CN116590370A - Cartilage peptide capable of promoting chondrocyte growth and preparation method thereof - Google Patents
Cartilage peptide capable of promoting chondrocyte growth and preparation method thereof Download PDFInfo
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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Abstract
The invention discloses a cartilage peptide capable of promoting chondrocyte growth and a preparation method thereof, belonging to the technical field of polypeptide preparation, and comprising the following steps: obtaining a cartilage crude extract; steam explosion to obtain a steam explosion material; grinding the steam explosion material and softened water, adding compound protease, inoculating fermentation strain after enzymolysis, and fermenting to obtain enzymolysis fermentation liquor; the invention obtains cartilage peptide with average molecular weight range of 300-3000 dalton, which is rich in chondroitin sulfate, hydroxyproline and hydroxylysine, has the advantages of safety, no toxic and side effect, easy digestion, complete dissolution in water, clear and transparent solution, acid resistance and high temperature resistance, can be used as raw materials of medicines, cosmetics, health care foods and foods, can specifically repair damaged cartilage cells, promote extracellular matrix generation, and play a role in preventing and improving osteoarthritis.
Description
Technical Field
The invention belongs to the technical field of polypeptide preparation, and particularly relates to a cartilage peptide capable of promoting chondrocyte growth and a preparation method thereof.
Background
Osteoarthritis (OA) is a chronic joint disease characterized by degeneration, destruction and hyperosteogeny of articular cartilage, also known as osteoarthropathy, degenerative osteoarthropathy, which affects most of the joints throughout the body including the cervical and lumbar vertebrae, knee joints, hip joints, interphalangeal joints, etc. Osteoarthritis is a progressively progressive and aggravated disease, and current treatments aim to delay the progression of the condition, relieve pain and improve joint function, with the trend of aging the population, the older the osteoarthritis will be, the more affecting the physical condition and quality of life of people.
There are many studies to date that cartilage peptides extracted from animal cartilage can relieve pain in osteoarthritis patients and increase joint flexibility.
Disclosure of Invention
The invention aims to provide a cartilage peptide capable of promoting the growth of cartilage cells and a preparation method thereof, and the obtained cartilage peptide can specifically repair damaged cartilage cells, promote the generation of extracellular matrixes and play a role in preventing and improving osteoarthritis.
The aim of the invention can be achieved by the following technical scheme:
a method for preparing cartilage peptide capable of promoting the growth of cartilage cells, comprising the following steps:
step S1, degreasing: soaking animal cartilage serving as a raw material in NaOH solution for 2-12 hours, taking out, washing with softened water for 3-5 times, adding 10 times of softened water into the material after washing to obtain solution a, inoculating staphylococcus saprophyticus and leuconostoc pseudomembranaceus, fermenting, discharging fermentation liquor, and washing to obtain a cartilage crude extract;
step S2, steam explosion: placing the above cartilage crude extract in ejection type steam explosion machine, controlling temperature at 180-270 deg.C, and explosion power density EPD (MW/m) 3 ) Maintaining the pressure for 5-100s at 20-50 s to obtain a steam explosion material;
step S3, cartilage peptide preparation: steam blasting materials and softened water are mixed according to a mass ratio of 1:10, adding into a colloid mill, grinding to fineness of 2-50 μm, adding compound protease, and performing enzymolysis for 2-8h at 45-60deg.C to obtain enzymolysis solution;
inactivating enzyme of the enzymolysis liquid in 90 ℃ environment for 30min, controlling pH to 6-7.5, inoculating fermentation strain at 35-40 ℃, fermenting for 2-6h to obtain enzymolysis fermentation liquid;
adding a clarifying agent into the enzymolysis fermentation liquor, standing for 2-8h to remove impurities, adding a decolorizing agent into the clarified feed liquor, decolorizing at 60 ℃ for 30-60min, filtering by a plate frame, filtering the obtained filtrate by a 2KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by a nanofiltration membrane, sterilizing by UHT, concentrating, spray drying, and carrying out air inlet at 180-220 ℃ and air outlet at 80-110 ℃ to obtain the cartilage peptide.
