Preparation method and application of bovine cartilage active polypeptide

Abstract

The invention relates to the technical field of biology, and provides a preparation method and application of bovine cartilage active polypeptide. The preparation method of the bovine cartilage active polypeptide comprises the following steps: performing degreasing, crushing and pulping, tissue homogenizing, enzymolysis, enzyme deactivation, filtration, concentration and sterilization on bovine cartilage to obtain the bovine cartilage active polypeptide. The enzymolysis is graded enzymolysis, proteases for the graded enzymolysis are different, the protease for the first-stage enzymolysis is alkaline protease, the protease for the second-stage enzymolysis is neutral protease, the protease for the third-stage enzymolysis is papain, and the protease for the fourth-stage enzymolysis consists of alkaline protease and neutral protease; protease used in the fifth-stage enzymolysis is composed of neutral protease and cartilage protease, and protease used in the sixth-stage enzymolysis is composed of alkaline protease and papain. According to the technical scheme, the problem of low yield of the bovine cartilage active polypeptide in related technologies is solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a preparation method and application of bovine cartilage active polypeptide. Background Art

[0002] Bovine cartilage is a part of the bone supply system in humans and vertebrates. The most important functions of cartilage are lubrication and support, providing blood and oxygen for the whole body joints, and being an important component of the whole body's bone system. Cartilage is a very special tissue organ in vertebrates. According to the theory of tonifying the viscera with the viscera in traditional Chinese medicine, bovine cartilage tubes must contain special functional factors. Analysis and detection show that fresh bovine cartilage contains nutrients, macroelements, trace elements, fatty acids and various amino acids required by the human body. It can be seen that bovine cartilage has extremely high nutritional value. The active polypeptide extracted from bovine cartilage has significant effects in promoting bone growth and repair, anti-inflammatory, antioxidant and immunomodulatory aspects, and shows great development value in industries such as medicine and health products.

[0003] However, there are many drawbacks in the traditional preparation method of bovine cartilage active polypeptide. One of the problems is the low yield of bovine cartilage active polypeptide, which will bring various adverse effects. In terms of resource utilization, the low yield of bovine cartilage active polypeptide means that a large amount of bovine cartilage resources are not fully and effectively utilized, resulting in waste of resources; from the perspective of product supply, due to the low yield, the product output is insufficient to meet the market demand for bovine cartilage active polypeptide products. Therefore, it is necessary to propose a preparation method of bovine cartilage active polypeptide with high yield. Summary of the Invention

[0004] The present invention proposes a preparation method and application of bovine cartilage active polypeptide, which solves the problem of low yield of bovine cartilage active polypeptide in the related technology.

[0005] The technical solution of the present invention is as follows: The present invention proposes a preparation method of bovine cartilage active polypeptide, which includes the following steps: After bovine cartilage is defatted, crushed and pulped, homogenized, enzymolyzed, inactivated of enzymes, filtered, concentrated and sterilized, bovine cartilage active polypeptide is obtained; the enzymolysis is hierarchical enzymolysis, and the proteases for the hierarchical enzymolysis are different. The protease used for the first-stage enzymolysis is alkaline protease, the protease used for the second-stage enzymolysis is neutral protease, the protease used for the third-stage enzymolysis is papain, the protease used for the fourth-stage enzymolysis is composed of alkaline protease and neutral protease, the protease used for the fifth-stage enzymolysis is composed of neutral protease and chondroprotease, and the protease used for the sixth-stage enzymolysis is composed of alkaline protease and papain.

[0006] As a further technical solution, the repair includes the following steps: cutting the bovine cartilage into strips with a length of 3 cm.

[0007] As a further technical solution, the temperature of each stage of enzymatic hydrolysis is independently 40~60°C, and the time of each stage of enzymatic hydrolysis is independently 10~50 min.

[0008] In the present invention, the enzymatic hydrolysis temperature is controlled at 40~60°C. This temperature range is an appropriate interval for various proteases to exhibit their optimal activities. Whether it is a single enzyme or a composite enzyme, they can maintain relatively high activities, thereby efficiently catalyzing the decomposition of bovine cartilage, which helps to improve the extraction efficiency of bovine cartilage active polypeptides. If the temperature is too high, it may cause protease inactivation and unable to effectively decompose bovine cartilage; if the temperature is too low, the protease activity is limited, and the enzymatic hydrolysis reaction rate is slow, affecting the production efficiency.

