FURTHER OBSERVATIONS ON YEAST MUTATION DURING CONTINUOUS FERMENTATION

It has been known for some time that conditions prevailing during continuous cultivation of micro-organisms favour the accumulation of mutant forms, a circumstance that is relevant to the stability of yeast behaviour and of beer quality during continuous fermentation. In an earlier paper the writer reported an investigation of the types of mutant appearing over an extended period of continuous cultivation of a particular strain of flocculent bottom fermenting brewery yeast: loss of flocculence and reduction of fermentation efficiency were the most commonly encountered mutations. Since nothing could be inferred as to the general validity of these results it was thought desirable to repeat the investigation with another strain of a similar type of yeast. While the occurrence of mutants was even more conspicuous than before, the overall picture was in some ways quite different: this strain appears to be less mutable in respect of flocculence, but more so in respect of growth rate, maltotriose fermentability and diacetyl production. The evidence so far available thus suggests that yeast strains are idiosyncratic as regards their mutational tendencies.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

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 .     

嗜杀酿酒酵母对海南山兰甜酒发酵品质的影响

以海南坡稻山兰为主要原料,在传统手工酿造山兰甜酒的工艺基础上添加嗜杀酿酒酵母作为发酵剂能保持山兰甜酒原始的酒醇甘甜微苦风味,维持独特的米脂芳香。控制和净化发酵醪中的酵母品质,在发酵过程中保持酒味纯正,使糖化与发酵得到同步,发酵力得到加强,酒味更加醇厚。同时发酵环境得到净化,发酵时间缩短,生产效率提高。嗜杀酵母发酵剂令山兰甜酒出现苦味物质的时间提前,同时相对传统工艺生成较高浓度的高级醇,可采取定时移除酒液的方法解决。     

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile‐filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture‐dependent methods and PCR‐DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR‐DGGE technique coupled with the culture‐dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by‐products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

IFA在酿酒污染野生酵母快速检测中的应用

用荧光抗体技术进行快速检测,方法简便、灵敏、快速。用三种混合血清可以检测出常见全部污染野生酵母。该技术在工厂中应用结果表明,酿酒种酵母中野生酵母污染现象是存在的,而且随着种酵母使用代数的增加而越趋严重。     

APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

Formation of higher alcohols by wine yeasts,and relationship to taste thresholds

The amounts of n-propanol, iso-butanol and iso-amyl plus active amyl alcohol produced during fermentation of grape juice were found to vary considerably according to the yeast used. The yeasts studied included 11 wine yeasts and one brewery yeast, all belonging to the genus Saccharomyces, and 4 yeasts belonging to genera which sometimes cause wine spoilage. Experiments were carried out in the laboratory and confirmed on pilot-plant scale. The average production of n-propanol in juices from 4 grape varieties varied from 13 to 106 ppm depending on the wine yeast used. The corresponding variations in the production of iso-butanol and isoamyl plus active amyl alcohol were 9 to 37 ppm and 115 to 262 ppm respectively,. Juices from different varieties' of grapes (Vitis vinifera) differed somewhat in the amounts of higher alcohols formed, irrespective of the yeast strain used. An increase in temperature of fermentation from 15° to 25° with four yeasts produced an average of 24% more iso-amyl plus active alcohol and 39% more iso-butanol, and 17% less n-propanol. A yeast/temperature interaction occurred. An increase in pH from 3.0 to 4.2 with four yeasts produced 28% more iso-amyl plus active amyl alcohol, 85% more iso-butanol, and 11% more n-propanol. A yeast /pH interaction occurred. Spoilage yeasts examined also formed higher alcohols and the amounts were not related to sugar consumed during the fermentation. Taste thresholds of the higher alcohols, with the exception of active amyl alcohol, were measured in dry white wine with 7 tasters. Iso-amyl alcohol thresholds ranged from 100 to 900 ppm (mean 300 ppm) and iso-butanol and n-propanol both exceeded 500 ppm. The average threshold of iso-amyl alcohol in distilled water was 4 ppm. For certain tasters strains of yeast can produce sufficient differences in iso-amyl alcohol to be detectable organoleptically, but differences in iso-butanol and n-propanol are not large enough to be detectable.     

FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR CONTINUOUS BEER FERMENTATION: A REVIEW

Recent fundamental research conducted on immobilised cells with a focus on continuous primary beer fermentation is presented in this review. The knowledge of whole-cell immobilisation, continuous fermentation, yeast biochemistry associated with beer flavour production, and bioreactor engineering design is required to apply immobilised yeast cells for industrial scale beer production. Understanding how immobilisation and continuous bioreactor operation affect yeast cell metabolism and viability will provide the groundwork for optimising beer quality. The latest studies on immobilised cell carriers, viability, vitality, mass transfer characteristics and bioreactor design indicate that an industrial scale immobilised cell system for primary beer fermentation may soon be a reality in the modern brewery .     

