Study On Formation Characteristics Of Vibrio Parahaemolyticus Biofilm In Aquatic Products

Biofilms, a self-protection growth pattern of bacteria, are defined as communitiesof microorganisms that attach to surfaces and are a prevalent mode of growth formicroorganisms in nature, which are different from planktonic cells. Pathogenicmicroorganisms can also attach to and grow on food surfaces, equipment and processingenvironments to form biofilms, which is very difficult for them to be completelydestroyed or removed. In addition, the occurrence of biofilms can increase energyconsumption, accelerate corrosion of metal surfaces and cause potential food safetyissues. Vibrio parahaemolyticus, a halophilic bacterium occurs widely in marine water,sediment and seafood, which is recognized as the most prevalent foodborne pathogen inaquatic product. In this experiment, the growth conditions, bacterial motility,two-dimensional electrophoresis analysis and other aspects of biological factors on V.parahaemolyticus biofilm formation were studied.（1）By biochemical experiments and automatic microorganism identification anddrug sensitivity analysis system (VITEK-2), a total of4suspected isolated strains wereidentified as V. parahaemolyticus. The stains were isolated from Pampus argenteus,Mactra veneriformis, sea catfish, Venerupis philippinarum, which were numbered asstrain A02, A08, A11, A15.（2）To optimize the formation conditions of V. parahaemolyticus biofilm,96-wellmicrotitre plate assay was used to stimulate biofilm formation and to determinate thebiomass.The results showed that, V. parahaemolyticus, with an initial bacterialconcentration of107cfu/mL, at28℃, under weak alkaline condition (pH8-9), with3%NaCl, formed stable biofilm after culturing for60h. It was observed that addition of2%saccharides (glucose, sucrose, lactose) and0.5%Ca2+would enhance the ability of V.parahaemolyticus to form biofilm.While addition of Mg2+, Cu2+, Fe3+inhibited theformation of biofilm.（3）The results indicated that, when V. parahaemolyticus was cultivated at28℃,pH8.0and3%-7%NaCl, three types of motilities were visibly observed. Adding0.20mmol/L EDTA,50mg/L sodium nitrate,15.0g/L magnesium chloride and1.0g/L teapolyphenol in V. parahaemolyticus culture medium showed significant inhibitory effect.In the early stage of biofilm formation, adding0.10mmol/L EDTA, or600mg/Lsodium nitrite, or2.0g/L tea polyphenols in culture medium would strongly inhibit biofilm formation. While addition of1.0%H2O2played a significant scavenging effecton matured biofilm.（4）The research on total bacterial protein of V. parahaemolyticus by SDS-PAGEelectrophoresis showed that20~30main protein bands were separated generally in therange from29to200KDa. The2-DE related techniques was constructed and optimizedin proteome of V. parahaemolyticus. The results showed that the resolution andreproducibility of2-DE profiles was significantly improved by using active rehydratingof17cm (pH4-7) IPG gel strips, loading the sample120μg, employing the salt bridge,increasing the voltage of isoelectric focusing to10000V, prolonging isoelectricfocusing time to80000vhr, preparing12.5%SDS-PAGE gel, and dyeing the gels bythe silver stained. The PDQuest software was used to analyze the2-DE images ofplanktonic and biofilm state of6strains of V. parahaemolyticus, which obtained46,58,46,44,51,33,30different protein spots separately. The results showed that, there weresome differences in bacterial biofilm state and planktonic state of protein expression.