Preparation method of yak bone collagen peptide for increasing bone mineral density and yak bone collagen peptide

Abstract

The invention discloses a preparation method of yak bone collagen peptide for increasing bone mineral density and the yak bone collagen peptide. The invention provides a preparation method of yak bone collagen peptide for increasing bone mineral density. The preparation method comprises the following steps: providing yak bone powder; mixing the yak bone meal with a phosphate buffer solution, adjusting the pH value to 5-5.5, mixing with compound bacteria, and carrying out demineralization treatment to obtain a demineralized product; mixing the demineralized product with a compound enzyme, and carrying out an enzymolysis reaction to obtain an enzymolysis product; heating, centrifugally filtering and freeze-drying the enzymolysis product to prepare yak bone collagen peptide; wherein the compound enzyme comprises trypsin, alkaline protease and flavourzyme, and the compound bacteria comprise lactobacillus plantarum and propionibacterium. Lactobacillus plantarum and propionibacterium in the compound bacteria are subjected to synergistic fermentation, and are combined with the enzymolysis reaction of the compound enzyme, so that collagen is decomposed into small molecule peptides more efficiently, the extraction rate of bone collagen peptide is increased, and the bone mineral density is increased.

Description

Technical Field

[0001] The present application relates to the field of protein extraction, and in particular to a method for preparing yak bone collagen peptide for increasing bone density and yak bone collagen peptide. Background Art

[0002] Yaks live in plateau areas, especially in the Qinghai-Tibet Plateau and the Himalayas. These extreme environments give yaks some special physiological adaptability, and their bones also show unique biological characteristics in structure and composition. Yaks have long adapted to high altitude, cold, and low oxygen environments, and need a strong and hard skeleton to support their survival in this harsh environment. Yak bones usually have a higher bone density, denser bone tissue, and the content of minerals (such as calcium and phosphorus) in yak bones is usually higher than that of ordinary cattle bones. This high mineral content enhances the strength of bones, but it also makes it more difficult to extract bone collagen and more difficult to obtain yak bone collagen peptides that can increase bone density.

[0003] At present, the preparation process of extracting collagen peptides from various common livestock and poultry bones such as pig bones, sheep bones, cattle bones, chicken bones and goose bones, the common methods of removing minerals include acid hydrolysis and enzyme method. Although the acid hydrolysis method is more thorough, and because the content of minerals such as calcium and phosphorus in yak bones is high, and the binding force with collagen is strong, it is difficult to untie these bonds by ordinary enzymatic hydrolysis, and it is necessary to increase the consumption of enzymes. Too long acid hydrolysis time or enzymatic hydrolysis time, as well as excessive acid or enzyme consumption will cause collagen peptide degradation or deformation, reduce its extraction efficiency and quality, and make the quality of the prepared bone collagen peptides poor. Summary of the invention

[0004] The present application is made in view of the above-mentioned problems, and its purpose is to obtain more low molecular weight collagen peptides from yak bones that are more conducive to increasing bone density, and the extraction process is efficient.

[0005] In order to achieve the above-mentioned purpose, the present application provides a method for preparing yak collagen peptide for increasing bone density, the preparation steps comprising: S1. Provide yak bone meal; S2, mixing the yak bone powder with a phosphate buffer, adjusting the pH value to 5-5.5, and adding a composite bacteria for fermentation reaction to obtain a demineralized product; S3, mixing the demineralized product with a composite enzyme to perform an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysis product; S4, heating, centrifugally filtering, and freeze-drying the enzymatic hydrolysis product to prepare yak bone collagen peptide; The composite enzyme comprises trypsin, alkaline protease and flavor protease, and the composite bacteria comprises plant lactobacillus and propionibacterium.

[0006] In some embodiments, the mass ratio of the trypsin, the alkaline protease and the flavor protease is 1:(1-3), the inoculation ratio of the Lactobacillus plantarum and the propionibacterium is 1:(1-2), and the inoculation amount of the composite bacteria is 3-5%.

[0007] In some embodiments, step S1 includes: washing, drying, defatting, and crushing yak bones to obtain yak bone powder.

[0008] In some embodiments, the degreasing agent includes acetone, ether or petroleum ether.

