Diastatic Power Enzymes Research Articles

Comparison of Diastatic Power Enzyme Release and Persistence during Modified Institute of Brewing 65°C and Congress Programmed Mashes

A key biochemical process in brewing is the hydrolysis of starch by diastatic power (DP) enzymes into fermentable sugars during the mashing stage of brewing. Efficient starch hydrolysis during mashing requires initially starch gelatinization at approximately 59–64°C and then sufficient DP enzyme activity to achieve starch hydrolysis. This investigation compared the persistence of DP enzyme activity during modified Institute of Brewing (MIoB, 1:3 grist/water ratio + Ca2+) mash with the conventional Congress (1:4 grist/water ratio) small-scale mash, using malt from three current Australian barley varieties. Traditionally, with the thermostability of DP enzymes, it is understood that α-amylase is relatively thermostable, whereas β-amylase and limit dextrinase (LD) are relatively thermolabile at conventional mashing temperatures. In addition, it is also known that β-amylase and in particular LD have bound and latent fractions that require release from binding proteins or inhibitors, respectively, before they are able to contribute to starch hydrolysis. One mechanism observed empirically was that heat applied during mashing at temperatures of approximately 55–60°C appears to liberate bound β-amylase and LD. The results in terms of DP enzyme release, thermostability, and activity were examined to understand the relative contributions of the three key DP enzymes to starch hydrolysis during different mashing conditions. Interestingly, we observed that both β-amylase and LD retained significant levels of activity (approximately 40% total activity), even after mashing for 60 min at 65°C. These observations are of critical importance to brewers in meeting beer quality specifications when they manipulate mash temperatures to cope with variations in malt quality, developing new products, or producing beers requiring different wort qualities for production. The implications from these results of the choice of small-scale mash protocol for malt quality evaluation, the targeting of DP enzyme alleles for barley malt quality improvement, and the selection of malt for brewing are discussed.

Maltose Effects on Barley Malt Diastatic Power Enzyme Activity and Thermostability at High Isothermal Mashing Temperatures: I. β-Amylase

The hypothesis that maltose would increase the thermostability of barley malt β-amylase activity during isothermal mashing was tested. Malts of the two-row cultivar Harrington and the six-row cultivar Morex were incubated in maltose concentrations of 0–500 mM at 68, 73, and 78°C and compared with isothermal mashing at 63°C. Incubations with additions of mannitol from 0 to 500 mM at the same temperatures were used to compare with incubations containing maltose. Incubations were sampled every 30 min for 120 min, and β-amylase activities were determined. In the absence of exogenous sugars, extracts of Harrington had higher initial β-amylase activities than did extracts of Morex at 63, 68, and 73°C but not at 78°C, for which the reverse was observed. In the absence of exogenous sugars, extracts of both cultivars had the highest activities at 63°C, and both exhibited maximal responses to sugar additions with 500 mM maltose (889% increase for Morex and 541% for Harrington) compared with 0 mM controls at 63°C. Activities in extracts of both cultivars were significantly influenced by maltose additions starting at 50–100 mM and as early as 30 min at 63°C, and these effects were pronounced at and above 200 mM maltose. The maximum % survival of β-amylase at 120 min compared with 0 min for both cultivars occurred at 63°C (85–93% for Morex and 90–91% for Harrington) when incubations were with 400 and 500 mM maltose. A comparison of β-amylase thermostability between the cultivars was calculated as the % activity detected under the most favorable condition (0 min at 63°C) and that detected under the least favorable condition (120 min at 78°C). Our study showed that β-amylase in Harrington extracts was more thermostable than that in Morex extracts (10.1 and 5.9%, respectively). β-Amylase in both Harrington and Morex extracts showed some increases in activity and thermostability when mannitol was included in incubations. Increases were most pronounced at 63°C with 500 mM mannitol. However, these effects were not as great or as consistent as seen with maltose additions to the incubations. The extent of the maximum response to both maltose and mannitol on the thermostability of β-amylase from Morex malts was 60% greater than that of the β-amylase from Harrington malts.

Maltose Effects on Barley Malt Diastatic Power Enzyme Activity and Thermostability at High Isothermal Mashing Temperatures: II. α-Amylase

