| Licorice（Glycyrrhiza uralensis Fisch.）is the most commonly used Chinese herbal medicine,which is sweet and flat.It returns to the heart,lung,spleen and stomach meridian,known as the king of medicine,and is one of the main local medicinal materials in Gansu Province.It has many pharmacological effects,such as antitussive,antiasthmatic,antibacterial,antioxidant,anticancer and whitening.Endophytes refer to a class of microorganisms that survive within part or all of their life history in healthy plant tissues without causing host plants to exhibit significant infection symptoms.Based on this,this study used Gansu local licorice as a material to obtain more specific cellulase by separating its endophytic bacteria,and applied it to the extraction of licorice active products.At the same time,the biological activity of endophytic secondary metabolites was studied in order to provide theoretical support for the deep application of licorice.The contents of this study are as follows:Screening cellulase-producing endophytic bacteria from licorice.The research used fresh licorice as raw material,designed the screening medium through licorice powder and cellulose as the substrate,and screened the cellulase-producing strain by Congo red staining.The cellulase activity of the strain was determined by 3,5-dinitrosalicylic acid colorimetric method（DNS method）.The glycyrrhizic acid in the licorice is extracted by bio-enzymatic method,and the content of glycyrrhizic acid is detected by high performance liquid chromatography（HPLC）.The cellulase-producing strain was identified by molecular biological methods.A total of 20endophytic bacteria were screened,and Congo red staining confirmed that 13 strains could produce cellulase.After measuring enzyme activity,there were 6 strains with enzyme activity exceeding 30U.The glycyrrhizic acid in licorice was extracted by bio-enzymatic method,and it was found that the cellulase produced by strains GG-3,G-0.5-1 and G-1-1 significantly promoted the extraction of glycyrrhizic acid,while the GG-3 strain produced Cellulase has the best promotion effect on extraction.Based on this,strain GG-3 was selected for assisting in the extraction of active ingredients in licorice.Based on this,strain GG-3 was selected for assisting in the extraction of active ingredients in licorice.Molecular biological identification of strain GG-3revealed that it has 99%homology with Bacillus pumilus.Fermentation of strain GG-3 and optimization of enzyme production conditions.The specific enzyme-producing strain GG-3 was used as the material to evaluate the effects of different factors on its growth and enzyme production,in order to obtain the most suitable growth and enzyme production conditions.The results showed that the optimal conditions for the growth of strain GG-3 were sodium carboxymethyl cellulose as the carbon source,beef extract as the nitrogen source,p H 7.5,and carbon to nitrogen ratio of 1:2.The optimal conditions for the production of the enzyme of GG-3 were as follows:the carbon source was sucrose,the nitrogen source was yeast extract,the p H was 7-8,and the ratio of carbon to nitrogen was 1:1.5.The enzymatic properties of the cellulase produced showed that the optimum temperature of the enzyme was 40℃,and the optimum p H was 8.The metal ion had an effect on the enzyme activity of the cellulase produced by the GG-3 strain,among which the impact of Zn2+is most significant.Study on the extraction of active ingredients from licorice by biological method.The crude enzyme solution prepared by fermentation of cellulase-producing strain GG-3 was used as a material to assist in the extraction of glycyrrhizic acid from licorice.The effects of different extraction conditions and different enzymatic conditions on the amount of glycyrrhizic acid extracted by enzyme-assisted extraction of glycyrrhizic acid were investigated.The extraction effect was the best when the p H of the enzymatic hydrolysis was determined to be 8,the enzyme amount was 24 U/g,the enzymatic hydrolysis temperature was 37℃,and single extraction was 2h,the ammonia content of ethanol in the solvent was 30%,the extraction temperature was90℃.The optimized process was used to extract glycyrrhizic acid from licorice.The results showed that the enzyme produced by strain G-G-3 had a significant effect on the extraction of glycyrrhizic acid.The cellulase produced in the control group had no effect on the extraction of glycyrrhizic acid from licorice.After extraction with GG-3enzyme,the extraction rate of glycyrrhizic acid was 32.5%higher than that of water treatment,which was 31.3%higher than that after commercial cellulase treatment.Better application prospects are displayed.Separation of endophytic fungi from licorice.In this study,nine kinds of mediums were designed by collecting fresh licorice.The endophytic fungi of licorice were separated by tissue grinding.The results showed that 128 endophytic fungi were isolated from licorice.Among them,88 strains are from licorice root,15 strains from stem parts,25 strains from leaf parts.The result of morphological and molecular biological identification namely included Cephalospporfum sp.,Absidia sp.,Trichderma sp.,Alternnrin sp.,Rhizoctonia sp.,Helminthoid sp.,Aspergillus sp.,Scopulariopsis Bainier.,Geotrichum sp.,Paecilomyces sp.,Aspergillus fumigatus.,Fusarium sp.,Uncultured fungus,Meyerozyma sp.,Nectria sp.,Pichia guilliermondii,Didymella macrostoma,Ceratobasidium sp.,which showed the rich diversity of endophytic fungi in licorice.Study on the activity of secondary metabolites of endophytic fungi.The antioxidant activity of endophytic fungi of licorice were evaluated by DPPH free radical（1,1-diphenyl-2-trinitrobenzene）scavenging method,hydroxyl radical scavenging method and reducing power method.The antibacterial activity of its secondary metabolites was evaluated by the filter paper method.The results showed that the clearance rates of DPPH free radicals in the GS-7 strain fermentation broth,GJ-5 and GJ-1 strain mycelium reached 96.87%,97.60%,95.62%,respectively,equaling the reference clearance rate（95.2%）;The clearance rates of-OH radicals in GS-12,GJ-5,YS-4,GS-6 and GS-10 mycelium samples reached 96.70%,98%,98.32%,99.17%,97.74%,respectively.All equaled the reference clearance rate（95.3%）,showing strong antioxidant capacity in vitro;total reduction of YS-2,YB-3,YB-4 fermentation broth and GJ-5 and GJ-1 mycelium is comparable to the reducing power of 0.1 mg/m L of Vc,showing a high research value.The antibacterial experiment results showed that the antibacterial activity of the sample YS-6fermentation broth against Escherichia coli reached 70%of streptomycin,and it also had good antibacterial activity against Bacillus licheniformis,reaching 67%of penicillin.The antibacterial activity against Staphylococcus aureus was 79%of penicillin;the GJ-5 mycelium had higher antibacterial activity against Staphylococcus aureus,reaching 81%of penicillin,which showed strong antibacterial activity. |
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