Constuction Of Yeast With High Yield Of 4-Vinylguaiaicoland Its Application In Soy Sauce Fermentation

Soy sauce is a condiment originated in China and spread all over the world with cultural exchanges.Due to the differences in people’s eating habits in different regions,soy sauce has gradually formed a flavor with non-local characteristics.Japanese like mellow compounds represented by heptanol,but people in China prefer the soy sauce flavor characterized by esters and phenols.However,the flavor of soy sauce on the market today is generally not high.In order to meet the preferences of Chinese consumers,in this study,the yeast strains added in the process of yeast fermentation were taken as the starting point from the perspective of its metabolism,improving the soy sauce flavor at the source of fermentation.This paper mainly studies the following aspects:(1)The tolerance of S yeast,T yeast and C yeast three different yeasts to ferulic acid was determined by gradient dilution experiment.It was found that the T yeast had the highest ferulic acid stress tolerance of0.1% ferulic acid and could not grow on 0.3% ferulic acid medium.Thus,T yeast was chosen and YPD medium with 0.3% ferulic acid was selected as the initial screening medium.(2)It was confirmed that the lethality of T yeast was maintained at 70%-80%under the condition that the mutagen concentration was 4.0% ethyl methanesulfonate(EMS)for 30 min.T yeast was chemically mutagenized under this condition,and M1 and M3 that could grow in 0.3% ferulic acid YPD medium were screened out through the primary screening medium,and they were used as the parents of the first round of genome rearrangement.(3)It was determined that the helicase concentration of 5 mg/m L and incubation at 30°C for 45 min were the optimal conditions for protoplast preparation.M1 and M3 were processed under these conditions to obtain protoplasts,and PEG-6000 was used to promote the DNA uptake of the strains for gene recombination.Through two rounds of genome rearrangement,the GS2-3 strain was screened on 0.4% ferulic acid YPD medium.(4)The RAPD experiment verified that the genomes of the strains obtained by genome rearrangement were different,and the ferulic acid resistance was proved by subculture.The GS2-3 strain had good genetic stability.(5)In a liquid YPD medium containing ferulic acid,the yield of 4-vinylguaiacol produced by GS2-3 strain was 116%higher than that by T yeast.(6)The gene FDC1 of T yeast and GS2-3 strain was sequenced and compared,the GS2-3 strain had insertion mutation after 576 bp,resulting in changes in amino acid sequence.(7)Using the starting strain T yeast as the control,GS2-3 strain was applied to soy sauce fermentation.By comprehensively comparing the determination results of amino acid nitrogen content,total nitrogen content,total acid content,flavor substances,sensory evaluation and other indicators in soy sauce fermentation,it was found that the phenolic content of soy sauce fermented with GS2-3 strain was higher than that of soy sauce fermented with T yeast,and the content of 4-Vinylguaiacol increased by 38.8%.In this paper,T yeast taken as the starting strain,a yeast strain GS2-3 with high yield of 4-Vinylguaiacol was constructed by genome rearrangement technology.The effectiveness and genetic stability of the strain constructed by genome rearrangement were verified by RAPD and subculture.It was applied to soy sauce fermentation.It was found that yeast strain GS2-3 can significantly improve the soy sauce flavor and overall quality of soy sauce.