[0009] In the present invention, the interval time of 10~50 min is set for each stage of enzymatic hydrolysis, which can provide a relatively independent and appropriate reaction process for each stage of enzymatic hydrolysis. On the one hand, after the completion of the previous stage of enzymatic hydrolysis, giving an appropriate interval time helps the reaction system to adjust to the optimal conditions required for the next stage of enzymatic hydrolysis, ensuring that the subsequent enzymatic hydrolysis process is not interfered by the residual reaction substances or conditions of the previous stage, enabling each protease to play its role in the most favorable environment and further enhancing the enzymatic hydrolysis effect. On the other hand, a reasonable interval time is also beneficial to controlling the rhythm of the entire preparation process, avoiding affecting the final yield of bovine cartilage active polypeptides due to too rapid or slow reactions.

[0010] As a further technical solution, the time of each stage of enzymatic hydrolysis is 30 min.

[0011] As a further technical solution, in the four-stage enzymatic hydrolysis, the mass ratio of alkaline protease to neutral protease is 1:1; in the five-stage enzymatic hydrolysis, the mass ratio of neutral protease to chondroprotease is 1:1; in the six-stage enzymatic hydrolysis, the mass ratio of alkaline protease to papain is 1:1.

[0012] In the present invention, the mass ratio of the two proteases in the four-stage, five-stage, and six-stage enzymatic hydrolyses is limited to 1:1, which is conducive to exerting the synergistic advantages of the two enzymes, enabling the proteins in bovine cartilage to be more fully and meticulously degraded, and enhancing the yield of bovine cartilage active polypeptides.

[0013] As a further technical solution, the pH of the first-stage enzymatic hydrolysis is 8~12, the pH of the second-stage enzymatic hydrolysis is 6~8, the pH of the third-stage enzymatic hydrolysis is 6~8, the pH of the fourth-stage enzymatic hydrolysis is 6~10, the pH of the fifth-stage enzymatic hydrolysis is 6~10, and the pH of the sixth-stage enzymatic hydrolysis is 6~8.

[0014] In the present invention, the pH is controlled during each stage of enzymatic hydrolysis. A suitable pH environment helps to maintain the activity and specificity of proteases and improve the enzymatic hydrolysis effect.

[0015] As a further technical solution, the dosage of protease for each level of enzymatic hydrolysis is 0.1% of the dry weight of bovine cartilage.

[0016] As a further technical solution, the degreasing is carried out in three times, and the solutions used for the three times of degreasing are different. The solutions used for the first and second degreasing are both sodium hydroxide solutions with a mass concentration of 0.1%, and the solution used for the third degreasing is an organic solution.

[0017] In the present invention, through three times of degreasing, the fat in bovine cartilage is gradually and comprehensively removed. The fat components in bovine cartilage are complex, and different fat structures and binding modes have different reactions to degreasing solutions. For the first and second degreasing, sodium hydroxide solutions with a mass concentration of 0.1% are used. Sodium hydroxide can react with fat to carry out saponification reaction, converting fat into soap and glycerol that are soluble in water, thereby removing part of the fat in bovine cartilage. After the first two times of degreasing, most of the easily saponifiable fat in bovine cartilage is removed. For the third degreasing, an organic solution is used. The organic solution can dissolve the remaining non-polar fat and some fat-soluble impurities that are difficult to be removed by sodium hydroxide solution, further reducing the fat content in bovine cartilage and creating favorable conditions for the subsequent extraction of active polypeptides from bovine cartilage.

[0018] As a further technical solution, the organic solution is petroleum ether.

