Study On Antibacterial Metabolites Produced By Strain Streptomyces Galilaeus AF1

The strain Streptomyces galilaeus AF1 was separated by our team,preliminary research found that the metabolites of S.galilaeus AF1 have antimicrobial activity against gram-positive bacterium such as S.aureus,S.Lugdunensis,especial with the same effect on drug-resistance bacteria such as MRSA?methicillin resistant S.aureus?.Preliminary studies on antibacterial mechanism showed that S.galilaeus AF1metabolites can destroy the cells of resting gram-positive bacteria,which mechanism of action is different from other antibiotics used as medicine obviously.But it have no bioactivity against gram-negative bacteria.The metabolites of S.galilaeus AF1 have the potential to develop new antibiotics.This paper studied the antibacterial activity and mechanism of S.galilaeus AF1 metabolites on S.agalactiae and MRSA,which used as indicator bacteria.The activity tracking method was used to isolate the metabolites,and the solid fermentation of strain AF1 spores in optimum conditions were studied.In order to provide a theoretical basis for the further development and utilization of S.galilaeus AF1.The antibacterial activity of the strain AF1 metabolites were evaluated by Plat stiletto method.The results showed that the extract of metabolites have different inhibitory effect on S.agalactiae,S.albus,S.aureus,Bacillus subtilis and MRSA,but without effect on Escherichia coli,Vibrio cholera,Ralstonia solanacearum.The MIC?Minimum inhibitory concentration?of strain AF1 metabolites to S.agalactiae was38?g/mL,and the MIC to MRSA was 68?g/mL.The inhibition results which compared with the certain antimicrobial agents ampicillin,cephalosporin,chloramphenicol and rifampicin against S.agalactiae showed that the metabolites of strain AF1 have good antibacterial activities on S.agalactiae.The growth curve method,direct counting and morphological observation method were used to study the bactericidal effect.The crude extract had certain bactericidal effect against S.agalactiae and MRSA.Lethal effect showed positive relationship of dose and time.The results of scanning electron microscopy showed that the morphology of the cells changed and seriously damaged by the metabolites of strain AF1.The permeability of cell wall by alkaline phosphatase content showed increased after treatment.There is no obvious change in the cell membrane potential measured by flow cytometry.The crude extracts of strain AF1 metabolites has destructive effect on the cell wall.The LC₅₀?96 h?value of the metabolites to zebrafish were 27.44 mg/L and 35.44 mg/L by static method and feeding method,respectively.The safety test showed that the crude extract was low toxic and safe to zebrafish.Using activity tracking methods,and the ethyl acetate extracts of S.galilaeus AF1fermentation was isolated by normal silica gel column chromatography,thin layer chromatography and high performance liquid chromatography.The C3 fraction with antibacterial activity was obtained.Further purification and NMR were made.It may be a dihydrophenanthrene based on the available data.The purified active substance had good antibacterial activity on S.agalactiae.To get a large number of spores with high quality,the solid fermentation conditions on producing spores for S.galilaeus AF1 were studied.A single factor experiment was conducted to screen the solid sporulation substrates?rice,corn flour,cassava residue,soybean flour,bran and oatmeal?.The effects of incubation time,temperature,liquid to solid ratio,inoculation amount,carbon source and nitrogen source on sporulation yield of S.galilaeus AF1 were tested.The conditions affecting the sporulation yield were optimized by orthogonal test based on single factor experiments.The results showed that the substrate for rice and the best fermentation conditions were as follows:the incubation temperature of 28?,the incubation time of 6 d,inoculation amount 10%?2 mL spore suspension inoculated in 20 g rice?,liquid?mL?to solid?g?ratio 1:1,1.5%?W/W?glucose as carbon source,1.5%?W/W?peptone as nitrogen source.Under above conditions,the amount of conidia reached 3.83×10⁹ g^-1,which was increased by 29%than original medium.The obtained S.galilaeus AF1 has high sporulation level and meets the requirement of large-scale inoculum.