Breeding Of Kiwifruit Wine Yeast With Aroma-producing Capacity And Killer Activity

In recent ten years, the kiwifruit winemaking industry has made a rapid development inChina. But the quality of kiwifruit wine is not so perfect，the fruity aroma is not enoughstrong, and the typicality is absent. And the contamination by other yeast in the early stage offermentation also leads the flavor of kiwifruit wine deteriorative. So, the purpose of this studyis to breed kiwifruit wine yeast with not only good aroma-producing performance but alsostrong killer activity. The yeast can produce more aromas and kill the wild yeasts duringfermentation, ensure pure-bred fermentation carry out smoothly and produce high qualitykiwifruit wine. The technique of protoplast electrofusion was used in the study. Theseparation of wild yeast from kiwifruits, sifting the aroma-producing yeast, preparing theparental protoplasts, determining the parameters of electrofusion, and screening andidentifying fusants were studied. The results showed as follows.（1） There are wild yeasts on the peel of kiwifruit. After isolation and culture bysmall-scale winemaking trials, a wild kiwifruit wine yeast with good fermentationperformance was selected from seven different varieties of kiwifruit.（2） The wild yeast and other12Saccharomyces cerevisiae were used to ferment kiwifruitjuice for wine. Through comparing the fermentation rate of all yeasts, physicochemicalindexes analysis and sensory evaluation of the kiwi wine, the yeast strain KD showed bettercharacteristics than others as follows: faster and smooth fermentation speed, higher alcoholproduction and content vitamin C, but lower volatile acid content. The kiwifruit winefermented by KD strain had high quality–light yellow color, mellow and refreshing taste,strong and pleasant fruit aroma, and pure alcoholic scent. The kiwifruit wine had harmoniousand typical fruit aroma.（3） GC-MS analysis showed that there were more kinds of aroma components and higherlevels of their content in the kiwifruit wine fermented by KD yeast than the wine fermentedrespectively by other6yeast strains. The former wine had48kinds of aroma volatile material,and the relative content of them was97.33percent. Include19kinds of ester,7kinds ofhigher alcohol and6kinds of organic acid, the relative content were respectively34.65 percent,15.85percent, and13.3percent. Yeast strain KD had strong capacity to producearoma substances, especially more kinds of ester and higher content. The kiwi wine had rich,strong and harmonious aroma. KD was a good yeast strain for kiwifruit winemaking.（4） S. cerevisiae KD, with capacity of produces more aromas, was non-autotrophic anddid not have killer activity. Killer yeast5045was autotrophic of histidine and was a mutant ofS. cerevisiae with defective for nuclear fusion. KD and killer yeast5045was parent. Based onProtoplast electrofusion, the killer plasmid from5045was leaded into the yeast strain KD tobreed yeast with killer activity and capacity to produce more aromas for kiwifruitwinemaking.（5） We studied the factors which effect the formation and regeneration of the protoplastof yeast5045, such as choice the pretreatment solution and the hypertonic buffer solution,parameters of enzymolysis and cultural methods for regeneration. The optimal conditionswere summarized as follows. The cells of5045cultured for4hours were washed withhypertonic buffer solution of0.8mol/L Mannitol, then pretreated with mixture of0.1%EDTA-Na₂solution and0.1%mercaptoethanol solution （1:1, V/V） for10minutes at30℃.The pretreatment solution was washed up with0.8mol/L Mannitol. The cells were hydrolyzedby2.5%Helicase solution for60minutes at35℃. As a result, the formation rate （FR） andregeneration rate （RR） of protoplast were respectively80.97percent and26.47percent, theproduct of FR and RR was21.43percent. Protoplast formation was the best.（6） The optimum conditions of protoplast formation of yeast strain KD were similar to5045. The cell of KD cultured for3hours also washed with hypertonic buffer solution of0.8mol/L Mannitol, then pretreated with mixture of0.1%EDTA solution and0.1%mercaptoethanol solution （1:1, V/V） for10minutes at30℃. The pretreatment solution waswashed up with0.8mol/L Mannitol. The cells were hydrolyzed by2.5%Helicase solution for30minutes at35℃. As a result, the formation rate （FR） and regeneration rate （RR） ofprotoplast were respectively81.15percent and10.4percent, the product of FR and RR was8.44percent.（7） The protoplasts of KD and the protoplasts of5045, mixed with the ratio of1to1,were electro fused on the conditions as follow. The pulse field strength was2KV/cm, pulsetime was40microseconds, and pulse interval was600microseconds and pulse3times. Therate of protoplast fusion was6.93×10^-5. After screening, rescreening and small-scalewinemaking, one fusant F16with excellent capability was obtained. The fusant had stablekiller activity in genetics.（8） Through comparing the cell shape and size, auxotrophic determination, killer activityidentification, detection the inhibition action of fusant F16to wild yeast during brewing process, aroma analysis and sensory evaluation, the results showed that fusant F16inheritedthe good fermentation characteristics of KD and the strong killer performance of5045. F16was yeast for kiwifruit winemaking with superior capacity to produce more aromas and finekiller activity, and the prospect using in kiwifruit wine industry was broad.