Further, the animal cartilage is chicken breast cartilage.
Further, the mass fraction of NaOH solution is 0.1-1%, and the inoculum size of Staphylococcus saprophyticus is 1×10 7 -8×10 7 cfu/g, leuconostoc pseudoenteroides inoculum size of 1×10 7 -8×10 7 cfu/g, calculated by taking mass of the solution a as a unit, fermentation temperature is 35-40 ℃ and fermentation time is 2-10h.
Further, staphylococcus saprophyticus is separated from Dong minority acid meat by itself, and is obtained through phenotype and biochemical identification, and leuconostoc pseudomembranaceus is purchased from Shanghai valley research industry Co.
Further, the dwell time during the steam explosion is preferably 30 to 60 seconds, the temperature is preferably 200 to 250 ℃, and the explosion power density EPD (MW/m 3 ) Preferably 28-48.
Further, the addition amount of the compound protease is 0.2% -2% of the mass of the steam explosion material, the enzymolysis pH value is 7.5-8.5, the compound protease consists of alkaline protease, ficin and kiwi fruit protease according to the mass ratio of 3:1-2:1-2, and the enzymolysis pH value is 7.5-8.5.
Further, the inoculum size of the fermentation strain is 1 multiplied by 10 9 -6×10 9 cfu/g, calculated by taking the mass of the steam exploded material as a unit, the fermentation strain is one or more of Lactobacillus helveticus, pediococcus cerevisiae and Lactobacillus buchneri according to any proportion.
Further, the adding amount of the clarifying agent is 0.2-5% of the mass of the enzymolysis fermentation liquor, and the clarifying agent is prepared from potassium polyaspartate, tamarind gum and karaya gum according to the mass ratio of 1: 1-5:2-4.
Further, the addition amount of the decoloring agent is 0.2-5% of the mass of the enzymolysis fermentation liquid, and the decoloring agent is composed of one or more of active carbon, diatomite and clay according to any proportion.
Further, a cartilage peptide capable of promoting the growth of cartilage cells is prepared by the preparation method.
The invention has the beneficial effects that:
the invention provides a cartilage peptide capable of promoting the growth of cartilage cells and a preparation method thereof, wherein the average molecular weight of the prepared cartilage peptide is 300-3000 daltons, and the cartilage peptide is rich in chondroitin sulfate, hydroxyproline and hydroxylysine, has the advantages of safety, no toxic or side effect, easy digestion, complete dissolution in water, clear and transparent solution, acid resistance and high temperature resistance, can be used as raw materials of medicines, cosmetics, health-care foods and foods, can specifically repair damaged cartilage cells, promotes the generation of extracellular matrixes, and has the effects of preventing and improving osteoarthritis.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for preparing cartilage peptide capable of promoting the growth of cartilage cells, comprising the following steps:
step S1, weighing 1000g of chicken breast cartilage, adding 10kg of NaOH solution with mass fraction of 0.3% for soaking for 10 hours, then washing with deionized water for 3 times, adding 10 times of volume water, and according to the weight of feed liquid and the inoculum size of 2.6X10 7 cfu/g inoculation of Staphylococcus saprophyticus and inoculum size 3.4X10 7 cfu/g leuconostoc mesenteroides, fermenting for 8 hours at 37 ℃, discharging fermentation liquor, and washing twice with deionized water to obtain a cartilage crude extract;
step S2, steam explosion: placing the above cartilage crude extract in ejection type steam explosion machine, controlling temperature to 240 deg.C, and explosion power density EPD (MW/m) 3 ) 30 g of steam explosion material is obtained after pressure is maintained for 60 s;
step S3, cartilage peptide preparation: steam blasting materials and deionized water are mixed according to a mass ratio of 1:10, adding the mixture into a colloid mill, grinding the mixture to the fineness of 2-50 mu m, adding 6g of neutral alkaline protease, 2g of ficin and 4g of kiwi fruit protease, and carrying out enzymolysis for 6 hours at the temperature of 55 ℃ to obtain an enzymolysis solution;
