Synergistic Antibacterial Mechanism Of Kojic Acid And Tea Polyphenols In Combination And Their Application On Chilled Aquatic Products

In order to solve the spoilage and food safety issues caused by the contamination and proliferation of foodborne bacteria in refrigerated aquatic products.In this study,in vitro antibacterial tests were carried out with the dominant spoilage bacteria and foodborne pathogens commonly found in chilled fish,which suggested that kojic acid(KA)and tea polyphenols(TP)exhibited synergistic antibacterial effects and were suitable for the applications in aquatic products.Hence,the preservation and pathogen elimination effects of KA and TP alone or in combination on chilled fish(sea bass and salmon)fillets were determined.In addition,the synergistic antibacterial mechanisms of KA and/or TP against Escherichia coli O157: H7 were also explored by phenotype analyses and RNA-seq.The specific research contents and conclusions were as follows.The minimal inhibitory concentrations(MICs),fractional inhibitory concentration index(FICI)and time-kill curves of kojic acid,phenyllactic acid,tea polyphenols and thymol against Shewanella putrefaciens and Pseudomonas fluorescens were determined to evaluate their combined antibacterial effects in vitro.The results showed that both KA and TP were effective in inhibiting these two bacteria,and their MICs were all below 2 mg/m L;The FICI of KA and TP in combination against S.putrefaciens and P.fluorescens were 0.5 and 0.25,respectively,indicating that KA and TP exhibited strong synergistic effects against two tested spoilage bacteria,and thier synergistic inhibition on P.fluorescens was much better;The synergistic effects of KA and TP were further verified by time-kill curves,which demonstated that KA combined with TP exhibited better antibacterial effects while reducing their dosage.Therefore,the preservation effects of KA and TP were further evaluated using chilled bass fillets as the model of aquatic products,which demonstrated that KA and/or TP could inhibit bacterial proliferation and lipid oxidation,thus effectively maintaining the freshness and physicochemical quality of fish.According to the results of all physical and chemical indexes and principal component analysis,the combination of KA and TP exhibited better preservation effect than their use individually,and extend the shelf-life of chilled bass fillets by 5-6 days,indicating that KA-TP could be used as a natural compound preservative for aquatic products.Similarly,the synergistic antibacterial effect of KA and TP against the common foodborne pathogen(i.e.,E.coli O157: H7)was verified by in vitro antibacterial tests.The MICs of KA and TP against E.coli O157: H7 were 1.6 and 1.0 mg/m L,respectively.The FICI of KA and TP in combination was 0.25,indicating that KA and TP had excellent synergistic inhibitory effect against E.coli O157: H7 in addition to the above two Gram-negative spoilage bacteria.Thus,it was speculated that KA and TP might have synergistic inhibitory effects on most Gram-negative bacteria.Therefore,the inhibitory mechanisms of KA and/or TP on E.coli O157: H7 was studied from three dimensions of cell membrane,genomic DNA and intracellular protein metabolism to explore the reasons behind the synergistic inhibitions of KA and TP on Gram-negative bacteria,and to provide some theoretical support for their optimization and application.The results suggested that KA and TP could attack the bacterial cell membrane cooperatively,thus disturbing membrane integrity and cell structure.Subsequently,KA could bind to bacterial genomic DNA by inserting base pairs to affect the biofilm formation and other physiological functions of E.coli O157: H7,thereby reducing its ability to resist external stress and eventually leading to bacterial lysis and death.Furthermore,the elimination effects of KA and/or TP on the contamination of E.coli O157: H7 in chilled salmon fillets were determined to verify their depuration effects on ready-to-eat(RTE)raw aquatic products.The results demonsrated that the combination of KA and TP performed better in elimination of pathogenic bacteria than them individually,and the clearance rate of E.coli O157: H7 contamination in chilled salmon fillets could reach 99.04% when treated with the combination of KA and TP at 2 MIC,suggesting that KA-TP was also suitable as natural compound disinfectants for the control of pathogenic bacteria in RTE raw aquatic products.In order to indirectly elucidate the antibacterial mechanism of KA or TP and the reasons behind their synergistic effect,this study revealed the response of E.coli O157: H7 to the stresses of sublethal KA and/or TP using high-throughput transcriptome sequencing technology.The results indicated that 978,135 and 232 differentially expressed genes(DEGs)were identified in KA,TP and their combined treatments,respectively.The enrichment analysis and discussion of these DEGs were conducted on the basis of the results of phenotypic experiments and previous reports,and specific conclusions are as follows: 1)KA inactivated E.coli O157:H7 not only by causing acid stress and oxygen starvation,but also by destroying cell membranes and genomic DNA,while DNA seemed to be the most important target;2)Under the stress of TP,E.coli O157: H7 confronted a series of adverse conditions,including oxidative stress,sulfate starvation and energy generation inhibition;3)Apart from the above two adverse effects caused by KA and TP individually,the hindered iron uptake pathways by KA-TP might be the reason behind the synergistic antibacterial effect of KA and TP,which would directly reduce the resistance of E.coli O157: H7 to external stress.