Alpha Amino Nitrogen and Fusel Alcohols of Sorghum Worts Fermented into Lager Beer

The levels of alpha amino nitrogen (AAN) and fusel alcohols during fermentations of lager worts produced from waxy sorghum grits either inoculated with yeast cultured in wort or yeast-malt media were performed. Worts produced from waxy sorghum grits had comparable AAN to commercial wort. The oxygen concentration in the reactor headspace changed from 20% at the beginning of fermentation to less than 1% after 72 hrs fermentation indicating a gradual change from aerobic to anaerobic conditions. The utilization of AAN for production of propanol, isobutanol and amyl-isoamyl alcohols from waxy sorghum grits was comparable to a control wort. Production of propanol, isobutanol and amyl-isoamyl alcohols followed the same trend over 144 hr fermentation. Isobutanol was produced in the lowest concentration. The initiation of propanol production occurred after 24 and 36 hr fermentation for worts inoculated with yeast cultured in wort and yeast-malt media, respectively. The final concentration of ethanol and fusel alcohols were within the expected range found in commercial beers. Worts produced from barley malt and waxy sorghum grits were an adequate substrate for Saccharomyces cerevisiae, and were comparable to a commercial wort. The utilisation of refined waxy sorghum grits as brewing adjuncts for lager beers was demonstrated.     

Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct

The sugar profile of wort from laboratory malted barley, malted sorghum, unmalted barley and unmalted sorghum grains mashed with commercial enzyme preparations were studied. Similar levels of glucose to maltose (1:7) were observed in wort of malted barley and malted sorghum. Mashing barley or sorghum grains with commercial enzymes changed the glucose to maltose ratio in both worts, with a greater change in wort from sorghum grains. Although hydrolysis with commercial enzymes released more glucose from maltose in sorghum wort, the same treatment retained more maltose in barley wort. Adding malted barley to sorghum grains mashed with commercial enzymes restored the glucose to maltose ratio in sorghum mash. Fermentation of wort produced from all barley malt (ABM) mash and commercial enzyme/barley malt/sorghum adjunct (CEBMSA) mash of similar wort gravity was also studied. ABM and CEBMSA worts exhibited similar glucose to maltose ratios and similar amino acid spectra. However, ABM released more individual amino acids and five times more proline than wort from commercial enzyme/barley malt/sorghum adjunct. ABM produced 27% more glucose and 7% more maltose than CEBMSA. After fermentation, ABM mash produced 9.45% ABV whilst commercial enzyme/barley malt/sorghum adjunct mash produced 9.06% ABV. Restoration of the glucose/maltose ratio in the CEBMSA mash produced wort with a sugar balance required for high gravity brewing. © 2020 The Institute of Brewing & Distilling     

ROLE OF ALPHA-GLUCOSIDASE IN THE FERMENTABLE SUGAR COMPOSITION OF SORGHUM MALT MASHES*

The cause of the high glucose to maltose ratio in sorghum malt worts was studied. Mashing temperature and pH strongly affected both the amount of glucose and the proportion of glucose relative to total fermentable sugars. The relative proportion of glucose was higher when mashing was performed. at pH 4.0, close to the pH optimum for sorghum alpha-glucosidase, than at the natural pH of the mash (pH 6.0–5.5). Mashing according to the EBC procedure using an enzymic malt extract with pre-cooked malt insoluble solids producing a wort containing maltose and glucose in an approximately 4:1 ratio, whereas mashing with a malt extract without pre-cooking the malt insoluble solids resulted in a wort with approximately equal amounts of maltose and glucose. Both treatments gave the same quantity of total fermentable sugars and amount of wort extract. Sorghum alpha-glucosidase was confirmed to be highly insoluble in water. All or virtually all activity was associated with the insoluble solids. Hence, it appears that the high amount of glucose formed when sorghum malt is mashed conventionally is due to alpha-glucosidase activity. Pre-cooking the malt insoluble solids inactivates the alpha-glucosidase, preventing the hydrolysis of maltose to glucose.     

