Preparation of small-molecular chondroitin sulfate preparation and application of small-molecular chondroitin sulfate preparation in protection of bone

Abstract

The invention discloses preparation of a small-molecule chondroitin sulfate preparation and application of the small-molecule chondroitin sulfate preparation to protection of bone joints of dogs and cats, and relates to the technical field of chondroitin sulfate preparation processing. The phospholipid has the advantages of improving the viscosity of enzymatic hydrolysate and improving the enzymolysis efficiency during chondroitin sulfate enzymolysis on one hand, and reducing the hygroscopicity of chondroitin sulfate enzymolysis products after enzymolysis on the other hand, improving the fluidity of the chondroitin sulfate enzymolysis products and improving the use convenience of the chondroitin sulfate enzymolysis products in product processing on the other hand; the obtained product can effectively maintain bone joint health of dogs and cats and relieve arthritis symptoms.

Description

Technical Field

[0001] This invention relates to the field of chondroitin sulfate processing technology, specifically to a method for preparing a small molecule chondroitin sulfate preparation with low hygroscopicity and high flowability, and its application in dog and cat food. Background Technology

[0002] Chondroitin sulfate is a naturally occurring glycosaminoglycan widely found in animal cartilage, synovial fluid, and connective tissue. Studies have shown that chondroitin sulfate plays a role in maintaining bone and joint health, such as improving arthritis, promoting bone and joint repair, and relieving joint pain. The small-molecule chondroitin sulfate obtained after enzymatic hydrolysis has a short molecular chain, good activity, and high bioavailability, attracting researchers' attention. However, chondroitin sulfate has strong hygroscopicity, increasing viscosity after absorbing water. This presents two problems: firstly, during enzymatic hydrolysis, the high viscosity of the chondroitin sulfate enzymatic hydrolysis system with high substrate concentration hinders efficient hydrolysis; secondly, after enzymatic hydrolysis, the chondroitin sulfate hydrolysate is prone to caking and clumping, resulting in poor flowability, reducing the convenience of chondroitin sulfate in production and processing, and thus limiting the application of chondroitin sulfate hydrolysate in related products.

[0003] Phospholipids are a general term for phosphoric acid lipids, including lecithin, cephalin, and inositol phospholipids, among others. Currently, there are no reports on the use of phospholipids to prepare low-hygroscopic, high-flowability small-molecule chondroitin sulfate. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a small molecule chondroitin sulfate preparation with low hygroscopicity and high flowability, and its application in dog and cat food.

[0005] In a first aspect, the present invention provides a method for preparing a small molecule chondroitin sulfate formulation with low hygroscopicity and high flowability, comprising the following steps: (1) Preparation of phospholipid ethanol solution: Add phospholipid powder to ethanol-water solution, stir and mix, centrifuge and keep the supernatant, which is the phospholipid ethanol solution; (2) Preparation of the mixture: Add the phospholipid ethanol solution from step (1) to the chondroitin sulfate solution and stir to mix; (3) Preparation of small molecule chondroitin sulfate preparation: Chondroitin sulfate enzyme is added to the mixture in step (2) for enzymatic hydrolysis. The enzymatic hydrolysis is assisted by stirring and ultrasound. After the enzyme activity of the obtained hydrolysate is inactivated by boiling water bath, it is concentrated and dried to obtain small molecule chondroitin sulfate preparation.

[0006] In step (1) above, the phospholipid ethanol solution is prepared by adding phospholipid powder to an ethanol-water solution at a ratio of 200~240g / L, wherein the volume percentage of ethanol in the ethanol-water solution is 90%~100%; when stirring and mixing, the temperature is 60~80℃ and the time is 30~60min.

[0007] Preferably, the phospholipid powder is egg yolk phospholipid powder prepared by processing egg yolk as the source.

[0008] In step (2) above, the chondroitin sulfate solution is prepared by adding chondroitin sulfate to water at a ratio of 200-300 g / L. The volume ratio of phospholipid ethanol solution to chondroitin sulfate solution is 1:10-1:20. The temperature and time for stirring and mixing are 60-80℃ and 10-20 min.

