THE EFFECTS OF MASHING TEMPERATURE AND MASH THICKNESS ON WORT CARBOHYDRATE

Transcription

1 J. Inst. Brew., March-April, 1991, Vol. 97, pp THE EFFECTS OF MASHING TEMPERATURE AND MASH THICKNESS ON WORT CARBOHYDRATE COMPOSITION By Robert Muller (Brewing Research Foundation, Lyttel Hall, Nutfield, Redhill, Surrey, RH1 4HY) Received 2 My 1990 Temperature and mash thickness are shown to affect both mash performance and enzyme activity. Alpha amylase was found to be considerably more resistant to heat inactivation than was beta amylase. This difference was reflected by changes in wort fermentability that were manifest at temperatures below those which affected levels of extract. Increasing the mashing temperature from 65 C to 80 C had only a slight effect on extract but reduced wort fermentability from over 70% to less than 30%. At 85 C and over, when temperature had a significant effect on alpha amylase, as well as on beta-amylase, extract was lost and starch was present in the wort. Diluting the mash with liquor had a similar effect to that of increasing temperature on both the amylolytic enzymes and on the mash performance. Thin mashes contained more starch and fewer fermentable sugars than did thick mashes at the same temperature. These changes can be related to the stability of the amylolytic enzymes. Key Words: Alpha amylase, beta amylase. mashing temperature, mash thickness. Introduction In recent years a significant amount of attention has been directed towards controlling the carbohydrate composition of wort Initially this interest centred on optimising mashing conditions to ensure complete extraction of the malt with a maximum conversion to fermentable sugars. The aim was to produce the greatest quantity of fermentable sugar so that the final beers would have high alcohol and low carbohydrate concentrations14'23. This work revealed that it was not possible, by simply using malt enzymes, to convert all the starch to fermentable sugars. This could only be achieved by the addition of enzymes, such as amyloglucosidase, to the brews prior to fermentation More recently, the use of superattenuating yeasts12-"-22 has by-passed the necessity for using exogenous enzymes. This allowed the appearance of a new type of product, the 'lite' or 'diet' beers which contained a much higher proportion of alcohol compared to carbohydrate than did normal beers. The process of mashing relies on the different biochemical characteristics of the enzymes involved. The enzyme alpha amylase is largely responsible for the breakdown of starch into lower molecular weight sugars and dextrins. Some of the sugars are fermentable but the majority, the dextrins, are not. The other important enzyme is beta amylase. Although beta amylase has a limited effect on starch, it can rapidly break down dextrins to form the fermentable sugar maltose'. The second important difference between these enzymes is their thermal stability. Alpha amylase is considerably more stable than beta amylase at high temperatures. These enzyme properties could be exploited to control the carbohydrate composition and fermentability of the final wort. However, the response of these enzymes to temperature is poorly character ized. Certainly, in the brewing literature, there is little infor mation concerning mash performance at temperatures over 75 C. In the past there has been little need to lower the fermentability of wort, usually the reverse was desired. There is now an interest in beers with reduced or low levels of alcohol, and production of these would benefit from controlled wort fermentability. Extraction of the malt must not be impaired, and starch must not appear in the final worts. The present study establishes mashing regimes that can be used to control wort fermentability from a maximum down to less than 10%. Experimental Small scale mashing was performed using a BRF mashing bath as described by Buckee et al.6 except that the mash temperature and thickness were varied. Mashing experiments using 1.5 kg of malt were performed on the BRF mashing column described by Bathgate et al3. The measurement of total carbohydrate levels in wort were performed by the method of Buckee and Hargitt5, and the levels of fermentable sugars in wort were determined using HPLC7. For the purposes of these experiments the levels of starch in worts were defined as carbo hydrate material that was precipitated from acidified solution by iodine14. Estimation of alpha amylase activity was by a modification of the method of Smith et al2i. Samples were taken from the B.R.F. mashing column and cooled rapidly on ice; the alpha amylase was then extracted using the buffers recommended by Smith etal21. The activity of this extract was determined using beta-limit dextrin (Rank Hovis) at 35'C. A standard curve of amylase over a temperature range from 15*C to 40 C was also prepared and this was used to extrapolate enzyme activity to 65'C. Over the lower temperature range the amylase was unlikely to suffer from thermal inactivation. Thus the initial activity at 65*C could be inferred, without encountering problems caused by heat and damage to the enzyme. To estimate beta amylase, samples of mash, or of malt, were extracted at an equivalent concentration of 50 g dry matter in 150 ml of mashing liquor. The liquor was adjusted to 30 mm sodium chloride, 3 mm dithio-threitol, 30 mm ethylenediamine tetraacetic acid (sodium salt) and 0.06 M acetate at ph 4.8, (enzyme buffer), by