Carbohydrate content and structure during malting and brewing: a mass balance study

A holistic view of the fate of barley starch, arabinoxylan and β-glucan throughout malting and brewing is largely missing. Here, an industrial scale malting trial and pilot brewing trial were performed, and the concentration and structural characteristics of carbohydrates were analysed at 28 key points in the process. The barley starch content decreased during malting from 75.0% to 69.7%. During mashing, malt starch was converted to fermentable sugars (75.3%), dextrin (22.8%) or was retained in spent grains (1.8%). Arabinoxylan was partially hydrolysed during malting. Despite mashing-in at 45°C, no further solubilisation of arabinoxylan was observed during mashing. However, the average degree of polymerisation of the soluble arabinoxylan fraction decreased slightly. During fermentation, the arabinoxylan content decreased to 2.5 g/L. The amount of barley β-glucan decreased gradually in time during malting. Of the solubilised β-glucan, 31% was retained in the spent grains during wort filtration, slightly lowering the β-glucan content in the wort. The β-glucan content remained at 0.5 g/L during fermentation. Sucrose was hydrolysed during mashing, probably by barley invertases. From the total amount of malt used, 41.0% was converted to fermentable sugars. This mashing yield could have been improved by the full hydrolysis to fermentable sugars of the present β-glucan (to 41.1%), the remaining starch in spent grains (to 42.0%) and dextrin in wort (to 50.3%). These results provide more insight into the carbohydrate conversions during malting and brewing and can act as a baseline measurement for future work. © 2020 The Institute of Brewing & Distilling     

β-GLUCAN AND β-GLUCAN SOLUBILASE IN MALTING AND MASHING

During malting the water-insoluble β-glucan of barley is diminished whilst water-soluble gum is little decreased. The amount of β-glucan surviving into malt depends on variety but barleys rich in glucan give malts with high β-glucan levels. The β-glucan content of barley depends on variety and growth site. β-Glucan solubilase survives mashing and catalyses the release of hemicellulose into solution. There is no correlation between the β-glucan content of malt and the amount released into wort. However, barley adjuncts containing high levels of β-glucan give worts rich in β-glucan. β-Glucan dissolution in mashing is dependent on time, temperature, grist particle size and liquor: grist ratio. Use of adjuncts derived from barley contribute relatively more β-glucan in wort, coinciding with reduced rates of wort separation, but these can be increased by using a β-glucanase produced by growing the fungus Trichoderma viride on spent grains.     

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP™, an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.     

The relationship between polypeptides and foaming during fermentation

The foam level during fermentations of hopped and unhopped wort and with fresh yeast and successive generations of yeast was examined. Simultaneously the total and hydrophobic polypeptide contents in worts, fermented worts and their concomitant foams were checked. It was shown that the total and hydrophobic polypeptide contents of the foam fraction and the foam level during fermentation were dependent on the generation number of yeast. The early generations of yeast (generations 1 and 2) promoted the formation of the largest amount of foam. It was also observed that a higher volume of foam occurred during fermentation of hopped wort in respect to unhopped one despite a higher concentration of polypeptides in unhopped wort. It could be a consequence of a higher foaming potential of polypeptides in hopped wort. The findings of the work may result in the limitation of foaming in the fermenters and consequently the increase of the brewery productivity without using additives and compromising the quality of the final product.     

SOME REASONS WHY HIGH GRAVITY BREWING HAS A NEGATIVE EFFECT ON HEAD RETENTION*

Our aim was to examine the effect of high gravity brewing on head retention with respect particularly to the effect of high gravity brewing on hydrophobic polypeptide levels. High gravity brewed beer had poorer head retention values when compared to a similarly brewed low gravity beer. Analysis of hydrophobic polypeptide levels in both high gravity wort (20° Plato) and low gravity wort (10° Plato) produced using a lauter tun, revealed that the high gravity wort contained 8% less hydrophobic polypeptide than the low gravity wort (undiluted basis). Analysis of hydrophobic polypeptides throughout the brewing process for these 10°P and 20°P brews demonstrated that the hydrophobic polypeptide content decreased, especially during the kettle boil and fermentation. Furthermore, the high gravity brewed beer suffered the greatest loss, leaving the final beer with approximately 40% less hydrophobic polypeptides than the low gravity beer. Brewing at 10°P and 20°P using a mash filter demonstrated that these filters can improve the head formation and stability of the resultant beers at sales gravity. However, the low gravity beer still produced a more stable foam (Rudin value 93 s) when compared to the high gravity beer (Rudin value 83 s). The mash filter slightly increased the hydrophobic polypeptide extraction. It is concluded that the mash filter produced higher hydrophobic polypeptide levels in the final beers, as well as having a positive effect on reducing the levels of foam negative compounds such as fatty acids in the wort, and therefore slightly improved head retention values .     

