740301 Method for producing dry beer from oats
WO2000024864 Preparation of wort and beer of high nutritional value, and corresponding products
CN1116652 Method for brewing health beer series not containing carcinogen but containing anticarcinogen
CN1401754 Enzymatic transglycosylation method for producing bifidus factor beer
US4355047 Low-calorie beer
EP949329 Gluten-free beer
CS236212 Method of manufacturing a light beer for diabetics by treating the mash with amylase
US4251630 Preparation of malt high in alpha-1,6-hydrolase
US4666718 Preparation of low calorie beer

1/17/2008 9:33:06 AM

commercial fungal glucoamylases) since they can retain
some activity even after beer pasteurization, thus increas-
ing the sweetness of the beer in the bottle at the expense of
residual dextrins (James and Lee, 1997). Therefore, yeast
thermolabile glucoamylases are usually applied.
These enzymes can be added individually or in enzymatic
mixtures that generally include other enzymes not directly
involved in starch hydrolysis (Souppe and Beudeker, 1998;
Annemueller et al., 2001; Annemueller et al., 2004) such
as glucanases and xylanases, which contribute to the filter-
ability of the wort (Dale et al., 1990) and to improve starch
solubilization through the hydrolysis of cell wall polymers
in the case that glucanases are added during the germina-
tion step (Grujic, 1998). Proteases are usually added with
amylases to increase nitrogen availability for fermentation.
The exogenous enzymes applied in brewing can be of
plant origin. Examples are the addition of Curculigo pilosa
as an exogenous -amylase source in the traditional elabo-
ration of sorghum beer in West Africa, which additionally
offers the advantage of high activity and stability, and the
ability to attack raw starch from wheat, corn, potato and
rice (Dicko et al., 1999), the employ of -amylase from
sweet potato for partial replacement of malt in brewing
( Jiang et al., 1994), or the use of multienzymatic prepa-
rations obtained from malted cereals (Annemueller et al.,
2001). Nevertheless, the exogenous enzymes applied in
brewing are usually of microbial origin due to the advan-
tages that fermentation offers in comparison to plant
enzymes recovery, as higher yields and less time-consuming
procedures. Microbial enzymatic preparations containing
one or several amylases are obtained from GRAS (“gener-
ally recognized as safe”) bacteria or fungi in cultures made
on starch, as bacterial and fungal
-amylases (Sobral and
De Vasconcelos, 1983; Imayasu et al., 1989; Goode et al.,
2002; Goode et al., 2003), bacterial -amylases (Xu et al.,
1994), yeast glucoamylases (Lowery et al., 1987) or bacte-
rial pullulanases (Odibo and Obi, 1989).
Several strategies can be applied for exogenous enzymes
enrichment. Generally, enzymes are added during mash-
ing to complete the hydrolysis of starch before the enzyme
deactivation that takes place during wort pasteurization. But
there is also the possibility to include exogenous enzymes
during fermentation (Dickscheit et al., 1973; Mizuno et al.,
2004) to progressively release glucose and/or maltose, thus
avoiding substrate inhibition of the yeast during the elabo-
ration of beers with high ethanol degree.
Finally, there are other possibilities for the use of exogenous
{AQ2} enzymes. They include the use of transgenic seeds and recom-
binant Saccharomyces strains, which will express heterologous
enzymes during malting in the first case, and during fermen-
tation in the second situation. The modification of barley
seeds by inclusion of a fungal thermotolerant endo-1,4-beta-
glucanase (EC 3.2.1.4) from microbial origin (Trichoderma
reesei) to reduce wort viscosity and improve beer filtration
(Nuutila et al., 1999) is yet reported. The construction
CH010.indd 121
Use of Amylolytic Enzymes in Brewing 121
of different amylolytic brewing yeasts (Saccharomyces cer-
evisiae and pastorianus) by transformation of the wild strains
with yeast and fungal
-amylase and glucoamylase genes
that allow growth on starch as sole carbon source for the
manufacture of low-carbohydrate diet beers (Hollenberg and
Strasser, 1990; Liu et al., 2004) is also described. The trans-
formation of laboratory strains of Saccharomyces cerevisiae
and brewers’ and distillers’ yeasts with both genes encoding
a glucoamylase from Saccharomyces diastaticus and an
-amy-
lase from Bacillus amyloliquefaciens, and the synergistic effect
of the co-expression of both genes into the same strain to
improve starch assimilation with an efficiency as high as 93%
is also reported (Steyn and Pretorius, 1991).
Starchy materials for brewing other
than barley
As commented before, addition of exogenous enzymes dur-
ing mashing allows to extent the possibilities of brewing
to starchy materials that show low DP and difficulties for
correct starch hydrolysis during mashing, whenever the fer-
mentability of the wort and the organoleptic quality of the
beer thus elaborated are adequate.
In these cases three strategies can be applied: partial addi-
tion of malted cereals as source of enzymes during mashing,
addition of exogenous enzymes, mainly during mashing or
even during the fermentation step, and use of an amylolytic
microorganism that grows on the starchy material in a first
stage, producing the amylases needed to hydrolyze in a sec-
ond stage the starch in fresh material. {AQ2}
Addition of malted barley as source of enzymes has the
