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 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 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.     

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.     

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     

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.     

Brewing with Rice Malt — A Gluten‐free Alternative

The preparation of beer‐like beverages with rice malt as the only raw material is reported. Several tests were performed on a laboratory scale and in a 25 L‐capacity pilot plant. Both the decoction and the infusion procedure were tested; malt and water were mixed in a ratio 1:3.5 for both methods and the mash was brewed without adding exogenous enzymes. The obtained worts were fermented using bottom fermenting yeasts, while “beers” were re‐fermented utilizing top fermenting yeasts and adding either sterile wort or sugar. A maximum ethanol of 4.5% vol. was obtained after the primary fermentation from an initial wort with an original gravity of 11.8°Plato. All parameters of the beer were found to be acceptable using a standard beer analysis. Owing to a suitable hop addition, an aroma very similar to that of a normal beer was obtained.     

Wheat Variety and Barley Malt Properties: Influence on Haze Intensity and Foam Stability of Wheat Beer

Laboratory wheat beers were brewed with different wheat varieties of different protein content (8.7–14.4%) and with five different barley malts, varying in degree of modification (soluble protein: 3.9–6.9%). In a first series of experiments, it was investigated whether wheat positively influences the foam stability, a major characteristic of wheat beers. NIBEM and Rudin (CO₂) foam analyses revealed that the effect of wheat on foam stability depended on the barley malt used for brewing. When using malt with high foaming potential, wheat exerts a negative influence. However, wheat added to over‐modified malt with less foam promoting factors, ameliorates beer foaming characteristics proving that wheat contains foam active compounds. In addition, Rudin (N₂) values suggested that wheat positively influences foam stability by decreasing liquid drainage, probably caused by a higher beer viscosity and/or a finer foam bubble size distribution. Furthermore, the haze in wheat beers, which is another important quality characteristic of these beers, was investigated. Permanent haze readings of the 40% wheat beers were lower than 1.5 EBC haze units. For 20% wheat beers, an inverse relation between the permanent haze (9.4–19.3 EBC haze units) and the protein content of the wheat was established. The barley malt used for brewing also influenced permanent haze readings. A positive correlation between the modification degree of the malt and the permanent haze intensity was found. It was concluded that the choice of raw materials for wheat beer brewing considerably influences the visual properties of the beer.     

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.     

Preparation of a Low Carbohydrate Beer by Mashing at High Temperature with Glucoamylase

A malt grist, supplemented with glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3), was mashed isothermally at 70°C to produce wort with a real fermentability of over 87% and beer with a residual carbohydrate content of less than 0.75% w/v at an original gravity of 42°S. The effects of varying mash pH, calcium content, glucoamylase addition rate and mash conversion time were investigated. The process was effective even with undermodified malt. Pilot scale brewing trials shozved no adverse effect on beer foam quality when the glucoamylase preparation had a specified range of protease side-activity. The process has the advantage of ensuring glucoamylase inactivation during wort boiling while being shorter than the alternative of enzyme treatment of wort prior to boiling.     

Physicochemical characterization of sahti,an ‘ancient’ beer style indigenous to Finland

Sahti, a strong, unhopped farmhouse beer flavoured with juniper, is still actively brewed in rural areas in Finland. Presented here is the first comprehensive analysis of the physical and chemical properties of this unique beer style. Twelve sahti samples from the southwest of Finland were analysed and, while properties varied, the beers generally had high levels of alcohol (mean = 7.9% ABV) and high residual extract (mean = 9.5°P). Foam stability was negligible, as is typical for the style, and glycerol concentrations at 3.1 – 4.7 g L^?1 were higher than in reference beers (commercial lager, wheat beer and porter). These features may be attributed to the very high gravity conditions employed in brewing sahti beers. Bitterness levels were relatively low (3–13 IBU) owing to the absence or moderate use of hops. All samples contained detectable levels of the clove‐like compound 4‐vinylguaiacol owing to the use of baker's rather than brewer's yeast for brewing. Concentrations of higher alcohols and esters were high, with many individual aroma compounds being above the normal flavour thresholds. Results have highlighted the uniqueness of this style of beer in comparison to commercially available beers and have contributed to our understanding of the reasons for the particular sensorial properties of this traditional beer style. Copyright © 2015 The Institute of Brewing & Distilling     

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.     

RAPID METHODS FOR DETERMINING THE HIGH MOLECULAR WEIGHT POLYPEPTIDE COMPONENTS OF BEER

Size exclusion chromatography and a coomassie blue dye-binding assay were used to investigate the high molecular weight polypeptide fractions of beer. Size exclusion chromatography using small colums of Sephadex G25, G50 (10 ml) and G75 (20 ml) together with an automated FPLC system enabled the rapid isolation of high molecular weight polypeptide fractions of beer. Size exclusion chromatography using small columns of Sephadex was investigated as a method for the determination of the total high molecular weight polypeptides in beer. The results from the analysis of a series of pilot brewery beers produced under standardised brewing conditions showed that the head retention value of a beer correlated with the content of high molecular weight polypeptide material regardless of the molecular weight fraction assayed. Both methods may be used to monitor the high molecular weight polypeptide content, predict the head retention value of a beer and are sufficiently rapid for routine use in the quality control laboratory.     

