阅读说明：本技术 一种提取硫酸软骨素的方法、硫酸软骨素及其应用 (Method for extracting chondroitin sulfate, chondroitin sulfate and application thereof ) 是由 赵元晖 王康宇 曾名湧 高瑞昌 白帆 汪金林 李钰金 薛长湖 毛相朝 薛勇 陈泽 于 2020-03-23 设计创作，主要内容包括：本发明属于硫酸软骨素提取技术领域,公开了一种提取硫酸软骨素的方法、硫酸软骨素及其应用,将鱼头软骨粉碎至颗粒状并晾干除去多余的水分作为蒸汽爆破原料,将部分软骨放于冻干机中冻干作为优化酶解工艺的原料并得到最优酶解工艺；干基汽爆鱼骨按照1：60料液比加入蒸馏水后加入1.2％的复合中性蛋白酶在55℃酶解4.5h；将酶解液放于90℃水浴10min后5000r/min离心10min,取上清液抽滤过滤纸和0.45μm滤膜除去大分子杂质,使用10kDa超滤膜重复循环分离6次得到硫酸软骨素和胶原蛋白肽,将所得溶液旋转蒸发除去多余的水分,再冻干得到产品。本发明是绿色无污染工艺,完全没有引入酸碱盐和使用乙醇,降低了生产成本。(The invention belongs to the technical field of chondroitin sulfate extraction, and discloses a method for extracting chondroitin sulfate, chondroitin sulfate and application thereof, wherein fishhead cartilage powder is crushed into particles and dried to remove redundant moisture to be used as a steam explosion raw material, and part of cartilage is put in a freeze dryer to be freeze-dried to be used as a raw material for optimizing an enzymolysis process, so that an optimal enzymolysis process is obtained; the dry-based steam-exploded fishbone is prepared according to the following steps of 1: adding distilled water according to the ratio of 60 materials to liquid, adding 1.2% of compound neutral protease, and performing enzymolysis at 55 deg.C for 4.5 h; placing the enzymatic hydrolysate in a 90 ℃ water bath for 10min, centrifuging for 10min at a speed of 5000r/min, taking supernate, filtering filter paper and a 0.45 mu m filter membrane to remove macromolecular impurities, repeatedly and circularly separating for 6 times by using a 10kDa ultrafiltration membrane to obtain chondroitin sulfate and collagen peptide, performing rotary evaporation on the obtained solution to remove excessive moisture, and freeze-drying to obtain the product. The invention is a green pollution-free process, and acid, alkali and salt are not introduced and ethanol is not used at all, so that the production cost is reduced.)

1. A method for extracting chondroitin sulfate, comprising:

firstly, crushing fish head cartilage powder into granules, airing the granules to remove redundant water to serve as a steam explosion raw material, putting part of cartilage into a freeze dryer, and freeze-drying the cartilage to serve as a raw material for optimizing an enzymolysis process to obtain an optimal enzymolysis process;

secondly, adding compound neutral protease into the dry-base steam-exploded fishbone according to the material-liquid ratio for enzymolysis;

and thirdly, centrifuging the enzymatic hydrolysate after water bath, taking supernate, filtering filter paper and a filter membrane to remove macromolecular impurities, repeatedly and circularly separating by using the ultrafiltration membrane to obtain chondroitin sulfate and collagen peptide, rotatably evaporating the obtained solution to remove redundant moisture, and freeze-drying to obtain the product.

2. The method for extracting chondroitin sulfate as claimed in claim 1, wherein the first step is to take sturgeon processing offal-fish head, put the sturgeon processing offal-fish head into water, heat the sturgeon processing offal-fish head for 10min with boiling water, wash off the excess part of the fish head with water to obtain skull, crush the cartilage of the fish head into particles, air-dry the crushed cartilage to remove excess water as a steam explosion raw material, and freeze-dry part of the cartilage in a freeze dryer as a raw material for optimizing enzymolysis parameters.

