The Application Of High Performance Liquid Chromatography To The Separation And Analysis Of The Bacteria In Red Kojic Rice

Fermented foods that made by microorganism with unique high nutritional value is a crucial support for food industry.For the purpose of ensuring the quality of fermented products,there is a great need for methods of separation and analysis of their microorganism.Focusing on the shortcomings of present methods to separate and analyze the microbes of fermented food,such as limited detection sensitivity and accuracy,complicated sample preparation procedure and unrecoverable microbial cells,the high performance liquid chromatography(HPLC)technique was innovatively used in this study.This study tried to lay a base for the application of HPLC to the separation,analysis and identification of Red Kojic Rice bacteria by the optimization of sample preparation procedure and chromagraphic conditions,recovery of bacteria,analysis of Denaturing Gradient Gel Electrophoresis(DGGE)and the identification of bacteria.The main contents and conclusions were listed as follows:Firstly,the procedure to prepare bacteria sample of Red Kojic Rice was investigated,based on the interactions between microbes and impurities,such as electrostatic interaction,hydrophobic interaction and hydrogen bond.Results indicated that the 0.02M,pH8.0 piperazine-HCl buffer together with 0.2 M NaCl was optical for sample preparation.Secondly,the optimization of HPLC conditions was conducted by using TSKgel Toyopearl SuperQ-650C anion exchange resin and an HPLC system connected to a UV monitor.It turned out that the optimal conditions for HPLC were setting the pH of buffer to be 8.0,the concentration of NaCl to be 1.0M,the enrichment of sample to be 40 times and the injection volume to be 500?L.Thirdly,on the basis of the optimization of the HPLC conditions,the chromatographic behavior of Red Kojic Rice bacteria was characterized in different gradient elution.20 fractions were detected and obtained,among which 19 were identified to contain bacteria cells by further confirmation with microscopy examination.Fourthly,the possibility of pure strain fractions among the 19 peaks was preliminarily analyzed by using culture-independent method-denaturing gradient gel electrophoresis(DGGE)of the V3 region of 16S rDNA.It showed that there were 9 fractions might be pure strains based on their electrophoretogram.Fifthly,the 16S rDNA of these 9 fractions were sequenced successfully.Then the sequences were compared with those listed in the NCBI database and the phylogenetic tree was constructed.Results indicated that the Red Kojic Rice were dominated by Bacillus circulans,Bacillus ginsengihumi,Pseudomonas sp.and Alicyclobacillus.These results indicated the HPLC technique could be a rapid,accurate and recoverable method for the separation of pure strain from Red Kojic Rice.The study realized an innovation of the methodology for the separation,analysis and identification of microecology of fermented food.