X-type collagen peptide for improving bone growth as well as preparation method and application of X-type collagen peptide

Abstract

The invention discloses an X-type collagen peptide for improving bone growth as well as a preparation method and application of the X-type collagen peptide, and relates to the technical field of biology. The preparation method comprises the following steps: S1, adding water into eggshell membrane powder, and extracting to obtain a crude extract; s2, enzyme is added into the crude extract for enzymolysis, and enzymatic hydrolysate is obtained; s3, bacillus natto powder is inoculated into the enzymatic hydrolysate, the inoculation amount is 0.05-1%, the temperature is 38-45 DEG C, and fermentation is conducted for 14-36 h; cooling to 25-30 DEG C, inoculating kluyveromyces lactis with the inoculation amount of 1-6%, and fermenting for 3-7 days to obtain eggshell membrane enzymolysis fermentation liquor; s4, centrifuging the eggshell membrane enzymolysis fermentation liquor, and adding a clarifying agent to obtain clarified liquor; and S5, adding a decolorizing agent into the clear liquid, and drying to obtain the X-type collagen peptide. The collagen peptide disclosed by the invention is relatively high in X-type collagen content, can promote the growth of osteoblasts, and has a wide application prospect in preparation of products for promoting growth.

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, and a preparation method and application thereof. Background Art

[0002] Type X collagen is a short-chain non-fibrous collagen. It is distributed in the extracellular matrix in a liquid homogeneous form and is an important component of the extracellular matrix. Its short chain is only 1 / 2 the length of ordinary collagen fibers. Type X collagen plays an important role in the growth and development of bones, especially in the endochondral ossification process. Type X collagen molecules aggregate in the cartilage matrix to form a super-large molecular hexagonal lattice structure, which provides a deposition 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, wherein 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 the eggshell membrane powder, heating to 85-100°C, and keeping the temperature for 10-30 minutes; (2) cooling, adding enzymes for enzymolysis, and obtaining eggshell membrane extract; (3) filtering, concentrating, and drying, wherein the enzymes used for enzymolysis comprise a mixture of serine protease, pectinase, cartilage sulfate extracting enzyme, keratinase, and aminopeptidase in a mass ratio of 15-35:5-15:15-25:10-20:5-15. The invention uses bovine collagen peptide, cartilage extract, eggshell membrane extract, turmeric, milk mineral salt, and sodium hyaluronate to significantly treat osteoporosis. Chinese patent CN118813744A discloses a method for optimizing ultrasound-assisted enzyme extraction of eggshell membrane protein based on response surface methodology, the method comprising: S1, peeling, cleaning and drying the eggshell membrane, and preparing the dried eggshell membrane into eggshell membrane powder by mechanical grinding; S2, immersing the eggshell membrane powder in distilled water with different solid-liquid ratios, and adjusting the pH with acetic acid to obtain an acid hydrolyzate; S3, ultrasonically treating the enzymatic hydrolysis supernatant on the basis of the optimal enzymatic hydrolysis conditions, collecting the enzymatic hydrolysis supernatant, adjusting the pH with NaOH solution, and salting out, centrifuging, dialyzing, and freeze-drying the supernatant to obtain the extracted eggshell membrane protein. The invention can improve the extraction effect and quality of collagen and reduce production costs. However, its effect on promoting bone growth and increasing the amount of type X collagen-containing white peptide 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] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a method for preparing a type X collagen peptide, the method comprising 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 natto Bacillus powder in the enzymatic hydrolysate 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 lactic acid Kluyveromyces at an inoculation amount of 1-6%, fermenting for 3-7 days, and obtaining eggshell membrane enzymatic fermentation liquid; S4, centrifuging the eggshell membrane enzymatic fermentation liquid, adding a clarifier, and obtaining a clarified liquid; S5. Add a decolorizing agent to the clarified liquid, dry it, and obtain the type X collagen peptide.

[0007] Specifically, in step S1, the protein membrane powder is ultrafinely crushed to 100-5000 mesh; preferably, crushed to 100-500 mesh.

[0008] 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.

[0009] Specifically, in step S1, the pH value is 8-14, the temperature is 75°C-95°C, and the heat preservation extraction is performed for 5-12 hours; 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; More preferably, in step S1, the pH value is 11, the temperature is 90° C., and the extraction is carried out for 12 hours.

[0010] Specifically, the enzyme added in step S2 includes pepsin and animal proteolytic enzyme.