3. The method for extracting chondroitin sulfate as claimed in claim 1, wherein the method for extracting chondroitin sulfate is steam-exploded: treating at 1.3MPa for 180 s;

the method for extracting the chondroitin sulfate is used for enzymolysis, then centrifugal filtration paper is carried out for 10min at the speed of 5000r/min, and then the chondroitin sulfate passes through a 0.45 mu m filter membrane and is separated by a 10kDa ultrafiltration membrane.

4. The method for extracting chondroitin sulfate as claimed in claim 1, wherein the second dry-based steam-exploded fish bone is prepared by mixing 1: adding 1.2% of compound neutral protease at a ratio of 60 materials to liquid, and performing enzymolysis at 55 deg.C for 4.5 h;

and thirdly, placing the enzymatic hydrolysate in a 90 ℃ water bath for 10min, centrifuging for 10min at a speed of 5000r/min, taking supernate, filtering filter paper and a 0.45-micrometer filter membrane to remove macromolecular impurities, repeatedly and circularly separating for 6 times by using a 10kDa ultrafiltration membrane to obtain chondroitin sulfate and collagen peptide, performing rotary evaporation on the obtained solution to remove redundant moisture, and freeze-drying to obtain the product.

5. Chondroitin sulphate extracted by the method for extracting chondroitin sulphate according to any one of claims 1 to 4.

6. Use of chondroitin sulfate as claimed in claim 5 in a medicament for treating joint diseases.

7. Use of chondroitin sulfate as claimed in claim 5 in anticoagulant and antithrombotic pharmaceutical products.

8. Use of chondroitin sulfate as claimed in claim 5 for the treatment of coronary heart disease.

9. Use of chondroitin sulfate as claimed in claim 5 as an additive in food.

10. Use of chondroitin sulfate as claimed in claim 5 in health products for enhancing physical fitness of human body and resisting bacteria, beautifying and aging.

Technical Field

The invention belongs to the technical field of extraction of chondroitin sulfate, and particularly relates to a method for extracting chondroitin sulfate, chondroitin sulfate and application thereof.

Background

The current methods for producing chondroitin sulfate mainly comprise three methods: a salt hydrolysis method, an alkaline hydrolysis method and an enzymolysis method are widely used in China, wherein dilute alkali or concentrated alkali and enzymolysis are combined to extract chondroitin sulfate, and the traditional process comprises the following steps: the production process of the chondroitin sulfate by adopting the alkaline enzymolysis has the defects of complicated steps, long period, generally 3-5 days, unstable product quality and yield, seriously damaged chondroitin sulfate in the processes of high-temperature stewing, alkaline extraction, enzymolysis and decoloration, low product yield, deepened solution color, high ethanol consumption, high environment damage and production cost, salt removal for producing a large amount of salt and prolonging the production time to cause environmental pollution. Therefore, the production process needs to be improved, the environmental pollution is avoided, the production cost is reduced, the resource waste is reduced, the production efficiency is improved, and the green, environment-friendly, economical and convenient production is realized.

Based on the above analysis of the prior art, there are problems and disadvantages:

(1) the traditional method for extracting chondroitin sulfate has the problem of environmental pollution caused by adding an acid-base reagent.

(2) The traditional method for extracting chondroitin sulfate has the defects that a large amount of high-purity ethanol is used in an alcohol precipitation process in a production process, so that the production cost and resource waste are increased.

The difficulty in solving the above problems and defects is: the purpose of enzymolysis is to change protein into polypeptide to expose chondroitin sulfate, but because chondroitin sulfate is glycosaminoglycan, polysaccharide and protein are connected with protein through O-glycosidic bond, and protease is difficult to break the glycosidic bond, adding dilute alkali is favorable for breaking the glycosidic bond, and alkali liquor is not introduced to greatly influence the purity of the chondroitin sulfate; the principle that the chondroitin sulfate is insoluble in ethanol is utilized to purify the chondroitin sulfate, so that the introduction of small molecular polypeptides is avoided, and the purity of the chondroitin sulfate is improved.