[0009] In some embodiments, in step S2: The fermentation temperature is 36~38℃ and the fermentation time is 36~48h.

[0010] In some embodiments, in step S3: The dosage of the composite enzyme is 1-3% of the mass of the demineralized product, the time of the enzymatic hydrolysis reaction is 3-5 hours, the temperature of the enzymatic hydrolysis reaction is 50-60° C., and the pH value of the enzymatic hydrolysis reaction is 7.0-8.0.

[0011] In some embodiments, the flavor protease includes at least one of papain, bromelain, and ficin.

[0012] The present invention also provides a yak bone collagen peptide for increasing bone density. The yak bone collagen peptide is prepared by any one of the above-mentioned methods for preparing the yak bone collagen peptide for increasing bone density.

[0013] The technical solution of the present invention utilizes the synergistic effect of microbial metabolism through the synergistic fermentation of plant lactobacillus and propionic acid bacteria in the composite bacteria, efficiently degrades yak bones and promotes calcium absorption and utilization. Specifically, plant lactobacillus can tolerate propionic acid, soften bone tissue, promote bone mineral dissolution, help release collagen, and provide a better substrate for subsequent enzymatic hydrolysis reactions, while propionic acid bacteria promote bone formation and increase bone density by regulating the activity of bone cells. In addition, calcium propionate generated by the reaction of propionic acid bacteria with free calcium can improve the utilization rate of calcium ions. After the demineralized product is hydrolyzed by the composite enzyme, the generated collagen peptide is more easily absorbed by the human intestine, further promoting the formation of bone calcium. The composite enzyme, specifically the synergistic effect of trypsin, alkaline protease and flavor protease, can gradually and more comprehensively and efficiently decompose collagen into small molecule peptides due to its different cleavage sites and modes of action, improve the extraction rate of collagen peptides after yak bone enzymatic hydrolysis, obtain more small molecular weight collagen peptides, and thus help increase bone density. DETAILED DESCRIPTION

[0014] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0015] At present, the preparation process of extracting collagen peptides from various common livestock and poultry bones such as pig bones, sheep bones, cattle bones, chicken bones and goose bones, the common methods of removing minerals include acid hydrolysis and enzyme method. Although the acid hydrolysis method is more thorough, and because the content of minerals such as calcium and phosphorus in yak bones is high, and the binding force with collagen is strong, it is difficult to untie these bonds by ordinary enzymatic hydrolysis, and it is necessary to increase the consumption of enzymes. Too long acid hydrolysis time or enzymatic hydrolysis time, as well as excessive acid or enzyme consumption will cause collagen peptide degradation or deformation, reduce its extraction efficiency and quality, and make the quality of the prepared bone collagen peptides poor.

[0016] In view of this, the present invention provides a method for preparing yak collagen peptide for increasing bone density, the preparation steps comprising: S1. Provide yak bone meal; S2, mixing the yak bone powder with a phosphate buffer, adjusting the pH value to 5-5.5, and adding a composite bacteria for fermentation reaction to obtain a demineralized product; S3, mixing the demineralized product with a composite enzyme to perform an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysis product; S4, heating, centrifugally filtering, and freeze-drying the enzymatic hydrolysis product to prepare yak bone collagen peptide; The composite enzyme comprises trypsin, alkaline protease and flavor protease, and the composite bacteria comprises plant lactobacillus and propionibacterium.

[0017] The technical solution of the present invention uses plant lactobacillus and propionic acid bacteria in the composite bacteria for synergistic fermentation. The lactic acid produced by the fermentation of plant lactobacillus can soften bone tissue and promote bone absorption. The lactic acid can tolerate propionic acid and help release collagen. The propionic acid can regulate the activity of bone cells and further promote bone formation. The synergistic effect of the two promotes the formation of osteoblasts and increases bone density. At the same time, a better substrate is provided for subsequent enzymatic hydrolysis reaction. At the same time, the yak bone can release more easily absorbable free calcium. The propionic acid bacteria can further react with the free calcium, thereby increasing the utilization rate of calcium ions. At the same time, the substances formed after enzymatic hydrolysis of the demineralized product are more easily absorbed by the human intestine and promote the formation of bone calcium. The composite enzyme is used to synergistically perform the enzymatic hydrolysis reaction. Since trypsin, alkaline protease and flavor protease have different cleavage sites and action modes, the synergistic effect can more comprehensively and efficiently decompose collagen into small molecular peptides, thereby not only improving the extraction rate of bone collagen peptides after enzymatic hydrolysis of yak bones, but also obtaining more small molecular weight bone collagen peptides, which is conducive to increasing bone density.

