Screening Endophytic Bacteria Of Producing Alkaloids From Lycoris Radiata And Studying Its Physiological Function

There are a variety of alkaloids contained in Lycoris radiata,and these alkaloids have important pharmacological effects.However,the yield of alkaloids extracted from Lycoris radiata is very low,and it can not meet the demand of current market.At present,isolating and screening endophytic bacteria which can produce the same active products from the host plant,and utilizing the fermentation of endophytic bacteria to accumulate secondary metabolites has become a alternative way to solve the lack of some plant active products.Meanwhile,there are few studies on endophytic bacteria of Lycoris radiata,we take the bulbs of Lycoris radiata as research materials in this experiment.Endophytic bacteria was isolated by Microbiology methods from Lycoris radiata,and fermented in liquid medium,then detect the content of alkaloids of fermentation broth by HPLC.Scanning the Endophyte which have a high yield.In this study,the determination the Physiological and biochemical reactions of endophyte,the degradation of Phenolic substances and the effects on inhibiting growth of crop pathogenic fungi and bacteria;the optimization of Fermentation medium composition and culture conditions by central composite deign.Meantime,we take Lycoris bulbs as explant to induce Adventitious buds through plant tissue culture.A culture system for the co-culture of adventitious buds and alkaloid-producing bacteria was established.The effects of different precursors on the growth and alkaloid accumulation of adventitious buds in the co-culture system were studied.Main results were concluded as below.1.18 different species endophytic bacteria were isolated from the bulbs of Lycoris radiata.And the alkaloid in the fermentation product is extracted by organic solvent after high-volume fermentation,the fermentation product was detected by thin layer chromatography and High Performance Liquid Chromatography.The results show Pseudomonas sp.and Acinetobacter sp.can yield alkaloid by fermentation,and Acinetobacter sp.has a higher-yield alkaloid than Pseudomonas sp.2.The optimal growth conditions of Acinetobacter sp.in the beef culture medium were determined.The results showed that the optimum culture temperature was 30?,the optimum rotation speed was 200 rpm,and the optimum growth pH was 7.0.Logarithmic growth phase appears in 12¹6h.3.Identification of physiological and biochemical reactions of Acinetobacter spp..The results showed that Acinetobacter sp.had denitrification and heterotrophic nitrification,and the result of MR test were positive.The results of gelatin hydrolysis test,indole test and VP test were negative.4.The degradation of Phenolic substances.The strain was subjected to repeated acclimation with phenol as the sole carbon source,and the concentration of the remaining phenol was determined by the 4-aminoimidine method.The degradation rate of repeated domesticated strain on catechol,hydroquinone,resorcinol,pyrogallol,phloroglucinol and2,4-dinitrophenol was determined.The results showed that the degradation rate of catechol,resorcinol and hydroquinone ware all more than 90%,and the degradation rate of pyrogallol and phloroglucin was 41.62%,52.33%,relatively;the degradation rate of2,4-dinitrophenol was only 10.27%.At the same time,the degradation rate of wild strain,one-domesticated strain and repeated-domesticated strain on 400 mg·L^-11 phenol was studied.It was found that the phenol was completely degraded by the repeated-domesticated strain for about 18 hours,and the degradation time was shortened by 12 h after about 30h.The kinetics of the degradation of different concentration phenol by repeated-domesticated strain was in accordance with Monod kinetic model.The reaction rate of K_m was 156.57 mg·L^-1phenol,the maximum reaction rate V_m is 32.34 mg phenol·?10⁸ bacteria·h?^-1.5.Inhibitory effects of fermentation broth of Acinetobacter sp.on crop pathogenic fungi and other bacteria.The in vitro antibacterial test of fermented broth was carried out by Oxford-cup method.The results showed that there was a strong bacteriostatic effect on Rhizoctonia solani,Fusarium oxysporum,Rhizoctonia Cerealis,Bacillus subtilis and Escherichia coli.6.Optimization of fermentation medium components and culture conditions of Acinetobacter spp.by central composite design methodology.The nutrients required for microbial growth and the economics of the culture medium are taking into account.We choose corn flour,yeast extract,beef paste,NaCl,MgSO₄,KCl,K₂HO₄,FeSO₄,glucose,and?NH₄?₂SO₄,MnSO₄ as medium components,and the effects of medium pH,temperature,Rotation speed,culture time on the fermention were studied.A 15-factors and two-levels was designed by minitab 17.0 software.The results show that medium components were Corn flour 15.0 g,yeast extract 25.0 g,beef extract 10.0 g,NaCl 1.0 g,MgSO₄ 0.2 g,KCl0.1 g,K₂HPO₄ 1.0 g,FeSO₄ 0.001 g,glucose 10.0 g,?NH₄?₂SO₄ 1.5 g,MnSO₄ 0.006 g;culture conditions were pH 7.4,speed 200 rpm,temperature 37?,culture time 72h.Under these conditions,the accumulation of alkaloids of Acinetobacter sp.could be the highest.7.Establishment of co-culture system of adventitious buds and Acinetobacter sp..The adventitious buds were induced from the bulbs of Lycoris radiata and transfered twice,and the medium was changed to MS liquid culture medium.At the same time,Acinetobacter sp.was domesticated by MS liquid medium.And at the 9th day,the domesticated strain was added to the system of adventitious buds liquid shaking medium,adding a series of different concentration of bacterial liquid to system.To observe the growth status of adventitious buds and to detect the content of alkaloids of adventitious buds.The results showed that the growth of adventitious buds was the best when adding 50?L of Acinetobacter sp.bacterial liquid,and the accumulation of alkaloids was the highest.8.Effects of precursors on the growth and bioaccumulation of co-culture system.We choose phenylalanine,tyrosine,tyramine,dopamine,protocatechuic aldehyde,4-hydroxyphenylpyruvic acid?4-HPPA?,p-hydroxybenzaldehyde as precursors,and study their effects.The results showed that the addition of 4-HPPA,p-hydroxybenzaldehyde and phenylalanine had no significant effect on the accumulation of alkaloids of adventitious buds.When adding tyrosine and tyramine with the concentration of precursors,the accumulation of alkaloids in adventitious buds increased,while the addition amount beyond 250?mol·L-¹,the accumulation of alkaloids slowed down;When added protocatechuic aldehyde 50?mol·L^-11 and dopamine 100?mol·L^-1,the adventitious buds has the fastest growing and the content of alkaloid was the highest in the experimental group.