Cartilage peptide capable of promoting growth of chondrocyte and preparation method thereof
By preparing chondroitin peptides rich in chondroitin sulfate, hydroxyproline, and hydroxylysine, the problem of lacking the ability to promote chondrocyte growth in existing technologies has been solved, achieving specific repair of damaged chondrocytes and improvement of osteoarthritis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI SEMNL BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-04-24
- Publication Date
- 2026-05-22
Smart Images
Figure SMS_1
Abstract
Description
[0001] This application is a divisional application of application number 202310448770.1 filed on April 24, 2023, entitled "A chondrocyte growth-promoting chondroitin peptide and its preparation method". Technical Field
[0002] This invention belongs to the field of polypeptide preparation technology, specifically relating to a chondroitin peptide that can promote chondrocyte growth and its preparation method. Background Technology
[0003] Osteoarthritis (OA) is a chronic joint disease characterized by degeneration and destruction of articular cartilage and bone hyperplasia. Also known as osteoarthropathy or degenerative osteoarthritis, it can affect most joints throughout the body, including the cervical and lumbar spine, knee, hip, and interphalangeal joints. Osteoarthritis is a progressive and worsening disease. Current treatments aim to slow disease progression, reduce pain, and improve joint function. With an aging population, osteoarthritis will increasingly impact people's physical condition and quality of life as they age.
[0004] Numerous studies have found that chondroitin peptides extracted from animal cartilage can alleviate pain and improve joint flexibility in patients with osteoarthritis. Summary of the Invention
[0005] The purpose of this invention is to provide a chondroitin peptide that can promote the growth of chondrocytes and its preparation method. The obtained chondroitin peptide can specifically repair damaged chondrocytes, promote the generation of extracellular matrix, and play a role in preventing and improving osteoarthritis.
[0006] The objective of this invention can be achieved through the following technical solutions:
[0007] A method for preparing a chondroitin peptide that can promote chondroitin cell growth includes the following steps:
[0008] Step S1, Degreasing: Using animal cartilage as raw material, soak it in NaOH solution for 2-12 hours and then take it out. Wash it with softened water 3-5 times. After washing, add 10 times the volume of softened water to the material to obtain solution a. Then inoculate it with Staphylococcus saprophyticus and Leuconostoc pseudoenteroides and ferment it. After that, drain the fermentation liquid, wash it, and obtain crude cartilage extract.
[0009] Step S2, Steam Explosion: Place the above-mentioned crude cartilage extract into a catapult-type steam explosion machine, control the temperature at 180~270℃, and achieve an explosion power density (EPD) of MW / m³. 3 The pressure is 20~50, and the pressure is maintained for 5~100s to obtain steam-exploded material;
[0010] Step S3, preparation of chondroitin peptides: Add steam-exploded material and softened water to a colloid mill at a mass ratio of 1:10, grind to a fineness of 2~50μm, add compound protease, and enzymatically hydrolyze for 2~8h at a temperature of 45~60℃ to obtain enzymatic hydrolysate;
[0011] The enzyme hydrolysate was inactivated at 90℃ for 30 min, then the pH was controlled at 6~7.5 and the temperature at 35~40℃, and fermentation was carried out for 2~6 h to obtain the enzyme hydrolysate fermentation broth.
[0012] A clarifying agent is added to the enzymatic fermentation broth, and the mixture is allowed to stand for 2-8 hours to remove impurities. A decolorizing agent is then added to the clarified broth, and the mixture is decolorized at 60°C for 30-60 minutes. The broth is then filtered through a plate and frame filter, and the resulting filtrate is filtered through a 2KD ultrafiltration membrane. Inorganic salts and water-soluble free amino acids are then removed through a nanofiltration membrane. The broth is then sterilized by UHT, concentrated, and spray-dried at an inlet air temperature of 180-220°C and an outlet air temperature of 80-110°C to obtain chondroitin peptides.
[0013] Furthermore, the animal cartilage is chicken breast cartilage.
