Extraction, Antibacterial Activity Test In Vitro And Toxicity Of Antibacterial Substance From Paenibacillus Polymyxa Cp-S316

Paenibacillus polymyxa Cp—S316 was antibiotic bacteria isolated from soils in Mountain Tai .The results of previous study showed that it had several good features such as broad antibiotic spectrum, good ferment characteristics and properties, stable ferment yield, and therefore it gave us an attractive prospect for development. This study included extraction, antibacterial activity test in vitro and toxicity of antibacterial substance from Cp—S316. The main results were as following:1. The exchange capacity of absorption and desorption of Cp—S316 antibacterial substance in 7 types of anion exchange resin and 4 types of cation exchange resin were compared, and macroporous weak-acid cation exchange resin D151 Was selected for further study, which had good consequence of absorption and desorption The further studies of D151 resin were as following: Absorption capacity, the optimum eluting solution, elution curve in static and dynamic state. The results indicated that in static condition, ferment solution(100mL) treated with D151 resin (1g), the absorption rate could reach 87.33%; both HCL (0.6mol/L)and H2SO4(0.5mol/L) were good eluting agents for antibacterial substance, and HCL(0.6mol/L) was finally selected as the optimum eluting agent; the rate of static elution (HCL0.6mol/L) to antibacterial substance was 58.04%[eluting agent: resin=6:1(mL/g)]; the rate of dynamic elution was 92.24%[collected solution: resin=4.3:1(mL/g)], andthe elution rate for higher concentration collected solution was 83.75%.Further more, the way to test the content of antibacterial substance was constructed .The pretreatment for ferment solution was tested, and it showed that the optimum content of oxalic acid was 0.4% in ferment solution.2. The minimum inhibitory concentration(MIC) and the minimum bacterial concentration(MBC) of antibacterial substance from Paenibacillus polymyxa Cp-S316 against Escherichia coli, Enterobacter aerogenes, Samlonella typhimurium , Pasteurella multocida , Salmonella pullorum , were studied by the double tube dilution method, and the time-killing curves had been plotted with the living bacteria number counted at different time intervals. In addition, the influence of different pH, bacterial concentration and Mg2+ concentration on MIC against Escherichia coli, Enterobacter aerogenes, Samlonella typhimurium were studied. Results showed that the MIC were 12.5μg/mL, 12.5μg/mL, 6.25μg/mL, 12.5μg/mL and 12.5μg/mL, and the MBC were 25μg/mL,25μg/mL, 12.5μg/mL, 50μg/mL,25μg/mL, respectively. As far as Staphylococcus aureus concerned, the MIC was 25μg/mL and the MBC was higher than 400μg/mL. Under double MIC, the three pathogenic bacteria-Escherichia coli, Enterobacter aerogenes, Samlonella typhimurium— could be killed in 12h, 24h, 24h, respectively, and for Staphylococcus aureus, it showed that Cp-S316 antibacterial substance only had inhibitory effect but no bactericidal effect; the changes of different pH and Mg2+ concentration on MIC were not obvious, and relatively, the bacterial concentrations affect MIC more obviously.3. Acute toxicity experiment and cumulative toxicity experiment were conducted, and results showed that LD50 of Cp-S316 antibacterial substancewas higher than 5g/Kg to mice and the cumulative toxicity coefficient K was higher than 5.