CN113234180A - Method for preparing chondroitin sulfate from squid cartilage - Google Patents
Method for preparing chondroitin sulfate from squid cartilage Download PDFInfo
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- CN113234180A CN113234180A CN202110439798.XA CN202110439798A CN113234180A CN 113234180 A CN113234180 A CN 113234180A CN 202110439798 A CN202110439798 A CN 202110439798A CN 113234180 A CN113234180 A CN 113234180A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses a method for preparing chondroitin sulfate by utilizing squid cartilage. Develops a green process and a new technology for extracting the chondroitin sulfate product by an enzymolysis method to obtain the squid chondroitin sulfate in different utilization ways. The method comprises the following steps: removing non-cartilage parts of squid cartilage, washing, crushing, adding 4% dilute alkali solution for degreasing, and repeatedly washing with distilled water to be neutral; adjusting pH to 2-3, performing enzymolysis for 24h, adjusting pH to neutral, adding sodium chloride to final concentration of 1.0mol/L, standing overnight, centrifuging for 30min, extracting supernatant, adjusting pH to 8.5-9, performing enzymolysis for 6h, heating, filtering, and repeatedly extracting filter residue once; precipitating with ethanol, redissolving the precipitate with distilled water, rotary evaporating, and freeze drying to obtain chondroitin sulfate product. The comprehensive extraction process adopted by the invention well keeps the natural structures and properties of collagen and chondroitin sulfate. Provides good technical support for better application of the composite material in preventing and treating diseases such as atherosclerosis, angina pectoris, hyperlipidemia and the like, and application of the composite material as biomedical engineering material in skin, bone and cartilage, nerve, heart and eye tissue engineering.
Description
Technical Field
The invention relates to the field of modern biotechnology, in particular to a method for preparing chondroitin sulfate by utilizing squid cartilage.
Background
Chondroitin sulfate is a sulfated acidic mucopolysaccharide, also called glycosaminoglycan, formed by repeatedly connecting D-glucuronic acid and N-acetyl-D-galactosamine through beta-1, 3 glycosidic bonds, and generally contains 50-70 disaccharide units. Chondroitin sulfate is widely distributed on extracellular matrix and cell surface of animal tissue, mostly exists in form of proteoglycan side chain in organism, is an important component for constituting combined tissue of animal cartilage, tendon, skin and the like, and mainly comes from tissue of animal cartilage, trachea, tendon, larynx and the like. Chondroitin sulfate has various physiological effects of resisting tumor, reducing blood fat, relieving arthritis, resisting blood coagulation activity and the like, and is widely applied to the fields of health-care food, biological medicine, cosmetics, nano materials and the like. The relative molecular mass, charge density, chemical properties and the like of chondroitin sulfate from different sources are obviously different, the squid chondroitin sulfate separated from squid cartilage has 2 components, namely polysaccharide with the weight-average molecular weight (Mw) of 393028 u and oligosaccharide with the weight-average molecular weight of 1079 u, and the content of the chondroitin sulfate in a sample is higher; monosaccharide composition analysis shows that monosaccharide compositions of the squid chondroitin sulfate and the shark chondroitin sulfate standard product are basically consistent, and the ratio of the aminouronic acid to the galactose sulfate is 1.034 to 1.265, which accords with the monosaccharide composition characteristics of the chondroitin sulfate. The results show that the invention retains the natural structure and properties of chondroitin sulfate. Provides good technical support for better application of the composite material in preventing and treating diseases such as atherosclerosis, angina pectoris, hyperlipidemia and the like, and application of the composite material as biomedical engineering material in skin, bone and cartilage, nerve, heart and eye tissue engineering.
Chondroitin sulfate is widely distributed in nature, and the extraction method mainly comprises an enzyme extraction method, an alkali extraction method, an enzymolysis-resin adsorption method, an ultrasonic extraction method and the like. The alkali extraction method has long preparation time and high cost, and the chondroitin sulfate is easy to degrade under the strong alkali condition and pollutes the environment; the enzymolysis-resin adsorption method saves energy, reduces environmental pollution, has high purity, but is not suitable for large-scale industrial production; the purity and extraction rate of chondroitin sulfate extracted by the ultrasonic extraction method are low.
