Alcohol-free chondroitin sulfate extraction process

Abstract

The invention belongs to the technical field of biology, and particularly relates to an alcohol-free chondroitin sulfate extraction process which comprises the following steps: (1) pretreatment of animal soft tissues: taking down the animal soft tissues, drying, crushing, sieving and ball-milling to obtain superfine powder; (2) preparing a deep eutectic solvent, namely mixing betaine and L-proline in water according to a molar ratio of 1: 3, and fully stirring at 60-90 DEG C until the solution is clear and transparent, so as to obtain the deep eutectic solvent; (3) enzymolysis extraction: adding the superfine powder into the deep eutectic solvent according to a solid-liquid ratio of 1: 30g / ml, and adding a compound enzyme for enzymolysis treatment; and (4) post-treatment: performing enzyme deactivation on the enzymatic hydrolysate obtained in the step (3), adding diatomite, and then performing filtration, membrane separation and spray drying to obtain chondroitin sulfate. According to the invention, betaine and L-proline are adopted to prepare the deep-eutectic solvent, and the high chondroitin sulfate extraction efficiency can be realized without ethanol precipitation under the condition that the deep-eutectic solvent is combined with enzymolysis in advance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a process for extracting chondroitin sulfate without alcohol. Background Art

[0002] Chondroitin sulfate is a white powder, odorless, hygroscopic, and sticky when in contact with water. It is a type of glycosaminoglycan that is covalently linked to proteins to form proteoglycans. It is widely present in animal tissues, cell surfaces, and between cell matrices. Chondroitin sulfate has a wide range of uses. It can be used as a dietary protectant to protect cartilage tissues such as joints, and can effectively relieve symptoms such as joint discomfort. At the same time, chondroitin sulfate has anticoagulant, sedative, anti-tumor, antioxidant, and lipid-regulating functions. Therefore, chondroitin sulfate is widely used in food, medical, cosmetics and other industries. In recent years, the demand for chondroitin sulfate has continued to increase, and products that use chondroitin sulfate as a research object have broad prospects.

[0003] Currently, chondroitin sulfate can be extracted from animal cartilage tissue. The animal connective tissue is destroyed by high-temperature cooking, and then chondroitin sulfate is extracted by alkaline hydrolysis or enzymatic hydrolysis. Common methods include water extraction, alkaline extraction and enzyme extraction. Among them, water extraction has a low product yield and high energy consumption; alkaline extraction has serious pollution and is prone to solvent residue; enzyme extraction has a higher extraction rate, but the use conditions are harsh and it is easily affected by the production environment.

[0004] Alcohol precipitation is a commonly used separation method that uses alcohol to separate impurities such as proteins and nucleic acids from the target product. However, this method has many limitations:

[0005] 1. Large amount of alcohol used: Alcohol needs to be used in large quantities as a separation medium, which not only increases production costs but also causes potential pollution to the environment.

[0006] 2. Product purity and recovery rate are limited: The separation effect of alcohol precipitation is easily affected by the type of extract, solution concentration and operating conditions, making it difficult to achieve the goals of high purity and high recovery rate.

[0007] 3. High safety risk: Alcohol is a flammable and explosive substance. Its storage, use and waste disposal during the production process require strict safety management, which increases the complexity of the process and potential risks.

[0008] 4. Insufficient demand for green production: Modern industry has put forward higher requirements for sustainable development and green environmental protection. The alcohol precipitation method is inconsistent with the development trend of low-energy consumption and low-pollution green processes.

[0009] In addition, the current industrial production of chondroitin sulfate basically uses the alcohol precipitation process. During the production process, a large amount of ethanol is used to precipitate and dehydrate the chondroitin sulfate, which results in a huge amount of ethanol used in the production workshop. During the storage, transportation and use of ethanol, if the operation is improper, it may cause fire or explosion, posing a serious safety hazard.

