Chondroitin sulfate extraction process

Abstract

The present invention discloses a chondroitin sulfate extraction process, comprising extracting chondroitin sulfate from cartilage raw material using a natural deep eutectic solvent, wherein the raw materials for preparing the natural deep eutectic solvent include betaine and urea. The natural deep eutectic solvent used in the present invention extracts chondroitin sulfate with a high extraction rate, is environmentally friendly, and highly efficient, and does not destroy the polysaccharide structure of chondroitin sulfate.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a chondroitin sulfate extraction process. Background Art

[0002] Bone tissue is rich in nutrients such as protein, polysaccharides, and bone minerals, and has enormous potential for utilization. Chondroitin sulfate (CS) is a sulfated glycosaminoglycan found in animal bone tissue. It consists of repeating disaccharide units composed of N-acetylgalactosamine (GalNAc) and D-glucuronic acid (GlcA) linked by β-1,4 glycosidic bonds. CS has multiple functional properties, including antioxidant, anti-inflammatory, and lipid-lowering activities. CS can interfere with the pro-inflammatory activation of monocytes and endothelial cells driven by TNF-α, preventing and alleviating bone tissue damage caused by most arthritis and multiple inflammatory conditions throughout the body, and preventing the formation of atherosclerotic plaques in humans.

[0003] Currently, common methods for extracting CS include water extraction, alkaline extraction, and enzymatic extraction. Water extraction suffers from low product yields and high energy consumption; alkaline extraction is highly polluting and prone to solvent residue; and enzymatic extraction offers high extraction yields but is subject to stringent conditions and is susceptible to environmental influences, making it less commonly used in production. Therefore, a technical solution that can overcome these drawbacks to some extent is needed. Summary of the Invention

[0004] One object of the present invention is to provide a chondroitin sulfate extraction process, which uses a natural deep eutectic solvent to extract chondroitin sulfate, has a high extraction rate, is environmentally friendly, and is highly efficient without destroying the polysaccharide structure of chondroitin sulfate.

[0005] To achieve these objectives and other advantages of the present invention, the present invention provides a chondroitin sulfate extraction process, comprising: extracting chondroitin sulfate from cartilage raw materials using a natural deep eutectic solvent, wherein the raw materials for preparing the natural deep eutectic solvent include betaine and urea.

[0006] Furthermore, the preparation method of the natural deep eutectic solvent comprises: mixing betaine and urea, adding 20-40% by weight of water, and stirring at 70-90° C. until the solution becomes clear and transparent, thereby obtaining the natural eutectic solvent.

[0007] Furthermore, the molar ratio of the betaine to the urea is 1.8-2.2:1.

[0008] Furthermore, the cartilage raw material is made into bone powder, mixed with the natural low eutectic solvent, heated for extraction, centrifuged, the supernatant is collected and filtered, ethanol is added, the mixture is allowed to stand, centrifuged to collect the precipitate, and dialyzed to obtain the chondroitin sulfate.

[0009] Furthermore, the bone powder is mixed with the natural deep eutectic solvent at a liquid-to-solid ratio of 20 to 50:1.

[0010] Furthermore, the cartilage raw material and the natural deep eutectic solvent are extracted at 90-110° C. for 2-2.5 hours.

[0011] Furthermore, ethanol is added until the volume ratio of ethanol reaches 70 to 80%.

[0012] Furthermore, the solution was dialyzed in ultrapure water using a 10,000 Da dialysis bag.

[0013] Furthermore, the method further comprises: taking the filtered supernatant, removing ethanol, and recovering the natural low eutectic solvent.

[0014] Furthermore, the cartilage raw material is bovine laryngeal cartilage.

[0015] The present invention has at least the following beneficial effects:

[0016] The natural deep eutectic solvent used in the present invention is used to extract chondroitin sulfate, with a high extraction rate and high purity. The raw materials for preparing the natural deep eutectic solvent include betaine and urea, which is easy to prepare, environmentally friendly, and highly efficient. It does not destroy the polysaccharide structure of chondroitin sulfate and can be recycled and reused. After multiple uses, the extraction rate of chondroitin sulfate is still relatively high, thereby reducing the extraction cost and the discharge of wastewater.

