A type X collagen peptide for improving bone growth and its preparation method and application

Abstract

The present invention discloses a type X collagen peptide for improving bone growth and its preparation method and application, relating to the field of biotechnology. The preparation method comprises the following steps: S1, eggshell membrane powder is extracted with water to obtain a crude extract; S2, enzyme is added to the crude extract for enzymolysis to obtain an enzymolysis solution; S3, natto Bacillus powder is inoculated in the enzymolysis solution, the inoculation amount is 0.05-1%, the temperature is 38-45 ° C, and the fermentation is 14-36h; the temperature is lowered to 25-30 ° C, lactic acid Kluyveromyces is inoculated, the inoculation amount is 1-6%, and the fermentation is 3-7 days to obtain eggshell membrane enzymatic fermentation liquid; S4, the eggshell membrane enzymatic fermentation liquid is centrifuged, a clarifier is added to obtain a clarified liquid; S5, a decolorizing agent is added to the clarified liquid, and it is dried to obtain a type X collagen peptide. The collagen peptide of the present invention has a high content of type X collagen, which can promote osteoblast growth and has broad application prospects in the preparation of growth-promoting products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a type X collagen peptide for improving bone growth, a preparation method and an application thereof. Background Art

[0002] Type X collagen is a short-chain, non-fibrous collagen that is distributed in a liquid, homogeneous phase within the extracellular matrix and is a key component of the extracellular matrix. Its short chains are only half the length of ordinary collagen fibrils. Type X collagen plays a crucial role in skeletal growth and development, particularly in endochondral ossification. Type X collagen molecules aggregate within the cartilage matrix, forming a hexagonal lattice structure that provides a framework for the deposition of new bone matrix during endochondral ossification.

[0003] Chinese patent CN116711862A discloses a composition for improving bone density and its application. The composition originally comprises 35-60 parts of bovine collagen peptide, 10-35 parts of cartilage extract, 0.2-0.8 parts of eggshell membrane extract, 0.1-2 parts of turmeric, 0.1-1.5 parts of milk mineral salt, and 0.05-0.15 parts of sodium hyaluronate. The extraction process of the eggshell membrane extract comprises: (1) adding water to eggshell membrane powder, heating to 85-100°C, and keeping the temperature for 10-30 minutes; (2) cooling, adding enzymes for enzymatic hydrolysis to obtain eggshell membrane extract; (3) filtering, concentrating, and drying to obtain the eggshell membrane extract. The enzymes used for the enzymatic hydrolysis comprise a mixture of serine protease, pectinase, cartilage sulfate extractase, keratinase, and aminopeptidase in a mass ratio of 15-35:5-15:15-25:10-20:5-15. This invention uses a combination of bovine collagen peptides, cartilage extract, eggshell membrane extract, turmeric, milk mineral salts, and sodium hyaluronate to significantly treat osteoporosis. Chinese patent CN118813744A discloses a method for optimizing ultrasound-assisted enzymatic extraction of eggshell membrane proteins based on response surface methodology. The method comprises: S1. peeling, washing, and drying the eggshell membranes, and mechanically grinding the dried eggshell membranes into eggshell membrane powder; S2. immersing the eggshell membrane powder in distilled water at varying solid-to-liquid ratios and adjusting the pH with acetic acid to produce an acid hydrolyzed solution; S3. ultrasonically treating the enzymatic hydrolysis solution under the optimal conditions, collecting the enzymatic supernatant, adjusting the pH with NaOH solution, salting out the supernatant, centrifuging, dialysis, and freeze-drying to obtain extracted eggshell membrane proteins. This invention improves the extraction efficiency and quality of collagen and reduces production costs. However, its effectiveness in promoting bone growth and increasing the production of type X collagen-containing peptides is poor.

