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Experimental Approach To Optimize the Use of
α-Amylases in Breadmaking
ARTICLE in JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY · MAY 2001
Impact Factor: 3.11 · DOI: 10.1021/jf010012j

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Nonetheless. high selectivity. in addition to the differences associated with their origins. The activity of different R-amylases was tested under different conditions (pH and temperature). The R-amylase is an endo-enzyme that randomly hydrolyzes the R-1. † Universidad de Buenos Aires. lipases. Argentina R-Amylases from different origins (wheat.O. Commercial R-amylases can be obtained from fungal. and crumb color. water activity. Spain and Departamento de Industrias y Quı´mica Orga´nica. Food Chem. breadmaking INTRODUCTION Nowadays. medium conditions (pH. E-mail: crosell@iata. because enzymes can promote effects similar to those of chemical additives but with the advantage of being considered as natural additives by regulators (1). Box 73. and relative humidity). However. The most extensively used enzymes are amylases. 46100-Burjassot. followed by those from cereal. stability. improvement of crumb grain. Lately. Universidad de Buenos Aires.00 © 2001 American Chemical Society Published on Web 05/19/2001 . their activity is greatly dependent on environmental conditions. the polysaccharides obtained from the hydrolytic activity also participate in the Maillard reactions that take place during baking. These results show the great variation of the R-amylase activity with the process conditions and the importance of its knowledge in the selection of the appropriate R-amylase for a specific breadmaking process. 18-20).J. cereal. because they are more susceptible to the enzyme attack. and (v) process conditions (pH. pentosanases. Nevertheless. These enzymes catalyze the hydrolysis of starch. activity. In baking there are enormous variations depending on (i) flour quality. and ionic strength) and the presence of different molecules that could modify their catalytic center (7. i. lipids. * To whom correspondence should be addressed. it is widely known that the activity and thermal stability of fungal. and proteins. the enzyme amount supplemented in relation to that of the flour should be optimized depending on the above-cited factors. it has been demonstrated that the antistaling effect of the R-amylases is due to their ability to retard the amylopectin retrogradation during bread storage (7-12). R-Amylases occur naturally in wheat flour but sometimes the endogenous activity is not sufficient to yield fermentable sugars. (iv) the process followed (sponge. to envisage the behavior of these R-amylases during the breadmaking process. Valencia. 2001. Fax: 34-96-363 6301. because of their increase of bread volume. (ii) ingredients used.csic. and in the presence of some bread ingredients (salt and sugar). resulting in short chains further fermented by yeast. Facultad de Ciencias Exactas y Naturales. 2973−2977 2973 Experimental Approach To Optimize the Use of r-Amylases in Breadmaking Cristina M. However. additives. the R-amylases are the enzymes most frequently utilized in bakeries. (2). 1428-Buenos Aires. only the bacterial R-amylases of intermediate thermal stability have been confirmed to be useful anti-staling additives by retarding the starch retrogradation (13-15). and flavor development promoted on the final product (3-6).1021/jf010012j CCC: $20. P. in this case it could be supposed that the R-amylase activity will vary according to both the process conditions and the enzyme origin. Keywords: R-Amylases. flours are frequently supplemented with exogenous R-amylases. malted barley. Enzymes are proteins characterized by their catalytic activity. Agric. and Carmen Benedito de Barber Instituto de Agroquı´mica y Tecnologı´a de Alimentos. crust. or microbial sources. and also catalyze the oxidation of the former compounds. it is necessary to have a thorough knowledge of the enzyme properties for their efficient application. nonstarch carbohydrates. and bacteria) are used extensively to improve breadmaking. In fact. The R-amylase activities were affected to a different extent by the addition of these compounds depending on the enzyme origin. Rosell. cereal. some differences are associated with their origin (21.4 glucosidic linkages in polysaccharides. Tel: 3496-390 0022. They have different thermal stabilities: fungal R-amylase is the most labile. (iii) presence of additives. because not doing so could result in under-dosage or over-dosage in the process. or sourdough).e. The objective of this study was to give some information that allows optimized use of R-amylases from 10. some breadmaking additives (ascorbic acid and sodium propionate). and the most stable are the R-amylases from bacterial sources (16. process conditions. and specificity. additives. fungi. 17). Therefore. 22). In general. are strongly affected by the process conditions and the presence of other compounds in the medium. temperature. straight dough. consequently. they can hydrolyze only damaged or gelatinized starch. etc.. and oxidases. Furthermore. However. Consejo Superior de Investigaciones Cientificas. Besides being necessary for fermentation. and some metabolites (organic acids and saccharides) generated during the fermentation step.* Monica Haros. In consequence.† Consuelo Escriva´. the use of enzymes has become a common practice in the baking industry. temperature. 49. the enzyme activities. the R-amylases from cereals (wheat and malted barley) were less sensitive to the presence of some ingredients. hemicellulases. and microbial R-amylases are pH-dependent.es. and metabolites. proteases.

