Antimicrobial Effect And Safety Of Lipopeptide Produced By Bacillus Subtilis FmbJ

Presently, more environmental amity and safer food antiseptic and no residual, nodrug-fast feed additives were hotly studied in food industry and stockbreeding. Comparingto traditional food antiseptics and feed additives, antibacterial peptide had many goodqualities, such as broad antibacterial spectrum, pollution free, biological degradation,synergistic effect, no drug resistance, good stability and broad effective scope etc. With theincreasing of living standard and the concentrating on health and longevity, people putforward safer and more environmental protective demands for food and feed additive whichcan accumulate directly and indirectly in the body,. So it is important to exploit natural andsafe food and feed additives.In the last few years, a food Bacillus subtilis fmbJ strain has been screened out in ourlab, which could secrete antibacterial active substances. These substances were stable toheat and some enzymes, and had very high antimicrobial activity and broad antibacterialspectrum. Present study demonstrated that the substances mainly included surfactant andfengycin, both of cyclolipopeptide. In order to provide theoretical base and technologicalsupport for reasonable utilization and application area expanding of antibacteriallipopeptide, the paper investigated its mechanism, some application function and foodpoison safe. Results were as follows:1. Sensibility and inhibition effect of lipopeptide of Bacillus subtilis fmbJ to E. coli wasstudied. The results showed that E. coli was very sensitive to it and MIC was 468.75μg/ml.Studies of growth curve of thalline, membrane permeability, SEM and TEM demonstratedthat lipopeptide could destroy integrity of cell membrane, which led to increasing ofmembrane permeability and leaking out of macromolecule in the cell, and then thallus cellsbreak. This phenomenon could be seen in thallus cells no matter which were in the stage ofgrowing or had integrity cell wall. In addition, lipopeptide could inhibit protein synthesis inE. coli cells, and lead to alteration of DNA maximum absorption and hypochromic effectand bathochromic effect by binding with DNA in vitro. Studies showed that lipopeptide had different extend effects on permeability of membrane interna and membrane externa andrespiratory metabolism of E. coli having integrity cell wall.2. Antibacterial activity of lipopeptide on Bacillus cereus in vitro was investigated and thepossible antibacterial effect was explained by the studies of morphological structure,growth-breeding, membrane permeability and protein synthesis etc. Inactivation effect tospore of Bacillus cereus was optimiTed by response surface method. Results demonstratedthat Bacillus cereus and its spore were very sensitive to lipopeptide, and their MIC was80μg/ml and 156.25μg/mL respectively. Lipopeptide could strongly inhibitgrowth-breeding of Bacillus cereus. Growth curve and electron microscope structureshowed lipopeptide could kill or lysis Bacillus cereus. TEM result showed that lipopeptidecould result in structure alteration no matter in resting spore or sprouting spore, but theeffect on the latter was more evident. Obvious leaking out of macromolecular withultraviolet absorption had not been observed by membrane permeability with absorptionmethod. But evident nucleic acid substance leaking could be seen by electrophoresis assay.Interaction between lipopetide and chromosome of Bacillus cereus in vitro indicated thatlipopetide could bind to DNA, resulting in alteration of DNA maximum absorption andhypochromie effect and bathochromic effect. After optimization, it caused a 2 log reductionin the number of spores of Bacillus cereus in the base under the conditions of temperature29.6℃, action time 7.6h and concentration 3.46mg/mL.3. Antifungal activity of lipopeptide to Penicillium notatum was examined. Resultsindicated that Penicillium notatum was very sensitive to it, and the MIC was 125μg/ml.Lipopeptide could strongly inhibit growth and spores germination of Penicillium notatum,and lyse spores of Penicillium notatum by the study of SEM and TEM. Activity of SDHand MDH could reduce markedly by the effect of lipopeptide. In addition, the adsorption ofcells nutrition substance was restrained or stopped when lipopeptide affected Penicilliumnotatum for different time.4. Inhibition activity of lipopeptide against Pseudorabies Virus, Porcine Parvovirus,Newcastle disease virus and Infectious Bursal Disease Virus was studied in vitro and thepossible mechanism was also discussed. Results showed that the TD₅₀ and TD₀ values oflipopeptide on Porcine Kidney cell and Chicken Embryo Fibroblasts cell were 47.57μg/mL,18.9μg/mL and 128.95μg/ml, 25.79μg/ml respectively. The lipopeptide could strongly inhibit the cell cytopathic effect induced by PRV, PPV, NDV as well as IBDV, andsignificantly increase the survival rate of cell (p＜0.05) and inactivate PRV, PPV, NDV andIBDV. In addition, lipopeptide could defend NDV lasota strain and IBDV H strain againstthe infection and inhibit multiplication. However, it could't inhibit the infection andmultiplication of PPV and PRV in PK-15 cell.5. Food safety of lipopeptide was investigated, and which provided toxicological safetybase for its application. Acute toxicity experiment and 30-day feeding experiment wereperformed according to GB15193—94《Procedures and methods for toxicologicalassessment on food safety》. Results demonstrated that died mouse and evident toxicsymptom weren't observed after mouse had been lavaged 5g/kg·BW one time for a week.Toxicological symptom and patho-symptom of liver, spleen, kidney weren't observed.Indexes of liver, spleen, and kidney had no significant difference. But growth of mouse hadabnormity at a higher dose (more than 1g/kg·BW). 30-day feeding experiment with Wistarrats showed that blood platelets count decreased significanfly(P＜0.05), content increasedsignificantly (P＜0.05), but gain weight, feed utilization rate, liver, spleen, kidney and theirindexes, other blood routine and biochemical indexes had no striking change at 250mg/kg.