内蒙古西部地区霞多丽葡萄自然发酵过程中酵母菌群研究

Wine yeasts in spontaneous fermentation of Chardonnay from West Inner Mongolia

【目的】研究葡萄汁自然发酵过程中酵母菌种类及其数量动态变化,为筛选特色酿酒酵母菌株提供科学依据。【方法】在无菌条件下对内蒙古西部地区霞多丽葡萄汁进行自然发酵,在发酵初、中、后期使用培养基涂布法分离酵母菌株,以WL营养琼脂对分离获得的酵母菌株进行形态学分类分析,采用基于酵母5.8S r RNA-ITS区域的RFLP分析对分类后的酵母菌株进行分子生物学鉴定,统计不同发酵时期发酵液中酵母菌株的种类及数量。【结果】共分离获得312株酵母菌株,分属于5个属的6个酵母菌种,分别是酿酒酵母(Saccharomyces cerevisiae)、浅黄隐球酵母(Cryptococcus flavescens)、葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、异常毕赤酵母(Pichia anomala)、克鲁维毕赤酵母(Pichia kluyveri)和星形假丝酵母(Candida stellata)。在自然发酵初期,葡萄汁有孢汉逊酵母和克鲁维毕赤酵母的数量占据优势,分别占这一时期分离总菌株数的50.69%和25.00%,随着发酵的进行,非酿酒酵母(non-Saccharomyces)菌株的数量均有所减少,酿酒酵母的数量逐渐增加,至发酵后期,发酵液中仅能检测到酿酒酵母。【结论】在内蒙古西部地区葡萄浆果表面野生酵母菌种较丰富,WL营养琼脂形态学分类法结合酵母5.8S r RNA-ITS区域的RFLP分析能够实现对野生酵母菌种的快速分类、鉴定,能实时监测发酵液中酵母菌群的动态变化趋势。

[Objective]The present experiment was conducted to investigate diversity and dynamic change of yeast during spontaneous fermentation of Chardonnay, in order to provide theoretical references for screening native specific yeast strain for wine brewing. [Method]The juice of Chardonnay, collected from West Inner Mongolia, was spontaneously fermented under aseptic condition, the spread plate method was used to isolate yeast strains at the beginning, middle and end of fermentation. Then, WL nutrient agar combined with streak plate method was used to conduct morphological classi- fication of isolated yeast strains, and the molecular biology of classified yeasts was identified by analysis of Restriction Fragment Length Polymorphism（RFLP） based on 5.8S rRNA-ITS region. [Result]The results showed that, a total of 312 isolated yeast strains was classified into 6 species of 5 genera, which were identified as Saccharomyces cerevisiae, Crypto- coccus tlavescens, Hanseniaspora uvarum, Pichia anomala, Pichia kluyved and Candida stellata, respectively. At the be- ginning of spontaneous fermentation, both H. uvarum and P. kluyven were dominated in the Chardonnay juice, and ac- counted for 50.69% and 25.00% of total isolated yeast strains, respectively. With the progress of fermentation, the amount of non-Saccharomyces strains decreased, while the amount of S. cerevisiae gradually increased. Moreover, at the end of fermentation, only S. cerevisiae existed in the fermentation broth. [Conclusion]Based on the above experimental results, it is inferred that there are plenty of wild yeast strains on grape fruit in West Inner Mongolia, and WL nutrient agar combined with RFLP analysis on the basis of 5.8S rRNA-ITS region is feasible in the classification and identification of wild yeasts, and can also be used to monitor dynamic changes of yeasts in the progress of fermentation.