MICROBIAL ENZYMES AND THEIR EFFECTS ON EXTRACT RECOVERIES FROM UNMALTED ADJUNCTS

Radial diffusion tests were used to detect β-glucan-, starch-, protein-, pentosan- and triglyceride-degrading enzyme activities in a variety of commercial enzyme preparations. Some implications of the presence of unexpected enzymes in particular preparations are discussed. A series of laboratory mashes were made with grists of milled barley, extruded barley, and extruded wheat, using various enzyme additions and temperature-time programmes. In addition a limited number of experiments were made with pale ale barley malt, and wheat flour pellets. Extract yields varied from acceptable to outstandingly good. The most intensive mashing system, with enzyme supplementation, increased the extract yield of an all-malt mash by 3·9% relative to the control mashing programme. Some worts were evaluated for Total Soluble Nitrogen (T.S.N.), Free Amino Nitrogen (F.A.N.) and viscosity. Slow wort-filtration rates occurred in some mashes although the viscosities of the worts from these mashes were low. This problem did not occur in any of the samples intensively mashed over a 5 h period. The results apparently exclude the possibility that a simple, reliable technique for estimating the extract yields of adjuncts could be devised, using these enzyme preparations.     

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.     

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.     

Effect of Amyloglucosidase on Wort Composition and Fermentable Carbohydrate Depletion in Sorghum Lager Beers

A three‐factorial experiment with a level of confidence of P < 0.05 was performed to study fermentable carbohydrate depletion and ethanol production during 144 h fermentations of lager beers produced with barley malt (BM), sorghum malt (SM), refined maize (MZ) or waxy sorghum (WXSOR) grits treated during mashing with or without amyloglucosidase (AMG). The percentage glucose, maltose and maltotriose, based on total fermentable carbohydrates for the BM wort was 20, 68 and 13% and for the SM wort 35, 48 and 17% respectively. Treatment with AMG increased wort glucose from 9.3 to 24.5 g/L wort and total fermentable sugar equivalents, expressed as g glucose/L, from 59.2 to 72.6 g/L wort. The SM worts had approximately 50% more glucose and 40% less initial maltose content respectively compared to the BM worts. The WXSOR grits produced worts and beers with similar properties to those produced from the MZ adjuncts. AMG addition led to a >2.5 fold increment in wort glucose and 23% in total fermentable carbohydrate content. Linear regression analysis determined that the consumption rate of fermentable carbohydrates during fermentation followed first order reaction kinetics. Depletion times to reach 50% of the initial concentrations of glucose, maltose and maltotriose were 49, 128 and 125 h, respectively, clearly indicating that the fermenting yeast preferred glucose. Maltose and maltotriose depletion times of the AMG treated worts were significantly faster and lower, respectively, when compared with the untreated worts. At the end of the fermentation, the BM beers contained higher ethanol levels (5.1% v/v) than the SM beers (3.9% v/v). For AMG treated beers, no significant differences in ethanol content were observed among samples mashed with BM and beers produced from SM and MZ grits. The results demonstrated that AMG could be used to increase the initial concentration of glucose and total fermentable carbohydrates thus decreasing dextrin levels, especially from sorghum mashes.     

NITROGENOUS COMPONENTS OF WORTS AND BEERS BREWED FROM ALL-MALT AND MALT PLUS WHEAT FLOUR GRISTS

Worts and beers prepared from grists containing malt and wheat flour contain more and larger proteins than those prepared from grists containing only malt. These properties are related to the superior foam stability possessed by malt plus wheat flour beers. Malt solubilizes some of the wheat flour protein during mashing without subjecting it to significant proteolysis. Only very limited enzyme inhibition is exhibited. Finings residues in draught beers increase the precipitability of protein by sodium chloride and are particularly effective with malt plus wheat flour beers. The proteinaceous material isolated in this way has marked foam-stabilizing properties. The relationship between the protein removed by chilling the beer and salt-precipi-table protein and foam stability is also examined. Non-fining hazes from certain malt plus wheat flour beers are related to other beer protein fractions and some recommendations for brewery practice are made.     

