Screening Of GD Stain Producing Antimicrobial Lipopeptide And Studying On Its Separation,Identification And Application

Antibacterial lipopeptides, essentially, are one of antimicrobial peptides and cyclic lipopeptides compounds that are produced by some of bacillus and other non bacillus through non-ribosomal synthesis in the stage of growth and reproduction. It has a broad spectrum antibiotic, good antibacterial effect and difficult to produce drug resistance and other characteristics, so it is an important class of antibiotic alternatives. Currently, more and more researchers will be transferred to the research on the development of these antibacterial substances. In our study, when some microbial resources preserved were verified, Bacillus subtilis GD strain was obtained with significant inhibition effect on pathogenic bacteria and a series of studies around the strain and its antibacterial substances were conducted including conventional physiological and biochemical and16S rDNA gene identification of Bacillus subtilis strain GD, and the antibacterial substances were separated, purified and identified. Meanwhile, it was preliminary studied on the effects of physical and chemical factors, and its mechanism of safety evaluation, animal experiment and industrial fermentation production process. The results showed that the metabolite produced by this strain has a good antibacterial effect and is a kind of green, safe and efficient antibacterial lipopeptide compounds.1. In order to get a strain of Bacillus producing antibacterial lipopeptide compounds as the material of a follow-up study, with E. coli O2from chickens as an indicator bacterium,4strains that inhibition zone diameter of strains fermentation products was above15mm were obtained from29Bacillus preserved and isolated through detecting antibacterial ability of metabolites produced by the strains in the research institute. Based on the study, antimicrobial spectrum and physicochemical stability test were carried out. It was found that the metabolite of the strain had good antimicrobial and bactericidal effect by detection on inhibitory spectroscopy to Gram-positive, negative and fungi with stable physical and chemical properties and20mm or more of the inhibition zone diameter to avian E.coli. Meanwhile, the strain was identified by systematic biochemistry and16S rDNA sequencing, and the results showed that the strain was Bacillus subtilis, called Bacillus subtilis GD strain.2. As the development and production of products in the future, it was necessary to study on types and characteristics of main ingredients of antibacterial lipopeptide compounds of Bacillus subtilis GD strain. After fermentation broth of Bacillus subtilis GD strain adsorbed and eluted by macroporous resin XD in the study, products obtained above and standards of surfactin was carried out by thin layer chromatography (TLC) analysis and comparison, antibacterial substances in strain GD metabolites was proved as cyclic lipopeptide compounds surfactin; and through analysis of content, molecule and structure of metabolites by means of liquid chromatography and mass spectroscopy (LC-MS), it was sure that main lipopeptide types of surfactin and concentration of77.39%, and molecular weights rung from994.6449to1036.6933. It was further proved that the metabolites of GD strains were homologs mixture of surfactin A, surfactin B and surfactin C.3. For study on inhibiting mechanism and characteristics of lipopeptides produced by GD strains on bacteria, fungi and yeast, it was analyzed through lipopeptides to avian Escherichia coli of bacterial cell membrane permeability, cell ultrastructure and cellular metabolic activity and so on. It was proved that the lipopeptides had high sensitivity to avian E. coli and the minimum inhibitory concentration of0.5mg/ml; The mechanism was that lipopeptides could cause that E. coli membrane integrity was damaged and then macromolecules within cells were let out, and bacterial cells were ruptured with decrease or loss of metabolism of bacterial cell. Based on this, lipopeptides play its bacteriostatic and bactericidal role.4. In order to strain GD and its metabolic products used in production, it was preliminary studied that acute toxicity and subacute toxicity in antimicrobial lipopeptides produced by the strain GD and the broiler production performance on feed conversion rate and survival rate and control effect on infection of E. coli O2were carried out. And the result revealed that ICR mice with the amount of lipopeptide crude to5g/kg.BW once had no any clinical symptoms and pathological changes compared to the control; the one with the amount to5g/kg.BW in30-day subacute toxicity test, beyond ICR mouse platelet number to rise (p<0.05), the other indexes were normal. It was proved that the metabolites of GD strains were safe to animal and no significant side effect. Simultaneously, according to the add amount to5mg/kg in broiler feed, feed meat ratio decreased by0.12compared to antibiotic group, and it was very significant difference (P<0.01), meanwhile, mortality rate was reduced by4.33%with significant different (P<0.05); it was found that that has the good effect in treatment group to the control, the antibacterial lipopeptide and the strain GD group was better than single antibacterial lipopeptide group. It was suggested that strain GD and its metabolites as feed additives on growth performance of broiler chickens with good regulation and promotion, has certain protective effect to infection of E.coli O2in observation of control infection of E.coli.5. In order to improve the production yields of GD strain and its metabolites and reduce production costs, it was screened and optimized from various nutritional factors and in the fermentation medium of Bacillus subtilis GD strain by means of the response surface analysis method through P-B, B-B and Taguchi method design by the Minitab analysis software for the impact on production of antimicrobial lipopeptide. The result showed that3important factors were, separately, corn syrup powder, starch, flour and yeast. Based on those results, it was carried out that it was obtained of fitting a mathematical model for high yielding lipopeptide antibiotic medium prescription of Bacillus subtilis GD strains using response surface analysis, and it was established that the main component ratio of industrial production medium of Bacillus subtilis GD strain:Plant Protein Powder25g/L, yeast extract,5.02g/L, starch,4.4g/L. it was6.82times to increase the yield of antimicrobial lipopeptide of the above-mentioned fermentation medium than the original. Greatly reduce the cost of production, laid the foundation for industrial production.