Release of phenolic flavour precursors during wort production: Influence of process parameters and grist composition on ferulic acid release during brewing

In this study, the effects of mashing variables such as mashing-in temperature, time and pH, mash thickness, grist coarseness and composition, and stirring regime on the release of ferulic acid were examined. Ferulic acid is a precursor for the formation of flavour-active volatile phenols and a potent natural antioxidant in beer. Given one barley malt variety, the multitude of choice in setting various process parameters and adding brewery adjuncts during brewhouse operations can give rise to worts with widely varying ferulic acid levels. A clear difference in temperature- and pH-dependence between the release of the water-extracted and the enzymatically hydrolyzed fraction was found. The T,t-dependencies of arabinoxylan-degrading enzyme activities were correlated with ferulic acid release during mashing. Results from laboratory-scale mashing experiments were validated with those from a pilot-scale (5 h) wort production process. Enhancing the enzymatic release of phenolic flavour precursors from bound forms during mashing can greatly enhance the phenolic aroma potential of wort. Optimising this precursor release during mashing may be a means for controlling final volatile phenol levels in beer.     

The Loss of Hydrophobic Polypeptides during Fermentation and Conditioning of High Gravity and Low Gravity Brewed Beer

The object of this study was to investigate the loss of hydrophobic polypeptides, which are important for foam quality and stability in finished beer. Loss of hydrophobic polypeptide due to fermenter foaming occurs during transfer of fermented wort since a gradient of hydrophobic polypeptides towards the surface is created during fermentation. Due to higher polyphenol levels in high gravity (20°Plato) wort, more hydrophobic polypeptides are lost due to cold break (cold trub) precipitation compared to low gravity (12°Plato) wort. Another important factor affecting the loss of hydrophobic polypeptides could be proteinase A activity during fermentation, especially in high gravity fermentation where the yeast is exposed the higher stress. During high gravity fermentation, where osmotic pressures are higher, ethanol levels become greater, and nitrogen‐carbohydrate ratios are lower, more proteinase A is released by the yeast. This release of proteinase A into fermenting wort could have implications for the foam stability of the finished product.     

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.     

DETERMINATION OF FATTY ACID HYDROPEROXIDES PRODUCED DURING THE PRODUCTION OF WORT

Linoleic and linolenic acid hydroperoxides in malt, mash, or wort were determined with high sensitivity and high selectivity by the chemiluminescence-high performance liquid chromatography (CL-HPLC) method using isoluminol-microperoxidase solution as a luminescing reagent. The determination limit of this method for both hydroperoxides was 0.1 μM in mash or wort. During the mashing in a laboratory mash bath, the hydroperoxides started to increase just after mashing-in, reached a maximum at 65°C, and then decreased. Though the hydroperoxides were detected in mash just before the lautering in a pilot scale brewing, they disappeared during the lautering and could not be detected during the subsequent stages of wort production. Therefore, it was thought that the mashing process is the most important of the lipid oxidation reactions during wort production. It is also expected that the CL-HPLC method can give useful information on lipid oxidation mechanisms during wort production.     