[0009] In step (3) above, the preparation of small molecule chondroitin sulfate preparation is as follows: the pH is 7.5~8.5 and the temperature is 30~40℃ during the enzymatic hydrolysis process; chondroitin sulfate enzyme is added to the enzymatic hydrolysate at a volume ratio of 0.03%~0.05%, and the enzyme activity of chondroitin sulfate enzyme is 5~10U / mL.

[0010] In step (3) above, the preparation of small molecule chondroitin sulfate preparation is as follows: during enzymatic hydrolysis, the stirring speed is 80~120r / min, the ultrasonic frequency is 20~40kHz, and the power density is 40~60W / L; the enzymatic hydrolysis time is 2~3h.

[0011] A second aspect of the present invention provides the application of a small molecule chondroitin sulfate preparation prepared by the above method in dog and cat food.

[0012] The application method is to feed the small molecule chondroitin sulfate preparation prepared by the above method to dogs and cats by direct feeding, mixing it into dog and cat food (or snacks), or adding it as a formula component to dog and cat food (or snacks).

[0013] Furthermore, the small molecule chondroitin sulfate preparations prepared by the above method are used in products that maintain the bone and joint health of dogs and cats.

[0014] Furthermore, the small molecule chondroitin sulfate preparations prepared by the above method can be used in other pets such as rabbits, foxes, and hamsters.

[0015] The present invention has the following beneficial effects: The method of this invention for preparing small molecule chondroitin sulfate formulations has the advantages of reducing the viscosity of high substrate concentration chondroitin sulfate enzymatic hydrolysate and reducing the hygroscopicity of chondroitin sulfate enzymatic hydrolysate products. The specific reasons are as follows: The existing problems are: (1) During the enzymatic hydrolysis of chondroitin sulfate, the viscosity of the hydrolysis system has a great influence on the hydrolysis efficiency. The viscosity of the chondroitin sulfate solution with low substrate concentration is low and has little impact on the hydrolysis efficiency. However, the viscosity of the chondroitin sulfate solution with high substrate concentration is high, which will reduce the flow and dispersion of chondroitin sulfate enzyme and the contact opportunity between the enzyme and the substrate, thereby reducing the hydrolysis efficiency. The reason for the high viscosity of the chondroitin sulfate hydrolysate with high substrate concentration is that chondroitin sulfate is composed of glucuronic acid and N-acetylgalactosamine, which are linked alternately. It contains a large number of hydrophilic hydroxyl, sulfonic acid and carboxyl groups. After absorbing water, it forms a hydrogel layer on its surface, thereby increasing the viscosity. (2) After the enzymatic hydrolysis of chondroitin sulfate, its dried product has strong hygroscopicity. It is easy to cause problems such as caking and poor flowability during storage and processing. This leads to problems such as inaccurate weighing, poor transportation, inefficient screening and uneven mixing in the production and processing of the product, which limits the application of chondroitin sulfate in related products. This is mainly because the surface of chondroitin sulfate hydrolysate contains a large number of hydrophilic hydroxyl, sulfonic acid and carboxyl groups, which absorb moisture from the air, thus making the chondroitin sulfate hydrolysate exhibit strong hygroscopicity.