[0017] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The recovery rate of chondroitin sulfate extracted from different NADES;

[0019] Figure 2 The basic composition of chondroitin sulfate extracted from different NADES;

[0020] Figure 3 This is the infrared spectrum of the extracted chondroitin sulfate;

[0021] Figure 4 This is a comparison chart of the infrared spectra of the extracted chondroitin sulfate and the chondroitin sulfate A standard;

[0022] Figure 5 The effect of liquid-solid ratio on the extraction rate of chondroitin sulfate is given;

[0023] Figure 6 The effect of water content on the extraction rate of chondroitin sulfate is given;

[0024] Figure 7The effect of extraction temperature on the extraction rate of chondroitin sulfate is given;

[0025] Figure 8 The effect of extraction time on the extraction rate of chondroitin sulfate is given;

[0026] Figure 9 The correlation between the physicochemical properties and recovery rates of different types of NADESs was given;

[0027] Figure 10 The physical and chemical properties analysis of NADES-3 with different water contents is given;

[0028] Figure 11 The effect of recycling times on the properties of deep eutectic solvents is given;

[0029] Figure 12 The effects of recycling times on the extraction rate and composition of chondroitin sulfate are given. DETAILED DESCRIPTION

[0030] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0031] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0032] The embodiment of the present application provides a chondroitin sulfate extraction process, comprising extracting chondroitin sulfate from a cartilage raw material using a natural deep eutectic solvent, wherein the raw materials for preparing the natural deep eutectic solvent include betaine and urea;

[0033] Natural deep eutectic solvents (NADES) are typically composed of two or three primary cellular metabolites. They are inexpensive, readily available, non-toxic, and biocompatible, meeting the requirements of green chemistry. In this embodiment, a natural deep eutectic solvent is prepared using raw materials including betaine and urea as an extraction solvent, which is then used to extract chondroitin sulfate from a cartilage raw material. The cartilage raw material can be selected from porcine cartilage, bovine cartilage, or fish cartilage. Preferably, the cartilage raw material is bovine laryngeal cartilage.

[0034] In another embodiment, the preparation method of the natural deep eutectic solvent comprises: mixing betaine and urea, adding 20-40% by weight of water, and stirring at 70-90° C. until the solution is clear and transparent, thereby obtaining the natural eutectic solvent; preferably, the molar ratio of the betaine to the urea is 2:1; preferably, 40% by weight of water is added; preferably, magnetic stirring is performed at 80° C. for 60.0 minutes until the solution is clear and transparent.

[0035] In another embodiment, the cartilage raw material is made into bone powder, mixed with the natural low eutectic solvent, heated for extraction, centrifuged, the supernatant is collected and filtered, ethanol is added, the mixture is allowed to stand, the precipitate is collected by centrifugation, and dialyzed to obtain the chondroitin sulfate; optionally, the bone powder is mixed with the natural low eutectic solvent at a liquid-solid ratio of 20 to 50:1, preferably, the liquid-solid ratio is 30:1; optionally, the cartilage raw material and the natural low eutectic solvent are extracted at 90 to 110° C. for 2 to 2.5 hours, preferably, at 110° C. for 2 hours; optionally, ethanol is added until the volume ratio of ethanol reaches 70 to 80%, preferably, the volume ratio is 75%; optionally, dialyzed in ultrapure water using a 10,000 Da dialysis bag, preferably, purified and dialyzed in ultrapure water at 4° C. for 2 days in a 10,000 Da dialysis bag, and the dialysate is collected and freeze-dried.

[0036] In another embodiment, the method further comprises: taking the filtered supernatant, removing ethanol, and recovering the natural deep eutectic solvent; optionally, removing ethanol from the filtrate on a rotary evaporator to recover the natural deep eutectic solvent.

[0037] In another embodiment, bovine laryngeal bone powder is added to NADES at a liquid-to-solid ratio of 30:1 and treated at 110°C for 2 hours. After cooling, the sample is centrifuged at 5,000 × g for 30 minutes, and the supernatant is collected by filtration through 6 layers of gauze. Ethanol is added to bring the ethanol content to 75%, and the mixture is allowed to stand at 4°C overnight. The precipitate is then collected by centrifugation at 10,000 × g for 15 minutes. The recovered chondroitin sulfate is placed in a 10,000 Da dialysis bag and dialyzed in ultrapure water at 4°C for 2 days. The dialysate is collected and lyophilized to obtain purified chondroitin sulfate.

[0038] The following is a description of specific examples and tests.