[0019] In the present invention, petroleum ether is selected as the organic solvent for the third degreasing. Petroleum ether has excellent solubility for non-polar fat and fat-soluble impurities. After the first two times of degreasing with sodium hydroxide solution, the remaining fat in bovine cartilage is mostly non-polar fat with a relatively complex structure. Petroleum ether can effectively dissolve with these fats by virtue of its non-polar molecular structure, thereby dissolving and removing them from bovine cartilage tissue, further reducing the fat content and significantly improving the yield of active polypeptides from bovine cartilage. Secondly, petroleum ether has a low boiling point and can volatilize rapidly at normal temperature or slightly heated conditions. This characteristic enables petroleum ether to be easily removed from bovine cartilage materials after degreasing and will not leave too much residue in the sample. Compared with some other organic solvents that are not easy to volatilize, using petroleum ether can reduce the difficulties brought by removing solvent residues in the subsequent treatment process, simplify the process flow, and also avoid the possible adverse effects of solvent residues on subsequent enzymatic hydrolysis and other reactions as well as the quality of the final product.

[0020] As a further technical solution, the temperature of degreasing is 0 - 25°C, and the mass ratio of the solution used for each degreasing to bovine cartilage is 100:1.

[0021] As a further technical solution, during the degreasing process, the degreasing time for each degreasing solution is 2 h.

[0022] As a further technical solution, the tissue homogenate includes the following steps: subjecting the pulverized liquid slurry to high-speed centrifugation, adding citric acid for acidolysis, and separating to obtain a protein solution.

[0023] In the present invention, citric acid can precisely adjust the pH value of the system, promote changes in the structure of protein molecules that are beneficial to separation, reduce the binding force between proteins and other components, and thus significantly improve the protein extraction rate.

[0024] As a further technical solution, the mass ratio of the added amount of citric acid to the mass of bovine cartilage is 1:100.

[0025] As a further technical solution, the centrifugation rate is 48000 r / min, and the centrifugation time is 30 min.

[0026] In the present invention, high-speed centrifugation can utilize a powerful centrifugal force field to quickly and efficiently preliminarily separate substances with different densities in the pulverized liquid slurry, making it easier to distinguish and separate protein components from other impurities, greatly improving the separation efficiency and shortening the processing time.

[0027] As a further technical solution, the temperature for enzyme inactivation is 95 - 100 °C, and the time is 30 min.

[0028] In the present invention, the temperature for enzyme inactivation is set at 95 - 100 °C. This temperature range can efficiently and thoroughly inactivate the target enzyme, avoiding problems such as a decline in product quality and component deterioration caused by residual enzyme activity.

[0029] As a further technical solution, the filtration is ultrafiltration, and the ultrafiltration membrane specification for the ultrafiltration is 10 KD.

[0030] The present invention also proposes a composition containing bovine cartilage active polypeptide, including the bovine cartilage active polypeptide obtained by the preparation method of the bovine cartilage active polypeptide described above.

[0031] The present invention also proposes a composition containing bovine cartilage active polypeptide, including the following components in parts by weight: 80 - 90 parts of bovine cartilage active polypeptide, 2 - 8 parts of bovine bone collagen peptide, 1 - 3 parts of fruit powder of pear, 0.5 - 2 parts of Chinese wolfberry, 0.5 - 2 parts of coix seed, 1 - 2 parts of papaya, 0.5 - 1.5 parts of wild jujube seed, 0.5 - 1.5 parts of polygonatum, 1 - 2 parts of poria, 0.5 - 1 part of angelica, 0.5 - 1 part of eucommia, 0.01 - 0.05 parts of konjac.

[0032] The present invention also provides a method for preparing a composition containing bovine cartilage active polypeptide, which includes the following steps: granulating and drying bovine cartilage active polypeptide, bovine bone collagen peptide, fruit powder pear extract, wolfberry extract, coix seed extract, papaya extract, wild jujube seed extract, polygonatum extract, poria cocos extract, angelica extract, eucommia extract, and konjac extract to obtain the composition containing bovine cartilage active polypeptide.

[0033] As a further technical solution, the drying temperature is 135°C.

[0034] As a further technical solution, the binder used during granulation is purified water.