advantage of contributing to color and flavor in the beer
if the main starch material gives no enough adequate orga-
noleptic profile to the beer. But even in that case, exog-
enous enzymes can be added to reinforce the amylolytic
ability of barley (Goode et al., 2000; Goode and Arendt,
2003). Addition of exogenous enzymes has the advan-
tage of allowing regulating the degree of starch hydrolysis
needed depending on the type of beer, as well as correcting
the levels of assimilable nitrogen and the filterability of the
wort if proteases and glucanases are also applied. The pos-
sibility of using amylolytic microorganisms constitutes the
basis for koji elaboration, as commented before.
Next, two examples of exogenous enzymes applications
in beer elaboration with starchy materials other than barley
are exposed.
Sorghum Beer A good example of exogenous enzymes
application in brewing is the case of sorghum beer elabo-
ration. Sorghum is the world’s fifth most important cereal
grain and constitutes a strategic natural food resource in
areas subject to hot and dry agroecologies, where it is dif-
ficult to grow other grains. In fact, 90% of the world’s sor-
ghum area lies in the developing countries, mainly in Africa
and Asia (FAO and ICRISAT, 1996).
1/17/2008 9:33:06 AM
 122 Beer Making, Hops and Yeast
For these reason sorghum has been widely used for several
traditional food purposes, including brewing of African beers
as burukutu (Faparusi, 1970; Novellie, 1977; Ogundiwin
and Tehinse, 1981). Recently, sorghum beer brewing has
also developed into a major industry for European-type
lager beer elaboration, especially in the case of Nigeria,
where barley importations have been restricted since 1988
(EtokAkpan, 2004).
There are several studies about the use of sorghum in
brewing (Owuama, 1997; Agu and Palmer, 1998; Jani
et al., 1999; Obeta et al., 2000; Okungbowa et al., 2002;
Goode and Arendt, 2003; EtokAkpan, 2005). As well
as the economic reasons, sorghum offers an interesting
potential as raw material for brewing due to its high starch
content and the absence of gluten, what makes it suitable
for the elaboration of gluten-free beers for celiac people.
Nevertheless, there are some inherent problems associated
with sorghum that must be solved to better compete with
barley in the elaboration of European-type beer.
A major disadvantage for the use of malted sorghum in
brewing is the usual low DP and amylolytic activity during
mashing. Although
-amylase isoenzymes are de novo syn-
thesized during sorghum germination, very low (Dufour
et al., 1992; Taylor and Robbins, 1993) or even total absence
(Uriyo and Eigel, 2000) of -amylase levels are detected,
causing incomplete saccharification of starch and low levels of
maltose in comparison to barley wort (Dicko et al., 2006). In
addition, the higher gelatinization temperature of sorghum
starch with regard to barley increases thermal deactivation of
the enzymes ( Jani et al., 1999).
Several studies have been developed to improve
- and
-amylase activities in malted sorghum in relation with
variety, cultivar, steep regime, steep liquor composition
and kilning temperature (Taylor and Robbins, 1993; Jani
et al., 1999; Obeta et al., 2000; Okungbowa et al., 2002).
Nevertheless, the best solution to reduce the levels of non-
fermentable sugars in sorghum worts is the use of mixtures
of malted barley with sorghum during mashing (Goode
and Arendt, 2003) or the addition of exogenous enzymes
like
-amylase and amyloglucosidase (Clayton, 1969),
-amylase from potato (Etim and EtokAkpan, 1992) or
microbial
-amylases (Goode et al., 2003) to unmalted
sorghum. In this last case it is also reported the con-
venience of including a percentage of malted sorghum
as source of endogenous proteases to avoid the need of
adding these enzymes since poor foam retention has been
associated to commercial proteolytic enzymes (Agu and
Palmer, 1998).
Chestnut in Brewing Among the above-mentioned seeds
used as raw materials for the preparation of fermented
drinks, other indigenous crops like chestnut can constitute
local alternative sources of starches for industrial purposes
(especially sugar and alcohol manufacture) and lead to con-
servation of agriculturally marginal lands. In fact, it is yet
CH010.indd 122
described the elaboration of a new Chinese chestnut beer
with beneficial health effects as anti-cancer, reducing blood
sugar and lowering blood fat activities (Li et al., 2005).
Therefore, chestnut is here studied as a source of starch for
brewing.
As sorghum, chestnut has no gluten but shows low
endogenous amylolytic activity. According to it, chestnut is
included in the elaboration of a kind of koji as a prior stage
to the ethanol fermentation for the elaboration of wine
(Iwasaki et al., 2002) or shochu (a Japanese popular distilled
light alcoholic drink produced from steamed cereals, mainly
rice and barley, but also from sweet potato soba (buck-
wheat), and chestnut in a lesser extent). Addition of malt
(Li et al., 2005) is also reported as a source of exogenous
enzymes to complete chestnut starch hydrolysis.
Another strategy for brewing chestnut consists on substi-
tuting koji by direct addition of exogenous enzymes. This
alternative takes as basis the two steps industrial process for
starch hydrolysis. In the first stage simultaneous gelatini-
zation and liquefaction of starch with a high-temperature

