| Ophiocordyceps longissima (Kobayasi) G.H. Sung et al., the teleomorph of Hirsutella longissima, which infects the nymph of cicada, is an important parasitic fungus. The bacteriostatic activity and partial components of cultured O. longissima were investigated in the paper for the first time, mainly including:submerged and solid culture of anamorphic strain RCEF 3891 of H. huangshanensis, four treatment measures used to extract the artificial cultured fruiting body of O. longissima and the mycelia by shake-flask culture, bacteriostatic tests for six kinds of bacteria and 1 kind of fungus by Oxford cup method, the indicator bacteria selected by determination of the bacteriostatic activity of the extract, separation, purification and structural identification of active component in the extract with high bacteriostatic activity and major effective constituents analysis of artificial culture and mass spectrometry analysis of methanol extract.The result of antimicrobial activity test showed that fruiting body of artificial cultured O. longissima, aqueous extract of mycelia and methanol extract have inhibitory action to Bacillus thuringiensis, Pseudomonas aeruginosa and Candida albicans and no bacteriostatic action to other tested bacteria; the bacteriostatic action to Candida albicans is the highest, thus, Candida albicans was selected as the optimum indicator microbe. Aqueous extract of the cultured Ophiocordyceps sinensis has weak bacteriostatic action to Staphylococcus aureus, Bacillus substilis, Escherichia coli and Pseudomonas aeruginosa and no bacteriostatic action to other tested bacteria. Ethyl acetate extract of three artificial cultures have no bacteriostatic activity to the tested bacteria. Fruiting body of artificial cultured O. longissima and extract of mycelia by shake-flask culture still have bacteriostatic activity after being heated in water bath at 80℃ which means the bacteriostatic constituents has the feature of heat resistance.The extract component was separated primarily by semi-preparative column in the test; using inhibition of Candida albicans as index, the activity of separated component is tested and one active component p-1 was confirmed; analytical column was used by the test to separate and such active component had been prepared successfully.Through analysis of LC-TOF-MS data and NMR data, it is known that molecular formula of component p-1 is C21H39NO6 and scientific name is 2S,3R,4R, 6E-2-aminogroup-3,4-dihydroxyl-2-hydroxyrnethy11-14-oxygen-6-eicosenoic acid, i.e. Myriocin. It is found firstly in O. longissima and its content is much higher.Effective constituent analysis of three kinds of artificial cultures of O. longissima, i.e.. stromata, synnemata and mycelia from solid plate, was determinated. The results showed that the maltose content of the three artificial cultures had no obvious difference, and other constituent contents varied a lot from each other; The contents of amino acids, total amino acids in the artificial cultures of O. longissima was in the following descending order: mycelia (26.68 g/100g)> synnemata (22.94 g/100g)> stromata(20.81 g/100g). Total essential amino acids in synnemata (8.03 g/100g) and stromata (7.97 g/100g) were higher than that in mycelia (7.4 g/100g).Through culture and optimization of myriocin content in liquid shake-flask cultured mycelia,12-15d was confirmed to be the optimum culturing time; through screening among five different kinds of fluid medium, nutrient solution V was selected to be the optimum liquid medium; And it was confirmed that wheat bran and yeast were optimum carbon source and nitrogen source, respectively.The constituents of methanol extract between mycelia and fruiting body (mixed with synnemata) of O. longissima possessed some common ingredients, both containing myriocin, stearic acid and sphingosine substances; The difference is in the mycelia of O. longissima containing octadecadienoic acid and neurine and other substances. Except these constituents, structures and activities of substances like C17H19N5, C22H47NO5 and C23H45NO3 owned by fruiting body and C25H32O8 and C21H39NO5 owned by mycelia are for further study. |
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