GAS-LIQUID CHROMATOGRAPHY OF WORT AND BEER CARBOHYDRATES I. THE CHARACTERIZATION OF WORT,BEER, AND BREWING SYRUP CARBOHYDRATES

Gas-liquid chromatography has been applied to the separation of the trimethylsilyl derivatives of wort, beer and corn syrup carbohydrates. The major carbohydrates identified in worts, beers, and corn syrups include fructose, glucose, sucrose, maltose, isomaltose, maltotriose and maltotetraose.     

SMALL SCALE MASHING EXPERIMENTS WITH GRISTS CONTAINING HIGH PROPORTIONS OF RAW SORGHUM

Small scale mashes (50 g total grist) with grists containing high proportions of raw sorghum (50%–80% malt replacement) showed high values of extract recovery and produced worts of lower total nitrogen, free amino nitrogen, viscosity and colour but higher values of pH compared to worts produced from all malt mashes. Increasing the proportion of raw sorghum in the grist relative to malt resulted in a decline in extract recovery, wort total nitrogen, free amino nitrogen and an increase in wort pH. Addition of industrial enzyme preparations to mashes containing raw sorghum resulted in higher values of extract recovery (enzyme preparations containing α amylase and β glucanase), higher values of wort total nitrogen and free amino nitrogen (enzyme preparations containing a neutral proteinase) and decreased wort viscosity (enzyme preparations containing β glucanase or cellulases) compared to worts produced from untreated mashes. Worts and beers were produced on a pilot brewery scale from 50% malt and 50% polished (whole) sorghum (single decoction mashing regime) and 20% malt and 80% raw sorghum supplemented with an industrial enzyme preparation (double mashing regime). Mashes comprising 50% malt and 50% polished sorghum showed comparable wort filtration behaviour (lautering) to that of control mashes (70% malt and 30% maize grists) whereas wort produced from 20% malt and 80% raw sorghum filtered slowly. Worts produced from grists containing sorghum were of high fermentability and showed lower levels of total nitrogen and free amino nitrogen compared to control worts. Analysis of worts produced from small scale mashes containing raw sorghum and a pilot brewery scale mash comprising 20% malt and 80% raw sorghum demonstrated that the levels of total nitrogen and free amino nitrogen were higher than expected from the reduction in the malt content of the mash, consistent with the release of nitrogenous components (polypeptides, peptides and amino acids) derived from sorghum into the wort. Beers produced from 50% malt and 50% polished sorghum and 20% malt and 80% raw sorghum were filtered without difficulty and were of sound flavour. Beers produced from 50% malt and 50% polished sorghum contained lower levels of isobutanol, 2-methylbutanol, dimethylsulphide and higher levels of n propanol and diacetyl compared to control beers.     

Corn grist adjunct – application and influence on the brewing process and beer quality

In the brewing industry, barley malt is often partially replaced with adjuncts (unmalted barley, wheat, rice, sorghum and corn in different forms). It is crucial, however, to preserve constant quality in the beer to meet the expectations of consumers. In this work, how the addition of corn grist (10 and 20%) influences the quality of wort and beer was examined. The following parameters were analysed: wort colour, dimethyl sulphide (DMS) and protein content, non‐fermentable extract, extract drop during fermentation, alcohol content and the attenuation level of the beer, together with filtration performance. The samples (all‐malt, and adjunct at 10 and 20% corn grist) were industrial worts and the beers produced in a commercial brewery (3000 hL fermentation tanks). The application of 10 and 20% corn grist had an effect on the wort colour, making it slightly lighter (11.1 and 10.5°EBC, respectively) than the reference barley malt wort (12.2°EBC). The free amino nitrogen level, DMS and non‐fermentable extract were significantly lower in the worts produced with the adjunct; the alcohol content and attenuation levels were higher in the beers produced with adjunct. The use of corn grist, at the level of up to 20% of total load, appears to affect some of the technological aspects of wort and beer production, but it does not significantly influence the final product characteristics. Copyright © 2014 The Institute of Brewing & Distilling     

ESTIMATION OF CARBOHYDRATES IN WORTS AND BEERS BY GAS CHROMATOGRAPHY

Modifications of existing methods using trimethylsilyl ethers for estimating wort and beer carbohydrates are described. Samples to which a known amount of phenyl β-D-glucopyranoside is added as internal standard are quickly dried in vacuo at 40° C. prior to trimethylsilylation and chromatography. Peak areas of separated sugars are measured relative to the standard and are computed as the concentrations present in the original samples by way of calibration factors, obtained by similar treatment of known amounts of pure sugars. Standard deviations of less than 5% are obtained for glucose, maltose and maltotriose from twelve replicate analyses of a given wort. The technique, applied to a number of worts and beers of varied composition, compares favourably with existing methods of estimating individual sugars in worts and beers.     

