【作者】 徐艳文；

【导师】 刘延琳；

【作者基本信息】 西北农林科技大学 ， 葡萄与葡萄酒学， 2007， 硕士

【摘要】 葡萄酒酿造是一个复杂的微生物学过程,其中由酵母菌主导的酒精发酵是葡萄酒生产最重要的阶段之一。酵母菌的生物多样性及微生物群体组成对葡萄酒的感观质量具有重要影响。葡萄酒相关酵母菌的生物多样性及分类鉴定是选育优良酿酒酵母菌种的基础和关键。而我国葡萄酿酒微生物的生物多样性研究尚未开始,葡萄酒相关酵母菌资源也被长期搁置。我国甘肃武威和高台葡萄产区属冷温带干旱区,正成为有竞争力的优质葡萄酒产区,同时也是未开启的葡萄酒相关酵母菌资源库。本研究以莫高葡萄酒厂和祁连葡萄酒厂的葡萄生产基地的葡萄浆果表面、土壤、设备表面等为分离源分离葡萄酒相关酵母菌。使用WL营养培养基的形态分类、经典分类学方法、26S rDNA-RFLP和26S rDNA D1/D2区和5.8S-ITS区序列分析,对所分离的酵母菌进行分类鉴定,揭示此地区的葡萄酒相关酵母菌的生物多样性。一、利用WL营养培养基进行聚类分析,将采集于莫高酒厂的234株葡萄酒相关酵母菌分为10个培养类型,将采集于祁连酒厂的酵母菌株166株分为7个培养类型,这说明两地的酵母菌种类有较大的差异。二、采用26S rDNA D1/D2区序列分析和5.8S-ITS区序列分析,结果表明莫高酒厂的葡萄酒相关酵母菌分属于Metschnikowia pulcherrima(美极梅奇酵母)、Hanseniaspora uvarum(葡萄有孢汉逊酵母)、Issatchenkia orientalis(东方伊萨酵母)、Pichia fermentans(发酵型毕赤酵母)、Pichia kluyveri var. kluyveri(克鲁维毕赤酵母)、Saccharomyces cerevisiae(酿酒酵母)、Cryptococcus magnu(s大隐球酵母)、Cryptococcus uzbekistanensis、Rhodosporidium kratochvilovae(红冬孢酵母属)、Rhodotorula mucilaginosa 10个种;祁连酒的葡萄酒相关酵母菌分别为Saccharomyces cerevisiae(酿酒酵母)、Issatchenkia orientalis(东方伊萨酵母)、Pichia anomala、Rhodotorula mucilaginosa(胶红酵母)、Cryptococcus magnus(大隐球酵母)、Cryptococcus flavescens(浅黄隐球酵母)、Hanseniaspora uvarum(葡萄汁有孢汉逊酵母)等7个种;三、莫高酒厂葡萄园土壤中的优势菌种为C.magnus和R. mucilaginosa,设备表面以红酵母和H.uvarum为优势菌种。祁连酒厂葡萄浆果表面的优势菌种为H. uvarum和I. orientalis。四、莫高葡萄酒厂在所调查的4个自然发酵过程中,其菌群变化如下:自然发酵初期的葡萄酒相关酵母菌主要有M.pulcherrima、H.uvarum、Pichia kluyveri var. kluyveri、I. orientalis,其中H.uvarum为启动发酵的最主要菌种。自然发酵中期时H.uvarum比例较初期有所下降甚至不存在,而M.pulcherrima较初期有所增长,同时S.cerevisiae也开始生长。此后S.cerevisiae为发酵的主要动力,主导发酵直至结束。五、26S rDNA D1/D2区序列分析表明菌株C248-K与Pichia fermentans的同源性为98.8%,存在7个碱基的差异,菌株C84与Metschnikowia pulcherrima的同源性为98.9%,存在5个碱基差异,这两个菌株的分类地位需要进行进一步研究。更多还原

【Abstract】 Alcoholic fermentation is one of the main phases in wine production. Yeasts play a central role in the fermentation process during winemaking. The diversity and the composition of the yeast micropopulation contribute to the sensory characteristics of wine significantly. The diversity and identification of the wine yeast is the basic of yeast screening and strain-breeding.No reports were found about the wine yeast diversity of our country, and the wine yeast resource has not discovered by far. The Wu-wei and Gao-tai grape ditrict are belong to cool-temperate arid region, which is abundant yeast resources and becoming to the most competitive of quality wine.Yeasts were isolated from these source: winery equipment surfaces, the grape surface and the spontaneous fermentation must of the Qi-lian and the Mo-gao wineries. WL Nutrient Medium culture and physiological observation and phylogenetic analysis were performed to identifify the isolates aming to reveal the diversity and the composition of the yeast micropopulation.1. These yeast stains were classed by WL Nutrient Medium first. There are 10 types of yeast strains isolated from MoGao winery, and the yeast strains isolated from QiLian winey were classed into 7 types. This indicated that there is big differences between the strains from the two defferent wineries.2. Similarity analysis based on 26S rRNA D1/D2 domain sequences and the sequence of 5.8S-ITS region indicated that the isolates from MoGao winery belong to 10 different species: Metschnikowia pulcherrima、Hanseniaspora uvarum、Issatchenkia orientalis、Pichia fermentans、Pichia kluyveri、Saccharomyces cerevisiae、Cryptococcus magnus、Cryptococcus uzbekistanensis、Rhodosporidium kratochvilovae、Rhodotorula mucilaginosa. and the isolates from QiLian winery belong to 7 different species: Saccharomyces cerevisiae、Issatchenkia orientalis、Pichia anomala、Rhodotorula mucilaginosa、Cryptococcus magnus、Cryptococcus flavescens、Hanseniaspora uvarum.3. C.magnus and R. mucilaginosa is the dominant yeast species in the soil of Mo-Gao vineyard, and the surface of equipment is predominanced by Hanseniaspora uvarum. And the surface of grape is predominanced by H. uvarum and I. orientalis in Qi-lian winery. 4. They are four spontaneous fermentation must were studied, and the changes of the wine-related yeast micropopulation is below:The early stage of the alcoholic fermentation are dominated by non-Saccharomyces yeasts such as M.pulcherrima、H.uvarum、Pichia kluyveri var. kluyveri、I. orientalis, and H.uvarum is the main species which started the fermentation. Then H.uvarum bagan to decline at middle stage, and M.pulcherrima together with S.cerevisiae increased comparing to the early stage. Later the non-Saccharomyces yeasts disappeared, and S.cerevisiae conducted the fermention to the end.5. 26S rDNA D1/D2 domain sequences analysis of strain C248-K reveals that it inhabits 98.8% identities with Pichia fermentans, while the strain C84 inhabits 98.9% identities with Metschnikowia pulcherrima. The identification of these two strains need further investigation.更多还原