酶解—膜分离耦合连续制备抗氧化性小分子透明质酸

Continuous preparation of antioxidant low molecular weight hyaluronic acid by coupling of enzymolysis and membrane separation

目的:构建一种酶解—膜分离耦合的方法用来提高小分子透明质酸的生产效率。方法:利用酶生物反应罐与平板式聚醚砜超滤膜分离组件组成的连续式酶解—膜分离耦合反应体系。以膜通量、膜污染、透过液透明质酸质量浓度、产率为评价指标,系统地考察各因素对酶膜耦合体系内两种不同相对分子质量透明质酸分离效果的影响,并以产率为指标进一步通过正交试验优化酶膜耦合反应的工艺参数。结果:酶膜耦合反应体系制备小分子透明质酸的最优工艺参数为搅拌速度200 r/min、跨膜压力0.15 mPa、酶解时间4.0 h、加酶量5 g/100 g。该条件下可以快速制备并分离出两种不同相对分子质量(Mr)的小分子透明质酸,实现一步法同时制备Mr在1万~5万的低相对分子质量透明质酸(LMW-HA)和Mr<3 000的透明质酸寡糖(O-HA),且这两种不同相对分子质量透明质酸均具有清除DPPH自由基和羟自由基的能力。结论:酶解—膜分离耦合法可以连续同步生产具有不同相对分子质量和一定抗氧化性的LMW-HA和O-HA。

Objective:This study aimed to construct an enzymolysis and membrane separation method to improve the production efficiency of low molecular hyaluronic acid(HA).Methods:A continuous enzymolysis and membrane separation coupling reaction system was designed,which consisted of an enzyme biological reaction tank and a flat polyethersulfone ultrafiltration membrane separation module.By evaluating membrane flux,membrane contamination,hyaluronic acid concentration,and yield,the effects of various factors on the separation of low molecular HA in the enzyme-membrane coupling system were systematically investigated.The process parameters of the enzyme-membrane coupling reaction were further optimized through orthogonal experiments.Results:The optimal process parameters for the preparation of low molecular hyaluronic acid by enzyme-membrane coupling reaction system were a stirring speed of 200 r/min,transmembrane pressure of 0.15 mPa,enzymolysis time of 4.0 h,and enzyme dosage of 5 g/100 g.In this method,two low molecular hyaluronic acids with different molecular weights(M r)can be quickly prepared and separated,the low molecular weight hyaluronic acid(LMW-HA)with Mw between 10000~50000 and the HA oligosaccharides(O-HA)with M r<3000 can be prepared rapidly by one-step approach.Both of these different molecular weights hyaluronic acid could scavenge DPPH and hydroxyl free radicals.Conclusion:The enzymolysis and membrane separation coupling method provides an efficient method for simultaneous and continuous production of LMW-HA and O-HA with different molecular weights and certain antioxidant properties.