[0039] 1 Materials and Methods

[0040] 1.1 Experimental materials and reagents

[0041] Bovine laryngeal cartilage raw material was purchased from Hebei Fucheng Wufeng Food Co., Ltd., thoroughly washed with tap water to remove residual beef, and then dried in an oven at 55°C for 48 h. The dried material was pulverized in a grinder and ball-milled to obtain bone powder, which was then sieved and sealed for later use. All reagents used in the experiment were purchased from Shanghai Yuanye Co., Ltd. and were of analytical grade.

[0042] 1.2 Determination of bovine laryngeal cartilage composition

[0043] The protein content of cartilage was determined by the Kjeldahl method according to GB / T 5009.5-2016. The fat content of cartilage was determined by Soxhlet extraction according to GB / T 5009.6-2016. The ash content of cartilage was determined by muffle furnace ignition according to GB / T 5009.4-2016. The total carbohydrate content of cartilage was calculated according to the formula (1) according to GB / Z 21922-2008:

[0044] Carbohydrate % = 100 - C pro -C fat -C ash (1)

[0045] Among them C pro 、C fat 、C ash are the contents of protein, fat and ash in dry cartilage (%) respectively.

[0046] 1.3 NADES preparation

[0047] The composition of NADES is as shown in Table 1. Betaine (Bet) or choline chloride (ChCl) is used as a hydrogen bond acceptor (HBA), and sugars, amides, and polyols are used as hydrogen bond donors (HBD) in the specified molar ratio. 20 wt% of water is added, and the mixture is magnetically stirred at 80°C for 60.0 min until the solution is clear and transparent. The mixture is then cooled and sealed for later use.

[0048] 1.4 NADES property determination

[0049] The NADESs at 25°C were weighed using a pycnometer and the density was recorded. The viscosity of the sample was measured using a rheometer at a steady state of 25°C. The pH electrode of a PHSJ-3F pH meter was immersed in a centrifuge tube containing NADESs to measure the pH value of the NADESs system at 25°C and record the data. The dye Nile red was dissolved in methanol, and an appropriate amount of the dye-methanol solution was mixed with the NADESs. The methanol was evaporated with nitrogen purge, and the maximum absorption wavelength λmax (nm) of the mixture was scanned using a UV-visible spectrophotometer. The total polarity E of the solution was calculated using (Equation 2) NR :

[0050] E NR =28591 / λmax (2)

[0051] 1.5 Comparison of extraction efficiency of CS using different NADES

[0052] The extraction efficiency of chondroitin sulfate from bovine laryngeal cartilage using 12 natural deep eutectic solvents was investigated. Water was used as a solvent instead of the natural deep eutectic solvent, and the treatment conditions remained unchanged to investigate the recovery rate of chondroitin sulfate from bovine laryngeal cartilage (Formula 3):

[0053] CS recovery rate (%) = (100-P pro )×M cs / M car (3)

[0054] Among them, M cs and P pro are the weight and protein content of CS samples, M car is the total weight of carbohydrates in the corresponding dry basis cartilage.

[0055] The extraction method includes: adding 2g (dry weight) of bovine laryngeal bone powder to NADES at a liquid-to-solid ratio of 30:1 and liquefying at 110°C for 2 hours. After cooling, the sample is centrifuged at 5,000×g for 30 minutes, and the supernatant is collected by filtration through 6 layers of gauze. Ethanol is added to bring the ethanol content to 75%, and the solution is allowed to stand at 4°C overnight. The precipitate is collected by centrifugation at 10,000×g for 15 minutes. The recovered chondroitin sulfate is placed in a 10,000Da dialysis bag and dialyzed in ultrapure water at 4°C for 2 days. The dialysate is collected and lyophilized to obtain purified chondroitin sulfate.

[0056] Table 1 12 natural deep eutectic solvents

[0057]

[0058]

[0059] 1.6 Comparison of the effects of different factors on extraction rate

[0060] Taking chondroitin sulfate content as an indicator and keeping other single factors unchanged, the effects of four single factors on the extraction of chondroitin sulfate, namely liquid-to-solid ratio (10:1, 20:1, 30:1, 40:1 and 50:1 mL / g), water content (5wt%, 20wt%, 40wt%, 60wt% and 80wt%), temperature (90, 100, 110, 120 and 130℃), and time (60, 90, 120, 150 and 180min), were investigated. The single factor variables were replaced with the corresponding conventional quantities in the process flow to investigate the influence of each factor on the extraction effect of lignan compounds. Each of the above treatments was repeated 3 times.

