| Vibrio parahaemolyticus is a typical marine foodborne pathogen. It’s the primarypathogens causing bacterial food poisoning hazard in coastal areas, seriously threats thehealth of people and causes huge economic losses. The priority must be given to solve thesecurity issues of Vibrio parahaemolyticus in seafood. First and formost, a sensitive, rapidand accurate detection technology should be established to identify and quantifytoxoggentic Vibrio parahaemolyticus. Then, a new green security control method need betried out to reduce the risk of foodborne disease caused by Vibrio parahaemolyticus.Immunomagnetic beads technique is a sensitive and rapid detection methodcombinated the immunological specificity and efficiency of magnetic separation.In thisthesis, S-type and D type beads coupled with anti-serum of the toxoggentic standard Vibrioparahaemolyticus respectively. The initial immunomagnetic beads preparation method ofVibrio parahaemolyticus established by using the beads with larger saturated adsorbingcapacity optimizing the coupling conditions such as activating agent, associated time andtemperature. The process is1mg S-type beads, activated with0.1mL0.005g/L EDC andNHS, coupled with0.2mL serum of Vibrio parahaemolyticus, then shaking lightly under25℃for24h. Under the optimal coupling conditions, the immunomagnetic beads have ahigh specificity and sensitivity and the sensitivity is5CFU/mL. Vibrio parahaemolyticusdetection by using immunomagnetic beads technique with easier operation and lower costcould shorten the experimental time. This technique is the foundation for future research.The established immunomagnetic beads technique and GB method were used toisolate Vibrio parahaemolyticus and identify the toxigenic strains from commerciallyavailable fresh, dried and frozen shrimp products in the local in summer and autumn. Thepollution levels of Vibrio parahaemolyticus in commercial shrimps assessed by the mostprobable number method, the infective dose of fresh shrimps and dried shrimps exceeds itspoisoning dose，effective measures must be taken to control it. Compared to GB,immunomagnetic beads method could shorten of the overall testing time, but it still needsfurther study to improve its applications in industrial.Vibrio parahaemolyticus hemolysin standards is vacant in domestic and foreign, inthis experiment, hemolysin was extracted from standard toxigenic Vibrio parahaemolyticusby means of enrichment, sonication, ammonium sulfate precipitation and DEAE cellulosecolumn chromatography,and the hemolytic activity of hemolysin is24.81HU/mL.On this basis, relative hemolytic activity quantitative methods of the hemolysin was established inthe range of0.6-1.1HU/mL, the contentlinear relationship between relative hemolyticactivity and hemolysin is y=0.0141x+0.4921,1relatively HU is equivalent to36.02μg H2.This method can be used to rapid macro-quantitative evaluation of hemolysin.The antibacterial spectrum of Bacillus antimicrobial peptides is wide, and the effectsof inhibiting of G+bacteria and G-bacteria are better. In this thesis, antimicrobial peptideextracted from cultivation of Bacillus NT-6, and Oxford cup method was used to verify theinhibition. The minimum inhibitory concentration of antimicrobial peptide is1.25mg/mL,its impact on the growth curve of Vibrio parahaemolyticus, bactericidal kinetics, as well aseffects of shrimp matrix, the results shows a certain concentration Bacillus antimicrobialpeptides NT-6can inhibit Vibrio parahaemolyticus reproduction obviously, especially inthe logarithmic phase. High concentration antimicrobial peptides have a strong bactericidaleffect on Vibrio parahaemolyticus, and the effects depend on concentration and reactiontime of Bacillus antimicrobial peptides. The Bacillus antimicrobial peptide of NT-6can beused for the control and prevention of Vibrio parahaemolyticus in shrimp products, toreduce the risk of poisoning caused by Vibrio parahaemolyticus. |
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