Quality characteristics of triticale malts and worts

Worts from triticale malts, in particular well modified malts, separated poorly from mashes. Worts prepared at 70° C had high viscosities (10–27 cSt) indicating that problems would occur during filtration in brewing. The viscosities of triticale worts were higher than those of worts from barley malts. In addition, worts from well modified malts were generally turbid. Proteinaceous material (partly degraded prolamins) was the primary cause of this turbidity. Although the degree of malt modification did influence the rate of wort separation, it had little effect on wort viscosity. High viscosity was caused by pentosans dissolved from the triticale malt during mashing. Oxidative gelation was not observed with these pentosans. Grains and malts were fractionated, and the high molecular weight fractions were analysed for their sugar and acyl components. All were rich in arabinose and xylose. There was a rough inverse correlation between the solubility of the poly saccharide fractions and the levels of substitution with acetyl and feruloyl residues. The poor wort separation from triticale malt grists appeared to be related to the particle size distributions, which were narrow. The sedimentation values of the grist ‘fines’ were high.     

AMINO ACID ANALYSIS OF BEER POLYPEPTIDES

The principles of amino acid analysis of proteins and polypeptides are reviewed. Analysis of the amino acid composition of dialysed beer material prepared from a wide variety of commercial and pilot brewery beers showed that the principal amino acids comprised glutamic acid/glutamine, proline, glycine and aspartic acid/asparagine. The results from the analysis of a series of pilot brewery beers brewed under standardised conditions showed that the composition of the grist may influence the amino acid composition of beer polypeptide fractions. Dialysed beer material prepared from beer brewed from grists containing torrified wheat, wheat flour and malted wheat contained greater proportions of glutamic acid/glutamine compared to material prepared from all malt beers. Further fractionation and analysis of dialysed beer material prepared from pilot brewery beers suggested that fractions MW>60000 contained polypeptide material derived from yeast mannan-protein. In addition fractions MW>60000 prepared from beer brewed from grists containing torrified wheat, wheat flour or all malted wheat may contain high molecular weight polypeptide material derived from wheat proteins. The results from the analysis of fraction MW 40,000–60000 prepared from beers brewed from grists containing all malt, 80% malt and 20% torrified wheat and 50% malt and 50% malted wheat are consistent with the presence of polypeptide material derived from cereal albumins and globulins whereas fractions MW 40,000–60000 prepared from beers brewed from 80% malt and 20% wheat flour and 100% malted wheat may contain polypeptide material derived from wheat prolamins and glutelins. The amino acid composition of fraction MW 20,000–40,000 from all pilot brewery beers investigated is consistent with the presence of polypeptide material derived from cereal prolamins and glutelins. The amino acid composition of beer polypeptide fractions may be used to detect the use of wheat adjuncts in beer brewing.     

Effect of unmalted oats (Avena sativa L.) on the quality of high-gravity mashes and worts without or with exogenous enzyme addition

Barley malt is the preferred brewing material these days because of its high extract content and high enzyme activities. However, when substituting malted barley with oats to create a unique beer flavor and aroma, endogenous malt enzymes become the limiting factor. Therefore, the objectives of this study were to evaluate the effect of 10–40 % unmalted oats on the quality of high-gravity mashes/worts and to investigate the limitations of endogenous malt enzymes as well as the benefits of the application of industrial enzymes. The enzyme mix Ondea^® Pro was found to be particularly suitable for mashing with unmalted oats and was therefore used in the present rheological tests and laboratory-scale mashing trials. In order to gain detailed information about the biochemical processes occurring during mashing, the quality of mashes was comprehensively analyzed after each mash rest using standard methods described by Mitteleuropäische Brautechnische Analysenkommission and Lab-on-a-Chip capillary electrophoresis. Mashing with up to 40 % oats resulted in increased mash consistencies, color/pH (20 °C) values, β-glucan concentrations, wort viscosities 12.0 %, and filtration times as well as decreased FAN and extract contents. The application of Ondea^® Pro enormously increased the color of worts despite lower pH values but considerably improved the quality and processability of 30 or 40 % oat-containing mashes/worts. However, the substitution of up to 20 % barley malt with unmalted oats can easily be realized without the addition of exogenous enzymes.     