[0061] 1.7 NADES Reusability

[0062] The ethanol in the filtrate was removed on a rotary evaporator to recover the NADES. The recovered NADES were reused for chondroitin sulfate extraction without further purification to assess their reusability. Samples designated Rn represent CS extracted from the NADES obtained from the nth recovery.

[0063] 1.8 Analytical methods

[0064] 1.8.1 Determination of protein, uronic acid and sulfate content

[0065] The protein content was determined by the BCA method, the uronic acid content in CS was determined by the sulfuric acid-carbazole method, and the sulfate content in CS was determined by the gelatin turbidimetry method.

[0066] 1.8.2 Monosaccharide composition analysis

[0067] The monosaccharide composition of the polysaccharides was determined by ion chromatography. 1 mL of 2 mol / L trifluoroacetic acid (TFA) was added to the sample and the mixture was nitrogen-sealed at 110°C for 4 h. The mixture was then dried under nitrogen and dissolved in ultrapure water. 130 μL of 0.3 mol / L sodium hydroxide solution and 150 μL of 70-phenyl-30-methyl-1-pyrazolone (PMP) reagent were added to each sample and standard mixture, and the mixture was derivatized at 70°C for 30 min. Chloroform was added to remove the PMP reagent. The upper aqueous phase was diluted threefold and filtered through a 0.2 μm filter. Chromatographic conditions: EC C18 (4.6 mm × 150 mm, 2.7 μm) analytical column, flow rate: 0.4 mL / min, wavelength: 245 nm.

[0068] 1.8.3 Infrared spectroscopy (FT-IR) analysis

[0069] A potassium bromide pellet containing 0.5% of the sample was prepared, and the infrared spectrum of the sample was obtained in the scanning range of 4000-400 cm-1.

[0070] 1.8.4 Molecular weight analysis

[0071] The molecular weight of CS was determined by liquid chromatography coupled with an 18-angle laser light scattering spectrometer and a differential detector (HPSEC-MALLS-RI), setting the refractive index increment dn / dc to 0.135. Chromatographic conditions: TSK gel G5000PWxl (7.8×300 mm) as the analytical column; the mobile phase was 1 mol / L sodium sulfate solution; and the flow rate was 0.5 mL / min.

[0072] 2 Results and Analysis

[0073] 2.1 Analysis of bovine laryngeal cartilage components

[0074] The results showed that the water content of bovine laryngeal cartilage was about 70.28% of its fresh weight. Analysis of the nutritional components of its dry matter showed that laryngeal cartilage had the highest protein content, accounting for about 63.65% of its dry weight, followed by carbohydrates at 17.41% (Table 2).

[0075] Table 2 Basic composition of bovine laryngeal cartilage

[0076]

[0077] 2.2 Properties of NADESs

[0078] Twelve NADESs with different molar ratios of HBA / HBD were synthesized from different hydrogen bond acceptors and hydrogen bond donors. During the screening process, the density, viscosity, pH value and polarity of NADESs were determined (Table 1).

[0079] The physical properties of NADESs, including density, viscosity, pH, and polarity, play an important role in the extraction process. The results showed that the viscosity of NADESs was negatively correlated with the recovery rate and positively correlated with the density ( Figure 9 ), lower viscosity contributes to higher solubility of CS. According to the hole theory, density and viscosity may affect the steric hindrance in NADESs. Since the average size of the hole is similar to the size of the corresponding ion, the smaller urea is easier to move in the solvent and transfer charges between other charge carriers compared to other HBDs, which increases the entropy of the system, thereby weakening the strength of hydrogen bonds between molecules and producing lower density and viscosity. After adding water for dilution, the density and viscosity of the solvent decrease, and the hydrogen bond interaction between NADESs components gradually weakens, enhancing the mass transfer movement of cartilage matrix to the solvent, thereby improving the recovery rate. Similarly, excess water molecules will destroy the hydrogen bond network, which is not conducive to the extraction process ( Figure 10 ), which is consistent with the results in the literature. The appropriate pH value can improve the stability of the target extract in a specific solvent. The pH is positively correlated with the recovery rate. This may be because the acidic CS and alkaline NADESs are more likely to form soluble salts, attacking the O-glycosidic bonds in the cartilage matrix, dissolving more polysaccharide components into the extract, and protecting the structural stability of the CS. NR Value (E NR The lower the value, the higher the polarity) is highly positively correlated with viscosity and negatively correlated with recovery rate, indicating that solvents with higher polarity have larger molecular dipole moments, lower viscosity, and stronger solubility for cartilage matrix. After adding polar water molecules, ion movement is easier and the polarity of the solvent is enhanced. The correlation analysis of the physicochemical properties of NADESs further confirmed its correlation with recovery rate. According to the final screening based on the physicochemical properties, it was determined that NADES-3 with a water content of 40wt% has a good mass transfer effect ( Figure 10 ).

