| In recent years, the widely use of antibiotics has resulted in the appearance of many drug-resistant strains, which has led into serious challenge for food safety and health care.Therefore, the need for stable and non-toxic antibacterial substances allows of no delay. The main purpose of this research is to find a new kind of antibacterial substance isolated from the bran fermentation by Paenibacillus bovis sp.nov. BD3526, and study the biological properties to provide the foundation for its future applications.In this article, the fermentation conditions of P. bovis BD3526 were optimized by response surface methodology (RSM). The optimum culture conditions for the antibacterial substances by P. bovis BD3526 were as follows: the culture temperature was 29℃, the culture time was 73 h, the inoculum amount was 3.2% and the concentration of wheat bran was 4.2%.Based on the results of RSM, the effects of different nutrient contents on the production of antibacterial substances by P. bovis BD3526 were also determined by orthogonal experiment.The results of orthogonal experiment showed that the 3.0% glucose, 1.0% peptone and 0.3%potassium dihydrogen phosphate were the optimal combination of nutrient components.Otherwise, the antibacterial substances produced by P. bovis BD3526 were separated and purified. In the preparation of crude extract, the separation efficiency of ammonium sulfate precipitation and organic solvent extraction were compared. The results showed that 60%ammonium sulfate precipitation was most suitable when compared the antibacterial effects of crude extracts from different ammonium sulfate concentrations. In the process of solvent extraction, the effect of chloroform, ethyl acetate, petroleum ether and n-butanol were compared and it was found that the n-butanol was the best. When using n-butanol as extraction, it was found that 4 times of extraction was the best. In order to compare the separation effect of ammonium sulfate precipitation and n-butanol extraction, MIC was detected during the different stages of separation, and the results showed that ammonium sulfate precipitation was better. In the preliminary purification, the purification effects of Sephadex G-15, Bio-Gel-p-2 and Sephadex LH-20 gel column chromatography were also compared. The results showed that Sephadex LH-20 gel column chromatography was more suitable. After compared the effect of different proportion of methanol-water, finally selected 75% methanol(v/v) as elution solvent for Sephadex LH-20 gel column chromatography. The thin layer chromatography was used for more information about the antibacterial substances and found that there were at least two active components in the sample. The sample was separated and purified by HPLC for further study, and the results showed that there were two kinds of antibacterial substances and the retention time were 23.56 min and 32.84 min,respectively.In order to identify the structure of the antibacterial substances, the mass spectrometry was used, and the molecular weight of the antibacterial substances are 882 Da and 910 Da,respectively. The antibacterial substance with retention time of 23.56 min was confirmed as LI-F04a after determined by tandem mass spectra. The antibacterial substance with retention time of 32.84 min was a derivative of LI-F04a after determined by tandem mass spectra and amino acid analysis, which named Bovisin.The biological characteristics and the antibacterial spectrum of LI-F04a and Bovisin were also stated in this paper. It was found that they both have good thermal stability, and they are also insensitive to acid, alkali, pepsin, trypsin, pronase and protease K. In the establishment of the antibacterial spectrum of LI-F04a and Bovisin,it was found that they both have antibacterial effect on some Gram-positive bacteria, and have no antibacterial effect on Gram-negative bacteria. |
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