[0010] As a kind of green solvent, deep eutectic solvents (DESs) have been widely used in the extraction of functional components such as polysaccharides, flavonoids, and polyphenols in recent years. DESs are mainly composed of hydrogen bond donor (HBD) and acceptor (HBA) molecules in a certain molar ratio. They are liquid solvents at room temperature and have strong eutectic behavior. The highly organized structure formed under the guidance of hydrogen bond interactions provides DES with significant advantages, such as biodegradability, low cost, solute stability, and simplicity of preparation. The solubility of different compounds in NADES is affected by the combination of HBD and HBA, enriching the design possibilities of its composition. This means that the target has a certain correspondence with the components of DES, and the extraction efficiency of the target can be improved by selecting the appropriate components and proportions. Although there are a few reports on the use of deep eutectic solvents for chondroitin sulfate extraction, its extraction efficiency still needs to be improved. Summary of the invention

[0011] In order to solve the above problems, the present invention provides a process for extracting chondroitin sulfate without alcohol, which comprises the following steps:

[0012] (1) Animal soft tissue pretreatment steps:

[0013] After the soft tissue of the animal is removed, it is first dried by hot air (temperature 50-60℃, time 1-2 hours), and then the dried tissue is mechanically crushed (time 30 seconds to 1 minute), sieved through 60 mesh to obtain coarse powder, and then ball milled (ball-to-material ratio 7:1, rotation speed about 500×g, ball milling time 2-3 hours) to obtain ultrafine powder with uniform particle size;

[0014] (2) Preparation steps of low eutectic solvent:

[0015] Betaine and L-proline were mixed in drinking water at a molar ratio of 1:3, and stirred at 60–90°C until the solution was clear and transparent, forming a low eutectic solvent with a certain polarity;

[0016] (3) Enzymatic extraction step:

[0017] Add the ultrafine powder obtained in step (1) to the eutectic solvent prepared in step (2) at a material-liquid ratio of 1:30 g / mL; first soak the ultrafine powder in drinking water (for 1 hour), then cook it with drinking water (for 3 - 4 hours), and then add an appropriate amount of sodium hydroxide for alkali hydrolysis (for 4 hours); finally, add pancreatin or alkaline protease under the adjusted required pH condition (hydrochloric acid can be added appropriately to adjust the pH), and continue the enzymatic hydrolysis treatment at the specified temperature (for 4 hours). After the enzymatic hydrolysis is completed, use a filtration device with a pore size of about 5 microns to preliminarily filter the reaction solution;

[0018] (4) Ion exchange and oxidation separation step:

[0019] Introduce the enzymatic hydrolysis product obtained in step (3) into an ion exchange column pre-packed with resin for treatment, and control the ion exchange conditions to make the exchange ratio reach about 1.0 - 1.1; subsequently, add an appropriate amount of sodium hydroxide, hydrochloric acid, and hydrogen peroxide to the material after ion exchange treatment in purified water, and carry out oxidation treatment under the condition that the pH is controlled at 9.0 - 10.0 for 4 hours, and use a filtration device with a pore size of about 5 microns for secondary filtration;

[0020] (5) Ultrafiltration, concentration, sterilization, and drying steps:

[0021] Use an ultrafiltration membrane with a molecular weight cut-off of 12 kDa to ultrafilter the mixed solution obtained in step (4) to separate low-molecular impurities; then, concentrate the ultrafiltrate at a temperature of 70 - 80 °C; then, sterilize the concentrated solution at a temperature not lower than 95 °C for 1 hour; finally, use a spray drying process for drying, where the inlet air temperature is set at 170 - 190 °C and the outlet air temperature is 80 - 100 °C to obtain dried chondroitin sulfate powder;

[0022] (6) Post-treatment and packaging steps:

[0023] Collect the powder product obtained by spray drying, perform necessary crushing and screening treatments, remove remaining impurities using appropriate metal separation techniques, and then carry out in-package and out-package before warehousing management.

[0024] Further, in the animal soft tissue pretreatment step, the drying condition is carried out at a temperature of 50 - 60 °C for 1 - 2 hours, the crushing time is 30 seconds to 1 minute, the sieving equipment used is a 60-mesh sieve, and the ball milling conditions are a ball-to-material ratio of 7:1, a rotation speed of about 500×g, and a ball milling duration of 2 - 3 hours.