[0014] Furthermore, the mass fraction of the NaOH solution is 0.1%–1%, and the inoculum size of *Staphylococcus saprophyticus* is 1 × 10⁻⁶. 7 ~8×10 7 cfu / g, the inoculum amount of Leuconostoc pseudometamorphosum was 1×10⁻⁶. 7 ~8×10 7 cfu / g, calculated based on the mass of solution a, fermentation temperature is 35~40℃, fermentation time is 2~10h.
[0015] Furthermore, *Staphylococcus saprophyticus* was isolated from Dong ethnic group's fermented meat and obtained through phenotypic and biochemical identification, while *Leuconostoc pseudoenteroides* was purchased from Shanghai Guyan Industrial Co., Ltd.
[0016] Furthermore, the preferred pressure holding time during steam explosion is 30-60 seconds, the preferred temperature is 200-250°C, and the preferred explosion power density is EPD (MW / m³). 3 The optimal value is 28~48.
[0017] Furthermore, the amount of compound protease added is 0.2% to 2% of the mass of the steam-exploded material, the enzymatic hydrolysis pH value is 7.5 to 8.5, and the compound protease is composed of alkaline protease, fig protease and kiwi protease in a mass ratio of 3:1 to 2:1 to 2, and the enzymatic hydrolysis pH value is 7.5 to 8.5.
[0018] Furthermore, the inoculum size for fermentation is 1×10⁻⁶. 9 ~6×10 9cfu / g is calculated in units of the mass of steam-exploded material, and the fermentation strains are one or more of Lactobacillus helveticus, Pediococcus brevis, and Lactobacillus buchneri in any proportion.
[0019] Furthermore, the amount of clarifying agent added is 0.2% to 5% of the mass of the enzymatic fermentation broth. The clarifying agent is composed of potassium polyaspartate, tamarind gum, and erythrina gum in a mass ratio of 1:1 to 5:2 to 4.
[0020] Furthermore, the amount of decolorizing agent added is 0.2% to 5% of the mass of the enzymatic fermentation broth, and the decolorizing agent is composed of one or more of activated carbon, diatomaceous earth and white clay in any proportion.
[0021] Furthermore, a chondroitin peptide that can promote chondrocyte growth is prepared by the above-described method.
[0022] The beneficial effects of this invention are:
[0023] This invention provides a chondroitin peptide that can promote chondrocyte growth and its preparation method. The prepared chondroitin peptide has an average molecular weight range of 300-3000 Daltons, is rich in chondroitin sulfate, hydroxyproline, and hydroxylysine, and is safe, non-toxic, easily digestible, completely soluble in water, produces a clear and transparent solution, and is resistant to acid and high temperatures. It can be used as a raw material for pharmaceuticals, cosmetics, health foods, and food products. It specifically repairs damaged chondrocytes, promotes the generation of extracellular matrix, and plays a role in preventing and improving osteoarthritis. Detailed Implementation
[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0025] Example 1
[0026] A method for preparing chondroitin peptides that can promote chondroitin cell growth, comprising the following steps:
[0027] Step S1: Weigh 1000g of chicken breast cartilage, add 10kg of 0.3% NaOH solution and soak for 10 hours. Then rinse three times with deionized water, add 10 times the volume of water, and inoculate according to the weight of the solution at an inoculation rate of 2.6 × 10⁻⁶. 7 CFU / g inoculation with Staphylococcus saprophyticus and an inoculation amount of 3.4 × 10⁻⁶ 7 CFU / g Leuconostoc pseudoenteritis was fermented at 37℃ for 8 hours. The fermentation broth was then discharged and washed twice with deionized water to obtain crude cartilage extract.
[0028] Step S2, Steam Explosion: The above-mentioned crude cartilage extract is placed in a catapult-type steam explosion machine, the temperature is controlled at 240℃, and the explosion power density is EPD (MW / m³). 3 The pressure was set at 30, and the pressure was maintained for 60 seconds to obtain 870g of steam-exploded material.
[0029] Step S3, preparation of chondroitin peptides: Add steam-exploded material and deionized water to a colloid mill at a mass ratio of 1:10 and grind to a fineness of 2~50μm. Add 6g of alkaline protease, 2g of fig protease and 4g of kiwi protease. Enzymatically hydrolyze at 55℃ for 6h to obtain the enzymatic hydrolysate.
