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10

Use of Amylolytic Enzymes in Brewing


N.P. Guerra, A. Torrado-Agrasar, C. Lpez-Macas, E. Martnez-Carballo, S. Garca-Falcn, J. Simal-Gndara 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
One of the main problems in the production of fermented alcoholic beverages from amylaceous raw materials is the efcient conversion of starch into fermentable sugars for Saccharomyces cerevisiae. -amylase, -amylase, -glucosidase and limit dextrinase are the enzymes responsible for the hydrolysis of starch into maltose and glucose in beer elaboration. Two different strategies are used in traditional brewing: to favor the activity of the endogenous enzymes that are present in the ingredients during the malting and mashing steps (beer in Western countries), or to use amylolytic microorganisms in a previous step to yeast fermentation (koji in Eastern countries). More recently, the development of technologies for the efcient production of enzymes, mainly of microbial origin, has allowed the application of exogenous amylases in beer elaboration to improve classical brewing by compensating enzymatic decits in poor malts and by reducing the needs of malt for mashing. But, moreover, the addition of exogenous enzymes has also allowed the use of new starchy materials with low amylolytic potential and the preparation of worts with adequate sugars composition to elaborate new kinds of beer with interesting functional properties, as low-caloric and gluten-free beers for celiac people. The importance of the amylolytic potential of the amylaceous material, the synergic activity of the three main enzymes ( -amylase, -amylase and limit dextrinase) involved in starch hydrolysis during mashing, and the need of a correct perforance of the malting and mashing steps to maximize the expression and activity of these enzymes and to minimize the losses of enzymatic activity due to the thermal treatments applied during brewing are here described, paying special attention to the use of barley and sorghum as the most used starchy substrates and sources of amylases. Finally, examples of the application of exogenous enzymes in brewing for the use of new starchy materials as chestnut, and for the elaboration of new kinds of functional beers, are also included.

List of Abbreviations
AA AAt AA a and b DP E EBC EC EU FAO GA3 GRAS I ICRISAT IoB IUBMB JECFA ka and kb Km Ks Total amylolytic activity in the mixture Total amylolytic activity (expressed as percentage referred to the initial total activity) for the amylases mixture at time t in equation (10.3) -Amylase activity (expressed as percentage referred to the initial total activity) in equation (10.3) Pre-exponential parameters in equation (10.3) for each glucoamylase form present in the commercial enzyme. Diastatic power Total enzyme concentration European Brewery Convention Enzyme Commission Enzymatic units Food and Agricultural Organization of the United Nations Gibberellic acid Generally recognized as safe Inhibitor (glucose) concentration in equation (10.2) International Crops Research Institute for the Semi-Arid Tropics Institute of Brewing International Union of Biochemistry and Molecular Biology Joint FAO/WHO Expert Committee on Food Additives First order constants in equation (10.3) for each glucoamylase form present in the commercial enzyme Operational MichaelisMenten constant in equation (10.2) Operational substrate inhibition constant in equation (10.2)
Copyright 2008 Elsevier Ltd All rights of reproduction in any form reserved

Beer in Health and Disease Prevention Volume 1 ISBN: 978-0-12-373891-2

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114 Beer Making, Hops and Yeast

K iC K iNC LD L R S S1, S2 SABS SDU V Vm WK

Operational competitive inhibition constant in equation (10.2) Operational non-competitive inhibition constant in equation (10.2) Limit dextrinase Degrees Lintner Ratio of -amylase/glucoamylase enzymatic units Substrate concentration in equation (10.2) Synergism terms in equation (10.3) South African Bureau of Standards Sorghum diastatic units Initial enzymatic reaction rate in equation (10.2) Maximal initial enzymatic reaction rate in equation (10.2) Degrees WindischKolbach

