| Soy sauce is an important and popular condiment that produced from soybean meal,wheat and other raw materials through fermentation involved with a variety of microorganisms.Safety,tasty and healthy soy sauce matches the demand of costumers for pursuing new traits of food,with the improvement of society.Usage of Aspergillus for making koji and addition of yeast significantly promotes the fermentation process and enhancement of the flavor of soy sauce.However,the mixed microorganism’s fermentation system sometimes is unstable.Contamination of spoilages bacteria also effects of the quality,flavor and safety of soy sauce.This is mainly related to poor understanding of the composition of the microflora and the function of dominant bacteria in soy sauce moromi.Weissella paramesenteroides is the main dominant bacteria in high-salt dilute soy sauce fermentation.It is important to disclose its function in soy sauce fermentation,and clarify its influences on other microorganisms,for accomplish optimization and stable control of soy sauce fermentation.In this study,the effects of synergistic fermentation of W.paramesenteroides on the quality and flavor of soy sauce,metabolism of microbial and composition of microbial community were studied,and the role of W.paramesenteroides in promoting the synthesis of flavor substances in soy sauce and the balance of microbe community in the fermentation was elucidated.The main contents and results of the study are as follows:(1)The role of synergistic fermentation of W.paramesenteroides in improving the quality and promoting the synthesis of flavors in soy sauce was revealed.The addition of W.paramesenteroides LCW28 during the fermentation of soy sauce which increased the number of W.paramesenteroides and yeasts in moromi by 1～2 and 4～5 magnitudes.The best way of synergistic fermentation was the simultaneous addition of W.paramesenteroides LCW28 and Z.rouxii ZQ01,which resulted in 95.44%and 36.77%higher amino acid nitrogen and total amino acid content in soy sauce than the control,respectively;The umami and sweet amino acids increased by 55.05%and 22.56%,respectively.In addition,synergistic fermentation with W.paramesenteroides LCW28significantly improved the salty and umami taste of the soy sauce.In addition to the significant increase in the content of 4-ethyl guaiacol,which contributes significantly to the umami of soy sauce,the content of various volatiles in soy sauce increased significantly to3229.39μg/L,which was 3.4 times higher than that of the control group.Among them,esters and alcohols increased by 11.86-folds and 1.39-folds compared to the control.(2)The mechanism of promoting the growth of Z.rouxii and its synthesis of volatiles in soy sauce(4-ethyl guaiacol)by W.paramesenteroides was elucidated.The co-culture revealed that W.paramesenteroides LCW28 increased the amount of Z.rouxii ZQ01 in the system by one order of magnitude to 1.3×10~8 CFU/m L.With the promotion of W.paramesenteroides LCW28,the expression levels of key genes such as pad1,tal,aro B,aro A,aro F,and tyr A and the enzyme activities of their encoded products in the pathway of synthesis of 4-ethyl guaiacol(4-EG)in soy sauce by Z.rouxii ZQ01 increased 2.6-fold to5.2-fold and 1.2-fold to 8.2-fold,respectively(p<0.05).In the co-culture system,W.paramesenteroides LCW28 increased the level of synthesis of 4-EG by Z.rouxii ZQ01 to21.3 mg/L compared with that of Z.rouxii ZQ01 alone which was 3.17 times higher than that of Z.rouxii ZQ01 cultured alone.This study also confirmed that Z.rouxii ZQ01 can synthesize 4-EG via the shikimic acid pathway.(3)The influence of functional microorganisms such as W.paramesenteroides on the keep the balance of flora and synthesis of volatiles was revealed by gradient salt-reduced fermentation of soy sauce.The decrease of salt in the moromi resulted in a significant decrease in the abundance and number of functional microorganisms and a significant increase in the abundance and number of spoilage bacteria.The numbers of functional bacteria(W.paramesenteroides,Z.rouxii and Candida versatilis)decreased by 2～5magnitudes,respectively,while the numbers of spoilage bacteria(Bacillus megaterium,Bacillus halodurans,Bacillus megaterium,Bacillus halodurans,Bacillus subtilis,Staphylococcus saprophyticus,Lactobacillus pobuzihii and Kurthia)increased by 2～4orders,respectively.When