Response Surface Methodology Paracin1.7 Fermentation Conditions Of The Initial Establishment Of The Bacteria Genetic Transformation System

The paracin 1.7 (AMP) produced by L.paracasei HD1.7 can inhibit not only G+, but also G- and yeast. It can be used as a new type of food preservative. By using Plackett-Burman and response surface methodology, we studied the medium optimization of Paracin 1.7 production from L.paracasei HD1.7. A Plackett-Burman design was first used to evaluate the influence of eight related faetors, and glueose,MgSO4,MnSO4 were affirmed the important faetors in the medium. Then, the path of steepest aseent and the Box-Behnken design were used for further optimization on these three faetors. By JMP 7.01 solving the quadratie regression model equation, the optimal concentrations of the variables were determined as (g/L):glueose 6.5685,MgSO4 0.454,MnSO4 0.01052. Under the optimal culture condition, the titre of the Paracin 1.7 was up to 141.648 AU/mL, this was improved more than 1.91 fold befor optimal.The optimized parameters affecting high-efficent electrotransforming of the suicide plasmid(pUC18-prcR-tet) into L.paracasei HD1.7 were studied preliminarily. It showed that a highest electro-transformation efficiency could be obtained. After L.paracasei HD1.7 had been cultivated 8h, the recipient cell was treated by 12.5μg/mL(final concentration) of benzylpenicillin and to be continued 1h. It washed by electrotransformation buffer of AEB1(0.3mol/L of sucrose), as well as the 1.8～2.0 kv of field strength and one time of pulse, then added it in MRS culture media quickly and recovered 5h, the high-efficent electrotransforming had been got.The process of antimicrobial peptide production showed the feature of quorum-sensing (QS). prcR is coding gene of regulatory factor in QS. The suicide plasmid, pUC18-prcR-tet, was imported to the L.paracasei HD1.7. By gene knock-out technique, the prcR mutant strain was screened by cultureing with tetracycline. PCR would be done to exmain the result. Finally proofed, the mutant strains showed resistance to tetracycline, pUC18-prcR-tet had been imported to the L.paracasei HD1.7, but homologous recombination was not occurred in the specified location. Antibacterial condition of mutant strains was observed by antibacterial test, the degree of mutant strains, antibacterial was weeker than the original strains, These showed that the related genes that affected produce of AMP was changed, thereby affected the generation of AMP.Research was done on the components of medium to optimize the production of the Paracin 1.7, and the generation of AMP was improved. The regulation gene (prcR) that effect on QS was explored by electrotransformation. These researches laid the foundation for the industrial production of the AMP in the future, and for it was widely used in food and drugs,to explore the QS of lactic acid bacteria and research the gene expression of the AMP.