[0030] The enzyme hydrolysate was inactivated at 90℃ for 30 min, then the pH was controlled at 6.5 and the temperature at 37℃. The material was calculated based on steam explosion, using a concentration of 3.2 × 10⁻⁶. 9 Inoculation with Lactobacillus helveticus at a dose of 1.6 × 10⁻⁶ cfu / g 9 Inoculation dose of CFU / g for Pediococcus beer, 1.2 × 10⁻⁶ 9 Inoculate with Lactobacillus buchneri at a CFU / g inoculum and ferment for 3 hours to obtain the enzymatic fermentation broth;
[0031] Add 20g of potassium polyaspartate, 60g of tamarind gum, and 80g of erythrina gum to the enzymatic fermentation broth. Let it stand for 4 hours to remove impurities. Add 10g of activated carbon and 15g of kaolin to the clarified broth. Decolorize at 60℃ for 30 minutes. Filter the broth using a plate and frame filter. Filter the filtrate using a 2KD ultrafiltration membrane. Then, remove inorganic salts and water-soluble free amino acids using a nanofiltration membrane. Sterilize using UHT, concentrate, and spray dry. The inlet air temperature is 180℃ and the outlet air temperature is 80℃ to obtain chondroitin peptides.
[0032] Among them, Staphylococcus saprophyticus was isolated from Dong ethnic group's fermented meat and identified as Staphylococcus saprophyticus by phenotypic and biochemical methods; Leuconostoc pseudoenteroides was purchased from Shanghai Guyan Industrial Co., Ltd.
[0033] The chondroitin peptide product obtained in Example 1 had an average molecular weight of 822, a protein content of 67.4%, a chondroitin sulfate content of 31.2%, hydroxyproline of 5.4%, and hydroxylysine of 0.96%. A 5% aqueous solution was clear and transparent, and no flocculation or precipitation occurred when heated at 121°C and pH 3.5 for 30 minutes.
[0034] Example 2
[0035] A method for preparing chondroitin peptides that can promote chondroitin cell growth, comprising the following steps:
[0036] Step S1: Weigh 1000g of chicken breast cartilage, add 10kg of 0.1% NaOH solution and soak for 2 hours. Then rinse three times with deionized water, add 10 times the volume of water, and inoculate at a rate of 1×10⁻⁶ based on the weight of the solution. 7 Inoculation with *Staphylococcus saprophyticus* at a concentration of CFU / g and an inoculum size of 1×10⁻⁶. 7 CFU / g Leuconostoc pseudoenteritis was fermented at 35℃ for 2 hours. The fermentation broth was then discharged and washed twice with deionized water to obtain crude cartilage extract.
[0037] Step S2, Steam Explosion: The above-mentioned crude cartilage extract is placed in a catapult-type steam explosion machine, the temperature is controlled at 180℃, and the explosion power density is EPD (MW / m³). 3 The pressure is set to 20, and the pressure is maintained for 10 seconds to obtain steam-exploded material;
[0038] Step S3, preparation of chondroitin peptides: Add steam-exploded material and deionized water to a colloid mill at a mass ratio of 1:10 and grind to a fineness of 2~50μm. Add 6g of alkaline protease, 2g of fig protease and 2g of kiwi protease. Enzymatically hydrolyze at 55℃ for 6h to obtain enzymatic hydrolysate.
[0039] The enzyme hydrolysate was inactivated at 90℃ for 30 min, then the pH was controlled at 6.5 and the temperature at 37℃. Based on the steam explosion calculation, the amount of material was calculated at 1×10⁻⁶. 9 Inoculate with Lactobacillus helveticus at a CFU / g inoculum and ferment for 3 hours to obtain the enzymatic fermentation broth;
[0040] Add 20g of potassium polyaspartate, 20g of tamarind gum, and 40g of erythrina gum to the enzymatic fermentation broth. Let it stand for 4 hours to remove impurities. Add 0.2% activated carbon (by weight of the enzymatic fermentation broth) to the clarified broth. Decolorize at 60℃ for 45 minutes. Filter the broth using a plate and frame filter. Filter the resulting filtrate using a 2KD ultrafiltration membrane, and then remove inorganic salts and water-soluble free amino acids using a nanofiltration membrane. Sterilize using UHT, concentrate, and spray dry at an inlet air temperature of 200℃ and an outlet air temperature of 100℃ to obtain chondroitin peptides.
