原生质体融合技术选育低甲醇果酒酵母

Reduction of Methanol Content by Protoplast Fusion in Fruit Wine Yeast

摘要:

【目的】利用原生质体融合技术选育低甲醇果酒酵母。【方法】以酵母LAB和酵母XR为亲本酵母，通过原生质体融合技术选育可在果酒发酵中产生低甲醇质量浓度、高乙醇体积分数的酵母菌株。以原生质体形成率、再生率和制备率为指标，通过单因素与正交试验优化酵母原生质体制备工艺；采用紫外线照射和加热2种方式对双亲原生质体进行灭活，并采用35 g/dL的聚乙二醇6000（PEG-6000）对灭活后的原生质体进行化学融合。【结果】原生质体最佳制备条件为：蜗牛酶质量浓度2.0 g/dL、酶解时间2.0 h、EDTA-巯基乙醇质量浓度1.25 g/dL、EDTA-巯基乙醇处理温度30.0 ℃。在此条件下，原生质体形成率为（93.61±0.86）%、再生率为（24.23±0.52）%、制备率为（22.68±0.29）%。经化学融合后共获得19株融合子，通过酿酒试验筛选出融合子lx3；与亲本酵母XR相比，其发酵果酒中甲醇质量浓度降低了50.87%，乙醇体积分数维持稳定。【结论】该研究成功选育出一株酿造低甲醇质量浓度、高乙醇体积分数百香果壳果酒的酵母菌株。

Abstract:

[Objective] This study aims to breed a fruit wine yeast strain with low methanol production by protoplast fusion.[Method] With yeast strains LAB and XR as parents, protoplast fusion was employed to breed the yeast strains for fruit wine fermentation with a low mass concentration methanol and a high volume fraction ethanol. The process of yeast protoplast preparation was optimized by single factor and orthogonal experiments with the formation rate, regeneration rate, and preparation rate of protoplasts as indicators. The parental protoplasts were inactivated by ultraviolet radiation and heating, and 35 g/dL PEG-6000 was used for chemical fusion after inactivation.[Result] The optimum conditions for protoplast preparation were as follows: a snailase mass concentration of 2.0 g/dL, enzymolysis time of 2.0 h, an EDTA-mercaptoethanol mass concentration of 1.25 g/dL, and an EDTA-mercaptoethanol treatment temperature of 30.0 ℃. Under these conditions, the formation rate, regeneration rate, and preparation rate of protoplasts were （93.61±0.86）%, （24.23±0.52）%, and （22.68±0.29）%, respectively. A total of 19 fusants were obtained by chemical fusion, and the fusant lx3 was screened out by the brewing experiment. The methanol mass concentration in the fermented fruit wine produced with lx3 was reduced by 50.87% on average compared with that produced with the parent strain XR, and the ethanol volume fraction was similar between lx3 and the parent strain.[Conclusion] This study bred a fruit wine yeast strain suitable for passion fruit shell fermentation with a low methanol mass concentration and a high ethanol volume fraction.