[0011] Furthermore, in step S2, the amount of pepsin added is 0.1-1%, and the enzymolysis time is 2-8h; Preferably, the amount of pepsin added in step S2 is 0.4-0.8%, and the enzymolysis time is 4-6h; Further preferably, in step S2, the amount of pepsin added is 0.5% and the enzymolysis time is 6 h.

[0012] Furthermore, in step S2, the amount of animal protein hydrolyzing enzyme added is 1-5%, and the enzymatic hydrolysis time is 2-8h; Preferably, in step S2, the amount of animal protein hydrolyzing enzyme added is 2-4%, and the enzymolysis time is 4-6h; Further preferably, in step S2, the amount of animal protein hydrolase added is 2%, and the enzymatic hydrolysis time is 4 hours.

[0013] Specifically, the percentage of added amount of pepsin and animal proteolytic enzyme in step S2 is mass volume percentage.

[0014] Furthermore, in step S2, pepsin is added first and then animal proteolytic enzyme is added.

[0015] Specifically, in step S2, before adding pepsin, the pH value is adjusted to 1.5-3 and the temperature is adjusted to 30-40° C.; Preferably, the pH value is adjusted to 2-2.5 and the temperature to 35-38° C. before adding pepsin in step S2; Further preferably, the pH value is adjusted to 2 and the temperature to 37° C. before adding pepsin in step S2.

[0016] Specifically, in step S2, before adding the animal proteolytic enzyme, the pH is adjusted to 7-8.5 and the temperature is adjusted to 50-60° C.; 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 proteolytic enzyme; Further preferably, the pH is adjusted to 7.5 and the temperature is adjusted to 50° C. before adding the animal proteolytic enzyme in step S2.

[0017] 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%; More preferably, in step S3, the inoculation amount of Bacillus natto powder is 0.4%, and the inoculation amount of Kluyveromyces lactis is 2%.

[0018] Preferably, after inoculating Bacillus natto powder in step S3, the mixture is stirred with ventilation, and the pH value is 7-8.5; Further preferably, in step S3, after inoculating Bacillus natto powder, ventilation and stirring are performed, and the pH value is 7.5.

[0019] Preferably, the fermentation temperature after inoculation of Bacillus natto powder in step S3 is 40-42° C., and the fermentation time is 16-32 h; More preferably, the fermentation temperature after inoculation of Bacillus natto powder in step S3 is 42° C. and the fermentation time is 24 h.

[0020] 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.

[0021] 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.

[0022] Specifically, in step S4, after centrifugation, the precipitate is removed and the pH is adjusted to 4.5-6, preferably, the pH is 5-5.5.

[0023] Specifically, the amount of the clarifier added in step S4 is 1-8%; preferably, the amount of the clarifier added is 2-5%.

[0024] Specifically, the percentage of the amount of the clarifier added in step S4 is a mass volume percentage.

[0025] 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.

[0026] Specifically, the clarifier in step S4 includes tragacanth gum, alginate and cassava polysaccharide.

[0027] Preferably, the ratio of tragacanth gum, alginate and cassava polysaccharide in step S4 is 1:1-5:1-5; Further preferably, in step S4, the ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1-3:1-3; Still more preferably, in step S4, the ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1:3.

[0028] 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%.

[0029] Specifically, the percentage of the amount of the decolorizing agent added in step S5 is a mass volume percentage.

[0030] Specifically, in step S5, the decolorizing agent includes activated carbon and diatomaceous earth; Preferably, the mass ratio of the activated carbon to diatomaceous earth is 2-10:1; Further preferably, the mass ratio of the activated carbon to diatomaceous earth is 3-6:1; Still further preferably, the mass ratio of the activated carbon to diatomaceous earth is 4:1.

[0031] Preferably, the activated carbon is walnut shell activated carbon.

[0032] Specifically, the temperature during decolorization in step S5 is 50-70°C, and the decolorization time is 30-90 minutes; 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.

[0033] Specifically, after decolorization in step S5, a step of removing inorganic salts and water-soluble free amino acids by liquid nanofiltration is also included.

[0034] Specifically, the drying in step S5 is spray drying.

[0035] In another aspect, the present invention provides a type X collagen peptide prepared by the above preparation method.

[0036] 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.

[0037] Specifically, the product is a medicine or a food.

[0038] Furthermore, the medicine may also include a pharmaceutically acceptable carrier.

[0039] Furthermore, the food may also include auxiliary materials acceptable in food science.

[0040] Specifically, the dosage form of the medicine or food is tablets, capsules, powders, granules, pills or oral liquids.

[0041] It should be noted that the application is not intended for the diagnosis or treatment of diseases.

