Preparation method of high-purity low-degradation chondroitin sulfate, chondroitin sulfate and application thereof

Abstract

This application relates to the field of chondroitin sulfate processing, specifically disclosing a method for preparing high-purity, low-degradation chondroitin sulfate, chondroitin sulfate and its applications. The method for preparing high-purity, low-degradation chondroitin sulfate includes the following steps: crushing animal cartilage and subjecting it to relaxation treatment to obtain pretreated cartilage raw material; placing the pretreated cartilage raw material in a water-alcohol two-phase system for stirring and dissolution treatment to obtain a primary extract; subjecting the primary extract to isoelectric point flocculation coupled with micro-filtration adsorption treatment, followed by centrifugation filtration to obtain a transparent and clear polysaccharide solution; subjecting the transparent and clear polysaccharide solution to alcohol precipitation treatment, followed by salt gradient fractionation treatment to obtain a refined polysaccharide solution; subjecting the refined polysaccharide solution to sodium salt replacement and shaping treatment to obtain a semi-finished product; subjecting the semi-finished product to ultrafiltration, aseptic filtration, and freeze-drying to obtain the finished product; applicable to food, health products, cosmetics, or biopharmaceutical fields; and possessing advantages such as low degradation, high purity, narrow distribution, and high stability.

Description

Technical Field

[0001] This application relates to the field of chondroitin sulfate processing, and more specifically, to a method for preparing high-purity, low-degradation chondroitin sulfate, chondroitin sulfate, and its applications. Background Technology

[0002] Chondroitin sulfate is a sodium salt of a natural high-molecular-weight glycosaminoglycan polysaccharide. It has excellent biocompatibility, moisturizing properties, joint care and anti-inflammatory activity, and has a wide range of applications.

[0003] Existing industrial preparation processes are mainly divided into three categories: high-temperature strong alkali hydrolysis, single enzymatic hydrolysis, and ordinary alcohol precipitation purification. However, high-temperature and strong acid-base systems easily break the glycosidic bonds of polysaccharides, resulting in severe product degradation, the generation of a large number of low-molecular-weight fragments, and a low proportion of effective high-molecular-weight polysaccharides. Conventional extraction systems have poor selectivity, with proteins, oils, and pigments dissolving simultaneously, leading to high pressure for subsequent impurity removal and difficulty in achieving high-end purity standards. Sodium salt conversion processes often involve direct strong alkali neutralization, resulting in drastic pH changes, secondary degradation, poor product stability, and a wide molecular weight distribution. Furthermore, the lack of fractional purification steps results in a mixture of high and low molecular weight products and significant batch-to-batch variations.

[0004] Therefore, how to prepare a new chondroitin sulfate with the advantages of low degradation, high purity, narrow distribution, and high stability is an urgent problem to be solved. Summary of the Invention

[0005] In order to prepare a new chondroitin sulfate with the advantages of low degradation, high purity, narrow distribution and high stability, this application provides a method for preparing high-purity low-degradation chondroitin sulfate, chondroitin sulfate and its applications.

[0006] In a first aspect, this application provides a high-purity, low-degradation chondroitin sulfate, employing the following technical solution: A method for preparing high-purity, low-degradation chondroitin sulfate includes the following steps: S1. The animal cartilage is crushed and then subjected to a relaxation process to obtain pretreated cartilage raw material; S2. Place the pretreated cartilage raw material in a water-alcohol two-phase system and stir to dissolve it, to obtain the initial extract; S3. The initial extract was subjected to isoelectric point flocculation coupled micro-filtration adsorption treatment, followed by centrifugation and filtration to obtain a transparent and clear polysaccharide solution. S4. The transparent and clear polysaccharide solution is first subjected to alcohol precipitation, and then subjected to salt gradient fractionation to obtain a refined polysaccharide solution. S5. The refined polysaccharide solution is subjected to sodium salt replacement and shaping treatment to obtain a semi-finished product; S6. The semi-finished product is subjected to ultrafiltration and aseptic filtration, followed by freeze drying to obtain the finished product.

[0007] By adopting the above technical solution, the pulverized animal cartilage undergoes a relaxation treatment, which gently breaks the non-covalent bonds of proteoglycans, making it less likely to damage the glycosidic bonds of polysaccharides. During the dissolution process, the chondroitin sulfate polysaccharide is dissolved using a water-alcohol biphasic process, inhibiting the dissolution of impurities such as proteins and lipids. Subsequent flocculation, filtration, and adsorption treatments specifically remove residual proteins, peptides, and pigment precursors. After centrifugation, a clear chondroitin sulfate polysaccharide solution is obtained. Then, alcohol precipitation removes coarse components, followed by a dual-effect purification process using salt gradient fractionation to remove low-molecular-weight degradation fragments and residual water-soluble impurities, enriching the high-molecular-weight intact polysaccharide components to obtain a narrow-distribution purified polysaccharide solution. Next, sodium salt replacement shaping treatment gently completes the steady-state transformation of chondroitin sulfate, controlling polysaccharide degradation and ensuring the yield of chondroitin sulfate. Finally, ultrafiltration further improves the purity of chondroitin sulfate, giving it the advantages of low degradation, high purity, narrow distribution, and high stability.

