FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

啤酒酵母的基因改良研究动态

近年来 ,利用基因工程进行酵母的育种在发酵广谱碳水化合物、提高糖化效率 ,改良酵母凝聚特性和改善啤酒风味方面取得了很大成绩。基因重组菌株将逐步应用到生产实践中。     

Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers

The burgeoning interest in archaic, traditional, and novel beer styles has coincided with a growing appreciation of the role of yeasts in determining beer character as well as a better understanding of the ecology and biogeography of yeasts. Multiple studies in recent years have highlighted the potential of wild Saccharomyces and non-Saccharomyces yeasts for production of beers with novel flavour profiles and other desirable properties. Yeasts isolated from spontaneously fermented beers as well as from other food systems (wine, bread, and kombucha) have shown promise for brewing application, and there is evidence that such cross-system transfers have occurred naturally in the past. We review here the available literature pertaining to the use of nonconventional yeasts in brewing, with a focus on the origins of these yeasts, including methods of isolation. Practical aspects of utilizing nondomesticated yeasts are discussed, and modern methods to facilitate discovery of yeasts with brewing potential are highlighted.     

西藏传统青稞酒酿造用藏曲中主要酵母菌的分离及酿造特性研究

采用WL酵母形态鉴别培养基从不同地域来源藏曲中分离筛选出48株典型酵母形态菌株,进一步通过分子鉴定为酿酒酵母(Saccharomyces cerevisiae)31株,扣囊复膜孢酵母(Saccharomycopsis fibuliger)9株,Saccharomycopsis malanga 3株。通过青稞汁培养基发酵研究了不同种类酵母的酿造性能,发现酿酒酵母是青稞酒发酵主要产酒微生物,S.malanga是主要产酸微生物。通过26种香气代谢物的分析比较3种酵母产香性能发现酿酒酵母是青稞酒发酵主要产香微生物,能够产丰富的酯类、醇类和有机酸类香气物质,扣囊复膜孢酵母也对青稞酒中微量香气物质具有重要贡献,而S.malanga产香能力较弱。该文系统研究了西藏传统酿造青稞酒酿造用藏曲中主要酵母菌的酿造性能和风味功能,对青稞酒酿造风味品质的科学控制提供了依据。     

Two-dimensional gel analysis of the proteome of lager brewing yeasts

Modern lager brewing yeasts used in beer production are hybrid strains consisting of at least two different genomes. To obtain information on the identity of the parental strains that gave rise to industrial lager yeasts, we used two-dimensional (2-D) gel electrophoresis and analysed the proteomes of different Saccharomyces species isolated from breweries. We found that the proteome of lager brewing yeasts and of the type strains of S. carlsbergensis, S. monacensis and S. pastorianus can be interpreted as the superimposition of two elementary patterns. One originates from proteins encoded by a S. cerevisiae-like genome. The other corresponds to a divergent Saccharomyces species whose best representative is a particular S. pastorianus strain, NRRL Y-1551. A map of industrial lager brewing yeasts has been established, with the individual origin of proteins and with identification of protein spots by comparison to known S. cerevisiae proteins. This 2-D map can be accessed on the Lager Brewing Yeast Protein Map server through the World Wide Web. This study provides the first example of the use of proteome analysis for investigating taxonomic relationships between divergent yeast species.     

Metabolic footprinting as a tool for discriminating between brewing yeasts

The characterization of industrial yeast strains by examining their metabolic footprints (exometabolomes) was investigated and compared to genome-based discriminatory methods. A group of nine industrial brewing yeasts was studied by comparing their metabolic footprints, genetic fingerprints and comparative genomic hybridization profiles. Metabolic footprinting was carried out by both direct injection mass spectrometry (DIMS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), with data analysed by principal components analysis (PCA) and canonical variates analysis (CVA). The genomic profiles of the nine yeasts were compared by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis and microarray comparative genome hybridizations (CGH). Metabolomic and genomic analysis comparison of the nine brewing yeasts identified metabolomics as a powerful tool in separating genotypically and phenotypically similar strains. For some strains discrimination not achieved genomically was observed metabolomically.     