[0080] 2.3 Screening of NADES

[0081] Based on the physicochemical properties analysis of NADES with different compositions, the extraction ability of chondroitin sulfate by NADES containing betaine or choline chloride as HBA and six different sugars, amides, and polyols as HBD was investigated and compared with the traditional extraction solvent (water).

[0082] This study compared the extraction of CS from laryngeal cartilage using the NADESs method with the traditional extraction solvent (water). When NADESs were applied to the cartilage matrix, new hydrogen bonds were formed competitively with carbohydrates, resulting in the destruction of the hydrogen bond network in the cartilage matrix. This allowed CS to separate from collagen and dissolve in the extract. The results showed that ( Figure 1 ), the recovery rates of CS extracted by different NADESs were significantly higher than those by the water extraction method, but there were also certain differences. Among them, NADES-3 (betaine / urea, Bet-U) had the highest recovery rate of 93.05% and a purity of 75.3%. The recovery rate of NADES-9 (choline chloride / urea, ChCl-U) was also high. As for HBDs, this may be due to the smaller molecular size of urea, which forms weaker hydrogen bonds in the solvent. Among them, the recovery rate of sucrose-based NADES was the lowest. The annular spatial structure of the sucrose molecule makes the molecular distance shorter, and the hydrogen bond interaction force formed is too strong, resulting in a decrease in the recovery rate. In addition, the recovery rate of the three-hydroxyl glycerol-based NADES was slightly lower than that of the 1,4-butanediol-based and ethylene glycol-based NADES. This may be because the more hydroxyl groups there are, the more complex the hydrogen bond network formed, resulting in differences in extraction effects, which is consistent with the literature. 1,4-Butanediol-based NADES is slightly lower than ethylene glycol-based NADES, possibly because molecules with long carbon chains have stronger London dispersion forces, which increases their flow resistance. As for HBAs, Bet acts as a good permeabilizer compared to ChCl, which can offset the denaturing effect of urea on proteins. This synergistic effect may be the reason for the higher recovery rate of NADES-3. The protein content, sulfate and uronic acid content in CS extracts were determined, and the results showed that the glycosidic bond breaking ability of NADESs was not much different from that of water ( Figure 2 ).

[0083] 2.4 Structural characterization and analysis of extracted products

[0084] Depend on Figure 3 It can be seen that the characteristic absorption bands of cartilage hydrolysate sample, amide A band, amide B band, amide I band, amide II band and amide III band can all be detected. The amide A band is related to NH stretching vibration and hydrogen bonding. The amide A band of cartilage hydrolysate sample is located at 3363 cm -1 The wavelength indicates that the NH group participates in hydrogen bond formation. The amide B band is located at 2980 cm -1The amide I band is located at a wavelength of 1658, which is caused by the stretching vibration of the C=O bond. The amide II band is located at 1554 cm -1 The wavelength is due to the bending vibration of the NH bond and the stretching vibration of the CH bond. The amide III band is located at 1253 cm -1 The wavelength is usually related to NH bending vibration and C=N stretching vibration. Compared with the infrared spectrum of chondroitin sulfate sample, the spectrum of cartilage hydrolyzate is at 1658cm -1 and 1554cm -1 New absorption peaks appeared at , which belonged to amide I band and amide II band respectively.