[0025] Further, the water content in the eutectic solvent is controlled at 40 - 70 wt% to meet the requirement of the mixed solution being clear and transparent.

[0026] Further, in the enzymatic hydrolysis extraction step, the addition amount of the complex enzyme is 1–2% of the mass of the ultrafine powder after the pretreatment of animal soft tissue.

[0027] Further, the complex enzyme includes alkaline protease, pepsin and papain.

[0028] Further, the mass ratio of the alkaline protease, pepsin and papain is 1:0.5:2.

[0029] Further, the enzymatic hydrolysis extraction step is carried out under the conditions that the pH is controlled at 6.5–7.5 and the temperature is 50–55 °C, and is treated for 2.5–3.5 hours under the condition of ultrasonic assistance (ultrasonic frequency is 20–25 kHz and power is 600–700 W).

[0030] Further, in the ion exchange and oxidation separation step, the ion exchange treatment is carried out in a preset resin column to make the ion exchange ratio reach about 1.0–1.1; the oxidation treatment is carried out under the condition that the pH value is regulated at 9.0–10.0, and the oxidation reaction lasts for 4 hours.

[0031] Further, in the ultrafiltration step, an ultrafiltration membrane with a cut-off molecular weight of 12 kDa is used to treat the mixed solution after ion exchange and oxidation treatment to separate and remove low-molecular impurities.

[0032] Further, in the concentration, sterilization and drying step, the concentration is carried out at 70–80 °C, the sterilization condition is that the temperature is not lower than 95 °C and lasts for 1 hour, the inlet air temperature during spray drying is 170–190 °C, and the outlet air temperature is 80–100 °C, so that the dried product meets the requirements of the predetermined moisture content.

[0033] Further, the post-treatment step includes powder collection, pulverization, screening and metal separation treatment of the dried chondroitin sulfate powder, and then it is warehoused and managed after inner packaging and outer packaging.

[0034] Further, in this process, an ion exchange and oxidation assistance step is directly adopted after enzymatic hydrolysis extraction, and the extraction, enrichment and purification of chondroitin sulfate are realized under the condition of no alcohol medium.

[0035] Advantages of the present invention

[0036] The present invention uses betaine and L-proline to prepare a deep eutectic solvent, and finds that it has the highest extraction efficiency of chondroitin sulfate among several tested deep eutectic solvents. In combination with enzymatic hydrolysis, it can further improve the extraction rate and extraction purity of chondroitin sulfate. Comparing with the extraction efficiency of chondroitin sulfate by the ethanol precipitation method, it is found that the extraction rate and extraction purity of chondroitin sulfate achieved by the method of the present invention are comparable to it. Description of the drawings

[0037] Figure 1 This is a schematic flow diagram of the process for extracting chondroitin sulfate without alcohol according to the present invention. Specific embodiments

[0038] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] Example 1

[0040] In this example, the following process steps are adopted to obtain chondroitin sulfate by a non-alcohol extraction method. The detailed operations are as follows:

[0041] S1 Pretreatment of animal soft tissues

[0042] Tissue collection and soaking: The animal soft tissues after removing impurities are placed in distilled water and soaked continuously for 2 hours to fully soften the cell tissues, facilitating subsequent impurity removal.

[0043] Manual tearing and drying: After soaking, obvious impurities attached to the tissue surface are carefully torn off by manual picking or mechanical assistance.

[0044] The treated soft tissues are placed in a hot air circulation oven and dried at 50 °C. The drying time is controlled within 2 hours until the moisture content drops to about 10% or less.

[0045] Crushing and screening: The dried soft tissues are put into a high-speed crusher for crushing. During the operation, the crushing time is controlled for 30 seconds to 1 minute to break the tissues into smaller particles. The crushed materials are screened through a 60-mesh standard sieve to remove larger lumps and obtain relatively uniform fine powder.

[0046] Washing and air drying: The sieved powder is washed with clear water to remove soluble impurities, and then air-dried naturally or dried at a low temperature (not exceeding 40 °C) to retain the active ingredients.