[0016] This invention utilizes purified phospholipids to simultaneously reduce the viscosity of high-substrate-concentration chondroitin sulfate hydrolysate and the hygroscopicity of the chondroitin sulfate hydrolysate. The preferred egg yolk phospholipid contains amphiphilic substances such as lecithin and cholesterol. Lecithin consists of a hydrophilic portion (choline) and a hydrophobic portion (fatty acid chain). Choline carries a positive charge and can adhere to the surface of chondroitin sulfate (carrying a negative charge) through electrostatic adsorption. Cholesterol consists of a hydrophilic portion (hydroxyl group) and a hydrophobic portion (steroidal ring and alkane side chain). The hydroxyl group has strong polarity and readily binds to the hydroxyl, carboxyl, and sulfonic acid groups on chondroitin sulfate through hydrogen bonds, thus adhering to the surface of chondroitin sulfate. The hydrophobic portions of lecithin and cholesterol face outwards. On the one hand, during enzymatic hydrolysis, this inhibits the formation of a hydrogel layer on the surface of chondroitin sulfate, thereby reducing the viscosity of the high-substrate-concentration chondroitin sulfate hydrolysate. On the other hand, after enzymatic hydrolysis, this makes the surface of the dried chondroitin sulfate product exhibit hydrophobic properties, effectively preventing the contact between moisture in the air and chondroitin sulfate, thus effectively reducing the hygroscopic and sticky nature of the dried product after enzymatic hydrolysis, overcoming the shortcomings of traditional small-molecule chondroitin sulfate products, such as high hygroscopicity and poor flowability. Furthermore, purification of phospholipids reduces the content of impurities such as phosphatidylcholine and inositol phospholipids that cannot effectively adsorb and attach to chondroitin sulfate, and increases the purity of lecithin and cholesterol. This contributes to the efficient enzymatic hydrolysis of chondroitin sulfate and effectively ensures the anti-hygroscopic effect of the hydrolysate.

[0017] In the enzymatic hydrolysis step of preparing small molecule chondroitin sulfate preparations according to the method of the present invention, the viscosity of the chondroitin sulfate enzymatic hydrolysate is significantly reduced, decreasing by 25.94% to 31.18% compared to the control (Control Example 3). The small molecule chondroitin sulfate preparations prepared according to the method of the present invention exhibit reduced hygroscopicity and improved flowability compared to chondroitin sulfate. Their hygroscopicity is reduced by 22.33% to 25.54% compared to the control (Control Examples 2-3), and their flowability test repose angle is reduced by 10.03% to 12.68% compared to the control (Control Examples 2-3). The small molecule chondroitin sulfate preparations prepared by the method of the present invention have a smaller molecular weight, with an average molecular weight 22.57% to 88.18% lower than the control (Control Examples 2-3). The small-molecule chondroitin sulfate preparation prepared according to the method of the present invention has functional activity in maintaining the health of canine and feline bones and joints. After using the small-molecule chondroitin sulfate preparation, the proliferation rate of osteoblasts in dogs and cats increased by 61.54%~75.97% and 54.86%~72.55% respectively compared with chondroitin sulfate (Control Example 2). After using the small-molecule chondroitin sulfate preparation, the joint swelling, joint temperature, and motor function impairment score of dogs with arthritis decreased by 6.82%~11.36%, 5.71%~7.30%, and 31.25%~50.00% respectively compared with chondroitin sulfate (Control Example 2). After using the small-molecule chondroitin sulfate preparation, the joint swelling, joint temperature, and motor function impairment score of cats with arthritis decreased by 8.89%~14.07%, 4.22%~5.52%, and 33.33%~50.00% respectively compared with chondroitin sulfate (Control Example 2). Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of the small molecule chondroitin sulfate formulation in this invention. Detailed Implementation

[0019] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention. Those skilled in the art can make non-essential improvements and adjustments to the invention based on the above description.

[0020] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The source, trade name, and components of the reagents used, if necessary, are indicated upon their first appearance, and subsequent use of the same reagents, unless otherwise specified, are identical to the initial indication.

[0021] The phospholipid raw materials used in this embodiment and the comparative example are commercially available, and are prepared by processing egg yolks.

[0022] In this embodiment and the control example, canine osteoblasts and feline osteoblasts were provided by the Pet Culture Collection Worldwide (PETCC).

[0023] The solution viscosity testing method in this embodiment and the comparative example: The viscosity of the solution was measured using a viscometer with a low-speed rotor selected. After stirring the sample solution in a water bath at 30°C for 20 minutes, it was transferred to the viscometer, ensuring the rotor was completely submerged. The rotor speed was set to 12 r / min, the instrument was started, and the viscosity reading was recorded.