[0008] Preferably, the specific steps of the relaxation treatment are as follows: using a sodium carbonate solution with a mass fraction of 0.3-0.8%, and treating at a constant temperature of 18-25℃ for 2-4 hours.

[0009] By adopting the above technical solution, using a 0.3-0.8% low-concentration sodium carbonate solution and treating at a low temperature of 18-25℃ for 2-4 hours, which is far lower than the traditional process conditions of long-term extraction with concentrated alkali, this method can gently break the O-glycosidic bonds between chondroitin sulfate and the core protein without damaging the β-1,3 and β-1,4 glycosidic bonds of the polysaccharide backbone. This avoids polysaccharide chain breakage and degradation from the source, ensuring the advantages of low degradation and high stability of chondroitin sulfate. Furthermore, the polysaccharide molecules are not easily excessively broken, and the molecular weight of chondroitin sulfate is also uniformly distributed. At the same time, the gentle treatment with low-concentration sodium carbonate can preliminarily saponify and dissolve impurities such as fat and connective tissue attached to the cartilage surface, preventing these impurities from entering the subsequent extraction process. This reduces impurity entrainment from the source and can improve the purity of chondroitin sulfate preparation.

[0010] Preferably, the volume fraction of ethanol in the water-alcohol biphase system is 12-22%, the extraction temperature is 22-30℃, and the stirring dissolution treatment time is 3-5h.

[0011] By employing the above technical solutions, limiting the volume fraction of ethanol and the temperature, the polysaccharide backbone in chondroitin sulfate is prevented from being destroyed, the dissolution time is limited, and the residence time of the active ingredient in the liquid phase is shortened, resulting in the advantage of low degradation. Furthermore, the water-ethanol biphase system can ensure the full dissolution of the target polysaccharide, and the solubility of impurities such as proteins is further reduced at low temperatures, thus reducing the solubility of impurities such as proteins, fats, and pigments and ensuring the purity of chondroitin sulfate. At the same time, the low-temperature treatment of the water-ethanol biphase system is less likely to cause random shearing of the long molecular chain of chondroitin sulfate, ensuring the integrity of the molecular chain, reducing the occurrence of a large number of small molecular fragments, ensuring molecular weight uniformity, and having the advantage of narrow distribution, as well as high structural stability.

[0012] Preferably, the isoelectric point flocculation pH is 5.6-6.3, and the micro-filtration adsorption treatment is carried out by using Moringa seed protein composite material as flocculant and diatomaceous earth as filter aid, flocculating and standing for 30-50 minutes, and centrifuging at a speed of 8500-10000 r / min.

[0013] By adopting the above technical solution, under the conditions of pH 5.6-6.3, the initial extract of chondroitin sulfate contains negatively charged impurities such as proteins and peptides, while moringa seed protein, rich in cationic amino acid residues, carries a positive charge under these weakly acidic conditions. Moringa seed protein attracts these impurities through electrostatic adsorption, causing them to aggregate and flocculate. Furthermore, the long-chain structure of moringa seed protein allows for the simultaneous adsorption of multiple impurity particles, forming a network of impurity-protein-impurity flocs. This increases the floc size and sedimentation rate. Combined with the binding and attraction of diatomaceous earth on the flocs, small flocs are prevented from passing through the filter layer. Centrifugation further reduces the impurity content and improves the purity of chondroitin sulfate. Simultaneously, the moringa seed protein peptide chain contains hydrophobic amino acid fragments, which interact hydrophobically with fat droplets and hydrophobic proteins in the impurities, efficiently removing fat-soluble impurities and resulting in high-purity chondroitin sulfate.

[0014] Preferably, the moringa seed protein composite is composed of quaternary ammonium salt modified moringa seed protein and tannin modified bacterial cellulose in a mass ratio of 1:0.5-1.

[0015] By adopting the above technical solution, moringa seed protein achieves surface charge modification through quaternary ammonium salt, enhancing its electrostatic adsorption capacity with anionic substances. Impurities such as miscellaneous proteins, peptides, and pigments are negatively charged, while the quaternary ammonium salt on the surface of moringa seed protein is positively charged. The quaternary ammonium salt can rapidly and stably adsorb impurities, solving the problem of insufficient amino protonation in natural moringa protein. This improves the adsorption stability and adsorption capacity of quaternary ammonium salt-modified moringa seed protein for negatively charged impurities, thereby reducing the impurity content.