[0018] It should be noted that the minerals in yak bones mainly include calcium, phosphorus, magnesium, sodium, potassium, sulfur and other elements, which mainly exist in the form of inorganic salts, forming minerals such as hydroxyapatite. Bone density, that is, bone mineral density, is an important indicator for measuring bone strength, reflecting the content of minerals (mainly calcium and phosphorus) in the bones and their compactness. Bone calcium, as one of the main components of bones, has a direct impact on bone density. The more calcium in the bones, usually means the higher the bone density, and the stronger and harder the bones are. This is because calcium is an important component of bone minerals, especially in the form of hydroxyapatite, which provides the necessary hardness and stability for bones.

[0019] Bone calcium refers to the calcium contained in human bones. It is the main mineral component of bones and is essential for maintaining bone strength and density. Calcium is the most abundant mineral in the human body, of which about 99% is stored in bones and teeth in the form of hydroxyapatite. This compound provides bone hardness. Bone calcium is not only essential for building and maintaining a strong skeletal system, but also participates in many other important physiological functions in the body, such as muscle contraction, nerve signal transmission, and blood coagulation.

[0020] In an embodiment of the present invention, the mass ratio of the trypsin, the alkaline protease and the flavor protease is 1:(1-3):(1-3), and the preferred inoculation range is 1:2:1 and 1:3:1. The reasonable ratio of different enzymes avoids excessive or insufficient amount of a certain enzyme and achieves the best synergistic effect. The three enzymes act together, trypsin can specifically cleave the carboxyl ends of lysine and arginine to generate medium-sized peptides, alkaline protease can non-specifically cleave hydrophobic amino acids to generate smaller peptides, and flavor protease further cleaves peptide chains to generate smaller small molecule peptides and amino acids. The three enzymes act together, trypsin generates medium-sized peptides, and alkaline protease and flavor protease further degrade to generate more small molecule collagen peptides.

[0021] In an embodiment of the present invention, the inoculation ratio of the plant lactobacillus and the propionibacterium is 1: (1~2), which can be 1: 1, 1: 2, and the inoculation amount of the composite bacteria is 3~5%. By adjusting the ratio and inoculation amount of the plant lactobacillus and the propionibacterium, the fermentation process can be optimized and the quality and yield of the collagen peptide can be improved. The inoculation amount is preferably 4%. This is because too much or too little lactobacillus will cause changes in calcium ion concentration, which will affect the growth and metabolism of lactic acid bacteria, delay growth and reduce the number of live bacteria, thereby reducing the total amount of bone calcium enrichment. Therefore, the inoculation ratio of the plant lactobacillus and the propionibacterium is 1: (1~2), which can not only optimize the fermentation process, improve bone density, but also increase bone calcium.

[0022] In an embodiment of the present invention, step S1 includes: washing, drying, defatting, and crushing yak bones to obtain yak bone powder.

[0023] Specifically, step S1 is: select fresh yak bones, remove tendons and meat, rinse with running water until there is no blood, then air-dry and defat the bones and crush them with a crusher to obtain yak bone powder. The bone powder is controlled between 200 mesh and 500 mesh, so as to ensure that the size of the aggregate is uniform, which can improve the permeability of acidic substances such as lactic acid in the subsequent fermentation process with composite bacteria and ensure that the demineralization treatment is sufficient.

[0024] In some embodiments, the degreasing agent includes acetone, ether or petroleum ether.

[0025] In an embodiment of the present invention, in step S2, the fermentation temperature is 36-38° C. and the fermentation time is 36-48 hours.