[0047] Ball milling treatment: The air-dried powder is put into a ball mill for fine grinding. The ball milling conditions are set as follows: the ball-to-material ratio is 7:1 (weight ratio), the rotation speed is 500×g, and the ball milling time is set at 150 minutes (2.5 hours) to obtain ultrafine powder with a uniform particle size distribution, which provides a sufficient reaction area for subsequent enzymatic hydrolysis.

[0048] S2 Preparation of eutectic solvent

[0049] Raw material ratio and mixing: Weigh betaine and L-proline and dissolve them in drinking water according to a molar ratio of 1:3.

[0050] In this embodiment, the water content in the low eutectic solvent is controlled to be 70 wt %, so that the target object in the solution has a stronger solubility.

[0051] Heating and stirring: Place the mixed solution in a reactor with a constant temperature function and stir it thoroughly at 80°C until the mixed solution becomes clear and transparent, ensuring that all components are completely dissolved to form a stable low-solvent system.

[0052] S3 enzymatic extraction

[0053] Preparation of enzymatic hydrolysis system: Add the ultrafine powder obtained in step S1 to the low eutectic solvent prepared in step S2 at a solid-liquid ratio of 1:30 g / mL. Use an instrument to adjust the pH of the solution to about 7.0 to provide good conditions for enzyme activity.

[0054] Pretreatment and water bath cooking: The mixed system is first pre-soaked for 1 hour to fully wet the solid particles, and then transferred to a water bath for cooking at a temperature set at 100°C (which can be slightly adjusted according to the actual equipment) for 3.5 hours to partially rupture the cell wall structure and initially release the target ingredients.

[0055] Alkaline hydrolysis treatment: After the cooking is completed, an appropriate amount of sodium hydroxide is added to the reaction system for alkaline hydrolysis treatment. The alkaline hydrolysis time is 4 hours to promote the hydrolysis of structural macromolecules and improve the efficiency of subsequent enzymatic hydrolysis.

[0056] Compound enzyme addition and enzymatic reaction: After the alkaline hydrolysis treatment, adjust the system pH to 7.0-7.2, then add compound enzyme according to 2% of the mass of animal soft tissue. The compound enzyme used is composed of pancreatic enzyme or alkaline protease, pepsin and papain, and the mass ratio is adjusted to 1:0.5:2. The system starts the enzymatic reaction at 50°C and continues the reaction for 3 hours under ultrasonic assisted conditions (ultrasonic frequency 20KHz, power 650W). Ultrasonic assistance helps to improve the efficiency of enzymatic hydrolysis and promote the effective release of the target chondroitin sulfate and its precursor.

[0057] Solid-liquid separation: After the enzymatic hydrolysis reaction is completed, a filter with a pore size of about 5 microns is used to separate the reaction liquid into solids and liquids, and the filtrate is collected for subsequent treatment.

[0058] S4 ion exchange and oxidation separation

[0059] Ion exchange treatment: The filtrate obtained in step S3 is directly fed into a pre-equilibrated resin ion exchange column. By adjusting the inlet flow rate and system conditions, the ion exchange ratio is adjusted to about 1.0-1.1, promoting the enrichment of chondroitin sulfate molecules on the resin.

[0060] Oxidation-assisted separation: Transfer the liquid after ion exchange into a reactor with temperature control and pH control functions, add purified water, and appropriately supplement sodium hydroxide, hydrochloric acid, and hydrogen peroxide to keep the system pH stable between 9.0 and 10.0; conduct an oxidation reaction under this condition, and control the reaction time to 4 hours. This step promotes the crystallization of chondroitin sulfate under alcohol-free conditions.

[0061] Secondary filtration: After the oxidation reaction is completed, filter the reaction solution again using a filtration device with a pore size of 5 μm to remove possible tiny insoluble impurities and ensure the purity of the material for subsequent processing.

[0062] S5 Ultrafiltration, concentration, sterilization, and drying

[0063] Ultrafiltration treatment: Apply an ultrafiltration membrane with a molecular weight cut-off of 12 kDa to the liquid filtered in step S4 for molecular-level separation, effectively removing low-molecular-weight impurities while retaining chondroitin sulfate molecules.