the salt content≤12%,the amino acid nitrogen content in soy sauce was lower than 0.3 g/100 m L,the umami amino acid content decreased by56.99%～61.57%,the sweet amino acid content decreased by 11.86%～12.85%,the unacceptable volatiles increased significantly,and the biogenic amine content increased by31.25-46.63 fold.Analysis of the correlation between volatiles and microorganisms showed that functional microorganisms such as W.paramesenteroides were significantly and positively correlated with main volatiles in soy sauce,while unacceptable volatiles were significantly and positively correlated with Bacillus megaterium and other spoilage microorganisms were significantly and positively correlated in soy sauce.(4)Salt-reduced soy sauce fermentation of was achieved through synergistic fermentation with functional microorganisms including W.paramesenteroides,based on balancing of microbe community.Two functional strains include W.paramesenteroides JL-5,which can inhibit Bacillus,Kurthia and Staphylococcus,and B.amyloliquefaciens JDF1,which can inhibit Lactobacillus pobuzihii,were isolated from moromi.Synergistic fermentation with B.amyloliquefaciens JDF1 and W.paramesenteroides JL-5 inhibited the growth of spoilage bacteria such as Bacillus megaterium,promoted the growth of functional microorganisms such as W.paramesenteroides,and resulted in successfully salt reduction fermentation of soy sauce.The amino acid nitrogen content of the synergistic fermentation salt-reduced soy sauce was 2.21 times higher than that of CL(12%Na Cl),reached 1.23g/100 m L;the biogenic amine content decreased by 70.52%;and the composition of volatiles was close to that of the high-salt control CH(18%Na Cl).In addition,the synergistic fermentation with W.paramesenteroides promoted the synthesis of isoamyl alcohol,which could increase the salinity of the reduced-salt soy sauce with a salt equivalent of 13.8 g/100 m L while the measured salt content was 8.3 g/100 m L.(5)A synthetic microbiome including W.paramesenteroides and Aspergillus oryzae and other functional microorganisms for soy sauce fermentation was established.Through preliminary analysis of the metabolism,growth and interactions of the dominant and functional microorganisms in morom.This study screened and identified five functional bacteria(W.paramesenteroides,B.amyloliquefaciens,Staphylococcus epidermidis,Pediococcus acidilactici and Lactobacillus plantarum)and five functional fungi(A.oryzae,Z.rouxii,C.versatilis,Pichia manshurica and Millerozyma faronisa)were identified as synthetic microbiomes for soy sauce fermentation.By optimizing the inoculation sequence of the synthetic microbiome in the simulated soy sauce fermentation system with reference to the microbial community succession pattern during the fermentation of high-salt dilute soy sauce.The synthetic microbiome inoculation pattern for soy sauce fermentation was determined as follows:non-lactobacilli bacteria(1.0×10~8 CFU/m L)at the beginning of fermentation,LAB(1.0×10~8 CFU/m L)on day 10,fungi(1.0×10~7 CFU/m L)on day 20.Preliminary fermentation of soy sauce with defined microbial composition was achieved using the established synthetic microbiome.(6)The role of synergistic fermentation of W.paramesenteroides in improving the flavor and quality of high-salt dilute soy sauce was verified at the fermenter(15 L)level.The synergistic fermentation of high-salt diluted soy sauce by W.paramesenteroides LCW28 significantly increased amino acid nitrogen by 23.48%to 1.63 g/100 m L,volatiles content by 61.37%,and 4-ethyl guaiacol by 2.45-fold to 325.41μg/L.Synergistic fermentation salt-reduced soy sauce with W.paramesenteroides JL-5 and B.amyloliquefaciens JDF1 for 60 days to reach 1.62 g/100 m L,and other quality and flavor indicators were close to those of the high-salt control soy sauce.Preliminary fermentation of soy sauce was achieved using a synthetic microbiome containing W.paramesenteroides,with amino acid nitrogen reaching 1.44 g/100 m L and volatiles type close to the control group.The results of sensory evaluation showed that the taste,aroma and overall evaluation of high-salt diluted soy sauce and reduced-salt soy sauce with synergistic fermentation with W.paramesenteroides were better than those without the addition of W.paramesenteroides. |
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