葡萄自然发酵过程中酵母的分离鉴定及优良葡萄酒酵母筛选
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摘要
酵母菌是葡萄酒酿造过程中的主要微生物,其酿造学特性直接影响葡萄酒的质量和风格,从葡萄品种原产地可以筛选得到充分展现该品种个性和地域特性的酵母。东北地区是我国葡萄酒的主产区之一,因而选育适合本产区葡萄特性的优良酵母显得尤为重要。本研究的目的是借助我国东北产区独特的地理及生态条件,挖掘东北葡萄酒产区的葡萄酒酵母资源。
本文以产自吉林省松原市‘双红’、‘双优’、‘赤霞珠’、‘黑赛必尔’4个葡萄品种的成熟果实为试材,对4个葡萄品种自然发酵过程中的酵母菌进行分离：应用WL培养基对分离的菌株进行初步分类；通过5.8S rDNA-ITS的PCR-RFLP分析对所分离菌株做进一步的分子鉴定。此基础上,对被鉴定为酿酒酵母的菌株进行筛选,以期从中得到具有优良发酵性状的葡萄酒酵母。
主要结论如下：
1、4个葡萄品种自然发酵过程中共分离葡萄酒相关酵母240株。通过菌株在WL培养基上的培养结果,依据菌落颜色及形态记录,可将全部酵母分为6类。
2、依据酵母菌5.8S rDNA-ITS区的PCR-RFLP酶切图谱,可将本试验分离得到的全部酵母鉴定为5类,分别是葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、酿酒酵母(Saccharomyces cerevisiae)、粘红酵母(Rhodutorula glutinis)、陆生伊萨酵母(Issatchenkia terricola)和假丝酵母属的Candida sorbosa。
3、试验证实WL培养基可以区分葡萄自然发酵过程中出现的大多数酵母,并且对酿酒酵母鉴定的准确度达到100%。
4、同一产区的山葡萄、欧亚种葡萄、欧美杂种葡萄,其自然发酵过程中的酵母菌群变化基本一致。在发酵前期,葡萄汁有孢汉逊酵母占主导地位；到发酵中期,酵母菌群的构成复杂化,含有本试验所分离鉴定的5种酵母；至发酵后期,酿酒酵母在菌群中占有绝对优势。
5、筛选得到2株能够快速启动发酵,且产酒精能力和发酵速率都明显好于活性干酵母的野生葡萄酒酵母,SYC1、HSB20。证明产地葡萄的自然发酵醪液中存在具有优良发酵性能的野生葡萄酒酵母。
Yeast is the main microbes in Wine Making, and it can directly affect the quality and style of wine. The yeasts which from grape original region can present variety characteristics and regional characteristics. Northeast China is one of the main wine production area. Therefore, it's important to breed the quality yeast which is suitable for local grape feature. The aim of this study is to develop the wine yeast resources of Northeast China, which by virtue of geographical condition and ecological condition of Northeast China.
In this study, yeasts were isolated from 4 different grape varieties in the city of Song Yuan: 'Shuang Hong','Shuang You','Cabernet Sauvignon','Seibel Noir', these yeasts were identified primarily by Wallerstein Laboratory Nutrient Agar (WL Agar); Then yeasts were further identified by 5.8S rDNA-ITS RFLP analysis; Based on this, all of the Saccharomyces cerevisiae used to select quality wine yeast.
The main results are as follows:
1.240 wine related yeasts were isolated from the spontaneous fermentation of 4 different grape varieties. The result indicates that there are 6 WL culture types of these yeasts.
2. According to the 5.8S-ITS RFLP analysis,5 kinds of yeasts were found in the fermentation broth of these 4 grape varieties:Hanseniaspora uvarum, Saccharomyces cerevisiae, Rhodutorula glutinis, Issatchenkia terricola, Candida sorbosa.
3. This work showed that WL Agar was useful in most part of wine yeast identification, which during the spontaneous fermentation of grap. And the WL Agar was effective for the identification of S. cerevisiae and the reliability is 100%.
4. The trend of the proportion of the flora in the spontaneous fermentation of Amur grape change is similar to which in Vitis vinifera and Europe America hybrid Vitis. H. uvarum plays a dominant role during the earlier spontaneous fermentation; The flora turn to be complex in the middle spontaneous fermentation, which contain 5 classes yeast species; S.cerevisiae plays a dominant role in the spontaneous fermentation anaphase.
