Purification And Quality Study Of Shikimic Acid In Fermentation Product

Shikimic Acid（3,4,5-trihydrox y-1-cyclohexene-1-carbox ylic acid,SA）is a natural organic acid,which is found in the dried mature fruits of Magnoliaceae and coniferae.It can be used as raw materials for most of the chiral drugs such as aromatic compounds,alkaloids and indole derivat ives,with high market value.In this paper,a process route for the separation and extraction of shikimic acid from fermentation broth was established,with the advantage of low cost,high yield and easy operation,.The ceramic membrane and nanofiltration membrane were used to remove most of the bacteria and proteins firstly.The factors affecting the decolorization of fermentation broth were determined by single factor experiment,and the best combination of decolorization conditions of shikimic acid fermentation broth was obtained by orthogonal experiment,which was that the dosage of activated carbon was 1.0%,the p H of sulfuric acid was 2.0,the decolorization temperature was 50℃,and the decolorization time was 1h,the decolorization effect was the best.Add anhydrous ethanol into the concentrated solution to desalinate the final ethanol concentration of 85%,filter and remove large amounts of alcohol insoluble matter,the ethanol solution of shikimic acid was concentrated and crystallized to obtain the crude shikimic acid,and shikimic acid with the purity of over 98.50%could be obtained by repeated crystallization.The production cost is far lower than the cost of plant extraction,and the market price of plant extraction is about 800-1000 yuan/kg,which has a good application prospect.Based on the role of shikimic acid as a starting material,general rules requirements of the The Pharmacopoeia of the People’s Republic of China,the REQUIREMENTS of the ICH guidelines,related determination methods were established.In this paper,the quality control indexes of shikimic acid were studied,which includes character,identification,inspection and content determination.In character study,the results showed that the appearance of self-developed shikimic acid is white powder,soluble in H₂O,slightly soluble in ethanol,the melting point is 183～188℃,the specific rotation is-180～-186°,which is consistent with the references’.In Identification study,by comparing the molecular weight,characteristic peaks in the infra-red spectrum,nuclear magnetic resonance spectra and crystal form of self-developed shikimic acid and reference substance,the results were consistent.In examination study,that includes loss on drying,KF water content,heavy metal and ignition residue,related substances,residual protein and residual DNA inspection.The results showed that the content of loss on drying,water and ignition residue were less than 0.50%respectively.The content of lead（Pb）,arsenic（As）,cadmium（Cd）and mercury（Hg）is less than 3ppm,2ppm,1ppm,and 0.1ppm respectively.The content of related substances,such as dehydroshishiic acid（DHS）,gallic acid（GA）,and protocatechuic acid（PA）,were less than0.10%respectively.What’s more,protein and DNA residue were also evaluated.The protein residue was less than 0.10%and the DNA residue was not detected.In the study of shikimic acid assay,a high performance liquid chromatography（HPLC）method were established,and the assay of shikimic acid was determined by external standard method,the assay of self-developed shikimic acid was not less than 98.50%.The assay method of shikimic acid was validated,the results showed that the linear range of the calibration curve was 0.025～0.150 mg/m L（r=1.0000）,the accuracy recovery was between 99.39～101.70%,the RSD of precision was 0.52%（n=6）.And the method of residual DNA was validated,the results showed that the linear range of the calibration curve was1.28～81.97 ng/m L（r=0.9990）,the accuracy recovery was104.59%,the RSD of precision was1.84%（n=6）,the limit of detection（LOD）was 0.64 ppm and the limit of quantification（LOQ）was 1.28 ppm.To sum up,this study can obtain shikimic acid products by extracting,purifying from fermentation broth.And a quality control system for shikimic acid was established.Compared with the methods of traditional plant extraction and preparation,this process route is simple and low cost,which can be used in large-scale production to meet the raw material requirements of drugs.