[0016] Moringa seed protein has a relatively short molecular chain. By utilizing the short, flexible polypeptide chains of moringa seed protein modified with quaternary ammonium salts, multiple destabilized micro-impurity particles are adsorbed, forming micro-flocs. Then, combined with the ultra-long nanofiber structure of bacterial cellulose modified with tannins, a long-chain bridging effect is provided, forming multi-level cross-linked flocs. This forms a cellulose network that intertwines and connects to form large-volume flocs, binding the micro-flocs. Bacterial cellulose acts as a connecting bridge, linking the micro-flocs together. Centrifugation further reduces the impurity content. At the same time, the large amount of tannins loaded on the surface of bacterial cellulose can further adsorb impurity molecules. The connection between tannins and moringa seed protein and impurities stabilizes the floc structure, making it less prone to secondary dispersion of flocs, further reducing the impurity content and improving the purity of chondroitin sulfate.

[0017] Tannins are polyhydroxy polyphenols, with each tannin molecule containing multiple hydroxyl groups. The numerous hydroxyl groups on the surface of bacterial cellulose can form multiple hydrogen bonds with the phenolic hydroxyl groups of tannins. Bacterial cellulose can not only load a large amount of tannin substances, but also improve the structural density and cross-linking density of bacterial cellulose. The loose macropores in bacterial cellulose are transformed into mesopores and micropores, which facilitate the binding of small molecule impurities such as proteins and peptides, and improve the binding effect on small molecule peptides. Tannins do not destroy the continuous nanofiber skeleton of bacterial cellulose, but only serve as a filler cross-linking component. Tannins attract small molecule peptides, and together with the complete three-dimensional network structure, they bind small molecule peptides, further improving the purity of chondroitin sulfate.

[0018] Preferably, the diatomaceous earth is loaded diatomaceous earth, which is obtained by activating diatomaceous earth, grafting it with silane coupling agent KH-560, and then loading it with β-cyclodextrin. The mass ratio of diatomaceous earth, silane coupling agent KH-560 and β-cyclodextrin is 100:1.5-2.5:25-35.

[0019] By adopting the above technical solution, diatomaceous earth itself possesses a porous physical structure with a high specific surface area. It can initially adsorb some large-particle pigment impurities in the system through van der Waals forces and physical sieving, providing sufficient sites for subsequent functional layer loading. Simultaneously, it acts as a flocculation framework to improve the overall settling velocity and prevent the suspension and dispersion of fine pigment particles. KH-570, as a silane coupling agent, hydrolyzes the trimethoxysilyl group at one end to generate silanol groups, which covalently bond with the hydroxyl groups on the surface of diatomaceous earth. The methacryloyloxy double bond at the other end can react with β-cyclodextrin through a cross-linking reaction. Active hydroxyl groups form stable linkages, building chemical bond bridges between inorganic diatomaceous earth and organic β-cyclodextrin, preventing the functional layer from detaching in chondroitin sulfate solution and ensuring the structural stability of the modified material. β-cyclodextrin is a cyclic molecule composed of 7 glucose units with a hydrophobic cavity of about 6 Å, which can encapsulate lipophilic and hydrophobic pigment molecules with matching molecular weights in chondroitin sulfate solution into the cavity, forming a stable host-guest inclusion complex. This anchors the originally dissolved and dispersed pigment molecules to the material surface, realizing the transfer from the liquid phase to the solid phase and binding pigment impurities.

[0020] The modified diatomaceous earth material retains a large number of active hydroxyl groups on its surface. Some unreacted amino sites can coordinate with polar groups such as carboxyl and phenolic hydroxyl groups in pigment molecules. At the same time, through surface charge regulation, it can neutralize some negatively charged anionic pigment colloids, breaking the stable dispersion state of pigment colloids in solution, promoting the destabilization and aggregation of tiny pigment particles, and forming large flocs that can settle. Furthermore, diatomaceous earth particles loaded with β-cyclodextrin can act as flocculation nuclei in solution. Through their own long-chain organic modification layer, they can connect and bridge multiple small pigment flocs that have been initially adsorbed, ultimately forming denser flocs with larger volume and better settling performance. This allows for rapid separation from the chondroitin sulfate system, avoiding pigment re-dissolution, improving decolorization efficiency, reducing impurities, and ensuring the purity of chondroitin sulfate.

[0021] Preferably, the alcohol precipitation treatment uses 35% to 45% ethanol by volume for initial precipitation; the salt gradient fractionation treatment uses 0.4 to 1.6 mol / L sodium chloride for fractional treatment.

[0022] By adopting the above technical solution, the polarity of the solution can be changed at this ethanol concentration, allowing large molecular weight polysaccharides such as chondroitin sulfate to preferentially precipitate from the system, achieving preliminary enrichment from the dilute extract and significantly reducing the volume of the liquid in subsequent processing; this low alcohol concentration will not cause all impurities to precipitate, and some large molecular weight proteins and large colloids with lower solubility can be separated in advance, avoiding the co-precipitation of a large number of impurities when high concentration alcohol precipitation occurs.

[0023] During the gradient salt washing process, impurities such as loosely bound proteins, small molecule pigments, and incompletely removed inorganic salts can be eluted stepwise, preventing impurities from remaining in the final product and improving the purity of the finished product.

