| 摘要: |
| 【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 rDNA D1/D2区域序列鉴定为发酵毕赤酵母(Pichia fermentans)，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pH 3.0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。 |
| 关键词: 野生酵母 β-葡萄糖苷酶 七叶苷培养基 白酒酒窖 |
| DOI： |
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| 基金项目:杨凌示范区科技计划项目(2016NY-23);陕西省科技计划一般项目（2017NY-143） |
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| Rapid screen of wild yeast with high β-glucosidase activity and evaluation of its glycosidase in winemaking |
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MA Decao,YOU Ling,LI Aihua,et al
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| Abstract: |
| 【Objective】This study established a semi-quantitative colorimetric method to screen high β-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 by β-glucosidase,a semi-quantitative colorimetric method was established to rapidly screen wild yeast strains with high β-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.H5Y1 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】The β-glucosidases of two selected stains (H5Y1 and Z9Y3) had good adaptation to the wine making environment. |
| Key words: wild yeast β-glucosidase esculin medium liquor brewing cellar |
