Selection And Breeding Of ARTP Mutagenesis In Highly Acid-tolerant Saccharomyces Cerevisiae And Its Acid Tolerance Mechanism

Wine fermentation can be divided into two stages:alcohol fermention（AF）and malolactic fermentation（MLF）.Malolactic fermentation is an indispensable part of red wine fermentation.Once alcohol fermentation is over,malolactic fermentation will begin.It can convert L-malic acid into L-lactic acid,reduce the acidity of wine.At the same time,it can also produce flavored substances and improve the flavor of wine.As the dominant bacteria of malolactic fermentation,Oenoccus oeni（O.oeni）often faces harsh wine environment,such as low p H value,high ethanol concentration,high sulfur dioxide and so on.Low p H value is the main adverse environment faced by O.oeni.In order to over-come this harsh environment,resistant strains need to be selected.The screening of traditional wild resistant strains is difficult to achieve in a short time,and mutation breeding can select the target strains in a short time.Therefore,in this study,mutation breeding was selected to improve the resistance of the strain.In this study,using O.oeni SD-2a as the starting strain,five acid-resistant mutants were selected by atmospheric pressure room temperature plasma mutagenesis.The fermentation abilities of five mutant strains in simulated wine and wine were compared and analyzed.Finally,the wine of the mutant strain ARTP-2 with the faster rate of malolactic fermentation was selected for aroma component analysis and transcriptome analysis.Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to explore the response mechanism of O.oeni to acid stress at the transcriptome level.The main conclusions are as follows:（1）Mutagenic breeding of O.oeni with high acid resistance by ARTP.Using O.oeni SD-2a as the starting strain,the optimum mutagenesis treatment time of 15 seconds was determined after ARTP mutagenesis treatment.Five acid-tolerant mutants with better growth performance and higherβ-glucosidase activity than the starting strain were obtained after subculture and screening under acid stress of p H 3.0.Among them,theβ-glucosidase activity of ARTP-1 and ARTP-2 was significantly higher than that of the starting strain.The cell surface structures of the starting strain SD-2a and five mutant strains were observed by scanning electron microscope.It was found that five mutant strains had good acid tolerance.By further exploring the tolerance of the five mutant strains to the lowest p H value and the highest ethanol concentration,it was found that five mutant strains could tolerate the lowest acid stress environment of p H 2.6,and could tolerate 16%high ethanol concentration.（2）Study on malolactic fermentation of mutant strain.The fermentation performance of the starting strain SD-2a and five mutants in simulated wine and wine were compared.In the simulated wine,the degradation amount of L-malic andβ-glucosidase activity of the five mutant strains in simulated wine were higher than those of the starting strain.Among them,the mutant ARTP-2 had the highest cumulative L-malic degradation andβ-glucosidase activity.In wine,the L-malic acid degradation of the five mutant strains was higher than that of the starting strain SD-2a.Among them,the mutant strain ARTP-2 completely degradation L-malic acid on the 18^th day,which was 12 days earlier than that of the starting strain SD-2a.Compared with the starting strain SD-2a,the variety and content of aroma components of wine inoculated with mutant strain ARTP-2 for malolactic fermentation increased significantly,and a total of 46 aroma components were detected,and the total content was 443.01 mg/L.According to the odor activity value of aroma substances,the main contributors of aroma components of mutant strain ARTP-2included esters,alcohols,aldehydes and ketones,acids and terpenes,which were more abundant than those of the starting strain SD-2a.（3）Transcriptome analysis of acid-tolerance mechanism of O.oeni.Through transcriptome sequencing analysis,it was confirmed that there were 1021 differentially expressed genes between primitive strain SD-2a and mutant strain ARTP-2 during acid stress,including 491 up-regulated genes and 530 down-regulated genes.In the Gene Ontology functional enrichment analysis,it was found that there were significant differences in gene expression in the cellular process,metabolism process,binding and catalytic activity.In the Kyoto Encyclopedia of Genomes pathway enrichment analysis,it was determined that the differential genes were mainly in fatty acid biosynthesis and metabolism,nucleotide metabolism,amino acid biosynthesis and metabolism and other pathyways.Through the analysis of differential gene expression in some modules,it was found that the up and down-regulated genes related to fatty acid synthesis,amino acid synthesis and metabolism and nucleotide metabolism pathway were related to acid tolerance of O.oeni.