[Objective] This study established a semi-quantitative colorimetric method to screen highβ-glucosidase activity wild yeasts rapidly and the fermented adaptability of β-glucosidase of the glycosidase extract were analyzed to provide reference for its application in winemaking.[Method] A total of 133 wild yeasts strains from Chinese liquor brewing cellar of Yibin were selected.Based on the dark brown appearance for the reaction of Fe3+ and esculin hydrolysate hydrolyzed byβ-glucosidase,a semi-quantitative colorimetric method was established to rapidly screen wild yeast strains with highβ-glucosidase activity according to the color level in 96-well plates with esculin medium.The adaptability of β-glucosidase of the selected yeasts in winemaking environment was also studied.[Result] Nineteen yeast strains with different dark degrees on esculin medium were selected initially from 133 strains.HSY1 and Z9Y3 yeasts were selected secondly due to their high β-glucosidase.These two strains were identified as Pichia fermentans by the sequence comparison of 26S rDNA D1/D2 domain,and the β-glucosidase activities of glycosidase extracts from the two strains were 39.71 mU/mL and 37.80 mU/mL,respectively.The β-glucosidases activity could maintain more than 51.1％ of the original enzyme activity when the glycosidase extracts were kept at 20 ℃ for 72 h,and maintain more than 90.0％ of the original enzyme activity after being kept at pH=3 for 12 h.Besides,the β-glucosidases activity could maintain more than 84.0％ and 90.0％ of the original enzyme activity under 2 different conditions of glucose concentrations (100-200 g/L) and alcohol degrees (5 ％-15 ％,V/V),respectively.[Conclusion] Theβ-glucosidases of two selected stains (H5Y1 and Z9Y3)had good adaptation to the wine-making environment.%[目的]建立快速筛选高产β-葡萄糖苷酶野生酵母的半定量显色法,并对酵母糖苷酶提取液中β-葡萄糖苷酶的酿造适应性进行分析,为其在酿酒中的应用提供参考.[方法]以从宜宾白酒酒窖中分离的133株野生酵母为试验菌种,根据β-葡萄糖苷酶可以将七叶苷水解为七叶苷原并与Fe3+作用显棕黑色的原理,在96孔板上设计含七叶苷培养基,根据培养液的显色深度建立快速筛选高产β-葡萄糖苷酶野生酵母的半定量显色法,并研究优选菌株β-葡萄糖苷酶对葡萄酒酿造环境的适应性.[结果]初筛获得19株在七叶苷培养基上显色水平有差异的野生酵母菌株,并最终优选2株β-葡萄糖苷酶活性较高且稳定的酵母菌株H5Y1和Z9Y3;2株酵母经菌落形态、细胞形态及26S rD-NA D1/D2区域序列鉴定为发酵毕赤酵母(Pichia ermentans),酵母糖苷酶提取液中β-葡萄糖苷酶活性分别为39.71和37.80 mU/mL,提取液在20℃保温72 h后,β-葡萄糖苷酶活性保持在初始酶活性的51.1％以上,在pH3.0条件下保持12 h后,β-葡萄糖苷酶活性保持在初始酶活性的90.0％以上,在100～200 g/L葡萄糖质量浓度和5％～15％(体积分数)酒精度的2个不同条件下,β-葡萄糖苷酶活性分别保持在初始酶活性的84.0％和90.0％以上.[结论]优选菌株H5Y1和Z9Y3 β-葡萄糖苷酶具有较好的葡萄酒酿造环境适应性.
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