[0042] The beneficial effects of the present invention are: The present invention provides a preparation method containing type X collagen peptide, comprising enzymolysis of a protein membrane extract by using pepsin and animal proteolytic enzymes at the same time, fermentation by using bacillus natto powder and lactic acid Kluyveromyces after enzymolysis, and then obtaining collagen peptide through clarification and decolorization steps. The collagen peptide obtained in the present invention has a high content of type X collagen, can significantly improve damaged osteoblasts, promote osteoblast growth, and has broad application prospects in the preparation of growth-promoting products. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the molecular weight detection spectrum of Example 1.

[0044] Figure 2 This is the molecular weight detection spectrum of Example 2.

[0045] Figure 3 This is the molecular weight detection spectrum of Example 3.

[0046] Figure 4 This is the molecular weight detection spectrum of Comparative Example 1.

[0047] Figure 5 This is the molecular weight detection spectrum of Comparative Example 2.

[0048] Figure 6 This is the molecular weight detection spectrum of Comparative Example 3.

[0049] Figure 7 This is the molecular weight detection spectrum of Comparative Example 4.

[0050] Figure 8 This is the molecular weight detection spectrum of Comparative Example 5.

[0051] Fig. 9 The effects 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

[0052] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0053] Sources of experimental materials: Pepsin and animal protein hydrolase were purchased from Nanning Pangbo Bioengineering Co., Ltd.; Bacillus natto powder (viable cell count: 7.9×10 8 cfu / g) was 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., activated carbon was purchased from Zhengzhou Yongkun Environmental Protection Technology Co., Ltd.; chicken type X collagen (Colx) ELISA kit was purchased from Shanghai Senpeptide Biotechnology Co., Ltd.

[0054] Example 1 Preparation of Type X Collagen Peptide A type X collagen peptide for improving bone growth, prepared by the following steps: Step S1, eggshell membrane crushing: weigh 100 grams of raw materials and crush them into 100-500 mesh; Step S2, preparation of crude eggshell membrane extract: adding 10 times of water to eggshell membrane powder, adjusting the pH value to 11, the temperature to 90° C., and heat preservation extraction for 12 hours; Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the total protein crude extract to a pH value of 2, a temperature of 37° C., adding 0.5% pepsin, and enzymolysis time of 6 h; then adjusting the pH value to 7.5, a temperature of 50° C., adding 2% animal protein hydrolase, and enzymolysis time of 4 h; Step S4, preparation of eggshell membrane enzymatic fermentation liquid: inoculate the eggshell membrane enzymatic fermentation liquid 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; temperature lowered to 30°C, pH adjusted to 7, inoculated with lactic acid Kluyveromyces (viable count: 2.4×10 6 cfu / g) 2%, fermentation for 5 days; Step S5, clarification of the eggshell membrane enzymatic fermentation broth: after centrifuging the eggshell membrane enzymatic fermentation broth, remove the precipitate, adjust the pH to 5, add 5% of a clarifier (the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1:3), let it stand at room temperature for 6 hours, and take the clarified liquid; Step S6, decolorization of eggshell membrane enzymatic fermentation liquid: adding 2% of mixed decolorizing agent walnut shell activated carbon and diatomaceous earth (mass ratio is 4:1) to the clarified liquid, decolorizing for 60 minutes at 60°C, and filtering by plate and frame; 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 then spray-dried to obtain type X collagen peptides.

[0055] Example 2 A type X collagen peptide for improving bone growth, prepared by the following steps: Step S1, eggshell membrane crushing: weigh 100 grams of raw materials and crush them into 100-500 mesh; Step S2, preparation of crude eggshell membrane extract: adding 8 times of water to eggshell membrane powder, adjusting the pH value to 8, the temperature to 75° C., and heat-preserving and extracting for 5 hours; 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 enzymolysis time for 2 hours; then adjusting the pH value to 7, the temperature to 50°C, adding 1% animal protein hydrolase, and enzymolysis time for 2 hours; Step S4, preparation of eggshell membrane enzymatic fermentation liquid: inoculating Bacillus natto powder into the eggshell membrane enzymatic hydrolysate, the inoculation amount is 0.05%, stirring with ventilation, pH7, temperature 38°C, and fermenting for 14 hours; lowering the temperature to 25°C, adjusting the pH to 7, inoculating 1% of Kluyveromyces lactis, and fermenting for 3 days; Step S5, clarification of the eggshell membrane enzymatic fermentation broth: after centrifuging the eggshell membrane enzymatic fermentation broth, remove the precipitate, adjust the pH to 4.5, add 1% of a clarifier (the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1:1), let it stand at room temperature for 2 hours, and take the clarified liquid; Step S6, decolorization of the eggshell membrane enzymatic fermentation liquid: 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 for 30 minutes at 60°C, and filtering by plate and frame; 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 then spray-dried to obtain type X collagen peptides.

