| Shikimic acid is an important starting material for chemical synthesis.Shikimic acid and its derivatives have antibacterial,anti-tumor,anti-inflammatory and analgesic effects.Shikimic acid is an essential and important intermediate product of the anti-influenza drug Tamiflu,which makes the position of shikimic acid particularly important.Shikimic acid was originally derived from plant extraction,but with the increase of demand,the high cost of plant extraction,limited raw materials and other shortcomings were exposed,thus ushering in the wave of using microbial fermentation to produce shikimic acid.Therefore,it is of strategic significance in economic development and medical field to develop a technological route for extracting shikimic acid from fermentation broth to provide technical support for industrial production.In this experiment,liquid chromatography was used to detect and analyze the components in fermentation broth,and a set of liquid detection method was established.The liquid phase was performed on a C18 column with the temperature set at 25?.The sample size was 5?L.The mobile phase consisted of a mixture of 5 m L acetonitrile,1 m L phosphoric acid and 994 m L highly pure water at the flow rate of 0.8 m L/min.Shikimic acid and other standard substances were used to establish a standard concentration curve,detect the peak area of a substance in the sample to be tested,and then the concentration of the substance in the sample could be calculated by putting it into the curve.After the fermentation liquid pour from the tank,it must be heated and sterilized first.The fermentation liquid is heated to 60? and kept warm for half an hour before cooling.Then,it is flocculated to settle most proteins and make the bacteria coagulate,which is convenient for the next ceramic membrane treatment.Through the screening of alum,polyacrylamide and chitosan,yellow prussiate and zinc sulfate flocculants and their combination,and finally determine add 2.5 g per liter fermented liquid yellow prussiate of zinc sulfate,2.5 g and 0.48 g chitosan flocculation,the flocculation conditions of protein in the fermented liquid,the best flocculation effect,the supernatant fluid protein is only 56mg/L.After flocculation,the fermentation broth was filtered by a ceramic membrane with an aperture of 50 nm to remove solids such as bacteria,and an appropriate amount of top washing water was added at the later stage of filtration to dilute the concentration of shikimic acid in the concentrated solution and improve the yield.The influence of different top washing water amounts,such as 20%,40%,60% and 80%,on the yield of ceramic membrane filtration was tested.Finally,the amount of top washing water added in the filtration process was determined to be 60% of the fermentation liquid,and the yield of shikimic acid in the filtration liquid could reach 93%.Shikimic acid,quinic acid fermented liquid containing dehydrogenation by-products,such as easy to remove the dehydrogenation shikimic acid,under the condition of heat can be converted into other substances,after testing found that the dehydrogenation of shikimic acid products for gallic acid,by testing different heating conditions of dehydrogenation of shikimic acid conversion rate,finally confirmed to keep the fermented liquid initial p H(5.5).All dehydroshikimic acid in the fermentation broth could be removed by heating the fermentation broth to 85? or above and holding for 4 hours.After pretreatment,the fermentation liquid was preliminally decolorized with activated carbon,and the decolorization effect was tried under different p H,temperature,decolorization time and the amount of activated carbon.Finally,the best decolorization conditions were determined as follows: the p H of the material liquid was adjusted to 2.5with sulfuric acid,the amount of activated carbon was 5 g/L,and the temperature was 60?for 30 min.After the decolorization of activated carbon,the color of the fermentation broth was improved from black to red,but the transmittance of the liquid was still not ideal,and the decolorization ability of activated carbon was limited,so it needed to be further decolorized by macroporous resin.Through static adsorption method,DM825 macroporous resin with better decolorization effect was screened out.After dynamic adsorption and decolorization,the light transmittance value of DM825 resin could reach 47.5%,and the residual soluble protein in fermentation broth could be basically removed.After being treated with macroporous resin,the fermentation is clear and transparent with a yellowish color,which also contains more inorganic salts and by-products such as quinic acid,etc.,which needs to be further removed by 717 resin.The effect of 717 resin on shikimic acid exchange capacity was observed by changing temperature,p H and other factors.The results showed that the exchange capacity of 717 resin was higher when the p H of fermentation broth was adjusted to 6.0 under normal temperature.The adsorption saturation time was about 2 hours,and the adsorption capacity of shikimic acid was about 1.2 g/10 g wet resin.The results showed that the solution of 5mmol/L sulfuric acid was the best solution,and the resolution rate was more than 90%.After determining the upper column conditions,dynamic adsorption was carried out and different upper column flow rates were controlled.The results showed that the maximum resin processing capacity was obtained when the flow rate was 0.5 BV/h,which was consistent with the results of static adsorption saturation time.In the dynamic elution process,it was found that the quinine acid could be eluted completely at the early elution stage,and the later eluent no longer contained quinine acid.The part of the later eluent that did not contain quinine acid was collected as the material liquid for the next treatment.Will 717 column under liquid containing no quinic acid part used to crystallization,material liquid enrichment to have just crystal precipitation,through flow with different amount of ethanol solution,after slow cooling to room temperature in 4 ? refrigerator,get the yield and purity of different crystals,ultimately determine the amount of ethanol to add 4 times best concentrate water volume,the crystallization yield can reach 95% under the condition.And the purity is about 99.66%.The obtained crystals were washed with ethanol and ethyl acetate respectively,and then the finished product of shikimic acid with 99.80% purity was obtained after drying.The purity and content of shikimic acid could reach 99.80% and 98.78%,and the final yield was about 47%.No toxic and harmful substances were used in the experiment,and the operation was simple,which provided a feasible idea for the industrial production of shikimic acid. |
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