Screening And Fermentation Condition Optimization Of The Saponin And Diosgenin-producing Endophytes Of Paris Polyphylla

Paris polyphylla,a kind of valuable medicinal plant belonging to Liliaceae family Paris genus,of which the dioscin,polyphyllin I,II,VI,VII and diosgenin are its main medicinal components and precursors,is mainly used for antisepsis and anti-inflammation,hemostasis,analgesia and anti tumor therapy.It is the important raw material of such Chinese patent medicines as “Yurman Baiyao” and “Gongxuening”.However,slow growth,long maturity cycle and artificial indiscriminate picking have led to the wild resources almost running out.In this study,the endophytic bacteria are separated from the tubers and seeds of Paris polyphylla,then the endophytes which can produce saponins and diosgenin are identified,followed by the strain identification and fermentation conditions optimization,as a result laying the foundation of further exploiting and utilizing Paris polyphylla endophytes resources and alleviating the present Paris polyphylla shortage problem.This thesis also aims to take up a preliminary selection of polyphylla endophytes with germination effect in the experiment.The main achievements are as follows:(1)149 strains of polyphylla endophytes were isolated from the tubers and seeds,among which 2 strains—clx-6 and clx-60 with the ability of producing polyphyllin VI;1 strain—clx-14 with the ability of producing polyphyllin I;1 strain—clx-74 with the ability of producing diosgenin were identified by the further use of liquid chromatography and mass spectrum.The strain identification of bacterial genus indicated that the clx-6 is Escherichia sp,the clx-14 is Enterobacter cloacae strain,the clx-60 is Bacillus amyloliquefaciens strain,and the clx-74 is Enterobacter aerogenes strain.(2)The preliminary screening of polyphylla endophytes with germination effect showed that the chloroform extraction of the endophytes borth can promote seed germination of winter wheat,while the aqueous phases did not,suggested that the active substance located in the organic phases.And the ability to promote seed germination differs in the organic phases extract of different endophytes: the petroleum-ether extract from Enterobacter sp.clx-74,the chloroform extract from Acinetobacter sp.clx-108,the chloroform extract from Bacillus sp.clx-138 and the ethyl acetate extract from Klebsiella sp.clx-149 possessed significant germination effect(p <0.05).The half dilution of the chloroform extract from Bacillus sp.clx-138 had the best germination effect(p <0.01).(3)In the experiment,the culture medium compositions and culture conditions of clx-14 which can produce polyphyllin I are optimized,the final composition of the fermentation culture medium is: Sucrose 12 g/L,Bean pulp 15 g/L,MgCl2 15 g/L;and the optimum culture condition is: The initial pH of culture medium is 6.0,fermentation temperature is 34 ?,table concentrator rotation rate is 150 r/min.As a result,under the optimum culture conditions mentioned above the yield of polyphyllin I of clx-14 is 0.338 mg/L,increased by 86.74% when compared with the initial yield of 0.181 mg/L.