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     

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.     

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.     

HOP SUBSTANCES AND YEAST BEHAVIOUR

Top fermentation yeast strains vary in their ability to form a yeast head in the absence of hop substances. However, the addition of isohumulone or humulone to unhopped wort stimulates yeast-head formation. A quantitative method for the estimation of hop substances bound to the yeast cell is described and is used to demonstrate that in top fermentation, most of the adsorbed bitter substances are present in the head. It is shown that most of the hop substances in the yeast head are transported there by bubbles of carbon dioxide; in bottom fermentation, considerable quantities are present in the surface foam. In comparison with hopped wort the fermentation of unhopped wort with Saccharomyces cerevisiae (N.C.Y.C. 240) is characterized by an increased amount of yeast in suspension during fermentation, although there is no increase in the rate of attenuation.     

HYDROPHOBIC POLYPEPTIDE EXTRACTION DURING HIGH GRAVITY MASHING— EXPERIMENTAL APPROACHES FOR ITS IMPROVEMENT

The aim was to establish if a substantial increase in hydrophobic polypeptides could be achieved during high gravity mashing. When worts with gravities ranging from 5–20°P were analysed for hydrophobic polypeptide content it was found that there was no appreciable increase in hydrophobic polypeptide levels. Remashing of the spent grains from low and high gravity mashes demonstrated that this resulted from inefficient extraction of hydrophobic polypeptide levels during the mashing process. For example, wort produced from remashed high gravity spent grains contained 150 mg/L hydrophobic polypeptides compared to only 10 mg/L in the low gravity remashed spent grains. Experiments were conducted, employing standard mashing techniques, in an attempt to increase the extraction of hydrophobic polypeptides during high gravity mashing. Thus the use of gypsum, proteolytic stands, varying liquor to grist ratios and wheat malt addition were all investigated for their effect on hydrophobic polypeptide extraction during high and low gravity mashing. Wort analysis demonstrated that none of the techniques employed had a significant effect on hydrophobic polypeptide extraction. When wort from remashed spent grains was used as mashing in liquor for a fresh mash and the resultant worts analysed for hydrophobic polypeptides it was observed that no increase in hydrophobic polypeptide extraction was achieved. For example, wort from the remashed high gravity spent grains, containing 140 mg/L hydrophobic polypeptides, when used as mashing-in liquor, produced no increase in hydrophobic polypeptide levels in the resultant high gravity wort (230 mg/L) when compared to a high gravity wort produced using distilled water as mashing-in liquor (255 mg/L). It is therefore concluded that a saturation point has been reached and no more hydrophobic polypeptides can be extracted during mashing regardless of the procedures employed.     

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 .     

OBSERVATIONS ON THE EFFECT OF LIPIDS ON HEAD RETENTION

Modification of the conventional method of filtering an infusion mash was found to increase the content of extractable head-negative substances, including phospholipids, in unhopped wort. The additional constituents were also present in the corresponding hopped worts and in beers at rack but largely disappeared during subsequent processing. Much of the additional fatty material was removed when finings were added: this process induced a substantial rise in head retention. The positive role of lipids in brewing processes is discussed.     

FERMENTATION OF WORTS MADE FROM 100% RAW SORGHUM AND ENZYMES

Worts made from raw sorghum and enzymes were successfully fermented even though the level of FAN present (51 mg/l) is well below that essential for fermentation of wort made from malted barley. Changes in typical fermentation parameters such as specific gravity, pH uptake of free amino nitrogen (FAN) and ammonium ions mirrored the increase in yeast cell concentration. Yeast viability remained high throughout the fermentation. Under identical fermentation conditions, malted barley worts showed typical fermentation profiles. However, malted barley worts with specific gravity maintained by the addition of D-glucose, but in which the FAN was diluted to a level similar to that found in a wort made from sorghum and enzymes, fermented more slowly and failed to attenuate fully. Five consecutive fermentations, using yeast cropped from the preceding to pitch the current fermentation were conducted. The specific gravity profiles were essentially the same in all five fermentations. Final values of pH, yeast in suspension, yeast viability and FAN were also indistinguishable. The yeast crop taken from fermentations of worts made from raw sorghum and enzymes represented a 5-fold increase over the initial pitching rate. When compared to commercial beers, the beers derived from fermentation of worts made from raw sorghum and enzymes contained lower levels of ethyl acetate, and higher levels of both 2- and 3-methyl butanol. In the beers derived from sorghum, isobutanol was always less than 20% of the total higher alcohol concentration.     

