The Applications Of Capillary Electrophoresis In The Analysis Of Bacterial Metabolites In Microbial Fuel Cell And Enzymolysis Products From Grain

Capillary electrophoresis(CE),also known as high-performance capillary electrophoresis(HPCE),is widely used as a separation technique with the properties of high speed,high efficiency and less sample consumption.CE is very suitable for the separation of amino acids and peptides.Amino acids are essential nourishment to creatures and they are the smallest units of proteins,having significant effect on the process of creature life.Many amino acids are also the metabolites of bacteria.In our work,Escherichia coli(E.coli)and Bacillus licheniformis were put into the microbial fuel cell(MFC)and provided with the source of nitrogen,carbon and so on.CE was used to separate and determine the metabolites produced by the bacteria and the metabolism process of the bacteria could be monitored by the power output of the MFC.The study showed that the bacteria could convert nutrition source to amino acids through their metabolism.In addition,capillary electrophoresis was also applied to analyze the exorphins and amino acid from the cereal protein after enzymatic hydrolysis and the results were satisfactory.There are four chapters in this paper.In the first chapter,the related research about this thesis were reviewed,including history of the CE development,the separation modes of CE,detection methods and sample derivation method and so on.This chapter also introduced the microbial metabonomics and microbial fuel cells.In the second chapter,CE-LIF was applied to separate and detect amino acid metabolites of E.coli in microbial fuel cells.A three-dimensional spontaneous sampling device was developed,it could effectively reduce the electricity discrimination effect of the analytes which generated in electric sampling,and the device had some advantages such as simple,durable,low cost and easy to operate.Combined the sampling device with CE-LIF,we successfully separated the two peptides(L-camosine and L-alanyl-glycine)and six amino acids(cystine,alanine,lysine,methionine,tyrosine and arginine)within 8 min under the optimum condition(pH=10.0,20 mM borax running buffer,separation voltage 3000 V).The detection limits(S/N)ranged between 20 and 300 nmol/L.Lastly,we applied this method to analyze the amino acid metabolites produced by E.coli in microbial fuel cell.L-camosine,L-alanyl-glycine and cystine were used as the nutrition source of carbon,nitrogen and sulfur.As a result,methionine was discovered as the metabolism prodct with the concention 23.3 μg/L.In the third chapter,a high-speed capillary electrophoresis system coupled with spontaneous injection device was applied to analyze the amino acid metabolites of bacillus licheniformis in microbial fuel cell.The injection device could realize thehigh speed,high efficiency separation with less sample consumption and shorter length of the capillary.peptides and amino acids were derived with FITC firstly,and then micellar capillary electrokinetic chromatography method was applied to separate the two peptides(L-camosine and L-alanyl-glycine)and four amino acids(tryptophan,valine,glycine and tyrosine).Under optimized separation conditions(separation voltage 3.0 kV,20 mmol/L borax buffer(pH=11.4)containing 15 mmol/L sodium dodecyl sulfate(SDS)),the mixture could be completely separated within 3 min.The detection limits(S/N = 3)ranged between 15 and 90 nmol/L.Finally,the method was succefully applied to separate and determine the amino acid metabolites of bacillus licheniformis in microbial fuel cell.L-carnosine and L-alanyl-glycine were use as the carbon source and nitrogen source for the bacteria.The results showed that valine was discovered as the metabolite produced by bacillus licheniformis with the concentration 3.84 × 10-11 mol/L.In the fourth chapter,CE-LIF was applied to separate and determine the exorphines(A5,B5 and C)and amino acids(arginine,glutamic acid,leucine and phenylalanine)digested from wheat protein after enzymatic hydrolysis gluten protein.Under the optimized condition(pH = 11.5,30 mmol/L borax buffer,separation voltag 3000V),the six analytes could be successfully separated within 6 min.The detection limit(S/N=3)ranged between 0.15 and 0.6 μmol/L.Finally,the method was applied in the separation and detection of the exorphines and amino acids digested from wheat protein after enzymatic hydrolysis.The recovery ranged between 82%and 120%.Results showed that the method was reliable.