[0024] Preferably, during the sodium salt replacement and shaping process: the system pH is controlled at 6.8-7.4, and a saturated sodium salt solution is added dropwise; ultrafiltration uses an 8-10 kDa ultrafiltration membrane, the freeze-drying temperature is -18 to -10℃, and the vacuum degree is 0.08-0.1 MPa.

[0025] By adopting the above technical solution, the free acidic groups on the chondroitin sulfate molecule are replaced by high-concentration sodium ions, which transform it into a more stable sodium chondroitin sulfate salt, greatly improving the water solubility of the product. At the same time, the charge distribution of the molecule is fixed, avoiding unstable phenomena such as molecular aggregation and precipitation in subsequent processes.

[0026] Ultrafiltration can remove residual small molecule proteins, colloidal fragments, small molecule pigments and excess salt, further improving product purity.

[0027] Secondly, this application provides a high-purity, low-degradation chondroitin sulfate, employing the following technical solution: A high-purity, low-degradation chondroitin sulfate, prepared using a method for preparing high-purity, low-degradation chondroitin sulfate.

[0028] By adopting the above technical solution, chondroitin sulfate with low degradation, high purity, narrow distribution, and high stability was obtained.

[0029] Thirdly, this application provides an application of high-purity, low-degradation chondroitin sulfate, employing the following technical solution: An application of high-purity, low-degradation chondroitin sulfate, and its application in the food, health product, cosmetic, or biopharmaceutical fields.

[0030] By adopting the above-mentioned technical solutions, they can play a role in food, health products, cosmetics, or biomedical insurance.

[0031] In summary, this application has the following beneficial effects: 1. After being pulverized, animal cartilage undergoes a relaxation process to gently break the non-covalent bonds of proteoglycans, minimizing damage to the glycosidic bonds of the polysaccharides. During the dissolution process, a water-alcohol biphasic approach is used to dissolve chondroitin sulfate polysaccharides, inhibiting the dissolution of impurities such as proteins and lipids. Subsequent flocculation, filtration, and adsorption treatments specifically remove residual proteins, peptides, and pigment precursors. Centrifugation yields a clear chondroitin sulfate polysaccharide solution. Then, alcohol precipitation removes coarse components, followed by a dual-effect purification process using salt gradient fractionation to remove low-molecular-weight degradation fragments and residual water-soluble impurities, enriching the high-molecular-weight intact polysaccharide components to obtain a narrow-distribution purified polysaccharide solution. Sodium salt replacement shaping treatment gently completes the steady-state transformation of chondroitin sulfate, controlling polysaccharide degradation and ensuring the yield of chondroitin sulfate. Finally, ultrafiltration further improves the purity of chondroitin sulfate, giving it the advantages of low degradation, high purity, narrow distribution, and high stability.

[0032] 2. In the initial extract of chondroitin sulfate, under pH 5.6-6.3 conditions, impurities such as proteins and peptides carry a negative charge, while moringa seed protein, rich in cationic amino acid residues, carries a positive charge under these weakly acidic conditions. It attracts these impurities through electrostatic adsorption, causing them to aggregate and flocculate. Furthermore, the long-chain structure of moringa seed protein allows for the simultaneous adsorption of multiple impurity particles, forming a network of impurity-protein-impurity flocs. This increases the floc size and sedimentation rate. Combined with the binding and attraction of diatomaceous earth on the flocs, small flocs are prevented from penetrating the filter layer. Centrifugation further reduces the impurity content and improves the purity of chondroitin sulfate. Simultaneously, the moringa protein peptide chains contain hydrophobic amino acid fragments, which interact hydrophobically with fat droplets and hydrophobic proteins in the impurities, efficiently removing fat-soluble impurities and resulting in high-purity chondroitin sulfate.

[0033] 3. Moringa seed protein undergoes surface charge modification via quaternary ammonium salts, enhancing its electrostatic adsorption capacity with anionic substances. Impurities such as miscellaneous proteins, peptides, and pigments carry negative charges, while the quaternary ammonium salts on the surface of moringa seed protein carry positive charges. The quaternary ammonium salts rapidly and stably adsorb impurities, solving the problem of insufficient amino protonation in natural moringa protein. This improves the adsorption stability and adsorption capacity of quaternary ammonium salt-modified moringa seed protein for negatively charged impurities, thereby reducing the impurity content. Detailed Implementation

[0034] The present application will be further described in detail below with reference to the embodiments.

[0035] Preparation example of quaternary ammonium salt modified Moringa seed protein The Moringa seed protein powder in the following ingredients was purchased from Fufeng Sinote Biotechnology Co., Ltd., while the other ingredients are food-grade commercially available.

[0036] Preparation Example 1: Quaternary ammonium salt modified Moringa seed protein was prepared using the following method: Moringa seed protein powder was ultrasonically dispersed in deionized water at a mass ratio of 1:30. The mixture was stirred at 45℃ and 150 r / min for 30 min to obtain a solution. The pH was adjusted to 8.6-9.0, and then a 65% (w / w) solution of 3-chloro-2-hydroxypropyltrimethylammonium chloride was added dropwise with water as the solvent. The mixture was stirred continuously for 30 min, and then the temperature was raised to 50℃. The mixture was stirred and reacted for 2 h. After cooling to room temperature, the mixture was vacuum dried at 40℃ to obtain the finished quaternary ammonium salt modified moringa seed protein, which was then passed through a 100-mesh sieve.

