Isolation And Purification Of Endophyte Fungus From Huperzia Crispata (Ching) And Study On Biologic Fermentation Technology Of Potassium Hydrogen Oxalate Hydrate

When we attempt to establish the plant tissue culture system,We find that the explant always infected no matter how strict to disinfect,so we speculate on the probability that endophyte is exist in the living plant tissure.It seems to be probable that some special endophyte fungus can produce some bioactive components which are similar to the components produced by its host plant as a result of inheritance information exchange between endophyte and host plants when they evolve cooperated with each other.We use the extraction of Huperzia crispata(Ching) Ching with chloroform exclusively to filter the fungus starting with isolation and purification of endophytic fungus.When we study on the endophyte variety of Huperzia crispata(Ching) Ching and bioactive metabolltes producing by these endophytes,We find out a kind of fungi,it can produce Potassium Hydrogen Oxalate Hydrate under appropriate fermentation condition.At the same time,We has explored the optimized fermentation condition to producing Potassium Hydrogen Oxalate Hydrate and obtained the following conclusion.1 Huperzia crispata(Ching) Ching holds abundant endophyte communityWe have obtained 32 kinds of endophytes separated from Huperzia crispata (Ching)Ching by using classical PDA solid culture medium,there are obvious contrast on the aspect of appearance,form and culture character among these 32 kinds of endophyte.These endophytes has been preliminary identified to be Achlya,Mucor, Aspergillus,Penicillium,Rhodotorula and so on.2 Fungi J 060918 possesses the capability to produce Potassium Hydrogen Oxalate HydrateWe have found that only 6 kinds fungi can grow up well on the hungriness culture medium which contains the Huperzia crispata(Ching) Ching chloroform extraction exclusively.We have found the fungi J 060918,It can produce Potassium Hydrogen Oxalate Hydrate with high yield(＞1.758g).This fungi has been preliminary identified to be Aspergillus niger.3 Optimized fermentation technics to produce Potassium Hydrogen Oxalate HydrateWe have optimized the fermentation condition from the aspects of carbon source variety,dosage of carbon source,culture time,primary pH,dosage of spore and so on. And designed orthogonal optimization experiment based on these experiment.The result indicate that the best fermentation condition is the following condition:PDA is the basal medium,the best concentration of fungi spore is 1×10~5/mL,used as 4 mL per 1 L to the medium.Dosage of sucrose is 10 g/L.nature pH,And the best culture time and temperature is 60 h and 40℃.120 r/min is the best Rolling rate.We can obtain crude Potassium Hydrogen Oxalate Hydrate sample 1.758 g/L at least under this optimized fermentation condition.