[0064] Concentration operation: Place the concentrated liquid obtained by ultrafiltration in a concentration device with a temperature control system, and conduct concentration treatment at 70 - 80 °C until the system volume is reduced to about 30% of the original volume.

[0065] Sterilization treatment: Sterilize the concentrated liquid by heating it to no less than 95 °C using high-temperature sterilization equipment and maintaining it for 1 hour to ensure the elimination of microorganisms.

[0066] Spray drying: Dry the sterilized concentrated liquid through a spray drying device, set the inlet air temperature to 170 - 190 °C, and control the outlet air temperature at about 80 - 100 °C to quickly form dry chondroitin sulfate powder.

[0067] S6 Post-treatment and packaging

[0068] Product collection and adjustment: Collect the dry powder obtained by spray drying using an automatic powder collection system, conduct fine crushing using a crushing device, and then adjust the particle size distribution through screening to meet the product quality requirements.

[0069] Impurity removal: Use magnetic separation or other applicable metal separation technologies to remove possible trace metal impurities in the product (CCP measure).

[0070] Packaging and warehousing: After inner packaging and outer packaging, conduct final quality inspection on the finally processed chondroitin sulfate powder, and store the qualified products in the warehouse.

[0071] In this embodiment, the settings of each key operation parameter are as follows:

[0072] Pretreatment of animal soft tissue: Soak for 2 h, dry for 2 h, crush for 30 s, and ball mill for 150 min;

[0073] Preparation of eutectic solvent: Betaine and L-proline at a molar ratio of 1:3, water content 70 wt%, stirred at 80 °C;

[0074] Enzymatic extraction: solid-liquid ratio 1:30 g / mL, pH adjusted to 7.0, composite enzyme addition amount 2%, reacted at 50 °C with ultrasonic assistance (20 KHz, 650 W) for 3 h;

[0075] Ion exchange: exchange ratio controlled at 1.0–1.1; Oxidation separation: pH 9.0–10.0, oxidized for 4 h;

[0076] Ultrafiltration: molecular weight cut-off 12 kDa; Concentration: concentrated to 30% at 70–80 °C; Sterilization: 95 °C for 1 h; Drying: inlet air temperature of spray drying 185 °C, outlet air temperature 90 °C.

[0077] Control experiment

[0078] To verify the influence of the parameters of the key steps in the process, the following control samples were prepared respectively. Except for the preparation of the eutectic solvent and some auxiliary conditions, the remaining steps were the same as those in Example 1:

[0079] Control 1

[0080] The molar ratio of betaine to L-proline in the eutectic solvent was adjusted to 1:1, and the other conditions were the same as those in Example 1.

[0081] Control 2

[0082] The molar ratio of betaine to L-proline in the eutectic solvent was adjusted to 1:2 (water content 70 wt%), and the other conditions were the same as those in Example 1.

[0083] Control 3

[0084] The molar ratio of betaine to L-proline in the eutectic solvent was adjusted to 1:4, and the other conditions were the same as those in Example 1.

[0085] Control 4

[0086] In the eutectic solvent, L-proline was replaced by urea, and the molar ratio of betaine to urea was set to 1:3 (water content 70 wt%), and the other steps were the same as those in Example 1.

[0087] Control 5

[0088] In the eutectic solvent, L-proline was replaced by glucose, the molar ratio of betaine to glucose was 1:3 (water content 70 wt%), and the other process parameters remained unchanged.

[0089] Control 6

[0090] Modify the pretreatment steps: The animal soft tissue powder that has been cleaned and air-dried is directly used for enzymatic hydrolysis extraction without ball milling, and the remaining steps are the same as in Example 1.

[0091] Comparative Example 7

[0092] Replace the deep eutectic solvent with physiological saline as the extraction solvent, and keep the remaining process steps the same as in Example 1.

[0093] Comparative Example 8

[0094] In the enzymatic hydrolysis extraction step, no complex enzyme is added, and only physical hydrolysis conditions are used for the reaction. The remaining steps are the same as in Example 1.

[0095] Comparative Example 9

[0096] In the enzymatic hydrolysis extraction process, ultrasonic assistance is not used, but stirring treatment is adopted. The stirring rate is set at 400 rpm, and the remaining steps are the same as in Example 1.

