Study On Inhibition And Eradication Effects Of Food Preservatives On The Biofilms Of Vibro Parahaemolyticus

Vibro parahaemolyticus is a halophilic Gram-negative bacteria. It is widely distributed in the marine or estuarine environments and frequently isolated from a variety of seafood and salted food. V. parahaemolyticus is also an important food-borne pathogen,which would cause food poisoning. Biofilms can be defined as a surface-attached community of microorganisms embedded and growing in a self-produced matrix of extracellular polymeric substances, which biofilms can resist to adverse conditions and lead persistent infection. Biofilms bring a serious risk in medical and food safety, because it is difficult to be eradicated by ordinary antibiotics and disinfectants, once they are formed.Therefore, the future course of action would be directed to natural and safe anti-biofilm agents, which can be treat biofilm-associated infections effectively.To explore the inhibition and eradication of V. parahaemolyticus biofilms by preservatives, crystal violet staining, plate counting, broth dilution method, XTT reduction method, phenol-sulfuric acid method, fluorescence microscope and confocal laser scanning microscope(CLSM) were used. The main results were as follows:The ability of biofilm formation of 24 strains isolated by ourselves and V.parahaemolyticus ATCC17802, J5421 was analyzed in vitro under different conditions. The results showed that the ability of NO.21 strain and NO.22 strain forming biofilm were stronger and the factors such as incubation time, temperature, p H, salinity, and adhesion materials could affect the formation of biofilms. Biofilms formation were better when p H were between 5.0 to 9.0. The larger biofilms of V. parahaemolyticus ATCC17802, J5421 and NO.22 strains were formed when the concentration of Na Cl reached 3.0%, while the lower salinity was beneficial to biofilm formation for NO.21 strain. Compared to the coverslip, stainless steel and fish scale, the adherence ability of crab shell was strongest.ATCC17802 and J5421 were selected for further research of inhibition effects of food preservatives on the biofilm formation. Among chitosan, potassium sorbate, sodiumdehydroacetate and ?-polylysine(?-PL), we found that the antibacterial activity of chitosan was strongest. Its MIC and MBC were 1.25 mg/m L and 2.5 mg/m L, respectively. When V.parahaemolyticus were cultivated in TSB with 1/2MIC, 1/4MIC and 1/8MIC chitosan 2 d,the bacterial inhibition rates could reach 68.37%, 34.12% and 14.38%, respectively; the metaboic activity reductions of biofilm were 47.74%, 34.34% and 17.17%, individually;and the decrease of extracellular polysaccharide(EPS) were 76.32%?62.57% and 29.38%respectively. Sub-MIC of chitosan could significantly inhibit biofilm formation and reduce the metabolic activities of bacteria. When incubated in TSB with 1/2MIC chitosan combined with 1/4MIC potassium sorbate, 1/2MIC sodium dehydroacetate, 1/4MIC?-polylysine, the inhibition rate of biofilm formation reached 79.54%, the reduction of metabolic activity was 59.72% and the inhibition rate of EPS was 85.44%. These indicated that the combination of Sub-MIC food preservatives could enhance the inhibitory effect of biofilm formation.The impacts of food preservatives on V. parahaemolyticus mature biofilms were also researched. We found that, eradication rates of biofilms were 86.94%?61.43%?46.94%respectively; metabolic activity reductions of bacteria reached 76.10%?63.11%?52.20%individually, and the EPS decreased 81.43%?69.63%?61.12% respectively, when mature biofilms were incubated in 4MIC, 2MIC, MIC chitosan for 4 h. It signified that high concentration of chitosan could eradicate mature biofilms effectively. Furthermore, the combination of 4MIC chitosan, MIC potassium sorbate, 2MIC sodium dehydroacetate and2 MIC ?-polylysine was used to eradication mature biofilms. The eradication rate of mature biofilms was 96.34%, reduction of metabolic activity was 85.64% and the EPS decreased89.71%. Compared to single preservatives, a pleasant surprise was the combination of food preservatives could enhance the eradication effect of the mature biofilms.In this study, we could observe the decrease of the numbers of viable cells in biofilms by fluorescence microscope. We are also able to find the reductions of EPS, dispersion and thinning of biofilms under the CLSM intuitively.