山葡萄自然发酵过程中酵母菌的分离鉴定
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摘要
葡萄酒酵母菌是葡萄酒酿造中最重要的微生物,其多样性和分离鉴定是选育优良酿酒酵母菌种的关键和基础。从东北地区特有山葡萄品种的自然发酵过程中筛选出可以充分展现该品种个性、地域特性的酵母菌,可以帮助酿酒企业酿出风格独特,且有品种典型性的山葡萄酒。
东亚种群的山葡萄,浆果本身酸高、单宁多酚高、干浸出物高、营养成分高、含糖量低、出汁率低、发酵季节温度低的这些特点决定了其酵母菌群与欧亚种的差异及酿造工艺的特殊性。针对这种现状,研究山葡萄相关酵母多样性,分离出特殊的能耐低pH、耐低温的酵母,可以保持山葡萄酒具有独特的风味、营养和资源环境特点且解决经常出现的发酵中止等问题。
从5个山葡萄品种自然发酵过程中分离出的酵母菌中随机选出300株酵母,应用WL培养基对采集菌种进行初步鉴定,同时,利用5.8S-ITS区的PCR/RFLP技术对所有采集菌种进行分子鉴定。所有被鉴定属于酿酒酵母属的菌株可为下一步的菌种筛选,至最终确定适合该产区特定葡萄品种、生态地理条件及工业条件、具有鲜明风格特征的优良酵母菌株提供大量实验基础。
通过本试验得到以下结果:
1.本试验共得到山葡萄酒酵母300株,所有菌株在WL培养基上培养5d所形成的菌落根据其颜色和形态分为4类。
2.根据5.8S-ITS区RFLP酶切图谱,山葡萄酒自然发酵过程中酵母菌的酶切图谱包括11个类型,酶切图谱中有类型5、6、8、9和11可以根据标准图谱鉴定到种,分别为Candida sorbosa、Rhodotorula mucilaginosa、Issatchenkia terricola、Hanseniasporauvarum、Saccharomyces cerevisiae。PCR产物大小范围为450-880bp。
3.本试验从山葡萄浆自然发酵过程共得到300株酵母菌,共得到5个遗传聚类群。
4.试验表明,WL培养基可以区分大多数山葡萄酒自然发酵过程出现的酵母菌,可以用于发酵过程菌群动力学研究和简化分子鉴定的辅助手段。
通过本试验,对东北产区山葡萄酒酵母菌群多样性有了初步的了解,为得到工艺学性状及发酵性状优良的葡萄酿酒酵母菌株提供试验基础。
Wine yeasts were the important microorganism during vinification, Wine yeasts diversity and identification are the foundation for further fine wine yeast selection. The yeasts which from Vitis.amurensis Rupr grape variety northeast region during the spontaneous fermentation can present variety characteristic, and region property, also can help brewaged corporation brewing original and typical Vitis.amurensis Rupr grape wine.
Due to the low total sugar and high acidity Vitis.amurensis Rupr grape possessed and low temperature during fermentation season, So autochthonic yeast must be selected to adapt to these factors and also to solve the stuck fermentation because the ordinary wine yeast can not endure low pH and high acidity must condition. In allusion to this actuality, separated the fine wine yeast from composition of wine yeast populations during spontaneous fermentation and keeped the Vitis.amurensis Rupr wine smell and nutrition was studied.
Five Vitis.amurensis Rupr grapes fermented respectively. 300 wine-related yeasts are isolated at random during spontaneous fermentation, preliminary assessment wine yeasts are carried out by Wallerstein Laboratory Nutrient Agar. At the same time, yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. All of the Saccharomyces strains can provide a great deal of experimental base for using to select fine wine yeast which can endure low pH, high acidity must condition and develop grape berry feature etc.
The results were as follows:
1.300 wine related yeasts were isolated from the Spontaneous fermentation of V.amurensis Rupr. Yeast species assessment was carried out by WL medium. 4 unique colony morphologies were identified.
2.By analysis of the restriction pattern of amplified 5.8S-ITS rRNA gene(RFLP)with the enzyme HinfⅠ, HaeⅢand HhaⅠ, respectively, 11 different genotypes were existed in fermentation. Thereinto, pattern 5、6、8、9 and 11 were respectively Candida sorbosa、Rhodotorula mucilaginosa、Issatchenkia terricola、Hanseniaspora uvarum、Saccharomyces cerevisiae。PCR product sizes vary from 450 to 880 bp.
3.By analysis of the restriction pattern of amplified 5.8S-ITS region(RFLP)5 different hereditary clusterings were found.
4.This work shows Wallerstein Laboratory Nutrient Agar(WL medium)was strengthened for using in wine fermentation to observe yeast populations and useful for predigesting in wine yeast molecular identification.