[0037] Example of preparation of tannin-modified bacterial cellulose All of the following ingredients are food-grade and commercially available.

[0038] Preparation Example 2: Tannin-modified bacterial cellulose was prepared using the following method: Tannic acid was dissolved in deionized water to obtain a 1% tannic acid solution. Bacterial cellulose powder was placed in the tannic acid solution at a mass ratio of 1:20 and ultrasonically dispersed for 10 min. Then, the mixture was stirred at 200 r / min and 40℃ for 2 h. The bacterial cellulose powder was then separated and washed with deionized water until no characteristic tannic acid absorption peak was detected by UV detection (280 nm). The product was then freeze-dried, crushed, and the finished tannic acid-modified bacterial cellulose was obtained. The finished tannic acid-modified bacterial cellulose was passed through a 100-mesh sieve.

[0039] Example of preparation of diatomaceous earth with loading material All of the following ingredients are food-grade and commercially available.

[0040] Preparation Example 3: The loaded diatomaceous earth was prepared using the following method: 100g of diatomaceous earth was calcined at 550℃ for 1h and then cooled to room temperature. It was then placed in 500mL of 0.2mol / L acetic acid solution and ultrasonically dispersed for 30min. The mass ratio of diatomaceous earth to acetic acid solution was 1:8. After washing with water until neutral, and drying, activated diatomaceous earth was obtained. 2g of silane coupling agent KH-560 was placed in 500mL of ethanol-water solution and stirred. The pH was adjusted to 4.8-5.0 to obtain a silane solution. The volume ratio of ethanol to water was 7:3. Activated diatomaceous earth was placed in the solution and ultrasonically dispersed for 30min. Then, it was refluxed and stirred for 2h at 150r / min and 60℃. After filtration and washing twice with ethanol, grafted diatomaceous earth was obtained. Take 30g of β-cyclodextrin and place it in 600mL of deionized water. Stir and mix evenly at 80℃ to obtain a mixture. Disperse the grafted diatomaceous earth in the mixture and stir for 2h at 200r / min and 50℃. Then let it stand at room temperature for 2h to separate the solid. Vacuum dry at 50℃ and air-jet break to obtain the finished diatomaceous earth.

[0041] Preparation Example 4: The difference between this preparation example and Preparation Example 3 is that: 100g of diatomaceous earth was calcined at 550℃ for 1h and then cooled to room temperature. It was then placed in 500mL of 0.2mol / L acetic acid solution and ultrasonically dispersed for 30min. The mass ratio of diatomaceous earth to acetic acid solution was 1:8. After washing with water until neutral, and drying, activated diatomaceous earth was obtained. 1.5g of silane coupling agent KH-560 was placed in 400mL of ethanol-water solution and stirred. The pH was adjusted to 4.8-5.0 to obtain a silane solution. The volume ratio of ethanol to water was 7:3. Activated diatomaceous earth was placed in the solution and ultrasonically dispersed for 30min. Then, it was refluxed and stirred for 2h at 150r / min and 60℃. After filtration and washing twice with ethanol, grafted diatomaceous earth was obtained. 25β-cyclodextrin was placed in 500 mL of deionized water and stirred at 80 °C until homogeneous to obtain a mixture. Grafted diatomaceous earth was dispersed in the mixture and stirred at 200 r / min and 50 °C for 2 h. The mixture was then allowed to stand at room temperature for 2 h to separate the solid. The solid was then vacuum dried at 50 °C and air-jet crushed to obtain the finished diatomaceous earth.

[0042] Preparation Example 5: The difference between this preparation example and Preparation Example 3 is that: 100g of diatomaceous earth was calcined at 550℃ for 1h and then cooled to room temperature. It was then placed in 500mL of 0.2mol / L acetic acid solution and ultrasonically dispersed for 30min. The mass ratio of diatomaceous earth to acetic acid solution was 1:8. After washing with water until neutral, and drying, activated diatomaceous earth was obtained. 2.5g of silane coupling agent KH-560 was placed in 600mL of ethanol-water solution and stirred. The pH was adjusted to 4.8-5.0 to obtain a silane solution. The volume ratio of ethanol to water was 7:3. Activated diatomaceous earth was placed in the solution and ultrasonically dispersed for 30min. Then, it was refluxed and stirred for 2h at 150r / min and 60℃. After filtration and washing twice with ethanol, grafted diatomaceous earth was obtained. Take 35g of β-cyclodextrin and place it in 650mL of deionized water. Stir and mix evenly at 80℃ to obtain a mixture. Disperse the grafted diatomaceous earth in the mixture and stir for 2h at 200r / min and 50℃. Then let it stand at room temperature for 2h to separate the solid. Vacuum dry at 50℃ and air-jet break to obtain the finished diatomaceous earth. Example

[0043] All of the following ingredients are food-grade and commercially available.