Introduction
Brewing is one of the best examples of the important role of enzymes in the elaboration of traditional food and beverages. Briey, beer can be dened as the alcoholic beverage elaborated by means of yeast fermentation with Saccharomyces cerevisiae of a starch-based material. The most used material is barley, although there are also beers made from wheat, rice, oats, rye, corn, sorghum, potato, cassava root or agave among others. Hop is added to give bitterness. Adjuncts can be also added as additional sources of fermentable sugars and nutrients for yeast development during the fermentation stage. Starch is the molecule of sugar storage in plants. It is the main component of cereal grains and tubercles. Starch can be dened as the mixture of two polymers of glucose: amylose, a linear molecule of D-glucose linked by
-amylase (endo -1,4-bond) O N-RE O O O O O O O N-RE O O O O -glucosidase glucoamylase (exo -1,4-bond) O N-RE O O O O O O O N-RE O O O

-1,4-glucosidic bonds, and amylopectin, a branched molecule of -1,4-glucose residues and -1,6-glucose bonds at the branching points (Figure 10.1). The inability of Saccharomyces to directly utilize the starch molecule as a carbon source makes necessary the previous hydrolysis of the polymer into smaller sugars, preferably the monomeric (glucose) and dimeric (maltose) molecules. The same happens with the proteins present in starchy materials since they must be hydrolyzed to amino acids to be used by the yeast as nitrogen sources. According to that, the rst steps in beer elaboration are directed to allow the hydrolysis of both starch and proteins. Amylases, limit dextrinase (LD) and proteases are the enzymes responsible for it. The fermentability of the wort (see Figure 10.2) and the nal levels of alcohol and 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 advantage of it stimulating the synthesis and activity of these endogenous enzymes. Still more, the election of barley as the most used material for brewing is not surprising considering the high content of this cereal in amylases. Figure 10.2 shows the main stages in beer elaboration, indicating those in which enzymes are implied for a correct brewing. During the rst stage (steeping) of barley malting, the grains are soaked in water for 13 days at 1015C to increase the humidity of the cereal. Afterwards, the steeping water is drained away and the grain is spread out to allow germination for 47 days with periodical aeration. During this phase proteases and the starch-degrading enzymes are accumulated. Proteases also contribute at this step to starch degradation releasing bound -amylase from starch (Loreti

Limit dextrinase glucoamylase ( -1,6-bond)

O O O O O

Limit dextrinase glucoamylase (-1,6-bond) O O O O -amylase (endo -1,4-bond) O O O N-RE OH

-amylase (endo -1,4-bond) -glucosidase (if no -glucosidase or glucoamylase glucoamylase hydrolysis) (exo -1,4-bond)

-amylase (endo -1,4-bond)

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

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Use of Amylolytic Enzymes in Brewing 115

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 avor by the Maillard reaction. In the case of malt storage, kilning also allows malt stabilization by stopping the enzymatic 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 treatment 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 germination, to allow next the fermentation. This stage ends heating at 7580C to inactivate the enzymes, and ltering 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 dextrins 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 ltered wort, which is nally boiled to inactivate residual enzymatic activities, to sterilize the medium, to coagulate proteins and to give avor by improving hop extraction and inducing Maillard reactions.

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 synthesis 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 difcult the operations of ltering (Georg-Kraemer et al., 2004; Kuusela et al., 2004).

Amylases in Traditional Brewing


The efcient extraction and transformation of starch into fermentable sugars is a critical step in brewing. Hence, amylases and limit dextrinase expression, stability and performance 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 denes 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 sugars 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 monosaccharide (glucose) and the lineal disaccharide (maltose) are readily fermentable by Saccharomyces cerevisiae, while the -1,6-disaccharide (isomaltose) and the lineal trisaccharide (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 concentrations 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-acting enzyme that rapidly and randomly attacks the -1,4glucosidic linkages of starch and related oligosaccharides to produce linear and branched oligosaccharides. The nal 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

Starchy material Malting steeping germination kilning Malt Adjuncts Mashing Wort Hops adjuncts Yeast Boiling Fermentation Enzymes Enzymes Gibberellic acid Enzymes (glucanases)

Maturation clarification

Clarification

Beer

Figure 10.2 Stages in beer elaboration.

