Study On The Effect Of Blueberry Extract On The Pathogenic Factors Of Three Foodborne Pathogens

Foodborne disease is a serious public health problem,the spread and infection of foodborne pathogens is one of the main causes of foodborne diseases in the world.Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella enteritidis are the most common foodborne pathogens.V.parahaemolyticus(VP)is the main cause of bacterial gastroenteritis related to seafood.L.monocytogenes(LM)and S.enteritidis(SE)can contaminate meat,eggs,milk,vegetables and other foods,which posing a greater threat to food safety and human health.Therefore,it is very important to control foodborne pathogens and reduce the pathogenic factors of foodborne pathogens for the prevention of foodborne diseases.Blueberry is an excellent source of flavonoids,proanthocyanidins,flavonols and phenolic acids.Blueberry has high antioxidant activity,cholesterol lowering and cardiovascular protection functions,and has antiviral effect.In the study,methanol formic acid extract of blueberry was used to study the effect of blueberry extract on pathogenic factors of foodborne pathogens.Firstly,the methanol formic acid method was used to prepare blueberry extract,and then the content of total phenol,total sugar and anthocyanin in blueberry extract was determined.Secondly,the antimicrobial effect of blueberry extract on V.parahaemolyticus,L.monocytogenes and S.enteritidis was determined,the sub-inhibitory concentration(SIC)without inhibiting the growth of bacteria was selected for subsequent experiments;then,the SICs was used to study the effects of blueberry extract on cell surface properties,motility,quorum sensing and biofilm formation of foodborne pathogens.Finally,RT-q PCR was used to investigate the effects of blueberry extract on motility,quorum sensing,biofilm formation and virulence gene expression of foodborne pathogens.This study provides a new method to reduce the pathogenic factors of foodborne pathogens,and provides a theoretical basisfor the development of antimicrobial agents with no toxic side effects and lower pathogenicity of pathogens.The main research contents and results are as follows:1.The effect of blueberry extract on motility of foodborne pathogensIn this study,the effects of blueberry extract on the swimming,swarming and twitching motility of foodborne pathogens were measured by using the swimming,swarming and twitching plates.The results showed that the SICs of blueberry extract significantly reduced the motility of three foodborne pathogens.Blueberry extract reduced the swimming,swarming and twitching diameter of V.parahaemolyticus to 5.0cm,0.9 cm,0.6 cm at most,and reduced the swimming,swarming and twitching diameter of L.monocytogenes to 4.9 cm,1.3 cm,1.0 cm.Blueberry extract completely inhibited the swimming and swarming of S.enteritidis.The diameter of swimming and swarming was reduced to zero,and the diameter of twitching was reduced to 0.8 cm.The motility inhibition effect of blueberry extract decreased the ability of foodborne pathogens to spread to a larger area.2.The effect of blueberry extract on cell surface properties of foodborne pathogensIn this study,the effect of blueberry extract on the hydrophobicity and auto-aggregation in foodborne pathogens was determined by microbial adhesion carbon hydrocarbon compound method and auto-aggregation method.The results showed that the SICs of blueberry extract significantly inhibited the hydrophobicity and auto-aggregation of the three foodborne pathogens.The hydrophobicity of V.parahaemolyticus,L.monocytogenes and S.enteritidis was reduced to 1.1%,5.0%,2.9% at most,and the autoaggregation was reduced to 2.1%,9.7%,4.7%.Blueberry extracts reduce the ability of foodborne pathogens to adhere to biological or abiotic surfaces.3.The effect of blueberry extract on the quorum sensing of foodborne pathogensIn this study,the Vibrio harveyi BB170 was used to determine the luminescent value produced by the three foodborne pathogens,explore the effect of blueberry extract on the quorum sensing of foodborne pathogens.The results showed that blueberry extract significantly reduced the luminescent value of three foodborne pathogens,that inhibited the production of AI-2 molecule.The inhibition rate of blueberry extract to V.parahaemolyticus,L.monocytogenes and S.enteritidis was 77%,68% and 70%.Blueberry extract significantly destroyed the quorum sensing of foodborne pathogens,which led to the influence of a series of biological behaviors including motility,biofilm formation and bacterial pathogenesis.4.The effect of blueberry extract on the biofilm of foodborne pathogensIn this study,crystal violet staining was used to explore the effect of blueberry extract on the biofilm formation and the eradication effect of mature biofilm.MTT method was used to determine the effect of blueberry extract on the metabolic activity of biofilm.The biofilm structure was observed by confocal laser scanning microscope.The results showed that blueberry extract inhibited the biofilm formation of three foodborne pathogens,The BFI of V.parahaemolyticus,L.monocytogenes and S.enteritidis were reduced to 0.1,0.6,0.4 at most.The percentage of biofilm formation of V.parahaemolyticus,L.monocytogenes and S.enteritidis was reduced to 20%,50%,40% after the mature biofilm was treated by blueberry extract.Blueberry extract reduced the metabolic activity of bacteria in the biofilm.The inhibition rate of biofilm metabolic activity of V.parahaemolyticus,L.monocytogenes and S.enteritidis was31.5%,64.4%,89.9%.Blueberry significantly destroyed the biofilm structure of foodborne pathogens and breaks down the dense biofilm structure into scattered bacteria.5.The effect of blueberry extract on gene expression of foodborne pathogensIn this study,RT-q PCR was used to investigate the effect of blueberry extract on the genes expression level of foodborne pathogens.The results showed that the expression of flagellum motility,quorum sensing and biofilm formation genes of three foodborne pathogens were significantly down-regulated by blueberry extract.Blueberry extract will inhibit the flagellum motility of pathogenic bacteria,reduce the production of quorum sensing signal molecules and inhibit the biofilm formation.After the treatment of blueberry extract,the expression of T3SS1 and T3SS2 related virulence genes was down-regulated,which may reduce the virulence of V.parahaemolyticus.Blueberry extract also significantly reduced the expression of virulence regulators of L.monocytogenes,so the toxicity of L.monocytogenes will be reduced.