Development Of Capillary Electrophoresis New Methods For Detection Signal Molecules And Inhibitors Of Quorum Sensing In Food Spoilage Bacterial

Microbial spoilage was the primary cause of food deterioration and caused huge economic losses and serious public health problems.The discovery of quorum sensing(QS)system got a new research direction for the control of food spoilage.How to detect the QS signal molecules and their inhibitors with high sensitivity and accuracy is the premise and foundation for the study of the QS corrosion mechanism and a new corrosion protection strategy.At present,the detection methods of QS signal molecules including biological bacteria reporting method,thin-layer chromatography,beta-galactosidase method,colorimetric method,GC-MS and HPLC-MS,etc.Capillary electrophoresis(CE)is a new kind of separation technology which has been widely used as a fast,efficient analytical separation technique,a low reagent consumption,almost no environmental pollution.However,the small size of capillary electrophoresis and the short detection of optical path,limiting its application in the analysis of trace pollutants in food.Quorum sensing system can be prevented through inhibit quorum-sensing signal molecules.The screening methods for enzyme inhibitors include spectrophotometry,radioisotope method,TLC method,electrochemical method and HPLC.These methods have limitations in many ways.Because the CE analysis time is short and the sample consumption is small,the reaction can be carried out continuously in the system and other characteristics.CE can be applied to the screening of enzyme inhibitors because of the short analysis time,the small sample consumption and the continuous reaction.In view of this,the followingmajor research work was performed in this dissertation.1.A method for detecting quorum sensing signal molecules by field amplification sampling reverse migration micelle electrophoresis.In this experiment,N-Dodecanoyl-L-homoserine lactone,N-(3-Oxotetradecanoyl)-L-homoserine lactone,N-(3-Oxododecanoyl)-L-homoserine lactone,N-Decanoyl-L-homoserine lactone and N-(3-Oxodecanoyl)-L-homoserine lactone are target analyte.The separation conditions(acetonitrile concentration,isopropanol concentration,SDS concentration)and enrichment conditions(injection voltage,injection time,injection time of water column and SDS concentration in samples)were investigated and optimized.Then,we further determined AHLs in fish,shrimp,CV12472 and PA01 under optimized experimental conditions,and verified the feasibility of using this method to detect AHLs through a series of evaluation methods.2.We established a new detection experiment of liquid-liquid dispersion microextraction combined with MECC for improving the sensitivity of CE detection of quorum sensing signal molecule AHLs.In this experiment,we review and optimize the terms of the influence of the extraction rate(the variety and dosage of extraction agent,the variety and dosage of dispersants,the variety and dosage of demulsifier)and the conditions of extraction,extraction time,concentration of salt in the sample solution).Then,we further determined AHLs in fish,shrimp,CV12472 and PA01 under optimized experimental conditions,and verified the feasibility of using this method to detect AHLs in complex matrix samples through a series of evaluation methods.3.Based on the separation mechanism of EMMA,We established a method for screening enzyme inhibitors by capillary electrophoresis.In this experiment,the separation conditions(concentration and pH of phosphate buffer solution,pH of HEPESbuffer solution,incubation time,substrate concentration,enzyme dosage and substrate solvent type)were investigated and optimized.A series of methods have been used to prove the possibility of this method for screening MTAN inhibitors.It is proved that this method is feasible for the screening of MTAN inhibitors by calculating the millisecond constant of MTAN based on the method established in 3.Then the effects of 21 Chinese herbs and compounds on enzyme activity were studied.Experimental results show that a variety of Chinese herbal medicine for MTAN with the reaction of the substrate inhibition,compounds only MTAN LD-14 degrees have inhibition,finally studied the inhibition mechanism of LD-14,proves that this method can be used for screening of quorum sensing inhibitors.