Intraspecific Diversity Of Saccharomvces Cerevisiae And Lactobacillus Sanfranciscensis Isolates From Chinese Traditional Sourdough And Their Interaction

Sourdough,is also known as Laomian,Jiaozi et al.in China.The steamed bread fermented with sourdough has always been preferred by consumers for its unique flavor and particular taste.However,there is a lack of systematic research on its dominant microorganisms and their interactions in China.In this work,the dominant microorganisms,Saccharomyces cerevisiae and Lactobacillus sanfranciscensis,were isolated from Chinese traditional sourdoughs and their intraspecific diversity were revealed based on both genetic and metabolic characteristics.In addition,the interaction between these two species during sourdough fermentation and its effect on the steamed bread quality were also investigated.The 98 L.sanfranciscensis strains isolated from 13 Chinese traditional sourdoughs in different regions were divided into 6 and 10 different types by multiplex-RAPD and MLST,respectively.MLST seems to have a higher discriminatory power compared with multiplex-RAPD since it produced more groups,but multiplex-RAPD could help to distinguish some strains in the same ST.Hence,an optimal genotypic characterization of L.sanfranciscensis was oobtained under the combination of MLST and multiplex-RAPD analysis.According to the result of MLST,it seems that the genotypic variations of L.sanfranciscensis strains have no relations to geographical origin,and most of the isolates have a close relationship.On the basis of the genotyping results,the metabolic characteristics of 28 L.sanfranciscensis strains were tested and compared.The proteolytic ability varied among L.sanfranciscensis strains.The variation was also found concerning the acid and volatile compounds of sourdough fermented with these strains.However,there was little difference in ESP produced by these strains.Compared the result of genotypic and metabolic characteristics,we found that no obvious relationship could be found between genetic diversity and metabolic properties of L.sanfranciscensis strains,indicating the molecular characterization obtained by multiplex-RAPD and MLST analysis might not reflect the heterogeneity of some specific regions of the genome which are more correlated with phenotypic traits,such as proteolytic ability,acid and volatile compounds production.One hundred and fifty isolates of S.cerevisiae from thirteen Chinese traditional sourdoughs in different regions were identified and typed into 5 clusters by RAPD analysis.According to the RAPD result,22 S.cerevisiae strains were selected to evaluate the fermentative properties.The leavening and glucose fermentation abilities varied among S.cerevisiae strains.The variation was also found concerning the texture and volatile compounds of steamed bread fermented with these strains.According the results,no obvious relationship could be found between genetic diversity and fermentative properties of S.cerevisiae strains,indicating the molecular characterization obtained by RAPD analysis might not reflect the heterogeneity of some specific regions of the genome which are more correlated with phenotypic traits,such as glycolysis route and Ehrlich pathway.When S.cerevisiae and L.sanfranciscensis were mixed with the ratio of 107:107 CFU/g,they tended to stabilize during sourdough fermentation at approximately 6h,which had a shorter time than other ratios.During the mixed-culture fermentation,the contents of maltose,glucose and fructose in the dough showed a trend of increasing first and then decreasing,while the content of sucrose decreased continuously.L.sanfranciscensis can influence the carbohydrate metabolism of S.cerevisiae.It released glucose during the use of maltose,which thereby increasing the metabolism of glucose but reducing the metabolism of maltose by S.cerevisiae.The total amount of amino acids in the dough showed a trend of decreasing first and then increasing during the cofermentation process,and it was higher than the single-culture samples fermented with S.cerevisiae and L.sanfranciscensis.Moreover,some amino acids that play an important role in flavor formation,such as valine,phenylalanine,tyrosine,cysteine,etc.,all increased at the later stage of cofermentation,and have higher content than the single-culture samples.Cofermentation of S.cerevisiae and L.sanfranciscensis also had a certain influence on the flavor composition of the sourdough,it produced a new volatile compound,isoamyl lactate,and promoted the synthesis of organic acids such as acetic acid and 2-methyl-hexanoic acid,but significantly reduced the content of some volatile substances which mainly produced by S.cerevisiae,like 2,3-pentanedione,2-methyl-1-propanol,3-methyl-1-butanol and phenylethyl alcohol.The transcriptomic analysis of the interaction between S.cerevisiae and L.sanfranciscensis showed that the different L.sanfranciscensis strains Sx14 and Ts1 have different effects on the gene expression of S.cerevisiae during co-culture fermentation.However,these two L.sanfranciscensis strains all resulted in the increase of glucose metabolism and the conversion of arginine to ornithine and the decrease of ethanol synthesis in S.cerevisiae.HXK2,CAR1 and PDC6 are the key genes in these pathway,and we validated their expression changes in S.cerevisiae during mixed fermentation by qPCR.In addition,the mixed fermentation also caused an increase in glycerol synthesis,amino acids metabolism and a decrease in biotin synthesis,glutathione metabolism,acetic acid synthesis,etc.,which also have a certain influence on the quality of steamed bread.When mixed fermentation with S.cerevisiae respectively,the related genes of diaminocnase,aminopeptidase and the synthesis of lactic acid and acetic acid had higher expression in L.sanfranciscensis strain Sx14 than strain Ts1,which seems that strain Sx14 would be more suitable for co-culturing with S.cerevisiae during sourdough fermentation.