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Table 10.1 Enzymes involved in starch degradation during brewing according to the IUBMB nomenclature Systematic name 1,4- -D-glucan glucanohydrolase EC 3.2.1.1 Random endohydrolysis of 1,4- -D-glucosidic linkages Starch, glycogen and related polysaccharides and oligosaccharides with three or more 1,4- -linked D-glucose units Starch, glycogen and related polysaccharides and oligosaccharides Polysaccharides Oligosaccharides (more rapidly) Code number Reaction Substrates Products Oligosaccharides, maltose, glucose (low amounts), -limit dextrin -maltose, -limit dextrin

116 Beer Making, Hops and Yeast

Trivial name

-Amylase, alpha-amylase, glycogenase, endoamylase, Taka-amylase A

-Amylase, beta-amylase, saccharogen amylase, glycogenase -D-glucoside glucohydrolase EC 3.2.1.20 Successive hydrolysis of terminal non-reducing 1,4-linked -D-glucose residues with release of -D-glucose

1,4- -D-glucan maltohydrolase

EC 3.2.1.2

Successive hydrolysis of 1,4- -D-glucosidic linkages to remove -maltose units from the non-reducing ends of the chains

-Glucosidase, maltase, glucoinvertase, glucosidoinvertase, maltase-glucoamylase, -glucoside hydrolase, glucosidosucrase, -Glucopyranosidase 1,4- -D-glucan glucohydrolase EC 3.2.1.3 Successive hydrolysis of terminal non-reducing 1,4-linked -D-glucose residues with release of -D-glucose Hydrolysis of terminal 1,6- -Dglucosidic linkages when the next bond in the sequence is 1,4 Hydrolysis of 1,6- -D-glucosidic linkages

-D-glucose

Glucoamylase, amyloglucosidase, glucose amylase, exo-1,4- -glucosidase, -amylase, acid maltase, Y-1,4-glucan glucohydrolase, lysosomal -glucosidase Dextrin -1,6glucanohydrolase EC 3.2.1.142

Polysaccharides (more rapidly) Oligosaccharides

-D-glucose

Limit dextrinase, R-enzyme, amylopectin-1,6-glucosidase

- and -limit dextrins of amylopectin (complete reaction), amylopectin (incomplete reaction)

Oligosaccharides, maltose (as the smallest sugar released)

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fast reduction of the viscosity of the medium. For that reason -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 nal 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 -1,4linkages 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 saccharifying amylases. Limit dextrinase (EC 3.2.1.142) is an endo-acting 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:

Next, the importance of every stage during brewing to enhance the activity of the enzymes and to obtain an adequate wort for beer elaboration is briey described. Malting: Germination and Kilning The rst step of germination 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 solubilization 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 2025C to 3035C 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 decit also inhibits -amylase synthesis (Hanson and Jacobsen, 1984; Loreti et al., 1998). Limit dextrinase in barley is partially synthesized following germination (Hardie, 1975) under the inuence 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 specic 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 reducing conditions that could improve the activity of cysteine proteases (McCafferty et al., 2000). In consequence, considering the high levels of limit dextrinase inhibitors usually 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

Degrees Lintner (L), dened 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 100L if 0.1 ml of a clear 5% infusion of the malt, acting on 100 ml of a 2% starch solution at 20C for 1 h, produces sufcient reducing sugars to reduce completely 5 ml of Fehlings 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-additives/specs/Monograph1/Additive-270.pdf. The DP is around 3540 for standard barley malts, but it can be as high as 100125 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 International, 2002, p. 29). degrees WindischKolbach (WK), used by the EBC (European Brewery Convention), which can be converted to Lintner units as follows: DP L WK 16 3.5

Sorghum diastatic units (SDU), used by the SABS (South African Bureau of Standards) especially sorghum and not easily comparable to L and WK (EtokAkpan, 2004).