the influence of the temperature on the hydrolytic activities of various R-amylases are shown. The pH rise led to the phenoxide colored form of pnitrophenol. Different carbohydrates (glucose. (24). MATERIALS AND METHODS A commercial blend of Spanish wheat flour (12.48% protein) from the local market was used to extract the endogenous wheat flour R-amylase. Vol. The cereal R-amylase assay reagent (from Megazyme International Ireland Ltd. salt. One unit of R-amylase activity was defined as the amount of enzyme which releases 1 µmole of p-nitrophenol per minute under the defined assay conditions. this study was complemented with the analysis of the effect of different metabolites. therefore it will be the enzyme with greatest activity during both the fermentation and initial baking steps. 15 °C) and the supernatant was filtered throughout glass wool. Extraction of r-Amylase from Both Wheat and Malt Flours.5. However. until the pH reached 6. the substrate reagent (30 µL) and then the enzyme extract (30 µL) were pipetted into individual wells of a 96-well microplate. Different behavior was observed with R-amylases from malted flour and bacteria: the first showed a bell-shaped activity curve reaching a maximum at pH 4. Agric. b. In addition. because it is more sensitive to any modification of the enzyme conformation. fungal. generated during the fermentation stage. the enzyme amount used was varied depending on the source in order to measure comparable activities. As expected.0. the enzyme activity increased with the temperature. were also added to the enzyme reaction medium in order to assess their effect on the R-amylase activity. additives. testing several final concentrations in the range customarily utilized in bread formulation. 2. Spain) referred to as bacterial. Other chemical reagents were analytical grade. The influence of sugar. sodium propionate) on R-amylase activity was determined by addition of those compounds to the enzyme reaction medium. the most common metabolites originated throughout the fermentation stage. 17). The fungal R-amylase and the endogenous one followed a similar trend. on the R-amylase activity. The pH affected the four R-amylases tested in different ways. below that a marked drop of activity was displayed. In Figure 2. bacterial. RESULTS AND DISCUSSION Effect of pH and Temperature on the Activity of Different r-Amylases. 6. Bioindustrial. 2001 Rosell et al. at that time 150 µL of 1% Trizma base solution was added to stop the reaction. there are some ingredients usually . Previous assays confirmed the stability of the R-amylase at 4 °C during the period of storage. Effect of Some Bakery Ingredients on r-Amylase Activity. even though it is not the natural substrate. but the trend was different depending on the enzyme origin: the bacterial R-amylase showed the highest increase with the temperature. Fungamyl 1500 BG (Novo Nordisk. because the activities of R-amylases from different origins were compared to that of the wheat flour endogenous R-amylase. Bioindustrial. The clear extract was kept at 4 °C for further enzyme assays. Effect of Some Ingredients and the Breadmaking Process on the r-Amylase Activity. Novamyl 1500MG (Novo Nordisk. but there is scarce information about the activity of these enzymes at the temperatures usually employed through the breadmaking process. A systematic study of the influence of some common ingredients.) was used to measure the R-amylase activity. and R-amylase from malted barley were tested. the enzyme reaction proceeded for 15 min at 30 °C. it is a specific substrate of R-amylase. the enzyme extracts were easily prepared by dissolving the commercial powder in distilled water. Fungal R-amylase. The greatest activities were observed at pHs higher than 4. using a Virtis homogenizer (3 × 10 s strokes at 20000 rpm). Spain) hereafter referred to as fungal. different origins. Briefly. Several reports described the inactivation temperatures of R-amylases from different sources (8. also. 9. In this study a short synthetic substrate was used to measure the enzyme activity. In the activity assays. The R-amylase activity was analyzed by measuring the enzyme activity in the range of temperature and pH usually found in the bread-making process. various R-amylases from different sources (cereal. and different additives (ascorbic acid. In the case of the fungal and microbial R-amylases. There are numerous formulations used in the breadmaking process depending on the type of bread desired. fungal. which was measured at 405 nm by using a microplate reader (Spectramax 190. malted flour. and process conditions (pH and temperature) on the hydrolytic activity of various R-amylases from different sources will be presented. The R-amylase activity was measured by using a blocked p-nitrophenyl maltoheptaosido (BPNPG7) as substrate following the method reported by McCleary and Sheenan (23) and further adapted to a microplate reader by Sirou et al. Molecular Devices). Determination of r-Amylase Activity. were also used. and bacterial) were analyzed at various pH levels and temperatures usually found through the breadmaking process. 