the addition of appropriate volumes of the concentrated solutions. The mixtures were shaken for one hour at 2*-4"C and undissolved material was removed by centrifugation at 2000 x g for 5 minutes. The supernatant was collected and diluted (with buffer) as required. After equilibration at 20 C, 1 ml of extract was mixed with 1 ml of buffer and 1 ml of 2% w/v Lintner starch. After 3 minutes at 20*C, 3 ml of DNS reagent was added. This reagent contained 1% w/v dinitrosalicylic acid (DNS), 1.6% w/v sodium hydrox ide and 30% w/v potassium sodium tartrate. The mixture was boiled for 5 minutes and cooled by the addition of 20 ml of cold water. The absorbance of the mixture was monitored at 540 nm, and the values for unknown samples compared with the appropriate blanks and standard solutions. Mash liquids frequently gave very high background values due to the presence of starch conversion products. These were effectively removed by dialysis overnight at 4 C, against enzyme buffer. As with alpha amylase activity, the initial beta amylase activity at 65 C was inferred from a standard curve. In this case

2 86 WORT CARBOHYDRATE COMPOSITION [J. Inst. Brew. the standards were obtained over the temperature range 10 C to 30"C. Laboratory scale fermentations, using one and a half litres of wort, were performed by the method of Brown and Kirsop4. Results The effect of different mashing temperatures on wort fermentabilily is due to the different properties of the two main amylolytic enzymes. Figure 1 shows that at 65 C (a typical mashing temperature) alpha and beta amylases have different stabilities. In order to achieve a mash stand temperature of 65'C, the liquor temperature was 72"C before striking. The temperature of the mash was monitored over the sampling period, the average value being 64.4 C. Samples of the mash were removed and the enzyme activity determined as described above. After 60 minutes approximately half of the alpha amylase activity remained, whilst less than ten per cent of the original beta amylase activity was present. The thermal decay of enzymes may be characterized by the equation A=Aoe~I", where Ao is the initial activity, A is the activity at time t, and t is the duration of the mash. From the data shown in Figure 1 the decay constants, k, of malt alpha and beta amylase activity were calculated. The value for alpha amylase was (r=0.96) and for beta amylase (r=0.98). This demonstrates that alpha amylase is considerably more stable than beta amylase at 65 C. Although there may be many potential inaccuracies with these measurements (see discussion), the equation can be integrated to give an indication of the total enzymic activity during the mash. The total potential activity of alpha amylase (adjusted to 65*C) was 87.9 g of starch digested per gram of malt, a level far in excess of that required to digest the amount of starch actually present. The total activity of beta amylase was considerably lower at 3.5 g of maltose produced per gram of malt. So not only was alpha amylase more stable than beta amylase at mashing temperatures, but loss of some alpha amylase could be predicted to have less effect on a mash than the loss of beta amylase. Figure 2 A-D compares the activity of alpha and beta amylases in mashes over a range of temperatures from 75 C to 90'C. Again l.s kg of malt were mashed with 3.75 L of water. The striking temperature of the liquor was adjusted each time to achieve the desired mash temperature, and was usually 6-TC higher than the final temperature17. At 75 C there was significant decay of both enzymes but considerable amounts of beta amylase were still present at the end of a 30 minute period. Conversely at 90 C there was extremely rapid breakdown of both enzymes. Almost all of the beta amylase was destroyed within 10 minutes but very little alpha amylase survived after 20 minutes. It remains unlikely that in such a mash there would be sufficient enzyme activity to completely digest all of the starch. The greatest difference in enzyme stability was observed at temperatures between 80 and 85 C. At these temperatures there was essentially complete inactivation of beta amylase but with sufficient alpha amylase activity surviving throughout the mash to ensure complete starch conversion. The effect on wort properties of a range of mashing tempera tures is shown in Figure 3 A-C. In view of the dramatic effects of very high temperatures on enzyme stability (shown in the previous figure) it was decided to study mashes over the slightly lower range of 70 C-85 C. In this case laboratory scale mashes were performed in the BRF mashing bath using SO g of malt with a range of mash thicknesses from 2:1 to 7:1. All mashes were cooled and subsequently adjusted to 450 g with water and filtered. The total carbohydrate, fermentable sugar and starch Alpha Amylase Activity 1.4r Beta Amylase Activity -i Time (mln) Fig. I. Inactivation of malt amylase mashed at 65*C. Samples of mash were taken from a standard run on the BRF Mashing column using l.s kg malt mashed with 3.75 litres of water (liquor to grist ratio 2.5:1). Alpha and beta amylases were extracted and their activity determined at a standard temperature. This activity was corrected to 65 C by extrapolation. Alpha amylase ( ) activity is presented as grams of limit dextrin hydrolysed per minute per gram of malt. Beta amylase (O O) activity is presented as grams of maltose produced per minute per gram of malt.

