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.     

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 .     

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.     

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.     

Changes in protein molecular weight during cloudy wheat beer brewing

To investigate the changes in protein in cloudy wheat beer (CWB) brewing, the molecular weight (M_w) of protein components in wort, fermenting wort, beer and beer foam were analysed by high‐performance size‐exclusion chromatography. It was found that the M_w of protein during brewing was mainly distributed between 100 and 0 kDa. From mash to the final beer, the most abundant protein component was the 7.6–2.1 kDa fraction, which occupied more than 40% of the total protein. The extraction of soluble protein from malts into wort mainly focused on the protein rest process, where the 100.0–32.1 and 7.6–2.1 kDa fractions increased significantly. In addition, the 100.0–13.2 kDa fraction was the predominant thermolabile protein that decreased during lautering and boiling. The dominant protein constituting the fine coagulum, the 100.0–13.2 kDa fraction, also decreased during fermentation and maturation. The CWB, defoamed CWB and CWB foam had similar protein M_w distributions, while the CWB foam concentrated much more protein and the 7.6–2.1 kDa fraction was the major protein in the CWB foam. Copyright © 2015 The Institute of Brewing & Distilling     

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.     

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.     

STUDIES ON THE MECHANISM OF ACTION OF COPPER FINING AGENTS (κ CARRAGEENAN)

Carrageenan mediated clarification of brewers' wort comprises two reactions: interaction between carrageenan and soluble wort polypeptides and flocculation of particulate material. The dose/response characteristics of interaction between carrageenan and soluble polypeptide material suggested that carrageenan exhibits selectivity with respect to specific polypeptide fractions. Size exclusion chromatography of worts demonstrated that carrageenan treatment resulted in reductions in fractions of relative molecular mass Mr 70 000, 40 000 and 12 000. Fining trials demonstrated that the reactivity towards soluble polypeptides is independent of the presence of insoluble particulate material. Furthermore, both interaction with soluble polypeptides and flocculation of particulate material occurred in systems fined under high (80°C) and low (20–25°C) initial temperatures and this suggested that the presence of carrageenan in a helical conformation is essential to fining action rather than conformational transitions in polysaccharide structure. Additionally, carrageenan has no effect on the levels of low molecular mass wort solutes such as nucleic acid derivatives, aromatic and heterocyclic amino acids.     

Acid Washing and Serial Repitching a Brewing Ale Strain of Saccharomyces cerevisiae in High Gravity Wort and the Role of Wort Oxygenation Conditions†

Acid washing pitching yeast is an effective method for removing bacterial contamination, but if the yeast is washed incorrectly decreased fermentation performance and beer quality problems may result. Various factors can affect the acid resistance of yeast strains during brewery fermentations. Yeast from shaking flask experiments was more resistant to the combination of high gravity and acid washing conditions than yeast cropped from static fermentations. Yeast harvested from static high gravity wort (20° Plato; 1.083 OG) fermentations was more adversely affected by acid washing than yeast from standard gravity (12° Plato; 1.048 OG) wort. Wort oxygenation resulted in enhanced yeast fermentation performance and healthier yeast crops when yeast was serially repitched into 20° Plato wort. Yeast cropped from fermentations with air saturated high gravity wort responded poorly when acid washed. These results suggest that the structure of the plasma membrane particularly the sterol and fatty acid composition, may have an important role in tolerating high gravity wort and acid washing conditions.     

IDENTIFICATION OF LAMBIC SUPERATTENUATING MICRO-ORGANISMS BY THE USE OF SELECTIVE ANTIBIOTICS

The microbial population present in Iambic beer after one year of spontaneous fermentation consists mainly of Brettanomyces yeasts, lactic acid bacteria and acetic acid bacteria. The density of the wort by that time has decreased to around 3.5° Plato. At that time a period of superattentuation is initiated, resulting in a Iambic with sometimes less than 1° Plato. Such old Iambics are used in the production of gueuze. In order to find out which organisms are really necessary for this process. Iambic attenuated to around 3.5° Plato was pasteurized and re-inoculated with a mixed microbial population obtained from fermenting Iambic. By the addition of the antibiotics actidione, pimaricin, gentamycin, oxytetracycline and nisin it was found that Brettanomyces was the main organism responsible for superattenuation, although this was less pronounced when Pediococcus was absent. Acetic acid bacteria were not involved. Bacteria alone were not really superattenuating. The process with Iambic wort which had reached the 3.5° Plato value by a natural spontaneous fermentation was slower than with a Iambic wort pre-fermented to 3.5° Plato with S. cerevisiae. It was found that Brettanomyces but not Saccharomyces survives well under the conditions normally found for a 1 year old Iambic.     

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.     

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.     

