Study On Optimization Of Preparation Process And Antioxidant Activity Ofchondroitin Sulfate Oligosaccharide From Raja Porosa

ObjectiveThe preparation,separation and purification of chondroitin sulfate oligosaccharides from Raja porosa were studied in this study.The yield,molecular weight and antioxidant activity of the products at different levels were compared.Response surface methodology was used to determine the optimum technological conditions for dilute alkali enzymatic method,which provided experimental basis for the research and development of chondroitin sulfate oligosaccharides.MethodsChondroitin sulfate was extracted from Raja porosa cartilage by three different extraction processes: concentrated alkali method,alkali-salt method and dilute alkali method.Then the quality differences of the products were compared by analyzing the yield of the products,the content of hexosamine and nitrogen impurities,in order to find the best extraction process.Response surface methodology was used to determine the optimum technological5 conditions for dilute alkali enzymatic method.After that,chondroitin sulfate oligosaccharides were further degraded,separated and purified from chondroitin sulfate products extracted by alkali salt method to obtain a single component of chondroitin sulfate oligosaccharides with high purity.Enzymatic hydrolysis and acidolysis were used in the degradation process.By comparing the biological activities of the degradation products,the enzymatic hydrolysis products were determined to enter the next stage of separation and purification.At the same time,the time of enzymatic hydrolysis was investigated,and the optimum time was 5.5 hours.After that,the primary products of chondroitin sulfate oligosaccharides hydrolyzed for 5.5 hours were used as raw materials to establish a separation and purification system.After separation by gel filtration,anion exchange separation,electropHoresis separation and AKTA Superdex peptide column separation,a high purity chondroitin sulfate oligosaccharide with relatively simple components was obtained.In this process,the molecular weight and antioxidant activity of each stage of separation products were tested.Response surface methodology was used to determine the optimum technological conditions for dilute alkali enzymatic method.ResultsAmong the three extraction processes,the yield of chondroitin sulfate product B obtained by alkali salt method is the highest,the content of hexosamine is the highest,and the content of nitrogen impurities is slightly higher than that obtained by dilute alkali method,but the purity is not very different.Considering comprehensively,alkali-salt method is the best technology for extracting chondroitin sulfate.The molecular weight and components of chondroitin sulfate enzymatic hydrolysate X and acid hydrolysate Y of Raja porosa have little difference,but the antioxidant activity of X is better than that of Y,and the color of X is milky white better than that of Y’s dark brown.Enzymatic hydrolysis should be the main method in chondroitin sulfate degradation of Raja porosa,and the optimal time of enzymatic hydrolysis should be controlled at 5.5 h.After the initial gel filtration and separation,two complex chondroitin sulfateoligosaccharide components of L1 and L2 were obtained.The antioxidant activity of L2 was higher than that of L1,while the weight average molecular weight of L1 was lower than that of L1.Chondroitin sulfate oligosaccharides,S1,S2 and S3,which are not single components,can be obtained by further anion exchange separation of L2 components.However,three kinds of chondroitin sulfate oligosaccharides,CS-a(536Da),CS-b(993Da)and CS-c(1357Da),were obtained by further preparation and electrophoresis and AKTA Superdex peptide column separation.The antioxidant activity of these three oligosaccharides showed a dose-effect relationship,but the difference between them was not obvious.ConclusionsThe results showed that there were three kinds of chondroitin sulfate oligosaccharides purified by the grading separation system established in this study.The components were relatively single,and their antioxidant activities showed a dose-effect relationship,but the difference between the three was not obvious.The optimum extraction conditions of chondroitin sulfate were sodium hydroxide concentration: 7.0%,temperature: 40 ℃,solid-liquid ratio: 5.0.The results of response surface methodology were as follows: the optimum extraction conditions of chondroitin sulfate were sodium hydroxide concentration: 7.0%,temperature: 40 ℃,solid-liquid ratio: 5.0.The optimum conditions of enzymatic hydrolysis were as follows: enzymatic hydrolysis time:4.3h,temperature: 44℃,dosage: 0.46%.