[0004] Based on this, there is an urgent need to develop a safe and efficient method for preparing collagen peptides that can increase bone density and type X collagen content. Summary of the Invention

[0005] The purpose of the present invention is to provide a type X collagen peptide for improving bone growth, and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] In one aspect, the present invention provides a method for preparing a type X collagen peptide, the method comprising the following steps:

[0008] S1. Extract eggshell membrane powder with water to obtain a crude extract;

[0009] S2, adding enzyme to the crude extract for enzymolysis to obtain enzymolysis solution;

[0010] S3, inoculating the enzymatic hydrolyzate with Bacillus natto powder at an inoculation amount of 0.05-1%, fermenting at a temperature of 38-45° C. for 14-36 hours; cooling to 25-30° C., inoculating Kluyveromyces lactis at an inoculation amount of 1-6%, and fermenting for 3-7 days to obtain eggshell membrane enzymatic fermentation liquid;

[0011] S4, centrifuging the eggshell membrane enzymatic fermentation broth, adding a clarifier, and obtaining a clarified liquid;

[0012] S5. Add a decolorizing agent to the clarified liquid and dry it to obtain type X collagen peptide.

[0013] Specifically, in step S1, the protein membrane powder is ultrafinely crushed to 100-5000 mesh; preferably, crushed to 100-500 mesh.

[0014] Specifically, the amount of water added in step S1 is 8-20 times the amount of water; preferably, the amount of water added is 10-16:1; further preferably, the amount of water added is 10:1.

[0015] Specifically, in step S1, the pH value is 8-14, the temperature is 75° C.-95° C., and the extraction is carried out for 5-12 hours;

[0016] Preferably, in step S1, the pH value is 10-12, the temperature is 80°C-90°C, and the heat preservation extraction is performed for 8-12 hours;

[0017] Further preferably, in step S1, the pH value is 11, the temperature is 90° C., and the extraction is carried out for 12 hours.

[0018] Specifically, the enzymes added in step S2 include pepsin and animal proteolytic enzymes.

[0019] Furthermore, in step S2, the amount of pepsin added is 0.1-1%, and the enzymatic hydrolysis time is 2-8 h;

[0020] Preferably, the amount of pepsin added in step S2 is 0.4-0.8%, and the enzymatic hydrolysis time is 4-6 hours;

[0021] Further preferably, the amount of pepsin added in step S2 is 0.5%, and the enzymatic hydrolysis time is 6 hours.

[0022] Furthermore, in step S2, the amount of animal protein hydrolyzing enzyme added is 1-5%, and the enzymatic hydrolysis time is 2-8 hours;

[0023] Preferably, in step S2, the amount of animal protein hydrolyzing enzyme added is 2-4%, and the enzymatic hydrolysis time is 4-6 hours;

[0024] Further preferably, in step S2, the amount of animal protein hydrolase added is 2%, and the enzymatic hydrolysis time is 4 hours.

[0025] Specifically, the percentages of the added amounts of pepsin and animal protein hydrolase in step S2 are mass volume percentages.

[0026] Furthermore, in step S2, pepsin is added first and then animal proteolytic enzyme is added.

[0027] Specifically, in step S2, the pH value is adjusted to 1.5-3 and the temperature is adjusted to 30-40° C. before adding pepsin;

[0028] Preferably, the pH value is adjusted to 2-2.5 and the temperature to 35-38° C. before adding pepsin in step S2;

[0029] Further preferably, the pH value is adjusted to 2 and the temperature to 37° C. before adding pepsin in step S2.

[0030] Specifically, in step S2, the pH is adjusted to 7-8.5 and the temperature is adjusted to 50-60° C. before adding the animal protein hydrolyzing enzyme;

[0031] Preferably, in step S2, the pH is adjusted to 7-7.5 and the temperature is adjusted to 50-55° C. before adding the animal protein hydrolyzing enzyme;

[0032] Further preferably, the pH is adjusted to 7.5 and the temperature is adjusted to 50° C. before adding the animal protein hydrolyzing enzyme in step S2.

[0033] Preferably, in step S3, the inoculation amount of Bacillus natto powder is 0.1-0.5%, and the inoculation amount of Kluyveromyces lactis is 2-5%;

[0034] Further preferably, in step S3, the inoculation amount of Bacillus natto powder is 0.4%, and the inoculation amount of Kluyveromyces lactis is 2%.