inactivating enzyme of the enzymolysis solution at 90deg.C for 30min, controlling pH to 6.5, and steam explosion at 37deg.C, according to 3.2X10 9 cfu/g inoculum size inoculating Lactobacillus helveticus, 1.6X10 9 cfu/g inoculum size inoculating Pediococcus cerevisiae, 1.2X10 9 Inoculating lactobacillus buchneri with cfu/g inoculum size, and fermenting for 3 hours to obtain enzymolysis fermentation liquor;
adding 20g of potassium polyaspartate into the enzymolysis fermentation liquor, 60g of tamarind gum and 80g of karaya gum, standing for 4 hours, removing impurities, adding 10g of active carbon and 15g of clay into the clarified feed liquid, decoloring for 30 minutes at 60 ℃, filtering by a plate frame, filtering the obtained filtrate by a 2KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by a nanofiltration membrane, performing UHT sterilization, concentrating, spray drying, and obtaining the cartilage peptide at the inlet air temperature of 180 ℃ and the outlet air temperature of 80 ℃.
Wherein staphylococcus saprophyticus is separated from Dong minority acid meat by itself, identified as staphylococcus saprophyticus by phenotype and biochemistry, and leuconostoc pseudomembranaceus is purchased from Shanghai valley research industry Co., ltd;
the detection shows that the cartilage titanium product obtained in example 1 has an average molecular weight 822, a protein content of 67.4%, a chondroitin sulfate content of 31.2%, hydroxyproline 5.4% and hydroxylysine 0.96%; the 5% aqueous solution was clear and transparent, and was heated at 121 ℃ and ph=3.5 for 30min without flocculation and precipitation.
Example 2
A method for preparing cartilage peptide capable of promoting the growth of cartilage cells, comprising the following steps:
step S1, weighing 1000g of chicken breast cartilage, adding 10kg of NaOH solution with mass fraction of 0.1% for soaking for 2 hours, then washing with deionized water for 3 times, adding 10 times of volume water, and according to the weight of feed liquid and the inoculum size of 1X 10 7 cfu/g inoculation of Staphylococcus saprophyticus and inoculum size 1×10 7 cfu/g leuconostoc mesenteroides, fermenting for 2 hours at 35 ℃, discharging fermentation liquor, and washing twice with deionized water to obtain a cartilage crude extract;
step S2, steam explosion: placing the above cartilage crude extract in ejection type steam explosion machine, controlling temperature to 180deg.C, and explosion power density EPD (MW/m) 3 ) 20, maintaining the pressure for 10s to obtain a steam explosion material;
step S3, cartilage peptide preparation: steam blasting materials and deionized water are mixed according to a mass ratio of 1:10, adding the mixture into a colloid mill, grinding the mixture to the fineness of 2-50 mu m, adding 6g of neutral alkaline protease, 2g of ficin and 2g of kiwi fruit protease, and carrying out enzymolysis for 6 hours at the temperature of 55 ℃ to obtain an enzymolysis solution;
inactivating enzyme of the enzymolysis solution at 90deg.C for 30min, controlling pH to 6.5, and steam explosion at 37deg.C according to 1×10 9 Inoculating lactobacillus helveticus with cfu/g inoculum size, and fermenting for 3 hours to obtain enzymolysis fermentation liquor;
adding 20g of potassium polyaspartate into the enzymolysis fermentation liquor, 20g of tamarind gum, 40g of karaya gum, standing for 4 hours, removing impurities, adding active carbon which is 0.2% of the mass of the enzymolysis fermentation liquor into the clarified feed liquor, decoloring for 45 minutes at 60 ℃, filtering by a plate frame, filtering the obtained filtrate by a 2KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by a nanofiltration membrane, sterilizing by UHT, concentrating, spray drying, and obtaining the cartilage peptide at an air inlet temperature of 200 ℃ and an air outlet temperature of 100 ℃.
