Breeding Of Indigenous Non-saccharomyces Cerevisiae With Low Ethanol Yield

In recent decades,global warming has increased the sugar content of grapes,which in turn has led to a gradual increase in wine alcohol.Ethanol in wine plays a key role in the balance of wine,but excessive ethanol will affect the flavor characteristics of wine and human health.Therefore,how to effectively reduce the alcohol content in wine has attracted more and more attention.Breeding native non-Saccharomyces yeasts with the ability to reduce alcohol is the most simple and economical strategy.In this study,we evaluated the ethanol production capacity and other fermentation performance of 111 non-Saccharomyces yeasts by simulated grape juice fermentation.It was verified that the ethanol volume and ethanol yield obtained form preliminary screening were lower than that of the control strain Saccharomyces cerevisiae NX11424.These strains were then evaluated for their oenological properties.In order to obtain non-Saccharomyces yeast strains with low ethanol production which can start fermentation quickly,the strains with low ethanol production and excellent brewing characteristics were mutated by ARTP.The main research results are as follows:(1)Four strains of Hanseniaspora with low ethanol yield were obtained.A total of 111indigenous non-Saccharomyces yeast strains from four different wine regions were identified and they were divided into eight yeast genera and fifteen yeast species.The 111 indigenous non-Saccharomyces yeasts were fermented by simulated grape juice,and 74 of them had lower ethanol production and ethanol yield than S.cerevisiae NX11424.By comprehensive analysis of the physical and chemical indexes of strains,four strains with lower ethanol yield and faster fermentation rate were screened,including Hanseniaspora opuntiae Ho A-1-3,Ho C-4-2 and Hanseniaspora uvarum Hu B-2-2,Hu C-3-2.Their ethanol yields were reduced by 26.03%,22.33%,23.77%and 32.05%,respectively.(2)Two strains of Hanseniaspora with low ethanol production and good brewing characteristics were screened.H.opuntiae Ho A-1-3,Ho C-4-2 and H.uvarum Hu B-2-2,Hu C-3-2 were used to simulate grape juice fermentation again to verify the alcohol reducing ability of the strains,and to explore their brewing characteristics.The results show that compared with S.cerevisiae NX11424,the ethanol yields of Ho A-1-3,Ho C-4-2,Hu B-2-2 and Hu C-3-2decreased by 23.87%,19.25%,10.74%and 18.74%,respectively.Four strains of Hanseniaspora all showed good growth ability and low foam-producing characteristics,and all of them had no killing activity against S.cerevisiae.Ho C-4-2 and Hu C-3-2 did not produce H₂S.In terms of tolerance,they can tolerate 400 g/L sugar,300 mg/L SO₂,p H 2.8,low temperature of 10?and high temperature of 40?.Ho A-1-3 and Ho C-4-2 can tolerate 6%(v/v)alcohol.Finally,two strains H.opuntiae Ho C-4-2 and H.uvarum Hu C-3-2 with low ethanol production and excellent brewing characteristics were obtained.(3)A strain of Hanseniaspora with high fermentation speed and low ethanol yield was selected by mutation breeding with ARTP.In order to further improve the fermentation rate of H.opuntiae Ho C-4-2 and H.uvarum Hu C-3-2,they were mutated by atmospheric and room-temperature plasma(ARTP).A total of 1063 mutant strains were obtained.Eight yeast strains with a higher fermentation rate than the mutagenesis of original strain were screened by using the cell growth(OD_600nm)as the preliminary screening index.Then,it was used to simulate grape juice fermentation,and the changes of ethanol yield were evaluated by measuring sugar consumption and ethanol yield.Finally,a strain Ho C32-2-72 with a faster fermentation speed than the starting strain and a 10.26%reduction in ethanol yield was obtained.