REGULATION OF AMYLOGLUCOSIDASE PRODUCTION BY SACCHAROMYCES DIASTATICUS

In wort fermentations, production of extracellular amyloglucosidase (AMG) by Saccharomyces diastaticus was greatest, and initiated earliest, when the requirement of yeast for oxygen or unsaturated lipid was incompletely satisfied. During such fermentations, maltotriose disappeared before maltose, glucose, the product of AMG action, accumulated and the wort was inadequately attenuated. In all circumstances examined, commencement of dextrin breakdown coincided with initiation of AMG excretion. Prior to excretion, active enzyme was associated with yeast cells but was located externally to the plasma-membrane. Dextrin or starch was not required to induce AMG production which is initiated, we conclude, when the carbon and energy-source becomes growth-limiting. S. diastaticus strains hydrolysed only a small proportion of wort dextrine and AMG produced in beer was unable to release glucose from pullulan. For production of low carbohydrate beer, strains which (i) synthesise significant amounts of a ‘debranching’ enzyme and (ii) are catabolite-derepressed for AMG production would be advantageous.     

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.     

STRAIN SPECIFIC RESPONSE OF BREWER'S YEAST STRAINS TO ZINC CONCENTRATIONS IN CONVENTIONAL AND HIGH GRAVITY WORTS*

The effect of zinc on a variety of yeast strains is extensively documented in the literature. However, due to the varied experimental protocols employed in each study there is little opportunity to directly compare the strain specificity of this ion. In the present study, the response of six yeast strains (three Saccharomyces cerevisiae (ale type) and three S.cerevisiae (lager type)) to altered zinc concentrations, in both high (1080 OG) and conventional (1048 OG) gravity worts, was investigated. Varying the initial wort zinc concentration in both gravities had an effect on the ethanol production, rate of fermentation, cell number and sugar uptake of all six strains studied. The extent of the response was found to be dependent upon the zinc concentration, the strain employed and wort gravity employed.     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

The Dynamic Changes of Proton Efflux Rate in Saccharomyces pastorianus Strains During High Gravity or Very High Gravity Brewing

The changes in the proton efflux rate (PER) during fermentation of normal gravity (NG), high gravity (HG) and very high gravity (VHG) wort by a lager yeast (Saccharomyces pastorianus) were monitored using an optimized PER test method. The values of the proton efflux rate in S. pastorianus decreased with increasing initial wort gravity. Moreover, the difference in the proton efflux rate values at the beginning of the fermentation was lower than at the end of fermentation from normal gravity to very high gravity brewing. These results demonstrated that the proton efflux rate in S. pastorianus was inhibited in the later stages of high gravity and very high gravity brewing. Furthermore, the changes of the proton efflux rate in S. pastorianus under the high ethanol concentration conditions appeared to depend on the concentration of ethanol in the fermentation liquid. A better negative correlation (P > 0.001, r = ?0.95) between the ethanol concentration at >4% (w/v) and the proton efflux rate was found. The changes of the proton efflux rate in the cells treated with exogenous ethanol confirmed that higher concentrations of ethanol could significantly inhibit proton efflux in S. pastorianus. This study offers a possible way to monitor and explain the performance of yeast in the complex environment of high gravity and very high gravity brewing.     

THE EFFECT OF CARBOHYDRATE ADJUNCTS ON BREWER'S WORT FERMENTATION BY SACCHAROMYCES UVARUM (CARLSBERGENSIS)*

The use of carbohydrate adjuncts such as sucrose, fructose and glucose in brewer's wort significantly modifies the initial wort sugar spectrum and also the pattern of sugar uptake during fermentation by a strain of Saccharomyces uvarum (carlsbergensis). Under these conditions, the concentration of glucose and fructose in the wort was observed to increase when compared to worts in which corn starch was employed as an adjunct and glucose was taken up at a faster rate than fructose. The increase in glucose concentration in the wort also resulted in severe repression of maltose and maltotriose utilization with significant levels of these sugars remaining in the beer produced.     

CHILL PROOFING BY A STRAIN OF SACCHAROMYCES CEREVISIAE SECRETING PROTEOLYTIC ENZYMES

The attempted exploitation of a strain of yeast with extracellular protease activity in the fermentation of brewers' wort is described. S. cerevisiae strain YS01 was previously shown to secrete proteases with activity against a variety of substrates. It is shown here that these included some of the tannin precipitable proteins involved in the formation of chill haze in beer and consequently, more stable beers resulted from incubation with added YS01 cell-free culture supernatants. The proteases were also produced in an active form at low levels during fermentation of brewers' wort using established commercial conditions. Concomitantly, a marginally more stable beer resulted. Respiratory deficient, auxotrophic rare-mating was used to transfer the extracellular proteolytic characteristic (epr1.1) to a commercial ale yeast. Segregants of the recombinants obtained showed great instability in protease secretion and were unsuitable for brewing purposes. The potential advantages and drawbacks to this approach are discussed.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