The significance of solving the problems and the defects is as follows: acid-base salt is avoided, and environment-friendly production is facilitated; the production cost can be reduced without adopting an alcohol precipitation process, the production time is shortened, and the production efficiency is improved.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for extracting chondroitin sulfate, the chondroitin sulfate and application thereof.

The invention is realized by a method for extracting chondroitin sulfate, which comprises the following steps:

firstly, crushing fish head cartilage powder into granules, airing the granules to remove redundant water to be used as a steam explosion raw material, and freeze-drying part of cartilage in a freeze dryer to be used as an enzymolysis raw material;

secondly, drying the basic fishbone according to the weight ratio of 1: adding distilled water to 60 feed liquid ratio, adding 1.2% composite neutral protease, and performing enzymolysis at 55 deg.C for 4.5 h. (ii) a

Thirdly, placing the enzymatic hydrolysate in a 90 ℃ water bath for 10min, centrifuging for 10min at a speed of 5000r/min, taking supernate, filtering filter paper and a 0.45-micron filter membrane to remove macromolecular impurities, repeatedly and circularly separating for 6 times by using a 10kDa ultrafiltration membrane to obtain chondroitin sulfate and collagen peptide, performing rotary evaporation on the obtained solution to remove redundant moisture, and freeze-drying to obtain the product.

Further, the first step is that the fish head which is the processing waste of the sturgeon is taken and put into water, the water is heated for 10min, the redundant part of the fish head is washed away by the water to obtain the skull, the cartilage powder of the fish head is crushed into particles and dried to remove the redundant water to be used as the steam explosion raw material, and part of the cartilage is put into a freeze dryer to be freeze-dried to be used as the raw material for optimizing the enzymolysis parameters.

Further, the enzymolysis process for extracting chondroitin sulfate comprises the following steps: the material-liquid ratio: 1: 60, adding a solvent to the mixture; enzyme addition amount: 1.2% (dry basis fishbone); enzymolysis temperature: 55 ℃; and (3) enzymolysis time: 4.5 h. The molecular weight of the polypeptide can be reduced as far as possible by optimizing the enzymolysis conditions to reach the maximum hydrolysis degree, so that the separation of chondroitin sulfate and collagen peptide is facilitated on one hand, and the yield of small-molecule collagen peptide is increased to improve the quality of the polypeptide on the other hand.

Further, the method for extracting chondroitin sulfate comprises the following steps of: treating at 1.3MPa for 180 s. The process of breaking the O-glycosidic bond by a physical method instead of a chemical method can be realized through the treatment of a steam explosion technology, so that the aim of not adding acid-base salt is fulfilled, and the environment-friendly production is favorably protected.

Further, the method for extracting chondroitin sulfate is used for separating the chondroitin sulfate through a 10kDa ultrafiltration membrane after filtering paper is centrifuged for 10min at 5000r/min after enzymolysis and then a 0.45 mu m filter membrane is passed. Insoluble substances can be separated through a series of membrane separation, a certain decolorizing purpose is achieved, and the joint production of collagen peptide and chondroitin sulfate is realized, so that the alcohol precipitation step is replaced, the production cost can be reduced, the production time is shortened, and the production efficiency is improved.

Another object of the present invention is to provide chondroitin sulfate extracted by the method for extracting chondroitin sulfate.

Another object of the present invention is to provide a use of the chondroitin sulfate in a medicament for treating joint diseases.

Another object of the present invention is to provide a use of said chondroitin sulfate in anticoagulant and antithrombotic medicaments.

The invention also aims to provide application of the chondroitin sulfate in treating coronary heart disease.

The invention also aims to provide the application of the chondroitin sulfate in food additives.

The invention also aims to provide application of the chondroitin sulfate in health-care products for enhancing human body constitution, resisting bacteria, beautifying and resisting aging.

