Inhibition Of Biofilm Information Of Two Food-borne Spoilage Bacteria By Carvacrol And Thymol

Enterobacter cloacae and Enterococcus faecalis are widely found in the environment and animals intestines,and they are vulnerable to become dominant spoilage bacteria in raw meat.The spoilage bacteria can adhere on the surface of equipment and food.After forming biofilms during food processin.The biofilms can cause spoilage of food products or outbreaks of foodborne diseases because they are not easily be inactivated.Carvacrol and thymol are isolated from natural plant essential oils.they are antibacterial agents with broad-spectrum antibacterial effects and are recognized as food additives by the European Commission and FDA.At present,there were few report of studies on the inhibition of biofilms formation of spoilage bacteria by carvacrol and thymol.In this paper,the effects of carvacrol and thymol on biofilm formation of two foodborne spoilage bacteria were studied,and the regulation mechanism was explored.The two trains of E.cloacae C4 and E.faecalis R612 which were isolated from low-temperature meat products were used as the target strains.This research provided a theoretical basis for application of carvacrol and thymol in controlling biofilm formation of food spoilage bacteria and a data support for regulation or disinfection of biofilm formation in the future.The specific research contents and results of this thesis were listed as follows:(1)The minimum inhibitory concentration(MIC)of carvacrol and thymol to E.cloacae C4 was both 256 ?g/m L.The growth of E.cloacae C4 planktonic bacteria was inhibited under by 1/2 MIC treatment of carvacrol and thymol and it was not affected under 1/4 MIC and 1/8 MIC treatment.Crystal violet staining determined that E.cloacae C4 has strong biofilm formation ability,and carvacrol and thymol can inhibit biofilm formation of E.cloacae C4.Plate counting results showed that the effect of bacteria colony counts in E.cloacae C4 biofilm by different concentrations of carvacrol and thymol.The refer to 1/2 MIC treatment significantly reduced the bacteria colony counts in E.cloacae C4 within 24 h,but other treatments had no significant affect.The change of exopolysaccharide content was determined by phenol-sulfuric acid method.We found that 1/2 MIC and 1/4 MIC treatment could significantly inhibit the secretion of exopolysaccharide.carvacrol and thymol can make the bacterial cells longer and slow the biofilm aggregation process then decrease the thickness of biofilm that was observed by SEM and CLSM images.When high concentration of carvacrol and thymol(2048 ?g/m L)treatment to biofilm of E.cloacae C4 has formed 3 d.After 30 min treatmented,the bacteria colony counts in the biofilm decreased to half.Quantitative Real-time PCR results showed that after 1/4 MIC treatment to E.cloacae C4 the relative expression of biofilm formation related curli synthesis genes(csg G,csg B,csg E,csg F)and cell division genes(Mra Z,fts A,fts L,fts Q,fts Z)and exopolysaccharide synthesis genes(wca A,wca M,wza,wzb,rcs A)were significantly down-regulated.(2)The MIC of carvacrol and thymol to E.faecalis R612 was both 512 ?g/m L.1/2 MIC of carvacrol inhibits the proliferation of E.faecalis R612 planktonic bacteria.The effects of thymol on E.faecalis R612 planktonic bacteria basically with same as carvacrol.However,1/4 MIC of thymol inhibited the growth of E.faecalis R612 planktonic bacteria in the first 6 h.E.faecalis R612 had a strong biofilm formation ability but weaker than E.cloacae C4 was determined by crystal violet staining.The bacteria colony counts in the biofilm of E.faecalis R612 was determined by Plate counting method.The results showed that compared with the control group,the counts of 1/2 MIC treatment were significantly reduced during the 72 h,and 1/4 MIC or 1/8 MIC treatment group had no significant affect.The change of exopolysaccharide content was determined by phenol-sulfuric acid method that 1/2 MIC and 1/4 MIC treatment could significantly inhibit the secretion of exopolysaccharides.1/8 MIC treatment has no significant effect on inhibiting the secretion of exopolysaccharides during the culture time.SEM and CLSM showed that carvacrol and thymol no impact on morphology of bacteria in biofilm,but the biofilm aggregation process was slowed and the structure of biofilm was damaged then the thickness was reduced.High concentration of carvacrol and thymol(2048 ?g/m L)treatment to biofilm of E.faecalis R612 has formed 3 d.After 15 min treatmented,the bacteria colony counts in biofilm decreased by half.The results of Quantitative Real-time PCR showed that the relative expression of pili synthesis genes(ebp R,ebp A,ebp C)and cell division genes(fts Q,fts A)and exopolysaccharide synthesis genes(epa A,epa B,epa E,epa G,epa H,epa M,epa Q,epa R)and Qurorum sensing genes(lux S)were significantly down-regulated.(3)High-throughput sequencing technology was used to sequence E.cloacae C4 whole genome and combined with bioinformatics analysis.We can find that the total length of E.cloacae C4 genome was 4,879,501 bp and the sequence was matched to obtain 4531 coding genes that are 88.7% in whole genome and the average length of the gene is 956 bp.Meanwhle,other gene elements were predicted about 3 prophage and 10 gene islands.E.cloacae C4 has all the genes who involved in the regulation system of the exopolysaccharide colanic acid synthesis of Enterobacter.(4)According to the change of relative expression of biofilm related genes in E.cloacae C4,it was hypothesised that carvacrol and thymol may affec t the synthesis of exopolysaccharide colanic acid by inhibiting the cascade of HNS ?rcs A?wca gene cluster and then inhibit synthesis of exopolysaccharides of E.cloacae C4.The recombinant plasmid hns-p DE 1 was constructed by using p DE 1 as the expression vector.Then the recombinant protein expression strain hns-p DE 1-BL21 was constructed.The recombinant protein HNS was over-expressed.HNS protein was obtained after purification ? desalting and concentration.Through the biomolecular interaction instrument,it was found that the HNS protein can specifically bind tightly to the Rcs A promoter.The kinetic equilibrium dissociation constant is 8.082E-9.This indicates that the global regulatory factor-HNS is involved in the exopolysaccharide synthesis cascade regulation pathway of E.cloacae C4,which regulates the synthesis of Rcs A at the transcriptional level.The results of the research indicated that carvacrol and thymol could inhibit the biofilm formation of E.cloacae C4 and E.faecalis R612 that mainly by affecting the initial adhesion of biofilm formation and the secretion of exopolysaccharides.The difference in the isomers of carvacrol and thymol does not produce regular differences in the inhibitory effect of the same strain.It is hypothesised that carvacrol and thymol affect the synthesis of E.cloacae C4 extopolysaccharide colanic acid through the cascade regulation of HNS?rcs A?wca gene cluster then it is preliminarily determined that HNS protein affected the synthesis of Rcs A protein at the transcriptional level.