| Hyaluronic acid(HA) is microbial polysaccharide formed by repeated connection of disaccharide unit that is composed of glucuronic acid and N-acetylglucosamine. As a kind of biological high-molecular polymer, HA is widely researched and applied in pharmaceuticals, cosmetics, food and health care, etc. because of its advantages. Molecular weight significantly affects the physiochemical characteristics and application effect of HA. HA with high molecular weight(Mr>2×106 Da) is usually applied in the field of pharmaceuticals while HA with low molecular weight(Mr<1×106 Da) is generally applied in the field of cosmetics and food. Generally, the higher the molecular weight, the higher the price.HA is mainly produced through microbiological fermentation. Taking Streptococcus zooepidemicus as fermentation strain, the factors that can significantly influence the molecular weight of HA was screened out by studying the effect of nutritional conditions, culture condition and membrane permeability on the fermentation of HA. And they were optimized through combination. Finally, a fermentation process with high yield and high molecular weight was obtained. This paper mainly studies the following aspects:Firstly, based on the characteristics of metabolic pathways of HA, this paper successively studied the effects of carbon source, nitrogen source, polyphosphates, and sodium pyruvate on molecular weight of HA. The result showed as follows: batch culture mode and higher level of glucose(70 g/L) can effectively relieve the competition on glucose between bacteria growth and HA synthesis, which was beneficial to the synthesis of HA with high molecular weight. The molecular weight of HA can reach to 1.63 MDa at maximum. Enhancing the concentration(start concentration: 10 g/L) of organic nitrogen source, i.e. yeast powder, and fed-batch ammonia water went against the synthesis of HA with high molecular weight. Competition on energy between bacteria growth and HA synthesis can be weakened through adding polyphosphates with higher degree of polymerization((NaPO3)6). When adding quantity of(NaPO3)6 were 3 g/L, the molecular weight can reach to 2.27 MDa. Competition on the common resource between glycolysis and HA synthetic route can be weakened through adding higher concentration of sodium pyruvate which can promote the synthesis of HA with high molecular weight. When the adding quantity of sodium pyruvate was 8 g/L, the molecular weight can reach to 2.14 MDa. The nutrient enrichment strategy combination mentioned above was applied to the fermentation of HA. Final yield and molecular weight respectively reached to 5.58 g/L and 2.35 MDa, which were respectively increased by 20% and 44.2% when compared with those before optimization.Secondly, this paper studied the effect of culture condition on the molecular weight of HA from four aspects including pH, temperature, ventilation and stirring speed. pH and temperature significantly affected the yield and molecular weight of HA. When pH was 7, yield of HA was highest, reaching to 4.71 g/L. When pH was 8, the molecular weight of HA was at maximum, reaching to 2.28 MDa. When the temperature was at 37℃, yield of HA was highest. When the temperature was at 31℃, the molecular weight of HA was at maximum, reaching to 2.34 MDa. Ventilation and stirring speed significantly affected the yield and molecular weight of HA. When stirring speed was 600 rpm and ventilation rate was 1 vvm, both yield and molecular weight of HA reached to the maximum values. Based on this, two-stage fermentation control strategy of pH and temperature was proposed, which significantly improved the yield and molecular weight of HA. Combining this strategy and nutrient enrichment strategy, final yield and molecular weight respectively reached to 5.65 g/L and 2.51 MDa.Thirdly, this paper studied the effect of surface active substance and phospholipids substances on the molecular weight of HA. The effects of cationic surfactant CPC, anionic surfactant SDS, nonionic surfactant tween-60 and phosphatidylcholine on the fermentation of HA were studied successively. The result showed as follows: the result of HA can be significantly improved by adding phosphatidylcholine. Adding 150 mg/L of phosphatidylcholine at the 8th hour of fermentation can make the yield and molecular weight reach to the highest value, i.e. 2.54 MDa. Finally, by combining adding phosphatidylcholine, nutrient enrichment and two-stage control culture strategy and applying them in the fermentation of HA, the yield and molecular weight of HA reached to 5.88 g/L and 2.71 MDa respectively, which realized the unification of high yield and high molecular weight. |
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