In summary, the advantages and positive effects of the invention are: the invention is a green pollution-free process, and then uses sturgeon production waste-skull as production raw material to reduce production cost; acid-base salt is not introduced and ethanol is not used, and a dialysis method is not needed for removing impurities, so that the environment is not damaged, and the production cost is reduced; the purification is carried out by adopting an efficient and simple ultrafiltration method, and the joint production mode of the chondroitin sulfate and the collagen peptide is realized.

Drawings

FIG. 1 is a flow chart of a method for extracting chondroitin sulfate according to an embodiment of the present invention.

Fig. 2 is a flow chart of the method for extracting chondroitin sulfate according to the embodiment of the present invention.

FIG. 3 is a schematic diagram of the scanning electron microscope and bone liquefaction rate of fish bone after steam explosion treatment under different conditions for extracting chondroitin sulfate according to an embodiment of the present invention.

FIG. 4 is an infrared spectrum of chondroitin sulfate extraction provided in the embodiments of the present invention.

FIG. 5 is a molecular weight and infrared spectrum of an isolated polypeptide from which chondroitin sulfate is extracted, as provided in an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a method for extracting chondroitin sulfate, the chondroitin sulfate and application thereof, and the invention is described in detail below with reference to the accompanying drawings.

As shown in fig. 1, the method for extracting chondroitin sulfate provided by the embodiment of the present invention includes the following steps:

s101: crushing the fish head cartilage powder into particles, airing the crushed fish head cartilage powder to remove redundant water to be used as a steam explosion raw material, and freeze-drying part of cartilage in a freeze dryer to be used as an enzymolysis raw material;

s102: the dry-basis fishbone is prepared according to the following steps of 1: adding 1.2% of compound neutral protease at a ratio of 60 materials to liquid, and performing enzymolysis at 55 deg.C for 4.5 h.

S103: placing the enzymatic hydrolysate in a 90 ℃ water bath for 10min, centrifuging for 10min at a speed of 5000r/min, taking supernate, filtering filter paper and a 0.45 mu m filter membrane to remove macromolecular impurities, repeatedly and circularly separating for 6 times by using a 10kDa ultrafiltration membrane to obtain chondroitin sulfate and collagen peptide, performing rotary evaporation on the obtained solution to remove redundant moisture, and freeze-drying to obtain the product.

The technical solution of the present invention is further described below with reference to the accompanying drawings.

The method for extracting chondroitin sulfate provided by the embodiment of the invention specifically comprises the following steps:

firstly, raw material pretreatment: taking processing leftovers of sturgeons, namely fish heads, putting the fish heads into water with a certain volume, heating the fish heads in boiling water for 10min, washing the redundant parts of the fish heads with the water to obtain skull, crushing the cartilage of the fish heads into granules, airing the crushed cartilage of the fish heads to remove the redundant parts of the cartilage to be used as a steam explosion raw material, and putting part of the cartilage into a freeze dryer to be freeze-dried to be used as a raw material for optimizing enzymolysis parameters.

Secondly, optimizing enzymolysis parameters: the enzymolysis parameters are optimized by adopting a single-factor method, the optimization conditions are respectively the feed-liquid ratio, the enzyme addition amount, the enzymolysis time and the enzymolysis temperature, and the scheme is as follows: respectively setting the ratio of material to liquid, the addition amount of enzyme, the enzymolysis time and the enzymolysis temperature as 1: 20. optimizing by taking 0.8%, 3h and 50 ℃ as the basis of enzymolysis conditions, firstly changing the ratio of material to liquid to be 1: 20. 1: 40. 1: 60. 1: 80. 1: 100, determining the optimal feed-liquid ratio by measuring the proteolysis degree by using an OPA method; the optimal enzyme addition amount is determined by measuring the degree of proteolysis by using an OPA method by changing the enzyme addition amount to 0.4%, 0.8%, 1.2% and 1.6% respectively; changing the enzymolysis time to 3h, 4.5h and 6h respectively, and determining the optimal enzymolysis time by measuring the proteolysis degree by using an OPA method; the optimum enzymolysis temperature was determined by measuring the degree of proteolysis with OPA method with the enzymolysis temperature changed to 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C, respectively. The optimal enzymolysis conditions obtained by determination are that the dry-base fishbone is prepared according to the following steps of 1: adding 1.2% of compound neutral protease at a ratio of 60 materials to liquid, and performing enzymolysis at 55 deg.C for 4.5 h.