Brewing Beer with Sorghum

Research reports on extracts, proteins, total nitrogen and free amino nitrogen content of sorghum malt and worts obtained from mashes indicate that sorghum is potentially an alternative substrate for conventional beer brewing in the tropics. Remarkable variations in biochemical characteristics among different sorghum cultivars affect their optimal malting conditions. Factors such as temperature and time of steeping and germinating of grains with their intrinsic enzymic activities, and kilning temperature determine the quality of malt. Further works on mashing, viscosity and fermentability of worts as well as the character of the resulting beers, such as alcoholic content, colour, taste and specific gravity tend to confirm the status of sorghum as a credible substitute for barley in beer brewing. This review reports on progress made in the use of sorghum for brewing beer.     

Determination of Prolamins in Beers by ELISA and SDS‐PAGE

Coeliac disease is triggered by exposure to the prolamin protein fraction of wheat, barley, or rye. The prolamin content of five lager beers and one wheat beer were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting and seven lager beers and three wheat beers were analyzed by enzyme‐linked immunosorbent assay (ELISA). Most of the lager beers were made from barley and some had varying amounts of rice or corn as adjuncts. One of the beers was “gluten‐free”, having been produced from corn and buckwheat without barley. The lager beer samples were gel‐filtered before ELISA or SDS‐PAGE analysis. Prolamin proteins were found in all but one beer which was made of corn, rice and barley and which was not the “gluten‐free” beer. ELISA analysis was done using a commercially available gluten assay kit. For lager beers, a barley prolamin standard for ELISA was propanol‐extracted from barley malt instead of using the prolamin standard of the gluten assay kit. As expected, the wheat beers contained much higher amounts of prolamins than the lager beers. The samples were studied by SDS‐PAGE to identify different prolamin fractions. Proteins having a relative molecular mass in the range of 8000–17,000 and 38,000 and above were detected in immunoblotting by the prolamin sensitive antibody in the lager beers.     

MALTING CHARACTERISTICS OF FINGER MILLET,SORGHUM AND BARLEY*

The malting characteristics of the finger millet variety Imele (FI), sorghum varieties Andivo (SA) and Ingumba (SI) and the barley variety Research (BR) were compared in relation to the brewing of traditional African opaque beer as well as conventional lager beer. The investigations include (a) the effect of steeping and germination conditions, (b) the influence of gibberellic acid and kilning temperature on the activity of important brewing enzymes and (c) an appraisal of the brewing potential of the worts obtained. FI, SA and SI malts were considered unsuitable as barley malt extenders for conventional lager beers, but FI and possibly SI malts would be suitable for tropical lager beer manufacture.     

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.     

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     

Unmalted triticale cultivars as brewing adjuncts: effects of enzyme activities and composition on beer wort quality

The applicability of three selected triticale cultivars (Trinidad, Lamberto, Fidelio) for use as brewing adjuncts was investigated in comparison with wheat adjunct and barley malt. Fermentable substance, crude protein and arabinoxylan levels of starchy materials were determined as well as their native potencies (amylolytic, proteolytic, pentosolytic) to solubilise and degrade grain components during mashing. Laboratory‐scale experiments were performed to evaluate the influence of the adjuncts (composition, enzyme potency) on beer wort quality by mashing mixed (1:1) grists of malt and adjunct. Barley malt was rated as the superior raw material, possessing considerably higher enzyme activities and yielding the lowest wort viscosity. Among the triticale cultivars cv Trinidad was identified as the most suitable to serve as a brewing adjunct due to its improved starch solubilisation properties and its ability to generate low wort viscosities. Compared with the potent malt enzymes, the enzyme activities of unmalted triticale (such as amylases, pentosanases and proteases) had little affect on the composition of the sweet worts. In contrast, the contents of crude protein and fermentable substance of the triticale varieties greatly affected wort quality. Furthermore, the adjunct moiety determined the level of wort viscosity when mashing a combination of malt and triticale. In general, the brewing properties of triticale cv Trinidad were comparable with those of wheat. Copyright © 2004 Society of Chemical Industry     

Brewing with up to 40% unmalted oats (Avena sativa) and sorghum (Sorghum bicolor): a review

Beer production with up to 40% unmalted cereals such as barley, wheat, rice and maize is legally allowed and thus practised in many European countries. The use of oats and sorghum as brewing adjuncts has great potential for creating new beer types/flavours and saving costs. In contrast to oats, sorghum is not as well known within Europe; however, its versatility makes it a very promising crop for exploitation in these temperate‐zone regions. This review describes the brewing‐relevant characteristics of unmalted oat and sorghum grain, investigates the role and properties of endogenous/exogenous enzymes during mashing, discusses the processability/quality of mashes, worts and beers produced with up to 40% oat or sorghum adjunct, and examines the effectiveness/limitations of endogenous enzymes as well as the benefits of the application of exogenous enzymes. Copyright © 2014 The Institute of Brewing & Distilling     

