Gene Expression And Regulation Of Nitric Oxide Synthase In Coagulase Negative Staphylococci And Its Application In Fermented Sausages

Color is an important indicator for consumers to evaluate the quality of meat products in which nitrite is vital for the color formation.However,the health threat associated with using nitrite has raised public concerns,since toxic N-nitrosamines can be generated from residual nitrite in the presence of secondary amine groups.Therefore,alternatives to substrate nitrite are urgently needed.The objective of this study was to investigate gene expression and regulation of nitric oxide synthase（NOS）in coagulase negative staphylococci（CNS）and its application in fermented sausages for nitrite replacement.The results showed that:（1）Expression and characterization of nitric oxide synthaseUnder normal physiological conditions,CNS were found to express NOS protein which can not only metabolizeinto NO but also form nitrosomyoglobin（NO-Mb）in meat products.Also,the nos gene from CNS species could be expressed in Escherichia coli BL21（DE3）and NOS protein was purified and identified.（2）Comparison of NOS activity and color-formation ability in different CNS species By comparing the amino acid sequences of different CNS strains,it was found that some key phosphorylation sites and dimer interface residues of NOS amino acid sequence may be the key factors that led to different NOS activities and NO-Mb formation ability among various CNS species.The results showed that the NOS activity of Staphylococcus equorum and Staphylococcus vitulinus was five times more than that of Staphylococcus carnosus.In fermented sausages,the results revealed that the content of NO-Mb in the sausages inoculated with S.vitulinus was three times more than that of the sausages inoculated with S.equorum or S.carnosus.（3）Effects of fermentation conditions on the production of NOSIt was found that adding different sugars could affect the NOS activity in S.vitulinus,which caused the different NO production.Compared with glucose,sucrose can enhance the level of oxidative stress in S.vitulinus,which led to the overexpression of nos and kat A gene,and thus the ability of oxidative stress resistance was up-regulated and the survival rate of S.vitulinus after H₂O₂ treatment was increased.In the fermented sausages,sucrose enhanced NO production of S.vitulinus,which promoted the generation of NO-Mb and color formation.It was illustrated that the up-regulation of nos gene in S.vitulinus could contribute to the color formation and achievement of nitrite substitution to improve the safety of meat products.