[0041] Among them, Staphylococcus saprophyticus was isolated from Dong ethnic group's fermented meat and identified as Staphylococcus saprophyticus by phenotypic and biochemical methods; Leuconostoc pseudoenteroides was purchased from Shanghai Guyan Industrial Co., Ltd.
[0042] The chondroitin peptide product obtained in Example 2 had an average molecular weight of 752, a protein content of 65.1%, a chondroitin sulfate content of 29.8%, hydroxyproline of 5.1%, and hydroxylysine of 0.84%. A 5% aqueous solution was clear and transparent, and no flocculation or precipitation occurred when heated at 121°C and pH 3.5 for 30 minutes.
[0043] Example 3
[0044] A method for preparing chondroitin peptides that can promote chondroitin cell growth, comprising the following steps:
[0045] Step S1: Weigh 1000g of chicken breast cartilage, add 10kg of 1% NaOH solution and soak for 10 hours. Then rinse three times with deionized water, add 10 times the volume of water, and inoculate at a rate of 8×10 based on the weight of the solution. 7 CFU / g inoculation with Staphylococcus saprophyticus and inoculation amount of 8×10 7 CFU / g Leuconostoc pseudoenteroides was fermented at 40℃ for 10 hours. The fermentation broth was then discharged and washed twice with deionized water to obtain crude cartilage extract.
[0046] Step S2, Steam Explosion: The above-mentioned crude cartilage extract is placed in a catapult-type steam explosion machine, the temperature is controlled at 270℃, and the explosion power density is EPD (MW / m³). 3 The pressure is set to 50, and the pressure is maintained for 100 seconds to obtain steam-exploded material;
[0047] Step S3, preparation of chondroitin peptides: Add steam-exploded material and deionized water to a colloid mill at a mass ratio of 1:10 and grind to a fineness of 2~50μm. Add 6g of alkaline protease, 4g of fig protease and 4g of kiwi protease. Enzymatically hydrolyze at 55℃ for 6h to obtain the enzymatic hydrolysate.
[0048] The enzyme hydrolysate was inactivated at 90℃ for 30 min, then the pH was controlled at 6.5 and the temperature at 37℃. The material was calculated based on steam explosion, according to 6×10... 9 Inoculate with Pediococcus beer at a CFU / g inoculum and ferment for 3 hours to obtain enzymatic fermentation broth;
[0049] Add 20g of potassium polyaspartate, 20-100g of tamarind gum, and 80g of erythrina gum to the enzymatic fermentation broth. Let it stand for 4 hours to remove impurities. Add 5% diatomaceous earth (based on the mass of the enzymatic fermentation broth) to the clarified broth. Decolorize at 60℃ for 45 minutes. Filter using a plate and frame filter. Filter the resulting filtrate using a 2KD ultrafiltration membrane, and then remove inorganic salts and water-soluble free amino acids using a nanofiltration membrane. Sterilize using UHT, concentrate, and spray dry at an inlet air temperature of 220℃ and an outlet air temperature of 110℃ to obtain chondroitin.
[0050] Among them, Staphylococcus saprophyticus was isolated from Dong ethnic group's fermented meat and identified as Staphylococcus saprophyticus by phenotypic and biochemical methods; Leuconostoc pseudoenteroides was purchased from Shanghai Guyan Industrial Co., Ltd.
[0051] The chondroitin peptide product obtained in Example 3 had an average molecular weight of 814, a protein content of 66.2%, a chondroitin sulfate content of 30.7%, hydroxyproline of 4.8%, and hydroxylysine of 0.92%. A 5% aqueous solution was clear and transparent, and no flocculation or precipitation occurred when heated at 121°C and pH 3.5 for 30 minutes.