[0085] Depend on Figure 4 It can be seen that the infrared spectra of the chondroitin sulfate sample prepared in the experiment are basically consistent with those of the chondroitin sulfate A standard. The chondroitin sulfate sample and the chondroitin sulfate A standard are at 3350 cm -1 and 3371cm -1 A broad absorption peak appeared at 2930 cm, indicating that the component contained hydroxyl structure of chondroitin sulfate. -1 A weak absorption peak appears near the CH2 or CH3 stretching vibration. The characteristic absorption peak at 1550cm-1 indicates the presence of carbonyl (-C=O-) and NH bond in the sample, indicating the presence of acetylamino structure in each component. -1 and 1045cm -1 The absorption peak at 850cm is due to the stretching vibration of SO and -COS. The above spectral characteristics are consistent with the literature reports. The sulfate group of type A CS (CSA) is at the C4 position, and the axial stretching vibration peak is at 850cm -1 Nearby, 850cm -1 The peak can be considered as chondroitin-4-sulfate, 820 cm -1 The peak of chondroitin sulfate is 852 cm -1 and 827cm -1 It showed that the sample was mainly composed of chondroitin sulfate A.

[0086] 2.5 Effect of liquid-solid ratio on CS extraction

[0087] NADES-3 (betaine-urea, Bet-U) was selected for the extraction of chondroitin sulfate in subsequent experiments. The effects of different liquid-to-solid ratios (10, 20, 30, 40, 50:1 mL / g) on the extraction of chondroitin sulfate were analyzed under fixed temperature (110°C), time (120 min), and water content (20 wt%). The results are as follows: Figure 5The results show that the recovery rate of the extract first increases significantly and then remains stable with the increase of NADES dosage. When the liquid-solid ratio increases to 20:1mL / g, the recovery rate of chondroitin sulfate is greatly improved. This is because the amount of bovine laryngeal cartilage powder dissolved in the solvent gradually increases with the increase of solvent amount. When the liquid-solid ratio is increased to 30:1mL / g again, the recovery rate of the target compound tends to be stable as a whole. When the NADES dosage is continued to be increased, the amount of chondroitin sulfate dissolved is the largest and the recovery rate does not increase. The content of uronic acid and sulfate in the extracted chondroitin sulfate was determined, and it was found that their change pattern was similar to the recovery rate. The increase of liquid-solid ratio has little effect on purity, which is about 75±2%. In order to effectively utilize NADES and avoid waste caused by excessive solvent, this application selects a liquid-solid ratio of 30:1mL / g.

[0088] 2.6 Effect of solvent water content on CS extraction

[0089] Water increases the polarity of NADES and reduces its viscosity, which can promote the dissolution of target compounds to a certain extent. Figure 6 As shown, the recovery rate of the extract first increases and then decreases with the increase of water content. When the water content is 5wt%, the recovery rate of chondroitin sulfate is 51.91%. This is because 5wt% NADES-3 will crystallize at room temperature and cannot fully contact with the cartilage powder and penetrate into the cells. When the water content of NADES-3 exceeds 40%, the excess water destroys the tight hydrogen bond structure of NADES-3, promotes the formation of a simple aqueous solution, and affects the extraction effect of the solvent. It was observed that the uronic acid content in the extracted chondroitin sulfate was not much different from the purity, and the change pattern of the sulfate content was similar to the recovery rate, indicating that the extraction effect of NADES-3 with a water content of 40wt% is better and the extracted chondroitin sulfate is of better quality.

[0090] 2.7 Effect of extraction temperature on CS extraction effect

[0091] Temperature can significantly reduce the viscosity of the solvent, accelerate the movement of ions in the solvent, and promote the solubility of the target compound. Figure 7 In the temperature range of 90-110°C, the yield of polysaccharides increases with the increase of temperature. The increase in temperature promotes the molecular diffusion rate. When the temperature rises to 110°C, the yield of polysaccharides decreases. This may be because the high temperature destroys the structure of the extracted chondroitin sulfate, causing the polysaccharide to decompose and the yield to decrease. In addition, the high temperature causes the purity, uronic acid and sulfate content of the extracted chondroitin sulfate to decrease. The Maillard reaction caused by high temperature may consume the target extract and increase the protein content in chondroitin sulfate. Therefore, this application selects 110°C as the optimal extraction temperature.

[0092] 2.8 Effect of extraction time on CS extraction effect

[0093] The longer the extraction time, the higher the cost. Figure 8 As can be seen, the extraction yield increases continuously with the passage of extraction time in the 60-120 min range, reaching its highest yield at 120 min. However, the extraction yield decreases between 120 and 180 min. This may be because polysaccharides continuously infiltrate the extract within a certain period of time, leading to a continuous increase in yield, which then reaches saturation with further increase in extraction time. Furthermore, polysaccharides are easily hydrolyzed at longer extraction times. Therefore, the extraction time selected in this application was 120 min.