[0097] Comparative Example 10

[0098] Modify the post-treatment steps: After filtration, an appropriate amount of absolute ethanol is added to the filtrate to precipitate chondroitin sulfate, replacing the membrane separation and spray drying methods. The remaining steps are kept the same as in Example 1.

[0099] Detect the chondroitin sulfate content according to GB / T 20365-2006, and the results are shown in the following table.

[0100] Table 1 Structural data of comparative tests

[0101]

[0102] As can be seen from the results in Table 1, the deep eutectic solvent prepared with betaine and L-proline at a molar ratio of 1:3 can be combined with enzymatic hydrolysis treatment to achieve the best extraction efficiency of chondroitin sulfate, and is comparable to the extraction efficiency of chondroitin sulfate achieved by ethanol precipitation.

[0103] From the results of Example 1 and Example 2, under the condition of a deep eutectic solvent with a molar ratio of betaine to L-proline of 1:3 and a water content of 70 wt%, supplemented with ultrasonic-assisted enzymatic hydrolysis, ion exchange and oxidation-assisted separation treatment, the extraction effect of chondroitin sulfate is relatively ideal, with a content of about 19 mg / g and a purity of about 89%, indicating that the process parameters under the reference conditions have a good effect on the release, enrichment and purification of chondroitin sulfate. In addition, in the comparative experiments, when the molar ratio deviates from 1:3, whether it is decreased (1:1) or increased (1:4), it will cause a decrease in the product content and / or purity, further indicating that the precise ratio of the deep eutectic solvent components is crucial for this process.

[0104] Further comparison with other comparative example conditions shows that although using urea to replace L-proline (Comparative Example 4) improves the purity to some extent, the overall extraction effect is still slightly lower than the baseline condition. Replacing it with glucose or physiological saline (Comparative Examples 5 and 7), however, significantly reduces the product quality. In addition, canceling the ball milling or ultrasonic-assisted treatment (Comparative Examples 6 and 9) also reduces the chondroitin sulfate content. Although using only non-composite enzymes or the ethanol precipitation method (Comparative Examples 8 and 10) shows improvement in some indicators, it is not completely consistent with the original intention of alcohol-free extraction. Generally speaking, the baseline conditions (Examples 1 and 2) have a better balance in terms of safety, environmental protection, and extraction efficiency, and are the best solutions in the application of this process.

[0105] It should be noted that the description of the present invention provides preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments do not serve as additional limitations to the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. Furthermore, the above technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as within the scope described in the description of the present invention. Further, for those of ordinary skill in the art, improvements or modifications can be made based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims of the present invention.