[0024] The hygroscopicity test method for materials in this embodiment and the comparative example: Accurately weigh 10.000g (recorded as m1) of the test material and spread it evenly in a dry glass petri dish with a diameter of 10cm. Place the petri dish open in a desiccator containing 500g of supersaturated sodium chloride solution, and place the desiccator in a 37℃ constant temperature oven for 20 hours for hygroscopicity testing. Remove the petri dish and weigh it again (recorded as m2), and calculate the hygroscopic rate according to the formula: Hygroscopic rate (%) = (m2-m1)×100 / m1.

[0025] The material flowability testing method in this embodiment and the comparative example: Take a test sample and perform a material flowability test according to the method of "GB / T11986-1989 Measurement of Angle of Repose of Surfactant Powders and Particles". The result is expressed as the angle of repose.

[0026] The method for testing the average molecular weight of materials in this embodiment and the comparative example: Take test samples and test the average molecular weight of the materials according to the method of "GB31645-2018 National Food Safety Standard Collagen Peptide".

[0027] The method for testing osteoblast proliferation in dogs and cats in this embodiment and the control example: Canine or feline osteoblasts were digested with trypsin and collected in centrifuge tubes. The cells were centrifuged at 1100 rpm for 4 minutes at 4°C, and the cell pellet was collected. 1 mL of complete culture medium was added. 20 μL of the cell resuspended cells were stained with trypan blue for cell counting, and then the cells were quantitatively diluted. 4Cells were seeded at a density of 1% (m / v) in 96-well plates and incubated at 37°C for 24 h. Test samples were dissolved in sterile physiological saline at 1% (m / v), shaken for 20 min, centrifuged, and the supernatant was filtered through a 0.22 μm filter to obtain the test sample solution. 100 μL of the test sample solution was added to each well, gently shaken, and incubated for another 24 h. After the incubation time was reached, the culture medium was removed, and 110 μL of 10% CCK-8 solution was added to each well, mixed, and incubated at 37°C for 3–4 h. The absorbance was measured at 450 nm. In the control group, sterile physiological saline was used instead of the test sample solution, with all other procedures performed identically. Cellless wells served as the blank group. Cell proliferation rate was calculated using the following formula: Cell proliferation rate (%) = (OD value of experimental group - OD value of blank group) × 100 / (OD value of control group - OD value of blank group). The cell viability of the control group is defined as 100%.

[0028] The feeding test method for dogs with rheumatoid arthritis in this embodiment and the control example: Dogs suffering from rheumatoid arthritis were recruited, including six each of the following breeds: Teddy, Schnauzer, Beagle, Pomeranian, Corgi, Chihuahua, French Bulldog, and Chinese Rural Dog. These dogs were evenly divided into six groups of eight (one dog of each breed) based on breed and weight. Each group was supplemented with 0.4 g / kg BW of dog food daily for 14 days.

[0029] The feeding test method for cats with rheumatoid arthritis in this embodiment and the control example: We recruited dogs suffering from rheumatoid arthritis, including six each of Ragdoll, Eastern Shorthair, American Shorthair, British Shorthair, Garfield, Maine Coon, Siamese, and Chinese domestic cat. These were evenly divided into six groups of eight cats each (one cat of each breed) based on breed and weight. Each group of cats was supplemented with 0.3g / kg BW of their food daily for 14 days.

[0030] The method for testing joint swelling in dogs and cats in this embodiment and the control example: Wrap a suture around the point of maximum swelling in the dog or cat joint and measure its length, which is the circumference of the swollen joint. Use the average circumference of a normal joint as a reference to calculate the degree of swelling. Swelling degree = (swollen joint circumference - average normal joint circumference) / average normal joint circumference.

[0031] The method for testing joint temperature in dogs and cats in this embodiment and the control example: During the measurement, the dog or cat was brought into a constant temperature and humidity room (temperature 25℃, humidity 50%) at 3 pm on the same day. After resting for 15 minutes to adapt to the environment, the temperature was measured by contacting the probe of the temperature measuring device with the fixed position of the joint.