[0035] Preferably, after inoculating Bacillus natto powder in step S3, the mixture is stirred with ventilation and the pH is 7-8.5;

[0036] Further preferably, in step S3, after inoculating Bacillus natto powder, ventilation and stirring are performed, and the pH is 7.5.

[0037] Preferably, the fermentation temperature after inoculation of Bacillus subtilis natto powder in step S3 is 40-42° C., and the fermentation time is 16-32 h;

[0038] More preferably, the fermentation temperature after inoculation of Bacillus natto powder in step S3 is 42° C., and the fermentation time is 24 h.

[0039] Preferably, the pH of the inoculation of Kluyveromyces lactis in step S3 is 7-8; further preferably, the pH of the inoculation of Kluyveromyces lactis is 7.

[0040] More preferably, in step S3, the fermentation is carried out for 5-6 days after the inoculation of Kluyveromyces lactis; and even more preferably, the fermentation is carried out for 5 days after the inoculation of Kluyveromyces lactis.

[0041] Specifically, in step S4, after centrifugation, the precipitate is removed and the pH is adjusted to 4.5-6, preferably, 5-5.5.

[0042] Specifically, the amount of the clarifier added in step S4 is 1-8%; preferably, the amount of the clarifier added is 2-5%.

[0043] Specifically, the percentage of the amount of the clarifier added in step S4 is a mass volume percentage.

[0044] Specifically, after adding the clarifier in step S4, the mixture is allowed to stand at room temperature for 2-8 hours; preferably, the mixture is allowed to stand at room temperature for 2-6 hours; further preferably, the mixture is allowed to stand at room temperature for 6 hours.

[0045] Specifically, the clarifier in step S4 includes tragacanth gum, alginate and cassava polysaccharide.

[0046] Preferably, the ratio of tragacanth gum, alginate and cassava polysaccharide in step S4 is 1:1-5:1-5;

[0047] Further preferably, in step S4, the ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1-3:1-3;

[0048] Still more preferably, in step S4, the ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1:3.

[0049] Specifically, the amount of the decolorizing agent added in step S5 is 0.5-5%; preferably, the amount of the decolorizing agent added is 1-3%; further preferably, the amount of the decolorizing agent added is 2%.

[0050] Specifically, the percentage of the amount of the decolorizing agent added in step S5 is a mass volume percentage.

[0051] Specifically, in step S5, the decolorizing agent includes activated carbon and diatomaceous earth;

[0052] Preferably, the mass ratio of the activated carbon to diatomaceous earth is 2-10:1;

[0053] Further preferably, the mass ratio of the activated carbon to diatomaceous earth is 3-6:1;

[0054] Still further preferably, the mass ratio of the activated carbon to diatomaceous earth is 4:1.

[0055] Preferably, the activated carbon is walnut shell activated carbon.

[0056] Specifically, the temperature during decolorization in step S5 is 50-70°C, and the decolorization time is 30-90 minutes;

[0057] Preferably, the temperature during decolorization in step S5 is 60° C., and the decolorization time is 40-60 min; more preferably, the decolorization time is 60 min.

[0058] Specifically, after decolorization in step S5, the process further includes removing inorganic salts and water-soluble free amino acids using liquid nanofiltration.

[0059] Specifically, the drying in step S5 is spray drying.

[0060] In another aspect, the present invention provides a type X collagen peptide prepared by the above-mentioned preparation method.

[0061] In another aspect, the present invention provides use of the above-mentioned type X collagen peptide in the preparation of a product for promoting bone growth in children.

[0062] Specifically, the product is a medicine or a food.

[0063] Furthermore, the medicine may also include a pharmaceutically acceptable carrier.

[0064] Furthermore, the food may also include supplementary materials that are acceptable in food science.

[0065] Specifically, the dosage form of the medicine or food is tablets, capsules, powders, granules, pills or oral liquids.

[0066] It should be noted that the application is not intended for the diagnosis or treatment of diseases.