Dry and Wet Milling of Malt. A Preliminary Study Comparing Fermentable Sugar,Total Protein,Total Phenolics and the Ferulic Acid Content in Non‐Hopped Worts

In this work, fermentable sugar, total protein, phenolics and ferulic acid content were estimated in sweet worts at different points of lautering. Transfer of these selected malt compounds into worts was analyzed in relation to the method of malt milling (wet milling of malt — the “test worts” or dry milling of malt — the “reference worts”). Glucose, maltose and maltotriose were more rapidly transferred into sweet worts at the early stages of lautering (40 hL and/or 80 hL of wort) after wet milling in comparison to dry milling. Total protein content in the test worts was significantly higher than in the corresponding reference worts at each stage of lautering. Transfer of phenolic compounds and ferulic acid (in the free as well as in the ester form) from the mash into sweet worts was significantly improved by dry milling, but not by wet milling. No difference in the total antioxidant activity was observed between the two types of worts. In conclusion, it can be stated that wet conditioning of malt before milling enhances the fast transfer of fermentable sugars and proteins from the mash into the sweet wort during lautering. Lautering is a time‐consuming process, and time reduction without the loss of wort quality should be a priority. Therefore, wet milling can be of interest to professionals in the field as an interesting alternative method to improve the mashing process.     

EXTRUDED SORGHUM AS A BREWING RAW MATERIAL

Small scale mashes (50 g total grist) with grists containing up to 50% by weight of extruded whole sorghum produced worts of high extract yield and low viscosity. Increasing the proportion of extruded sorghum in the grist resulted in decreasing wort filtration volume, total nitrogen and free amino nitrogen content. The wort filtration behaviour of mashes containing sorghum extruded at 175°C was superior to that of mashes containing sorghum extruded at 165°C or 185°C. The results from such small scale mashing experiments suggested that extruded sorghum compared favourably to extruded barley and extruded wheat as a brewing adjunct. Worts and beers were produced on a pilot brewery scale (100 1) from grists comprising 70% malt + 30% extruded sorghum and 100% malt under isothermal infusion mashing conditions. Mashes containing sorghum extruded at 175°C showed comparable wort filtration behaviour to that of the all malt control mash whereas mashes containing sorghum extruded at 165°C or 185°C showed poor wort filtration behaviour. Worts produced from grists containing extruded sorghum fermented more quickly than the control wort and attained lower values of final gravity. The resulting beers were filtered without difficulty. Beers produced from grists containing extruded sorghum contained lower levels of total nitrogen and free amino nitrogen compared to the control beer consistent with extruded sorghum contributing little or no nitrogenous material to the wort and beer. Beers brewed from grists containing extruded sorghum were of sound flavour and showed reasonable foam stability behaviour.     

SOME EFFECTS OF WORT COMPOSITION ON THE RATE AND EXTENT OF FERMENTATION BY BREWING YEASTS

The time required to ferment worts of varied composition to a given extent is dependent upon the extent of exponential growth in the early stages of fermentation; in the worts studied this is determined by the concentration of assimilable nitrogen. When the concentration of all the non-carbohydrate nutrients in malt wort is halved by dilution with carbohydrate, the addition of appropriate quantities of serine or arginine restores the rate of fermentation to that of the malt wort. Minor nutrients, other than amino acids specifically required by the yeasts used, are thus present in at least two-fold excess in the malt wort. The yeast produced during exponential growth in malt wort (sp.gr. 1·040) is able to ferment rapidly much greater quantities of fermentable carbohydrate than are present in that wort. The majority of the strains of yeast examined ferment equally well when either glucose or maltose is added to malt wort and do so whether the sugar is added prior to fermentation or towards the end; however, one strain fails to ferment satisfactorily if a substantial quantity of glucose is added to wort prior to fermentation, because of the subsequent failure of the yeast to adapt to ferment maltose. It is suggested that most brewing strains do not require to adapt to maltose utilization during the fermentation of wort.     

