Characterization of the yeast ecosystem in grape must and wine using real-time PCR

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.     

β-Glucanases as a Tool for the Control of Wine Spoilage Yeasts

ABSTRACT:  In this study, the antimicrobial activity of a commercial β-glucanase preparation against wine spoilage yeasts such as  Cryptococcus albidus ,  Dekkera bruxellensis ,  Pichia membranifaciens ,  Saccharomyces cerevisiae ,  Zygosaccharomyces bailii , and  Zygosaccharomyces bisporus  has been evaluated. Yeast species tested showed different sensitivities to the enzyme preparation.  In vitro  assays in laboratory medium (GPY) showed inhibition by the β-glucanase preparation of  D. bruxellensis  and  Z. bailii  growth with IC₅₀ and MIC values approximately 3 to 4-fold greater than the recommended dose for improving wine filtration. Wine spoilage experiments showed antimicrobial action against  D. bruxellensis  and  Z. bailii  although with reduced effectiveness to that showed in laboratory medium. Under the conditions tested, the addition of β-glucanase did not affect wine enological parameters. Our data suggest the potential use of β-glucanases as antimicrobial agents in wine and indicate that the application of antimicrobial enzymes for yeast spoilage control deserves further investigation.     

RAPID DETERMINATION OF THE PURITY OF YEAST CULTURES BY IMMUNOFLUORESCENCE AND FLOW CYTOMETRY

Flow cytometry has been shown to be capable of quantifying the fluorescence response of wild yeasts detected by immunofluorescence techniques.     

Sulphite binding in ciders

Summary The extent of sulphite binding was measured in commercial ciders, and experimentally observed binding curves were compared with theoretically derived curves based on assessment of the levels of individual sulphite binding compounds determined in the ciders. Subsequently, experimental ciders were fermented under various controlled conditions using nine different strains of cider yeasts. The results indicated considerable differences both in the levels of sulphite binding compounds produced, and in the ability of different yeast strains to produce SO₂ in the cider. Juice concentration and the presence of cloud in juice had little or no effect on sulphite binding. Other factors which affected sulphite binding included the type and condition of juice (especially the effect of pectinase treatment) and, in some instances, the use of added yeast nutrients. Significant sulphite binding was also attributable to unaccountable components, probably derived from poor quality fruit, which were present in the apple juice prior to fermentation.     

废啤酒酵母制取酵母抽提物的酶促工艺研究

为了获得废啤酒酵母制取酵母抽提物的最佳工艺条件,通过单因素试验获得外加酶法制取酵母抽提物的最佳工艺条件,并比较其与传统工艺条件的效果;使用GC—MS对最终产物进行风味分析。通过优化得到最佳工艺条件为添加中性蛋白酶,外加酶0.20%,pH 6.0,酶促溶时间36h,得到的该抽提物中含有多种酸类、酯类和醇类等物质。研究表明,外加中性蛋白酶法是开发利用啤酒废酵母制备酵母提取物的较好方法,得到的产物营养丰富。     

RELATIONSHIP BETWEEN YEAST CELL PROLIFERATION AND INTRACELLULAR ESTERASE ACTIVITY DURING BREWING FERMENTATIONS

Flow cytometry was used for the determination of the intracellular esterase activity of unstressed and stressed Saccharomyces cerevisiae cells using fluorescein diacetate as substrate during brewing fermentations in EBC tubes. The determination of intracellular esterase activity by flow cytometry was compared with in vitro assays for determination of yeast esterase activity. The method was regarded valid with a high degree of reproducibility. Intracellular esterase activity during brewing fermentations was dependent on the yeast strain applied but independent of the wort compositions applied within this study. Further, the intracellular esterase activity during fermentation was correlated with cell proliferation determined by DNA staining and flow cytometry and by calculating the percentage of G1-phase cells. Yeast esterase activity in both unstressed and ethanol stressed cells followed a similar pattern during brewing fermentations. Furthermore, this pattern could be correlated with the percentage of G1-phase cells during fermentation indicating that the esterase activity was in some way related to cell cycle progression .     

The Use of Fatty Acid and Sterol Analyses as Quality Control Methods in the Brewing Industry

Fatty acid and sterol analyses were evaluated as alternative quality control methods to conventional differentiation and characterisation systems in the brewing industry. The presence of lino‐leic acid (18:2) in brewing yeast could be used to distinguish these from closely related yeast species. Furthermore, the absence of lanosterol and stigmasterol enabled differentiation of the brewing yeast from the rest of the closely related species tested. However, both fatty acid and sterol methods were not sensitive enough to detect mutants (variants) of brewing yeast. Conventional brewing identification tests proved sensitive enough to detect variants at low concentrations.     

ANTIGENIC STRUCTURE OF BREWING YEASTS AND ITS RELATIONSHIP TO NON-SEROLOGICAL METHODS OF CLASSIFICATION

Antigen extracts of 43 brewing strains of Saccharomyces cerevisiae were examined using micro immuno-electrophoresis. The data obtained was analysed by numerical taxonomic techniques and a serological classification of brewing yeasts was constructed based on the results. This classification is compatible with our earlier taxonomic study based on characters of practical brewing importance.⁵ Significant relationships were observed between certain antigens and the fermentation properties of head formation and deposit formation.     

The microbiology of cocoa fermentation and its role in chocolate quality

The first stage of chocolate production consists of a natural, seven-day microbial fermentation of the pectinaceous pulp surrounding beans of the tree Theobroma cacao. There is a microbial succession of a wide range of yeasts, lactic-acid, and acetic-acid bacteria during which high temperatures of up to 50 degrees C and microbial products, such as ethanol, lactic acid, and acetic acid, kill the beans and cause production of flavor precursors. Over-fermentation leads to a rise in bacilli and filamentous fungi that can cause off-flavors. The physiological roles of the predominant micro-organisms are now reasonably well understood and the crucial importance of a well-ordered microbial succession in cocoa aroma has been established. It has been possible to use a synthetic microbial cocktail inoculum of just 5 species, including members of the 3 principal groups, to mimic the natural fermentation process and yield good quality chocolate. Reduction of the amount of pectin by physical or mechanical means can also lead to an improved fermentation in reduced time and the juice can be used as a high-value byproduct. To improve the quality of the processed beans, more research is needed on pectinase production by yeasts, better depulping, fermenter design, and the use of starter cultures.