Method for increasing the extraction rate of chondroitin sulfate prepared from tilapia skull

A method for increasing the extraction rate of chondroitin sulfate prepared from tilapia skull comprising:

• enzymatic hydrolyzing tilapia skull powder by subtilisin protease (SAVINASE 16L) to form a first enzymatic hydrolysate;
• re-enzymatic hydrolyzing the first enzymatic hydrolysate by an alkaline protease (2709 ALKALINE PROTEASE) to form a second enzymatic hydrolysate;
• then separating and purificating the second enzymatic hydrolysate by ethanol and cetylpyridine chloride to obtain the chondroitin sulfate;
• wherein
• a mass-volume ratio of the tilapia skull powder to the subtilisin protease (SAVINASE 16L) is 1:15 to 1:25 in g:μL;
• a mass-volume ratio of the alkaline protease (2709 ALKALINE PROTEASE) to the subtilisin protease (SAVINASE 16L) enzymatic hydrolysate is 5:1 to 6:1 in mg:mL;
• and wherein
• the method comprises:
• adding the tilapia skull powder into 50 mM Na2CO3 solution, adjusting pH of the mixed solution to neutral, and enzymatic hydrolyzing the mixed solution at 55° C. for 4 hours;
• then adding the subtilisin protease (SAVINASE 16L) into the mixed solution and stirring the mixed solution in a water bath at 55° C. for 4 h;
• inactivating enzymes to the mixed solution to obtain a first enzymatic hydrolysate;
• adding alkaline protease into the first enzymatic hydrolysate, enzymatic hydrolyzing the first enzymatic hydrolysate at 50° C. for 2 hours, and then inactivating enzymes to reacted solution to obtain a second enzymatic hydrolysate;
• adding trichloroacetic acid into the second enzymatic hydrolysate, stirring and then standing the second enzymatic hydrolysate for 3 h, obtaining supernatant by centrifugation;
• adding absolute ethyl alcohol into the supernatant and alcohol precipitating at 4° C.′, then collecting a first precipitate by centrifugation;
• dissolving the first precipitate into 20 mM Na2SO4 solution, subsequently, slowly adding cetylpyridium chloride solution into the Na2SO4 solution, then collecting a second precipitate by centrifugation;
• dissolving the second precipitate into NaCl-ethanol aqueous solution, adding ethyl alcohol and then standing the solution at 4° C., collecting a third precipitate by centrifugation;
• dissolving the third precipitate into water for clarification, and then freeze-drying the precipitate to obtain chondroitin sulfate;
• and wherein
• the NaCl-ethanol aqueous solution is a mixture of 2M NaCl solution and ethyl alcohol, and the volume ratio of the 2M NaCl solution to the ethyl alcohol is 100:15.