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118 Beer Making, Hops and Yeast

more effective to optimize those malting and mashing conditions 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 germination, 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 temperatures will lead to dramatic losses of the amylolytic ability of the malt. The thermostability of the synthesized and activated enzymes during germination is also of great importance for correct mashing. It depends on the enzyme, its origin 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 inhibitor-bound forms of this last enzyme are more resistant to thermal degradation (Sissons et al., 1995). Mashing At this stage, starch hydrolysis must be completed to produce wort with an adequate composition of fermentable sugars. The use of malt with high DP will be the rst 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 signicantly 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 contributes 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 differences in the levels of non-fermentable sugars in the wort (MacGregor et al., 1999). In effect, in spite of the importance 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 oligosaccharides of adequate molecular weight for this saccharifying 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

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 equilibrium between - and -amylase and free limit dextrinase, as reects the empirical model obtained by MacGregor et al. (1999) including the main effects of - and -amylase, 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 rst of all on the genotypic characteristics of the starchy material, the variety, the cultivar 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 56 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 60C 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 specic activities at each temperature and on their resistant to thermal 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 70C). -Amylase and limit dextrinase show maximum enzymatic activities at 6062.5C (Stenholm and Home, 1999), but while limit dextrinase {AQ1} retains most of its activity at these temperatures, especially 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 during mashing (MacGregor et al., 1999). In this sense, there is already some evidence that improving the heat stability of -amylase may be more benecial than selecting barleys with higher -amylase levels to increase the levels of fermentable sugars in the wort (MacGregor et al., 1999). Mashing can be performed isothermally at approximately 65C (infusion mash) or by using a ramped temperature prole from approximately 45C to 70C (temperature

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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 prole to improve the activity of the enzymes that control the extent of starch hydrolysis (Brandam et al., 2003), according to the desired nal 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 alcohol or a low-caloric content, different mashing temperature proles 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 brewers the choice of the kind of malt and the mashing conditions 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, including 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 alcoholic beverage of Japan made from rice), soy sauce or sufu (soybean cheese). There are different kinds of koji depending on the material used (mainly rice and soybean, but also barley), the mold inoculated (mainly Aspergillus oryzae, but also Aspergillus avus and species belonging to Zygomycetous group) and the nal use of the koji. In all cases, microorganisms growth is not only a source of enzymes, but also of vitamins and avors that give the particular organoleptic character to the nal food or beverage.

during all the brewing process). As well as the optimization of temperature and time conditions during the enzymatic steps, the addition of exogenous enzymes, especially thermostable amylases, has been introduced in brewing to compensate enzymatic decits in case of incomplete germination 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 materials with low DP, as sorghum (Maccagnan et al., 1999). The use of exogenous enzymes has also allowed elaborating 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 calculated approximately as follows (Lewis and Young, 1995): Calories in 10 cl 4 % (w/v) solids % (w/v) alcohol 7 (10.1)

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 rst enzymatic aid kit to solve different problems that can appear

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 necessary amylolytic enzymes to avoid the synthesis of -glucanases during germination is also described as a strategy to elaborate functional beers, mainly from oats or barley, with benecial 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 materials, 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 -1,6linkages can be performed by glucoamylase, which offers the advantage over -glucosidase of also hydrolyzing -1,6glucosidic bonds when the next bond is -1,4. Care must be taken in the use of thermostable glucoamylases (many

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Table 10.2 List of patents including the use of exogenous enzymes in brewing for different purposes Title

120 Beer Making, Hops and Yeast

Patent number

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 RU2190012 Method for manufacture of beer Arsenalnoe Temnoe 4 FR2203875 Manufacturing beer DE2352906 Enzymes in beer wort manufacture ZA7004735 Brewers wort for making beer NL7713669 Brewers 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 Methods for isolation from malted grain of enzymes and enzyme mixtures and their application in beer and food production Preparation of enzymes and culture media components from malted grain for use in beverage and food industries Enzyme granulate for use in food technology Isolation and characterization of barley endoxylanase gene and products and their use to enhance arabinoxylan degradation in brewing processes Thermostable variants of Bacillus amyloliquefaciens -amylase and their preparation and use

Enzymatic preparations DE10027915 DE10241647 WO9742839 US6031155 FR2676456

Special new beers with nutritional benets 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 bidus 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