[0056] Example 3 A type X collagen peptide for improving bone growth, prepared by the following steps: Step S1, eggshell membrane crushing: weigh 100 grams of raw materials and crush them into 100-500 mesh; Step S2, preparation of crude eggshell membrane extract: adding 20 times of water to eggshell membrane powder, adjusting the pH value to 14, the temperature to 95° C., and extracting at this temperature for 12 hours; Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the total protein crude extract to a pH value of 3, a temperature of 40° C., adding 1% pepsin, and enzymatic hydrolysis for 8 h; then adjusting the pH value to 8.5, a temperature of 60° C., adding 5% animal protein hydrolase, and enzymatic hydrolysis for 8 h; Step S4, preparation of eggshell membrane enzymatic fermentation liquid: inoculating Bacillus natto powder into the eggshell membrane enzymatic hydrolysate at an inoculation amount of 1%, stirring with ventilation, pH 8.5, temperature 45° C., and fermenting for 36 hours; lowering the temperature to 30° C., adjusting the pH to 8, inoculating 5% of Kluyveromyces lactis, and fermenting for 6 days; Step S5, clarification of the eggshell membrane enzymatic fermentation broth: after centrifuging the eggshell membrane enzymatic fermentation broth, remove the precipitate, adjust the pH to 6, add 8% of a clarifier (the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:5:5), let it stand at room temperature for 6 hours, and take the clarified liquid; Step S6, decolorization of the eggshell membrane enzymatic fermentation liquid: 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; 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 then spray-dried to obtain type X collagen peptides.

[0057] Comparative Example 1 A type X collagen peptide for improving bone growth, prepared by the following steps: Step S1, weigh 100 grams of raw material and grind it into 100-500 mesh; Step S2, preparation of crude eggshell membrane extract: adding 10 times of water to eggshell membrane powder, adjusting the pH value to 11, the temperature to 90° C., and heat preservation and extraction for 12 hours; Step S3, preparation of eggshell membrane enzymatic hydrolysate: adjusting the total protein crude extract to a pH value of 2, a temperature of 37° C., adding 0.5% pepsin, and enzymolysis time of 6 h; then adjusting the pH value to 7.5, a temperature of 50° C., adding 2% animal protein hydrolase, and enzymolysis time of 4 h; Step S4, clarification of the eggshell membrane hydrolysate: after centrifuging the eggshell membrane hydrolysate, remove the precipitate, adjust the pH to 5, add 5% of a clarifier (the mass ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1:3), let it stand at room temperature for 6 hours, and take the clarified liquid; Step S5, decolorization of eggshell membrane enzymatic fermentation liquid: adding 2% of mixed decolorizing agent walnut shell activated carbon and diatomaceous earth (mass ratio of 4:1) to the clarified liquid, decolorizing for 60 minutes at 60°C, and filtering by plate and frame; 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 then spray-dried to obtain type X collagen peptides.

[0058] Comparative Example 2 Compared with Example 1, the only difference is that step S3 is different. In Comparative Example 2, step S3 is: Preparation of eggshell membrane hydrolysate: adjust the pH value of the total protein crude extract to 2, the temperature to 37°C, add 0.5% pepsin, and the hydrolysis time is 6 hours; then adjust the pH value to 7.5, the temperature to 50°C, add 2% papain, and the hydrolysis time is 4 hours.

[0059] Comparative Example 3 Compared with Example 1, the only difference is that step S3 is different. In Comparative Example 3, step S3 is: Preparation of eggshell membrane hydrolysate: adjust the pH value of the total protein crude extract to 2, the temperature to 37°C, add 2% pepsin, and the hydrolysis time is 6 hours; then adjust the pH value to 7.5, the temperature to 50°C, add 6% animal protein hydrolase, and the hydrolysis time is 4 hours.

[0060] Comparative Example 4 Compared with Example 1, the only difference is that step S4 is different. In Comparative Example 4, step S4 is: Preparation of eggshell membrane enzymatic fermentation broth: Lactobacillus bulgaricus powder (purchased from Xi'an Dongfeng Biotechnology Co., Ltd.) was inoculated into the eggshell membrane enzymatic hydrolysate, with an inoculation amount of 0.4%, and fermented for 24 hours with ventilation and stirring, pH 7.5, and temperature 42°C; the temperature was lowered to 30°C, the pH was adjusted to 7, and 2% of brewer's yeast (purchased from Jinan Jinyuyuan Biotechnology Co., Ltd.) was inoculated and fermented for 5 days.