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.     

THE METABOLISM OF PUTRESCINE,SPERMIDINE AND SPERMINE BY YEAST IN RELATION TO THE AVAILABILITY OF MAGNESIUM

During the lag period immediately after inoculation of yeast into a defined salts medium rapid changes in the internal concentrations of the oligoamines occurred when the medium was deficient in magnesium or high in calcium. Pronunced changes in the oligoamine content of yeast also occurred after inoculation into a range of worts and the extent of the effect appeared to depend on the magnesium content of the wort. The amount of magnesium in all the worts was much higher than was found to be saturating in a minimal medium and it therefore seems that the magnesium in worts is not freely available to the yeast cells. Putrescine was the only oligoamine found in wort and was present at about 1 mg/litre. The putrescine concentration in the wort always increased during fermentation and the period of most rapid increase corresponded to the period of decline in the internal oligoamine pools.     

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.     

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 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.     

The Yeast Vacuole ‐A Scanning Electron Microscopy Study During High Gravity Wort Fermentations

The yeast vacuole has been shown to exhibit morphological responses to environmental conditions when exposed to worts of different gravity during fermentation. Marked effects of high gravity wort (20° Plato) on yeast morphology compared to more conventional wort gravity (12° Plato) were observed. High gravity worts caused vacuolar enlargement compared to conventional gravity wort. These results suggested that yeast cells experienced severe alterations with the vacuolar tonoplast when exposed to high osmotic pressure and elevated levels of ethanol.     

Comparative evaluation of the formations of gamma‐aminobutyric acid and other bioactive amines during unhopped wort fermentation

This study aimed to investigate the effect of Saccharomyces cerevisiae on the formation of gamma‐aminobutyric acid (GABA) and the other bioactive amines during wort fermentation. Within 8 days of fermentation, GABA concentration increased to 182.80 and 534.10 mg/L in unspoiled and spoiled worts, respectively. Although formation of tyramine and histamine did not occur in unspoiled wort, 142 mg/L of tyramine and 130 mg/L of histamine were found in spoiled wort at the end of fermentation. Decreased concentrations of tyrosine and histidine were associated with increased concentrations of tyramine and histamine, respectively, in spoiled wort. The results indicated that S. cerevisiae is a causative agent for the accumulation of GABA in wort during fermentation. Therefore, occurrence of GABA in beers should not be considered as one of the indicators of microbial contamination differently from tyramine and histamine.

Practical applications

Bioactive amines have important metabolic and physiological roles in the body. Their formation in foods is generally related to microorganisms having decarboxylase activity. It was found in this study that spoilage microorganisms produced tyramine and histamine while both spoilage microorganisms and Saccharomyces cerevisiae are responsible for the formation of gamma‐aminobutyric acid during unhopped wort fermentation.     

采用复合诱变选育低分泌蛋白酶A啤酒酵母菌株的研究

啤酒酵母在发酵过程中分泌胞外的蛋白酶A是影响纯生啤酒泡沫稳定性的关键因素，本研究通过采用亚硝基胍（TNG）和甲基黄酸乙酯（EMS）复合诱变选育满足食品安全性的低蛋白酶A、发酵性能正常的优良啤酒酵母菌株，为解决纯生啤酒泡沫稳定性奠定基础。     

啤酒发酵过程中影响蛋白酶A分泌的因素

纯生啤酒中残存的蛋白酶A严重影响泡沫稳定性,制约了纯生啤酒的质量提升。为了探索啤酒发酵过程中影响蛋白酶A分泌的因素,作者分别考察了菌种、酵母生理状态、酵母代数、麦汁浓度、发酵时间等对蛋白酶A分泌的影响。结果发现,蛋白酶A分泌量高的菌株,处于稳定期之后的酵母、较高的酵母代数、较高的原麦汁浓度和在发酵阶段末期都会导致发酵液中蛋白酶A活性偏高。建议在实际生产中,采用蛋白酶A分泌量少的菌种、调整酵母生理状态、使用小于3代的酵母、采用18°P以下的麦汁发酵和尽早结束发酵都会对降低蛋白酶A的分泌量起到积极作用。     

