| Methicillin-resistant Staphylococcus aureus(MRSA)is a highly lethal pathogen throughout the world,called of "super bacteria",MRSA infections have exceeded AIDS,tuberculosis and viral hepatitis become the main death reason of patients.This disease pose a great threaten to the public health.The phenomenon that MRSA resistant to common antimicrobial is serious.Vancomycin had been isolated drug-resistant strains as the first choice of clinical use.This grim outlook promoting us to find some new anti-MRSA lead compounds to replaced Vancomycin and developing new antimicrobial drugs.This study is based on screening bacillus strains that can inhibit MRSA significantly and studied on fermentation process,purification of effective components,structure identification,vitro activity and content determination,the mainly work is described as follows:1.One of 45 Bacillus strain SC-2 showed the strongest activity for inhibit MRSA with a good stability,considered the morphological observation,physiological and biochemical determination,combined with the sequence of 16 S rDNA determination,SC-2 was identified as Bacillus subtilis.2.Optimize the best fermentation medium of SC-2 strains: 2.0% glucose,0.5% soybean powder,0.05%calcium carbonate,p H7.0;The optimal seed fermentation conditions was temperature 36 ℃,inoculation quantity 5%(V/V),fluid volume is 1000 m L/ 2000 mL bottle,cultivate 16h;50L fermentation craft: temperature 36℃,inoculation amount 5%(V/V),150 rpm,ventilation 25L/min,tank pressure 0.05 MPa.700 L fermentation: temperature36 ℃,inoculation quantity 7%(V/V),rotation speed 150 rpm,ventilation 21 m3 / h,tank pressure 0.05 MPa.3.The preparation methods of active extracts from SC-2 fermented liquid was filtered by nanofiltration membrane enrichment,this step determines the main active ingredients of SC-2 molecular weight scope is 200-1000;Screen out HP20 macroporous adsorption resin for SC-2 membrane concentrate liquid decoloration,technical parameters: sampling amount 7 BV,adsorption rate 2BV/h,solvent decortion is 50% ethanol contained 2% hydrochloric acid;Screen out AmberliteXAD16 N macroporous adsorption resin for extracted the active substances from SC-2 destaining solution,technical parameters: adsorption quantity 3.5 BV,adsorption rate is BV/h,elution solvent is 20%,elution volume 3.5BV,elution rate 1BV/h;use 75% ethanol precipitation to remove impurities Finally.This process is simple,noise rate reached 80%,crude extractings equivalent to antimicrobial activity of vancomycin on the same concentration.4.One active substance named G2-Ⅱ was obtained by use of silica gel column chromatography,gel G10 filtering and HPLC preparation.It was identified as a peptide compound,molecular weight is 376.3,composed of Ala-Lys-APPA,formula as C14H25N4O6 P by mass spectrometry,amino acid N-terminal sequencing,amino acid composition analysis and compared with the literature product reported.5.Established a reversed phase HPLC method to determine the content of active substance G2-Ⅱ in fermented liquid.The chromatographic conditions included column of Welchrom LP-C18(250mm×4.6mm,5um,);Wavelength:204nm;Mobile phase: methanolwater(1:99→3:97,0-30min);Flow velocity: 0.8mL · min-1;Column temperature: 25℃.The results showed that a good linear relationship was established in the range 5.0μg~25.0μg for compound G2-Ⅱ with the regression equation Y=2E+06x+870660,r=0.9996.The content of G2-Ⅱ is about 0.90~0.93mg/m L in SC-2 fermented liquid by this method.6.Compound G2-Ⅱ antimicrobial spectrum included staphylococcus aureus,resistant acinetobacter baumannii,acne propionic acid bacillus,e.coli,salmonella,h.pylori bacteria,candida albicans,Pityrosporum,etc.It has a wide antimicrobial spectrum.The G2-Ⅱ MIC of anti-MRSA value is 8 ug/m L,superior to vancomycin 16 u g/m L;Compound G2-Ⅱ antibacterial activity for MRSA was a growing tendency in the concentration of 32 ug/mL-3mg/mL and the antibacterial effect was obviously superior vancomycin in vitro. |