Thirdly, optimizing steam explosion parameters: optimizing steam explosion parameters by adopting a single-factor method, putting fish bone particles into a steam explosion device QBS-80SE produced by genuine bioenergy companies in the city of the wall of the crane to be respectively treated for 120s, 150s, 180s, 210s and 240s under the conditions of 1, 1.3, 1.45 and 1.6MPa, collecting cartilage solution subjected to steam explosion treatment, carrying out freeze-drying treatment on the cartilage solution, carrying out enzymolysis under the most enzymolysis parameters, putting the enzymolysis solution in a 90 ℃ water bath for 10min, centrifuging for 10min at 5000r/min, taking supernate, filtering and filtering filter paper and a 0.45 mu m filter membrane to remove macromolecular impurities, repeatedly and circularly separating for 6 times by using a 10kDa ultrafiltration membrane to obtain chondroitin sulfate (greater than 10kDa) and collagen peptide (less than 10kDa), rotatably evaporating the obtained solution to remove excessive moisture, freeze-drying the obtained product to obtain the optimal content of the chondroitin sulfate as a steam explosion parameter for judging standards.

In the preferred embodiment of the invention, the hybrid sturgeon "sturgeon 1" (variety registration number: GS-02-002-.

In a preferred embodiment of the present invention, the Steam Explosion technique (ICSE) (simply Steam Explosion) is a physicochemical treatment method. The steam explosion technology can improve the utilization efficiency of raw materials and has the advantages of short action time, low energy consumption, high efficiency, no pollution, suitability for industrialization and the like. At present, the method is mainly applied to extracting bioactive substances from the byproducts of agricultural product processing, improves the added value of the byproducts and is rarely applied to the treatment of the byproducts of animal processing.

In a preferred embodiment of the invention, the cartilage: the waste, namely the skull, is produced by adopting sturgeon I. Steam explosion: treating at 1.3MPa for 180 s. Enzymolysis: 1.2% of enzyme addition, 55% of enzymolysis temperature and 1: 60. the enzymolysis time is 4.5 h. And (3) ultrafiltration: after enzymolysis, the mixture is centrifuged at 5000r/min for 10min, filtered by a filter paper with the diameter of 0.45 mu m and separated by a 10kDa ultrafiltration membrane.

The chondroitin sulfate is used as a medicine for treating joint diseases in a medical main application way, is matched with glucosamine, has the effects of relieving pain and promoting cartilage regeneration, and can fundamentally improve the joint problems. Chondroitin sulfate can reduce pain of osteoarthritis patients, improve joint function, reduce joint swelling and effusion, and prevent gap stenosis at knee joints and hand joints. Provides a cushion effect, relieves the impact and friction during movement, can absorb water into proteoglycan molecules, thickens cartilage, and increases the amount of synovial fluid in joints. One of the important functions of chondroitin is to act as a transport conduit, transporting important oxygen supplies and nutrients to the joint, helping to remove waste from the joint, and removing carbon dioxide and waste. Since articular cartilage is not supplied with blood, all oxygenation, nourishment and lubrication comes from the synovial fluid.