-amylase are performed. The maltodextrins thus gener-
ated are then saccharified with a glucoamylase in the sec-
ond stage (Slominska, 1993). Optimal reaction conditions
depend on the amylases used, as well as on the starch ori-
gin, which affects the composition, viscosity and degrada-
tion resistance of the polysaccharide.
In our laboratory, we have optimized the enzymatic
hydrolysis of chestnut starch in solid (chopped) and sub-
merged (aqueous liquid pastes) operation in a single step
with an enzymatic mixture of a commercial heat resist-
ant
-amylase (Termamyl 120L(S)) and glucoamylase
(AMG 300L), both purchased by Novo Nordisk A/S
Industries (López et al., 2004, 2005, 2006). Some details
of these enzymatic processes are next briefly described as an
example of practical application and optimization of the
addition of exogenous enzymes in brewing of new starchy
materials.
The submerged process of hydrolysis was performed at
70ºC to reduce the viscosity of the purée. In these condi-
tions total conversion of starch to glucose in a single step
was only achieved when an adequate concentration and
composition of the enzymatic mixture of
-amylase/
glucoamylase was present in the reaction medium (López
et al., 2004). Figure 10.3 shows the linear increasing effect
of the concentration of the enzymes on chestnut starch sol-
ubilization and hydrolysis, being the best the higher value
assayed (60 EU/g raw chestnut, corresponding to 10.5 EU/ml),
and the second order effect of the ratio of
-amylase/
glucoamylase, which implies the existence of an optimum
value corresponding to glucoamylase enriched mixtures
(
-amylase/glucoamylase ratio of 0.35/0.75 EU). Sugars
profile in the hydrolyzate corresponded to glucose (as the
only final product of starch hydrolysis) and saccharose
(present in chestnut) in concentrations of 70 and 15 g/l
respectively.
1/17/2008 9:33:06 AM
 Use of Amylolytic Enzymes in Brewing 123

100 100 80
% Solubilization

% Hydrolysis

Glucose (g/l)
50 50 45

0 0 10

1.5
1.5
1

1.5
1.5
1
0 0 0 0 0 0
E 1.5 R E 1.5 R E 1 R

Figure 10.3 Combined effect of the enzyme concentration and the ratio of
-amylase/glucoamylase (in codified values) on the solubili-
zation, hydrolysis, and glucose release from chestnut purée with a mixture of amylases. E, enzyme concentration; R, ratio of
-amylase/
glucoamylase in the mixture. Published with permission of the American Chemical Society (Copyright 2004).