Wherein staphylococcus saprophyticus is separated from Dong minority acid meat by itself, identified as staphylococcus saprophyticus by phenotype and biochemistry, and leuconostoc pseudomembranaceus is purchased from Shanghai valley research industry Co., ltd;
the detection shows that the cartilage titanium product obtained in example 2 has average molecular weight 752, protein content 65.1%, chondroitin sulfate content 29.8%, hydroxyproline 5.1% and hydroxylysine 0.84%; the 5% aqueous solution was clear and transparent, and was heated at 121 ℃ and ph=3.5 for 30min without flocculation and precipitation.
Example 3
A method for preparing cartilage peptide capable of promoting the growth of cartilage cells, comprising the following steps:
step S1, weighing 1000g of chicken breast cartilage, adding 10kg of NaOH solution with mass fraction of 1% for soaking for 10 hours, then washing 3 times with deionized water, adding 10 times of water according to the weight of feed liquid and the inoculum size of 8 multiplied by 10 7 cfu/g inoculation of Staphylococcus saprophyticus and inoculum size of 8X10 × 7 cfu/g leuconostoc mesenteroides, fermenting for 10 hours at 40 ℃, discharging fermentation liquor, and washing twice with deionized water to obtain a cartilage crude extract;
step S2, steam explosion: placing the above cartilage crude extract in ejection type steam explosion machine, controlling temperature to 270 deg.C, and explosion power density EPD (MW/m) 3 ) 50, maintaining the pressure for 100s to obtain a steam explosion material;
step S3, cartilage peptide preparation: steam blasting materials and deionized water are mixed according to a mass ratio of 1:10, adding the mixture into a colloid mill, grinding the mixture to the fineness of 2-50 mu m, adding 6g of neutral alkaline protease, 4g of ficin and 4g of kiwi fruit protease, and carrying out enzymolysis for 6 hours at the temperature of 55 ℃ to obtain an enzymolysis solution;
inactivating enzyme of the enzymolysis solution at 90deg.C for 30min, controlling pH to 6.5, and steam explosion at 37deg.C according to 6×10 9 Inoculating Pediococcus cerevisiae with cfu/g inoculum size, and fermenting for 3 hr to obtain enzymolysis fermentation liquor;
adding 20g of potassium polyaspartate into the enzymolysis fermentation liquid, 20-100g of tamarind gum and 80g of karaya gum, standing for 4 hours, removing impurities, adding 5% diatomite which is the mass of the enzymolysis fermentation liquid into the clarified feed liquid, decoloring for 45 minutes at 60 ℃, filtering by a plate frame, filtering the obtained filtrate by a 2KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by a nanofiltration membrane, performing UHT sterilization, concentrating, spray drying, and obtaining the cartilage peptide at an air inlet temperature of 220 ℃ and an air outlet temperature of 110 ℃.
Wherein staphylococcus saprophyticus is separated from Dong minority acid meat by itself, identified as staphylococcus saprophyticus by phenotype and biochemistry, and leuconostoc pseudomembranaceus is purchased from Shanghai valley research industry Co., ltd;
the detection shows that the cartilage titanium product obtained in example 3 has average molecular weight 814, protein content 66.2%, chondroitin sulfate content 30.7%, hydroxyproline 4.8% and hydroxylysine 0.92%; the 5% aqueous solution was clear and transparent, and was heated at 121 ℃ and ph=3.5 for 30min without flocculation and precipitation.
