Fermentation Process Control, Isolation And Purfication Of Sialic Acid

Sialic acids, existed on the surface of mammal cells, exhibited very important biological functions in the process of cell interactions. In medical applications, sialic derivatives were used as antivirus, diagnosis reagent, and medicine fro treating neuropathic malady.Different factors influencing the cell growth and polysialic acid synthesis were investigated, and the results showed that the polysialic acid synthesis was coupled with the cell growth. During the stationary phase of fermentation, cell productivity was slightly reduced. MgSO₄ and tryptone have the positive effect on the product synthesis and cell growth respectively. Based on these conclusions, MgSO₄ and tryptone were added to the feed medium to enhance the production of polysialic acid. Variable feed rate policies were applied to the fed-batch fermentation process and the final biomass and polysialic acid production were both increased by 40 percent.Polysialic acid, as a linear polymer of 100-300 sialic acid monomers, was different from the impurity in molecular weight and other physical properties. Therefore, the implement of polysialic acid purification is easier than that of sialic acid. The main impurity other than polysialic acid in the broth were proteins and micromolecular metablites. Two processes for removing protein from broth were developed. The two processed were named as ethanol fractional precipitation and ethanol precipitation-filtration, respectively. Comparing the two new developed method with general methods such as Sevage method and salt out method, it was found that ethanol precipitation-filtration was the optimal process. Finally, the optimal conditions of protein removing process were gained, i.e. 30 liters ethanol was added into the 10 liters of polysialic acid broth. The supernatant was removed by centrifugation and 150 g diatomite filter aid was added to the precipitate, 800 ml deionized water was added to dissolve the polysialic acid in the precipitate. The polysialic acid solution was obtained by filtration. The recovery yield of polysialic acid is 94.5%, and 89.5 % of protein was removed. Then gel filtration, positive ion resin absorption and isoelectric deposition were applied for polysialic acid purification process. The purity of polysialic acid was above 90 % with neutralisation deposition method and the recovery yield of polysiac acid was 55.8%.During the process of polysialic acid hydrolyzation, a large mount of pigment was produced and interfered the successive separation process. The effect of various decoloring agents on decoloraion efficiency and the recovery yield of sialic acid. The results showed that attapulgite clay was the most suitable decloring agent comparing with others. The decoloraion efficiency was 91.7% and the recovery yield of sialic acid was 96.7%. Finally,the sialic acid crystalling process was devoloped, i.e. polysialic acid was dealt with hydrolyzation, neutralization, decoloration, and freeze-drying, 3 g sialic acid powder was added to 100 ml ethanol at 45 °C, centrifugated and the precipitate was removed, the solution was condensed by decompression, until little precipitate was fomed, 50ml ethyl acetate was added to the solution, the precipitate was removed, more ethyl acetate was added until no more precipitate deposited out. Sialic acid crystal was obtained through centrifugation and drying. The purity of the sialic acid product was 92%. The infrared spectrum of the sialic acid we had purified was very similar to that of the sialic acid standard sample. (Sigma, type 111).