FLOCCULATION MECHANISMS OF TOP AND BOTTOM FERMENTING BREWING YEAST

The flocculation behaviour of a large set of top and bottom fermenting brewing yeasts was investigated. Bottom and top fermenting strains flocculated according to different mechanisms. Bottom strains flocculated in the stationary growth phase in the presence of sufficiently high Ca²⁺ and sufficiently low sugar concentrations; these strains possessed a lectin-mediated flocculation mechanism. Top strains flocculated in the stationary growth phase without addition of Ca²⁺, only in the presence of sufficiently high concentrations of ethanol. Some of the top strains were inhibited with mannose, but not with sucrose or galactose, while others were not inhibited by any of these sugars. The different sensitivity of flocculation of top and bottom strains with respect to ethanol may be related to the hydrophobicity of the cell surface. Flocculation models for bottom and top fermenting yeasts were proposed. It is suggested that, besides the sugar inhibition pattern, the sensitivity of flocculation with respect to ethanol should be included as an additional parameter for classification of brewing yeasts .     

THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.     

BREWING YEAST IDENTIFICATION AND CHROMOSOME ANALYSIS USING HIGH RESOLUTION CHEF GEL ELECTROPHORESIS

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis has been used to study the karyotypes of a range of Saccharomyces cerevisiae yeast strains. The time required from sampling yeast cultures to CHEF analysis was achieved within six hours, making this procedure very useful in reference and quality control work in the brewing industry. Regions of the chromosome profiles were closely studied by adjusting electrophoresis conditions to increase resolution between bands. Both ale and lager strains of brewing yeasts were studied alongside haploid laboratory strains. By comparing different regions of the profiles even very closely related strains of lager yeast could be distinguished. Brewing strains consistently had significantly more chromosome bands than haploid laboratory strains. The electrophoretic karyotypes of brewing yeasts were represented as groups of bands on CHEF gels which apparently comigrated with their haploid chromosomal counterparts.     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

THE SPORULATION AND MATING OF BREWING YEASTS

A study has been made of the sporulating behaviour of twenty selected brewing strains of yeast, and the mating activity of the products of sporulation. ‘Lager’ yeasts (strains of Saccharomyces carlsbergensis) in general sporulated to a lesser degree and more slowly than ‘ale’ yeasts (strains of Saccharomyces cerevisiae) and produced 1-or 2- spored asci compared with 2-or 3- spored asci for the latter yeasts. Most of the parent strains of S. cerevisiae were shown to be heterozygous for mating type, and they were all probably either triploid or aneuploid. Two of the strains of S. carlsbergensis were apparently homozygous for mating type and also triploid or aneuploid. The compatibility system favours outbreeding of yeasts, ‘ale’ yeasts being more compatible with ‘lager’ yeasts than with other ‘ale’ yeasts.     

2μm DNA PLASMID IN BREWERY YEASTS

Using an agarose gel screening procedure, 2 μm DNA plasmid was detected in all of 10 brewing strains of Saccharomyces yeast examined and in both of two non-brewing, dextrin-utilising strains. Plasmid DNA was identified in yeast grown with access to air in MYGP medium or in hopped wort, and in yeast harvested from 3-day wort fermentations. The yeast plasmid is a suitable self-cloning vector for the genetic manipulation of brewing yeasts by transformation.     

Enhancing the Microbiological Stability of Malt and Beer — A Review

While beer provides a very stable microbiological environment, a few niche microorganisms are capable of growth in malt, wort and beer. Growth of mycotoxin‐producing fungi during malting, production of off‐flavours and development of turbidity in the packaged product due to the growth and metabolic activity of wild yeasts, certain lactic acid bacteria (LAB) and anaerobic Gram negative bacteria, impact negatively on beer quality. It follows that any means by which microbial contamination can be reduced or controlled would be of great economic interest to the brewing industry and would serve the public interest. There has been an increasing effort to develop novel approaches to minimal processing, such as the exploitation of inhibitory components natural to raw materials, to enhance the microbiological stability of beer. LAB species, which occur as part of the natural barley microbiota, persist during malting and mashing, and can play a positive role in the beer‐manufacturing process by their contribution to wort bioacidification or the elimination of undesirable microorganisms. Other naturally occurring components of beer that have been valued for their preservative properties are hop compounds. It may be possible to enhance the antimicrobial activities of these compounds during brewing. Some yeast strains produce and excrete extracellular toxins called zymocins, which are lethal to sensitive yeast strains. Yeast strains resistant to zymocins have been constructed. Imparting zymocinogenic activity to brewing yeast would offer a defence against wild yeasts in the brewery. Thus, the antimicrobial properties of naturally occurring components of raw materials can be exploited to enhance the microbial stability of beer.     

