Breeding Of Superior Chinese Rice Wine Yeast And Mechanisms Study Of Related Ethanol Tolerance

As yeast is important in Chinese rice wine-making,its performance greatly affect the productivity and flavor quality of Chinese rice wine.Breeding yeast with excellent phenotype,not only can improve the productivity,but also provide microbial resource for brewing differentiated Chinese rice wine.During Chinese rice wine brewing,the ethanol content can reach to 14-20%?v/v?,which is toxic to yeast cell and therefore lower yield and quality of the resulting wine.In this paper,yeasts exhibited excellent stress-resistance were screened out from traditional fermented foods.Technologies of haploidization,ultraviolet mutagenesis?UV?,directed evolution and protoplast fusion were applied for breeding aroma-enhancing yeast,which showed superior ethanol tolerance and fermentation activity.High throughput sequencing,transmission electron microscope?TEM?,fluorescence polarization,GC-MS and molecular biology were used to study the mechanism of ethanol tolerance.The selected yeast was applied for Chinese rice wine brewing and the oenological properties were analyzed.In addition,effects of post-treatment process on Chinese rice wine quality were studied.The major results were:?1?Four yeast strains which exhibited excellent initial fermentation capacity,alcohol-producing ability and fermentation performance were isolated from fermented mash,yeast starter and lees of Chinese rice wine.Through 5.8S rDNA-ITS sequence analysis,the four yeasts were identified as Saccharomyces cerevisiae.Tolerance of the four yeast to high concentration of ethanol,glucose,lactic acid and osmotic pressure were analyzed.The results showed that BR 20 and BR30 strains exhibited superior overall performance.BR20 showed the best ethanol tolerance,which could tolerant to 18%?v/v?ethanol.While BR30 exhibited the highest fermentation activity,which the reduction of CO₂ in BR30 strain was 10.03%higher than BR20 in 7 days.?2?Haploid strains of 20-Ha4 and 30-Ha5,which respectively generated from diploid strains BR20 and BR30,were selected as candidates for ethanol domestication and UV mutagenesis.Then,haploid strain Et20,which could tolerant to 20%?v/v?ethanol was obtained.Haploid strain CTZ30 exhibited significantly higher fermentation activity by UV mutagenesis and clotrimazole-resistance selection was acquired.The haploids Et20 and CTZ30 were used as parental strains for protoplast fusion.Finally,diploid hybrid BR129f23?F23?showed a cell viability of 6.2%under 25%?v/v?ethanol was acquired,whereas its diploid parental strains could not survive under 20%?v/v?ethanol.During Chinese rice wine-making,compared to the diploid parents,F23 produced 7.07-12.44%higher yield of ethanol and 19.99-26.55%higher content of flavor compounds.Specifically,F23 exhibited higher capacity in producing 2-phenylethanol,short-chain and long-chain fatty-acid ethyl-ester than diploid parents.Compared to diploid parents,F23introduced more flavor contributors with odor activity values?OAVs?above one to Chinese rice wine.The flavor characteristic of F23 wine was similar to each of parent wine.Additionally,sensory evaluation showed that F23 wine was highly assessed for its intensive levels in fruit-aroma,alcohol-aroma and mouthfeel.The breeding strategies not only generated yeast with superior ethanol tolerance and fermentation activity,but also could provide potential“mixed-like”starter to enrich wine style and differentiation.?3?Genome-wide denovo sequencing was applied for BR20 and F23,and single nucleotide polymorphisms?SNP?between the two strains were analyzed.Different SNP and Indel variants between BR20 and F23 were respectively accounted for about 5%and4.5%of the total variation data.The differential SNP variations mainly occurred in the exon region,while Indel variations mainly occurred in the upstream?1 Kb?,downstream?1 Kb?,and junctions between upstream and downstream.Gene annotation and metabolic pathway of differential SNP variants between BR20 and F23 were analyzed by GO and KEGG databases,respectively.The results indicated that the phenotypic differences between BR20 and F23 was resulted by metabolisms of lipid,carbohydrate,amino acid and nucleotide,the biological process and cell components.The differential SNP variants and annotation results provided critical resources for studying the ethanol tolerance mechanisms of F23.?4?Cell membrane permeability of BR20 and F23 under ethanol stress were analyzed,and the membrane permeability was improved with the increase of ethanol content.The two strains maintained normal growth while cell membrane permeability increased by no more than 20%.However,once the membrane permeability increased by more than 30%,cell growth limited by ethanol was observed.Under different ethanol stress,increased rate of membrane permeability in BR20 was higher than F23 strain,which exhibited better protection against ethanol than BR20 strain.Ethanol stress induced the decline of cell membrane fluidity and total content of unsaturated fatty acids?UFA?.Total content of UFA in F23 was higher than that of BR20,and decrease rate of membrane fluidity was slower than BR20.The OLE1 gene,which encoded desaturase,was analyzed by q-PCR.Under 10%?v/v?ethanol stress,the expression of OLE1 in F23 increased by13.36-fold,while that of BR20 increased only 3.65-fold.Under 18%?v/v?or higher ethanol stress,OLE1 showed little expression in BR20 and yeast cells stopped growing,whereas the OLE1 expression in F23 was still 2-4 times higher than the control.F23 strain with overexpressed OLE1 was constructed,and its OLE1 expression and content of UFA were significantly higher than that of F23 strain under ethanol stress.Besides,the decrease rate of cell membrane fluidity was slower than that of F23.The above results demonstrated that,under ethanol stress,yeast enhanced its ethanol tolerance by increasing the expression of OLE1 to reduce the decrease rate of UFA content,slow down the decrease rate of cell membrane fluidity,and thereby weakening the adverse essects of ethanol on cell growth and metabolism.?5?F23 strain was applied for brewing 20 L-scale Chine rice wine,which provided a reference for the industrial application of F23 strain.As fermentation time prolonged,the content of reducing sugar gradually decreased,while total acidity,ethanol,and amino acid nitrogen contents increased.The contents of reducing sugar,total acidity,ethanol,and amino acid nitrogen in the final wine mash were 2.51 g/L,5.20 g/L,146.04 g/L,and2.98 g/L,respectively.F23 strain fermented fast in the initial fermentation and slowly in the secondary fermentation,indicating that the way to successful brewing is ensuring growth advantage of F23 in the initial fermentation.Effects of traditional boiling,UHT and HHP on wine quality were investigated.Thermal-treatment resulted in decrease of total FAA content,whereas HHP-treatment promoted for the increase.However,UHT-and HHP-treatments had little effect on FAA with sweet and umami-like taste.Wine processed at 400 or 600 MPa for 10 min exhibited similar flavor characteristic to the untreated wine,whereas thermal-treated wines showed different flavor characteristic.Sensorial attributes and flavor compounds were used as variants for partial least squares regression?PLSR?.The results indicated that sensory attributes of cereal-aroma and astringency were highlighted after thermal-treatment,while fruit-aroma,continuation and full-body were dominant after HHP-treatment.HHP-treatment at 600 MPa for 10 min,which greatest improved the content of aromatic compounds,short-chain and long-chain fatty acid ethyl esters,could notably enhance the sensory profiles of Chinese rice wine.