[0044] Example 1: A method for preparing high-purity, low-degradation chondroitin sulfate: S1. The animal cartilage is crushed and treated with a 0.5% sodium carbonate solution at 22°C for 3 hours to complete the relaxation process and obtain pretreated cartilage raw material. S2. Place the pretreated cartilage raw material in a water-alcohol biphase system and stir to dissolve it. The volume fraction of ethanol in the water-alcohol biphase system is 18%, the extraction temperature is 26℃, and the stirring and dissolving time is 4h to obtain the initial extract. S3. The initial extract was subjected to flocculation coupled with micro-filtration adsorption under the condition of isoelectric point pH 5.6-6.3. 0.2% of the total amount of the initial extract was added with Moringa seed protein composite material as flocculant and 1 g / L of the initial extract volume was added with diatomaceous earth prepared in Preparation Example 3 as filter aid. The mixture was allowed to stand for 40 min for flocculation and centrifuged at 9000 r / min to obtain a transparent and clear polysaccharide solution. The Moringa seed protein composite material was composed of quaternary ammonium salt modified Moringa seed protein prepared in Preparation Example 1 and tannin modified bacterial cellulose prepared in Preparation Example 2 in a mass ratio of 1:0.8. S4. The transparent and clear polysaccharide solution is first initially precipitated with 40% ethanol (volume fraction) to complete the alcohol precipitation treatment, and then treated with 1 mol / L sodium chloride gradient in stages to obtain the refined polysaccharide solution. S5. The purified polysaccharide solution is kept at pH 7.0-7.2, and a saturated sodium salt solution is added dropwise to complete the sodium salt replacement and shaping treatment, resulting in a semi-finished product. S6. The semi-finished product is subjected to ultrafiltration using a 9kDa ultrafiltration membrane. After sterile filtration, it is freeze-dried at a freeze-drying temperature of -15℃ and a vacuum degree of 0.09MPa to obtain the finished product.

[0045] Example 2: The difference between this example and Example 1 is that: S1. The animal cartilage is crushed and treated with a 0.3% sodium carbonate solution at 18°C ​​for 4 hours to complete the relaxation process and obtain pretreated cartilage raw material. S2. Place the pretreated cartilage raw material in a water-alcohol biphase system and stir to dissolve it. The volume fraction of ethanol in the water-alcohol biphase system is 12%, the extraction temperature is 22℃, and the stirring and dissolving time is 5h to obtain the initial extract. S3. The initial extract was subjected to flocculation coupled with micro-filtration adsorption treatment at an isoelectric point of pH 5.6-6.3. Moringa seed protein composite was used as a flocculant, and diatomaceous earth prepared in Preparation Example 4 was used as a filter aid. After flocculation and standing for 30 min, the mixture was centrifuged at 8500 r / min to obtain a transparent and clear polysaccharide solution. The moringa seed protein composite was composed of quaternary ammonium salt modified moringa seed protein prepared in Preparation Example 1 and tannin modified bacterial cellulose prepared in Preparation Example 2, with a mass ratio of 1:0.5. S4. The transparent and clear polysaccharide solution was first initially precipitated with 35% ethanol (volume fraction) to complete the alcohol precipitation treatment, and then treated with a gradient of 0.4 mol / L sodium chloride to obtain the refined polysaccharide solution. S5. The purified polysaccharide solution is kept at pH 6.8-7.0, and a saturated sodium salt solution is added dropwise to complete the sodium salt replacement and shaping treatment, resulting in a semi-finished product. S6. The semi-finished product is subjected to ultrafiltration using an 8kDa ultrafiltration membrane. After sterile filtration, it is freeze-dried at a freeze-drying temperature of -18℃ and a vacuum degree of 0.08MPa to obtain the finished product.

[0046] Example 3: The difference between this example and Example 1 is that: S1. The animal cartilage is crushed and treated with a sodium carbonate solution of 0.8% by mass at 25°C for 2 hours to complete the relaxation process and obtain pretreated cartilage raw material. S2. Place the pretreated cartilage raw material in a water-alcohol biphase system and stir to dissolve it. The volume fraction of ethanol in the water-alcohol biphase system is 22%, the extraction temperature is 30℃, and the stirring and dissolving time is 3h to obtain the initial extract. S3. The initial extract was subjected to flocculation coupled with micro-filtration adsorption treatment at an isoelectric point of pH 5.6-6.3. Moringa seed protein composite was used as a flocculant and diatomaceous earth prepared in Preparation Example 5 was used as a filter aid. After flocculation and standing for 50 min, the mixture was centrifuged at 10000 r / min to obtain a transparent and clear polysaccharide solution. The moringa seed protein composite was composed of quaternary ammonium salt modified moringa seed protein prepared in Preparation Example 1 and tannin modified bacterial cellulose prepared in Preparation Example 2, with a mass ratio of 1:1. S4. The transparent and clear polysaccharide solution was first initially precipitated with 45% ethanol (volume fraction) to complete the alcohol precipitation treatment, and then treated with a 1.6 mol / L sodium chloride gradient in stages to obtain the refined polysaccharide solution. S5. The purified polysaccharide solution is kept at pH 7.2-7.4, and a saturated sodium salt solution is added dropwise to complete the sodium salt replacement and shaping process, resulting in a semi-finished product. S6. The semi-finished product is subjected to ultrafiltration using a 10kDa ultrafiltration membrane. After sterile filtration, it is freeze-dried at a freeze-drying temperature of -10℃ and a vacuum degree of 0.1MPa to obtain the finished product.

