US-12618090-B2 - Method for increasing the extraction rate of chondroitin sulfate prepared from tilapia skull

US12618090B2US 12618090 B2US12618090 B2US 12618090B2US-12618090-B2

Abstract

This present disclosure provides a method for preparing chondroitin sulfate from tilapia skull, including enzymatic hydrolyzing tilapia skull powder by Savinase 16L to obtain Savinase 16L enzymatic hydrolysate, re-enzymatic hydrolyzing the Savinase 16L enzymatic hydrolysate by 2709 alkaline protease, then separating and purificating the solution by ethanol and cetylpyridine chloride to obtain the chondroitin sulfate. The present disclosure adopts a compound enzymatic hydrolyzing method by Savinase 16L and 2709 alkaline protease to effectively achieve the efficient extraction of chondroitin sulfate of tilapia skull powder, and further achieve the purification of chondroitin sulfate by ethanol and cetylpyridine chloride, so that tilapia by-products can be utilized with high value.

Inventors

Saiyi ZHONG
Gege ZUO
Jing Chen
Jianping Chen
Rui Li
BingBing Song
Kangjian CHEN
Xiaofei Liu
Xuejing JIA

Assignees

GUANGDONG OCEAN UNIVERSITY

Dates

Publication Date: 20260505
Application Date: 20220324
Priority Date: 20211115

Claims (3)

1 . 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 Na 2 CO 3 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 Na 2 SO 4 solution, subsequently, slowly adding cetylpyridium chloride solution into the Na 2 SO 4 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.
2 . The method of claim 1 , wherein the condition of inactivating enzymes to obtain the first enzymatic hydrolysate and the condition of inactivating enzymes to obtain the second enzymatic hydrolysate are all 100° C. for 10 min and solution are all cooled by ice-water bath; the condition of centrifugation in each step are all 8000 r/min for 20 min at 4° C.
3 . The method of claim 1 , wherein the mass concentration of the cetylpyridium chloride solution is 6%.

Description

TECHNICAL FIELD The present disclosure belongs to the field of bioengineering technology and relates to a method for improving the extraction rate of chondroitin sulfate prepared from tilapia skull. BACKGROUND Chondroitin Sulfate (CS) is a highly sulfated glycosaminoglycan consisting of alternating D-glucuronic acid and N-acetyl-d-galactosamine linked disaccharide unit. Chondroitin sulfate has many isomers according to the number and types of sulfate groups linked at different positions of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). Based on existing research, chondroitin sulfate has a variety of biological activities such as anti-inflammatory, antioxidant, anticoagulation, lipid-lowering and anti-tumor, and can effectively inhibit thrombosis, hinder the formation of atherosclerotic plaque and reduce the lipid index by regulating growth factors, thus can prevent cardiovascular diseases. In addition, chondroitin sulfate can also be used as a food additive, a health food and a dietary supplement. Commercial chondroitin sulfate was mainly extracted from bovine cartilage, pig cartilage, shark cartilage, but due to the mad cow disease, the foot-and-mouth disease and other diseases, the development of terrestrial sources of chondroitin sulfate was limited. In recent years, for shark resources have become increasingly scarce and are expensive, so it is urgent to find a new source of chondroitin sulfate to replace shark raw materials. Tilapia is a tropical teleost fish characterized as fast growth, high yield, strong fecundity, etc. The total production of freshwater tilapia in China is increasing, and the processing by-products of tilapia are also increasing, such as fish skull, fish tail, fish bone, fish scale, fish skin and internal organs, etc., which are usually discarded or indiscriminately processed into low-value products such as feed, making a large number of resources have not been fully developed and utilized. The extraction of chondroitin sulfate from tilapia skull is one of the effective measures to transform low-value tilapia processing by-products to high-value products. However, it should be pointed out that chondroitin sulfate is not only easy to degrade under the condition of high temperature and strong acid, so that its molecular weight is reduced, but also can be degraded under the action of chondroitin sulfate lyase. Traditional extraction methods of chondroitin sulfate include alkali extraction method, salt extraction method, alkali salt method, ultrasonic assistance method, etc. Among them, the alkali salt method will pollute the environment and will also make chondroitin sulfate degradation, the ultrasonic assistance method have a high demand for time and may produce impurities, and the extraction efficiency of chondroitin sulfate by the neutral salt method is low. Separation and purification methods of chondroitin sulfate include chromatography method, electrophoretic ultrafiltration method, anion exchange resin method, etc. However, these methods have the problems of complex steps, long production cycle and high cost. SUMMARY The purpose of the present disclosure is to provide an efficient method for preparing chondroitin sulfate from fish skull. For the above purposes, this application resolves this requirement in the field by providing a method for increasing the extraction rate of chondroitin sulfate prepared from tilapia skull. On the one hand, the present disclosure relates to a method for increasing the extraction rate of chondroitin sulfate prepared from tilapia skull including: enzymatic hydrolyzing tilapia skull powder by subtilisin protease (sold under the trademark SAVINASE 16L) to form a first enzymatic hydrolysate; re-enzymatic hydrolyzing the first enzymatic hydrolysate by an alkaline protease (sold under the trademark 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 (sold under the trademark SAVINASE 16L) is 1:15 to 1:25 in g:μL; a mass-volume ratio of the alkaline protease (sold under the trademark 2709 ALKALINE PROTEASE) to the first enzymatic hydrolysate is 5:1 to 6:1 in mg:mL. Further, in the method of improving the extraction rate of chondroitin sulfate prepared from tilapia skull provided by the present disclosure, the enzymatic hydrolyzing tilapia skull powder by the subtilisin protease (sold under the trademark SAVINASE 16L) to form the first enzymatic hydrolysate includes: adding the tilapia skull powder into Na2CO3 solution; enzymatic hydrolyzing the mixed solution; inactivating enzymes to obtained solution to form the first enzymatic hydrolysate. Further, in the method of improving the extraction rate of chondroitin sulfate prepared from tilapia skull provided by the present disclosure, the re-enzymatic hydrolyzing t