| Currently,there are increasing studies on ethanol degradation by traditional Chinese medicine in China.However,the studies on ethanol degradation by endophytes of Siraitia grosvenorii remains to be rare.Therefore,endophytes were isolated from Siraitia grosvenorii by traditional culture isolation method in this study.Plate method and potassium dichromate-sulfuric acid method to determine ethanol tolerance were used to screen the strains with ethanol degradation ability,which were identified by morphological observation and molecular biology technology.Ethanol degradation rate was taken as the evaluation index,the fermentation conditions were optimized by single factor experiment and orthogonal experiment.By studying the rapid ethanol degradation ability and gastric and intestinal fluid resistance test,whether it has the potential of living bacteria preparation could be judged,so as to provide a new way for the research and development of products with ethanol degradation function and lay a foundation for industrial production of products.The main research contents are as follows:(1)Isolation of endophytic strains from Siraitia grosvenoriiA total of 68 endophytic strains were isolated from roots,stems,leaves and fruits,including27 strains from roots,15 strains from stems,5 strains from leaves and 21 strains from fruits.(2)Screening of ethanol-degrading strainsAmong the 68 strains,17 strains were tolerant to ethanol.According to the growth of endophytic strains on the plate,14 strains were selected for fermentation and re-screening,and finally the numbered G-1 strain purified from the roots performed well.When strain G-1fermentation broth stock solution was treated with 8 %(v/v)ethanol(mixed in PDB medium,the same below,omitted)for 1 h,the ethanol degradation rate reached(9.1±0.7)%;when the concentration of 10 %(v/v)ethanol was treated for 24 h,the ethanol degradation rate was(16.7±0.3)%;when the concentration of 20 %(v/v)ethanol was treated for 24 h,the ethanol degradation rate was(13.2±1.2)%.Therefore,strain G-1 was selected for strain identification and follow-up research.(3)Optimization of ethanol degradation and fermentation conditions for strain G-1 in fermentation broth and its identificationThrough single factor experiment and orthogonal experiment,the effects of various factors on ethanol degradation rate of strain G-1 fermentation broth were explored.The results showed that the best fermentation conditions of strain G-1 are as follows: inoculation amount for 7 %,liquid volume 200/500 m L triangular flask,fermentation temperature for 32 ? and fermentation time for 3 days.The results showed that the ethanol degradation rate of strain G-1 was(29.8±0.7)%,which was 2.2 times higher than that of strain G-1 when the ethanol concentration was 20 % and the strain was re-screened before the optimal fermentation conditions(13.2±1.2)%.The morphological identification results of the strain are as follows: the surface of the colony is protruding,the colony is flocculent with 3?5 mm in height,and the hyphae are white and dense;the back is pink and white,slightly purple;when observed under a microscope,conidia are attached to the hyphae and gather into an oval shape at the top.The results of molecular biology showed that the sequence length was 534 bp,and its homology with Fusarium was more than 99 %.Based on the above results,strain G-1 belongs to Fusarium.(4)Determination on fast ethanol degradation ability of strain G-1 fermentation broth and analysis on gastric and intestinal fluid resistanceThe results of rapid ethanol degradation ability of strain G-1 fermentation broth showed that with the increase of time,the ethanol degradation rate of strain G-1 fermentation broth increased.The results showed that the fermentation broth of strain G-1 had the characteristics of rapid ethanol degradation and could effectively degrade ethanol in a short time.The results of simulated gastric juice environment showed that the degradation rate of ethanol by fermentation broth of strain G-1 decreased with the decrease of p H value.Compared with the blank control group,the fermentation broth of strain G-1 can maintain a certain degree of ethanol degradation within 2 h under the environment of low acid(p H=3.0?4.0).The degradation rate of ethanol by the fermentation broth is equivalent to 72.5 % ? 85.0 % of the blank control group at 1 h,and it is equivalent to 52.6 % ? 65.5 % of the blank control group at 2h.The results of simulated intestinal fluid environment showed that the three cholate experimental groups had the same trend,and the ethanol degradation rate decreased with time,which indicated that cholate would affect the ethanol degradation of fermentation broth with time.Compared with the blank control group,it still has a better ethanol degradation effect within the range of 4h.The ethanol degradation rate is equivalent to 69.3 %?75.9 % of the blank control group at 1h,and it is equivalent to 57.5 % ? 67.5 % of the blank control group at 4 h.Based on the above experimental results,it is concluded that the fermentation broth of strain G-1 can maintain a certain ethanol degradation ability under simulated human internal environment conditions,and has the potential to become a living bacteria preparation.(5)Screening of active ingredients for ethanol degradation and pre-experiment of their chemical constituentsThe results showed that when the concentration of samples in each phase was 1 mg/m L,the activity of n-butanol phase was the highest,and the degradation rate of ethanol was 13.5 %.After chemical composition pre-experiment,it was found that the phase contained protein,polysaccharide and flavonoids.(6)Purification of active components and preliminary identification of active componentsThe n-butanol phase was preliminarily separated by macroporous adsorption resin D101 column chromatography.The results showed that the sample eluted with 10 % ethanol had the highest ethanol degradation rate,reaching(17.6±0.5)%.The samples eluted with 10 % ethanol were further purified by silica gel column chromatography.The results showed that the ethanol degradation rate of group 4 was the best,which was(18.1±0.6)%.The results of purification on silica gel column were analyzed by HPLC,and the results showed that a single peak appeared at2 min.Through the pre-experiment of its chemical composition,it was preliminarily identified as a flavonoid compound.In this study,the strain G-1(Fusarium)with ethanol degradation ability was screened from the endophytic strains of Siraitia grosvenorii.It can maintain the ethanol degradation ability to certain degree under simulated human internal environmental conditions,and it is preliminarily determined that flavonoids play a role. |
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