THE PROPERTIES,COMPOSITION AND FERMENTABILITIES OF WORTS MADE FROM 100% RAW SORGHUM AND COMMERCIAL ENZYMES

A mashing regime was developed using 100% raw sorghum which enabled commercially acceptable hot water extracts to be obtained in 85 minutes with minimal use of a heat stable α-amylase and proteolytic enzymes. This gave worts of HWE 295 1°/kg, with FAN levels of about 40 mg/l and ammonium ion concentration of about 60 mg/l. Higher, but commercially unacceptable, levels of proteolytic enzymes gave worts with FAN from 84.5 to 95 (mg/l). Addition of an amyloglucosidase as the commercial preparation Amylo300L, was required to convert the HWE to fermentable extract. The addition of Amylo300L, increased the DP1, DP2 and DP3 carbohydrate fractions of the worts from 22% to more than 90% of the total, compared to about 80% for a wort made from malted barley without the use of enzymes. Two different proteolytic enzymes gave different extracts and FAN contents presumably reflecting either differences in susceptibilities of the sorghum to the two enzymes or the presence of different additional enzyme activities in the different preparations. The level of ammonium ions in malted barley worts was 86 mg/l and up to 88 mg/l in worts produced from sorghum and enzymes. Enzyme addition produced increased levels of ammonia. The content of Group A (the most readily assimilated) amino acids was proportionally higher in sorghum worts compared to malted barley wort. Worts made from raw sorghum and enzymes, containing as little as 40 mg/l FAN, were fully attenuated. The yeast consumed about 35 mg/l FAN and 45 mg/l ammonium ions. Under identical fermentation conditions, the same yeast, fermenting a malted barley wort of comparable extract consumed 104 mg/l FAN and 37 mg/l ammonium ions.     

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.     

THE VISCOSITY OF WORTS IN RELATION TO THEIR CONTENT OF β-GLUCAN

A procedure is given for assessing that proportion of wort viscosity which is attributable to β-glucan. Worts obtained from unkilned samples of malt which have been processed for 54 or 72 h show enhanced viscosity. This is principally due to β-glucan although the contribution of other constituents, absent from the wort of fully modified malt, is of significance. Barley variety is shown to have a pronounced effect on wort viscosity. Insoluble β-glucan is brought into solution in mashes at 65° C. The β-glucan isolated from malt which has been inactivated using aqueous ethanol prior to extraction at 65° C, is of higher specific viscosity than that isolated from control worts prepared at the same temperature. The introduction of a rest by mashing initially at 40° C results in the production of wort of lower viscosity, a decrease in the β-glucan content of the wort and a reduction in the specific viscosity of the β-glucan. There is no apparent relationship between the endo-β-glucanase content of the malts and either the viscosity of derived worts or the degree of breakdown of β-glucan which occurred during malting and mashing. Abrasion of barley, which is a factor assisting the distribution of enzymes during malting, acts to reduce wort viscosity.     

Effects of mashing on total phenolic contents and antioxidant activities of malts and worts

Effects of mashing on total phenolic contents (TPC), radical‐scavenging activity, reducing power and metal chelating activity of malts and corresponding worts were clarified in this study. Results showed that there were significant variations in TPC and antioxidant activity across malts and worts. An 8.8% of increase in TPC was observed during the early stage of mashing, accompanied by an increase in antioxidant activity. However, 2.6% of decrease in TPC and inconsistent changes in antioxidant activity were found during the mashing at higher temperature. Overall, mashing resulted in 6.0% and >10.0% of increases in TPC and antioxidant activity, respectively. Moreover, Pearson correlation analysis revealed that there were good correlations (ranging from 0.622 to 0.735, P < 0.01) between TPC and antioxidant activity of malts and worts. Additionally, mashing resulted in more positive correlations between TPC and antioxidant activity, emphasising the key role of malt phenolic compounds for wort antioxidant activity.     

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     

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.     

High Gravity Brewing by Continuous Process Using Immobilised Yeast: Effect of Wort Original Gravity on Fermentation Performance

The present work evaluated the influence of all‐malt wort original gravity on fermentative parameters and flavour‐active compound formation during primary fermentation of high gravity brewing by a continuous process using a lager yeast immobilised on a natural carrier obtained from brewer's spent grain (the main brewery by‐product). The all‐malt worts with original gravity (OG) ranging from 13.4 to 18.5°Plato were prepared by diluting a very‐high‐gravity wort (20°Plato) with sterile brewery water. The continuous assay was carried out in a bubble column bioreactor with a total working volume of 5.2 litres, at 15°C, using a constant gas flow rate of 250 mL/min (200 mL/min of CO₂ and 50 mL/min of air) and a dilution rate of 0.04 h^?1 (residence time of 25 h). The results indicated that as the wort OG was increased, the ethanol concentration of the outflowing beer increased. On the other hand, the continuous fermentation of the most concentrated worts (16.6 and 18.5°Plato) resulted in beers with unbalanced flavour profiles due to excessive ethyl acetate formation. The immobilised cell concentration appeared to be nearly independent from increasing wort OG.     

