慕萨莱思酵母菌YPD表型多样性及其酿酒酵母δ序列遗传多样性分析

利用YPD稀释涂布法,从新疆阿瓦提地区6个不同采样点46份样液(原料、预处理液,不同时期发酵液)中分离出700株酵母菌,具有15种表型特征。挑选141株酿酒酵母代表株,与7株不同年份酿酒酵母,采用δ序列分析,得到41种基因型,显示阿瓦提慕萨莱思相关酵母菌,在不同采样点之间及同一采样点的原料批次,不同发酵期,不同发酵容器,不同年份之间均存在多样性。     

Interdelta PCR指纹图谱法区分鉴定沙城产区酿酒酵母

运用Interdelta PCR 指纹图谱分析技术,对从沙城产区龙眼葡萄相关的葡萄园土壤、酿酒设备和自然发酵过程中分离得到的54 株酿酒酵母(Saccharomyce cerevisiae)进行亚种水平的区分鉴定,研究不同酿酒酵母的分布和变化规律。结果表明：54 株酿酒酵母产生7 种指纹图谱,代表7 种不同的基因类型,基因型Ⅰ～Ⅶ分别占所分离菌株的31.5%、11.1%、7.4%、42.6%、3.7%、1.85% 和1.85%。自然发酵中后期有4 种基因类型的酵母菌,酿酒设备表面3 种,葡萄园土壤中2 种。通过聚类分析软件处理所得数据作出树形图,可直观地看出亚种第Ⅰ和第Ⅳ的亲缘关系最近,而第Ⅶ与其他亚种的亲缘关系最远。     

利用微卫星标记分析酿酒酵母的遗传多样性

为研究本土酿酒酵母的种内多态性,以及本土野生酵母与商业酵母之间的差异性,本实验采用4个微卫星标记（ScAAT1、YOR267C、C11、YPL009C）分析18株商业酵母和28株陕西泾阳分离的野生酿酒酵母的遗传多样性,利用PopGen32进行遗传参数的分析。结果表明：4个位点共检测出66个等位基因,每个位点等位基因数为16～18个;平均多态信息含量0.862 3,均为高多态位点;46株菌表现出39种基因型,分辨率为98.94%;观测杂合度0.478 3～0.658 5。野生酵母和商业酵母均具有丰富的遗传多样性,两个群体各具有自己独特的等位基因,且两者在聚类图中有较清晰的界线。     

葡萄自然发酵过程中酵母菌的研究

目的:筛选性状优良的野生酿酒酵母,对葡萄自然发酵过程中的酵母菌进行分离鉴定,探讨葡萄自然发酵期间酵母菌群的变化.方法:利用WL培养基,对分离自吉林松源的"双红"、"双优"、"赤霞珠"和"黑塞比尔"4个葡萄品种自然发酵液的245株酵母进行初步鉴定,并对其中的120株进行分子鉴定.结果:利用WL培养基可将245株酵母分为6个营养类型.结合酵母菌菌株5.8S-ITS区域的RFLP分析,4个葡萄品种的自然发酵液中共有5种酵母,分别是葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、酿酒酵母(Saccharomyces cerevisiae)、粘红酵(Rhodutorula glutinis)、陆生伊萨酵母(Issatchenkia terricola)和假丝酵母属的Candida sorbosa.结论:自然发酵过程中酵母菌群的比例是不断变化的,"双优"品种的自然发酵液中可能存在性状优良的野生酿酒酵母.     

河北产区优选酿酒酵母的中试研究

将分离自河北产区的3株本土优良葡萄酒酿酒酵母菌株NJ5、N19、NJ17用于葡萄酒中试发酵,考查不同酵母菌株的发酵性能、所发酵葡萄酒的理化特征、多酚类物质含量和挥发性香气成分组成.结果表明,3株本土酿酒酵母具有良好的发酵性能,可生产具有典型地域特征的葡萄酒,有望于用于河北地区特色葡萄酒的生产.     