In coastal areas with abundant squid resources, a large amount of squid leftover squid cartilage is regarded as waste, and particularly, highly sulfated chondroitin sulfate is found in the squid, so that the squid cartilage has unique activity in the aspects of protecting and repairing neurons and the like. The leftovers of the squid processing are fully utilized, and the waste of resources and the pollution to the environment are avoided.
Disclosure of Invention
In order to solve the problems of environmental pollution, long treatment time, high cost and degradation of chondroitin sulfate in the process of extracting the chondroitin sulfate, the invention provides the method for preparing the chondroitin sulfate by using the squid cartilage as the raw material, which has the advantages of reasonable preparation process, short required time and simple operation, and keeps the natural structure and properties of the chondroitin sulfate.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:
(1) pretreatment of raw materials: removing non-cartilage components from squid cartilage, cleaning, and pulverizing; degreasing the crushed bone meal with 4% dilute alkali solution for 4-6 hours, and repeatedly washing the crushed bone meal with distilled water to be neutral;
(2) taking supernatant after enzymolysis: adjusting the pH value of pretreated fishbone to 2-4 with hydrochloric acid, adding 0.1-0.2% pepsin, performing enzymolysis for 18-24 h at 4-6 ℃, adjusting the pH value to be neutral, slowly adding ground fine NaCl to the final concentration of 1.0mol/L, standing overnight, centrifuging at 10000 r/min for 30-40 min, and taking supernatant;
(3) extracting the chondroitin sulfate solution: and continuously adjusting the pH value of the supernatant to 8.5-9.0, and adding 2% trypsin by mass at the temperature of 60-66 ℃ for enzymolysis for about 6-8 hours. And then heating to 80-90 ℃, cooling, filtering, repeatedly extracting filter residues once under the same conditions, and combining the two filtrates. And continuously stirring and adding ethanol into the obtained hydrolysate until the volume fraction of the ethanol in the solution system is 90%, standing for more than 9-12 h, collecting precipitates, and washing for 3 times by using absolute ethyl alcohol. Redissolving the precipitate with distilled water, and dialyzing with distilled water for 3 d until the conductivity of the dialysate is not changed. Obtaining chondroitin sulfate solution;
(4) and (3) finished product: and (4) carrying out rotary evaporation on the solution obtained in the step (3) at the temperature of 30-60 ℃, and then carrying out freeze drying to obtain a dried squid chondroitin sulfate product.
Preferably, the crushed bone meal in the step (1) is degreased by 4% dilute alkali solution for 4-6 h.
Further optimizing, adding hydrochloric acid to adjust the pH to 2-4 before enzymolysis in the step (2); the enzymolysis temperature is 4-6 ℃, and the time is 18-24 h.
Further optimizing, centrifuging and taking supernatant after enzymolysis in the step (2), wherein the centrifugation conditions are as follows: centrifuging at 10000 r/min for 30-40 min.
Further optimizing, adjusting the pH value of the supernatant obtained in the step (3) to 8.5-9.0, and adding 2% trypsin by mass for enzymolysis for about 6-8 hours at the temperature of 60-66 ℃.
Further optimizing, wherein the rotary evaporation temperature of the chondroitin sulfate solution obtained in the step (4) is controlled to be 30-60 ℃.
The invention adopts the enzymolysis method to extract the chondroitin sulfate in the squid cartilage, the enzymolysis method has mild reaction conditions, the chondroitin sulfate structure is not easy to be damaged, the biological activity of the chondroitin sulfate is favorably kept, the period is shortened, and simultaneously, higher product quality is obtained, the chondroitin sulfate can be better applied to preventing and treating atherosclerosis, angina pectoris, hyperlipidemia and other diseases, and can be used as a biomedical engineering material to provide good technical support for skin, bone and cartilage, nerve, heart and eye tissue engineering. Meanwhile, the method is suitable for industrial mass production.