THE EFFECTS OF GLUCOSE ADJUNCT IN HIGH GRAVITY FERMENTATION BY SACCHAROMYCES CEREVISIAE 2036

Laboratory fermentations of 16°Plato glucose adjunct worts by Saccharomyces cerevisiae 2036 demonstrated the absence of “glucose repression” of maltose and maltotriose uptake. However, when compared to worts in which maltose syrup was employed as an adjunct, residual glucose was present at the end of fermentation, maltose and maltotriose uptake rates were enhanced, fructose uptake was blocked and the sequence of sugar uptake was changed. These findings partially explain residual glucose and fructose that sporadically appear in commercial beers. Further research suggests that the physiological quality of the yeast is of prime importance in carbohydrate metabolism, and that critical concentrations of glucose vary with different physiological conditions for this brewing strain in 16°P wort .     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

GAS LIQUID CHROMATOGRAPHY OF WORT AND BEER CARBOHYDRATES II. QUANTITATIVE DETERMINATION OF WORT,BEER, AND BREWING SYRUP CARBOHYDRATES

A method for the preparation and gas chromatography of the trimethylsilyl derivatives of carbohydrates was applied to the quantitative analysis of carbohydrates in worts, beers, and brewing syrups. The derivatives can be easily and quickly prepared from lyophilized worts or beers and aliquots of the reaction mixtures can be injected directly into the gas chromatograph. Gas chromatographic separations of the mono- to tetra-saccharide fractions were achieved in less than thirty minutes through the use of linear temperature programming. Recoveries of carbohydrates added to wort ranged from 97 to 105%. Quantitative data for replicate wort analyses showed relative standard deviations of from 1·7% for glucose to 5·3% for maltose. The silylation reaction can be applied to the volatilization and subsequent study of a wide variety of compounds occurring in brewing materials.     

Effect of varying starch properties and mashing conditions on wort sugar profiles

The aim of this research was to investigate the relationship between starch composition in barley and its malted counterpart alongside malt enzyme activity and determine how these factors contribute to the fermentable sugar profile of wort. Two Australian malting barley varieties, Commander and Gairdner, were sourced from eight growing locations alongside a commercial sample of each. For barley and malt, total starch and gelatinisation temperature were taken, and for malt, α‐ and β‐amylase activities were measured. Samples were mashed using two mashing profiles (infusion and Congress) and the subsequent wort sugar composition and other quality measures (colour, original gravity, soluble nitrogen) were tested. Variety had no significant (<0.05) effect on any barley, malt, enzyme or wort characteristics. However, growing location impacted gelatinisation temperature, colour, malt protein content and original gravity. The gelatinisation temperature in malt samples was higher, by ~0.8°C, than in the equivalent barley sample. Several malt samples, even with protein contents <12.0%, had gelatinisation temperature >65°C. The fermentable sugars measured in the malt prior to mashing showed a higher proportion of maltose than glucose or maltotriose. In addition, there were significant differences in the amount of sugar produced by each mashing method with the high temperature infusion producing a higher amount of sugar and proportionally more maltose. There is scope for further research on the effect of genetics and growing environment on gelatinisation temperature, mash performance and fermentable sugar development. Routinely measuring gelatinisation temperature and providing this information on malt specification sheets could help brewers optimise performance. © 2019 The Institute of Brewing & Distilling     

Infrared spectral analysis of sugar profiles of worts from varying grist to liquor ratios using infusion and ramping mash styles

Fermentability is an important trait for the brewing industry. Current industry methods lack the predictive capacity to accurately estimate how well a wort will ferment in the brewhouse. Wort from two mashing styles (high‐temperature infusion and low‐temperature ramping), and under varying grist to liquor (G:L) ratios, were produced and the differences in maltose, maltotriose and glucose were measured. The two mashing styles showed differences in original extract (Plato) values between the G:L ratios with a 1:2 G:L having the highest original extract. Maltose was the most abundant sugar in all sample types. All worts were scanned using Fourier transform infrared (FTIR) and the spectra also showed differences between the original extract and final extract with most of the changes around the spectral region associated with carbohydrates, a result of sugar utilization by the yeast. FTIR calibrations for extract and fermentable sugars all had r² values >0.98, with ratio of standard error of prediction to standard deviation >5. The results indicated changing fermentable sugar levels, thus making a prediction of fermentability possible. The FTIR also provided a rapid measure of changes in the fermentable sugar profile, which could assist maltsters and brewers in monitoring malt and beer quality. Copyright © 2016 The Institute of Brewing & Distilling     