[0026] Specifically, step S2 includes: the phosphate buffer is selected as 0.1 mol / L phosphate buffer, in which yak bone powder is mixed with the phosphate buffer, and the pH value is adjusted to between 5 and 5.5 to prevent the death of the bacteria due to excessive acidity during the fermentation process. The pH value is suitable for the growth of the composite bacteria, and then the composite bacteria are added for fermentation reaction to obtain a demineralized product. The fermentation temperature is 36 to 38°C and the fermentation time is 36 to 48 hours. The fermentation effect is better, and the demineralized product can soften bone tissue, help release collagen, and promote the dissolution of calcium in bones and increase bone calcium content.

[0027] In an embodiment of the present invention, in step S3, the amount of the complex enzyme is 1-3% of the mass of the demineralized product; the time of the enzymatic hydrolysis reaction is 3-5 hours, the temperature of the enzymatic hydrolysis reaction is 50-60°C, and the pH value of the enzymatic hydrolysis reaction is 7.0-8.0. By controlling the amount of enzyme, temperature, time and pH conditions, the release of collagen peptides can be maximized to ensure the efficiency and stability of the enzymatic hydrolysis process.

[0028] Specifically, step S3 includes: mixing the demineralized product with the complex enzyme and performing an enzymatic hydrolysis reaction at 55°C and pH 7.0-8.0 for 4 hours to obtain an enzymatic hydrolysis product, wherein the enzymatic hydrolysis can decompose the large-molecule collagen peptide into small-molecule peptides. The molecular weight of the small-molecule peptides is usually in the range of 1000-5,000Da, and they have good biological activity, can promote osteoblast proliferation, differentiation and mineralization, and thus help increase bone density. Peptides with too small a molecular weight may lack sufficient biological activity, while peptides with too large a molecular weight may be difficult to be effectively absorbed and utilized.

[0029] It should be noted that the heating and freeze-drying steps in step S4 can inactivate the protease, effectively remove excess water and stabilize the structure of the peptide, and remove insoluble matter by centrifugal filtration to ensure the quality of the final collagen peptide.

[0030] The specific step S4 includes: heating the enzymatic hydrolysis product to 85°C for 5-10 minutes to terminate the enzymatic hydrolysis, centrifuging at 10,000 rpm for 20 minutes to remove insoluble impurities, filtering the supernatant with a filter membrane, and then freeze-drying at -40°C to -50°C for 48 hours to ensure complete drying to obtain yak bone collagen peptide.

[0031] In an embodiment of the present invention, the flavor protease includes at least one of papain, bromelain and ficin. Flavor protease has a strong proteolytic ability and can effectively decompose collagen molecules. When mixed with trypsin and alkaline protease, it can release more smaller peptides, which helps to improve the bioavailability of the product and increase bone density.

[0032] The present invention also provides a yak bone collagen peptide, wherein the yak bone collagen peptide is prepared by any one of the above-mentioned methods for preparing the yak bone collagen peptide for increasing bone density.

[0033] Example The following describes the embodiments of the present application. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0034] Implementation Materials Papain (500,000 U / g protein, Shanghai Livzon Dongfeng Biotechnology Co., Ltd.), trypsin (400,000 U / g protein, Shanghai Livzon Dongfeng Biotechnology Co., Ltd.), alkaline protease (400,000 U / g protein, Shanghai Livzon Dongfeng Biotechnology Co., Ltd.); Lactobacillus plantarum was purchased from Shaanxi Shuoyang Biotechnology Co., Ltd., catalog number SY663855; Propionic acid bacteria were purchased from Shaanxi Shuoyang Biotechnology Co., Ltd., product number SY3258758; The components of MRS medium are: peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, Tween-80 0.1%, ammonium citrate 0.2%, pH 5.5-6.0, sterilized at 115℃ for 30min; Components of PAB medium: yeast extract: 5.0 g / L, trypsin 10.0 g / L, sodium propionate 3.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, calcium carbonate 5.0 g / L.