Drawings
FIG. 1 is a block diagram of a process flow for preparing chondroitin sulfate by using squid cartilage under the assistance of microwave;
FIG. 2 is a Fourier transform infrared spectrum of chondroitin sulfate.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
A method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:
(1) pretreatment of raw materials: removing non-cartilage components from squid cartilage, cleaning, and pulverizing; defatting the crushed bone powder with 4% dilute alkali solution for 4 hr, and repeatedly washing with distilled water to neutrality;
(2) taking supernatant after enzymolysis: adjusting the pH value of pretreated fishbone to 2-4 with hydrochloric acid, adding 0.1-0.2% pepsin, performing enzymolysis at 4 ℃ for 18h, adjusting the pH value to be neutral, slowly adding ground fine NaCl to the final concentration of 1.0mol/L, standing overnight, centrifuging at 1000 r/min for 30min, and taking supernatant;
(3) extracting the chondroitin sulfate solution: the supernatant was further adjusted to pH8.5 and digested with 2% trypsin at 60 ℃ for about 6 h. Heating to 80 deg.C, cooling, filtering, extracting the residue under the same conditions, mixing the filtrates, adding ethanol into the obtained hydrolysate under stirring until the volume fraction of ethanol in the solution is 90%, standing for 9 hr, collecting precipitate, and washing with anhydrous ethanol for 3 times. Redissolving the precipitate with distilled water, and dialyzing with distilled water for 3 d until the conductivity of the dialysate is not changed. Obtaining chondroitin sulfate solution;
(4) and (3) finished product: and (4) carrying out rotary evaporation on the solution obtained in the step (3) at the temperature of 30 ℃, and then carrying out freeze drying to obtain a dried squid chondroitin sulfate product.
The method for preparing the chondroitin sulfate by using the squid cartilage provided by the embodiment has the advantages of reasonable preparation process, short required time and high effect, and can keep the natural structure and properties of the chondroitin sulfate.
Example 2
A method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:
(1) pretreatment of raw materials: removing non-cartilage components from squid cartilage, cleaning, and pulverizing; defatting the crushed bone powder with 4% dilute alkali solution for 5 hr, and repeatedly washing with distilled water to neutrality;
(2) taking supernatant after enzymolysis: adjusting the pH value of pretreated fishbone to 2-4 with hydrochloric acid, adding 0.1-0.2% pepsin, performing enzymolysis at 4 ℃ for 20h, adjusting the pH value to neutral, slowly adding ground fine NaCl to the final concentration of 1.0mol/L, standing overnight, centrifuging at 1000 r/min for 35min, and taking supernatant;
(3) extracting the chondroitin sulfate solution: the supernatant was further adjusted to pH9 and hydrolyzed by addition of 2% trypsin at 63 ℃ for about 7 h. Then heating to 85 ℃, cooling and filtering, extracting the filter residue once again under the same condition, and combining the two filtrates. Continuously stirring the obtained hydrolysate, adding ethanol until the volume fraction of the ethanol in the solution system is 90%, standing for 10 h, collecting precipitate, and washing with anhydrous ethanol for 3 times. Redissolving the precipitate with distilled water, and dialyzing with distilled water for 3 d until the conductivity of the dialysate is not changed. Obtaining chondroitin sulfate solution;
(4) and (3) finished product: and (4) carrying out rotary evaporation on the solution obtained in the step (3) at the temperature of 40 ℃, and then carrying out freeze drying to obtain a dried squid chondroitin sulfate product.
The method provided by the embodiment can improve the yield of the chondroitin sulfate by increasing the enzymolysis time of the trypsin and properly increasing the heating temperature, does not influence the structure of the chondroitin sulfate, and has good color and luster and excellent biological activity.
Example 3
A method for preparing chondroitin sulfate by utilizing squid cartilage comprises the following process steps:
(1) pretreatment of raw materials: removing non-cartilage components from squid cartilage, cleaning, and pulverizing; defatting the crushed bone powder with 4% dilute alkali solution for 5 hr, and repeatedly washing with distilled water to neutrality;
(2) taking supernatant after enzymolysis: adjusting the pH value of pretreated fishbone to 2-4 with hydrochloric acid, adding 0.1-0.2% pepsin, performing enzymolysis at 6 ℃ for 24h, adjusting the pH value to neutral, slowly adding ground fine NaCl to the final concentration of 1.0mol/L, standing overnight, centrifuging at 1000 r/min for 40min, and taking supernatant;
(3) preparation of chondroitin sulfate solution: the supernatant was further adjusted to pH9 and hydrolyzed by the addition of 2% trypsin at 66 ℃ for about 8 h. Then heating to 90 ℃, cooling and filtering, extracting the filter residue once again under the same condition, and combining the two filtrates. Continuously stirring the obtained hydrolysate, adding ethanol until the volume fraction of the ethanol in the solution system is 90%, standing for 12 h, collecting precipitate, and washing with anhydrous ethanol for 3 times. Redissolving the precipitate with distilled water, and dialyzing with distilled water for 3 d until the conductivity of the dialysate is not changed. Obtaining chondroitin sulfate solution;
(4) and (3) finished product: and (4) carrying out rotary evaporation on the solution obtained in the step (3) at the temperature of 60 ℃, and then carrying out freeze drying to obtain a dried squid chondroitin sulfate product.