CHARACTERISATION OF AMYLOLYTIC BREWING YEAST

Amylolytic brewing yeast can be used for the production of low carbohydrate beer and for maximizing fermentation efficiency. In this paper we describe the characterisation of amylolytic brewing yeast in which the STA2 (DEXI) gene, which codes for an extracellular glucoamylase, was cloned under two different promoters; PGK (phosphoglycerate kinase) and GPD1 (sn-glycerol-3-phosphate dehydrogenase) present on episomal plasmids. Both amylolytic strains were shown to ferment and degrade wort as efficiently as the control strain supplemented with an exogenous commercial glucoamylase, despite reduced intracellular glycogen levels (30% of wild-type). However, the nature of the promoter on the expression plasmid was shown to influence both the growth rate of the amylolytic strains and the stability of the plasmids during non-selective growth. One of the strains containing plasmid pDVX4 (GPD promoter) was found to show high levels of stability when tested in ten successive pilot scale (8Hlitre) fermentations.     

Production of Beer with a Genetically Engineered Strain of S. cerevisiae with Modified Beta Glucanase Expression

This study used a recombinant Saccharomyces cerevisiae strain, which expressed both β‐glucanase enzyme and reduced Pro‐teinase A expression during wort fermentations. The genetic stability and fermentation features of the strain were examined. The recombinant strain's proteinase A activity was reduced compared to the parent strain; β‐glucanase was produced throughout the fermentation. The fermentation with the recombinant S. cerevisiae strain exhibited a larger reduction in β‐glucan content than what was observed with the control strain, with β‐glucan degradation above 80%. The foam stability period was reduced when the beer produced by the recombinant S. cerevisiae was stored for 3 months. SDS‐PAGE analysis of the beer proteins indicated that lipid transfer protein 1 had disappeared. Fermentation studies indicated that based on the parameters examined, this recombinant strain was suitable for industrial beer production.     

Evaluation of the potential of commercial non-Saccharomyces yeast strains of Torulaspora delbrueckii and Lachancea thermotolerans in beer fermentation

This work evaluated the potential of three commercial non-Saccharomyces yeast strains Torulaspora delbrueckii (Biodiva and Prelude) and Lachancea thermotolerans (Concerto) for beer fermentation. The fermentation performance, volatile and non-volatile profiles were compared. Growth behaviours of all three yeast strains exhibited similar trends during the initial fermentation phase although a marked population decline was detected in strains Prelude and Concerto, which also showed a rapid utilisation of maltose, while strain Biodiva was unable to consume maltose and consumed lesser amounts of amino acids. Additionally, terpenoids inherently absent in the wort such as β-caryophyllene and geranyl acetone were produced in all beers, significantly higher in beers fermented with strain Prelude. For volatile profiles, Prelude and Concerto produced more ethanol and significantly higher amounts of acetate esters and long-chained ethyl esters. Strain Biodiva, on the other hand, produced higher amounts of isoamyl alcohol and ethyl butanoate.     

高浓高温对啤酒酵母发酵性能的影响

以啤酒酵母S-6为实验菌株,研究了主发酵温度和原麦汁浓度对啤酒发酵的残糖、酒精度、风味物质和絮凝性等性能指标的影响。结果表明,原麦汁浓度一定时,主发酵温度对高级醇和乙酸酯的含量影响较大,主发酵温度由10 ℃提高至16 ℃时,高级醇含量提高了10%～20%,乙酸酯含量提高了8%～16%,但CO2累积质量损失、残糖、酒精度和絮凝性基本不受温度的影响;主发酵温度一定时,原麦汁浓度对酵母絮凝性影响较大,原麦汁浓度越高,酵母絮凝性越低,将高浓（18 °Bx）发酵液稀释50%至常浓（12 °Bx）,残糖、酒精度和高级醇的含量与常浓发酵液基本相同。该研究为选育高温高浓发酵低产高级醇同时强絮凝性酵母菌株提供了重要依据。     

Comparative lactic acid fermentation with five Lactobacillus strains of supernatants made of extruded and saccharified chickpea flour

Glucose-rich supernatants were developed based on the enzymatic hydrolysis of raw and extruded chickpea flours first treated with alcalase® and then with a combination of alpha-amylase and amyloglucosidase or only amyloglucosidase. The optimum treatments were the extruded hydrolysates further treated with amyloglucosidase ‘EF(AMG)’, or the raw chickpea flour treated with a combination of alpha-amylase and amyloglucosidase ‘RF (Amyl+AMG)’. The resulting supernatants were further fermented with five different strains of Lactobacillus and monitored for 24 h in terms of pH, titratable acidity, and microbial counts. Also, the glucose content and oligosaccharides, total protein, and viability were determined. Both supernatants presented a successful fermentation as estimated by the pH drop (<4.2), lactic acid produced (>0.7%), and microbial counts 9 log (CFU mL⁻¹) which increased up to 5 log during the 24 h fermentation. However, the EF(AMG) supernatant induced fewer reductions in glucose and protein contents in comparison to the RF (Amyl+AMG) counterpart. Among bacteria, the strain Lp 29 in EF(AMG) showed the fastest growth rate (K) and the highest residual protein. In all cases, the proposed technology enhanced the growth of five Lactobacillus strains by taking advantage of the hydrolyzed chickpea chemical composition.     

