Screening For The Claviceps And Analysis Of The Metabolites Of The Claviceps

Ergot alkaloids are products of tryptophan by prenylation reaction, the compounds have similar structure with neurotransmitters such as5-hydroxy tryptamine, dopamine and a-noradrenaline. And they also show agonists, partial agonists or antagonists on the active receptor sites, thus affecting immune system, reproductive system, circulatory system and nervous system. The biotechnology of ergot alkaloids abroad has reached the level of industrialization, but the domestic was still unable to carry out industrial production.At present, the ergot alkaloids used in the research of production in our countries were provided by culture collection center. Fermentation of DSM No.833has been reported. Our group has used DSM No.833obtained from foreignal culture collection center to research the fermentation of the strain, and we also analyzed the products of fermentation. Through the methods of chromogenic reaction, TLC, ESI-MS and1HNMR, we finally determined that fermentation products of the strain didn’t contain ergot alkaloids.In natural, ergot fungu infect with pistil of flowering plants, and then form sclerotia at the ovary. Our group has stimulated the natural environment and used the medium of DSM No.883, DSM No.884, DSM No.885and DSM No.833to infect rye to observe these four strains used rye seed embryoor or seeds as medium could be actived or not. The research suggests that rye seeds can not be infected to form sclerotia, DSM No.883, DSM No.884, DSM No.885and DSM No.833also weren’t converted into active strains.It has been reported that C.paspali could infect with Paspalum distichum L. and form sclerotia. Our group has studied the effective components in sclerotia of Paspalum distichum L.. Through chromogenic reaction, TLC, ESI-MS and1HNMR, we inferred that the sclerotia of Paspalum distichum L. contain ergot alkaloids.There were not reported screening experiments about the ergot fungus from Paspalum distichum L., Oplismenus compositus (Linn.) Beauv., and fescue. This research screened ergot alkaloids producing strains from these three plants and then studied fermentation of the strains. In the process of screening, we examined the effects of different medium and antibiotics. The results showed that SP solid medium containing chloramphenicol and gentamycin was more conductive to separation of ergot fungus.We finally separated250white ergot fungu.We used48deep well-plate to instead the usual screening of shake flask fermentation and produced a high-flux method to screen alkaloid productivity by using double wave ultraviolet spectrophotometry that based on96well-plate microplate reader. We have screened180white fungu having the alkaloid producing ability from250white fungu and used freeze-dried preservation method to maintain the strains.We choosed a white fungi numbered F50to ferment. Lastly, after a series of analysis methods including color reaction, TLC, ESI-MS and1HNMR, we infered that YHW-49-D was ergometrine, YHW-49-B maybe α-hydroxyethyl lysergic acid amide, and YHW-49-E maybe lysergic acid amide.To improve the production, we choosed natural selection and UV mutagenesis selection for F50, and the alkaloid production of the strain have been improved to about200ug/ml from30-40ug/ml. It also retained its ability of producing alkaloid after several times to the future generation. After that, we investigated the relationship between fermentation time and biomass, alkaloid production of F50.It showed that the total alkaloid peaked at the11th day.