This study was conducted to determine if maltose, the primary product of starch degradation during mashing, would affect the activity of or increase the thermostability of barley malt α-amylase activity at high temperatures used in mashing and temperatures above those normally used in mashing. Malts of the two-row cultivar Harrington and the six-row cultivar Morex were mashed for 2 h with maltose concentrations of 0–500 mM at temperatures from 63 to 78°C. Worts were sampled every 30 min for 120 min and α-amylase activities determined utilizing the Megazyme Ceralpha assay. Mannitol, a polyhydric alcohol, was used for comparison. Polyhydric alcohols are known to enhance enzyme thermostability. At 63°C both cultivars had relatively thermostable α-amylase without the addition of maltose or mannitol. However, Morex, and to a lesser extent Harrington, α-amylase activity was significantly elevated by high levels of either maltose or mannitol, indicating a stimulatory effect on activity in which thermostability was apparently not involved. At 73 and 78°C increasing concentrations of maltose conferred considerable and highly significant (LSD analysis, P < 0.0001) thermal protection of α-amylase in both cultivars. For example, at 30 min of mashing at 73°C, maltose concentrations of 400 and 500 mM resulted in α-amylase activity in Harrington of 227 and 252% and in Morex 349 and 314%, respectively, of the 0 mM maltose activity. At 60 min of mashing at 73°C, concentrations of 400 and 500 mM maltose resulted in α-amylase activities of Harrington of 290 and 431% and Morex of 489 and 501%, respectively, of the 0 mM maltose activity. High mannitol incubations at higher temperatures such as at 73°C rendered lesser α-amylase thermal protection than maltose; however, it was also considerable and highly significant (P < 0.0001) for both cultivars (e.g., 30 min incubation at 73°C, with 400 and 500 mM maltose, respectively, α-amylase activities of Harrington were 163 and 174% and Morex were 188 and 260% of the 0 mM maltose activity). These data suggest that the production of maltose, as could be expected in high-gravity mashes, significantly increases the activity and thermostability of barley malt α-amylase.

Relationship between levels of diastatic power enzymes and wort sugar production from different barley cultivars during the commercial mashing process of brewing

The fermentable carbohydrate composition of wort has a direct influence on yeast fermentation efficiency and resultant beer quality. In this study, the relationship between diastatic power enzymes (DPE) and their wort sugars products during the course of small-scale, emulated commercial mashing was investigated. Malts derived from 13 barley cultivars were mashed and assayed at five time points during mashing for the levels of DPE and fermentable sugars. Comparisons of the patterns of DPE activity and wort sugar production showed that the activity levels of β-amylase and limit dextrinase (LD) during mashing were variable between the 13 cultivars, in comparison to the level of α-amylase and resultant composition of wort sugars. Moreover, comparison of peak DPE activities indicated that α-amylase correlated positively and significantly with LD, while no obvious correlation was found between β-amylase and either α-amylase or LD, indicating that activity pattern of α-amylase and LD was closely related during mashing. Multiple linear regression models, based on levels of the DPE as various time points during mashing, thermostability of β-amylase and malt Kolbach index, were able to explain 42.9%, 91.9%, 94%, and 73.2% of wort maltotriose, maltose, glucose, and fermentable sugar composition, respectively. A combination of these insights into the dynamics of starch hydrolysis during mashing will assist brewers in malt cultivar selection and the adjustment of mashing conditions so as optimize the sugar content for the efficient production of high quality beer.

The Impact of Malt Blending on Lautering Efficiency, Extract Yield, and Wort Fermentability

The impact of malt blending on brewing performance in terms of extract, lautering, and fermentability performance was examined in a series of small-scale mashing trials. Malts were blended so that 40–60% of the grist consisted of malt with lower levels of one or more of these malt quality characteristics. With extract, blending resulted in additive improvements between malts of lower and higher levels. In contrast, improvements were synergistic for lautering performance when there was a substantial difference between the lautering performance of the two malts. The markers for synergism in lautering performance were the level of β-glucanase and wort viscosity. Fermentability performance also typically showed synergism between low and high fermentability malts, with the exception of one malt sample whose blend combination showed an unexplained antagonism. The synergism in fermentability performance was achieved when there was a deficiency between the malts for Kolbach index, the diastatic power enzymes, or β-amylase thermostability that compensated for the lack of these components in the lower-fermentability malts. The importance of these blending interactions was discussed in relation to malt quality specifications and achieving consistent and predictable brewing outcomes.

Refining the Prediction of Potential Malt Fermentability by Including an Assessment of Limit Dextrinase Thermostability and Additional Measures of Malt Modification, Using Two Different Methods for Multivariate Model Development

