低双乙酰酿酒酵母的DNA标记研究

对15株酿酒酵母菌株的外观发酵度、双乙酰生成和还原能力进行了比较,通过随机扩增多态性DNA(RAPD)和HSP150编码基因的长度多态性对不同酿酒酵母菌株的基因组DNA分子差异进行了分析。结果显示,菌株YZU-APK和SS-05的外观发酵度分别为77.2%和76.8%,双乙酰峰值分别为0.26mg/L和0.25mg/L,并在发酵第8d均降至0.09 mg/L。在46条随机引物中筛选出的2条引物P07和P42能够扩增出具有较好多态性的RAPD指纹图谱,可以将15个酵母菌株分成9个遗传型;对细胞壁热休克蛋白HSP150编码基因的PCR扩增,并根据扩增条带长度的不同可以将15株酵母分成6个遗传型。综合2种分析结果,并对低双乙酰酿酒酵母菌株YZU-APK和SS-05进行DNA分子标记,确定了其基因型分别是(P07:1,P42:1,hsp150:1)和(P07:1,P42:5,hsp150:2)。     

酿造条件对酿酒酵母发酵香气的影响

采用顶空固相微萃取结合气相色谱-质谱联用技术,比较不同酿酒酵母菌株（CY3079、LA-FR、LA-RA、MST、OFC）在模拟葡萄汁中发酵产香性能,并分析不同酿造条件下各类发酵香气的变化规律。结果表明：LA-FR酵母菌株产酯类、萜烯类物质能力最强,产香性能最好,因而选用LA-FR酵母菌株研究氮源质量浓度、发酵温度、pH值、SO2添加量对各类香气物质的影响。当氮源质量浓度从1.0 g/L增加至2.0 g/L时,LA-FR菌株所产生的醇类、萜烯类物质含量分别降低36.41%和42.56%,酯类、酸类物质含量分别增加72.92%和26.86%;当发酵温度在18～28 ℃范围内变化时,较低温度发酵有利于酯类、萜烯类物质的积累,但不利于醇类、酸类物质的合成;当pH值从3.2升高至3.8时,酯类、萜烯类含量分别增加19.50%和67.43%,醇类、酸类含量分别降低30.24%和34.16%;SO2添加量与醇类、酯类合成量呈正相关,与酸类、萜烯类合成量呈负相关。多元回归分析结果表明,氮源质量浓度对醇类和酯类香气含量影响最大,发酵温度对酸类物质含量影响最大,SO2添加量对萜烯类香气含量影响最大,该研究结果可为深入研究复合因素对发酵香气的影响及葡萄酒香气风味调控提供理论依据。     

低双乙酰啤酒酵母激光诱变条件的研究

以激光为诱变剂照射啤酒酵母，得到1株双乙酰值低的啤酒酵母。用该菌株酿造的啤酒，发酵液中双乙酰含量为0．0836mg／L，比用原菌株酿造的啤酒下降了53％。激光诱变的条件为：波长623．8nm(红色)、功率6mW的He—Ne激光，用滤纸片法照射啤酒酵母，照射时间为5min。     

固定化后期添加啤酒酵母菌降低啤酒中双乙酰含量的探讨

为探索用后期添加固定化酵母菌降低啤酒中双乙酰的工艺,通过单因素和正交试验,发现后期接入固定化啤酒酵母菌降低双乙酰含量的最佳工艺为:发酵温度为12℃,接入时间为发酵第16d,发酵再次接入固定化酵母菌的接种量为1.0%,此时双乙酰的量为0.083mg/L,啤酒口感达到9.2分。固定化的酵母菌可以重复利用3次,啤酒的双乙酰值和口感维持稳定。该研究表明利用固定化细胞的后期添加可以降低啤酒中的双乙酰含量。     

The chromosomal constitution of wine strains of Saccharomyces cerevisiae

A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial-laboratory strain hybrids. The multiply auxotrophic haploid testers used carried a dominant erythromycin-resistance marker, allowing hybrids to be selected in mass matings with spores produced by the wild-type industrial strains. Analysis of a number of independent crosses between the haploid testers and an unselected population of spores of each wine strain distinguished between disomic, trisomic and tetrasomic chromosomal complements in the parents. Possible explanations for a significant class of aberrant segregation frequencies are discussed. Results of the analysis indicate that UCD Enology 522 (Montrachet) is diploid and possibly trisomic for chromosome VII; 522X is diploid; UCD Enology 505 (California Champagne) is disomic for chromosome XVI, trisomic for chromosomes I, II, III, VI, VIII, IX, X, XII, XV, tetrasomic for chromosomes IV, XI, XIII, XIV and either trisomic or tetrasomic for chromosomes V and VII; and that UCD Enology 595 (Pasteur Champagne) is disomic for chromosomes I, II, III, IX, XVI, trisomic for chromosomes IV, VI, X, XII, XIV, XV, tetrasomic for chromosomes V, VIII, XI, XIII and either disomic or tetrasomic for chromosome VII.     