[0047] Example 4: The difference between this example and Example 1 is that: The flocculant is Moringa seed protein powder.

[0048] Example 5: The difference between this example and Example 1 is that: In the flocculant, the quaternary ammonium salt modified moringa seed protein was replaced with an equal mass of moringa seed protein.

[0049] Example 6: The difference between this example and Example 1 is that: No tannin-modified bacterial cellulose was added to the flocculant.

[0050] Example 7: The difference between this example and Example 1 is that: Tannin-modified bacterial cellulose was replaced with an equal mass of tannin in the flocculant.

[0051] Example 8: The difference between this example and Example 1 is that: The filter aid is diatomaceous earth.

[0052] Comparative Example Comparative Example 1: The difference between this comparative example and Example 1 is that: No alcohol precipitation or salt gradient fractionation was performed.

[0053] Application examples Application Example 1: The chondroitin sulfate prepared in Example 1 is used in the fields of food, health products, cosmetics, or biomedicine.

[0054] Performance testing 1. Purity testing The finished chondroitin sulfate was prepared using the methods of Examples 1-8 and Comparative Example 1, respectively. The purity was determined by high performance liquid chromatography, and the protein residue was determined by BCA method. The absorbance was determined by UV-Vis spectrophotometry at 450 nm. The lower the absorbance value, the less pigment residue. The data were recorded.

[0055] 2. Degradation rate detection The finished chondroitin sulfate was prepared using the methods of Examples 1-3 and Comparative Example 1, respectively. The degradation rate of the polysaccharide backbone was detected by enzymatic hydrolysis-high performance liquid chromatography, and the data were recorded.

[0056] 3. Molecular weight uniformity test The finished chondroitin sulfate was prepared using the methods of Examples 1-3 and Comparative Example 1, respectively. The PDI was detected by gel permeation chromatography, and the data were recorded.

[0057] Table 1 Performance Test Table ("-" in the table indicates that there is no data for the corresponding embodiment and comparative example)

[0058] As can be seen from Examples 1-3 and Table 1, the chondroitin sulfate prepared in this application has the advantages of high purity, low impurity protein content, low pigment content, and low degradation rate, and has a uniform molecular weight with a narrow distribution effect.

[0059] Combining Examples 1 and 4-8 with Table 1, it can be seen that the flocculant in Example 4 is Moringa seed protein powder. Compared with Example 1, the purity of Example 4 is lower than that of Example 1, the protein residue is higher than that of Example 1, and the absorbance is higher than that of Example 1. This indicates that the combination of quaternary ammonium salt modified Moringa seed protein and tannin modified bacterial cellulose can efficiently and stably adsorb impurities and polypeptides, remove flocs, and improve the purity of the finished chondroitin sulfate.

[0060] In Example 5, the quaternary ammonium salt-modified moringa seed protein was replaced with an equal mass of moringa seed protein in the flocculant. Compared with Example 1, the purity of Example 5 was lower than that of Example 1, the protein residue was higher than that of Example 1, and the absorbance was higher than that of Example 1. This indicates that the quaternary ammonium salt on the surface of moringa seed protein carries a positive charge. The quaternary ammonium salt can quickly and stably adsorb impurities, solving the problem of insufficient amino protonation of natural moringa protein. This improves the adsorption stability and adsorption capacity of quaternary ammonium salt-modified moringa seed protein for negatively charged impurities, thereby reducing the impurity content and improving the purity of chondroitin sulfate.

[0061] In Example 6, no tannin-modified bacterial cellulose was added to the flocculant. Compared to Example 1, the purity of Example 6 was lower than that of Example 1, the protein residue was higher, and the absorbance was higher. This indicates that the bacterial cellulose in the tannin-modified bacterial cellulose can not only load a large amount of tannin substances, but also improve the structural density and cross-linking points of bacterial cellulose. The loose macropores in bacterial cellulose are transformed into mesopores and micropores, which facilitates the binding of small molecule impurities such as proteins and peptides. Combined with the connection between tannin and Moringa seed protein and impurities, it stabilizes the floc structure, making it less prone to secondary dispersion of flocculent substances, further reducing the impurity content and improving the purity of chondroitin sulfate.