[0067] The beneficial effects of the present invention are:

[0068] The present invention provides a method for preparing type X collagen peptides, comprising enzymatically hydrolyzing a protein membrane extract using pepsin and animal proteolytic enzymes, fermenting the extract with Bacillus natto powder and Kluyveromyces lactis, and then clarifying and decolorizing the extract to produce the collagen peptides. The collagen peptides produced in this invention have a high content of type X collagen and can significantly improve damaged osteoblasts and promote their growth, thus possessing broad application prospects in the preparation of growth-promoting products. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the molecular weight detection spectrum of Example 1.

[0070] Figure 2 This is the molecular weight detection spectrum of Example 2.

[0071] Figure 3 This is the molecular weight detection spectrum of Example 3.

[0072] Figure 4 This is the molecular weight detection spectrum of Comparative Example 1.

[0073] Figure 5 This is the molecular weight detection spectrum of Comparative Example 2.

[0074] Figure 6 This is the molecular weight detection spectrum of Comparative Example 3.

[0075] Figure 7 This is the molecular weight detection spectrum of Comparative Example 4.

[0076] Figure 8 This is the molecular weight detection spectrum of Comparative Example 5.

[0077] Figure 9 The effect of collagen peptides in each group on cell activity; compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0078] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0079] Sources of experimental materials:

[0080] Pepsin and animal protein hydrolase were purchased from Nanning Pangbo Bioengineering Co., Ltd.; Bacillus subtilis natto powder (viable cell count: 7.9×10 8 cfu / g) were purchased from Takahashi, Japan; Kluyveromyces lactis (viable count: 2.4×10 6 cfu / g) was purchased from Shaanxi Angxu Biotechnology Co., Ltd.; tragacanth gum was purchased from Hebei Baipin Biotechnology Co., Ltd.; alginate was purchased from Qingdao Qingshan Bioengineering Co., Ltd.; cassava polysaccharide was purchased from Yangling Ciyuan Biotechnology Co., Ltd.; diatomaceous earth was purchased from Hebei Runhuabang New Materials Technology Co., Ltd., and activated carbon was purchased from Zhengzhou Yongkun Environmental Protection Technology Co., Ltd.; the chicken type X collagen (Colx) ELISA kit was purchased from Shanghai Senpeptide Biotechnology Co., Ltd.

[0081] Example 1 Preparation of Type X Collagen Peptide

[0082] A type X collagen peptide for improving bone growth is prepared by the following steps:

[0083] Step S1, eggshell membrane crushing: weigh 100 g of raw material and crush it into 100-500 mesh;

[0084] Step S2, preparation of crude eggshell membrane extract: adding 10 times the amount of water to eggshell membrane powder, adjusting the pH to 11, temperature to 90° C., and heat-extracting for 12 hours;

[0085] Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the pH value of the crude total protein extract to 2, the temperature to 37°C, adding 0.5% pepsin, and enzymatic hydrolysis for 6 hours; then adjusting the pH value to 7.5, the temperature to 50°C, adding 2% animal protein hydrolase, and enzymatic hydrolysis for 4 hours;

[0086] Step S4, preparation of eggshell membrane enzymatic fermentation broth: inoculate the eggshell membrane enzymatic hydrolyzed broth with Bacillus natto powder (viable cell count: 7.9×10 8 cfu / g), inoculation amount 0.4%, ventilation stirring, pH 7.5, temperature 42 ° C, fermentation for 24 hours; the temperature was lowered to 30 ° C, pH was adjusted to 7, and lactic acid Kluyveromyces was inoculated (viable count: 2.4 × 10 6 cfu / g) 2%, fermented for 5 days;

[0087] Step S5, clarification of the eggshell membrane enzymatic fermentation broth: After centrifugation of the eggshell membrane enzymatic fermentation broth, the precipitate was removed, the pH was adjusted to 5, 5% of a clarifier (the mass ratio of tragacanth gum, alginate, and cassava polysaccharide was 1:1:3) was added, and the mixture was allowed to stand at room temperature for 6 hours, and the clarified liquid was collected;

[0088] Step S6, decolorization of the eggshell membrane enzymatic fermentation broth: adding 2% of a mixed decolorizing agent of walnut shell activated carbon and diatomaceous earth (mass ratio of 4:1) to the clarified liquid, decolorizing at 60°C for 60 minutes, and filtering by plate and frame;

[0089] Step S7, preparation of type X collagen peptides for improving bone growth: after decolorization, the liquid is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain type X collagen peptides.