Prediction of malt fermentability (apparent attenuation limit — AAL) by measurement of the diastatic power enzymes (DPE), α-amylase, total limit dextrinase, total β-amylase, β-amylase thermostability, and the Kolbach index (KI or free amino nitrogen — FAN) is superior to the conventional use of diastatic power (DP) alone. The thermostability of β-amylase is known to be an important factor in determining fermentability, thus the thermostability of the other relatively thermolabile enzyme, limit dextrinase, was investigated to determine if it was also useful in predicting fermentability. To facilitate this aim, methods were developed for a rapid and cost efficient assay of both β-amylase and limit dextrinase thermostability. Internationally important Australian and international malting varieties were compared for their total limit dextrinase and β-amylase activity and thermostability. Interestingly, the level of limit dextrinase thermostability was observed to be inversely correlated with total limit dextrinase activity. The prediction of malt fermentability was achieved by both forward step-wise multi-linear regression (MLR) and the partial least squares (PLS) multivariate model development methods. Both methods produced similar identifications of the parameters predicting wort fermentability at similar levels of predictive power. Both models were substantially better at predicting fermentability than the traditional use of DP on its own. The emphasis of this study was on the identification of predictive factors that can be consistently used in models to predict fermentability, because the model parameter estimates will subtly vary depending on mashing conditions, yeast strain/fermentation conditions and malt source. The application of these multivariate model development methods (PLS and MLR) enabled the identification of further potential fermentability predicting factors. The analyses divided the predictive parameters into those defined by DP enzymes and those associated with modification (KI, FAN, fine/coarse difference, wort β-glucan and friability). Surprisingly, limit dextrinase thermostability was not a substantial predictor of fermentability, presumably due to its negative correlation with total limit dextrinase activity. The application of these insights in the malting and brewing industries is expected to result in substantial improvements in brewing consistency and enable more specific quality targets for barley breeder's progeny selection cut-off limits to be more precisely defined.

Improved Prediction of Malt Fermentability by Measurement of the Diastatic Power Enzymes β-Amylase, α-Amylase, and Limit Dextrinase: II. Impact of Barley Genetics, Growing Environment, and Gibberellin on Levels of α-Amylase and Limit Dextrinase in Malt

The determination of the levels of the diastatic power enzymes (DPEs) β-amylase, limit dextrinase, and α-amylase has previously been shown to predict barley malt fermentability. Using micromalted samples of barley from different genotypes and growing environments, it was demonstrated that both these factors were important in determining the level of DPEs in malt. In terms of genotypic effects, a trial of breeders' lines and varieties showed substantial variation in the levels of total β-amylase (means 455–914 U/g), total limit dextrinase (means 268–603 U/kg), and α-amylase (means 154– 316 U/g). The application of gibberellin (GA) during malting resulted in substantial increases in the levels of total limit dextrinase and α-amylase and the extent of modification (KI). However, the level of total β-amylase was relatively unchanged. It was also observed that the levels of total limit dextrinase and α-amylase and extent of KI generally were highly correlated (r ≈ 0.6–0.7), which was attributed to the sensitivity of each of these malt quality factors to GA. The results are discussed in terms of their practical importance to barley breeders and maltsters seeking to supply malt that satisfies the malt fermentability requirements of their brewing customers.

Improved Prediction of Malt Fermentability by Measurement of the Diastatic Power Enzymes β-Amylase, α-Amylase, and Limit Dextrinase: I. Survey of the Levels of Diastatic Power Enzymes in Commercial Malts

In this study, more than 1,000 commercial malting samples from Australia and elsewhere, primarily malted in 2005 and 2006, were analyzed for their levels of the diastatic power (DP) enzymes α-amylase, β-amylase, and limit dextrinase. The survey showed that there was more variation within a variety for DP and DP enzymes than between varieties. The data were evaluated, and a micromalting experiment was conducted to ascertain whether the wide range of malt qualities observed was the result of customer specifications, environmental conditions under which the barley was grown, variety, or malting practices. The evaluation of malt used by two breweries over the course of a year suggested that the conventional brewery customer specifications for variety, Kolbach index, and DP are somewhat successful in constraining potential fermentability variation. The conditions under which barley for malting was grown were also plausible factors that could explain the observed differences in DP enzyme levels. However, micromalting barley sourced from different regions showed that malting conditions had a strong influence on the malt levels of α-amylase and limit dextrinase. When combined, the observations and conclusions of this study further support our previous recommendations that the routine measurement of individual DP enzymes would most likely improve the consistency and predictability of the potential fermentability of malts supplied to brewers.

A More Cost- and Labor-Efficient Assay for the Combined Measurement of the Diastatic Power Enzymes β-Amylase, α-Amylase, and Limit Dextrinase

An improved, combined extraction protocol in an 8 × 12 microwell format has been devised that satisfactorily estimates the levels of β-amylase, α-amylase, and limit dextrinase in malt. The improved protocol enables one person to undertake the assessment of 46 samples and 1 control in duplicate for each of the Ceralpha, Betamyl, and Limit DextriZyme assays, which require 5.5 hr of labor over 2 days, including an overnight extraction. The scaling down of the method resulted in the use of between one-quarter and one-fifth of the amount of substrate per assay recommended by the manufacturer, providing substantial cost savings. The increased labor and cost efficiencies described in this paper will increase the potential for diastatic power enzyme analyses to be undertaken as routine assays by malting quality evaluation laboratories, including breeding programs, maltsters, and grain traders.