Comparative example 1
A method for preparing cartilage peptide capable of promoting the growth of cartilage cells, comprising the following steps:
weighing 1000g of chicken breast cartilage, adding 10kg of 0.3% NaOH solution by mass fraction for soaking for 10h, then washing with deionized water for 3 times, performing steam explosion for 60s at 240 ℃ for EPD (MW/m) 3 ) 30, obtaining a steam explosion product according to the mass ratio of 1:10 adding deionized water, grinding the mixture to the fineness of 2-50 mu m by a colloid mill, adding 12g of alkaline protease, and carrying out enzymolysis for 6 hours at the temperature of 55 ℃ to obtain an enzymolysis liquid; inactivating enzyme at 90deg.C for 30min, adding chitin 160g, standing for 4 hr, removing impurities, adding activated carbon 10g and clay 15g into clarified feed liquid, decolorizing at 60deg.C for 30min, plate-frame filtering, filtering filtrate with 2KD ultrafiltration membrane, nanofiltration to remove inorganic salt and water-soluble free amino acid, UHT sterilizing, concentrating, and spray drying to obtain cartilage peptide.
The average molecular weight of the product is 1067, the protein content is 51.2%, the chondroitin sulfate content is 20.7%, the hydroxyproline is 2.9%, and the hydroxylysine is 0.22% through detection. The 5% aqueous solution is in the form of emulsion, opaque, and has flocculation and precipitation after heating at 121deg.C and pH3.5 for 30 min.
The chondropeptides obtained in examples 1 to 3 and comparative example 1 were tested for their effect on chondrocyte activity and extracellular matrix gene expression, and specific test procedures were as follows:
1. after the chondrocytes were plated, the cells were collected into a 10mL centrifuge tube, centrifuged at 1200rpm for 3min, the supernatant was removed, fresh cell culture medium was added for pipetting uniformly and counted with a blood cell counting plate, 104 cells were plated in 96 well plates in a total volume of 200. Mu.L per well, and at 37℃5% CO 2 Culturing overnight under the condition to obtain chondrocyte liquid, then adding 800 mug/mL of the chondropeptide of the example and the chondropeptide of the comparative example respectively, wherein the treatment time is set to be 24 hours, then treating for 3 hours by using 600 mu M of hydrogen peroxide, then adding a culture solution containing 10% of CCK-8 into each hole for reaction for 2 hours, measuring the absorbance of 450nm by using an enzyme-labeling instrument, wherein SW1353 human chondrocyte serial numbers are purchased from the GmbH of the marpranopsis life sciences, and CCK-8 reagent is purchased from MCE;
2. after the chondrocytes were plated, the cells were collected into a 10mL centrifuge tube, centrifuged at 1200rpm for 3min, the supernatant was removed, fresh cell culture medium was added, and the cells were blown down uniformly and counted with a blood cell counting plate, and 2.5X10 cells were plated per well in a 12-well plate 5 Culturing cells overnight to obtain chondrocyte liquid, adding 800 mug/mL of the chondropeptide of the example and the chondropeptide of the comparative example respectively, adding 400 mu M of hydrogen peroxide for treatment for 6 hours, extracting cell RNA according to the steps of the specification, carrying out reverse transcription to synthesize cDNA, preparing reagents according to the following reaction system, carrying out RT-qPCR experiments, and obtaining cDNA synthesis kit from the company of the following holy biotechnology;
the test results are shown in table 1:
TABLE 1
Note that: the "×" represents the chondrocyte liquid which was treated without hydrogen peroxide and to which no cartilage peptide was added, the blank group was the chondrocyte liquid which was treated with hydrogen peroxide and to which no cartilage peptide was added, the example 1 was the chondrocyte liquid which was treated with hydrogen peroxide and to which the cartilage peptide obtained in example 1 was added, the example 2 was the chondrocyte liquid which was treated with hydrogen peroxide and to which the cartilage peptide obtained in example 2 was added, the example 3 was the chondrocyte liquid which was treated with hydrogen peroxide and to which the cartilage peptide obtained in example 3 was added, and the comparative example 1 was the chondrocyte liquid which was treated with hydrogen peroxide and to which the cartilage peptide obtained in comparative example 1 was added, which was extremely significant difference (P < 0.01) compared to the control group.