5. After 3 steps selection, the quality wine yeast strain named SYC1 and HSB20, which with quick start fermentation had received. These 2 wild yeasts had the high ability of produce alcohol, and its alcohol fermentation rate was obviously higher than active dry yeast. Meanwhile, this study showed that there were quality wild wine yeasts during the spontaneous fermentation of local grape.
本文以产自吉林省松原市‘双红’、‘双优’、‘赤霞珠’、‘黑赛必尔’4个葡萄品种的成熟果实为试材,对4个葡萄品种自然发酵过程中的酵母菌进行分离：应用WL培养基对分离的菌株进行初步分类；通过5.8S rDNA-ITS的PCR-RFLP分析对所分离菌株做进一步的分子鉴定。此基础上,对被鉴定为酿酒酵母的菌株进行筛选,以期从中得到具有优良发酵性状的葡萄酒酵母。
主要结论如下：
1、4个葡萄品种自然发酵过程中共分离葡萄酒相关酵母240株。通过菌株在WL培养基上的培养结果,依据菌落颜色及形态记录,可将全部酵母分为6类。
2、依据酵母菌5.8S rDNA-ITS区的PCR-RFLP酶切图谱,可将本试验分离得到的全部酵母鉴定为5类,分别是葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、酿酒酵母(Saccharomyces cerevisiae)、粘红酵母(Rhodutorula glutinis)、陆生伊萨酵母(Issatchenkia terricola)和假丝酵母属的Candida sorbosa。
3、试验证实WL培养基可以区分葡萄自然发酵过程中出现的大多数酵母,并且对酿酒酵母鉴定的准确度达到100%。
4、同一产区的山葡萄、欧亚种葡萄、欧美杂种葡萄,其自然发酵过程中的酵母菌群变化基本一致。在发酵前期,葡萄汁有孢汉逊酵母占主导地位；到发酵中期,酵母菌群的构成复杂化,含有本试验所分离鉴定的5种酵母；至发酵后期,酿酒酵母在菌群中占有绝对优势。
5、筛选得到2株能够快速启动发酵,且产酒精能力和发酵速率都明显好于活性干酵母的野生葡萄酒酵母,SYC1、HSB20。证明产地葡萄的自然发酵醪液中存在具有优良发酵性能的野生葡萄酒酵母。
Yeast is the main microbes in Wine Making, and it can directly affect the quality and style of wine. The yeasts which from grape original region can present variety characteristics and regional characteristics. Northeast China is one of the main wine production area. Therefore, it's important to breed the quality yeast which is suitable for local grape feature. The aim of this study is to develop the wine yeast resources of Northeast China, which by virtue of geographical condition and ecological condition of Northeast China.
In this study, yeasts were isolated from 4 different grape varieties in the city of Song Yuan: 'Shuang Hong','Shuang You','Cabernet Sauvignon','Seibel Noir', these yeasts were identified primarily by Wallerstein Laboratory Nutrient Agar (WL Agar); Then yeasts were further identified by 5.8S rDNA-ITS RFLP analysis; Based on this, all of the Saccharomyces cerevisiae used to select quality wine yeast.
The main results are as follows:
1.240 wine related yeasts were isolated from the spontaneous fermentation of 4 different grape varieties. The result indicates that there are 6 WL culture types of these yeasts.
2. According to the 5.8S-ITS RFLP analysis,5 kinds of yeasts were found in the fermentation broth of these 4 grape varieties:Hanseniaspora uvarum, Saccharomyces cerevisiae, Rhodutorula glutinis, Issatchenkia terricola, Candida sorbosa.
3. This work showed that WL Agar was useful in most part of wine yeast identification, which during the spontaneous fermentation of grap. And the WL Agar was effective for the identification of S. cerevisiae and the reliability is 100%.
4. The trend of the proportion of the flora in the spontaneous fermentation of Amur grape change is similar to which in Vitis vinifera and Europe America hybrid Vitis. H. uvarum plays a dominant role during the earlier spontaneous fermentation; The flora turn to be complex in the middle spontaneous fermentation, which contain 5 classes yeast species; S.cerevisiae plays a dominant role in the spontaneous fermentation anaphase.
5. After 3 steps selection, the quality wine yeast strain named SYC1 and HSB20, which with quick start fermentation had received. These 2 wild yeasts had the high ability of produce alcohol, and its alcohol fermentation rate was obviously higher than active dry yeast. Meanwhile, this study showed that there were quality wild wine yeasts during the spontaneous fermentation of local grape.
引文
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