Establishment Of Detection Methods Of Escherichia Coli O157:H7 Based On Hydrolysis Of Penicillin And Study On Chlorinated Antimicrobial Film For Eliminating Escherichia Coli

Microbial contamination is considered to be the primary cause of food safety issues.Escherichia coli which is widespread in the environment is an important index indicating the quality of food.Some pathogenic Escherichia coli?for example E.coli O157:H7?cause serious illness,even death.It is easy for food to be contaminated by Escherichia coli during production,packaging and transportation process.It is very meaningful to control the food contamination of Escherichia coli and establish a fast and sensitive detection method of pathogenic Escherichia coli for food safety.In this paper,two methods were established to detect Escherichia coli O157:H7 fast and sensitively based on the hydrolysis of penicillin by?-lactamase with enzyme-linked immunosorbent assay?ELISA?and lateral flow immunoassay.These methods could improve the detection sensitivity of Escherichia coli O157:H7 compared with the traditional method,and the time needed for detection was shorten.In order to control the contamination of E.coli in food,chitosan was mixed with poly?vinyl alcohol?to obtain the blended film,then the blended film was chlorinated.The chlorinated CH-PVA film could effectively eliminate Escherichia coli and achieve effective control of Escherichia coli in the food.In the first chapter of this study,the general information of Escherichia coli,different detection methods and the prevention and control strategies of Escherichia coli were reviewed.In the second chapter of this study,cascade signal amplification in ELISA involving double-antibody sandwich ELISA?ds-ELISA?and indirectly competitive ELISA?ic-ELISA?was established to sensitively detect Escherichia coli O157:H7 based on the hydrolysis of penicillin.In the ds-ELISA,anti-E.coli O157:H7 polyclonal antibodies were coated in the wells,E.coli O157:H7 cells were fixed in the wells by immunoreaction and combined with biotinylated anti-E.coli O157:H7 monoclonal antibody,streptavidin was added in the wells,and then biotinylated?-lactamase was added.If there were some E.coli O157:H7 cells in the sample,there would be some?-lactamase in the wells.The penicillin solution was added into the wells and hydrolyzed by?-lactamase.The hydrolysate was transferred to the wells of ic-ELISA.The concentration of penicillin could be sensitively detected in ic-ELISA.In ds-ELISA,increased the concentration of E.coli O157:H7 resulted in more?-lactamase and less penicillin.The concentration of streptavidin,the addition of biotinylated?-lactamase and the p H of hydrolyzation were optimized,and hydrolysis mechanism of penicillin was analyzed.The detection sensitivity of E.coli O157:H7,which was 20 CFU/m L with the cascade signal amplification in ELISA,was 1,000-fold higher than that of traditional ELISA.Furthermore,the novel method can be used to detect E.coli O157:H7 in milk?2CFU/g?.In the third chapter,a fast detection method with lateral flow immunoassay based on the hydrolysis of penicillin was established to sensitively detect Escherichia coli O157:H7.Firstly,E.coli O157:H7 cells were captured by immunomagnetic nanoparticles,magnetic nanoparticles-anti-E.coli O157:H7 polyclonal antibodies-E.coli O157:H7?Complex A?was combined with anti-E.coli O157:H7 monoclonal antibodies-gold nanoparticles-?-lactamase?Complex B?by immunoreaction.The penicillin solution was mixed with the complex?magnetic nanoparticles-anti-E.coli O157:H7 polyclonal antibodies-E.coli O157:H7-anti-E.coli O157:H7 monoclonal antibodies-gold nanoparticles-?-lactamase?,after the hydrolysis of penicillin,the supernatant was separated from the complex by magnetic separation,and then the concentration of penicillin was detected by lateral flow strip.In the detection system,increasd the concentration of E.coli O157:H7 resulted in more?-lactamase and less penicillin.The amount of anti-E.coli O157:H7 monoclonal antibody Complex B and the addition of Complex B were optimized.The detection sensitivity of E.coli O157:H7 which was 2×10² CFU/m L with the novel method was 570-fold higher than that of traditional lateral flow strip.In the fourth chapter,chitosan?CH?was mixed with poly?vinyl alcohol??PVA?in different compositions to obtain antimicrobial films.Upon chlorine bleach treatment,amino groups in chitosan were transformed into N-halamine structures,antimicrobial films which had excellent antimicrobial efficiency were gained.Film-bacteria interactions were imaged using a scanning electron microscope?SEM?,the results showed that the E.coli cell established an irreversible interaction with the chlorinated film.The effect of the quality ratio of CH/PVA on the antibacterial efficiency,mechanical properties,thermal stability and water vapor permeability?WVP?were studied.When the quality ratio of CH/PVA was 4:6,the elongation at break of the chlorinated film?Cl OCH4PVA6?was 24.646%,which was higher than pure chlorinated chitosan film.The Cl OCH4PVA6 film provided total elimination of 10⁸ colony forming units?CFU?of E.coli in 30 min.The thermal analyse showed that Cl OCH4PVA6 film had a good themal stability.The WVP of Cl OCH4PVA6 film was 4.173×10^-11 g·m^-2·s^-1·Pa^-1,that showed Cl OCH4PVA6 film had a good water resistance performance.The Cl OCH4PVA6 film could elimate E.coli cells.This study provided technique support for controling food safety.In the fifth chapter,glycerol,xitol,sorbitol,tween 80,span 80,and PEG-6000 were added into the blended film as plasticizer,respectively.The effects of different plasticizers on the antibacterial properties and physical properties of belended films were studied.The addition of different plasticizers had no effect on the antibacterial properties of chlorine films.The enlongation at break of belended film with glycerol?Cl OCH4PVA6G0.5?is 36.442%,which is higher than the elongation at break of Cl OCH4PVA6.The different addition of glycerol was evaluated,different addition of glycerol could not cause the antibacterial efficiency of the films.When 0.5 m L of glycerol was added into the film?Cl OCH4PVA6G0.5?,the elongation at break?EB?of the film was biggest than the EB of other films with different addition of glycerol.The the WVP of Cl OCH4PVA6 film without glycerol.Cl OCH4PVA6G0.5 film has excellent moisture resistance and is suitable for food packaging.The comtamination of E.coli is one of the most important factors which endanger food safety.In this paper,the detection methods could detect E.coli O157:H7 fast and sensitively,the chlorinated film could elimate E.coli effectively,this project is very meaningful for the food safety.