| Endophytic fungi are highly diverse microbial groups,that can synthesize the same or similar chemical components of host plantsand the secondary metabolites are rich and diverse.Siraitia grosvenori is a traditional medicine and food plant in China.The saponins,polysaccharides and flavonoids in Siraitia grosvenori fruit have good antioxidant activity.This indicates that the endogenous fungus may produce metabolites with antioxidant activity,which may be an important source of antioxidant activity.This paper,use the method of active tracking to study the endophytic fungi and their metabolites in longsheng county,guilin.The contents include: the separation of the endogenous bacteria of fresh Siraitia grosvenori;Research on antioxidant activity;Tracking and separation of active components.Main content and result analysis:1.Separation and chemical composition pretest of endogenous fungi in Siraitia grosvenoriIn this paper,15 strains of endophytic fungi were isolated from the fresh Siraitia grosvenori,and 10 strains were selected to observe the colony morphology.And strain to the fermentation medium of 26 ?,170 r/min table fermentation 6 d,after the fermentation,vacuum suction filter separation fermented liquid and mycelium.Fermented liquid enrichment to extract,mycelium 50 drying,crushing,the? n extracted with petroleum ether,ethanol and water respectively,extracts of chemical composition test,a preliminary understanding of the chemical content of strains of metabolites.The results show that most endophytic fungi contain polysaccharides,flavonoids,anthraquinones and organic acids.2.Screening and identification of active strainsThe removal of 1,1-diphenyl hydrazine-2-bitter free radical(DPPH ·),remove hydroxyl free radical(· OH)and the total reducing power three methods of 10 strains of endophytic fungi on antioxidant activity of fermented product selection.Results show that the strain F-5 fermentation product concentration of 1 mg/m L,the fermented liquid of DPPH · and · OH clearance were 74.09% and 64.74% respectively,the clearance rate of mycelium were 73.56% and 55.32% respectively,less than with the concentration of Vc on DPPH · clearance rate(99.36%)and the · OH clearance(95.77%).The total reduction force of fermentation liquid and mycelium was 1.547 and 1.241 respectively,greater than the total reduction force of the same concentration Vc 1.145.The data indicates that the f-5 strain had strong antioxidant activity,so the molecular biological identification of the f-5 strain was carried out.Selected strain F-5,on the top of free radical clearance on PDA medium,26 4?-5 d,colony growth faster,loose texture,mycelium growth,for the first white with yellow,then into a greenish brown.The DNA of strain f-5 was extracted,and the PCR amplification of 18 s r DNA gene was carried out by using universal primers.The results show that the sequence length is 576 bp.Sequence by BLAST and compare the Gen Bank existing sequence,and construct phylogenetic tree,which shows the strain with Aspergillus oryzae 99% homology,combined with the morphological observation,it is concluded that the strain of Aspergillus oryzae,Aspergillus oryzae).3.Optimization of strain fermentation conditionsUsing single factor experiment,research of carbon source,nitrogen source,the fermentation time of strain effect of antioxidant activity,and on the basis of single factor experiment results,the selection of contents of glucose,peptone,sodium chloride content and initial p H value of the four factors orthogonal experiment design,further optimize the strain condition of antioxidant activity.Results show that the best nitrogen source is peptone,the best carbon source is glucose,the best fermentation time is 6 d,initial p H value of 6 ~ 7,to the strains of the influence factors of scavenging free radicals in the order: sodium chloride content > glucose content > peptone content > initial p H value.The optimal conditions of f-5 are A4B1C1D2,namely glucose 35 g,peptone 2.0 g,Na Cl 0.5g,and initial p H value of 6~7.In this condition,the bacterial strain f-5 was tested for fermentation,and its clearance rate was 87.53% and 84.43%,respectively.The optimum conditions for the antioxidant activity of the strain f-5 fermentation