[0035] The working principle and beneficial effects of the present invention are as follows: In the present invention, the entire preparation process covers the pre-treatment processes such as defatting, pulverizing and pulping, and tissue homogenization of bovine cartilage, ensuring the scientificity and rationality of raw material treatment, laying a foundation for subsequent enzymatic hydrolysis and efficient progress of each step. The hierarchical enzymatic hydrolysis method is adopted, and different proteases are selected to carry out enzymatic hydrolysis in sequence. From the ingenious combination of single enzyme to compound enzyme, bovine cartilage can be fully and accurately decomposed at different stages, effectively improving the extraction efficiency of bovine cartilage active polypeptide and greatly increasing the yield of bovine cartilage active polypeptide. Finally, through steps such as ultrafiltration, concentration, and ultra-high temperature sterilization, not only the product quality is further improved, but also the safety and stability of the product are ensured. Detailed implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0037] In the following examples and comparative examples: Alkaline protease: Bacillus licheniformis protease, enzyme activity is 200,000 U / g; Neutral protease: Bacillus subtilis protease, enzyme activity is 100,000 U / g; Papain: enzyme activity is 100,000 U / g; Chondroprotease: cartilage proteolytic enzyme, enzyme activity is 300,000 U / g.

[0038] Example 1 A method for preparing bovine cartilage active polypeptide, comprising the following steps: thaw the frozen bovine cartilage at room temperature, cut 80 g of the thawed bovine cartilage into strips with a length of 3 cm, add it to 800 g of a sodium hydroxide aqueous solution with a mass concentration of 1%, stir and degrease at 0 °C for 2 h, pulverize and beat into pulp after degreasing, centrifuge the pulverized slurry at a high speed of 48000 r / min for 30 min, add 0.8 g of citric acid for acidolysis, separate to obtain a protein solution, perform fractional enzymolysis on the protein solution, inactivate the enzyme at 95 °C for 30 min, then separate and purify the active polypeptide through a 10 KD ultrafiltration membrane, collect the filtrate, and obtain 20.6 g of bovine cartilage active polypeptide after concentration and ultra-high temperature sterilization; Enzymolysis process: For the first-stage enzymolysis, the protease used is alkaline protease, the enzymolysis temperature is 50 °C, the enzymolysis time is 40 min, the dosage is 0.08 g, and the enzymolysis pH is 8; For the second-stage enzymolysis, the protease used is neutral protease, the enzymolysis temperature is 45 °C, the enzymolysis time is 30 min, the dosage is 0.08 g, and the enzymolysis pH is 6; For the third-stage enzymolysis, the protease used is papain, the enzymolysis temperature is 60 °C, the enzymolysis time is 60 min, the dosage is 0.08 g, and the enzymolysis pH is 6; For the fourth-stage enzymolysis, the protease used is composed of alkaline protease and neutral protease (mass ratio 1:1), the enzymolysis temperature is 45 °C, the enzymolysis time is 40 min, the total dosage is 0.08 g, and the enzymolysis pH is 6; For the fifth-stage enzymolysis, the protease used is composed of neutral protease and chondroprotease (mass ratio 1:1), the enzymolysis temperature is 40 °C, the enzymolysis time is 50 min, the total dosage is 0.08 g, and the enzymolysis pH is 6; For the sixth-stage enzymolysis, the protease used is composed of alkaline protease and papain (mass ratio 1:1); the enzymolysis temperature is 50 °C, the enzymolysis time is 60 min, the total dosage is 0.08 g, and the enzymolysis pH is 6; A method for preparing a composition containing bovine cartilage active polypeptide, comprising the following steps: mix 80 parts of the bovine cartilage active polypeptide obtained by the above preparation method, 2 parts of bovine bone collagen peptide, 1 part of fruit powder pear extract, 0.5 part of wolfberry fruit extract, 0.5 part of coix seed extract, 1 part of papaya extract, 0.5 part of wild jujube seed extract, 0.5 part of polygonatum extract, 1 part of poria cocos extract, 0.5 part of angelica extract, 0.5 part of eucommia bark extract, and 0.01 part of konjac extract evenly, add purified water as a binder for granulation, and dry at 135 °C to obtain the composition containing bovine cartilage active polypeptide.