Biodiversity of brewery yeast strains and their fermentative activities

We investigated the genetic, biochemical, fermentative and physiological characteristics of brewery yeast strains and performed a hierarchical cluster analysis to evaluate their similarity. We used five different ale and lager yeast strains, originating from different European breweries and deposited at the National Collection of Yeast Cultures (UK). Ale and lager strains exhibited different genomic properties, but their assimilation profiles and pyruvate decarboxylase activities corresponded to their species classifications. The activity of another enzyme, succinate dehydrogenase, varied between different brewing strains. Our results confirmed that ATP and glycogen content, and the activity of the key metabolic enzymes succinate dehydrogenase and pyruvate decarboxylase, may be good general indicators of cell viability. However, the genetic properties, physiology and fermentation capacity of different brewery yeasts are unique to individual strains. Copyright © 2014 John Wiley & Sons, Ltd.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

Isolation and Characterization of Shochu Yeasts with Superior Brewing Ability from Shochu Mashes

Seven yeasts with superior ability in terms of alcohol fermentation and aromatic ingredient generation were isolated from 272 wild yeasts obtained from shochu mashes of shochu breweries. These seven yeasts were examined for their fermentation ability with rice and sweet potato using small scale of fermentation tests. Moreover, their thermotolerance was evaluated by growth tests and fermentation tests with barley koji. Among the isolated seven yeasts, the shochu yeast “MF062” was superior in the characteristics of fast fermentation, high alcohol production, aromatic ingredient generation and thermotolerance. It was named the “Heisei Miyazaki Yeast”. From the results of analyses of physiological and genetic characteristics of MF062, it was shown to be a Saccharomyces cerevisiae, but it showed different characteristics from the industrial yeasts used in shochu production. Using a sweet potato shochu brewing test, with 60 kg of raw materials and yeast MF062, it was confirmed that a high alcohol production yield and a high quality shochu could be obtained.     

Population dynamics of yeasts inhabiting bioethanol production with cell recycling

Seeking to understand the dynamics of the yeast population in a bioethanol fermentation process that uses cell recycling, the yeast inhabiting the fermentation tanks throughout the production season were monitored. A total of 26 yeasts were isolated from tanks in a Brazilian bioethanol distillery plant during six different periods of the season. These yeasts were evaluated with regard to fermentative capacity and all yeasts were qualified to be used for bioethanol production. Based on the numerical taxonomy, it was possible to say that they were all representative of Saccharomyces sensu stricto. A total of 10 different banding patterns were obtained from the 16 strains isolated. This work has shown that the yeast introduced at the beginning of the season was quickly replaced by one or more native yeast strains. It was also shown that the replacement of these strains is not always harmful to the process and isolating such yeasts found in the fermentation tanks could be an interesting strategy for new strain selection. Copyright © 2015 The Institute of Brewing & Distilling     

传统四川泡菜中酵母菌的动态变化规律

为研究传统四川泡菜发酵过程中酵母菌的动态变化规律,对传统四川泡菜自然发酵过程中的酵母菌进行分离、鉴定和计数。从自制四川泡菜样品中共分离到5 株不同菌落特点的酵母菌,采用26S rDNA D1/D2区序列分析法鉴定,酿酒酵母（Kazachstania exigua）4 株、膜璞毕赤酵母（Pichia membranefaciens）1 株。结果表明：2 种酵母菌都存在于泡菜发酵前期,初始数量为104～106 CFU/mL,随着发酵进行,泡青菜中2 种酵母菌数量持续减少,泡萝卜中2 种酵母菌数量先增加后减少,泡白菜中没有检测到膜璞毕赤酵母,酿酒酵母数量先增加后减少。2 种酵母菌在泡菜主要发酵时期的第3～5天减少最快,5 d后消失,不同发酵原料和盐水中pH值变化是影响2 种酵母菌动态变化的重要原因。     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

Quality assessment of lager brewery yeast samples and strains using barley malt extracts with anti-yeast activity

Membrane active anti-yeast compounds, such as antimicrobial peptides and proteins, cause yeast membrane damage which is likely to affect yeast vitality and fermentation performance, parameters which are notoriously difficult to analyse. In this work the sensitivity of lager brewery yeast strains towards barley malt extracts with anti-yeast activity was assessed with an optimised assay. It was found that yeast, obtained directly from a brewery, was much more sensitive towards the malt extracts than the same yeast strain propagated in the laboratory. Sensitivity to the malt extracts increased during the course of a laboratory scale fermentation when inoculated with brewery yeast. As the assay was able to differentiate yeast samples with different histories, it shows promise as a yeast quality assay measuring the yeast's ability to withstand stress which can be equated to vitality. The assay was also able to differentiate between different lager yeast strains of Saccharomyces cerevisiae propagated in the laboratory when challenged with a number of malt extracts of varying anti-yeast activity. The assessment of yeast strains in the presence of malt extracts will lead to the identification of yeast strains with improved quality/vitality that can withstand malt-associated anti-yeast activity during brewery fermentations.     

The Effects of Osmotic Pressure and Ethanol on Yeast Viability and Morphology

The selection of a brewing yeast strain with the required fermentation and recycling characteristics is critical. The yeast strain will influence the rate and extent of fermentation, the flavour characteristics and the overall quality and stability of the finished beer, and consequently, the economic viability of the brewery. Since high gravity worts can have a deleterious effect on yeast fermentation performance, it is imperative that the strain selected be suitable for this environment, which includes a capacity to withstand high osmotic pressures and elevated ethanol levels. Under controlled in vitro osmotic and ethanol induced stresses, there was a decline in mean cell volume in both lager and ale yeast strains. Whilst significant reductions in viability were observed in the lager strains, the ale strains studied were not affected. Cell surface investigations revealed shrinkage of the yeast cells and crenation of the outside envelope under both stresses, although exposure to ethanol had a more marked effect on the yeast cell surface than sorbitol‐induced elevated osmotic pressure.