The chondroitin sulfate has a protective effect on corneal collagen fibers, can promote the growth of fibers in a matrix, enhance the permeability, improve the blood circulation, accelerate the metabolism, promote the absorption of penetrating fluid and the elimination of inflammation; the polyanion has strong water retention, can improve the water metabolism of corneal tissues of eyes, has strong affinity to the cornea, can form a layer of breathable water retention film on the surface of the cornea and improves the dry symptom of the eyes. By promoting the generation of stroma, a framework is provided for the migration of cells, and the migration of corneal epithelial cells is facilitated, so that the healing of corneal wound, the absorption of exudate and the elimination of inflammation are promoted. Can be used for treating nervous headache, trigeminal neuralgia, coronary heart disease, angina pectoris, myocardial anoxia, cardiovascular and cerebrovascular diseases, arthralgia, atherosclerosis, hepatitis, etc., wherein CS has anticoagulant and antithrombotic effects, and can also be used for adjuvant treatment of auditory disorder and liver function injury caused by streptomycin and adjuvant treatment of hyperlipemia. Can be used as additive in health product and food, and has effects in improving body constitution, resisting bacteria, caring skin, and resisting aging. Improve hearing and dry skin. Can inhibit the absorption of lipid and glucose in small intestine in vivo to achieve weight reducing effect. CS is widely found in human and animal cartilage tissue. The medicinal preparation mainly contains two isomers of chondroitin sulfate A and chondroitin sulfate C, and the contents of chondroitin sulfate in the cartilages of different varieties and ages of animals are different. The pharmacological action is shown as follows: CS can remove lipid and lipoprotein in blood, remove cholesterol in peripheral blood vessel of heart, prevent and treat atherosclerosis, and increase intracellular conversion rate of lipid and fatty acid. CS can effectively prevent and treat coronary heart disease. The compound has the effects of resisting atherosclerosis and atherogenic plaque formation on an experimental arteriosclerosis model; increase the coronary artery branch or collateral circulation of atherosclerosis, and accelerate the healing, regeneration and repair of myocardial necrosis or degeneration caused by experimental coronary arteriosclerosis or embolism.

The chondroitin sulfate of the invention can increase the biosynthesis of messenger ribonucleic acid (mRNA) and deoxyribonucleic acid (DNA) of cells and has the function of promoting the metabolism of the cells. Has low anticoagulant activity. Chondroitin sulfate has a mild anticoagulant effect, and each l of chondroitin sulfate A is equivalent to the anticoagulant activity of 0.45U of heparin. This anticoagulant activity does not depend on antithrombin III, and it can exert anticoagulant activity through the fibrinogen system. Chondroitin sulfate also has antiinflammatory, wound healing promoting and antitumor effects. Chondroitin sulfate is used for treating neuralgia, nervous migraine, arthralgia, arthritis, scapular arthralgia, abdominal postoperative pain, etc. Preventing and treating auditory disorder caused by streptomycin, and auditory dysesthesia, tinnitus, etc. caused by various noises, with remarkable effect. It can be used for the adjuvant treatment of chronic nephritis, chronic hepatitis, keratitis and corneal ulcer. In addition, chondroitin sulfate is also used in cosmetics, wound healing agents, and the like. Chondroitin sulfate is an acidic mucopolysaccharide, is one of important components in eye tissues, has the effects of promoting corneal water metabolism and improving cornea, and is suitable for asthenopia and xerophthalmia.

TABLE 1 chondroitin sulfate extraction product parameters provided in the examples of the invention

Fig. 3 is a scanning electron microscope and a schematic diagram of bone liquefaction rate of fish bones subjected to steam explosion treatment under different conditions for extracting chondroitin sulfate provided in the embodiment of the present invention (it is proved that steam explosion improves the liquefaction rate of fish bones by crushing the fish bones, which is beneficial to improving enzymolysis efficiency and promoting separation of chondroitin sulfate and protein).

FIG. 4 is an infrared spectrum of chondroitin sulfate extract provided in the present invention (comparing with infrared spectrum of a standard sample, it is proved that the structure of the obtained chondroitin sulfate product is not changed much).

Table 2 schematic amino acid composition of isolated chondroitin sulfate-extracted polypeptide provided in the examples of the present invention (proving that the obtained polypeptide is collagen peptide).

TABLE 2

Amino acid composition of steam-exploded fishbone separated polypeptide

FIG. 5 is a molecular weight and infrared spectrum of the isolated polypeptide from which chondroitin sulfate was extracted according to the present invention (it was confirmed that the molecular weight of the polypeptide was mainly below 5kDa, which is a high quality collagen peptide).

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.