接种不同嗜杀特性的酿酒酵母对赤霞珠发酵中酵母多样性的影响

以赤霞珠葡萄为原料,分别接种不同嗜杀特性的酿酒酵母(Saccharomyces cerevisiae)菌株NXU17-26(中性)、UCD522(敏感菌株)和UCD2610(嗜杀菌株),并以自然发酵为对照,研究各菌株对赤霞珠葡萄酒的发酵特征及发酵中酵母菌多样性的影响。结果表明,接种发酵在启酵和发酵速度上显著快于自然发酵。WLN培养基将分离到的480株酵母菌鉴定为7种类型,26S rDNA D1/D2序列分析进一步将其鉴定为4属5种:葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、克鲁维毕赤酵母(Pichia kluyveri)、伯顿丝孢毕赤酵母(Hyphopichia burtonii)、S.cerevisiae、库徳毕赤酵母(Pichiakudriavzevii)。这4属5种的酵母均存在于自然发酵中,而接种发酵中仅有H.uvarum和S.cerevisiae两种酵母,接种发酵中酵母菌多样性较低。Interdelta指纹图谱分析表明,所接种的酿酒酵母菌株是相应发酵中的优势菌株:接种中性酵母NXU17-26的发酵中,NXU17-26的基因型占比为63.46%;接种敏感菌株...     

沙城龙眼葡萄园土壤中酵母菌多样性研究

为分离筛选具有产区特色的菌种,本实验采用梯度稀释和划线分离技术,从沙城地区3 个龙眼葡萄园土壤中共分离得到210 株酵母菌。按照形态表型分为9 类,又利用核糖体5.8S rDNA-ITS 区域RFLP 方法将这9 类酵母在分子水平上进行了区分,其中两类酵母为常见酵母,分别为东方伊萨酵母(Issatchenkia orientalis)和热带假丝酵母(Candida tropicalis)。实验证明了该地区土壤中酵母菌的多样性。     

优良本土酿酒酵母的酿造特性研究

开发优良的本土酵母对充分发挥我国葡萄酒的风土特征,改变国外商业酵母垄断的局面具有重要意义。本研究通过小试发酵从11株本土酵母中筛选出2株发酵和产香性能良好的本土酵母60258和60268,应用2个品种的葡萄(白葡萄品种“雷司令”和红葡萄品种“西拉”)进行中试酿造。结果表明:60258可以稳定的高产酯类物质,在“雷司令”和“西拉”中的乙酸酯总量分别是商业酵母的3倍和1.6倍,其乙酯产量是商业酵母乙酯总量的近2倍,可以显著提升葡萄酒的花香和果香风味特征;60268可以高产甘油,更适合提升红葡萄品种的香气品质。2株酵母在实际生产中具有较大的应用前景。     

分离自烟台白玉霓种植园的酿酒酵母发酵特性研究

以烟台白玉霓葡萄种植园分离筛选的3株酿酒酵母为研究对象,在发酵力、产酒精能力、残糖量、产酸量以及耐酒精度、耐SO2、耐酸能力等方面与商品化活性干酵母进行比较分析.结果表明,本土酵母ZYYT-2具有较好发酵能力,产酒精能力强,产酸量适中,并且能耐受16％vol的酒精度、150mg/L的SO2和2.5％的酸,在葡萄酒酿造条件下发酵良好.     

Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae

We describe a new procedure for the generation of plasmids containing a large promoter and terminator region of a gene of interest, useful for gene disruption. In a two-step polymerase chain reaction (PCR), a fragment, corresponding to the terminator and promoter regions separated by a 16 bp sequence containing a rare restriction site (e.g. AscI), is synthesized (T-P fragment). This PCR fragment is cloned in vectors presenting a rare blunt-end cloning site and a yeast marker for selection in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plasmids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approach was used to disrupt three open reading frames identified during the sequencing of COS14–1 from chromosome XIV of S. cerevisiae.     