15 min. 49. control.. Various R-amylases of different sources. whereas the bacterial type displayed a continuous increase of activity as the pH increased. In all cases four replicates were assayed for each experimental point. It is known that the bacterial R-amylase of intermediate thermal stability also exhibits the highest thermal stability (8). microbial R-amylase of intermediate thermostability. The homogenate was centrifuged (12000 rpm. Extracts were prepared by homogenizing 10 g of wheat flour or malted flour in 50 mL of water. Symbols: (. and maltose) and organic acids (lactic and acetic).5.2974 J. No.0.5-5. The enzyme activities of Figure 1. keeping their activity constant at pHs higher than 4. which means that it is not hydrolyzed by β-amylases. The effect of pH on the R-amylase activities is shown in Figure 1. Food Chem. gifts from several companies. fructose. Effect of pH on the R-amylase activities from different sources. The extract obtained from wheat flour was referred to hereafter as the control.

malted flour. bacterial. Ascorbic acid is an oxidant usually added in breadmaking to strengthen dough through the process (for detailed information see Grosch’s review. 9. The addition of salt promoted a decrease of the R-amylase activity in the range of salt assayed. in fact. malted flour. In consequence. 2. Symbols: (.0 mM sucrose. Food Chem. confers color to bread crust. which showed a pronounced increase of its activity as the ascorbic acid concentration was augmented (Figure 5). Effect of sodium chloride on the activity of various R-amylases. These ingredients could affect the activity of the endogenous R-amylase together with the R-amylase supplemented as a commercial additive. 49. Influence of temperature on various R-amylases from diverse origins. whereas fungal R-amylase retained 30% of its activity at the same concentration. as less than 15% of its activity remained at 1. 9. No. However.Activity of R-Amylases from Different Sources Figure 2. fungal. Influence of Some Bakery Additives on r-Amylase Activities from Different Sources. Figure 3. The R-amylase activity showed a continuous decrease with increasing concentrations of sucrose. they proposed the use of salt as a way to control the enzyme activity in J. 28). again. respectively. bacterial. we observed a diverse grade of inhibition.0 mM sucrose. control. fungal. control. 2001 2975 Figure 4. 27). were exhibited at 5 mM salt. Only 40% of the enzyme activity in the case of bacterial R-amylase. b. who reported the inhibition promoted by the addition of growing concentrations of sucrose on the R-amylase of Aspergillus oryzae. Effect of sucrose on the activity of some R-amylases.88%. Symbols: (. Symbols: (. Agric. The endogenous and malted flour R-amylases exhibited 52% and 69% of their activity at 3. when doughs were supplemented with fungal or bacterial R-amylases this inhibition should be considered in order to adjust the enzyme dosages. The direct effects of sodium chloride and sucrose on the R-amylase activity was assayed at the different concentrations usually utilized in baking. b. b. However. This behavior was already envisaged by Adams (18). with the exception of the R-amylase from malted flour. fungal. malted flour. 6. This behavior was explained by . furnishes fermentable sugars to yeast.. 2. employed in baking such as salt or sugar. Bacterial R-amylase was drastically inhibited by sucrose. This inhibition agrees with previous results reported by Harinder and Bains (25) showing the increased rate of the falling activity number as the salt concentration increased from 0 to 1. The R-amylases from cereals were less inhibited by salt than those from fungi or bacteria. Sucrose is another ingredient frequently used in baking. Sucrose provides sweet taste. 2. and improves the texture. retaining only 50% of its activity at 5 mM NaCl (Figure 3). control. The effect of the presence of sucrose in the reaction media on the R-amylase activity has also been analyzed (Figure 4). The presence of growing concentrations of ascorbic acid hardly affected the R-amylase activity. Vol. high R-amylase flours in order to improve the loaf quality when using sprouted wheat in bakery (26. R-amylases from cereals were inhibited in less proportion than R-amylases from fungi or bacteria. or less than 20% in fungal R-amylase. when the activities of R-amylases from different origins were compared. bacterial. 9.