[0035] Example 1 S1. Select fresh yak bones, remove the meat, rinse with tap water and dry for 48 hours, use petroleum ether as a degreasing solvent for the dried bone raw material, ensure that the operating environment is well ventilated, take out the bones from the petroleum ether after drying, repeat the operation several times, grind the bones into bone powder, and control the bone powder between 200 mesh and 500 mesh to obtain yak bone powder; S2, take 100g yak bone powder, add 0.1mol / L phosphate buffer, stir evenly, adjust the pH to 5.5 with 0.1mol / L NaOH to obtain a mixed solution suitable for the growth of composite bacteria, sterilize the mixed solution at 121℃ for 15min, and cool to 37℃ for use; Lactobacillus plantarum was cultured on MRS medium and Propionibacterium spp. was cultured on PAB medium until the logarithmic growth phase, until the concentration of both reached 2x10 9 cfu / mL, and then mixed in a ratio of 1:1, and after mixing, added to the cooled mixed solution at a 4% inoculum volume (v / v), inoculated under a sterile environment, placed in a 37°C constant temperature shaker, and shaken for 36 hours. After the fermentation is completed, the demineralized product needs to be heated to 80°C for 10 minutes to terminate the fermentation enzyme activity, centrifuged, and the supernatant is taken as the demineralized product for later use; S3, using 0.1 mol / L NaOH to adjust the pH of the demineralized product to 8, and adding a buffer to maintain the pH value, adding a composite enzyme to the demineralized product, wherein the amount of the composite enzyme added is 2% of the mass of the demineralized product, and the mass ratio of the trypsin, the alkaline protease and the papain in the composite enzyme is 1:2:1, and enzymolysis is performed at 55° C. for 4 hours to obtain an enzymolysis product; S4. The enzymatic hydrolysate was heated to 85° C. for 10 min to inactivate the enzyme activity, and then centrifuged at 10,000 rpm for 20 min. The supernatant was filtered through a filter membrane, and freeze-dried at -40° C. to -50° C. for 48 h to ensure complete drying to obtain yak bone collagen peptide.

[0036] Example 2 The preparation method of Example 2 of the present application is similar to that of Example 1, except that the mass ratio of the trypsin, the alkaline protease and the papain is 1:1:3.

[0037] Example 3 Example 3 of the present application is a preparation method similar to that of Example 1, except that the mass ratio of the trypsin, the alkaline protease and the papain is 1:3:3.

[0038] Example 4 Example 4 of the present application is a preparation method similar to that of Example 1, except that the mass ratio of the trypsin, the alkaline protease and the papain is 1:2:2.

[0039] Example 5 The preparation method of Example 5 of the present application is similar to that of Example 1, except that the inoculation ratio of Lactobacillus plantarum to Propionibacterium is 1:2.

[0040] Comparative Example 1 The preparation method of Comparative Example 1 of the present application is similar to that of Example 1, except that papain is not added.

[0041] Comparative Example 2 The preparation method of Comparative Example 2 of the present application is similar to that of Example 1, except that trypsin is not added.

[0042] Comparative Example 3 Comparative Example 3 of the present application has a similar preparation method to Example 1, except that flavor protease is not added.

[0043] Comparative Example 4 Comparative Example 4 of the present application has a preparation method similar to that of Example 1, except that the plant lactobacillus is not added.

[0044] Comparative Example 5 Comparative Example 5 of the present application has a preparation method similar to that of Example 1, except that the propionic acid bacteria are not added.

[0045] Performance Test: (1) The yak collagen peptides for increasing bone density prepared in Examples 1 to 5 and Comparative Examples 1 to 5 were subjected to the following tests. The test results are shown in Table 1: 1. Bone density measurement: Refer to the national health food method for the assessment of increasing bone density function).

[0046] Rats were divided into 10 groups and fed with food according to various formulas, except that each group was fed with the collagen peptides of Examples 1 to 5 and Comparative Examples 1 to 5 for 9 weeks. The activities of the rats were observed during the period. After the last feeding, the rats were killed, and the femurs on both sides were quickly peeled off. The femurs on both sides were immersed in physiological saline and stored at -40°C for testing. During the measurement, the left femur was thawed and the surface moisture was removed, and then evenly arranged on a muscle simulation board. The entire left femur was scanned with a LUNAR Prodigy dual-energy X-ray absorptiometry, and the average bone density of the bone represented the bone density of the bone. The data were analyzed by GE Lunar software, as shown in Table 1.

[0047] 2. Determination of bone calcium content: The determination method was carried out according to the national standard GB / T5009.92-2003 using a SpectrAA-220FS atomic absorption spectrophotometer, as shown in Table 1.