3 Vol. 97, 1991] Figure 2A 75 C WORT CARBOHYDRATE COMPOSITION Figure 2B 80 C Time (minutes) Time (minutes) Figure 2C 85 C Figure 2D 90'C 120 r <> Time (minutes) Time (minutes) Fig. 2. Enzyme stability in mashes of different temperatures. As for figure I except that the striking temperature of the liquor was adjusted to give mash stand temperatures of 75'C, 80 C, 85 C and 90'C. In this case the enzyme activities are expressed as a percentage of the original so that a direct comparison can be made. Alpha amylase ( ): beta amylase (O O).

4 88 WORT CARBOHYDRATE COMPOSITION [J. Inst. Brew. Figure 3A [Carbohydrate] % 12r Water (ml) / Malt (50g) Figure 3B Fermentabllity % 80r Figure 3C Starch % Water (ml) / Malt (50g) Water (ml) / Malt (60g) Fig. 3. Effect of mashing temperature on the carbohydrate contents of wort. Samples of malt (50 g) were mashed with varying amounts of water in the BRF mashing bath. After 1 hour at the temperatures shown, the mashes were cooled and adjusted to 450 g with cold water. After filtration the total carbohydrate levels (3A) were measured by the anthrone reaction. Fermentable sugar levels (3B) were determined by HPLC and are presented as a percentage of the total carbohydrate level. Wort starch (3C) was measured by iodine precipitation followed by anthrone reaction. Temperatures: 70 C ( ); 75"C (O O); 80 C (A A); 85 C ( D).

5 Vol. 97, 1991] WORT CARBOHYDRATE COMPOSITION 89 levels in the wort were measured using techniques described above. The temperature range studied had only a slight effect on the level of total soluble carbohydrate that could be extracted into the wort (see Figure 3A). At the lower water to malt ratios (thicker mashes) there seemed to be more variation with the lower temperatures yielding carbohydrate levels between 8 and 9 g/100 ml and with the higher temperatures yielding levels between 9 and 10 g/100 ml. At higher ratios there was less variation. The situation was very different where fermentability was concerned (Figure 3B). At each mash thickness a 5'C increase in temperature significantly reduced the fermentability of the final wort. Decreasing the mash thickness also resulted in wort with lower fermentability. This latter effect was small at 70'C but a difference of 26% fermentability was obtained at 75'C between worts from mashes at ratio 2:1 and 7:1. Indeed the fermentability of wort produced at 75 C and ratio 7:1 (30.3%) was lower than wort produced at 80 C and a mash thickness of 2:1 (33.8%). A thin mash (7:1) at 85 C yielded wort of only 6% fermentability, despite of producing good extract levels (Figure 3A). The results for fermentability were mirrored by the wort starch measurements in thinner mashes where each S'C rise in temperature increased the levels of wort starch, the difference between 80 and 85'C being most dramatic (Figure 3C). In addition at 80' and 85 C there was a significant increase of starch with decreasing mash thickness, with the effects at 85'C again being most noticeable. However between mash ratios 2:1 to 3:1 at 70', 75* and 80'C there was very little difference in wort starch levels. The effects of mashing temperature on wort sugars were investigated further using the BRF mashing column. Thus 1.5 kg of malt were mashed with 4.5 litres of water (ratio 3:1) at 85'C. During the mashing-in process the temperature fell to 80 C, where it was held for 15 minutes. Extending the mashing time for periods up to 60 minutes did not have any beneficial effect. Probably by this time most of the starch had already been digested, and most of the enzymes destroyed. Figure 4 shows the carbohydrate profiles of worts run from the BRF mashing column over time. The run off from the mash was begun after 15 minutes with sparging at the usual temperature of 72 C. The three parameters shown are total carbohydrate (as % concentration), wort fermentability (as fermentable sugars/total carbohydrate * 100%) and the pro portion of starch (as starch/total carbohydrate x 100%). The elution profile of total carbohydrate run from the mash showed that the maximum concentration of carbohydrate (21.4 g/ 100 ml) was achieved after 40 minutes from the opening of the taps, and declined thereafter. The fermentability also rose to a maximum, at 40 minutes, before declining. A second peak of fermentability occurred at 60 minutes. This second peak coincided with the passage of the sparge front through the mash and into the