[0061] Comparative Example 5 Compared with Example 1, the only difference is that step S5 is different. In Comparative Example 5, step S5 is: Clarification of eggshell membrane enzymatic fermentation broth: After centrifugation of the eggshell membrane enzymatic fermentation broth, 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 take the clarified liquid.

[0062] Experimental Example 1 The samples were tested for protein content, molecular weight, and type X collagen content. The protein content was determined in accordance with GB5009.5 National Food Safety Standard for Determination of Protein in Food, the molecular weight was determined in accordance with GB31645 Collagen Peptide, and the type X collagen content was determined in accordance with the instructions for the enzyme-linked immunosorbent assay kit. The test results are shown in Table 1 and Figure 1-Figure 8 : Table 1

[0063] It can be seen from the above table that the protein content and type X collagen content of Examples 1-3 are significantly higher than those of Comparative Examples 1-5.

[0064] Experimental Example 2 Effect of Type X Collagen Peptide on the Growth of Osteoblasts 1 Experimental methods Step 1: After culturing osteoblasts (Procell CL-0202) in a cell culture incubator for 3 h, the medium was changed and cultured for another 24 h. After that, the culture medium was removed and 0.25% trypsin was added for digestion for 5 min. The same volume of cell culture medium was added to terminate the digestion. The cells were collected into a 10 mL centrifuge tube and centrifuged at 1200 rpm for 3 min. The supernatant was removed and new cell culture medium was added and evenly mixed. The supernatant was added to 3 culture dishes and the culture medium was replenished. When the cells grew to 80% of the plate, the above operation was repeated for passage. The culture medium for osteoblasts was a purchased special culture medium for Saos-2 cells.

[0065] Step 2: Effect of collagen peptide on cell activity: After the cells are spread on the plate, collect the cells into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 3 min, remove the supernatant, add new cell culture medium, blow evenly and count with a hemocytometer. 4 cells in a total volume of 200 μL at 37°C with 5% CO 2 Conditions were used for overnight culture. Then, Example 1 and Comparative Example 2 were added to treat the cells, respectively, so that the final concentrations were 400, 800, and 1200 μg / mL, respectively. Four replicate wells were set for each concentration, and the polypeptide treatment time was set to 24 hours. Then, 400 μM hydrogen peroxide was 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 an enzyme reader, and then the cell survival rate was calculated. No hydrogen peroxide was added to the blank control group, and the control group was treated with 400 μM hydrogen peroxide without peptide treatment.

[0066] 2 Experimental Results The experimental results are as follows Fig. 9 As shown, from Fig. 9 It can be seen that Example 1 can significantly improve damaged osteoblasts and promote their growth.

[0067] 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 principle of the present invention should be included in the protection scope 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 natto Bacillus powder in the enzymatic hydrolysate 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 lactic acid Kluyveromyces at an inoculation amount of 1-6%, fermenting for 3-7 days, and obtaining eggshell membrane enzymatic fermentation liquid; S4, centrifuging the eggshell membrane enzymatic fermentation liquid, adding a clarifier, and obtaining a clarified liquid; S5. Add a decolorizing agent to the clarified liquid, dry it, and obtain the type X collagen peptide.

2. The preparation method according to claim 1, characterized in that: The enzymes added in step S2 include pepsin and animal proteolytic enzymes.

3. The preparation method according to claim 2, characterized in that: In step S2, the amount of pepsin added is 0.1-1%, and the enzymolysis time is 2-8h.

4. The preparation method according to claim 2, characterized in that: In step S2, the amount of animal protein hydrolase added is 1-5%, and the enzymolysis time is 2-8 hours.

5. 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%.

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

7. The preparation method according to claim 6, characterized in that: The amount of the clarifier added in step S4 is 2-5%.

8. The preparation method according to claim 1, characterized in that: The clarifying agent in step S4 includes tragacanth gum, alginate and cassava polysaccharide.

9. The preparation method according to claim 8, characterized in that: The ratio of tragacanth gum, alginate and cassava polysaccharide in step S4 is 1:1-5:1-5.

10. The preparation method according to claim 9, characterized in that: In step S4, the ratio of tragacanth gum, alginate and cassava polysaccharide is 1:1-3:1-3.

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

12. The type X collagen peptide prepared by the preparation method according to any one of claims 1 to 11.

13. Use of the type X collagen peptide according to claim 12 in the preparation of a product for promoting bone growth in children.

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