Performance of Husked, Acid Dehusked and Hull-less Barley and Malt in Relation to Alcohol Production

Studies carried out on normal husked barley, normal hull-less (naked) barley, acid dehusked barley and acid dehusked hull-less barley, as well as the malts derived from them, showed that when acid dehusked barley samples (obtained from either husked or hull-less barley), were processed using commercial enzyme preparations, they produced more alcohol when compared with the alcohol yield obtained from the barley samples from which the acid dehusked samples were derived. When the husked (Optic) control, acid dehusked and hull-less barley samples were malted, Optic control barley produced malt that gave higher dextrinising units (DU) and diastatic power (DP), whilst acid dehusked Optic and hull-less barley produced malts that gave similar DU results on day 5 of the germination time. When mashed, acid dehusked (Optic) barley malt produced wort that filtered faster than the wort obtained from the malt made from hull-less barley. This observation is very important because it shows that the husk of the barley is not the only factor that determines the filtration performance of the malted barley, since both the malt samples made from husked and acid dehusked barley had similar filtration rates on day 5 of the germination time. The slow filtration rate observed for the wort made from hull-less barley suggests that other factors play some role during the filtration of the mash made from hull-less barley malt. Although hull-less malt appeared to develop lower DU and DP enzyme activities, when compared with the values obtained for the Optic control, hull-less barley malted faster and produced optimum predicted spirit yield (PSY) at day 4 of the germination time. In contrast, the control husked Optic barley malt that had higher DU and DP produced equivalent (optimum) predicted spirit yield one day later at 5 days germination time. This is an advantage for hull-less barley, both in terms of time and energy saving during the malting of barley. Although the acid dehusked Optic barley produced more alcohol than the husked control when commercial enzyme preparation was used to process barley, it was surprising that when the derived malt was assessed, it gave a lower predicted spirit yield than the husked control, even though it produced a higher amount of hot water extract (HWE). The higher extract yield and lower predicted spirit yield obtained from the malt made from acid dehusked malt confirmed that high extract yield is not necessarily associated with high fermentable extract.

Assessing the Impact of the Level of Diastatic Power Enzymes and Their Thermostability on the Hydrolysis of Starch during Wort Production to Predict Malt Fermentability

In this study, commercially produced malts were used for small-scale simulated mashing trials to investigate the impact of differences in the level and thermostability of malt diastatic power (DP) enzymes on the resultant wort fermentability. A modified European Brewery Convention/American Society Brewing Chemists mashing protocol was used with mash-in temperatures ranging between 45 and 76°C for full-malt and 30% rice adjunct mashes. Malt extract yield varied little with mashing temperature for most varieties in this temperature range. However, the fermentability, maltose content, and free amino nitrogen of that extract was considerably affected by mashing temperature with 65°C achieving the highest fermentability for all malt varieties. Multilinear regression analysis of full-malt and rice adjunct mashing trials at 65°C using 43 commercial malts showed that the level of α-amylase and total limit dextrinase activity, Kolbach Index, and the total β-amylase activity level and thermostability were the most important malt quality predictors of wort fermentability. These conclusions suggest that the conventional DP assessment could be replaced with the measurement of its component enzymes outlined above so that maltsters could better satisfy brewers malt quality expectations by blending and defining their malt quality in terms of these fermentability predicting factors. This information would be particularly useful to brewers who brew with multiple varieties and blends from different suppliers. The focus on individual enzyme characteristics by barley breeders is likely to provide selection targets that are more accurate and achievable.

The Impact of the Thermostability of α-Amylase, β-Amylase, and Limit Dextrinase on Potential Wort Fermentability

Diastatic power (DP), a measure of the level of activity of starchhydrolyzing enzymes, is a term that refers to the combined activity levels of β-amylase, α-amylase, limit dextrinase, and α-glucosidase. The result of the action of these enzymes during brewing is to break down starch into component sugars that are readily fermented into alcohol by yeast. Although DP is a reasonable predictor of fermentability, it does not always accurately estimate the level of fermentable sugars generated during mashing or the subsequent fermentability of the resultant wort. Previously, it has been shown that allelic variation in β-amylase thermostability has a significant impact on wort fermentability. The thermostability of the DP enzymes is critical in determining fermentable sugar yield during mashing, where the mash temperature profile is a balance between the temperature required for starch gelatinization to enable efficient hydrolysis and the rate of thermal inactivation of the DP enzymes. This study demonstrates that there is significant variation in the thermostability of α-amylase and limit dextrinase in malts made from a wide range of Australian and international malting barley varieties. This variation in α-amylase and limit dextrinase thermostability combined with that of β-amylase provides an opportunity for barley breeders to produce divergent malting varieties that better fulfill brewers' fermentability requirements. The implications of the varying DP enzyme thermostability on the selection of malt by brewers to optimally suit different brewing styles and regimens are outlined.