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commercial fungal glucoamylases) since they can retain some activity even after beer pasteurization, thus increasing 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 lterability 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 germination 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 elaboration 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 preparations obtained from malted cereals (Annemueller et al., 2001). Nevertheless, the exogenous enzymes applied in brewing are usually of microbial origin due to the advantages 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 (generally 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 bacterial pullulanases (Odibo and Obi, 1989). Several strategies can be applied for exogenous enzymes enrichment. Generally, enzymes are added during mashing 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 elaboration of beers with high ethanol degree. Finally, there are other possibilities for the use of exogenous enzymes. They include the use of transgenic seeds and recom{AQ2} binant Saccharomyces strains, which will express heterologous enzymes during malting in the rst case, and during fermentation in the second situation. The modication of barley seeds by inclusion of a fungal thermotolerant endo-1,4-betaglucanase (EC 3.2.1.4) from microbial origin (Trichoderma reesei) to reduce wort viscosity and improve beer ltration (Nuutila et al., 1999) is yet reported. The construction

of different amylolytic brewing yeasts (Saccharomyces cerevisiae 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 transformation of laboratory strains of Saccharomyces cerevisiae and brewers and distillers yeasts with both genes encoding a glucoamylase from Saccharomyces diastaticus and an -amylase from Bacillus amyloliquefaciens, and the synergistic effect of the co-expression of both genes into the same strain to improve starch assimilation with an efciency 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 during mashing allows to extent the possibilities of brewing to starchy materials that show low DP and difculties for correct starch hydrolysis during mashing, whenever the fermentability of the wort and the organoleptic quality of the beer thus elaborated are adequate. In these cases three strategies can be applied: partial addition 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 rst stage, producing the amylases needed to hydrolyze in a sec{AQ2} ond stage the starch in fresh material. Addition of malted barley as source of enzymes has the advantage of contributing to color and avor in the beer if the main starch material gives no enough adequate organoleptic prole to the beer. But even in that case, exogenous 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 advantage 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 lterability of the wort if proteases and glucanases are also applied. The possibility 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 elaboration. Sorghum is the worlds fth most important cereal grain and constitutes a strategic natural food resource in areas subject to hot and dry agroecologies, where it is difcult to grow other grains. In fact, 90% of the worlds sorghum area lies in the developing countries, mainly in Africa and Asia (FAO and ICRISAT, 1996).

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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 synthesized 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 saccharication 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 nonfermentable 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 convenience 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 conservation of agriculturally marginal lands. In fact, it is yet

described the elaboration of a new Chinese chestnut beer with benecial 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 (buckwheat), 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 substituting koji by direct addition of exogenous enzymes. This alternative takes as basis the two steps industrial process for starch hydrolysis. In the rst stage simultaneous gelatinization and liquefaction of starch with a high-temperature -amylase are performed. The maltodextrins thus generated are then saccharied with a glucoamylase in the second stage (Slominska, 1993). Optimal reaction conditions depend on the amylases used, as well as on the starch origin, which affects the composition, viscosity and degradation resistance of the polysaccharide. In our laboratory, we have optimized the enzymatic hydrolysis of chestnut starch in solid (chopped) and submerged (aqueous liquid pastes) operation in a single step with an enzymatic mixture of a commercial heat resistant -amylase (Termamyl 120L(S)) and glucoamylase (AMG 300L), both purchased by Novo Nordisk A/S Industries (Lpez et al., 2004, 2005, 2006). Some details of these enzymatic processes are next briey 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 70C to reduce the viscosity of the pure. In these conditions 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 (Lpez et al., 2004). Figure 10.3 shows the linear increasing effect of the concentration of the enzymes on chestnut starch solubilization 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 prole in the hydrolyzate corresponded to glucose (as the only nal product of starch hydrolysis) and saccharose (present in chestnut) in concentrations of 70 and 15 g/l respectively.

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100 % Solubilization % Hydrolysis

100 Glucose (g/l) 1.5 0 R E 1.5 0 R

80

50

50

45

0 1.5 0 E 1.5 0

1.5

0 1.5

10 1 0 E 1 0 R

Figure 10.3 Combined effect of the enzyme concentration and the ratio of -amylase/glucoamylase (in codied values) on the solubilization, hydrolysis, and glucose release from chestnut pure 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 % AA (EU/ml)

75 % AA (EU/ml)

50

50

25

25

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

0 200

Figure 10.4 Thermal loss of amylolytic activity of a mixture of -amylase and glucoamylase (respective ratio: 0.35/0.65) at 70C in (a) 50 mM citricphosphate buffer pH 4.75 and (b) 225 g/l pH 4.75 buffered chestnut pure. 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 ttings to a rst order model and to model (10.3).