Influence of temperature on continuous high gravity brewing with yeasts immobilized on spent grains

Flavor compounds’ formation and fermentative parameters of continuous high gravity brewing with yeasts immobilized on spent grains were evaluated at three different temperatures (7, 10 and 15 °C). The assays were performed in a bubble column reactor at constant dilution rate (0.05 h⁻¹) and total gas flow rate (240 ml/min of CO₂ and 10 ml/min of air), with high-gravity all-malt wort (15°Plato). The results revealed that as the fermentation temperature was increased from 7 to 15 °C, the apparent and real degrees of fermentation, rate of extract consumption, ethanol volumetric productivity and consumption of free amino nitrogen (FAN) increased. In addition, beer produced at 15 °C presented a higher alcohols to esters ratio (2.2–2.4:1) similar to the optimum values described in the literature. It was thus concluded that primary high-gravity (15°Plato) all-malt wort fermentation by continuous process with yeasts immobilized on spent grains, can be carried out with a good performance at 15 °C.     

Effects of β‐Glucans and Environmental Factors on the Viscosities of Wort and Beer

This paper reports on the influence of molecular weight and concentration of barley β‐glucans on the rheological properties of wort and beer. Environmental conditions such as pH, maltose level in wort, ethanol content of beer, shearing and shearing temperature were also examined for their effects on wort and beer viscosities. In the range of 50–1000 mg/L, β‐glucans increased solution viscosity linearly with both molecular weights (MW) of 31, 137, 250, 327, and 443 kDa and concentration. The influence of MW on the intrinsic viscosity of β‐glucans followed the Mark‐Houwink relationship. Shearing wort and beer at approximately 13,000 s^?1for 35 s was found to increase the wort viscosity but reduce beer viscosity. Shearing wort at 20°C influenced β‐glucan viscosity more than shearing at 48°C and 76°C whereas the shearing temperature (0, 5 and 10°C) did not effect the viscosity of beer. At lower pHs, shearing was found to reduce the viscosity caused by β‐glucans in wort but had no effect in beer. Higher concentrations of maltose in wort and ethanol in beer also increased the viscosity of β‐glucan polymers. It was found that β‐glucans had higher intrinsic viscosities in beer than in wort (5°C), and lower critical overlap concentrations (C^*) in beer than in wort.     

Differences in protein content and foaming properties of cloudy beers based on wheat malt content

To investigate differences in protein content, all barley malt beer, wheat/barley malt beer and all wheat malt beer were brewed, and the protein during mashing, wort, fermentation and beer determined. It was shown that protein was mainly extracted during mashing and the protein rest phase, decreased in the early stages of fermentation and remained almost steady during wort boiling and cooling, in the middle and late stages of fermentation. By separating beer foam from beer, similar protein bands of 51.7, 40.0, 27.3, 14.8, 6.5 and < 6.5 kDa appeared in the three beers, defoamed beers and beer foams using the sodium dodecyl sulphate polyacrylamide gel electrophoresis. Quantitatively, protein bands of 6.5–14.8 and <6.5 kDa had the highest contents in the three beers. Unique bands at 34, 29.2, 23.0, 19.7 and 17.7 kDa were found in beer, defoamed beer and beer foam from wheat beer and all‐wheat malt beer, respectively. Wheat beer foam showed the best foam stability and the protein in all barley malt beer showed the best migration to the foam. The beer foam properties were influenced by not only protein content but also protein characteristics and/or origin. It is suggested that the barley malt contributed the beer foam ‘skeleton protein’ while protein components from wheat malt kept the foam stable. © 2018 The Institute of Brewing & Distilling     

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.     

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.     

Structural characterisation and antioxidant activity of melanoidins from high-temperature fermented apple

High-temperature fermented apple is a new fermented product with characteristic black appearance, which is prepared through natural fermentation of whole fresh apple at controlled high temperature (60–90 °C) and humidity (relative humidity of 50–90%). In this work, melanoidins from high-temperature fermented apple were obtained by ultrafiltration and subsequent diafiltration. The structural characterisation of melanoidins was investigated in the present study. Results showed that the molecular weight of the obtained melanoidins was about 179 kDa and the melanoidins were comprised primarily of furans by pyrolysis–gas chromatography–mass spectrometry. Antioxidant activities of the high-temperature fermented apple and its melanoidins were evaluated by ABTS, DPPH, hydroxyl radicals, ferric reducing antioxidant power and Fe²⁺-chelating assays. Both high-temperature fermented apple and its melanoidins showed extremely high antioxidant activities in scavenging hydroxyl radicals. High correlation between the antioxidant activity assayed methods and the physicochemical characteristics of melanoidins was obtained. The contribution of melanoidins to the total antioxidant capacity of high-temperature fermented apple was determined.     

Amino Acid Profiles of Fermenting Wheat Sour Doughs

The free amino acid patterns of unfermented and fermented wheat sour doughs started with Lactobacillus plantarum were related by factor analysis to dough yield, ash content of flour, fermentation temperature and presence/absence of yeast, from data corresponding to a central composite design of samples. Individual levels of hydrophobic, acid and basic amino acids as well as total amino acid content positively correlated with extraction rate of flours. The presence of yeast promoted metabolism of histidine, aspartic acid and asparagine, particularly in samples made with whole and wholemeal flours. Proliferation of predominant amino acids by enzymatic release was more notable at higher fermentation temperatures and wholemeal samples. Sour doughs with maximum levels of hydrophobic and basic amino acids were started with no yeast bacterial cultures, made with whole and wholemeal flours and fermented at 35°C.