Screening And Identification Of Marine Actinomyces And Study On Of Its Antimicrobial Active Substances

With the extensive use of antibiotics,pathogen resistance gradually increases;existing antibiotics cannot deal with this situation.In order to overcome these problems,the continued development of new bioactive substances is an urgent need for the current pharmaceutical industry.The marine is rich in microbial resources,and the unique environment allows marine microorganism to produce many novel biologically active substances.Actinomycetes are the main species of produced biologically active substances.Screening of actinomycetes producing biologically active substances from the marine is a hotspot in recent years,and many new marine bioactive substances have good application prospects.In this study,an actinomycetes isolated from sea mud of the East China Sea had wide antimicrobial spectrum and had strong antibacterial activity,and identified the actinomycete.The physicochemical properties of the antimicrobial substances produced by this actinomycete were studied,and the fermentation medium was optimized.Finally,the mechanism of action of this actinomycete on Listeria monocytogenes ATCC19114 was studied.The specific studies are as follows:1.Screening and identification of marine actinomycetesThe target bacteria from sea mud samples was primarily screened with agar block method,and further screened with punching diffusion method.A total of 12 strains showing antimicrobial activities to antagonistic strains were isolated,and the antimicrobial activity of Y10,Y11,Y15,Y16 and Y21 were stronger,and it can produce antimicrobial active substances in the four fermentation medium.The antimicrobial spectrum analysis showed that Y15 had the most extensive antimicrobial spectrum.The Y15 could be against 18 of the 20 kinds of indicator strains,especially for L.monocytogenes,S.aureus and A.veronii.Therefore,the Y15 was identified as target strain.Y15 was identified as target strain by antimicrobial spectrum analysis.This strain Y15 was identified as Micromonospora by using 16 S rRNA gene sequence homology analysis and morphological analysis,and obtained Gen Bank accession number: KU517850.2.Studing on the properties of Y15 antimicrobial substanceChange of antimicrobial substance during Y15 fermentation and Y15 passages stability were studied.The temperature stability pH stability,UV stability,enzyme hydrolysis stability and polarity of Y15 antimicrobial substance were analyzed.Activity of Y15 antimicrobial substance reached a maximum of 480 AU/m L in 144 h,maintained 320 AU/mL from 168 h to 216 h.The strain Y15 in the 48 h to 216 h for the growth of the logarithmic period,in the 216 h to 288 h for the growth of stable period.The strain Y15 passage stability is better,in the first generation to the seventh generation of strain to maintain stability.Antimicrobial activity of Y15 antimicrobial active substances remained stable from-20 °C to 60 °C,and gradually declined from 80 °C to 120 °C;antimicrobial activity remained stable from pH 7.0 to 10.0,and from pH 2.0 to 6.0 and pH 11.0 to 12.0 antimicrobial activity declined;Y15 antimicrobial substances processed in the UV and four kinds of enzymes were stable,there were non-protein and non-peptide antimicrobial substances.The Y15 antimicrobial substance is easy to be extracted by organic reagents,and ethyl acetate extraction effect is the best,after the activity of up to 5200 AU/mL,indicating that the antimicrobial substance has a moderate polarity.3.Optimizing of Y15 fermentation mediumThe incubation conditions of strain Y15 were optimized by single factor experiment,and the composition of Y15 fermentation medium was optimized by response surface methodology.The optimum culture conditions were determined as follows: temperature was 30 °C,medium initial pH was 7.20,inoculation size was 4% and shaking speed was 180 rpm.The single factor experiment results suggested that the optimal medium components were soluble starch,beef extract,peptone,NaCl,K2HPO4,MgSO4,and FeSO4,while the Plackett-Burman experiment demonstrated that beef extract,K2HPO4 and soluble starch were significant factors affecting antimicrobial activity.The steepest ascent experiment results further showed the central point of three significant factors were 1.00 g/L of K2HPO4,25.00 g/L of soluble starch and 18.00 g/L of beef extract,respectively.The CCD revealed that the optimum values of the tested significant variables for the production of antimicrobial metabolites were 1.03 g/L of K2HPO4,27.67 g/L of soluble starch,18.30 g/L of beef extract,7.50 g/L of tryptone,0.02 g/L of FeSO4,0.38 g/L of Na Cl,0.75 g/L of MgSO4,accordingly.Under this optimal condition,the antimicrobial activity of the fermented medium was 1236.79 ± 14.56 AU/m L,which increased 3.86 times compared with the initial medium.4.Preliminary study on antimicrobial mechanism of Y15 antimicrobial ActiveSubstanceThe mechanism of Y15 antimicrobial substance on antimicrobial of L.monocytogenes ATCC19114 was discussed.The results showed that Y15 antimicrobial substance had significant effect on the growth curve of the indicator bacteria.There was no damage to the cell wall,the intracellular ion was leaked and the macromolecules did not leak,suggested that the antimicrobial substance has little damage to the cell membrane.The SEM observation showed that the experimental group indicating bacteria cell morphogenesis shrinkage,bending,but did not cause cell lysis,confirm the above inference.On the other hand,the antimicrobial substance of Y15 had no effect on the DNA,but decreased the expression of soluble protein.Therefore,it is concluded that the mechanism of action of Y15 may be inhibited the expression of intracellular soluble protein and change cell membrane permeability,and the specific mechanism remains to be further studied.Micromonospora is a rare actinomycete,and it produced biologically active substances second only to Streptomyces,has important development value.Y15 showed closest relationship with Micromonospora endolithica,which was a new species identified.Up till now,there has been no report about Micromonospora endolithica's ability to produce antimicrobial metabolites,but Y15 has excellent antimicrobial activity.The Y15 has a broad antimicrobial spectrum,and it can be aganist to aquatic product pathogens such as L.monocytogenes,S.aureus,A.veronii and V.parahaemolyticus.Therefore,Y15 in the prevention and control of aquatic products also has certain application significance.The results of this study can provide theoretical basis for the separation and purification of antimicrobial substances in the later stage,practical application and mechanism research based on molecular biology.