APPLYING YEAST GENETICS IN BREWING. A CURRENT ASSESSMENT

The challenges for the yeast geneticist in brewing are threefold: (i) to monitor and control changes in the genetic make-up of brewing yeast strains, (ii) to ‘tailor’ yeast strains to brewing needs and (iii) to use genetics as an approach to understand, and hence better utilise, yeast behaviour. Mutation and selection provide a simple direct means of genetic alteration but the potential for genetic improvement is restricted without recombination of characters from different strains. Sexual production of recombinants by involving the haploid/diploid life cycle has proved to be a difficult and unfruitful task with brewing strains of Saccharomyces. Recently, however, the ‘mating-type barrier’ to recombination has been overcome in novel ways—by use of the ‘rare-mating’ technique, by spheroplast fusion and by transformation. Transformation now offers the greatest potential for the construction of novel or improved brewing strains; when coupled with recombinant DNA technology, highly specific changes in genome can be attempted, thereby avoiding the introduction of undesirable characteristics or the loss of strain stability.     

昌黎产区产酶酵母多样性及其应用潜力分析

利用优选酵母混菌发酵,是改善葡萄酒品质及增香的有效途径。以赖氨酸培养基、WL培养基和产酶筛选培养基为基础,分析了昌黎4个葡萄园土样中产酶酵母的多样性,结果表明,该地区产酶酵母丰富,50%的产酶酵母能同时产2种或2种以上的酶。对产酶酵母酿造因子耐性分析表明,菌株GY1、M9和J24具有高糖耐性,适合于高糖低醇果酒酿造;菌株GY13和M9具有高酒精耐性,酒精含量为15.000%时活性较强;菌株J24和GY1具有高酸耐性,pH 2.5时具有较高活性;7株菌15℃时具有较高活性。综合分析发现,菌株M9(Torulaspora delbrueckii)同时产β-葡萄糖苷酶、果胶酶和纤维素酶,除酸耐性较差外,具有高糖、高酒精度和低温耐性,在高糖低醇果酒酿造中具有很大应用潜力。     

烟台贵人香葡萄自然发酵醪中本土酿酒酵母的选育及酿造特性研究

自葡萄原料生产地选育优良酿造特性的本土酵母是开发地方特色葡萄酒品种的重要措施。本研究以烟台地区贵人香葡萄自然发酵醪为材料,筛选酿酒酵母并对其发酵特点和耐受能力进行测定,进而选育本土酿酒酵母用于霞多丽干白和赤霞珠干红葡萄酒的酿造试验。采用孟加拉红选择性培养基筛选本土酵母菌,并经WL鉴定培养基和5.8S ITS序列鉴定获得12株酿酒酵母。通过测定发酵后残糖量和酒精度及耐受能力(酒精度、SO2、酸、高糖),选育4株酵母用于酿造霞多丽干白葡萄酒,对酒样指标、主要挥发物和香气组分分析,显示菌株YGF2、YGF5和YGF10的酿造特性和发酵指标较好,并用于酿造赤霞珠干红葡萄酒。结果表明菌株YGF2酿造的葡萄酒质量较好,香气浓郁,部分指标优于商品酿酒酵母,具备生产区域特色葡萄酒的潜力。     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

YEAST CLASSIFICATION*

Following a discussion of the need for systems of classification, which are inevitably to some extent subjective, a survey is made of primary and auxiliary diagnostic tests used in yeast classification. The possibilities and limitations of numerical taxonomy are discussed and an assessment is made of the value of current techniques used for identifying strains of brewing yeasts. Methods are described for detecting wild yeasts and for evaluating the suitability of different strains of brewing yeast. Reasons are given for retaining Saccharomyces carlsbergensis as a species in its own right rather than as a synonym of Sacch. uvarum.     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.