[0032] The scoring method for motor dysfunction in dogs and cats in this embodiment and the control example: The motor dysfunction index (MDI) is used to evaluate the motor function of dogs and cats using a 4-point rating system (0-3 points). The higher the score, the more severe the motor dysfunction. 0 points: The dog's paws can touch the ground normally, and the gait is normal; 1 point: The swollen paws touch the ground less frequently, the movement speed is slow, and there is mild lameness; 2 points: The swollen paws are often flexed, only briefly touching the ground, the movement speed is slow, and there is moderate lameness; 3 points: The swollen paws are completely off the ground, the willingness to move actively is low, and there is severe lameness.

[0033] Compare with Example 1 In the feeding trials for dogs and cats, they were given their normal daily rations without any supplemental feeding.

[0034] After the canine feeding trial, the joint swelling degree of the dogs with arthritis was 1.42, the joint temperature was 32.4℃, and the motor dysfunction score was 2.4.

[0035] After the cat feeding experiment, the joint swelling degree of the cat with arthritis was 1.44, the joint temperature was 32.1℃, and the motor dysfunction score was 2.3.

[0036] Compare with Example 2 Chondroitin sulfate was used as the test sample. Chondroitin sulfate had a moisture absorption rate of 33.16%, a flowability test angle of repose of 44.26°, and an average molecular weight of 32680 Da.

[0037] After the canine feeding trial, the joint swelling degree of the dogs with arthritis was 1.32, the joint temperature was 31.5℃, and the motor dysfunction score was 1.6.

[0038] After the cat feeding experiment, the joint swelling degree of the cat with arthritis was 1.35, the joint temperature was 30.8℃, and the motor dysfunction score was 1.8.

[0039] Compare with Example 3 Chondroitin sulfate solution was prepared by adding 200 g / L of chondroitin sulfate to water and stirring (at 80℃ for 10 min). Chondroitin sulfate enzyme (5 U / mL activity) was added to the chondroitin sulfate solution at a volume ratio of 0.05%. Enzymatic hydrolysis was performed at pH 7.5 and 30℃ for 2 h. During hydrolysis, the stirring speed was 120 r / min, and ultrasonication was applied simultaneously at a frequency of 20 kHz and a power density of 60 W / L. After hydrolysis, the enzyme activity was inactivated by boiling in a water bath. The hydrolysate was then concentrated and dried to obtain the chondroitin sulfate hydrolysate.

[0040] When preparing chondroitin sulfate hydrolysate, the viscosity of the chondroitin sulfate solution was 667 mPa·s before adding chondroitin sulfate enzyme.

[0041] Chondroitin sulfate hydrolysate was used as the test sample. The hygroscopicity of chondroitin sulfate hydrolysate was 32.34%, the angle of repose in the flowability test was 43.59°, and the average molecular weight was 5578 Da.

[0042] After the canine feeding trial, the joint swelling degree of the dogs with arthritis was 1.21, the joint temperature was 29.4℃, and the motor dysfunction score was 0.9.

[0043] After the cat feeding experiment, the joint swelling degree of the cat with arthritis was 1.18, the joint temperature was 29.3℃, and the motor dysfunction score was 1.1.

[0044] Example 1 Egg yolk phospholipid powder was added to an ethanol solution at a concentration of 240 g / L (ethanol volume percentage 100%), stirred and mixed (at 80℃ for 30 min), and the supernatant was collected by centrifugation; this is the phospholipid ethanol solution. Chondroitin sulfate was added to water at a concentration of 200 g / L to prepare a chondroitin sulfate solution. The phospholipid ethanol solution was then added to the chondroitin sulfate solution (phospholipid ethanol solution to chondroitin sulfate solution volume ratio 1:10) and stirred and mixed (at 80℃ for 10 min). Chondroitin sulfate enzyme (enzyme activity 5 U / mL) was added to the mixture at a volume ratio of 0.05%. Enzymatic hydrolysis was performed at pH 7.5 and temperature 30℃ for 2 h. During enzymatic hydrolysis, the stirring speed was 120 r / min, and ultrasonication was applied simultaneously at a frequency of 20 kHz and a power density of 60 W / L. After enzymatic hydrolysis, the enzyme activity was inactivated by boiling water bath. The hydrolysate was then concentrated and dried to obtain a small molecule chondroitin sulfate preparation.

