Constructing Hybrid Yeasts Through Protoplast Fusion For Lowering Ethanoland Enhancing Aroma

This study aimed to construct new yeast hybrid strains for improving aroma and flavor of cider but reducing alcohol content with protoplast fusion.To date,no studies have been carried.Saccharomyces cerevisiae WLS21 and Candida ethanolica SJ03,the protoplasts of which have been selected from a large number of cider yeasts for good aroma-producing ability and alcohol-reducing ability respectively,were prepared and fused.Screened fusants were used to ferment apple juice.The results are as follows:(1)On the basis of the Box-Behnken design at 3 factors and 3 levels,the protoplasts of Saccharomyces cerevisiae and Candida ethanolica were prepared under the optimized condition of ultrasonic and enzymatic hydrolysis treatment(Not reported).When Snail enzyme concentration was 4.0%,temperature was 28.7°C,ultrasonic time was 44.8 min and Snail enzyme concentration was 4.5%,temperature was 29.0°C,ultrasonic time was 46.7 min,the highest value of the product of protoplast formation rate and regeneration rate was obtained of respectively.The formation rates of WLS21 and SJ03 were 83.15%and 86.43%,respectively and the regeneration rates were 23.13%and 24.42%,respectively.The results showed that protoplast formation rate and regeneration rate was significantly improved compared with previous results,processing time shortened by nearly 3 times and conducive to the integration of protoplasts.Carried out in the presence of PEG-6000,the fusion frequency of 1.1×10~-22 was obtained,better than the previous results.(2)We picked out 24 fastest growing regenerated colonies.By performing Durham's medium fermentation,TTC medium fermentation and re-screening identification,9 stable hybrid strains were inoculated in subsequent fermentation.(3)The alcoholic degree of ciders fermented by 6 hybrids achieved less than 2%(v/v),reduced about 17%(v/v)compared with the one fermented by parental strain WLS21.(4)9 yeast hybrid strains,parental strains and mixed strains were further compared through GC-MS and fuzzy comprehensive evaluation.The total aroma of cider fermented by R6 was 459.22 mg/L,which is 104.88 mg/L more than cider fermented by WLS21.Hybrid R6 scored highest,displayed superior performance and desirable properties of both parents.