8 ( 6.5 ( 3.6 134. The unique exception to this behavior was the R-amylase from malted barley flour.0 158.2 ( 1.2976 J. The values are means of four replicates ( SD.6 ( 5.9 ( 2.5 ( 4. 2.3 94. Effect of Different Acids and Sugars on r-amylase Activities from Various Sources acetic acid Figure 5.4 ( 2.3 ( 4.7 ( 0.0 122.4 ( 5. No. retaining more than 50% of its activity at 2.1 ( 6. with the R-amylases from malted flour and bacteria showing the highest increase.1 115.0 ( 12.3 fructose 8 106. fungal.3 ( 6. this result was likely related to approaching its optimum pH (as was observed with the addition of ascorbic acid).3 ( 3.9 128.9 84.6 154.0 123.8 112. other additives commonly used in baking include anti-microbial agents such as different salts of propionic acid.5 138.2 ( 3.9 ( 9. To determine the possible effect of some compounds generated through the fermentation stage.5 ( 2..0 a The R-amylase activity was referenced to the activity of the control (R-amylase activity of the wheat flour).0 ( 1. Agric.5 110 ( 2.7 ( 9. fungal. fructose.2 145. malted flour. The effect of various concentrations of sodium propionate (NaProp) on R-amylase activity was also investigated (Figure 6). but large differences were observed regarding both the acid added and the R-amylase origin.5 92. who found that ascorbic acid inhibited β-amylases but did not affect the R-amylases of plants.1 ( 0.0 mM NaProp).3 90 101.0 ( 2.1 115.0 108.4 107. it is not known how these salts affect different R-amylases from various sources. A drastic inhibition by this salt was observed with the other R-amylases.7 103.7 ( 2. The addition of sodium propionate inhibited the R-amylase activity but to differing extents depending on the enzyme origin.7 ( 3.1 25 101.5 ( 0. Effect of Different Metabolites Generated during Breadmaking on the r-Amylase Activity.9 ( 9.9 104.9 ( 2.7 100. Food Chem. A slight enhancement of the R-amylase activity was observed. Selection of the range of concentrations to be assayed was based upon the previous results reported by Barber et al.4 230.2 159.2 ( 5. In addition.8 ( 0.7 33 97.0 ( 4. Effect of ascorbic acid on the activity of some R-amylases from various sources.1 ( 2. 9.6 ( 5.1 224.9 50 102.7 lactic acid 25 105.9 ( 1. Vol.4 ( 3.9 ( 2.3 ( 1.2 129.7 ( 13.9 80 107. because the reaction medium was not buffered (results not shown). control.1 ( 9.3 193.9 111.3 17 105.3 20 100.6 100.9 maltose 15 104.6 ( 0.6 96. 49.1 glucose 6 105. b.9 205. These results are in agreement with those previously reported by Purr (29).2 120.2 133. 6.9 182. This effect was mainly attributed to its presence rather than to its concentration.1 ( 2. The presence of acids led to an increase of the R-amylase activity. control.0 ( 1. which showed higher activity at growing acid concentrations.9 ( 2.8 109.8 117.7 100.6 110.7 30 105.7 196.0 ( 0. Table 1. 2.0 90.0 ( 2. (30).8 ( 5.0 ( 0.3 ( 5. also explained by the effect of the acid upon the pH. The endogenous R-amylase was the most stable. An inhibitory effect of the enzyme activity should be expected because the salts of propionic acid are used as anti-microbial agents.2 ( 1. b. bacterial. who analyzed the acid compositions in several doughs prepared by using sourdough. Symbols: (.6 121.3 ( 9. (µM) control malted flour fungal bacterial 0 40 80 125 100.8 112.3 111.0 114.4 ( 6. which showed a steady increase of activity with the acid concentration.4 ( 0.0 ( 0. with the fungal R-amylase being the most affected (retaining only 9% of its activity at 2. except for the R-amylase from malted barley flour.0 105. moving that to the malted flour R-amylase pH optimum.4 ( 7. malted flour.1 ( 2. but R-amylase