Isolation,Identification And Application Of Flavor Functional Bacteria In Soy Sauce

Soy sauce is a traditional fermented condiment widely favored by consumers.The fermentation process of high-salt liquid fermented soy sauce is open to the air,so the types of microorganisms involved in the process are rich and diverse,and they are one of the main contributors to the formation of soy sauce flavor.Nowadays,there are many domestic and foreign researches on the dominant bacteria in the fermentation process of soy sauce,such as Tetragenococcus and Weissella,but less attention has been paid to microorganisms with low relative abundance in soy sauce.In this thesis,high-throughput sequencing and GC-MS technologies were used to systematically analyze the relationship between bacterial communities and flavor substances in soy sauce samples,and to explore potential flavor-functional bacteria.Based on the team's early research and the fermentation model of single and mixed microorganism,the effects of potential flavor functional bacteria were verified by combining sensitivity assessment,physical and chemical analysis,and flavor analysis.Main results of this thesis were summarized as follows:(1)Through the next-generation sequencing,the bacterial composition of different soy sauce residues was determined by 16S rDNA sequence analysis,and there were 16 genera with relative abundance more than 0.5%.The dominant bacteria(?50%)were Staphylococcus and Weissella.The relative abundance(<10%)of Lactobacillus,Tetragenococcus,Pediococcus and Bacillus was low,but the detection rate was high in different samples.The results of flavor analysis showed that lactic acid,succinic acid and acetic acid were the most organic acids in soy sauce,and their total contents accounted for about 70%of the organic acids.Glutamic acid,leucine and aspartic acid were the main free amino acids.The analysis results of aroma-active compounds showed that the aroma compositions of the five samples were different,and SS2had the highest total contents of aromas,especially alcohols,acids,aldehydes and furanones.(2)Based on the results of the microflora,physicochemical and flavor analysis in Chapter2,principal component analysis(PCA)and cluster analysis(CA)were performed and the results showed that free amino acids had the certain correlation with bacterial flora,while organic acids and volatile substances had correlations with bacterial flora.Therefore,an O2PLS(bidirectional orthogonal partial least squares)model was established for the composition of bacteria,organic acids and volatile substances in the soy sauces.Under the screening conditions of VIP_pred?1.0and the percentage of bacteria in any group higher than 0.5%,15 strains of potential flavor functional bacteria with influence on metabolites were obtained.Among then,it was found that some bacteria had mild positive correlations(r>0.5)with the content of lactic acid,including T.halophilus,Enterococcus faecalis,W.paramesenteroides and S.sciuri.Lactobacillus were positive correlated(r>0.9)with the content of succinic acid.In terms of volatile substances,bacteria that were positively correlated with the contents of HDMF,HEMF,and phenylacetaldehyde,included T.halophilus,Leuconostoc fallax and W.confusa.In addition,three kinds of Lactobacillus(L.fermentum,L.delbrueckii and L.salivarius)showed positive correlation with the contents of ethyl acetate,phenethyl alcohol and 2-furfuryl alcohol.(3)According to the characteristics of colony morphology and microscopic examination,41 strains were selected from the isolated and purified strains for 16S gene sequence analysis,and a total of 7 genera and 13 species of bacteria were identified.A total of 7 strains were identified(B.amyloliquefaciens,B.subtilis,S.saprophyticus,Kocuria kristinae,T.halophilus,L.fermentum and W.confusa)as representatives for subsequent analysis.Using the rapid simulation fermentation model,it was found that K.kristinae and T.halophilus had the strongest ability to produce acid,and S.saprophyticus had the fastest growth rate.In terms of the organic acid composition,T.halophilus produced the highest content of lactic acid,and L.fermentum produced the highest content of succinic acid.It was consistent with the conclusion that T.halophilus had a middle positive correlation(r=0.68)with the contents of lactic acid and L.fermentum had a highly positive correlation(r=0.95)with the contents of succinic acid in the Chapter 2.In terms of the influence on the aroma composition of the fermentation model,L.fermentum can significantly increase the content of 2-furfuryl alcohol,methionol,phenethyl alcohol,and 4-hydroxy-3-methoxystyrene,which was consistent with the correlation analysis results in Chapter 2.Sniffing sensory evaluation results showed that T.halophilus,L.fermentum and W.confusa had the most obvious enhancing effects,among which L.fermentum could enhance key aroma such as floral and roasted potato,while T.halophilus could make the whole aroma composition softer.(4)Different representative bacteria(7 strains)and yeasts(4 strains)were mixed in pairs and added into the rapid fermentation simulation model.The results showed that the yeasts generally promoted the proliferation of bacteria,while bacteria generally inhibited the proliferation of yeasts,among which there was a certain degree of mutualism between Z.rouxii and some bacteria,such as T.halophilus and L.fermentum.Combined with the analysis of PCA,it was found that L.fermentum could interact with Z.rouxii better,and their effects were significantly different from that of the single addition of bacteria or yeasts.They showed the outstanding performance in increasing the content of ethyl acetate.Besides,compared with the blank,L.fermentum could enhance the taste of fermented soy sauce,especially in umami and kokumi.It also could increase the intensities of floral and fruity,cooked-potato like and caramel-like notes,significantly.It means L.fermentum has a strong potential for fermentation application.