100 100

75 75
% AA (EU/ml)

% AA (EU/ml)
50 50

25 25

0 0
0 50 100 150 0 50 100 150 200
Time (min)
(a) (b)

Figure 10.4 Thermal loss of amylolytic activity of a mixture of
-amylase and glucoamylase (respective ratio: 0.35/0.65) at 70ºC in
(a) 50 mM citric–phosphate buffer pH 4.75 and (b) 225 g/l pH 4.75 buffered chestnut purée. AA, total amylolytic activity expressed as
percentages referred to the initial value (1.35 EU/ml). Symbols represent the mean values of two experimental points; dashed and solid
lines represent, respectively, the fittings to a first order model and to model (10.3).

Incomplete starch conversion at low enzyme concentra- operational Michaelis–Menten and substrate inhibition
tions was attributed to product and substrate inhibition as constants, I is the inhibitor (glucose) concentration, and
well as thermal deactivation (López et al., 2006). In effect, K iC and K iNC are operational competitive and non-com-
substrate and product inhibition were confirmed and petitive product inhibition constants, respectively.
modeled by equation (10.2), this derived from Michaelis– Anyway, substrate and glucose inhibition was not strong
Menten and Briggs–Haldane’s models considering the enough to explain the low percentage of hydrolysis reached
simultaneous competitive and non-competitive product at low enzymes concentration. Consequently, thermal deac-
inhibition as well as substrate inhibition: tivation of the
-amylase and glucoamylase mixture at low
enzyme concentration was also studied in presence and
vm S absence of substrate (Figure 10.4), and the losses of amylo-
v (10.2)
⎛ ⎞⎛
⎜⎜  ⎛⎜ I ⎞⎟
⎟⎟  S  K S S 2 ⎟⎟⎟ ⎜⎜1  I
⎞⎟
⎟⎟
lytic activity were fitted to model (10.3), based on Aymard
⎜⎜ K m ⎜⎜1   ⎟⎟⎠ ⎟⎟ ⎜⎜  ⎟⎟⎠ and Belarbi’s one for an enzymatic mixture (Aymard and
⎜⎝ ⎜⎝ K iC ⎠⎝ K iNC Belarbi, 2000), and modified to include a synergistic effect
between these two enzymes (López et al., 2006):
where v and v m are respectively initial and maximal initial
reaction rates of the enzymes mixture in chestnut purée, S is AAt  AA
 ae
kat  b e
kbt
the substrate concentration, K m and K s represent respectively  S1 AA
(ae
kat )  S2 AA
(b e
kbt ) (10.3)