[0045] When preparing small molecule chondroitin sulfate preparations, the viscosity of the mixture before adding chondroitin sulfate enzyme is 472 mPa·s.

[0046] The small molecule chondroitin sulfate formulation was used as the test sample. The moisture absorption rate of the small molecule chondroitin sulfate formulation was 25.12%, the angle of repose in the flowability test was 39.22°, and the average molecular weight was 4319 Da.

[0047] After the canine feeding trial, the joint swelling degree of the dogs with arthritis was 1.17, the joint temperature was 29.5℃, and the motor dysfunction score was 0.8.

[0048] After the cat feeding experiment, the joint swelling degree of the cat with arthritis was 1.23, the joint temperature was 29.1℃, and the motor dysfunction score was 1.2.

[0049] Example 2 Egg yolk phospholipid powder was added to an ethanol solution (90% by volume) at a concentration of 200 g / L. The mixture was stirred and mixed (60℃ for 60 min), then centrifuged, and the supernatant was collected as the phospholipid ethanol solution. Chondroitin sulfate was added to water at a concentration of 300 g / L to prepare a chondroitin sulfate solution. The phospholipid ethanol solution was then added to the chondroitin sulfate solution (volume ratio of phospholipid ethanol solution to chondroitin sulfate solution was 1:20) and stirred and mixed (60℃ for 20 min). Chondroitin sulfate enzyme (10 U / mL activity) was added to the mixture at a volume ratio of 0.03%. Enzymatic hydrolysis was performed at pH 8.5 and temperature 40℃ for 3 h. During hydrolysis, the stirring speed was 80 r / min, and ultrasonication was applied simultaneously at a frequency of 40 kHz and a power density of 40 W / L. After hydrolysis, the enzyme activity was inactivated by boiling in a water bath. The hydrolysate was then concentrated and dried to obtain a small molecule chondroitin sulfate preparation.

[0050] When preparing small molecule chondroitin sulfate formulations, the viscosity of the mixture before adding chondroitin sulfate enzyme is 494 mPa·s.

[0051] Small molecule chondroitin sulfate formulation was used as the test sample. The moisture absorption rate of the small molecule chondroitin sulfate formulation was 24.83%, the angle of repose in the flow test was 38.65°, and the average molecular weight was 3864 Da.

[0052] After the canine feeding trial, the joint swelling degree of the dogs with arthritis was 1.20, the joint temperature was 29.7℃, and the motor dysfunction score was 1.1.

[0053] After the cat feeding experiment, the joint swelling degree of the cat with arthritis was 1.16, the joint temperature was 29.5℃, and the motor dysfunction score was 0.9.

[0054] Example 3 Egg yolk phospholipid powder was added to an ethanol solution (95% ethanol by volume) at a concentration of 220 g / L. The mixture was stirred and mixed at 70°C for 45 min, then centrifuged, and the supernatant was collected as the phospholipid ethanol solution. Chondroitin sulfate was added to water at a concentration of 250 g / L to prepare a chondroitin sulfate solution. The phospholipid ethanol solution was then added to the chondroitin sulfate solution (volume ratio of phospholipid ethanol solution to chondroitin sulfate solution was 1:15) and stirred and mixed at 70°C for 15 min. Chondroitin sulfate enzyme (enzyme activity 10 U / mL) was added to the mixture at a volume ratio of 0.03%. Enzymatic hydrolysis was performed at pH 8.0 and temperature 35°C for 2.5 h. During hydrolysis, the stirring speed was 100 r / min, and ultrasonication was applied simultaneously at a frequency of 35 kHz and a power density of 50 W / L. After hydrolysis, the enzyme activity was inactivated by boiling in a water bath. The hydrolysate was then concentrated and dried to obtain a small molecule chondroitin sulfate preparation.