[0062] In Example 7, tannin-modified bacterial cellulose was replaced with an equal mass of tannin in the flocculant. Compared to Example 1, the purity of Example 7 was lower than that of Example 1, the protein residue was higher, and the absorbance was higher. This indicates that the ultra-long nanofiber structure of bacterial cellulose provides a long-chain bridging effect, forming multi-level cross-linked flocs. The cellulose network is entangled and connected to form large flocs, which bind small flocs. Bacterial cellulose acts as a connecting bridge to connect the small flocs. Centrifugation further reduces the impurity content and improves the purity of chondroitin sulfate.

[0063] In Example 8, the filter aid was diatomaceous earth. Compared to Example 1, the purity of Example 8 was lower than that of Example 1, the protein residue was higher, and the absorbance was higher. This indicates that diatomaceous earth particles loaded with β-cyclodextrin can act as flocculation nuclei in solution. Through their own long-chain organic modification layer, multiple small pigment flocs that have been initially adsorbed are connected and bridged to each other, eventually forming dense flocs with larger volume and better sedimentation performance. This allows for rapid separation from the chondroitin sulfate system, avoiding pigment re-dissolution, improving decolorization efficiency, and reducing pigment impurity content.

[0064] Combining Example 1 and Comparative Example 1 with Table 1, it can be seen that Comparative Example 1, which did not undergo alcohol precipitation and salt gradient fractionation, had lower purity, higher protein residue, higher absorbance, higher degradation rate, and higher PDI compared to Example 1. This indicates that alcohol precipitation and salt gradient fractionation can improve the purity of the finished chondroitin sulfate, accurately remove degraded small molecules, enrich intact high molecular weight polysaccharides, and significantly improve product uniformity.

[0065] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing high-purity, low-degradation chondroitin sulfate, characterized in that, Includes the following steps: S1. The animal cartilage is crushed and then subjected to a relaxation process to obtain pretreated cartilage raw material; S2. Place the pretreated cartilage raw material in a water-alcohol two-phase system and stir to dissolve it, to obtain the initial extract; S3. The initial extract was subjected to isoelectric point flocculation coupled micro-filtration adsorption treatment, followed by centrifugation and filtration to obtain a transparent and clear polysaccharide solution. S4. The transparent and clear polysaccharide solution is first subjected to alcohol precipitation, and then subjected to salt gradient fractionation to obtain a refined polysaccharide solution. S5. The refined polysaccharide solution is subjected to sodium salt replacement and shaping treatment to obtain a semi-finished product; S6. The semi-finished product is subjected to ultrafiltration and aseptic filtration, followed by freeze drying to obtain the finished product.

2. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 1, characterized in that: The specific steps of the relaxation treatment are as follows: use a sodium carbonate solution with a mass fraction of 0.3-0.8% and treat at a constant temperature of 18-25℃ for 2-4 hours.

3. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 1, characterized in that, The water-alcohol biphase system has an ethanol volume fraction of 12-22%, an extraction temperature of 22-30℃, and a stirring dissolution treatment time of 3-5 hours.

4. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 1, characterized in that, The isoelectric point flocculation pH is 5.6-6.

3. Micro-filtration adsorption treatment: Moringa seed protein composite material is used as flocculant and diatomaceous earth is used as filter aid. Flocculation is allowed to stand for 30-50 minutes and centrifuged at 8500-10000 r / min.

5. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 4, characterized in that, The moringa seed protein composite is composed of quaternary ammonium salt modified moringa seed protein and tannin modified bacterial cellulose in a mass ratio of 1:0.5-1.

6. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 4, characterized in that, The diatomaceous earth is loaded diatomaceous earth, which is obtained by activating diatomaceous earth, grafting it with silane coupling agent KH-560, and then loading it with β-cyclodextrin. The mass ratio of diatomaceous earth, silane coupling agent KH-560 and β-cyclodextrin is 100:1.5-2.5:25-35.

7. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 1, characterized in that, The alcohol precipitation treatment uses 35%–45% ethanol by volume for initial precipitation; the salt gradient fractionation treatment uses 0.4–1.6 mol / L sodium chloride for fractional treatment.

8. The method for preparing high-purity, low-degradation chondroitin sulfate according to claim 1, characterized in that, During the sodium salt replacement and shaping process: the pH of the system is controlled at 6.8-7.4, and a saturated sodium salt solution is added dropwise; ultrafiltration is performed using an 8-10 kDa ultrafiltration membrane, the freeze-drying temperature is -18 to -10℃, and the vacuum degree is 0.08-0.1 MPa.

9. A high-purity, low-degradation chondroitin sulfate, characterized in that, Chondroitin sulfate prepared by the method for preparing high-purity, low-degradation chondroitin sulfate according to any one of claims 1-8.

10. An application of high-purity, low-degradation chondroitin sulfate, characterized in that, The application of chondroitin sulfate prepared by the method for preparing high-purity, low-degradation chondroitin sulfate according to any one of claims 1-8, or chondroitin sulfate prepared according to claim 9, in the fields of food, health products, cosmetics, or biomedicine.

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