The Antimicrobial Peptides Produced By Bacillus Subtilis ZJU15

A bacterial strain ZJU15, isolated from rice leaf in Hangzhou, China and was identified as Bacillus subtilis ZJU15 according to the conventional physiological biochemistry properties, electron microscope and 16S rDNA sequence analysis. The cell-free supernatant (CFS) of B.subtilis ZJU15 showed antimicrobial activity against several important Gram-positive pathogenic and food-spoilage bacteria, which including Enterococcus feacalis, Listeria monocytogenes, Micrococcus luteus, meticillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis, but it was neither inhibitory to the Gram-Negative bacteria nor the closely related Bacillus bacteria such as B. subtilis, B. pumilus and B. licheniformis. The CFS could also inhibit the growth of some fungal strains such as Aspergillus flavus, Penicillium digitatum and Rhizoctonia solani, but could not inhibit the growth of Candida spp.The comparison of different culture mediums indicated that CM culture medium was most suitable for the production and extraction of the antibacterial peptides of strain ZJU15. Through the growth curve and the activity detection, it was found that the biggest inhibiting activity reached after the culture of 30 hours. The inhibiting activity of suspension could reach maximum after ammonium sulfate precipitation with 20%-30% saturateion. Based on these results, strain ZJU15 was incubated in CM medium under shake cultivation for 30 hours at 35℃. Three AMPs designated ZJU15A, ZJU15B and ZJU15C were purified from the CFS by sequential ammonium sulphate precipitation with 20% saturateion, gel filtration and reverse phase high-performance liquid chromatography (RP-HPLC).Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), Electrospray ionization mass spectrometry (ESI/MS) and Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was performed at the Chinese People's Liberation Army Academy of Military Medical Sciences Biomedicine Analysis Center and the results revealed that the ZJU15A, ZJU15B, and ZJU15C had molecular mass of 1021.70 Da, 1021.71 Da, and 1035.69 Da with the sequences of nine amino acid residues:LLELLVPLL for ZJU15A, LLELLVPLL for ZJU15B and LTHMLVPLL for ZJU15C respectively. There had no significant homology to the known peptides in the database, in our knowledge which indicating that they may be novel antimicrobial peptides. The cell-free culture of strain ZJU15 lost the inhibiting activity completely after the treatment of proteinase K, and was sensitive to the proteinase E partially, but was stable after the treatment of trypsin, pepsin. The inhibiting activity of the cell-free culture of ZJU15 was invariable at pH2-10, and droped obviously at pHl land pH12. The inhibiting activity of the cell-free culture of strain ZJU15 maintains invariable after treatment at 37℃,60℃,80℃,100℃or 121℃for 30min.The culture of Listeria monocytogenes at early expression phase was added with different concentration of the Crude Antimicrobial Peptides Supernatant (CAPS) of strain ZJU 15, and the colony-forming units (CFU)/ml was detected after 24h. The results showed that 256AU/ml of the CAPS was the minimum bactericidal concentration (MBC) and 512AU/ml of the CAPS was the minimum inhibitory concentration (MIC). The cells edge blurring, yacuole, wall flaw was observed by transmission electron microscope after the addtion of 512AU/ml of the CAPS to the culture of L. monocytogenes. This result indicated that the CAPS of strain ZJU 15 could cause the cell wall of L. monocytogenes to burst or to form the hole, which resulted in the contents divulging and the cell death finally.The results indicated that the CFS of strain ZJU 15 and the nisin have the coordination synergized action.The acute oral toxicity on the mouse showed that the CAPS of strain ZJU15 was not caused death to the tested mouses at the dosage of 50ml/kg.In order to estimate the preservation efficiency of the antimicrobial peptides produced by ZJU 15 strain on foods, cured meat, fresh soya-bean milk and milk were tested by adding the crude antimicrobial peptides of strain ZJU15. The cured meat, which were treated with 1024 AU/g and 512 AU/g as final concentration of the CAPS or dH2O (as control), were kept at 37℃. After 7d, white microbial spots appeared on the cured meat with the CAPS at 1024 AU/g or 512 AU/g, which was four days later than the time white microbial spots appeared on the cured meat with dH2O. The fresh soya-bean milk, which was treated with 1024 AU/g or 512 AU/g as final concentration of the CAPS or dH2O (as control), was kept at 37℃. After cultivation for16h, no microorganisms was detected in the fresh soya-bean milk with the crude antimicrobial peptides at 1024 AU/g or 512 AU/g. The commercial pure milk, which was treated with 1024 AU/g or 512 AU/g as final concentration of the CAPS or dH2O (as control), was kept at 37℃. After cultivation for 20h, the number of microorganisms was less greatly in the fresh soya-bean milk which incubated with the crude antimicrobial peptides at 1024 AU/g than other treatments.