Studies On The Extraction Process Of Chondroitin Sulfate On The Basis Of Removing Protein By Enzymatic Hydrolysis Method

Chondroitin sulfate(ChS),a kind of acid glycosaminoglycan,is extensively present in theconnective tissue of animal and linked with core protein as a kind of proteoglycans existing in the cartilage through covalent bonds. As a kind of an important biological macromolecules,ChS has been proven to posses various biological activities such as ease arthritis,hypolipidemic,anti-oxidation and anti-angiogenesis functions,etal.Which has been used in food,medicine,cosmetics and other areas and has broad development prospects.At present,varieties of extraction technology of chondroitin sulfate have been founded but,for the most part,the extraction rates are not high and processes are complicated.So a lower cost,highly more efficient production technology is a basic goal in the study.The present research studied pre-treatment of chondroitin sulfate material (bovine nose cartilage) by enzymatic hydrolysis method to dispose of some protein at frist.Then the best conditions of alkaline extraction and ethanol deposition were explored by single factor and orthogonal experiments. As a result,the process was simplified and theyield of ChS was improve. Major results of this paper are as following:Fristly, the pre-treatment of chondroitin sulfate material (bovine nose cartilage) by enzymatic hydrolysis method was studied in this paper. The cartilage was digested by pepsin to eliminate muscle、fat and connective tissue from cartilage surface. The optimal enzymatic hydrolysis conditions of cartilage were explored by single factor and orthogonal experiments to be enzymatic hydrolysis time of8h, hydrolysis temperature of35℃,pepsin dosage of0.2%,hydrolysis pH of2.5and the weight proportion of1:5(material:liquid). Under the optimal conditions, the yields of cartilage and chondroitin sulfate were85.12%and28.98%and the removal rate of protein was10.3%.Secondly,ChS was extracted with alkaline solution and sedimentated with ethanol. The optimal alkaline hydrolysis conditions of cartilage powder were explored by single factor and orthogonal experiments to be the ratio of solid to liquid of1:6, alkali solution of4%,temperature of alkali extraction of42℃and the duration of alkali extraction of5h.Under the optimal conditions,the yield was12.58%,the content of Glucuronic acid was32.44%and the purity quotient of ChS was82%.Thirdly,the ethanol deposition technology of ChS was studied in the paper, which was extracted from bovine nose cartilage hydrolyzed by alkaline solution.The optimal ethanol deposition conditions of ChS were explored by single factor and orthogonal experiments to be ethanol solution of65%,sodium acetate solution of4.5%,pH6.0.Under the optimal conditions,the yield was14.81%;the content of Glucuronic acid was33.36%;the purity quotient of ChS was85.9%.Lastly,ethanol precipitation,isoelectric point precipitation and ion-exchange method were applied to purify chondrointin sulfate.Compared with physical and chemical indexes,sensory indexes of products. The results showed that ethanol precipitation method was superior to other methods.The purity was the highest achieved90.3%and more than export standard;The purity of isoelectric point precipitation was86%and ion-exchange method was86.3%. Others indexes also meet with standard. In order to further verificated the purity of the products, the ultraviolet and infrared spectrum analysis were used. UV analysis revealed that the absorption peak of polysaccharide appeared at196nm,while characteristic absorption peaks of protein (280nm) and nucleic acid (260nm) were not found.Typical absorption peaks of ChSA could be found by IR.