| Echinacea purpurea is a perennial herb of the genus Echinacea,which can enhance immunity and anti-inflammatory effects.There are many active ingredients in Echinacea purpurea,mainly including phenolic acids,polysaccharides,alkyl amides,unsaturated ketones,volatile oils,alkaloids and polyacetylenes.Chicory acid is the most important active ingredient in phenolic acid,it can enhance the pharmacological effects of immunity,anti-inflammation and anti-oxidation.The quality of Echinacea purpurea and its preparations is usually based on the content of chicory acid.At present,the extraction methods of chicory acid mainly include organic solvent reflux extraction,ultrasonic assisted extraction,ultrasonic synergistic extraction and solvent extraction.However,there have been no reports on the study of enzymatic hydrolysis and microbial fermentation of chicory acid.Therefore,this paper mainly studied the effects of enzymatic hydrolysis,microbial fermentation and microbial fermentation combined with ethanol reflux extraction on the yield of chicory acid extraction,and provide new research methods and ideas for the extraction of chicory acid from Echinacea purpurea.The main research content of this paper:1?Firstly,we establish a high performance liquid chromatography?HPLC?method for determination of chicory acid with chicory acid standards.It lays a foundation for qualitative analysis of chicory acid and determination of extraction yield.According to the yield of chicory acid from the extract of Echinacea purpurea as an index,the effects of cellulase,hemicellulase and two enzymes on the extraction of chicory acid from Echinacea purpurea were applied.The effects of enzyme dosage,temperature and time on the yield of chicory acid were discussed.Univariate and orthogonal experiments showed that the combination of cellulase and hemicellulase could significantly increase the yield of chicory acid.The optimal extraction conditions for chicory acid from Echinacea purpurea by enzymatic hydrolysis were as follows:cellulase 2808 U·mL-1,hemicellulase 900 U·mL-1 enzymolysis temperature 55°C,enzymolysis time 2 h,and enzymatic hydrolysis pH 6.0,Under these conditions,the yield of chicory acid was47.6%.2?On the basis of extracting chicory acid from Echinacea purpurea by enzymatic hydrolysis,strains with high cellulase yield were screened.Optimization of bacterial production conditions,The optimum conditions for enzyme production were 2%CMC,initial pH 6.0,inoculum 10%,shaker speed 180 r·min-1,culture temperature 37?,culture time 72 h.The cellulose activity of Q3 was 18.667 U·mL-1,which was 2.26times that of the enzyme production conditions.There is no positive correlation between the amount of bacterial growth and the amount of enzyme produced,which is a guiding significance for the fermentation of chicory acid from Echinacea purpurea.3?The process conditions for bacterial fermentation of chicory acid were optimized by single factor test and response surface analysis.The bacteria are highly tolerant to chicory acid,and the concentration of chicory acid is 200-1600 ug·mL-1,which does not inhibit the growth of bacteria.The optimum conditions for the extraction of chicory acid from Echinacea purpurea by microbial fermentation were 1.0%CMC addition,initial pH 7.0,10%bacterial inoculation,36°C fermentation temperature and 33 h fermentation time.The yield of chicory acid under this fermentation condition was66.2%.4?The effect of organic solvent reflux extraction conditions on the extraction yield of chicory acid from Echinacea purpurea.The effects of different organic solvents,concentration of organic solvents,reflux temperature,reflux time and reflux times on the extraction yield of chicory acid from Echinacea purpurea were studied.Based on the analysis of the results and considering the cost factors,it was determined that the ratio of solid to liquid of 1:20,and refluxing at 87°C with 20%ethanol solution was the optimal extraction condition.On this basis,the optimal extraction conditions were further optimized to obtain an optimal reflux extraction time of 2 h and a reflux extraction time of 2 times.The extraction yield of chicory acid from Echinacea purpurea by ethanol reflux was 74.9%.Finally,we combined microbial fermentation with ethanol reflux extraction of chicory acid from Echinacea purpure.The extraction rate of chicory acid from the second batch of Echinacea purpurea was 57.3%,and the extraction rate of chicory acid from Echinacea purpurea by ethanol reflux was 63.5%.The yield of reflux extraction was 72.8%.After analysis,it was found that the results of ethanol reflux extraction after microbial fermentation were significantly different from those of microbial fermentation?P<0.01?,and there was significant difference between ethanol extraction and extraction?0.01<P<0.05?,indicating that the test results were significant difference,and preliminary determination of ethanol reflux extraction after microbial fermentation is a practical method of extraction of chicory acid. |
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