receiving vessel. Wort collected in the first 5 minutes after the taps had been opened contained some undigested starch. It is likely that this was due to the presence of starch underneath the false bottom of the mash bed, rather than the high mashing temperature since cloudy first runnings were a feature of 65 C mashes also. Undigested starch could be largely eliminated by recycling the first 5% of wort to the top of the mash bed, giving the mash sufficient time to form a good filter bed, and ensuring that all the wort had the opportunity to pass through this filter. The carbohydrate composition of the wort could be further modified by maintaining it between 70 and 80*C before boiling. Figure 5 shows the destruction of alpha and beta amylases in thin solution rather than in a thick mash. At 70'C tch2o;fermentabllity % Starch/tCH2O t 20 Taps open Time (mln) Fig. 4. Analysis of wort run-on*. The BRF mashing column was used to mash 1.5 kg of malt at 80'C. After IS minutes the taps were opened and Tractions of wort were collected over a period of 80 minutes. From these fractions the total carbohydrate (by anthrone reaction), fermentable sugars (by HPLC) and starch (by Iodine precipitation) were determined. The fermentable sugars contents are presented here as fermeniability (fermentable sugars/total carbohydrate) and the starch content as a starch Traction (starch content/total carbohydrate). Total carbohydrate % ( ): Termentability % (D ); starch/total carbohydrate % ((A A).

6 90 WORT CARBOHYDRATE COMPOSITION [J. Inst. Brew Time (minutes) Fig. 5. Stability of malt amylases in solution. Alpha and beta amylases were extracted from malt using cold buffers. One hundred millilitre volumes were maintained at the appropriate temperature and sampled for enzyme analysis at various times. Alpha amylase 70*C ( ): beta amylase 70 C (O O); alpha amylase 80 C (A A); beta amylase 80*C (D D). beta amylase was rapidly inactivated but more than 80% of alpha amylase activity remained after 30 minutes at this temperature. At 80 C however the breakdown of alpha amylase was much more rapid. The results shown in Figure 3B and 3C have already indicated that the amylase enzymes are less stable in thinner mashes resulting in lower fermentabilities and higher levels of starch. A comparison of Figures 2 and 5 shows that amylases are stabilized by the thicker mash and are very unstable in a solution such as wort. By maintaining the wort at a temperature higher than 70 C but lower than 80 C any remaining beta amylase was rapidly destroyed before it could produce significant amounts of maltose whilst the alpha amylase retained sufficient activity to breakdown any remain ing starch. Figure 6 shows an HPLC analysis of a wort produced by the methods described above. Malt (1.5 kg) was mashed with 4.5 L of water at either 65*C or at 80 C and after a 15 minute mash stand 10 L of wort was collected over a period of 1 hour. Both worts were boiled for I h. The worts contained the same quantity of total carbohydrate measured by an anthrone reaction and both had a specific gravity of However, the quantity of ferment able sugars present in the wort produced at 85 C was consider ably lower than in the worts produced at 65OC. The total quantity of glucose in the low fermentability wort was 30% of that in the normal wort. Similarly the quantity of maltotriose was 23% of the normal wort. These two sugars give the best indication of the inactivation of alpha amylase, since they were not products of beta amylase activity. The low fermentability wort contained 8.6% of the maltose found in the normal wort. Maltose is largely a product of beta amylase activity which was the main target of the elevated temperatures. Thus it was the maltose concentration which was most severely affected. Interestingly the sucrose level was only reduced to 71.0% since it is largely a product of the malting process. The two worts described above were fermented using a standard BRF ale yeast, NCYC In addition a quantity of Quantity Maltose Concentrations % Low Fermentablllty Normal 1 Glucose Sucrose Sucrose Maltose Glucose Maltotrlose Maltotriose Totals Fraction number Fig. 6. Wort sugar analysis. Two worts (both gravity) were prepared from the BRF mashing column, at 65 C and at 80 C. The individual fermentable sugars were determined by HPLC in conjunction with standard sugar solutions. Normal wort ( ); wort produced at high temperature ( ).