Incomplete starch conversion at low enzyme concentrations was attributed to product and substrate inhibition as well as thermal deactivation (Lpez et al., 2006). In effect, substrate and product inhibition were conrmed and modeled by equation (10.2), this derived from Michaelis Menten and BriggsHaldanes models considering the simultaneous competitive and non-competitive product inhibition as well as substrate inhibition: v K m 1 I K iC vm S S K S S 2 1 (10.2)

I K iNC

operational MichaelisMenten and substrate inhibition constants, I is the inhibitor (glucose) concentration, and K iC and K iNC are operational competitive and non-competitive product inhibition constants, respectively. Anyway, substrate and glucose inhibition was not strong enough to explain the low percentage of hydrolysis reached at low enzymes concentration. Consequently, thermal deactivation of the -amylase and glucoamylase mixture at low enzyme concentration was also studied in presence and absence of substrate (Figure 10.4), and the losses of amylolytic activity were tted to model (10.3), based on Aymard and Belarbis one for an enzymatic mixture (Aymard and Belarbi, 2000), and modied to include a synergistic effect between these two enzymes (Lpez et al., 2006): AAt AA ae kat b e kbt S1 AA (ae kat ) S2 AA (b e
kbt )

where v and v m are respectively initial and maximal initial reaction rates of the enzymes mixture in chestnut pure, S is the substrate concentration, K m and K s represent respectively

(10.3)

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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, preexponential parameters and rst order constants for each glucoamylase 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 reects 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 nally proposed as an alternative to reduce the need of high concentrations of enzymes, especially of glucoamylase (Lpez et al., 2004). Two main factors prevent the achievement of highglucose 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 pure 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 pure, carried out by using the hydrolyzate obtained in the rst cycle free of solids to make a new chestnut pure for the next cycle of hydrolysis, led to a 65% increase of the glucose concentration in the hydrolyzate obtained after three cycles of operation (Lpez 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 (Lpez et al., 2005). Applying the same optimal enzyme concentration and -amylase/glucoamylase ratio dened for the submerged operation, total hydrolysis was achieved operating in solid state at 70C (Lpez 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 possibility 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 consequence of mass transfer restrictions (Lpez et al., 2005).

100 80 % Hydrolysis 60 40 20 0 0 10 20 30 Time (min) 40 50 60

Figure 10.5 Kinetics of the one-step solid-state hydrolysis of chestnut with a mixture of 0.35/0.65 -amylase/glucoamylase and 60 EU/g of raw chestnut at three temperatures: 70C (triangles), 30C (circles) and 17C (squares). Published with permission of the American Chemical Society (Copyright 2005).

Summary Points

Enzymes are the underlying responsible for all the biochemical reactions that take part in the elaboration of many foodstuffs. Beer is an example of that. Amylases are, together with limit dextrinase and proteases, 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 nal levels of alcohol 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 inadequate conditions of pH, temperature and time of operation during malting and mashing can lead to low-quality beers. The prole 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, providing a readily fermentable substrate for the yeast. The latter 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

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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 benets increasing the wort fermentability of poorly modied malts, reducing 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 characteristics 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 during mashing to reduce the amount of fermentable sugars in the wort. Beers for diabetics, light or diet beers, which are characterized by low levels of residual sugars in the nal product, can be elaborated increasing the extent of starch hydrolysis during mashing by adding amylaserich multienzyme preparations. Beers for celiac people can be elaborated employing gluten-free starchy sources with low DP (as sorghum) in combination with the addition of exogenous enzymes.

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Author Queries {AQ1} Please note that Stenholm (1999) has been changed to Stenholm and Home, (1999) as per the reference list. {AQ2} Please check this paragraph. Is it ok? {AQ3} Please note that Brandam et al. (2002) has not been cross-referred in the text. {AQ4} Please provide the name of the publisher and the place. {AQ5} Please provide the Chapter title.

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