[0094] 2.9 NADES Recyclability Analysis

[0095] One of the most advantageous advantages of DES extraction is its superior recyclability compared to traditional extraction methods. In this study, NADES-3 with 40 wt% water content was used as an example to evaluate the recyclability of CS extraction by NADES. Figure 11 ), with the increase of repeated use, the viscosity of NADES-3 gradually increases, the pH value decreases, and the E NR The value increased (polarity weakened), which may be due to the fact that some collagen remained in the solvent during the multiple extraction processes, increasing the steric hindrance between molecules and weakening the fluidity of the solvent. Figure 12 After being reused five times, NADES-3 still maintained a good recovery rate (about 91.26%). As the number of recovery times increased, the purity and uronic acid content of the extracted CS gradually decreased. After being reused five times, the purity only decreased by 7.51%. This may be because the large molecular protein impurities in the regenerated NADES-3 weakened the hydrogen bond interaction force in the solvent, indicating that the NADES-3 prepared in this application has recycling and reuse value in the extraction of CS.

[0096] 3 Summary

[0097] In summary, the highest chondroitin sulfate recovery rate of 96.24% was achieved using NADES-3 (betaine / urea, 1:2 molar ratio), 40 wt% water content, a liquid-to-solid ratio of 30:1 mL / g, an extraction temperature of 110°C, and an extraction time of 2 h. Using a conventional water-based extraction method, the chondroitin sulfate recovery rate was 11.86% under the same conditions. The extraction efficiency using NADES was 711.47% higher than that of the conventional water-based extraction method. The effects of the physicochemical properties of NADES on CS recovery were analyzed, revealing a negative correlation between recovery and viscosity, a positive correlation with pH and polarity, and a weak correlation with density. Ultimately, it was determined that NADES-3 with a 40 wt% water content exhibited lower density, lower viscosity, stronger alkalinity, and higher polarity, resulting in improved mass transfer for CS extraction. After five cycles of reuse using this NADES-3, the CS recovery rate remained at 91.26%. Therefore, using NADES-3 for extraction offers low cost and minimizes wastewater discharge.

[0098] The number of equipment and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the chondroitin sulfate extraction process of the present invention will be readily apparent to those skilled in the art.

[0099] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A chondroitin sulfate extraction process, characterized in that: include: A natural deep eutectic solvent is used as an extraction solvent to extract chondroitin sulfate from a cartilage raw material. The raw materials for preparing the natural deep eutectic solvent include betaine and urea.

2. The chondroitin sulfate extraction process according to claim 1, wherein The preparation method of the natural deep eutectic solvent comprises: Betaine and urea are mixed, 20-40% by weight of water is added, and the mixture is stirred at 70-90° C. until the solution becomes clear and transparent, thereby obtaining the natural eutectic solvent.

3. The chondroitin sulfate extraction process according to claim 2, wherein The molar ratio of the betaine to the urea is 1.8-2.2:

1.

4. The chondroitin sulfate extraction process according to claim 1, wherein The cartilage raw material is made into bone powder, mixed with the natural low eutectic solvent, heated for extraction, centrifuged, the supernatant is collected and filtered, ethanol is added, the mixture is allowed to stand, centrifuged to collect the precipitate, and dialyzed to obtain the chondroitin sulfate.

5. The chondroitin sulfate extraction process according to claim 4, wherein The bone powder is mixed with the natural deep eutectic solvent at a liquid-to-solid ratio of 20 to 50:

1.

6. The chondroitin sulfate extraction process according to claim 4, wherein The cartilage raw material and the natural deep eutectic solvent are extracted at 90-110° C. for 2-2.5 hours.

7. The chondroitin sulfate extraction process according to claim 4, wherein Add ethanol until the volume ratio of ethanol reaches 70~80%.

8. The chondroitin sulfate extraction process according to claim 4, wherein The solution was dialyzed in ultrapure water using a 10,000 Da dialysis bag.

9. The chondroitin sulfate extraction process according to claim 4, wherein: Also includes: The filtered supernatant is taken, ethanol is removed, and the natural deep eutectic solvent is recovered.

10. The chondroitin sulfate extraction process according to claim 4, wherein: The cartilage raw material is bovine laryngeal cartilage.

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