7 Vol. 97,1991] WORT CARBOHYDRATE COMPOSITION 91 Yeast weight (g) 0.1 r Time (hours) 60 Fig. 7. Yeast growth in low fermentability wort. The two worts from Figure 6 were fermented in 1.5 litre stirred fermenlers using NCYC In addition 10 ml of amyloglucosidase (Novo) was added to a third fermenter containing 80 C wort. During the growth period, 10 millilitre samples were removed, the yeast washed, and the dry weight measured. Normal wort (A A); wort produced at high temperature ( - ); wort produced at high temperature and with amyloglucosidase (O O). amyloglucosidase was added to a third fermentation vessel containing wort from an 80"C mash. The growth of yeast was monitored over a period of 72 hours. The fermentation profiles of the normal wort and wort from an 80 C mash began in a similar manner (Fig. 7). However, by 24 hours, when the yeast in the normal wort was growing vigorously, fermentation in the wort from the 80 C mash was almost complete. The fermen tation in the normal wort continued for another 24 hours. This experiment clearly demonstrates that not only does wort from a high temperature mash appear to be less fermentable from its chemical analysis but that it indeed supports less yeast growth. However, when treated with amyloglucosidase, this wort supported as much, or slightly more, yeast growth as a normal wort. The action of amyloglucosidase is to generate fermentable glucose units from the non-fermentable dextrins. (This can be demonstrated by HPLC analysis but is not presented here.) This experiment confirms that a large part of the extract obtained during high temperature mashing was not fermentable by normal yeast but demonstrates also that some brewing enzymes, or superattenuating yeast may be able to utilize the dextrins. The faster rate of growth in the treated wort was probably due to the greater proportion of glucose formed by amyloglucosidase activity. Discussion Although mashing regimes differ widely between breweries, in general a brewer does not alter his mashing process on a routine basis. Certainly a lager malt is mashed differently from an ale malt, but within these broad groups a brewer will probably use a standard mashing regime. In fact mashing para meters can be altered to achieve a desired wort composition. The two enzymes which are largely responsible for the carbo hydrate composition of wort are alpha and beta amylase16. The sole product of beta amylase activity (in the presence of alpha amylase) is maltose whilst alpha amylase produces a variety of different molecules9. These range from the lower molecular weight sugars, glucose, maltose and maltotroise, which are fermentable by most brewing yeasts, to high molecular weight dextrins which are not. Figure I shows that these two enzymes have different stabilities at high temperatures. It is important to realize, in the wider context of mashing, that it is alpha amylase that is an unusually stable enzyme at high temperatures. The other enzymes in the mash, including beta amylase, have much lower thermal stabilities2-23. The data in Figure I can be used to assess to total enzymic activities available during the mash. There are a number of potential inaccuracies in such an exercise; (i) alpha amylase activity was measured at 35 C and extrapolated to 65 C using a standard curve; (ii) its substrate was limit dextrin rather than starch; (iii) beta amylase was measured at 20 C, and also extrapolated to 65 C; (iv) its substrate in a mash would include dextrins rather than just starch; (v) both enzymes were measured separately but would act in concert during mashing and (vi) both were measured in thin solution rather than a thick mash. Nevertheless these differences would tend to result in an under estimate, rather than an over-estimate, of available enzyme activity. The results shown in Figure 1 suggest that there is considerably more alpha amylase activity than is needed to completely digest all the starch in a mash. The total beta amylase activity would be sufficient for a normal mash but more susceptible to inactivation by high temperatures as shown in Figure 2. The difference was most marked between 80 C and 85 C when beta amylase was rapidly inactivated but alpha amylase retained considerable activity. The predicted effect of raising the mashing temperature would be a shift in wort composition away from the products of beta amylase (i.e. a mixture of fermentable and non-fermentable sugars). Figure 3 shows that there was very little difference in