[0090] Example 2

[0091] A type X collagen peptide for improving bone growth is prepared by the following steps:

[0092] Step S1, eggshell membrane crushing: weigh 100 g of raw material and crush it into 100-500 mesh;

[0093] Step S2, preparation of crude eggshell membrane extract: adding eggshell membrane powder to 8 times water, adjusting the pH to 8, temperature to 75°C, and heat-extracting for 5 hours;

[0094] Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the pH value of the crude total protein extract to 1.5, the temperature to 30°C, adding 0.1% pepsin, and enzymatic hydrolysis for 2 hours; then adjusting the pH value to 7, the temperature to 50°C, adding 1% animal protein hydrolase, and enzymatic hydrolysis for 2 hours;

[0095] Step S4, preparing eggshell membrane enzymatic fermentation broth: inoculating the eggshell membrane enzymatic hydrolyzed broth with Bacillus natto powder at an inoculum level of 0.05%, stirring under ventilation, at a pH of 7 and a temperature of 38° C., and fermenting for 14 hours; lowering the temperature to 25° C., adjusting the pH to 7, and inoculating 1% Kluyveromyces lactis, and fermenting for 3 days;

[0096] Step S5, clarification of the eggshell membrane enzymatic fermentation broth: After centrifugation of the eggshell membrane enzymatic fermentation broth, the precipitate was removed, the pH was adjusted to 4.5, 1% clarifier (tragacanth gum, alginate, and cassava polysaccharide in a mass ratio of 1:1:1) was added, and the mixture was allowed to stand at room temperature for 2 hours, and the clarified liquid was collected;

[0097] Step S6, decolorization of the eggshell membrane enzymatic fermentation broth: adding 0.5% of a mixed decolorizing agent of walnut shell activated carbon and diatomaceous earth (mass ratio of 2:1) to the clarified liquid, decolorizing at 60°C for 30 minutes, and filtering on a plate and frame;

[0098] Step S7, preparation of type X collagen peptides for improving bone growth: after decolorization, the liquid is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain type X collagen peptides.

[0099] Example 3

[0100] A type X collagen peptide for improving bone growth is prepared by the following steps:

[0101] Step S1, eggshell membrane crushing: weigh 100 g of raw material and crush it into 100-500 mesh;

[0102] Step S2, preparation of crude eggshell membrane extract: adding eggshell membrane powder to 20 times water, adjusting the pH to 14, temperature to 95° C., and heat-extracting for 12 hours;

[0103] Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the pH value of the crude total protein extract to 3, the temperature to 40°C, adding 1% pepsin, and enzymatic hydrolysis for 8 hours; then adjusting the pH value to 8.5, the temperature to 60°C, adding 5% animal protein hydrolase, and enzymatic hydrolysis for 8 hours;

[0104] Step S4, preparing eggshell membrane enzymatic fermentation broth: inoculating the eggshell membrane enzymatic hydrolyzed broth with Bacillus natto powder at an inoculum amount of 1%, stirring under ventilation, at a pH of 8.5, at a temperature of 45° C., and fermenting for 36 hours; lowering the temperature to 30° C., adjusting the pH to 8, inoculating 5% Kluyveromyces lactis, and fermenting for 6 days;

[0105] Step S5, clarification of the eggshell membrane enzymatic fermentation broth: After centrifugation of the eggshell membrane enzymatic fermentation broth, the precipitate was removed, the pH was adjusted to 6, and 8% of a clarifier (the mass ratio of tragacanth gum, alginate, and cassava polysaccharide was 1:5:5) was added. The broth was allowed to stand at room temperature for 6 hours, and the clarified liquid was collected;

[0106] Step S6, decolorization of the eggshell membrane enzymatic fermentation broth: adding 5% of a mixed decolorizing agent of walnut shell activated carbon and diatomaceous earth (mass ratio of 10:1) to the clarified liquid, decolorizing at 60°C for 90 minutes, and filtering by plate and frame;

[0107] Step S7, preparation of type X collagen peptides for improving bone growth: after decolorization, the liquid is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain type X collagen peptides.