[0039] Example 2 A preparation method of bovine cartilage active polypeptide, comprising the following steps: thaw the frozen bovine cartilage at room temperature, cut 85 g of the thawed bovine cartilage into strips with a length of 3 cm, add it to 850 g of an aqueous sodium hydroxide solution with a mass concentration of 1%, stir and degrease at 15 °C for 2 h, pulverize and beat into a pulp after degreasing, centrifuge the pulverized slurry at a high speed of 48000 r / min for 30 min, add 0.85 g of citric acid for acidolysis, separate to obtain a protein solution, perform fractional enzymatic hydrolysis on the protein solution, inactivate the enzyme at 98 °C for 30 min, then separate and purify the active polypeptide through a 10 KD ultrafiltration membrane, collect the filtrate, and obtain 20.9 g of bovine cartilage active polypeptide after concentration and ultra-high temperature sterilization; Enzymatic hydrolysis process: For the first-stage enzymatic hydrolysis, the protease used is alkaline protease, the enzymatic hydrolysis temperature is 50 °C, the enzymatic hydrolysis time is 40 min, the dosage is 0.085 g, and the enzymatic hydrolysis pH is 10; For the second-stage enzymatic hydrolysis, the protease used is neutral protease, the enzymatic hydrolysis temperature is 45 °C, the enzymatic hydrolysis time is 30 min, the dosage is 0.085 g, and the enzymatic hydrolysis pH is 7; For the third-stage enzymatic hydrolysis, the protease used is papain, the enzymatic hydrolysis temperature is 60 °C, the enzymatic hydrolysis time is 60 min, the dosage is 0.085 g, and the enzymatic hydrolysis pH is 7; For the fourth-stage enzymatic hydrolysis, the protease used is composed of alkaline protease and neutral protease (mass ratio 1:1), the enzymatic hydrolysis temperature is 45 °C, the enzymatic hydrolysis time is 40 min, the total dosage is 0.085 g, and the enzymatic hydrolysis pH is 8; For the fifth-stage enzymatic hydrolysis, the protease used is composed of neutral protease and chondroprotease (mass ratio 1:1), the enzymatic hydrolysis temperature is 40 °C, the enzymatic hydrolysis time is 50 min, the total dosage is 0.085 g, and the enzymatic hydrolysis pH is 8; For the sixth-stage enzymatic hydrolysis, the protease used is composed of alkaline protease and papain (mass ratio 1:1); the enzymatic hydrolysis temperature is 50 °C, the enzymatic hydrolysis time is 60 min, the total dosage is 0.085 g, and the enzymatic hydrolysis pH is 7; A preparation method of a composition containing bovine cartilage active polypeptide, comprising the following steps: mix 85 parts of the bovine cartilage active polypeptide obtained by the above preparation method, 6 parts of bovine bone collagen peptide, 1 part of fruit powder pear extract, 1 part of wolfberry extract, 1 part of coix seed extract, 1.5 parts of papaya extract, 1 part of wild jujube seed extract, 1 part of polygonatum extract, 1.5 parts of poria cocos extract, 0.8 part of angelica extract, 0.8 part of eucommia ulmoides extract, and 0.03 part of konjac extract evenly, add purified water as a binder for granulation, and dry at 135 °C to obtain the composition containing bovine cartilage active polypeptide.

