Preliminary Study On The Secondary Metabolites Of Bacillus Subtilis UMBR1027 And Streptomyces Somaliensis UMBR2015

Staphylococcus aureus is a serious gram-positive pathogenic bacteria to human and animal.S.aureus was condition pathogenic bacteria which can cause pneumonia,sepsis,endocarditis and other deadly disease.At present,the antibiotic treament was the most effective treatment to S.aureus inhibit.However,as the abuse of antibitics by people,the drug resistance appear gradually,especially the emergence of MRSA.At the same time,the new effective antibiotics which had been approve by government was reducing year by year.So it was very important to explore a new generation of antibiotics including antibacterial protein drugs which has active to S.aureus.This paper contains two parts of research:The first part was on isolation and identification of the active protein which can supprese the s.aureus from marine Bacillus subtilis UMBR1027 and optimization of fermentation process by the response surface method.The active proteins was extracted by ammonium sulfate precipitation,and separated by SephadexLH-20 column chromatography and SDS-PAGE,then identified by ABI 4800 plus MALDI-TOF.The results showed that active protein including alkaline serine protease,subtilisin,endo-β-1,3-1,4 glucanase,alaph amylase were identified.The bacteriostasis activity was assessed by the filter paper diffusion method which’s indicators is the bacteriostatic circle diameter.The three key factors including the pH,salinity and temperatue of the culture medium were evaluated respectively to optimize the fermentation conditions by design Box-Behnken experiments.The optimal culture conditions were that medium pH was 6.95,salinity was 10.38%and the tempreture was 34.95℃.Selecting experiment under the culture conditions were that pH was 6.95,salinity was 10.38%and the tempreture was 35℃,the result is that the bacteriostatic circle diameter was 20.4 mm,which was closed to the predicted value.The second part was on extracting the active secondary metabolism based on active track from Stretomyces somaliensis UMBR2015 which was derived of mangrove forests from Beihai.The active substance was adsorbed from the fermented liquid of S.somaliensis UMBR2015 by macroporous resin HP20,and further purified by Sephadex LH-20 column chromatography and semi-preparation HPLC.Then,the structure of the active compound were determined on the spectroscopic analysis including 1H-NMR and 13C-NMR.The result showed that the active compound was Cyclo-(Leu-Val).The research was carried on in order to shorten the period of biological fermentation of S.somaliensis UMBR2015.The influence of the high-pressure wood pulp sponge to S.somaliensis UMBR2015 was explored by dropping different number of high-pressure wood pulp sponge which volume was 1 cm3 into the medium.The results showed that the high-pressure wood pulp sponge can shorten the producing time of the active metabolites.This discovery provides a new train of thought for the problem that how to shorten the period of biological fermentation.