[0108] Comparative Example 1

[0109] A type X collagen peptide for improving bone growth is prepared by the following steps:

[0110] Step S1: weigh 100 g of raw material and grind it into 100-500 mesh;

[0111] Step S2, preparation of crude eggshell membrane extract: adding 10 times the amount of water to eggshell membrane powder, adjusting the pH to 11, temperature to 90° C., and heat-extracting for 12 hours;

[0112] Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the pH value of the crude total protein extract to 2, the temperature to 37°C, adding 0.5% pepsin, and enzymatic hydrolysis for 6 hours; then adjusting the pH value to 7.5, the temperature to 50°C, adding 2% animal protein hydrolase, and enzymatic hydrolysis for 4 hours;

[0113] Step S4, clarification of the eggshell membrane hydrolysate: After centrifugation of the eggshell membrane hydrolysate, the precipitate was removed, the pH was adjusted to 5, 5% of a clarifier (tragacanth gum, alginate, and cassava polysaccharide in a mass ratio of 1:1:3) was added, and the solution was allowed to stand at room temperature for 6 hours, and the clarified solution was collected;

[0114] Step S5, decolorization of the eggshell membrane enzymatic fermentation broth: adding 2% of a mixed decolorizing agent of walnut shell activated carbon and diatomaceous earth (mass ratio of 4:1) to the clarified liquid, decolorizing at 60°C for 60 minutes, and then filtering on a plate and frame;

[0115] Step S6, preparation of type X collagen peptides for improving bone growth: after decolorization, the liquid is nanofiltered to remove inorganic salts and water-soluble free amino acids, and spray-dried to obtain type X collagen peptides.

[0116] Comparative Example 2

[0117] Compared with Example 1, the only difference is that step S3 is different. In Comparative Example 2, step S3 is:

[0118] Preparation of eggshell membrane hydrolysate: adjust the pH of the crude total protein extract to 2, the temperature to 37°C, add 0.5% pepsin, and hydrolyze for 6 hours; then adjust the pH to 7.5, the temperature to 50°C, add 2% papain, and hydrolyze for 4 hours.

[0119] Comparative Example 3

[0120] Compared with Example 1, the only difference is that step S3 is different. In Comparative Example 3, step S3 is:

[0121] Preparation of eggshell membrane hydrolysate: adjust the pH value of the crude total protein extract to 2, the temperature to 37°C, add 2% pepsin, and hydrolyze for 6 hours; then adjust the pH value to 7.5, the temperature to 50°C, add 6% animal protein hydrolase, and hydrolyze for 4 hours.

[0122] Comparative Example 4

[0123] Compared with Example 1, the only difference is that step S4 is different. In Comparative Example 4, step S4 is:

[0124] Preparation of eggshell membrane enzymatic fermentation broth: Eggshell membrane enzymatic hydrolysate was inoculated with Lactobacillus bulgaricus powder (purchased from Xi'an Dongfeng Biotechnology Co., Ltd.) at an inoculum level of 0.4% and fermented for 24 h at a pH of 7.5 and a temperature of 42°C under ventilation and stirring. The temperature was then lowered to 30°C, the pH adjusted to 7, and 2% Saccharomyces cerevisiae (purchased from Jinan Jinyuyuan Biotechnology Co., Ltd.) was inoculated and fermented for 5 days.

[0125] Comparative Example 5

[0126] Compared with Example 1, the only difference is that step S5 is different. In Comparative Example 5, step S5 is:

[0127] Clarification of the eggshell membrane enzymatic fermentation broth: After centrifugation, remove the precipitate, adjust the pH to 5, add 5% clarifier (the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:6:6), let it stand at room temperature for 6 hours, and collect the clarified liquid.