Claims

1. A process for extracting chondroitin sulfate without alcohol, characterized in that, The process includes the following steps: (1) Pretreatment step of animal soft tissue: After taking down the animal soft tissue, it is first dried by hot air at a temperature of 50–60 °C for 1–2 hours, then the dried tissue is mechanically pulverized for 30 seconds to 1 minute, and coarse powder is obtained through a 60-mesh sieve. Subsequently, ball milling treatment is carried out with a ball-to-material ratio of 7∶1, a rotation speed of about 500×g, and a ball milling time of 2–3 hours to obtain ultrafine powder with uniform particle size; (2) Preparation step of deep eutectic solvent: Betaine and L-proline are mixed in drinking water at a molar ratio of 1:3 and stirred at 60–90 °C until the solution is clear and transparent to form a deep eutectic solvent with a certain polarity; (3) Enzymatic hydrolysis extraction step: The ultrafine powder obtained in step (1) is added to the deep eutectic solvent prepared in step (2) at a material-liquid ratio of 1:30 g / mL; first, the ultrafine powder is soaked in drinking water for 1 hour, then steamed in drinking water for 3–4 hours, and then an appropriate amount of sodium hydroxide is added for alkaline hydrolysis for 4 hours; finally, trypsin or alkaline protease is added under the condition of adjusting to the required pH, and the pH is adjusted with an appropriate amount of hydrochloric acid, and enzymatic hydrolysis treatment is continued at a specified temperature for 4 hours. After the enzymatic hydrolysis is completed, the reaction solution is preliminarily filtered with a filtering device with a pore size of about 5 microns; (4) Ion exchange and oxidation separation step: The enzymatic hydrolysis product obtained in step (3) is introduced into an ion exchange column pre-filled with resin for treatment, and the ion exchange conditions are regulated to make the exchange ratio reach about 1.0–1.1; Subsequently, an appropriate amount of sodium hydroxide, hydrochloric acid, and hydrogen peroxide are added to the material after ion exchange treatment in purified water, and oxidation treatment is carried out under the condition that the pH is regulated to 9.0–10.0 for 4 hours, and secondary filtration is carried out with a filtering device with a pore size of about 5 microns; (5) Ultrafiltration, concentration, sterilization, and drying steps: Ultrafiltration is carried out on the mixed solution obtained in step (4) with an ultrafiltration membrane with a cut-off molecular weight of 12 kDa to separate low-molecular impurities; Then, the ultrafiltrate is concentrated at a temperature of 70–80 °C; Then, the concentrated solution is sterilized at a temperature not lower than 95 °C for 1 hour; Finally, spray drying process is used for drying, in which the inlet air temperature is set at 170–190 °C and the outlet air temperature is 80–100 °C to obtain dried chondroitin sulfate powder; (6) Post-treatment and packaging steps: The powder product obtained by spray drying is collected, subjected to necessary pulverization and screening treatment, and the remaining impurities are removed by appropriate metal separation technology, and then stored in the warehouse after inner packaging and outer packaging.

2. The process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, In the pretreatment step of the animal soft tissue, the drying condition is carried out at a temperature of 50–60 °C for 1–2 hours, the pulverization time is 30 seconds to 1 minute, the sieving equipment used is a 60-mesh sieve, and the ball milling conditions are a ball-to-material ratio of 7∶1, a rotation speed of about 500×g, and a ball milling duration of 2–3 hours.

3. The process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, The water content in the deep eutectic solvent is controlled at 40–70 wt% to meet the requirement of a clear and transparent mixed solution.

4. A process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, In the enzymatic hydrolysis extraction step, the addition amount of the composite enzyme is 1–2% of the mass of the ultrafine powder after pretreatment of the animal soft tissue.

5. A process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, The composite enzyme includes alkaline protease, pepsin and papain.

6. The process for extracting chondroitin sulfate without alcohol according to claim 5, characterized in that, The mass ratio of the alkaline protease, pepsin and papain is 1:0.5:

2.

7. A process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, The enzymatic hydrolysis extraction step is carried out under the conditions that the pH is controlled at 6.5–7.5 and the temperature is 50–55 °C, and is treated for 2.5–3.5 hours under the condition of ultrasonic assistance (ultrasonic frequency is 20–25 kHz and power is 600–700 W).

8. A process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, In the ion exchange and oxidation separation step, the ion exchange treatment is carried out in a preset resin column so that the ion exchange ratio reaches about 1.0–1.1; the oxidation treatment is carried out under the condition that the pH value is regulated at 9.0–10.0, and the oxidation reaction lasts for 4 hours.

9. A process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, In the ultrafiltration step, an ultrafiltration membrane with a molecular weight cut-off of 12 kDa is used to treat the mixed solution after ion exchange and oxidation treatment to separate and remove low-molecular impurities.

10. The process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, In the concentration, sterilization and drying step, the concentration is carried out at 70–80 °C, the sterilization condition is that the temperature is not lower than 95 °C and lasts for 1 hour, the inlet air temperature during spray drying is 170–190 °C, and the outlet air temperature is 80–100 °C, so that the dried product meets the requirements of the predetermined moisture content.

11. A process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, The post-treatment step includes powder collection, pulverization, screening and metal separation treatment of the dried chondroitin sulfate powder, and then it is stored in the warehouse after inner packaging and outer packaging.

12. The process for extracting chondroitin sulfate without alcohol according to claim 1, characterized in that, This process directly adopts ion exchange and oxidation assistance steps after enzymatic hydrolysis extraction, and realizes the extraction, enrichment and purification of chondroitin sulfate under the condition of no alcohol medium.

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