In the technical scheme provided by the embodiment, the raw material pretreatment time is prolonged, the parameters in the chondroitin sulfate preparation process are changed, the production efficiency is effectively improved, and the structure of the chondroitin sulfate is not damaged. Meanwhile, the experimental process is economic, efficient, green and environment-friendly, and does not cause pollution to the environment.
Claims (6)
1. A method for preparing chondroitin sulfate by utilizing squid cartilage is characterized by comprising the following process steps:
(1) pretreatment of raw materials: removing non-cartilage components such as fascia and meat from squid cartilage, cleaning to obtain clean cartilage, and pulverizing; degreasing the crushed bone meal for 4-6 hours by using a 4% dilute alkali solution to remove fat and non-collagen components and prevent endogenous protease from influencing chondroitin sulfate, and repeatedly washing the crushed bone meal to be neutral by using distilled water;
(2) taking supernatant after enzymolysis: adjusting the pH value of pretreated fishbone to 2-4 with hydrochloric acid, adding 0.1-0.2% pepsin, performing enzymolysis for 18-24 h at 4-6 ℃, adjusting the pH value to be neutral, slowly adding ground fine NaCl to the final concentration of 1.0mol/L, standing overnight, centrifuging at 10000 r/min for 30-40 min, and removing supernatant;
(3) extracting chondroitin sulfate: continuously adjusting the pH value of the supernatant to 8.5-9.0, adding 2% trypsin by mass at 60-66 ℃ for enzymolysis for 6-8 h, heating to 80-90 ℃, cooling, filtering, repeatedly extracting filter residues once under the same conditions, combining two filtrates, continuously stirring and adding ethanol into the obtained hydrolysate until the volume fraction of the ethanol in a solution system is 90%, standing for more than 9-12 h, collecting precipitates, washing the precipitates for 3 times by using absolute ethanol, redissolving the precipitates by using distilled water, and dialyzing for 3 d by using the distilled water until the electrical conductivity of the dialysate is unchanged;
(4) and (3) finished product: and (4) carrying out rotary evaporation on the solution obtained in the step (3) at the temperature of 30-60 ℃, and then carrying out freeze drying to obtain a dried squid chondroitin sulfate product.
2. The method for preparing chondroitin sulfate from squid cartilage according to claim 1, wherein the method comprises the following steps: and (2) degreasing the crushed bone meal in the step (1) for 4 hours by using a 4% dilute alkali solution.
3. The method for preparing chondroitin sulfate from squid cartilage according to claim 1, wherein the method comprises the following steps: and (3) performing enzymolysis for 24 hours by using 0.1-0.2% pepsin at 4 ℃ in the step (2), and adding NaCl into the solution after enzymolysis, wherein the concentration is controlled at 1.0 mol/L.
4. The method for preparing chondroitin sulfate from squid cartilage according to claim 1, wherein the method comprises the following steps: and (3) in the step (2), the centrifugation rate of the enzymolysis liquid after standing overnight is controlled to be 1000 r/min, and the centrifugation time is 30 min.
5. The method for preparing chondroitin sulfate from squid cartilage according to claim 1, wherein the method comprises the following steps: adjusting the pH value of the supernatant fluid after centrifugation in the step (3) to 8.5-9.0, controlling the temperature of enzymolysis to 60-66 ℃ and the time to 6 hours; and after enzymolysis, the heating temperature is controlled to be 80-90 ℃.
6. The method for preparing chondroitin sulfate from squid cartilage according to claim 1, wherein the method comprises the following steps: in the process of precipitating the ethanol and the salinization liquid in the step (3), the volume fraction of the ethanol in a solution system is controlled to be 90%, and the obtained precipitate is redissolved by distilled water; the rotary evaporation temperature of the chondroitin sulfate solution is controlled at 50 ℃.
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| CN113429494A (en) * | 2021-08-15 | 2021-09-24 | 四川农业大学 | Method for extracting chondroitin sulfate from pig trachea |
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| CN113429494A (en) * | 2021-08-15 | 2021-09-24 | 四川农业大学 | Method for extracting chondroitin sulfate from pig trachea |
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