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.
