Production of yeast hybrids for improvement of cider by protoplast electrofusion

• Ultrasonication was used to degrade cell wall to obtain protoplasts efficiently.
• The fusion of protoplasts of Saccharomyces cerevisiae and Candida krusei was achieved.
• Conditions of electrofusion were optimized.
• Newly generated yeast fusants were screened by fuzzy comprehensive evaluation.
• The fusant R4 was superior to both parental strains and was perfect for cider-making.

This study aimed to construct new yeast hybrid strains for introducing flavor and aroma diversity to ciders. The inactivated protoplasts of Saccharomyces cerevisiae and Candida krusei were electric-induced fused under the optimized electric condition of pulse field density 2200 V/cm, pulse time 20 μs, pulse number 2 times and pulse interval 1 s, and 69 fusants were initially obtained. By performing Durham's fermentation for ten generations, 9 stable hybrid strains were screened. The chemical analysis showed that the alcoholic degree of ciders fermented by R2, R4, R5, R6 and R8 achieved about 12% (v/v), which was statistically the same level as the one fermented by parental strain WF1. The GC–MS results showed different strain generated totally different aroma profiles. R4 produced significant higher concentration of 2-methyl-butanoic acid ethyl ester, 2-methyl-1-propanol, 3-methyl-butanol acetate, 1-butanol, acetic acid hexyl ester, 1-hexanol and 1-octanol. The 9 hybrid yeast strains and parental strains were further compared through fuzzy comprehensive evaluation combining sensory score and aroma components content. The results showed that the hybrid R4 scored highest and displayed desirable properties of both parents.