FRACTIONATION OF HIGH MOLECULAR WEIGHT POLYPEPTIDES FROM BEER USING TWO DIMENSIONAL GEL ELECTROPHORESIS

High molecular weight polypeptides from beer were fractionated by a two dimensional gel electrophoretic method comprising isoelectric focusing in the first dimension followed by gradient gel electrophoresis in the second dimension. Silver stained gels revealed a complex pattern of spots which is consistent with individual polypeptide species having both discrete isoelectric points and molecular weights. The pattern of silver stained spots was reproducible both within and between beers and was independent of the sample preparation method or the composition of the sample solubilisation mix. Beers produced from grists containing malt plus 20% wheat flour were shown to contain four polypeptides of approximate molecular weight 30 000, 30 000, 16 000 and 15 000 which were absent from all malt control beers.     

OXYGEN-SCAVENGING ENZYMES IN BARLEY AND MALT AND THEIR EFFECTS DURING MASHING

Off the enzymes that may be involved in the scavenging off oxygen radicals in barley and malt, superoxide dismutase, catalase and peroxidase all increase their specific activities during malting, whereas polyphenol oxidase decreases to zero. Of these, however, only the isoenzymes of peroxidase survive (in part) in the mash, and are responsible for the oxidation of polyphenolic materials. The concentration of hydrogen peroxide normally found in wort limits their action. Addition of hydrogen peroxide to the mash or its generation via a glucose oxidase system greatly increases haze formation, decreases the polyphenol content and causes the development of a red coloration. When the same amount of the different malt peroxidase isoenzymes was added to mashes, the intensity of the red colour varied according to the isoenzyme used. The worts produced by enhanced peroxidation afford more colloidally stable beers. The effects however are limited by the dissolved oxygen concentration in the wort.     

Brewing with 100% green malt – process development and key quality indicators

Brewing with undried, germinated (green) malt has the potential to lower energy and water usage in the malting and brewing chain. However, doing so introduces technical and biochemical (flavour) challenges. Beers were brewed using 100% green malt (n = 3) or kilned pilsner malt (n = 3), prepared from the same batch in each case, utilising the pilot brewery at KU Leuven (2.5 hL). Three further pairs of beers were brewed whereby the green malt was pre-steeped under deaerated water for 1 hour; this procedure was previously shown to lower LOX activity in green malt. Six green malt beers were brewed with acceptable specifications in terms of pH, alcohol content, foam stability and colour. No significant taints or obvious defects were detected in green malt beers. Increased S-methyl methionine levels were measured in worts and beers made from green malt, however DMS concentrations in the finished beers did not differ significantly from the reference beers. Furthermore, the results demonstrated promising indicators for flavour stability, such as reduced TBI, lower residual FAN and trihydroxy fatty acid (THFA) levels in brews using untreated green malt. Using re-steep water in green malt brewing (for reasons of water economy), however, increased THFA levels, possibly because oxygen uptake was not adequately controlled at this step. Whilst further process optimisations are undoubtedly required, it is shown that an acceptable lager style beer could be brewed to a specification not dissimilar to that of a kilned malt control beer, using 100% green malt with intact rootlets. © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

WORT-BOILING IN RELATION TO BEER QUALITY

Comparisons of beers from worts mashed with pale ale malts (nitrogen 1·3%) at 150°F. (65·5°C.) and boiled for 1/2, 1 and 2 hr. show that increase in boiling time leads to slightly increased hop-utilization, decreased head-retention, improved non-biological stability and slight differences in flavour. Findings were very similar when the mashing temperature was either 145°F. (63°C.) or 155°F. (68°C.) except that, in the latter case, no difference in flavour was detectable between beers from long and short boiled worts. Stirring can replace vigorous boiling to secure normal hop-utilization and shelf-life but simmered worts give beers of characteristic flavour probably as a result of enhanced contents of hop oil. Use of a lightly kilned malt of high nitrogen content (1·8%) produced a characteristically different flavour from that obtained with pale-ale malt and reduced the utilization of hop substances by provoking a greater loss of this material during fermentation.     

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.