[0052] Comparative Example 1
[0053] A method for preparing chondroitin peptides that can promote chondroitin cell growth, comprising the following steps:
[0054] Weigh 1000g of chicken breast cartilage, add 10kg of 0.3% NaOH solution and soak for 10h. Then rinse three times with deionized water, and perform steam explosion at 240℃ for 60s. EPD (MW / m³) 3 30, steam explosion product was obtained, deionized water was added at a mass ratio of 1:10, and the mixture was ground to a fineness of 2~50μm using a colloid mill. 12g of alkaline protease was added, and the mixture was enzymatically hydrolyzed at 55℃ for 6h to obtain the hydrolysate. The enzyme was inactivated at 90℃ for 30min, 160g of chitin was added, and the mixture was allowed to stand for 4h to remove impurities. 10g of activated carbon and 15g of bleaching clay were added to the clarified solution, and the solution was decolorized at 60℃ for 30min. The solution was filtered through a plate and frame filter, and the filtrate was filtered through a 2KD ultrafiltration membrane. Inorganic salts and water-soluble free amino acids were then removed by nanofiltration. After UHT sterilization, concentration and spray drying were performed to obtain chondroitin.
[0055] The product was tested and found to have an average molecular weight of 1067, a protein content of 51.2%, a chondroitin sulfate content of 20.7%, hydroxyproline of 2.9%, and hydroxylysine of 0.22%. A 5% aqueous solution was milky and opaque. Heating at 121°C and pH 3.5 for 30 minutes resulted in flocculation and precipitation.
[0056] The chondroitin peptides obtained in Examples 1-3 and Comparative Example 1 were tested to assess their effects on chondrocyte activity and extracellular matrix gene expression. The specific test procedures are as follows:
[0057] 1. After spreading chondrocytes onto a plate, collect the cells into 10mL centrifuge tubes, centrifuge at 1200rpm for 3min, discard the supernatant, add fresh cell culture medium, mix well by pipetting, and count cells using a hemocytometer. Seed 10⁴ cells per well in a 96-well plate, for a total volume of 200μL. Incubate overnight at 37℃ and 5%CO₂ to obtain chondrocyte slurry. Then, add 800μg / mL of chondroitin from the examples and comparative examples, respectively, for 24h. After treatment with 600μM hydrogen peroxide for 3h, add culture medium containing 10% CCK-8 to each well and react for 2h. Measure the absorbance at 450nm using a microplate reader. SW1353 human chondrocytes were purchased from Wuhan Pronosei Biotechnology Co., Ltd., and CCK-8 reagent was purchased from MCE.
[0058] 2. After spreading the chondrocytes onto a plate, collect the cells into 10mL centrifuge tubes, centrifuge at 1200rpm for 3min, discard the supernatant, add fresh cell culture medium, mix well by pipetting, and count the cells using a hemocytometer. Then, seed 2.5 × 10⁶ cells into each well of a 12-well plate. 5 One cell was cultured overnight to obtain chondrocyte slurry. Then, 800 μg / mL of chondrocyte peptide from the examples and comparative examples was added, followed by treatment with 400 μM hydrogen peroxide for 6 h. Cell RNA was extracted according to the instructions, and cDNA was synthesized by reverse transcription. RT-qPCR experiments were performed according to the following reaction system. The cDNA synthesis kit was purchased from Yisheng Biotechnology Co., Ltd.
[0059] The test results are shown in Table 1:
[0060] Table 1. Results of chondrocyte testing
[0061]
[0062] Note:" "This indicates a highly significant difference compared to the control group (" P <0.01), the blank group was chondrocyte fluid without hydrogen peroxide treatment and without the addition of chondroitin, the control group was chondrocyte fluid treated with hydrogen peroxide but without the addition of chondroitin, Example 1 was chondrocyte fluid treated with hydrogen peroxide but with the addition of chondroitin obtained in Example 1, Example 2 was chondrocyte fluid treated with hydrogen peroxide but with the addition of chondroitin obtained in Example 2, Example 3 was chondrocyte fluid treated with hydrogen peroxide but with the addition of chondroitin obtained in Example 3, and Comparative Example 1 was chondrocyte fluid treated with hydrogen peroxide but with the addition of chondroitin obtained in Comparative Example 1.