Antimicrobial activity of Enterococcus faecium L50, a strain producing enterocins L50 (L50A and L50B), P and Q, against beer-spoilage lactic acid bacteria in broth, wort (hopped and unhopped), and alcoholic and non-alcoholic lager beers

Enterococcus faecium L50 produces enterocin L50 (L50A and L50B) (EntL50, EntL50A and EntL50B), enterocin P (EntP) and enterocin Q (EntQ) and displays a broad antimicrobial spectrum against the most relevant beer-spoilage lactic acid bacteria (LAB) (i.e., Lactobacillus brevis and Pediococcus damnosus), which is mainly due to the production of EntL50 (EntL50A and EntL50B). Bacteriocin assays using in vitro-synthesized EntL50 (EntL50A and EntL50B) showed that both individual peptides possess antimicrobial activity on their own, EntL50A being the most active, but when the two peptides were combined a synergistic effect was observed. The only virulence genes detected in E. faecium L50 were efaAfm (cell wall adhesin) and ccf (sex pheromone), and this strain was susceptible to most clinically relevant antibiotics. E. faecium L50 survived but did not grow nor showed antimicrobial activity in hopped and unhopped wort, and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) commercial lager beers. However, when unhopped wort was supplemented with 50% (v/v) MRS broth, E. faecium L50 grew and exerted antimicrobial activity similarly as in MRS broth. The enterocins produced by this strain were bactericidal (5 log decrease) against P. damnosus and Lb. brevis in a dose- and substrate-dependent manner when challenged in MRS broth, wort (hopped and unhopped), and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) lager beers at 32 degrees C, and no bacterial resistances were detected even after incubation for 6-15 days. The enterocins in wort and lager beer (5% ethanol, v/v) withstood the heat treatments commonly employed in the brewing industry during mashing, wort boiling, fermentation, and pasteurization, and retained most of their antimicrobial activity in lager beer (5% ethanol, v/v) after long-term storage at 8 and 25 degrees C.     

COMPOSITION OF NON-STARCH POLYSACCHARIDES IN WORT AND SPENT GRAIN FROM BREWING TRIALS WITH MALT FROM A GOOD MALTING QUALITY BARLEY AND A FEED BARLEY

Worts and spent grains were obtained from pilot-scale brews using malts of two barley varieties differing in malting quality. Triumph was used as an example of a good malting quality barley and Golf as a typical feed grade barley. Arabinoxylan concentrations were similar in worts of both varieties, whereas (1–3), (1–4)-β-glucan concentrations were much higher in wort prepared from Golf malt than in wort from Triumph malt. From the worts, polysaccharide fractions were isolated by ethanol precipitation and characterised. Results indicated the presence of high molecular weight arabinoxylans and (1–3), (1–4)-β-glucans. The arabinose to xylose ratios in the precipitates were considerably higher than in total worts. Methylation analysis showed little differences between precipitated wort arabinoxylans from both varieties. In comparison to water-insoluble arabinoxylans extracted from barley and malt, the precipitated wort arabinoxylans were richer in xylopyranose residues substituted with arabinose residues at both 0–2 and 0–3. Viscosities of the hopped worts of both varieties decreased after treatment with endoxylanase 1 from Aspergillus awamori. This confirms that arabinoxylans play a role in determining wort viscosity, possibly through interactions with (1–3), (1–4)-β-glucans.     

Modelling of Yeast in Suspension During Malt Fermentation Assays

Premature yeast flocculation (PYF) is a poorly understood condition leading to attenuation of fermentation and poor alcohol yields. The yeast in suspension in fermenting worts prepared from either normal or premature flocculating malts was continually measured during the small 15 mL fermentation test recently developed in the Dalhousie laboratories. A simple inexpensive monitoring system was constructed from a laser level, photometric cell and data logger. This system allowed non‐destructive data collection of absorbance data (over 700 data points per fermentation) at ?650 nm. A non‐linear modeling technique was applied to the data, and yielded two best‐fitting logistic models. The model parameters were quantitatively compared to determine if statistical differences between PYF and normal wort were apparent. A student's t test of the logistic parameters indicated a significant difference (p > 0.025) between control and PYF worts for the increase in absorbance at the beginning of the fermentation. A significant difference (p > 0.0001) in the inflection time of absorbance down‐curve between the control and PYF wort was also noted.