Systematic analysis of sporulation phenotypes in 624 non-lethal homozygous deletion strains of Saccharomyces cerevisiae

A new high throughput mutant screening procedure for the detection of sporulation mutants was developed and used to analyse a set of 624 non-lethal homozygous deletion mutants created in the European joint research program EUROFAN. The screening procedure involved determination of LL- and DL-dityrosine, sporulation-specific compounds, which were shown to be robust markers of the extent and arrest stage of sporulation mutants. Secondary screens consisted of light microscopy to detect mature and immature spores and DAPI staining to monitor the progress of meiotic nuclear divisions. We discovered new phenotypic classes of mutants defective in spore wall synthesis that were not discovered by previous screens for sporulation mutants. The genes corresponding to the sporulation mutants fell in several functional classes, some of which were previously unknown to be involved in spore formation. Peroxisomes seem to play a role in spore wall synthesis. Mitochondria play a role in sporulation that is not simply restricted to supply of ATP from respiratory metabolism. The deletion mutants included in the set were functionally unknown at the start of EUROFAN; however, within the last few years the importance to sporulation of some of them was also reported by other authors. Taken together, about 8% of all single gene deletion mutants of non-essential genes of Saccharomyces cerevisiae seem to display a clear and reproducible sporulation phenotype.     

紫外诱变筛选低高级醇和双乙酰含量的啤酒酵母

根据酵母相关代谢途径，将降低高级醇和双乙酰含量为目的进行育种。采用紫外诱变方法对啤酒酵母进行处理，用乳酸平板、麦芽汁-碳酸钙平板、TTC上层平板进行显色分离，最终得到1株高级醇产量降低约30％、双乙酰含量亦较低的新菌株。     

Detection method for polygalacturonase-producing strains of Saccharomyces cerevisiae

In the presence of glycerol or ethanol, SCPP (a strain of Saccharomyces cerevisiae that expresses pectinolytic activity) is capable of utilizing galacturonic acid or pectins for growth purposes. We now establish a relationship between the pectinolytic power of various strains of S. cerevisiae and their ability to grow on a pectin/glycerol-based medium. This property is further exploited for the detection of polygalacturonase-producing strains of S. cerevisiae.     

Relatedness of medically important strains of Saccharomyces cerevisiae as revealed by phylogenetics and metabolomics

Ten medically important Saccharomyces strains, comprising six clinical isolates of Saccharomyces cerevisiae and four probiotic strains of Saccharomyces boulardii, were characterized at the genetic and metabolic level and compared with non-medical, commercial yeast strains used in baking and wine-making. Strains were compared by genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis, by ribosomal DNA ITS1 sequencing and by metabolic footprinting using both direct injection mass spectrometry (DIMS) and gas chromatography-time of flight-mass spectrometry (GC-ToF-MS). Overall, the clinical isolates fell into different groupings when compared with the non-medical strains, with good but not perfect correlation amongst strains at both the genetic and metabolic levels. Probiotic strains of S. boulardii that are used therapeutically to treat human gastro-intestinal tract disorders showed tight clustering both genetically and metabolically. Metabolomics was found to be of value both as a taxonomic tool and as a means to investigate anomalous links between genotype and phenotype. Key discriminatory metabolites were identified when comparing the three main groups of clinical, probiotic and non-medical strains and included molecules such as trehalose, myo-inositol, lactic acid, fumaric acid and glycerol 3-phosphate. This study confirmed the link between a subset of clinical isolates and baking or probiotic strains but also highlighted that in general the clinical strains were more diverse at both the genomic and metabolic levels.     

The effects of grape must fermentation conditions on volatile alcohols and esters formed by Saccharomyces cerevisiae

Changes in aroma compounds synthesised from grape must during fermentation carried out by Saccharomyces cerevisiae, in semi-aerobic, anaerobic, short aeration conditions and after adding ergosterol and oleic acid to the must were studied. The biosynthesis of these aroma compounds was strongly dependent on the fermentation conditions and on the growth of the yeast. Ethanol, isoamyl alcohols, isobutyl alcohol, phenethyl alcohol and isoamyl, butyl and hexyl acetates were produced in greater concentrations in semiaerobic conditions, mainly during cellular growth. 1-Butanol and 1-pentanol were produced in greater levels in anaerobic conditions, when cellular growth was lower. Ergosterol and oleic acid added to the musts generally increased the levels of the aroma compounds in wine compared to those obtained in anaerobic conditions. © 1997 SCI.     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

Endomitotic diploidization of Saccharomyces cerevisiae by heat treatment during spore germination

Diploid cells with ability to mate, hereafter referred to as diploid mater cells, were obtained at significant frequencies by the heat treatment of haploid spores at the early germination stage in Saccharomyces cerevisiae heterothallic strain CG5M ( a /α diploid cells heterozygous for five auxotrophic markers). The highest frequency (ca. 11%) of diploidization was obtained from viable cells after heat treatment at 55°C for 10 min when spores were precultivated for 30 min in liquid medium to initiate the germination. The diploid mater cells obtained were homozygous for mating type and for the auxotrophic markers. The diploidization of a spore is thus concluded to be due to endomitotic events in germinating heat-treated spores.     

