Screening Of Tropical Marine Streptomyces Griseus And Its Metabolically Active Products

Actinomycetes can produce natural products with biological activity and play an important role in the development of microbial drugs.The marine environment is characterized by high pressure,low oxygen,high salt and oligotrophin,which makes marine actinomycetes produce metabolic pathways different from those of terrestrial actinomycetes,and then produce metabolites with novel structure.Therefore,marine actinomycetes become a research hotspot of natural medicine.This paper screened actinomycetes from mangrove soil in Dongzhaigang Mangrove,haikou,and obtained a strain with strong activity.The main research results are as follows:(1)The mangrove soil samples in Dongzhaigang Mangrove of haikou were treated by air drying,a total of 45 actinomycetes were isolated from sample using four different media.The test origanisms were used as Staphylococcus aureus,Escherichia coli,Gas bacillus,Proteus,Aspergillus flavus,and the antimicrobial activity was studied by cup plate method,17 strains of actinomycetes were found with antibacterial activity,3 strains showed inhibitory effect on Staphylococcus aureus,8 strains showed inhibitory effect on Aerobacter aerogenes,14 strains showed inhibitory effect on Proteus,1 strain showed inhibitory effect on Escherichia coli,4 strains showed inhibitory effect on Aspergillus flavus.The results showed that the mangrove soil inDongzhaigang Mangrove of haikou contained rich actinomycetes resources,and 37.78% of the strains had antibacterial activity,which was an effective source of the products of new natural products.The strain HN60 with good antibacterial activity was selected for subsequent experiments.The strain HN60 was identified as Streptomyces griseoplanus by morphological observation,physiological and biochemical experiments and molecular identification.(2)The culture conditions of Streptomyces griseoplanus HN60 were optimized by single factor experiments.The experimental results showed that when the culture time was 6 days,the initial pH of the culture medium was 7.0,the culture temperature was 30?,and the inoculation amount was 8%,the yield was relatively high.After optimization,the inhibition zone of Staphylococcus aureus,Aerobacter aerogenes and Proteus were increased by 7 mm,6 mm and 7 mm respectively.The stability of the antimicrobial substance produced by Streptomyces griseoplanus HN60 was studied from four aspects: temperature,acidity,alkalinity,genetic and light time.The results showed that the antibacterial activity retention rate of fermentation broth decreased to 80%?85% after 100? treatment for 1h.There was no significant difference in the retention rate of antibacterial activity between the fermentation broth and the control group after treatment with a lower pH(2,3),while the retention rate of antibacterial activity after treatment with a higher pH(11,12,13)was significantly lower than that of the control group,dropping to less than 80% and as low as 29%;There was no significant effect of different light time and the number of strain generations on the retention rate of antibacterial activity.The results showed that the antibacterial substances produced by Streptomyces griseoplanus HN60 had good light stability and genetic stability,and could exist stably under acidic conditions,but had poor stability under high temperature and alkali environment.The fermentation broth has a good inhibitory effect on Aspergillus flavus,and the biological control experiment of maize shows that the fermentation broth has a good biological activity in practical application,which is the theoretical basis for it to become a new biological control agent.(3)Liquid fermentation was carried out on Streptomyces griseoplanus HN60.The ethyl acetate phase metabolites of strain HN60 were isolated and purified by means of silica gel column chromatography,reverse-phase silica gel column chromatography,and Sephadex LH-20 and two compounds were obtained.1D-NMR and 2D-NMR were used to determine the structural formula of the two compounds and the relative molecular mass of the two compounds was determined by the combination of LC-MS.The results showed that compound 1 was actinomycin V and compound 2 was actinomycin D.The hydrophase metabolites of Streptomyces griseoplanus HN60 were isolated and purified by methyl alcohol precipitation,isoelectric precipitation and Sephadex G-25 column chromatography,and complex S-1 was obtained.(4)The activity of actinomycin V and actinomycin D against Staphylococcus aureus was determined by the minimum inhibitory concentration test.The results showed that the minimum inhibitory concentrations of actinomycin V and actinomycin D against Staphylococcus aureus were 0.195 ?g/mL and 0.391 ?g/mL,respectively.Effects of compounds on the growth curve of Staphylococcus aureus it was found that actinomycin V and actinomycin D could significantly inhibit the growth of Staphylococcus aureus.By transmission electron microscopy,scanning electron microscope to observe the effects of compound of Staphylococcus aureus morphology,found actinomycin V,actinomycin D all can affect the morphology of Staphylococcus aureus and the effect of effect similar to that in low drug concentration in the form of Staphylococcus aureus,high drug concentration of Staphylococcus aureus cells pyknosis phenomenon in a matrix.The above experimental results indicated that actinomycin V and actinomycin D could inhibit the growth of Staphylococcus aureus by changing its cell structure.