were A4B1C1D2.4.Screening of antioxidant active sitesStrain F-5 after fermentation,the fermentation liquor by liquid-liquid extraction with petroleum ether,chloroform,ethyl acetate,three kinds of organic solvent extraction,according to polarity from small to large in fermentation liquid recycling organic solvent to obtain concentrate,three extraction phase and water phase five parts.Three methods were used to determine the antioxidant capacity of 5 kinds of extracts and to pretest the chemical composition of them.According to the results,when the sample concentration was 1mg/ml,ethyl acetate phase and water phase,respectively,were 76.88% and 73.53% of DPPH.The removal rate of OH was 62.21% and 61.98%;The total reduction capacity is 0.736 and 0.615.The removal of free radical ability was higher than that of the other two extraction phases,indicating that the endogenous bacteria had certain antioxidant activity,and the active sites were mainly concentrated in ethyl acetate and aqueous phase.5.Purification of antioxidant active sites and tracking and structure identification of active componentsWhen water is condensed,matter is precipitated out as compound A.The water phase and ethyl acetate phase of the active site were separated by AB-8 macroporous adsorption resin.When the concentration of water phase of the eluent is equivalent to 500 mg/m L heavy concentrate,water phase of the eluent has stronger antioxidant activity and DPPH · and · OH clearance rate were 80.85% and 66.01% respectively,less than the clearance rate of Vc(1 mg/m L)were 99.36% and 95.77% respectively;The water eluent of ethyl acetate phase was only 51.79% and 41.56% of DPPH and ooh.In the water phase,80% of the ethanol eluent was 70.11% and 46.25% respectively.80% ethanol eluent of ethyl acetate was 38.79% and 23.42% of DPPH.In this paper,the optimal water phase water eluting solution of DPPH and ·OH clearance was studied.With 80% ethanol,the aqueous solution of water phase was reacted with alcohol precipitation,the sediment was sample 1,the alcohol concentration was obtained sample 2,and the antioxidant activity was determined as sample 1> sample 2.When the concentration of the sample 1 is equivalent to 500 mg/m L concentrate heavy,on DPPH · clearance rate was 90.6%,the · OH clearance rate was 79.69%,the clearance rate of sample 1 close to Vc(1 mg/m L),on DPPH · clearance rate was 99.36%,the · OH clearance rate was 95.77%.Sample 2 was 38.89% and 23.42%,respectively,for DPPH.When using anhydrous ethanol to remove monosaccharide from sample 1,the concentrated alcohol solution obtained compound B,and the insoluble substance was crude polysaccharide.The crude polysaccharides were separated by glucan gel and obtained compound C.The compound A,B,and C were determined by using agilent UV spectrum scanning(UV),400 MHz superconducting nuclear magnetic resonance spectrometer,infrared spectrometer and other structural identification methods.Found that compounds A and B respectively the results are determined by 1 HNMR and 13 CNMR and literature reports of mannose,maltose show 1 H NMR,13 CNMR spectrum data are consistent,that compound for mannitol,A the second is compound B for maltose.Compound C of ultraviolet(UV)spectral scanning results showed that in 260 nm and 280 nm are no peak,that does not contain nucleic acids and proteins,infrared spectrum(IR)show that compound C contains four polysaccharide material characteristic absorption peak,compound C for polysaccharide substance.The antioxidant capacity of compound A,B and C was determined,and the DPPH clearance rate was 46.29%,12.36% and 96.35% respectively.The removal rate of OH was 35.48%,3.18% and 92.85% respectively.The total reduction force is 0.496,0.009 and 1.23 respectively.Polysaccharide compounds have the strongest antioxidant capacity,compound B,and the compound A is the lowest,so the compound C can provide A theoretical basis for the development and utilization of natural antioxidant.In this study,we obtained a polysaccharide compound with strong antioxidant activity produced by endometriosis endosperm,Aspergillus oryzae.This study can provide a basis for the further development of antioxidants. |
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