As can be seen from table 1, the cartilage peptides obtained in examples 1, 2 and 3 can specifically repair damaged cartilage cells, promote gene expression of extracellular matrix, thereby promoting generation of extracellular matrix, preventing and improving osteoarthritis, and have significant differences from the control group; whereas the cartilage peptide obtained in example 2 was not significantly different from the control group.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present 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 therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method for preparing cartilage peptide capable of promoting the growth of cartilage cells, which is characterized by comprising the following steps:
step S1, taking animal cartilage as a raw material, soaking the animal cartilage for 2-12 hours by using a NaOH solution, taking out the animal cartilage, adding 10 times of volume of softened water into the material after cleaning to obtain a solution a, inoculating staphylococcus saprophyticus and leuconostoc pseudoenteroides, fermenting, discharging fermentation liquor, and cleaning to obtain a cartilage crude extract;
s2, placing the cartilage crude extract in an ejection type steam explosion machine, controlling the temperature to be 180-270 ℃, controlling the explosion power density to be 20-50, and maintaining the pressure for 5-100S to obtain a steam explosion material;
step S3, steam explosion materials and softened water are mixed according to a mass ratio of 1:10, adding into a colloid mill, grinding to fineness of 2-50 μm, adding compound protease, and performing enzymolysis for 2-8h at 45-60deg.C to obtain enzymolysis solution;
inactivating enzyme of the enzymolysis liquid in 90 ℃ environment for 30min, controlling pH to 6-7.5, inoculating fermentation strain at 35-40 ℃, fermenting for 2-6h to obtain enzymolysis fermentation liquid;
adding a clarifying agent into the enzymolysis fermentation liquor, standing for 2-8h to remove impurities, adding a decolorizing agent into the clarified feed liquor, decolorizing at 60 ℃ for 30-60min, plate-frame filtering, filtering the obtained filtrate by adopting a 2KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by adopting a nanofiltration membrane, sterilizing by UHT, concentrating and spray-drying to obtain the cartilage peptide.
2. The method for producing a cartilage peptide for promoting the growth of chondrocytes according to claim 1, wherein the mass fraction of the NaOH solution is 0.1 to 1%.
3. The method for producing a cartilage peptide for promoting chondrocyte growth according to claim 1, wherein the inoculum size of Staphylococcus saprophyticus is 1X 10 7 -8×10 7 cfu/g, leuconostoc pseudoenteroides inoculum size of 1×10 7 -8×10 7 cfu/g, calculated as mass of solution a.
4. The method for preparing cartilage peptide for promoting chondrocyte growth according to claim 1, wherein the fermentation temperature in the step S1 is 35-40 ℃ and the fermentation time is 2-10h.
5. The method for preparing the cartilage peptide capable of promoting the growth of chondrocytes according to claim 1, wherein the addition amount of the composite protease is 0.2% -2% of the mass of the steam exploded material, and the composite protease consists of alkaline protease, ficin and actinidin according to the mass ratio of 3:1-2:1-2.
6. The method for producing a cartilage peptide for promoting chondrocyte growth according to claim 1, wherein the inoculum size of the fermentation strain is 1X 10 9 -6×10 9 cfu/g, calculated by taking the mass of the steam exploded material as a unit, the fermentation strain is one or more of Lactobacillus helveticus, pediococcus cerevisiae and Lactobacillus buchneri according to any proportion.
7. The method for preparing cartilage peptide capable of promoting chondrocyte growth according to claim 1, wherein the adding amount of a clarifying agent is 0.2-5% of the mass of the enzymolysis fermentation liquor, and the clarifying agent is prepared from potassium polyaspartate, tamarind gum and karaya gum according to the mass ratio of 1:1-5: 2-4.
8. The method for preparing cartilage peptide capable of promoting chondrocyte growth according to claim 1, wherein the adding amount of the decoloring agent is 0.2-5% of the mass of the enzymolysis fermentation broth, and the decoloring agent is composed of one or more of active carbon, diatomite and clay according to any proportion.
9. A cartilage peptide for promoting chondrocyte growth, characterized by being prepared by the method of any one of the above claims 1-8.
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