CH010.indd 123 1/17/2008 9:33:06 AM

 124 Beer Making, Hops and Yeast
where AAt represents the total amylolytic activity (expressed
as percentage referred to the initial total activity) for the
mixture at time t, AA
is the
-amylase activity expressed
as percentage, a and b, and ka and kb are, respectively, pre-
exponential parameters and first order constants for each glu-
coamylase form present in the commercial enzyme (Amirul
et al., 1996), and S1 and S2 are the synergism terms between
the
-amylase and each glucoamylase form.
Experimental and modeled data showed a strong decrease
of the enzymatic activity in both cases, what reflects the
important role of enzymes thermal deactivation as a reason
for incomplete starch hydrolysis. Glucoamylase, following a
biexponential kinetics pattern, was only responsible for the
thermal deactivation of the mixture, while the
-amylase
kept 100% of the initial activity at the end of the incubation
in these conditions. Therefore, the slightly retarded addition
of glucoamylase with regard to
-amylase, to reduce the
exposure of this enzyme to high temperatures while taking
advantage of the synergic action of both enzymes working
together, was finally proposed as an alternative to reduce
the need of high concentrations of enzymes, especially of
glucoamylase (López et al., 2004).
Two main factors prevent the achievement of high-
glucose hydrolyzates from chestnut, they condition the
utility of chestnut as an adequate starchy material for use
in brewing or as adjunct. They are the low concentration of
starch in raw chestnut (around 30%) and the impossibility
of working with chestnut purée concentrations higher than
300 g/l due to their viscosity. Therefore, two procedures
were developed to increase the glucose levels in chestnut
hydrolyzates.
In a way, the elaboration of hydrolyzates in consecutive
cycles of submerged one-step hydrolysis of chestnut purée,
carried out by using the hydrolyzate obtained in the first
cycle – free of solids – to make a new chestnut pureé for
the next cycle of hydrolysis, led to a 65% increase of the
glucose concentration in the hydrolyzate obtained after
three cycles of operation (López et al., 2004) with regard to
the simple one-step operation.
On the other hand, solid state hydrolysis, performed with
chopped-wet chestnut without free water, also showed the
suitability of this mode of operation to increase the levels
of glucose in the hydrolyzates obtained after one-step solid
state operation and posterior aqueous extraction (López
et al., 2005). Applying the same optimal enzyme concen-
tration and
-amylase/glucoamylase ratio defined for the
submerged operation, total hydrolysis was achieved operat-
ing in solid state at 70ºC (López et al., 2005) (Figure 10.5),
what led to a 50% increase of the glucose concentration in
the hydrolyzate comparing to the submerged process. The
assays performed at lower temperatures to check the possi-
bility to develop a process of simultaneous hydrolysis and
fermentation showed the inability of the system to achieve
total hydrolysis of the chestnut starch (Figure 10.5) as a con-
sequence of mass transfer restrictions (López et al., 2005).
CH010.indd 124
100
80
% Hydrolysis
60
40
20
0
0 10 20 30 40 50 60
Time (min)
Figure 10.5 Kinetics of the one-step solid-state hydrolysis
of chestnut with a mixture of 0.35/0.65
-amylase/glucoamy-
lase and 60 EU/g of raw chestnut at three temperatures: 70°C
(triangles), 30°C (circles) and 17°C (squares). Published with per-
mission of the American Chemical Society (Copyright 2005).
Summary Points
●
Enzymes are the underlying responsible for all the bio-
chemical reactions that take part in the elaboration of
many foodstuffs. Beer is an example of that.
●
Amylases are, together with limit dextrinase and pro-
teases, the main enzymes implicated in brewing. Their
action during the stages of malting and mashing allows
the degradation of the starch and proteins present in the
cereal grain to transform them into assimilable sugars
and amino acids for the yeasts.
●
The fermentability of the wort and the final levels of alco-
hol and remaining sugars in beer are strongly dependent
on the activity of the starch degrading enzymes (amylases
and limit dextrinase). Low levels of enzymes or inad-
equate conditions of pH, temperature and time of opera-
tion during malting and mashing can lead to low-quality
beers.
●
The profile of cereal endogenous starch degrading
enzymes includes
-amylase, -amylase,
-glucosidase
and limit dextrinase. It depends mainly on the cereal,
although variety, cultivar and malting conditions also
affect the levels of the enzymes synthesized. They are
accumulated during the malting step by de novo synthesis
or by transformation into active forms by proteases.
●
The main amylases in barley and sorghum are - and

-amylase. The former is the major amylase in barley. It is
an exoenzyme that liberates
-maltose from starch, provid-
ing a readily fermentable substrate for the yeast. The lat-
ter is the major amylase in sorghum. It is an endoenzyme
that reduces starch viscosity breaking the starch molecule
in lower weight non-directly assimilable oligosaccharides,
maltose, and glucose in low amounts. The only action of

-amylase on starch provides low levels of fermentable
1/17/2008 9:33:06 AM

sugars. Therefore, this enzyme needs the following action
of -amylase or
-glucosidase as saccharifying amylases.
●
Limit dextrinase is the debranching enzyme responsible
for the hydrolysis of the
-1,6-linkages in
- and -limit
dextrins resulting from the exhaustive action of
- and
-amylases on starch. The activity of this enzyme during
mashing is usually low in malted cereals, making up less
than 20% of its potential total activity due to the major
presence of limit dextrinase as inactive forms of the
enzyme bound to endogenous proteinaceous inhibitors.
●
Exogenous amylases (mainly
-amylase,
-glucosidase
and glucoamylase) can be added during the mashing stage
to reinforce the action of the endogenous enzymes. This
strategy has interesting operational benefits increasing
the wort fermentability of poorly modified malts, reduc-
ing the need of malt for mashing, and allowing the use of
other non-easily maltable starchy sources as chestnut.
●
New kinds of beers with interesting nutritional charac-
teristics can be obtained by different strategies related to
the role of amylases:
– Beers with low ethanol content can be obtained by
limiting the activity of the endogenous enzymes dur-
ing mashing to reduce the amount of fermentable
sugars in the wort.
– Beers for diabetics, light or diet beers, which are char-
acterized by low levels of residual sugars in the final
product, can be elaborated increasing the extent of
starch hydrolysis during mashing by adding amylase-
rich multienzyme preparations.
– Beers for celiac people can be elaborated employ-
ing gluten-free starchy sources with low DP (as sor-
ghum) in combination with the addition of exogenous
enzymes.
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