[0063] As shown in Table 1, the chondroitin peptides obtained in Examples 1, 2 and 3 can specifically repair damaged chondrocytes, promote gene expression in the extracellular matrix, and thus promote the production of the extracellular matrix, thereby preventing and improving osteoarthritis, with significant differences compared to the control group; while the chondroitin peptides obtained in Example 2 group showed no significant difference compared to the control group.
[0064] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0065] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a chondroitin peptide that can promote chondrocyte growth, characterized in that, The steps include the following: Step S1: Using animal cartilage as raw material, soak it in NaOH solution for 2-12 hours, take it out, wash it, add 10 times the volume of softened water to the material to obtain solution a, inoculate it with Staphylococcus saprophyticus and Leuconostoc pseudoenteroides, ferment it, then discharge the fermentation liquid, wash it, and obtain crude cartilage extract. Step S2: Place the cartilage crude extract into a catapult-type steam explosion machine, control the temperature at 180~270℃, the explosion power density at 20~50, and hold the pressure for 5~100s to obtain steam-exploded material. Step S3: Add the steam-exploded material and softened water to a colloid mill at a mass ratio of 1:10, grind to a fineness of 2~50μm, add the complex protease, and enzymatically hydrolyze for 2~8h at a temperature of 45~60℃ to obtain the enzymatic hydrolysate. The enzyme hydrolysate was inactivated at 90℃ for 30 min, the pH was controlled at 6~7.5, the temperature was controlled at 35~40℃, and the fermentation strain was inoculated. Fermentation was carried out for 2~6 h to obtain the enzyme hydrolysate fermentation broth. Add a clarifying agent to the enzymatic fermentation broth, let it stand for 2-8 hours to remove impurities, add a decolorizing agent to the clarified broth, decolorize at 60℃ for 30-60 minutes, filter by plate and frame, filter the filtrate through a 2KD ultrafiltration membrane, and then remove inorganic salts and water-soluble free amino acids through a nanofiltration membrane. After UHT sterilization, concentrate and spray dry to obtain clear and purified chondroitin. The complex protease is composed of alkaline protease, fig protease, and kiwi protease in a mass ratio of 3:1~2:1~2; The amount of clarifying agent added is 0.2% to 5% of the mass of the enzymatic fermentation broth. The clarifying agent is composed of potassium polyaspartate, tamarind gum and erythrina gum in a mass ratio of 1:1 to 5:2 to 4. The inoculum size for fermentation is 1×10⁻⁶. 9 ~6×10 9 cfu / g is calculated in units of the mass of steam-exploded material, and the fermentation strains are one or more of Lactobacillus helveticus, Pediococcus brevis, and Lactobacillus buchneri in any proportion.
2. The preparation method according to claim 1, characterized in that, The amount of compound protease added is 0.2% to 2% of the mass of the steam-exploded material.
3. The preparation method according to claim 1, characterized in that, The amount of decolorizing agent added is 0.2% to 5% of the mass of the enzymatic fermentation broth. The decolorizing agent is composed of one or more of activated carbon, diatomaceous earth and white clay in any proportion.
4. The preparation method according to claim 1, characterized in that, The mass fraction of the NaOH solution is 0.1% to 1%.
5. The preparation method according to claim 1, characterized in that, The inoculum size for *Staphylococcus saprophyticus* is 1 × 10⁻⁶. 7 ~8×10 7 cfu / g, the inoculum amount of Leuconostoc pseudometamorphosum was 1×10⁻⁶. 7 ~8×10 7 cfu / g, calculated in units of the mass of solution a.
6. The preparation method according to claim 1, characterized in that, In step S1, the fermentation temperature is 35~40℃ and the fermentation time is 2~10h.
7. A chondroitin peptide that can promote chondrocyte growth, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 6.