[0048] 3. Determination of collagen peptide content and corresponding molecular weight: The method for detecting the content of the collagen peptides prepared in Examples 1 to 5 and Comparative Examples 1 to 5 is carried out with reference to the method for determining the content of the collagen peptides described in Appendix B of GB / T 22492-2008 and GB / T 22729-2008; the method for determining the relative molecular weight distribution of the collagen peptides is carried out with reference to Appendix A of GB / T 22492-2008. The test results are shown in Table 2.

[0049] 4. The test results are as follows: Table 1

[0050] From the test results in Table 1, it can be seen that the present embodiment first uses lactobacillus fermentation and then uses enzymatic hydrolysis. Since organic acids such as lactic acid are produced during the fermentation process, it helps to promote the dissolution of bone minerals, increase their solubility and bioavailability, and the enzymatic hydrolysis reaction can also obtain more specific small molecule peptides, thereby promoting the improvement of bone density and bone calcium. However, in the process of preparing yak collagen peptides, comparative examples 1 to 5 lack the components of composite enzymes or composite bacteria, resulting in poor fermentation and enzymatic hydrolysis reactions, thereby reducing the bone density and bone calcium content.

[0051] Table 2

[0052] As can be seen from Table 2, from the test results in Table 1, it can be seen that the present application embodiment first uses lactobacillus fermentation and then uses enzymatic hydrolysis reaction, which can specifically cut the peptide bonds in the collagen and decompose it into smaller peptide segments or even amino acids, which not only improves the yield of collagen peptides, but also obtains more small molecule peptides, which are more easily absorbed and promote the increase of bone density. However, in the process of preparing yak collagen peptides, Comparative Example 1 lacks the components of composite enzymes or composite bacteria, which makes the fermentation reaction and enzymatic hydrolysis reaction poor, thereby reducing the content of collagen peptides and the content of small molecule peptides.

[0053] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the subject matter of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing yak collagen peptide for increasing bone density, characterized in that: The preparation steps include: S1. Provide yak bone meal; S2, mixing the yak bone powder with a phosphate buffer, adjusting the pH value to 5-5.5, and then mixing with a composite bacteria for demineralization to obtain a demineralized product; S3, mixing the demineralized product with a composite enzyme to perform an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysis product; S4, heating, centrifugally filtering, and freeze-drying the enzymatic hydrolysis product to prepare yak bone collagen peptide; The composite enzyme comprises trypsin, alkaline protease and flavor protease, and the composite bacteria comprises plant lactobacillus and propionibacterium.

2. The method for preparing yak collagen peptide for increasing bone density according to claim 1, characterized in that: The mass ratio of the trypsin, the alkaline protease and the flavor protease is 1:(1-3):(1-3), the inoculation ratio of the plant lactobacillus and the propionic acid bacteria is 1:(1-2), and the inoculation amount of the composite bacteria is 3-5%.

3. The method for preparing yak collagen peptide for increasing bone density according to claim 1, characterized in that: Step S1 includes: washing, drying, defatting and crushing the yak bones to obtain yak bone powder.

4. The method for preparing yak collagen peptide for increasing bone density according to claim 3, characterized in that: The degreasing agent includes acetone, ether or petroleum ether.

5. The method for preparing yak collagen peptide for increasing bone density according to claim 1, characterized in that: In step S2: The demineralization temperature is 36~38℃, and the demineralization time is 36~48h.

6. The method for preparing yak collagen peptide for increasing bone density according to claim 1, characterized in that: In step S3: The dosage of the complex enzyme is 1-3% of the mass of the demineralized product, the time of the enzymatic hydrolysis reaction is 3-5 hours, the temperature of the enzymatic hydrolysis reaction is 50-60° C., and the pH value of the enzymatic hydrolysis reaction is 7.0-8.

0.

7. The method for preparing yak collagen peptide for increasing bone density according to claim 1, characterized in that: The flavor protease includes at least one of papain, bromelain and ficin.

8. A yak collagen peptide for increasing bone density, characterized in that: The yak bone collagen peptide is prepared by the preparation method of the yak bone collagen peptide for increasing bone density according to any one of claims 1 to 7.

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