| Soy sauce is a traditional condiment originated in China and brewed from soybeans and wheat through microbial fermentation.Except the role of Aspergillus oryzae in soy sauce fermentation,the synergistic effect of other function microorganisms is the key to the formation of flavor and quality.Acid-producing bacteria in soy sauce brewing can ferment at high-salt condition to produce organic acids,which contribute to the formation of palatable sourness,sweetness and mellowness of soy sauce.The formation of esters from organic acids and alcohols is beneficial to the formation of sauce aroma.Staphylococcus is an acid-producing bacterium often found in fermented foods,with the ability of acid production,free amino acid accumulation,color protection and fragrance enhancement,which can enhance the flavor and quality of fermented foods.However,few studies have been reported on moromi Staphylococcus,and the its performance and effect on soy sauce fermentation needs to be studied in depth.In this paper,Staphylococcus with excellent acid-producing performance was screened and identified from high-salt liquid-state moromi.And its fermentation performance,the effect on soy sauce flavor and quality,and the effect of co-fermentation with ester-producing yeast on high salt liquid-state soy sauce were studied,in order to lay a foundation for the industrial application of Staphylococcus in soy sauce brewing.The main results of this paper are as follows:(1)In order to obtain salt-tolerant Staphylococcus with excellent acid production,the 34 Staphylococcus spp.were isolated from moromi using bromocresol purple-MRS plates.The strains with pH value of fermentation broth lower than 4.5 and the accumulation of lactic acid greater than 2.0 g/L were screened,and three strains with good acid production capacity and high lactic acid content named JL04,JL15 and JL17 were obtained.16S rDNA sequence analysis combined with physiological and biochemical tests shows that the strains were identified as S.condimenti,S.piscifermentans and S.carnosus.The hemolysis test of the three strains was negative,and they were preliminarily determined to be safe food fermentation strains,which can be used for further research.(2)In order to investigate the properties of salt-tolerant Staphylococcus in soy sauce brewing,the salt-tolerance,growth,acid production,antibacterial effect and heat resistance of the screened Staphylococcus were studied.The results showed that all of the three strains grew well in liquid MRS medium with 80～160 g/L NaCl.The analysis of MRS fermentation broth with 160 g/L NaCl indicated that the three strains could metabolize to produce L-malic acid,lactic acid,acetic acid,citric acid,succinic acid and fumaric acid.The strain with the best acetic acid production performance was JL04(acetic acid accumulation of 9.06 g/L),and the strain with the best lactic acid production performance was JL15(lactic acid accumulation of 2.36 g/L).The study of antibacterial effect showed that the strains could effectively inhibit the growth of contaminated bacteria in soy sauce fermentation.The antibacterial effect types of fermentation broth were divided into acid inhibition and non-acid inhibition,which had synergistic promoting effect.In addition,the three strains reached 100%lethality at 65℃ for 5 min,and can be killed by pasteurization without affecting the biostability of the soy sauce.In conclusion,the three strains can be used for soy sauce brewing.(3)In order to explore the effect of salt-tolerant Staphylococcus on the flavor and quality of high-salt liquid-state soy sauce,the strains JL04,JL15 and JL17 were added to high-salt liquid-state soy sauce for fermentation studies.The physicochemical indexes,organic acids and volatile flavor compounds of soy sauce were analyzed.The results showed that all three strains of salt-tolerant Staphylococcus could promote the accumulation of total acid and ammonia nitrogen in soy sauce,among which JL04 elevated the total acid of soy sauce most significantly with 17.03 g/L,which was 12.41%higher compared with the control group.The JL17 elevated the ammonia nitrogen of soy sauce most significantly with 9.92g/L,which was 7.71%higher than the control group.During the fermentation process,the accumulation of lactic acid,acetic acid and succinic acid was affected by the addition of 3 strains,and metabolic accumulation pathway analysis revealed they were up-regulated organic acid.Among them,the JL04 had the most significant improvement on these three organic acids,and their contents were increased by 26.17%,28.01%and 51.24%,respectively,compared with the control.The SPME-GC-MS analysis showed that the fermentation of the three strains increased the types and contents of volatile flavor compounds in soy sauce,with the most significant increase in the contents of acetate and lactate ester.The sensory evaluation results of soy sauce showed that the addition and fermentation of the three Staphylococcus were all beneficial to the improvement of the aroma and taste for soy sauce.Therefore,the three strains of salt-tolerant Staphylococcus can be used as brewing flavor bacteria in the soy sauce fermentation.(4)In order to analyze the effect of the co-fermentation of Staphylococcus and esterproducing flavor yeast CS2.42(Zygosaccharomyces rouxii)for the flavor and quality of soy sauce,the strain JL04,JL15,JL17 were added to moromi to co-fermentation with CS2.42,using the moromi added CS2.42 alone as the control.The physicochemical indixes,organic acids and volatile flavor compounds of soy sauce were analyzed.The results showed the contents of total acid and ammonia nitrogen in soy sauce co-fermented with 3 strains of Staphylococcus and CS2.42 were higher than that in soy sauce fermented with CS2.42 alone.The total contents of 6 characteristic organic acids in co-fermented soy sauce were increased by 30.68%,23.20%and 24.10%than the control.SPME-GC-MS analysis showed that the co-fermentation of three strains with CS2.42 significantly increased the content of esters associated with characteristic organic acids metabolized by salt-tolerant Staphylococcus in soy sauce,among which the cofermentation of JL04 and CS2.42 contributed the most to it.Sensory scores showed that the taste,aroma and total score of soy sauce co-fermented with Staphylococcus and CS2.42 were higher than those fermented by Staphylococcus or CS2.42 alone,indicating that the cofermentation of Staphylococcus and ester-producing flavor yeast was more conducive to the formation of good flavor in soy sauce.This research enriched the strain resources of flavor bacteria for soy sauce brewing,explored the role of salt-tolerant Staphylococcus in high-salt liquid-state fermentation,clarified the effect of its co-fermentation with ester-producing flavor yeast on the flavor of soy sauce,and laid the foundation for the application of salt-tolerant Staphylococcus in high-salt liquidstate soy sauce brewing for acid production and flavor enhancement. |
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