[0055] When preparing small molecule chondroitin sulfate preparations, the viscosity of the mixture before adding chondroitin sulfate enzyme is 459 mPa·s.

[0056] Small molecule chondroitin sulfate formulation was used as the test sample. The moisture absorption rate of the small molecule chondroitin sulfate formulation was 24.69%, the angle of repose in the flow test was 38.84°, and the average molecular weight was 4155 Da.

[0057] After the canine feeding trial, the joint swelling degree of the dogs with arthritis was 1.23, the joint temperature was 29.2℃, and the motor dysfunction score was 1.0.

[0058] Following the cat feeding experiment, the joint swelling degree of the cat with arthritis was 1.19, the joint temperature was 29.4℃, and the motor dysfunction score was 1.1.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a small molecule chondroitin sulfate formulation, characterized in that... Follow these steps: (1) Preparation of phospholipid ethanol solution: Add phospholipid powder to ethanol-water solution, stir and mix, centrifuge and keep the supernatant, which is the phospholipid ethanol solution; (2) Preparation of the mixture: Add the phospholipid ethanol solution from step (1) to the chondroitin sulfate solution and stir to mix; (3) Preparation of small molecule chondroitin sulfate preparation: Chondroitin sulfate enzyme is added to the mixture in step (2) for enzymatic hydrolysis. The enzymatic hydrolysis is assisted by stirring and ultrasound. After the enzyme activity of the obtained hydrolysate is inactivated by boiling water bath, it is concentrated and dried to obtain small molecule chondroitin sulfate preparation.

2. The method for preparing the small molecule chondroitin sulfate preparation according to claim 1, characterized in that, In step (1), the phospholipid ethanol solution is prepared by adding phospholipid powder to an ethanol-water solution at a ratio of 200-240 g / L, wherein the volume percentage of ethanol in the ethanol-water solution is 90%-100%; when stirring and mixing, the temperature is 60-80℃ and the time is 30-60 min.

3. The method for preparing the small molecule chondroitin sulfate preparation according to claim 1, characterized in that, In step (1), the phospholipid ethanol solution is prepared by processing egg yolk phospholipid powder.

4. The method for preparing the small molecule chondroitin sulfate preparation according to claim 1, characterized in that, In step (2), the mixture is prepared by adding chondroitin sulfate to water at a ratio of 200-300 g / L to prepare a chondroitin sulfate solution; the volume ratio of phospholipid ethanol solution to chondroitin sulfate solution is 1:10-1:20; the temperature is 60-80℃ and the time is 10-20 min when stirring and mixing.

5. The method for preparing the small molecule chondroitin sulfate preparation according to claim 1, characterized in that, In step (3), the preparation of small molecule chondroitin sulfate preparation is carried out at pH 7.5-8.5 and temperature 30-40℃ during enzymatic hydrolysis. Chondroitin sulfate enzyme is added to the hydrolysate at a volume ratio of 0.03%-0.05%, and the enzyme activity of chondroitin sulfate enzyme is 5-10 U / mL.

6. The method for preparing the small molecule chondroitin sulfate preparation according to claim 1, characterized in that, In step (3), the preparation of small molecule chondroitin sulfate preparation is carried out as follows: during enzymatic hydrolysis, the stirring speed is 80~120r / min, the ultrasonic frequency is 20~40kHz, and the power density is 40~60W / L; the enzymatic hydrolysis time is 2~3h.

7. The application of the small molecule chondroitin sulfate preparation prepared by the method of claims 1-6, which is fed to dogs and cats by direct feeding, mixing into dog and cat food (or snacks), or added as a formula component to dog and cat food (or snacks).

8. The small molecule chondroitin sulfate preparation prepared by the method of claims 1-6, used in products for maintaining bone and joint health in dogs and cats.

9. The small molecule chondroitin sulfate preparation prepared by the method of claims 1-6 is applied to other pets such as rabbits, foxes, and hamsters.

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