| Objective: in this study,a large number of probiotics for medicine and food were selected to ferment Astragalus,and the best fermentation strain and technology were selected.Through the fermentation experiment,the extraction rate of effective components in the fermentation system was improved.In order to understand and explain the material basis of Astragalus fermentation system,the best fermentation technology was determined.Methods: 1.In this study,according to the determination method of Astragalus in Pharmacopoeia 2015,different batches of Astragalus were determined,and the Astragalus with the highest content of pistil isoflavone glucoside and astragaloside a was selected.2.To establish the ultraviolet spectrophotometer method for the determination of total astragaloside,total flavone and crude polysaccharide;to establish the HPLC-DAD method for the determination of glucoside,isoflavone,anthocyanin and anthocyanin;to establish the HPLC-ELSD method for the determination of astragaloside I,astragaloside II and astragaloside a;3.In this study,the total astragaloside,total flavone,crude polysaccharide and isoflavone were used The comprehensive scores of glucoside,pistil isoflavone,anthocyanin,anthocyanin,astragaloside ?,astragaloside ? and Huang Qijia were taken as the indexes,including Lactobacillus bulgaricus,Leuconostoc mesenteroides,Bacillus coagulans,Lactobacillus plantarum,Lactobacillus fermentum,Streptococcus thermophilus,Bacillus subtilis,Paecilomyces cicadae,Penicillium,Monascus,Aspergillus niger,Cordyceps militaris,Sophorae fungus 4 The orthogonal experiment was used to optimize the enzymolysis process of Astragalus membranaceus fermentation,and the optimal factors were enzyme type,enzymolysis time,enzyme amount and enzymolysis temperature;then the response surface experiment was used to optimize the Astragalus membranaceus fermentation process,and the optimal factors were: fermentation time,initial pH,and the amount of Astragalus and astragalus;5.Sulfuric acid anthrone method was used to determine the crude polysaccharide,rutin colorimetry was used to determine the total flavonoids In addition,there are also uhplc-qtof-ms methods to study the quality of Astragalus fermentation products.Results: 1.According to pharmacopoeia method,we evaluated the content of Astragalus membranaceus glycosides and astragaloside a in Astragalus membranaceus.The results showed that the content of astragaloside and astragaloside a in purchased Astragalus medicines met the requirements of Pharmacopoeia.Among them,the content of astragaloside and astragaloside a in Hunyuan Wansheng was the highest,0.0652% and 0.1158% respectively.Therefore,Astragalus medicines in Hunyuan Wansheng were selected as experimental materials.2.To establish the evaluation method of Astragalus membranaceus fermentation products,the method of sulfuric acid anthrone was used for crude polysaccharide,rutin colorimetry was used for total flavonoids,and sulfuric acid glacial acetic acid method was used for total saponins.The standard curves of the three methods were y = 0.0103 x + 0.0115,y = 0.0163 x + 0.013,y = 0.017x-0.0348,the correlation coefficients were all greater than 0.999,and the linearity was good.The accuracy,stability and repeatability of the first-order sampling recovery meet the requirements;the HPLC-DAD content determination method established in the experiment can simultaneously determine four flavonoids,namely,glucoside,isoflavone,anthocyanin and anthocyanin.The HPLC-ELSD content determination method can simultaneously determine three saponins,namely astragaloside a,astragaloside I and astragaloside II The regression equations were y = 6205.4x-11.21,y = 13095x-78.307,y = 15598x-151.23,y = 17506 x + 47.458,y = 148493 x + 4297.5,y = 162934 x + 7795.6,y = 47307 x + 2131.8,and the correlation coefficient r was greater than 0.999.The RSD values of the precision,stability and repeatability experiments were less than 3%.The recovery of the samples were 100.54%,100.25%,100.86%,99.37%,100.13% respectively,100.15%,100.17%,RSD were 1.37%,0.56%,1.20%,1.62%,0.06%,0.34% and 0.06% respectively.Meanwhile,a method based on uhplc-qtof-ms for the determination of Astragalus components was established.3.The Astragalus fermentation strains were screened from Lactobacillus bulgaricus,Leuconostoc enteromembrane subspecies,Bacillus coagulans,Lactobacillus plantarum,Lactobacillus fermentans,Streptococcus thermophilus,Bacillus subtilis,Paecilomyces Cicadidae,Penicillium,Monascus,Aspergillus niger,Cordyceps militaris,Sophora fungus and other strains.Finally,the Astragalus fermentation strains were determined as Bacillus subtilis,Bacillus natto.4.Orthogonal experiment was used to optimize the enzymatic hydrolysis process of Astragalus membranaceus.Firstly,according to the type and amount of enzyme added.The best type of enzyme was selected from the single factors,such as the time and temperature of enzymatic hydrolysis,as the mixed enzyme system of cellulase and pectinase.The time and temperature of enzymolysis were the experimental factors,and the orthogonal experiment of three factors and three levels was designed.The final enzymolysis process was 0.1% enzyme addition,60 min enzymolysis time and 50 ? enzymolysis temperature.The response surface method was used to optimize the fermentation process of Astragalus.First,the single factor experiment was carried out,including Astragalus dosage,initial pH,fermentation time,bacteria amount and carbon According to the results of single factor experiment,the factors that have no obvious influence on the fermentation results of Astragalus are removed.Finally,the three-level response surface is designed to optimize the fermentation process of Astragalus.The final optimal fermentation process is 13% of Astragalus,7 days of fermentation,3 mL of bacteria,and 6.50 of initial pH.5.The results showed that the polysaccharide content of Astragalus fermentation products was lower than that before fermentation,the total saponin was increased by 9.21%,and the total flavone content was increased. |
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