After this study, the preliminary ideas of Vitis.amurensis Rupr grape wine yeast diversity in northeast China were established. And it was settled a base for the fine wine yeast which could endure low temperature and low pH.
东亚种群的山葡萄,浆果本身酸高、单宁多酚高、干浸出物高、营养成分高、含糖量低、出汁率低、发酵季节温度低的这些特点决定了其酵母菌群与欧亚种的差异及酿造工艺的特殊性。针对这种现状,研究山葡萄相关酵母多样性,分离出特殊的能耐低pH、耐低温的酵母,可以保持山葡萄酒具有独特的风味、营养和资源环境特点且解决经常出现的发酵中止等问题。
从5个山葡萄品种自然发酵过程中分离出的酵母菌中随机选出300株酵母,应用WL培养基对采集菌种进行初步鉴定,同时,利用5.8S-ITS区的PCR/RFLP技术对所有采集菌种进行分子鉴定。所有被鉴定属于酿酒酵母属的菌株可为下一步的菌种筛选,至最终确定适合该产区特定葡萄品种、生态地理条件及工业条件、具有鲜明风格特征的优良酵母菌株提供大量实验基础。
通过本试验得到以下结果:
1.本试验共得到山葡萄酒酵母300株,所有菌株在WL培养基上培养5d所形成的菌落根据其颜色和形态分为4类。
2.根据5.8S-ITS区RFLP酶切图谱,山葡萄酒自然发酵过程中酵母菌的酶切图谱包括11个类型,酶切图谱中有类型5、6、8、9和11可以根据标准图谱鉴定到种,分别为Candida sorbosa、Rhodotorula mucilaginosa、Issatchenkia terricola、Hanseniasporauvarum、Saccharomyces cerevisiae。PCR产物大小范围为450-880bp。
3.本试验从山葡萄浆自然发酵过程共得到300株酵母菌,共得到5个遗传聚类群。
4.试验表明,WL培养基可以区分大多数山葡萄酒自然发酵过程出现的酵母菌,可以用于发酵过程菌群动力学研究和简化分子鉴定的辅助手段。
通过本试验,对东北产区山葡萄酒酵母菌群多样性有了初步的了解,为得到工艺学性状及发酵性状优良的葡萄酿酒酵母菌株提供试验基础。
Wine yeasts were the important microorganism during vinification, Wine yeasts diversity and identification are the foundation for further fine wine yeast selection. The yeasts which from Vitis.amurensis Rupr grape variety northeast region during the spontaneous fermentation can present variety characteristic, and region property, also can help brewaged corporation brewing original and typical Vitis.amurensis Rupr grape wine.
Due to the low total sugar and high acidity Vitis.amurensis Rupr grape possessed and low temperature during fermentation season, So autochthonic yeast must be selected to adapt to these factors and also to solve the stuck fermentation because the ordinary wine yeast can not endure low pH and high acidity must condition. In allusion to this actuality, separated the fine wine yeast from composition of wine yeast populations during spontaneous fermentation and keeped the Vitis.amurensis Rupr wine smell and nutrition was studied.
Five Vitis.amurensis Rupr grapes fermented respectively. 300 wine-related yeasts are isolated at random during spontaneous fermentation, preliminary assessment wine yeasts are carried out by Wallerstein Laboratory Nutrient Agar. At the same time, yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. All of the Saccharomyces strains can provide a great deal of experimental base for using to select fine wine yeast which can endure low pH, high acidity must condition and develop grape berry feature etc.
The results were as follows:
1.300 wine related yeasts were isolated from the Spontaneous fermentation of V.amurensis Rupr. Yeast species assessment was carried out by WL medium. 4 unique colony morphologies were identified.
2.By analysis of the restriction pattern of amplified 5.8S-ITS rRNA gene(RFLP)with the enzyme HinfⅠ, HaeⅢand HhaⅠ, respectively, 11 different genotypes were existed in fermentation. Thereinto, pattern 5、6、8、9 and 11 were respectively Candida sorbosa、Rhodotorula mucilaginosa、Issatchenkia terricola、Hanseniaspora uvarum、Saccharomyces cerevisiae。PCR product sizes vary from 450 to 880 bp.
3.By analysis of the restriction pattern of amplified 5.8S-ITS region(RFLP)5 different hereditary clusterings were found.
4.This work shows Wallerstein Laboratory Nutrient Agar(WL medium)was strengthened for using in wine fermentation to observe yeast populations and useful for predigesting in wine yeast molecular identification.
After this study, the preliminary ideas of Vitis.amurensis Rupr grape wine yeast diversity in northeast China were established. And it was settled a base for the fine wine yeast which could endure low temperature and low pH.
引文
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