Study On Preparation Of Hyaluronic Acid By Fermentation And Its Composite Modification

Hyaluronic acid (HA) is an important medicine material, a cosmetic material and a nevel resource food. In recent years, instead of extracting HA from animal tissue, microorgnisim fermentation has become the main method to prepare HA.In the process of preparing HA, HA content in large number of samples need to be measured.The common method, Bitter-Muir method (BMM), has some hazard because of using concentrated sulfuric acid and carbazole as the reagent, and its measuring procedure is complicated. This study explored the characters of reaction systems of HA and CTAB (cetyltrimethylammonium bromide), which was a cationicsurfactant, and established a quick and safe method, CTAB Turbidimetric method (CTM). In this method, 25 mg/ml CTAB reagent was used as the reaction reagent, 400 nm was selected as the measuring wavelength. In room temperature, made the purified sample solution react with the CTAB reagent for exact 10 min, and controled the measuring time error in 10 s, then, calculated the HA content through the calibration line.The results indicate that CTM was superior to BMM in terms of accuracy, precision and sensitivity. Except protein, other impurities had less influence on the determination. This method not only reduced time consumption but also improved processing safety. The suitable molecular weight bound for the method was higher than 50 000 Da.In this study, a mutant designated NC168 which did not produce streptolysin was selected from wild-type Streptococcus equisimilis by ultraviolet ray combined with 60Co-γray mutation. This selected mutant NC168 had higher HA yield, 150.61 mg/L, and kept higher relative molecular weight (Mr) of HA, 677 000 Da. Furthermore, its HA output and HA Mr maintained genetic stability after 10 generations and there was no reverse mutation on hemolysis.Aiming at the mutant NC168, the quadratic rotary combination design and the response surface was used to optimize the culture medium and the established mathematical model was as follows:Y=174.76168+6.09122X3-9.26943X12-12.92694X22-14.36237X32-12.20569X42 The model estimated that, a maximal yield of HA could be obtained when the concentrations of yeast extract, peptone, glucose, and MgSO4 were set at 30 g/L, 20 g/L, 5 g/L and 1.5 g/L, respectively. The model prediction output of HA was 174.76 mg/L and the verified experiment output got 178.5 mg/L. Using the optimum culture medium mentioned above, batch fermentation experiments were conducted in 5 L fermenter. The results show that, the yield of HA was significantly influenced by pH value, initial concentration of glucose, temperature and rotate speed. The optimum culture conditions were as follows: pH, 7.0; initial concentration of glucose, 30 g/L; temperature, 35℃; rotate speed, 200 r/min.The yield of HA could got 3.0 g/L by fermentation with 5 L fermentor. In the basis of optimum fermentation conditions, the biomass and the output of HA were simulated by Logistic, Slogistic and SGompertz models. The Logistic model mostly suited to the biomass, the simulation equation was as follows: y = (?), the correlation coefficient got 0.999. The best simulated model of HA output was Slogistic model, the simulation equation was as follows: y =(?), the correlation coefficient was 0.996. The glucose consume model was founded with DoseResp model, which correlated experiment well with of R2 of 0.991, the simulation equation was y = 2. 085+1 +1206.05.165x6?2.80.CTAB sedimention combined with ultrafiltration was adopted to separate and purify HA. The fermentation broth was diluted to 0.6-0.8 mg/L HA. The CTAB solution, which was 15% of sample solution volume, was added into sample solution. Under 35℃, the solution was sedimented for 1h, and then the sediment was separated. The sediment was dissolved in 1.5 mol/L NaCl solution for 4 h, and 70% ethanol was used to precipitate HA. The HA yield could got 93.8%, the protein impurities reduced to 1.5%. The 100 KDa ultrafiltration membrane was selected to further purification. The sample solution was diluted to 0.02%-0.05% HA, the pressure through membrane was controlled at 11 psi.The results show that the yield of HA could got 93.5% and the removing rate of protein got 94.0%. After seperation and purification, HA got Pharmaceutical Grade and the leading indicators were as follows: the total yield of HA got 87.7%, the content of protein in HA sample decreased to 0.09% and the content glucuronic acid got 43.5%,.In order to obtain novel polymeric derivatives of HA with improved physicochemical properties, composite hydrogels were prepared by cross linking sodium alginate (SAL) and HA, which Mr was 1500 000 Da. The modification used acetate buffer solution as reagent to adjust pH, EDC as a carboxyl activation reagent, ADH as the cross-linker. The expected amide linkages in the hydrogels were confirmed by FT-IR, and the microstructures were observed by SEM. Texture profile analysis indicates that the hardness of hydrogels was enhanced with the increase of the polymers concentration, while declined with the increase of HA/SAL molar ratio. The swelling capacity of hyadrogels were positively correlated with HA content while negatively correlated with EDC amount. The resistance against hyaluronidase was improved with the increased proportion of SAL in hydrogels and EDC amount. The novel hydrogels might have great potentials to be applied in dermal fillers and tissue engineering scaffolds because of its better swelling capacity, physical-chemical stability, natural biological compatibility and suitable biodegradability.