COMPOSITION OF BREWING SYRUPS—A REVIEW

The composition of various syrups derived from barley, malt, maize and wheat and of worts prepared from them has been surveyed. Wider variations in the concentrations of fermentable carbohydrates occurred in these worts than in malt worts. Total nitrogen contents in the diluted barley and malt syrups examined were generally similar to those of wort. Certain amino acids were present in greater amounts in barley syrups than in wort but it is doubtful whether the differences are of practical significance. Only very limited information was found on the lipid, tannin and mineral contents of syrups.     

Effect of mashing procedures on brewing

The effect of the double decoction mashing method (method A) and the single decoction plus infusion mashing method (method B) on brewing were compared. The trials were carried out with the same raw material (malt and a minor amount of corn as adjunct) on an industrial-scale plant. The effects of mashing methods A and B were evaluated in wort and beer samples obtained with the high gravity system. The analytical parameters of the worts and beers produced and the economic aspects of production (yield, beer quality, time and energy) were discussed. The results showed no considerable differences in beer quality, while a significant difference was observed in the composition of fermentable sugars of worts. Method B gave a wort with a higher content of fermentable sugars which were converted to alcohol during fermentation; therefore, it allowed to obtain a higher beer volumetric yield of the same quality while saving time and energy.     

THE EFFECT OF CARBOHYDRATE ADJUNCTS ON BREWER'S WORT FERMENTATION BY SACCHAROMYCES UVARUM (CARLSBERGENSIS)*

The use of carbohydrate adjuncts such as sucrose, fructose and glucose in brewer's wort significantly modifies the initial wort sugar spectrum and also the pattern of sugar uptake during fermentation by a strain of Saccharomyces uvarum (carlsbergensis). Under these conditions, the concentration of glucose and fructose in the wort was observed to increase when compared to worts in which corn starch was employed as an adjunct and glucose was taken up at a faster rate than fructose. The increase in glucose concentration in the wort also resulted in severe repression of maltose and maltotriose utilization with significant levels of these sugars remaining in the beer produced.     

INHIBITOR OF FLUORESCENCE REACTIONS BETWEEN CALCOFLUOR AND β-(1,3)(1,4)-D-GLUCAN IN BEER AND WORT

An inhibitor of calcofluor fluorescence reaction in beer and wort was shown by analyzing beers and worts containing no β-glucan. The inhibitory activity was enhanced against the fluorescence reaction which is intensified by linkage of calcofluor with β-glucan compared to the fluorescence of calcofluor itself. The investigation indicated that the inhibitor was derived from malt and is produced during the germination process. The inhibitory activity in beer and wort changed with the degree of malt modification and the ratio of malt in the grist. Japanese beers had a wide range of inhibitory activities against the calcofluor fluorescence, lowering the apparent β-glucan contents by 12 to 20%. The inhibitor in beer and wort appeared to be an acidic compound with a molecular weight of less than 1000 daltons.     

I. CONDITIONS OF MASHING AND CARBOHYDRATE COMPOSITION OF THE WORT

Experimental mashings of ungerminated barley and 5–10% of malt with addition of the debranching enzyme pullulanase have been carried out. Worts with high attenuation are obtained in good yield. Of the fermentable sugars, there is less glucose, and more maltose and maltotriose than normally observed. The dextrin pattern is different from, but not necessarily inferior to, that traditionally seen. The worts resulting from the action of pullulanase are deprived of the dextrins with 8–14 glucose units, whereas the amounts of dextrins with 4–6 glucose units are close to those normally observed. The pullulanase preparations used are accompanied by proteolytic activity. It is suggested that debranching enzymes such as pullulanase offer an alternative choice of carbohydrases to be used in brewing from unmalted cereals.