Production Of Low Molecular Weight Hyaluronic Acid By Two-Stage Semi-Continous Fermentation

Hyaluronic acid is a multifunctional valuable biological macromolecular polysaccharide that has wide applications in cosmetics,health food,and orthopedics.Different molecular weights of hyaluronic acid have different applications,and low molecular weight hyaluronic acid（1×10⁴-1×10⁶ Da）is more widely used in cosmetics and food,which can be obtained by hydrolysis of high molecular weight hyaluronic acid.Streptococcus zooepidemicus ATCC39920 is an excellent natural production strain that produces hyaluronic acid with good quality.However,its titer is lower and cannot meet the demand for higher production.In this study,the strain was subjected to UV mutagenesis to obtain a high titer hyaluronic acid strain;the culture conditions were optimized;a semi-continuous fermentation process was constructed to improve the productivity of hyaluronic acid;and the effect of reducing the viscosity of the fermentation broth to improve the dissolved oxygen（DO）and mass transfer conditions in the bioreactors on hyaluronic acid production was studied.（1）To improve the titer of hyaluronic acid in S.zooepidemicus ATCC 39920,the wide type strain was UV mutagenized to obtain hyaluronic acid high-titer strain SZ07.after 36 h of shake flask fermentation,the hyaluronic acid titer of SZ07 was achieved 1.41 g·L^-1,which was 54.9%higher compared with the WT strain.To enhance the productivity of strain SZ07,its culture conditions were optimized,including nitrogen source,initial p H of the medium,and phosphate concentration.After 36 h of shake flask fermentation,the titer of hyaluronic acid was achieved 4.2 g·L^-1,which was 197.9%higher than the initial titer.The titer of hyaluronic acid was increased to 13.3g·L^-1 with a yield of 0.19 g·g^-1 when batch fermentations were performed in bioreactors.（2）In order to further enhance the productivity of hyaluronic acid,a semi-continuous fermentation process of Streptococcus zooepidemicus was constructed.The production of hyaluronic acid in the 2^nd stage bioreactors could reach the level of 13.3 g·L^-1 at 22 h of the batch fermentation strategy in 14 h,and the semi-continuous fermentation could be carried out stably for more than 6 batches,and the productivity was increased by 68.3%compared with that of the batch fermentation.In order to reduce the limitation of high viscosity fermentation broth on hyaluronic acid production,the hyl B gene encoding hyaluronidase from Streptococcus zooepidemicus was expressed in E.coli BL21,and the induction conditions were optimized.The p ET-22b plasmid was used for the highest expression of hyaluronidase,and the maximum enzyme activity could reach 1.59×10⁵ U·m L^-1.（3）The addition of hyaluronidase to the two-stage semi-continuous fermentation process reduced the viscosity of the fermentation broth and improved the conditions of mass transfer and DO in the fermenter.Compared to semi-continuous fermentation without the addition of hyaluronidase,the fermentation time in the 2^nd bioreactors was extended to 24 h with the addition of hyaluronidase.The addition of hyaluronidase was optimised and the maximum hyaluronic acid titer of 29.38 g·L^-1 was achieved when the hyaluronidase was added at 300U·m L^-1.As the addition of hyaluronidase continues to be increased and hyaluronic acid titer is no longer elevated.In this study,through the strategies of UV mutagenesis,optimization of culture conditions,construction of a semi-continuous fermentation process and degradation of fermentation broth viscosity,the titer and productivity of hyaluronic acid were greatly improved.And this study provideds a reference for the production of other polysaccharide products.