An improved method for whole protein extraction from yeast Saccharomyces cerevisiae

A new method for protein extraction from yeast Saccharomyces cerevisiae cells is described. This method involves the use of LiAc and NaOH to enhance the permeability of yeast cell wall prior to protein extraction with SDS-PAGE sample buffer. It was safe and efficient compared to other methods reported so far in the literature. The proteins extracted with this new method retained their immunoreactive properties and are suitable for most applications in molecular biology studies.     

不同酿酒酵母发酵特性及抗氧化特性的比较

该文比较了不同来源的5株葡萄酒酿酒酵母(2株野生酿酒酵母CC17、SC5,2株实验室常用工程菌株BY4743、INVSC1,以及1株工业酿酒酵母菌株CICC1450)的生长曲线、泡沫产生能力、H2S产生能力、CO2产生能力,结果表明该实验室分离得到的野生酿酒酵母CC17具有良好的发酵特性.经不同浓度的氧化性物质H2O2和甲萘醌分别处理后得知,CC17在抗氧化性能上具有明显优势,说明该菌株在实际生产中具有开发利用的潜力.     

Fatty acid-containing lipids of the yeast Saccharomyces cerevisiae during post-fermentation decline in viability

On exhaustion of the sugar supply in the medium, cells of the yeast Saccharomyces cerevisiae maintained at 30°C lost most of their phospholipid content and their viability, as assessed by methylene blue staining, within a few days. Both processes occurred more rapidly in cells which had fermented glucose rather than maltose. During this period of decline the cell content of other fatty acid esters, mainly t riacylglycerols, increased. This seemed greater in cells grown with maltose than with glucose but the latter cells also synthesised distinct amounts of waxes. The total quantity of lipid per cell did not change significantly during loss of viability although the composition of the lipid fraction did.     

不同酿酒酵母发酵对干红葡萄酒品质的影响

为得到优良的本土酿酒酵母（Saccharomyces cerevisiae）,以商业酿酒酵母（DS、CECA、CEC01）为对照,采用本土优良酿酒酵母WJ1、Q12发酵赤霞珠干红葡萄酒,测定其发酵过程中总酸、残糖、酒精度、花色苷、单宁和总酚含量的变化,并对葡萄酒进行感官评价。结果表明,不同酿酒酵母发酵赤霞珠干红葡萄酒过程中总酸、残糖、酒精度、花色苷、单宁和总酚含量变化趋势一致。本土酿酒酵母WJ1发酵的赤霞珠干红葡萄酒酸度较高,为6.92 g/L,残糖、酒精度、花色苷、单宁和总酚含量分别为3.32 g/L、15.0%vol、282.35 mg/L、204.95 mg/L、3 110.04 mg/L,感官评分为89分,显著高于商业酿酒酵母（P＜0.05）。酿酒酵母Q12发酵的赤霞珠干红葡萄酒酸度、残糖、酒精度分别为3.87 g/L、3.5 g/L、14.8%vol,花色苷、单宁和总酚含量分别为192.22 mg/L、205.01 mg/L、2 215.37 mg/L,感官评分为70分,显著低于商业酿酒酵母（P＜0.05）。因此,本土酿酒酵母WJ1可用作发酵赤霞珠干红葡萄酒的商业酵母。     

New modules for the repeated internal and N-terminal epitope tagging of genes in Saccharomyces cerevisiae

Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.     

Computational approaches for the genetic and phenotypic characterization of a Saccharomyces cerevisiae wine yeast collection

Within this study, we have used a set of computational techniques to relate the genotypes and phenotypes of natural populations of Saccharomyces cerevisiae, using allelic information from 11 microsatellite loci and results from 24 phenotypic tests. A group of 103 strains was obtained from a larger S. cerevisiae winemaking strain collection by clustering with self‐organizing maps. These strains were further characterized regarding their allelic combinations for 11 microsatellites and analysed in phenotypic screens that included taxonomic criteria (carbon and nitrogen assimilation tests, growth at different temperatures) and tests with biotechnological relevance (ethanol resistance, H₂S or aromatic precursors formation). Phenotypic variability was rather high and each strain showed a unique phenotypic profile. The results, expressed as optical density (A₆₄₀) after 22 h of growth, were in agreement with taxonomic data, although with some exceptions, since few strains were capable of consuming arabinose and ribose to a small extent. Based on microsatellite allelic information, naïve Bayesian classifier correctly assigned (AUC = 0.81, p < 10^?8) most of the strains to the vineyard from where they were isolated, despite their close location (50–100 km). We also identified subgroups of strains with similar values of a phenotypic feature and microsatellite allelic pattern (AUC > 0.75). Subgroups were found for strains with low ethanol resistance, growth at 30 °C and growth in media containing galactose, raffinose or urea. The results demonstrate that computational approaches can be used to establish genotype–phenotype relations and to make predictions about a strain's biotechnological potential. Copyright © 2009 John Wiley & Sons, Ltd.