activity (%)a conc.8 100. and glucose) were selected as the most representative metabolites. Figure 6.6 ( 4.6 ( 0.5 109.9 214. bacterial.5 ( 0.8 ( 1.4 ( 0.0 161.9 10 100.0 ( 2.1 ( 5.4 98. because only slight variations were observed at increasing concentrations of acetic acid. 9.7 ( 1.2 ( 4. The effect of these compounds on the activity of the different R-amylases tested can be observed in Table 1.0 ( 0. Symbols: (.8 ( 0.6 103. two acids (lactic and acetic) and various saccharides (maltose. Acetic acid yielded an increase of the R-amylase activity. Influence of sodium propionate on the R-amylase activities from different origins. The effect of lactic acid was less pronounced than that of the acetic acid. 2001 Rosell et al.7 142.9 106.6 206.3 103.3 ( 3. . the effect of ascorbic acid on lowering the reaction medium pH.1 ( 12.6 109.4 104.3 ( 0.5 ( 2.8 122.6 ( 1.5 ( 4.0 ( 0.0 mM NaProp.2 60 98.1 111.

23. 1965. E. 1985. Food Technol. Leatherhead Food RA Food Ind. 20-27.. Eds. Influence of alpha-amylases on bread staling and on retrogradation of wheat starch models. JF010012J . 133-149. K.. Otzen... 223-232. A. (21) Bird. Starch/Staerke 1998. 19. J. CONCLUSION From the above results it could be concluded that. 71. Brathen. Baker’s Dig. C. Buleon. 1FD97-0671-C02-01) and Consejo Superior de Investigaciones Cientificas (CSIC.. C. 1996. L. Rev... Bread Staling: X-ray diffraction studies on bread supplemented with R-amylases from different sources. 7. Cereal Chem. 570-572. 239-241. B. (19) Rubenthaler. Firming of starch gels and amylopectin retrogradation as related to dextrin production by R-amylase. 4. 212. Technol. Food Chem... Escriva. M.. 36-41. J.. E. (16) Miller. R-amylases commonly employed in baking are selected as a function of their thermostability. The action of some R-amylases on amylose.. Galliard. Revised manuscript received March 22. G. Eur. 68. The influence of vitamin C (ascorbic acid) on plant and animal amylases. A. A. Palmer. Benedito de Barber. D. Martı´nez. Food Chem. (24) Sirou. Kulp. 58. Use of intermediate temperature stability enzymes for retarding staling in baked goods. (18) Adams. Antistaling additive effect on fresh wheat bread quality. (11) Champenois. G.7 angstrom resolution. 28. C. Biochem J. acid and salt on the rheological properties of dough. 139-144. Pomeranz.. 1987. 32. V. P. J.. S. T. Currently. Received for review January 2.. Colonna. T. C. Cereal Foods World 1991. Food Chem.. S.. Y. R. Cereal Chem. Z. C. A. A. D. Europe.. Biochem. although enzymes are really useful in food technology. Vol. Finney. 131-134. 1997. however higher activities resulted when the R-amylases were from malted flour and fungi. W. K. Lecommandeur. salt and L-cysteine HCl in the dough. the five-domain “maltogenic” amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1. (5) Sahlstro¨m. 310-314.. K. 38. L. A. Lebensm.. Dura´n. 1999. fungal. A. 471-486. 425-435. Bowles. R. (12) Dura´n. Planchot. Effects of enzyme preparations for baking. Beier. 2001.. (2) Martı´nez-Anaya. Finney. 619-624. T. 35. 6. Andreu. Schafer.. Barber.. Devesa. J. E. M. Aliment. Cereal Chem. for the postdoctoral grant. (3) Armero. R. E. Norman. J. Cereal Chem. 1997. 3842. C. Effect of low molecular weight dextrins on gelatinisation and retrogradation of starch.. Accepted March 22.. acid and salt on paste characteristics. Brzozowsk. Sci. In consequence. Int. Ponte. Christensen. 2001 2977 (13) Hebeda. V. Food Technol. L. Andreu. 1980. 3. Unters. Agroquim. Y. Aliments 1999. J.. J. J. Food Sci.. Int... Collar. K. R.. 86-99. Collar. The Royal Society of Chemistry: London. Blanschard. D. Bains. 1986. 