Regulation Of Biogenic Amines Associated With Spoilage In Mackerel Mediated By Quorum Sensing Signal Molecules DKPs

Due to abundant protein and amino acid,mackerel is prone to spoilage and deterioration under microbial growth and metabolism,and especially the formation and accumulation of biogenic amines is a serious threat to food safety and human health.Quorum-sensing signaling molecules regulate a variety of microbial functions including the biogenic amine-producing capacity and the spoilage ability.At present,the research on biogenic amines associated with scombroid spoilage is still focused on the ecological factors that affect the activities of related enzymes,and lacking in the deeper regulatory mechanisms.Therefore,this study initially explored the correlation of DKPs signaling molecules on biogenic amines and spoilage of mackerel.In order to provide theoretical basis and new ideas for storage and preservation of mackerel,the key biogenic amine-producing bacteria Morganella morganii and Klebsiella oxytoca were taken as the research objects,and the regulation mechanism of DKPs signaling molecules on biogenic amines and spoilage was investigated.The main results of this study are as follows:(1)By measuring the typical spoilage indexes(TVB-N value,TVC,pH,TBA and water holding capacity)of mackerel at different temperatures,it was found that the mackerel reached the end of shelf life at 4℃ for 8 d and 25℃ for 36 h,respectively.During the storage process,the biogenic amine contents increased continuously,and the growth rate of biogenic amines at 25℃ was much higher than that at 4℃.The histamine content of mackerel exceeded the histamine limit of American aquatic products(50mg/kg)when stored at 4℃ for 8 d and 25℃ for 12 h,respectively.This is also consistent with high concentrations of precursor amino acids,particularly histidine,detected in mackerel.In addition,cyclo-(L-Leu-L-Leu),cyclo-(L-Pro-L-Leu)and cyclo-(L-Pro-L-Phe)signal molecules were detected in mackerel by HPLC-MS.Correlation analysis showed that there was a significant correlation between biogenic amine contents and spoilage indexes of mackerel,and the contents of DKPs was also significantly correlated with biogenic amine contents and spoilage indexes.Then,two key biogenic amine-producing strains,Morganella morganii and Klebsiella oxytoca,were isolated from mackerel by plate method combined with high performance liquid chromatography(HPLC).(2)The key amine-producing strains,M.Morganii and K.oxytoca,were cocultured,and it was found that the ability of biogenic amine production and spoilage were significantly increased in the co-culture system,and the expression of related genes were also significantly up-regulated.Both strains were detected to produce three DKPs signal molecules,cyclo-(L-Leu-L-Leu),cyclo-(L-Pro-L-Leu)and cyclo-(L-ProL-Phe),and the signal molecules production of co-culture system was significantly enhanced,compared with that of single culture system.In addition,by adding supernatant of M.morganii and K.oxytoca and exogenous DKPs,the biogenic amine production and spoilage potential were significantly increased,and the expression levels of related genes were upregulated to varying degrees,among which cyclo-(LLeu-L-Leu)signal molecules had the most obvious promoting effect.This further confirmed that DKPs signaling molecules can promote the biogenic amine-producing capacity and spoilage capacity of M.morganii and K.oxytoca by regulating the transcription of related genes.(3)Through genome analysis and homology comparison,it was found that Klebsiella oxytoca had potential NRPS gene encoding non-ribosomal peptide synthase,and the ΔNRPS knockout strain was constructed.By comparing the phenotypic between the knockout strains and the wild strain,it was found that the deletion of NRPS gene had no effect on the growth of the knockout strain,while the contents of DKPs signal molecules in the knockout strain was significantly reduced,as well as the production of putrescine and cadaverine and the yield of biofilm.In addition,the TVB-N value and pH value of sterile mackerel fillets inoculated with the knockout strain were significantly lower than those of the wild strain during storage,and the expression of related genes also showed varying degrees of down-regulation after gene NRPS deletion.These results further confirmed that DKPs signaling molecules could regulate the biogenic amine-producing capacity and the spoilage capacity of bacteria,and then affect the biogenic amine production and spoilage process of mackerel.Finally,compared with the co-culture of M.morganii and K.oxytoca,there were no significant changes in the production of DKPs signaling molecules,as well as the biogenic amines and spoilage related phenotypes in the co-culture of M.morganii and ΔNRPS.It is speculated that K.oxytoca may have signal molecule eavesdropping ability,which can use the signal molecules produced by M.morganii to regulate its physiological function.The results of this study provide reference for the targeted inhibition of biogenic amines and spoilage in aquatic products based on quorum sensing.