Comparison of beer quality attributes between beers brewed with 100% barley malt and 100% barley raw material

BACKGROUND: Brewing with 100% barley using the Ondea^® Pro exogenous brewing enzyme product was compared to brewing with 100% barley. The use of barley, rather than malt, in the brewing process and the consequences for selected beer quality attributes (foam formation, colloidal stability and filterability, sensory differences, protein content and composition) was considered. RESULTS: The quality attributes of barley, malt, kettle‐full‐wort, cold wort, unfiltered beer and filtered beer were assessed. A particular focus was given to monitoring changes in the barley protein composition during the brewing process and how the exogenous OndeaPro^® enzymes influenced wort protein composition. All analyses were based on standard brewing methods described in ASBC, EBC or MEBAK. To monitor the protein changes two‐dimensional polyacrylamide gel electrophoresis was used. CONCLUSION: It was shown that by brewing beer with 100% barley and an appropriate addition of exogenous Ondea^® Pro enzymes it was possible to efficiently brew beer of a satisfactory quality. The production of beers brewed with 100% barley resulted in good process efficiency (lautering and filtration) and to a final product whose sensory quality was described as light, with little body and mouthfeel, very good foam stability and similar organoleptic qualities compared to conventional malt beer. In spite of the sensory evaluation differences could still be seen in protein content and composition. Copyright © 2011 Society of Chemical Industry     

Study on the Membrane Filtration of Mash with Particular Respect to the Quality of Wort and Beer

The quality of a membrane filtered wort, and the beer from it, is different from that produced with a lauter tun or mash filter. A membrane is capable of producing a clear wort in terms of turbidity while solid contents, in particular larger molecular substances, are retained. The selectivity of separation is not directly set by the membrane's pore size but rather by the formation of the fouling layer. Thus an interaction between technical filtration parameters and selectivity determines the quality of the resulting wort and beer. The filterability of a beer produced with a membrane filter system for lautering was considerably better when a poorly modified malt was used and foam stability was worse when a normally modified malt was used. For all other properties examined in the beers, no significant differences between lauter tun and membrane filtration including sensory characteristics were observed.     

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.     

THE INFLUENCE OF CONDITIONING ON LAGER BEER QUALITY

The relative contributions to lager beer quality of malt, conditioning, and other parts of the brewing process have been assessed by analytical and sensory evaluation. Backed beers, brewed and fermented under various conditions, were stored at temperatures of 12°C, 4°C or 0°C for periods ranging from 2 to 38 days before filtration and bottling. No evidence could be found to justify a need for long periods of beer storage. Although prolonged storage can provide a remedial process, correcting aspects of beer quality such as diacetyl levels, final gravity, and removal of ‘young beer’ flavour, all these features can be more rapidly, effectively and economically controlled by closer attention to materials quality and primary fermentation procedure.     

The Impact of Malt Derived Proteins on Beer Foam Quality. Part II: The Influence of Malt Foam-positive Proteins and Non-starch Polysaccharides on Beer Foam Quality

Pilot (50 litre) and small scale (700 mL) brewing trials conducted using, similar brewing protocols with 25 different malts, indicated that differences in malt quality influenced foam stability (Rudin head retention value) by up to 24%. In addition to conventional measures of malt quality, enzyme-linked immunosorbent assays (ELISA) were used to measure the level of the putative foam-positive proteins, BSZ4 (protein Z4), BSZ7 (protein Z7), BSZ7b and lipid transfer protein 1 (LTP1). Regression analysis performed on the combined pilot and small scale data sets identified that malt BSZ4, wort β-glucan and wort viscosity, and beer protein, β-glucan and arabinoxylan were positively correlated with foam stability, while malt Kolbach index (KI), and beer FAN were negatively correlated with foam stability. Potentially foam-positive proteins such as BSZ7 and LTP1 were not correlated with foam stability. The negative correlation of BSZ4 level with KI suggested an additional role for BSZ4 in influencing protein modification. Step-wise multiple regression indicated that up to 82% of the variation in foam stability could be predicted from the malt and beer characteristics measured, demonstrating that there are a number of inter related malt derived factors that influence beer foam stability.     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

低浓度淡爽型啤酒的酿造

以改善低浓度淡爽型啤酒品质为目的 ,提出了一种新颖的低浓度淡爽型啤酒的酿造方法 .采用二次煮出二段式糖化法 ,用 70 %麦芽和 3 0 %大米的原料配比 ,提高麦芽汁中糖与非糖的比值 ,并在糖化过程中添加啤酒酵母提取物作啤酒发酵的补充氮源 .所酿造的啤酒口味纯正 ,泡沫洁白细腻 ,持久挂杯 .