36. X-ray structure of Novamyl. W. and bacterial alpha-amylases as supplements for bread-making. Studies on baking of high alpha-amylase flours: effect of pH. 8385-8392. R. M. C. Effects of the combination of starters and enzymes in regulating bread quality and shelf life. V. Varriano-Marston. A.. 7580. 2001. the addition of the sugars tested did not modify the activities of the R-amylases from wheat flour (control) and the bacterial R-amylase. H. 1953. High R-amylase flours: effect of pH. S.Activity of R-Amylases from Different Sources With respect to the sugars influence. Bains. 56. Storage of wheat breads with hydrocolloids. M. Among the sugars studied. 49.. 64. 25. High R-amylase flours: effect of pH. F. (30) Barber. J. J. Johnson.. (25) Harinder. 1994. Zooming in on enzymes. Bains. enzymes and surfactants: anti-staling effects. a thermostable alpha-amylase. (20) Pomeranz. S. Christensen. (10) Leo´n. Haros thanks the Rene´ Hugo Thalmann Program of the Universidad de Buenos Aires. A. E. S. Leo´n. LITERATURE CITED (1) Penstone. Food Sci. Wilson. R. S. 2. Cambios en los a´cidos orga´nicos vola´tiles C2-C5 durante la fermentacio´n de masas panarias preparadas con masas madre comerciales y con cultivos puros de microorganismos. Lauriere. (4) Qi Si. C. E. (9) Akers. Argentine. 98-101. G. 453-457. 205. B. 481-490. Water-soluble dextrins from R-amylase treated bread and their relationship to bread firming. Cereal Sci. 2... Bowles. Z. A. Cereal Foods World 1990.. Food Technol.. G. 11411148. A comparison of cereal. 1990. 1994. Frazier. 588-594. L. 1998. J. S. I. G. E. 1999. V. (23) McCleary. Agric. 2001. 1996.. F. Nahrung 1988.. (6) Martı´nez.. D. Valle. Y. Hoseney. 49. 39-45. Enzymatic activity during bread baking. 359-363. (14) Christophersen. K. (28) Grosch. 38. K. mixing time and resting time on bread quality and bread staling. J. Comparison to fungal and bacterial enzymes and an emulsifier in white pan bread. Agric. Sheenan. Technol. 1987. thorough knowledge about the system where the enzyme will act is necessary before selecting its source. M. M. This study was financially supported by the European Union and Comisio´n Interministerial de Ciencia y Tecnologia Project (FEDER. P.. Borchert. Measurement of cereal R-amylase: a new assay procedure.. 6. B. W.. Food Technol. 1996. M.. 35-38. No. 7. 2001. New enzymes for the baking industry. G. S. Teague. E. 1934... K. 1975. Forsch. P. 1990. 1954. 67. S. A. Enzymatic characterisation of NovamylRegistered. Dauter. Spain). (15) Dauter. Biochem. G. but as has been exposed through this study. 323-333. 1987. 60-64. K.. Collar. (26) Harinder. Developments in enzymes for retarding staling of baked goods. K. J.. 45.. Amylases: their kinds and properties and factors which influence their activity. T. 171-174. T. R. Specific enzymatic micro-assays of R-amylase and β-amylase in cereals. Studies on a rawstarch digesting enzyme. Benedito de Barber. C. (7) Dragsdorf. (29) Purr. Cereal Foods World 2000.. 57. Redox systems in dough.. 50. Alberola. Cereal Chem. 203-207. Food Rev. prior to selecting a specific enzyme it is necessary to have thorough knowledge of its properties and also of the environmental conditions under which it is going to be added.. Teague... P. Food Res. Technol.. M. H. Sugars in breadmaking.. C.. (22) Valjakka. pp 155-169. Martı´nez-Anaya. Tecnol. (8) Hebeda. P. Davies. In Chemistry and Physics of Baking. B. all the compounds present and the medium conditions will affect the final enzyme activity. 19. Y. K. maltose produced the highest enzyme activation. (27) Harinder. C.. ACKNOWLEDGMENT M. (17) Mathewson. Benedito de Barber. Hopkins. 1953. 237-251. E..