FERMENTATIVE CAPACITY OF YEASTS WHICH METABOLIZE MALTOTRIOSE RAPIDLY

A strain of Saccharomyces carlsbergensis which removes maltotriose particularly rapidly from wort exhibits a very high rate of fermentation of this sugar in isolation. This rate is quickly established after pitching and is maintained throughout the greater part of a batch fermentation despite a rapid decline in the ability of the cells to ferment glucose. This same strain and certain others of the same species which also remove maltotriose readily from wort are able to ferment the trisaccharide as fast as or even faster than maltose after growth on the disaccharide as sole carbon source; this property is usually accompanied by repression of glucose fermentation. Evidence is presented which indicates the presence, in these strains, of two or more α-glucosidases having different relative affinities for the oligosaccharides.     

Impact of buffering capacity on the acidification of wort by brewing‐relevant lactic acid bacteria

Acidified wort produced biologically using lactic acid bacteria (LAB) has application during sour beer production and in breweries adhering to the German purity law (Reinheitsgebot ). LAB cultures, however, suffer from end product inhibition and low pH, leading to inefficient lactic acid (LA) yields. Three brewing‐relevant LAB (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7) were examined during batch fermentation of wort possessing increasing buffering capacities (BC). Bacterial growth was progressively impaired when exposed to higher LA concentrations, ceasing in the pH range of 2.9–3.4. The proteolytic rest (50°C) during mashing was found to be a major factor improving the BC of wort. Both a longer mashing profile and the addition of an external protease increased the BC (1.21 and 1.24, respectively) compared with a control wort (1.18), and a positive, linear correlation (R ² = 0.957) between free amino nitrogen and BC was established. Higher levels of BC led to significant greater LA concentration (up to +24%) after 48 h of fermentation, reaching a maximal value of 11.3 g/L. Even higher LA (maximum 12.8 g/L) could be obtained when external buffers were added to wort, while depletion of micronutrient(s) (monosaccharides, amino acids and/or other unidentified compounds) was suggested as the cause of LAB growth cessation. Overall, a significant improvement in LA production during batch fermentation of wort is possible when BC is improved through mashing and/or inclusion of additives (protease and/or external buffers), with further potential for optimization when strain‐dependent nutritional requirements, e.g. sugar and amino acids, are considered. Copyright © 2017 The Institute of Brewing & Distilling     

Alpha Amino Nitrogen and Fusel Alcohols of Sorghum Worts Fermented into Lager Beer

The levels of alpha amino nitrogen (AAN) and fusel alcohols during fermentations of lager worts produced from waxy sorghum grits either inoculated with yeast cultured in wort or yeast-malt media were performed. Worts produced from waxy sorghum grits had comparable AAN to commercial wort. The oxygen concentration in the reactor headspace changed from 20% at the beginning of fermentation to less than 1% after 72 hrs fermentation indicating a gradual change from aerobic to anaerobic conditions. The utilization of AAN for production of propanol, isobutanol and amyl-isoamyl alcohols from waxy sorghum grits was comparable to a control wort. Production of propanol, isobutanol and amyl-isoamyl alcohols followed the same trend over 144 hr fermentation. Isobutanol was produced in the lowest concentration. The initiation of propanol production occurred after 24 and 36 hr fermentation for worts inoculated with yeast cultured in wort and yeast-malt media, respectively. The final concentration of ethanol and fusel alcohols were within the expected range found in commercial beers. Worts produced from barley malt and waxy sorghum grits were an adequate substrate for Saccharomyces cerevisiae, and were comparable to a commercial wort. The utilisation of refined waxy sorghum grits as brewing adjuncts for lager beers was demonstrated.     

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.     

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.     

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts. The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. Lambic and Gueuze are especially rich in caprylic (C₈) and capric (C₁₀) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of lambic and gueuze.     

THE OCCURRENCE OF TWO GEOMETRICAL ISOMERS OF 2,4,5-TRIMETHYL-1,3-DIOXOLANE IN BEER

Two geometrical isomers of 2,4,5-trimethyl-1,3-dioxolane have been detected in a range of commercial and experimental beers at levels up to ca 0.1 ppm. These compounds, plus a number of other dioxolanes, are present in unboiled wort but are lost by evaporation during wort boiling. The trimethyldioxolanes are then reformed during subsequent fermentation. The flavour threshold of a mixture of the two trimethyldioxolanes was found to be ca 0.9 ppm, at which concentration it produced in beer ‘phenolic’ and ‘astringent/drying’ flavour notes. However, these compounds are not present in sufficiently high concentrations to make a significant contribution to the flavour of beer.