THE FATE OF HUMULONE DURING WORT BOILING AND COOLING

Variations in the nature of the grist, mashing temperature, gravity and content of anthocyanogens of wort influenced the yield of break obtained after boiling with humulone, but not the bitterness of the resulting worts. Only small amounts of resin were found on the break and although increased break formation resulted in increased adsorption of resin this did not lead to measurable differences in the bitterness of wort. The bitterness of the cooled wort was independent of the degree of aeration and speed of cooling.     

The Effect of Proteinase A on Foam‐Active Polypeptides During High and Low Gravity Fermentation

The ability of beer to produce good foam is influenced by the level of foam‐active polypeptides. Specific polypeptides with hydrophobic domains, such as Lipid Transfer Protein (LTP1), are important components of beer foam. Although, high gravity brewing is a commercially viable technique, it has the disadvantage of producing beer with less foam stability compared to lower gravity brewed counterparts. It is thought that proteinase A plays a key role in the degradation of these hydrophobic polypeptides responsible the beer foam stability. The object of this study was to compare and quantify the loss of hydrophobic polypeptides and specifically foam‐LTP1 during high gravity (20°Plato) and low gravity (12°Plato) wort fermentations and to evaluate the effect of proteinase A on these polypeptides. The losses of hydrophobic polypeptides and foam‐LTP1 were generally greater in high gravity brews. Furthermore, the results obtained suggest that proteinase A alters the hydrophobicity of these polypeptides rather than their molecular size. Approximately 20% of hydrophobic polypeptides and approximately 57% of foam‐LTP1 appeared to be proteinase A resistant. These differential losses of hydrophobic polypeptide and foam‐LTP1 could have implications for the foam stability of the finished product.     

PROCESSES FOR PRODUCING BEER WITH A LOW TENDENCY TO FORM HAZE

It was confirmed that wort from malt resteeped in a solution of formaldehyde (1000 mg./litre), had a low level of anthocyanogens. It was shown that beer brewed from this malt had a lesser tendency to form haze than beer brewed from a malt resteeped in water. Malt yielding wort with usefully reduced levels of anthocyanogens could be prepared by adding formaldehyde (500–1000 mg./litre), to the final steep, in an otherwise conventional malting sequence. The rapid rate of haze formation that occurred in beers to which formalin had been added was shown to be a useful, quick guide to their stability under different storage conditions. When hydrogen peroxide (100 mg./litre) mashing liquor was added to mashes it reduced the anthocyanogen levels of the wort. The beers prepared from treated mashes were remarkably slow to form haze. The effect was greatest when hydrogen peroxide was added in small increments throughout the mashing period. The other alterations in wort characteristics resulting from this process, including marginal increases in colour and decreases in fermentability, were small. In most trials the treatment did not significantly alter the flavour of the beer. Charcoal (Norit, N.K.), added to the mash (500 mg./kg. grist), or to the copper (500 mg./kg. grist), reduced the anthocyanogen contents of the worts; the final beers had greatly enhanced shelf-lives. Charcoal was most effective when spread over the surface of the mash at the start of sparging.     

Investigation of foam‐active polypeptides during beer fermentation

During ale fermentation there was an accumulation of total and hydrophobic polypeptides in the foam relative to the wort. Comparisons were made not only of the total and hydrophobic polypeptide contents but also of the molecular weights of these polypeptides present in wort, partially fermented wort and its concomitant foam. Wort, fermented wort and foam fractions had very similar polypeptide compositions with a major group having molecular weights of 40–43 kDa. Material of molecular weight in the range of 5–17 kDa and at 66 kDa was also detected. The polypeptides accumulated in foam displayed both hydrophobic and non‐hydrophobic character. The presence of yeast polypeptides in foam was confirmed. Comparison was also made between the fermentations of 10°Plato and 15°Plato wort. The results of the work may contribute to a better understanding of the mechanism of foam formation during beer fermentation, leading to reduced foaming and enabling an increase in the working capacities of fermenters. Copyright © 2004 Society of Chemical Industry