Construct Of Killer Yeast For Kiwi-Fruit Wine Using Protoplast Electronic-Induced Fusion

In wine fermentation,wine is often polluted by wild yeasts which led to turbidity,precipitation, abnormal smell etc. and that affectes the normal fermentation. Low quality kiwi-wine featured with poor banquet, less fruit aroma, weak typicality mainly resultes from lacking of specific yeasts. In this study, one excellent yeast for kiwi-wine was selected from several wine yeasts, and then electronic protoplast fusion was carried out between the selected yeast and a killer yeast S.cerevisiae 5045 (his4,Karl-1,KIL-K1). A well kiwi-wine yeast with killing activity was constructed successfully. The results showed that:1. One excellent yeast S.cerevisiae KD for kiwifruit wine was selected from 12 fine wine yeasts through comparing their fermentation capacity, fermentation speed, chemical and sensory quality, the main aroma and flavoring substances, and using the methods of microbiological testing, chemical determination, sensory evaluation and SPME-GC/MS. 2. Technical parameters for S.cerevisiae 5045 protoplast preparation : high exudate of 0.8mol/L Mannitol, pretreatment solution of 0.1%EDTA+0.1%2-Mercaptoethanol, 2% Snailase in PBS pH5.8, treatment for 60min. at 30℃.3. For S.cerevisiae KD ,the suitable enzymatic time was 30min,and the inactivation condition was 55℃for 21min,when the inactivated rate was 99.11%.4.Mixed the protoplast of KD and 5045 ,at the ratio of 1:1,and electronic fusion was conducted according to the following parameters:pulse strenth of 2KV/cm,pulse time of 40μs,pulse interval of 800ms,pulse number of one. 56 fusants was detected, when the fusion rate was 6.93×10-5,through nutrient demanding and killing activity identification, and the fusion rate was 6.93×10-5.5. The well kiwi-wine yeast with killing activity named fusant E16 was selected through comparing the genetic stability, fermentation capacity, and sensory quality and chemical parameters.6. Identification results showed that E16 had the similar characteristics with the parental S.cerevisiae KD like cell morpherlogy,, growth rate and nutritional requirement. Besides, it had killing activity,similar to the parent S.cerevisiae5045.7. Inoculated E16 and sensitive yeast with ratio of 1:1 and 1:5, and cultured with different times, respectively. The results were the cell number of sensitive yeasts decreased and the growth inhibited. It showed E16 can kill the wild yeast effectively during the fermentation process.8.The fermentation experiment showed that,the kiwiwine made by E16 had high chemical and sensory quality. The aroma components of kiwifruit wine were analyzed by SPME-GC/MS,and 46 components were identified,which represented 99.18% of the total peak area, which were composed of esters (22 kinds, 78.01%), higher alcohols(5 kinds, 11.48%), fatty acids(6 kinds, 7.89%), and micro-content of alkyls, aldehydes and ketones, E16 is a fine yeast that is suitable for Kiwifruit wine brewing.