Isolation And Idenfication Of Moss Endophytes And The Study Of Their Metabolites

Moss plants have had a long history on the application of medicine, and they areone of the pioneers in plant kingdom, literature from home and broad show that Mossendogenous bacteria can be used as a new type of microorganism resources, somemetabolites have the ability of antitumor and antibiosis, and we had gotten a series ofnovel active compounds from these metabolites.This study focused on the separation of20genera of moss endogenous bacteriafrom Guangxi Guilin. Firstly the endogenous bacteria isolation method was optimized.The main steps included surface disinfection, grinding, dilution, coating, thermostaticculture and got237endophytes. The colony and cell morphology of33endophyteswhich have big difference on phenotypic characteristics was anlysised. The results showthat the moss endogenous has rich diversity in the area. We used staphylococcus aureusas indicator bacteria to screen endogenous bacteria which have good activity, then wedisscussed the differences of carbon source, nitrogen source,inorganic salt on theinfluence of bacteria biomass and activity, later, we anlysised the diversity of metabolicproducts of endogenous bacteria from the moss. After that we used Gram positivebacteria staphylococcus aureus and Gram negative bacteria E.coli as indicator bacteriato preliminary screen the antibiosis of these moss, and found that moss1-01、122-02、z12、0409-1and1108-09have the strongest antibiosis activity, thus studied thebacteriost at of these five endophytes, and found that0409-1and122-02have the broadspectrum antibacterial activity; we conducted a small-scale fermentation and oreparedcoarse extraction liquid sample of10strains of moss endogenous bacteria, used PC3、MCF-7and HepG-2as target cell to executed antitumour experiment, and it can be seenthat the sample of7-6-4or122-02have good activit to cells. The preliminary studiedshowed that there were a certain exploration values of moss endogenous bacteria7-6-4、122-02、1507-7、041721-03、1023-01. So we can follow up the purification andidentification of active metabolites.The aims of this study was the exploration of mossendophytes isolation method, the construction of moss endophyte resources platform,the probability of finding antibacterial or antitumor activity ingredients. The specificcontent of this paper as follows:(1) The establishment of endogenous bacteria separation methods: Through comparisonto the Ultravioket radiation、75%of ethanol immersion and diluted one hundred times 84disinfectant disinfection effect，the test showed ultravioket radiation and75%ofethanol immersion have good surface disinfection effect. And the cost was so low thatwe can matched them in use. The best way to release endogenous bacteria was todistribute them after grinding. PDA medium and CYM medium were proved to besuitable for most endogenetic bacterial growth. The optimum growth temperature was30℃. Eventually we separated237Endophytic bacteria from20genera of moss leaves,which collected from the Guilin area of Guangxi by this method.(2)We screened out33endophytes which have big difference on phenotypiccharacteristics from237endophytes, studied the diversity of endogenous bacteria byanalysisng the colony morphology and cells form of these strains. By comparing thecolony morphology and cells form, found that they had high morphological diversity.We used staphylococcus aureus as indicator bacteria to screen endogenous bacteriawhich have antibacterial activity, then we anlysised the diversity of nine endogenousbacteria expending of carbon source, nitrogen source, inorganic salt which have goodactivity.(3)We identified six endophytes which have good activity on staphylococcus aureus bymolecular techniques, and identified that z12is bacillus megaterium,1-1belongs toendosporous species,110-02and122-02belong to smoke of aspergillus,122-03belongs to mucor,7-6-4belongs to portuguese stem root of mould.(4)In order to find the active material in moss endogenous bacteria, we choosed strainswhich have good activity on staphylococcus aureus to analysis the characteristics ofmetabolic products by TLC and HPLC. From this we can see the rich varieties ofmetabolites of moss endogenous bacteria, different moss endogenous bacteria havedifferent metabolic products. We can lay the foundation for the next study of activematerial.(5)We used E.coil and staphylococcus aureus as preliminary screening indicatorbacteria, and carried out antimicrobial activity screen on the33strains which have bigdifferences on phenotypic characteristics, we found that11strains have differentdegrees of inhibiton,18strains have inhibition on staphylococcus aureus. The fivestrains which have the strongest bacteriostat are1-01、122-02、z12、0409-1、1108-09,and we did the further research of the antibacterial ability. The result showed that thethree strains,122-02、z12、0409-1, displayed goog braking effect on mutiples ofindicator bacteria.(6)We had studied coarse extraction liquid sample of10strains which have goodacvitity, PC3、MCF-7and HepG-2had been used as target cells, we can get that the sample of7-6-4and122-02have good activity on cells, where in1507-7、041721-03、1023-01showed better activity on HepG-2, thus metabolin of moss endogenous bacteria,7-6-4、122-02、1507-7、041721-03、1023-01have certain development potential.Thereby we can carry on active metabolite purification and identification, and thenprovide valuable resources for seeking the new active natural products.