Effects Of Curcumin-mediated PDT On Vibrio Parahaemolyticus In Shrimp

Our country has a vast sea area and a rich variety of aquatic products,making it the world’s largest exporter of aquatic products.However,aquatic products carry a large number of pathogenic microorganisms and are a major factor in the initiation of human foodborne diseases.Vibrio parahaemolyticus is a typical food-borne pathogen in aquatic products,but also the primary bacterial food poisoning bacteria in coastal areas.In this paper,we first investigated the contamination status,virulence genes and drug resistance of Vibrio parahaemolyticus（Vp）in four major types of aquatic products（shrimp,fish,shellfish and crab）sold in Hangzhou.Then,the bactericidal effect of photodynamic technology（PDT）combined with Citric acid（CA）on Vp and its bactericidal mechanism were studied with standard strain ATCC 17802 as the research object,which provided a theoretical basis for its application in aquatic products.Prawns have high nutritional value,delicious taste,but high protein and water content in the body,perishable and susceptible to Vibrio parahaemolyticus infection.And coastal areas often have the habit of raw aquatic products,such as raw shrimp,without heating sterilization is more likely to be contaminated by Vp and other pathogenic bacteria,resulting in food poisoning incidents.Therefore,in this paper,a strain 2MX1-1,which was isolated from aquatic products and contained the most toxic force genes and had the strongest drug resistance,was infected with prawns.The bactericidal effect of Vp 2MX1-1 on prawns was studied when the photodynamic technology combined with citric acid treatment was actually applied to prawns,and the changes in its quality at 4℃were tracked.The main research results are as follows:（1）A total of 106 aquatic products of 24 types（shrimp,fish,shellfish,crab）were collected from supermarkets and farmers’markets from April to July.30 Vp isolates were obtained by Gram staining microscopy,biochemical identification and 16Sr RNA isolation,with a total detection rate of 28.30%.The distribution of virulence genes was further analyzed by PCR.The tdh gene was not detected,one strain was positive for trh gene,the detection rates of tlh,fla A and fur genes were 100.00%,the Exs A gene detection rate was 96.67%and tox R detection rate was 83.33%.The drug resistance analysis of 30 Vibrio parahaemolyticus isolates showed that most of the isolates were resistant to the antibiotics widely used in the early stage,and the phenomenon of multiple drug resistance was prominent.（2）Curcumin-mediated PDT combined with CA treatment has good bactericidal effect on Vp and can effectively remove biofilm.The bactericidal effect was enhanced with the increase of curcumin concentration,the extension of illumination time and citric acid（0.5 mg/m L）.Under PBS condition,curcumin concentration was 2μM,citric acid concentration was 0.5 mg/m L,and blue LED light with wavelength of429.5 nm and irradiance of 18.34 m W/cm²was illuminated for 2 min.Under the optimal treatment condition,all Vibrio parahemolyticus were killed at 7.43 lg CFU/m L.When activated by blue light,photosensitor curcumin produces ROS with strong oxidation through electron transfer and energy transfer.ROS levels in bacteria continuously increase,and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase decrease by 97.98%and 97.34%,respectively.In the supernatant,AKP enzyme activity increased by 0.28 King unit/100m L,the bacteria underwent serious deformation and even tended to be flat,the intracellular antioxidant enzyme CAT enzyme activity decreased to 0,SOD enzyme activity decreased by 89.75%,DNA bands darkened or even disappeared,and finally led to cell death.（3）In order to study the effects of curcumin-mediated photodynamic technology and citric acid treatment on the quality of prawn,prawn was used as experimental material.Microbial indexes（total number of colonies,Vibrio parahaemolyticus number）,physical and chemical indexes(p H,color difference,texture,volatile base nitrogen value,thiobarbituric acid value,water activity,water retention,Ca²⁺-ATPase activity)and sensory evaluation were used to analyze the quality changes of shrimp within 4 days.The results showed that 99.97%of Vibrio parahaemolyticus and99.53%of bacteria could be inactivated by curcumin photodynamic combined with citric acid when the illumination time was 20 min and the concentration of curcumin was 40μM.These conditions were used for the subsequent preservation experiment of prawns at 4℃.After PDT combined with citric acid treatment,the total number of colonies,Vibrio parahaemolyticus,p H,texture,water activity,water retention,volatile base nitrogen value,thiobarbituric acid value,Ca²⁺-ATPase activity and sensory evaluation of shrimp were better than those of the control group within 4days.In summary,the detection rate of Vibrio parahaemolyticus in aquatic products is very high,and the problem of bacterial drug resistance is prominent,which is resistant to at least one antibiotic.Photodynamic bactericidal technology and citric acid treatment can effectively kill Vibrio parahaemolyticus,and will not lead to drug resistance.The photodynamic sterilization technology and citric acid treatment were applied to prawns,which effectively inhibited the growth of microorganisms and slowed down the quality deterioration of prawns.