[0128] Experimental Example 1

[0129] The samples were tested for protein content, molecular weight, and type X collagen content. Protein content was determined in accordance with GB5009.5 National Food Safety Standard for Determination of Protein in Foods, molecular weight was determined in accordance with GB31645 Collagen Peptide, and type X collagen content was determined in accordance with the instructions for the enzyme-linked immunosorbent assay kit. The test results are shown in Tables 1 and Figures 1-8 :

[0130] Table 1

[0131]

[0132] As can be seen from the above table, the protein content and type X collagen content of Examples 1-3 are significantly higher than those of Comparative Examples 1-5.

[0133] Experimental Example 2 Effect of Type X Collagen Peptide on Osteoblast Growth

[0134] 1 Experimental Methods

[0135] Step 1: Osteoblasts (Procell CL-0202) were cultured in a cell culture incubator for 3 hours, then the culture medium was changed and cultured for an additional 24 hours. The culture medium was then removed and digested with 0.25% trypsin for 5 minutes. The digestion was terminated by adding an equal volume of cell culture medium. The cells were harvested into a 10 mL centrifuge tube and centrifuged at 1200 rpm for 3 minutes. The supernatant was removed and fresh cell culture medium was added, pipetting evenly to distribute the cells. The cells were then evenly distributed among three culture dishes and topped up with culture medium. When the cells reached 80% confluence, the above procedure was repeated for passage. The osteoblast culture medium used was a commercially available Saos-2 cell-specific medium.

[0136] Step 2: Effect of collagen peptide on cell activity: After the cells are fully plated, collect the cells into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 3 min, remove the supernatant, add new cell culture medium, pipette evenly and count with a hemocytometer. 4cells in a total volume of 200 μL and cultured overnight at 37°C under 5% CO2. Then, Example 1 and Comparative Example 2 were added to treat the cells, respectively, to a final concentration of 400, 800, and 1200 μg / mL, respectively. Four replicates were set for each concentration, and the polypeptide treatment time was set to 24 hours. 400 μM hydrogen peroxide was then used for treatment for 3 hours, and then a culture solution containing 10% CCK-8 was added to each well for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and then the cell viability was calculated. No hydrogen peroxide was added to the blank control group. The control group was treated with 400 μM hydrogen peroxide and no peptide treatment was added.

[0137] 2 Experimental Results

[0138] The experimental results are as follows Figure 9 As shown, from Figure 9 It can be seen that Example 1 can significantly improve damaged osteoblasts and promote their growth.

[0139] 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 in the scope of protection of the present invention.

Claims

1. A method for preparing type X collagen peptide, characterized in that: The preparation method comprises the following steps: S1. Extract eggshell membrane powder with water to obtain a crude extract; S2, adding enzyme to the crude extract for enzymolysis to obtain enzymolysis solution; S3, inoculating the enzymatic hydrolyzate with Bacillus natto powder at an inoculation amount of 0.05-1%, fermenting at a temperature of 38-45° C. for 14-36 hours; cooling to 25-30° C., inoculating Kluyveromyces lactis at an inoculation amount of 1-6%, and fermenting for 3-7 days to obtain eggshell membrane enzymatic fermentation liquid; S4, centrifuging the eggshell membrane enzymatic fermentation broth, adding a clarifier, and obtaining a clarified liquid; S5. adding a decolorizing agent to the clarified solution and drying to obtain type X collagen peptide; The enzymes added in step S2 are pepsin and animal protein hydrolase. First, 0.1-1% pepsin is added, and the enzymatic hydrolysis time is 2-8 hours. Then, 1-5% animal protein hydrolase is added, and the enzymatic hydrolysis time is 2-8 hours. The clarifier in step S4 is composed of tragacanth gum, alginate and cassava polysaccharide, and the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1-5:1-5.

2. The preparation method according to claim 1, characterized in that In step S3, the inoculation amount of Bacillus natto powder is 0.1-0.5%, and the inoculation amount of Kluyveromyces lactis is 2-5%.

3. The preparation method according to claim 1, characterized in that The amount of the clarifier added in step S4 is 1-8%.

4. The preparation method according to claim 3, characterized in that The amount of the clarifier added in step S4 is 2-5%.

5. The preparation method according to claim 1, characterized in that In step S4, the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1-3:1-3.

6. The preparation method according to claim 1, characterized in that The amount of the decolorizing agent added in step S5 is 0.5-5%.

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