8 92 WORT CARBOHYDRATE COMPOSITION [J. Inst. Brew. the amount of extract obtained when mashing over the range of temperatures 70 C to 85 C. The sugar spectrum, however, changed considerably, giving lower fermentabilities at higher temperatures. Furthermore, decreasing mash thickness also reduced the wort fermentability. It is known that dilution of enzyme systems increases enzyme instability, but, as with temperature, beta amylase was more susceptible to dilution than alpha amylase (Figure 3B). This data confirms and considerably extends the work of MacWilliam el a/.13-14, and of Young and Briggs23. The activity of mash enzymes is more susceptible to heat in a thin solution than in a mash as shown in Figure 5. Even at 80 C very little alpha amylase survived beyond 10 minutes (unlike the mash). Nevertheless, this would be sufficient time for the enzyme to remove any remaining starch. The beta amylase would be so rapidly inactivated that it would have no effect on fermentability. As may be expected from the fermentability data, the wort starch content increases with inactivation of the enzymes. The biggest changes were seen at 85 C perhaps because at other temperatures alpha amylase activity compen sated for loss of beta amylase. This would also explain why the increase in wort starch was considerably less than the decrease in wort fermentability for the same temperature change. Between 65 C and 80 C any loss of beta amylase would be compensated for by alpha amylase attack on the remaining starch. However, at 85 C the alpha amylase is also unstable (Fig. 2) so that changes in mash thickness have a greater effect on wort starch (Fig. 3C). MacWilliam el a/ found similar results working at temperatures up to 75 C. They found that wort fermentability fell at temperatures over 63 C, to about 30% at 75 C, and being 90% of the maximum at 67 C. However, these workers used different malts (rather than a single one) and did not specify mash thickness, nor did they extend their work beyond 75 C. Most of the other work on high temperature mashing has been performed using temperature programmed mashing systems10-1' 20, in which high temperatures were arrived at slowly. The process of starch conversion can be extremely rapid and if temperatures are raised slowly then beta amylase would have sufficient time to convert dextrins to fermentable sugars. The work described above does not apply to temperature programmed mashing systems. Figures 6 and 7 showed typical analytical and process data for worts obtained by high temperature mashing. All worts used in these two experiments were of the same gravity although the wort produced at 80 C contained considerably less fermentable sugar, and was unable to support full yeast growth. Such a wort could be converted to a more normal type by the addition of amyloglucosidase. Superattenuating yeast would also be able to utilize this wort fully. Conclusions The experiments described illustrate how readily wort parameters, such as total carbohydrate, fermentability and undigested starch, can be influenced by changes in mashing temperature and mash thickness. The appearance of starch in the wort and the loss of extract would be readily noted by brewers and yet these are not the most easily influenced parameters. Wort fermentability however is readily altered by mashing temperature or thickness because of the lability of beta amylase. Such changes would not be apparent until the end of fermentation. The relationship between malt enzymes, malt starch and the final wort composition is complex and affected by parameters other than mash temperature and mash thick ness. Further work is required to elucidate such interactions and to establish the importance of starch availability, gelatinization characteristics and the role of starch associated proteins. Acknowledgements The author would like to thank Mrs D. Cairncross for excellent technical assistance, Drs E. D. Baxter and D. R. J. Laws for advice and discussion, and the Director of the Brewing Research Foundation for permission to publish this work. References 1. Bamforth, C. W. European Brewery Convention Symposium on Wort Production, Maffiiers, 1986, Monograph (XI), Bamforth, C. W. The Brewer, 1986,72, Bathgate, G. N., Bennett, H. O. & Crabb, D. Journal of the Institute of Brewing, 1975,81, Brown, B. H. & Kirsop, M. L. Journal of the Institute of Brewing, 1972,78, Buckee, G. K. & Hargitt, R. Journal of the Institute of Brewing, 1977,85, Buckee, G. K., Hickman, E. & Bennett, H. O. Journal of the Institute of Brewing, 1978,84, Buckee, G. K. & Long, D. Journal of the American Society of Brewing Chemists, 1982, 40, Dickenson, C. J. The Brewer, 1987,73, Dixon, M. & Webb, E. C. The Enzymes, London: Longman, Enevoldsen, B. S. 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