不同菌龄酿酒酵母细胞壁蛋白差异性分析

以酿酒酵母为研究对象,比较了完整细胞提取法、稀碱缓冲液提取法及溶菌酶和β-葡聚糖酶复合酶法等三种酵母菌细胞壁蛋白提取方法,分析了不同菌龄酵母细胞壁差异性蛋白。结果表明:溶菌酶和β-葡聚糖酶复合酶液水解纯化好细胞壁提取蛋白的方法具有所得胞壁蛋白条带较多,且纯度较高的优点,确定了此方法为提取酵母细胞壁蛋白的最佳提取方法。同时,通过SDS-PAGE电泳分析发现,不同菌龄酵母细胞壁蛋白存在着较大的差异性,并确定了分子质量在36 ku、17 ku和12 ku为不同酵母代数细胞壁的3个主要差异性蛋白,其中36 ku、17 ku处条带蛋白随着菌龄的增加酵母细胞壁蛋白表达量逐渐减少,而12 ku处条带蛋白随着菌龄的增加酵母细胞壁蛋白表达量逐渐增加。     

PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.     

Saccharomyces cerevisiae Biodiversity During the Brewing Process of an Artisanal Beer: A Preliminary Study

In this study we investigated the biodiversity of Saccharomyces cerevisiae during the brewing of an artisanal beer, as well as during its storage in the bottle for 107 days at 20°C. After inoculation with an active dried yeast (ADY), the yeast counts were followed during fermentation and after bottling. Yeast loads remained stable at 10⁶–10⁷ colony forming units (cfu)/mL, and only after day 21, were they were reduced to 10⁴ cfu/mL. After three months in the bottle they spanned 10²–10⁵ cfu/mL. Almost all isolated yeasts were identified as S. cerevisiae and after molecular characterization, unexpected results were obtained. The ADY did not take over the fermentation process and only after 21 days did isolates from the beer share similarities with the inoculated strain. During storage, a high diversity was found, underlining that each bottle developed its own micro‐ecosystem. This study highlighted the necessity for better investigations of S. cerevisiae population dynamics during artisanal brewing. Even when the chemical parameters measured confirmed a correct fermentation process, the inoculated strain was not the main yeast involved in the fermentation and consequently, the final product may have different sensory characteristics from the ones expected by the producers.     

Functional analysis reports. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction

We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.     

Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae

The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.     

23 株酿酒酵母ISSR指纹图谱分析及SCAR标记的建立

目的：构建酿酒酵母菌株的简单重复序列间多态性指纹图谱数据库并建立序列特异性扩增区（sequencecharacterized amplified region,SCAR）标记技术,为酿酒酵母菌株的分类、遗传亲缘关系鉴定及菌种专利保护提供可靠的DNA分子标记技术依据。方法：在简单重复序列间多态性（inter-simple sequence repeat,ISSR）指纹数据分析基础上进行聚类分析并对菌种进行分类鉴定,同时将酿酒酵母菌株9号和15号中扩增获得的ISSR特异性DNA带转化为可以直接用于菌株快速鉴定的SCAR分子标记。结果：构建23 株酿酒酵母的ISSR指纹图谱,并在相似系数为0.85水平上将23 个供试菌株分为3大类,其中,1、2、4、7、15、16、17、19、20、21、23聚为第一类;10、11、12、13、14、18号菌株聚为第二类且10号和11号菌为同一菌株;3、5、6、8、9、22号菌聚为第三类且属于同一菌株。此外,利用所获得的2 个特异性条带成功转化为序列特异性扩增区分子标记。结论：在生产上酿酒酵母菌株遗传背景差异不大,常存在同物异名现象,而采用ISSR指纹及其SCAR分子标记技术快速鉴定酿酒酵母菌株在工业生产上具有重要意义。