[0040] Example 3 A preparation method of bovine cartilage active polypeptide, comprising the following steps: thaw the frozen bovine cartilage at room temperature, cut 90 g of the thawed bovine cartilage into strips with a length of 3 cm, add it to 900 g of an aqueous sodium hydroxide solution with a mass concentration of 1%, stir and degrease at 25 °C for 2 h, pulverize and beat into pulp after degreasing, centrifuge the pulverized slurry at a high speed of 48000 r / min for 30 min, add 0.9 g of citric acid for acidolysis, separate to obtain a protein solution, perform fractional enzymolysis on the protein solution, inactivate the enzyme at 100 °C for 30 min, then separate and purify the active polypeptide through a 10 KD ultrafiltration membrane, collect the filtrate, collect the filtrates of different components, and obtain 21.0 g of bovine cartilage active polypeptide after concentration and ultra-high temperature sterilization; Enzymolysis process: For the first-stage enzymolysis, the protease used is alkaline protease, the enzymolysis temperature is 50 °C, the enzymolysis time is 40 min, the dosage is 0.09 g, and the enzymolysis pH is 12; For the second-stage enzymolysis, the protease used is neutral protease, the enzymolysis temperature is 45 °C, the enzymolysis time is 30 min, the dosage is 0.09 g, and the enzymolysis pH is 8; For the third-stage enzymolysis, the protease used is papain, the enzymolysis temperature is 60 °C, the enzymolysis time is 60 min, the dosage is 0.09 g, and the enzymolysis pH is 8; For the fourth-stage enzymolysis, the protease used is composed of alkaline protease and neutral protease (mass ratio 1:1), the enzymolysis temperature is 45 °C, the enzymolysis time is 40 min, the total dosage is 0.09 g, and the enzymolysis pH is 10; For the fifth-stage enzymolysis, the protease used is composed of neutral protease and chondroprotease (mass ratio 1:1), the enzymolysis temperature is 40 °C, the enzymolysis time is 50 min, the total dosage is 0.09 g, and the enzymolysis pH is 10; For the sixth-stage enzymolysis, the protease used is composed of alkaline protease and papain (mass ratio 1:1); the enzymolysis temperature is 50 °C, the enzymolysis time is 60 min, the total dosage is 0.09 g, and the enzymolysis pH is 8; A preparation method of a composition containing bovine cartilage active polypeptide, comprising the following steps: mix 90 parts of the bovine cartilage active polypeptide obtained by the above preparation method, 9 parts of bovine bone collagen peptide, 3 parts of fruit powder pear extract, 2 parts of wolfberry extract, 2 parts of coix seed extract, 2 parts of papaya extract, 1.5 parts of wild jujube seed extract, 1.5 parts of polygonatum extract, 2 parts of poria cocos extract, 1 part of angelica extract, 1 part of eucommia extract, and 0.05 part of konjac extract evenly, add purified water as a binder for granulation, and dry at 135 °C to obtain the composition containing bovine cartilage active polypeptide.

[0041] Example 4 Compared with Example 3, Example 4 is different in that the degreasing process is carried out in two steps. The solutions for the first degreasing and the second degreasing are both 900 g of an aqueous sodium hydroxide solution with a mass concentration of 0.1%, and the time for each degreasing is 2 h. 24.3 g of bovine cartilage active polypeptide was obtained in this example.

[0042] Example 5 Compared with Example 3, Example 5 is different in that the degreasing process is carried out in two steps. The solution for the first degreasing is 900 g of an aqueous sodium hydroxide solution with a mass concentration of 0.1%, and the solution used for the second degreasing is 900 g of petroleum ether. The time for each degreasing is 2 h. 23.5 g of bovine cartilage active polypeptide was obtained in this example.

[0043] Example 6 Compared with Example 3, Example 6 is different in that the degreasing process is carried out in three steps. The solutions for the first degreasing and the second degreasing are both 900 g of an aqueous sodium hydroxide solution with a mass concentration of 0.1%, and the solution used for the third degreasing is 900 g of petroleum ether. The time for each degreasing is 2 h. 26.9 g of bovine cartilage active polypeptide was obtained in this example.

[0044] Example 7 Compared with Example 3, Example 7 is different in that the degreasing process is carried out in three steps. The solution for the first degreasing is 900 g of an aqueous sodium hydroxide solution with a mass concentration of 0.1%, and the solutions used for the second degreasing and the third degreasing are both 900 g of petroleum ether. The time for each degreasing is 2 h. 24.0 g of bovine cartilage active polypeptide was obtained in this example.

[0045] Comparative Example 1 Compared with Example 3, Comparative Example 1 is different in that the protease used in the secondary enzymatic hydrolysis is papain, and the protease used in the tertiary enzymatic hydrolysis is neutral protease. 19.2 g of bovine cartilage active polypeptide was obtained in this comparative example.

[0046] Comparative Example 2 Compared with Example 3, Comparative Example 2 is different in that only the primary enzymatic hydrolysis, secondary enzymatic hydrolysis, and tertiary enzymatic hydrolysis are retained. 7.0 g of bovine cartilage active polypeptide was obtained in this comparative example.

[0047] Comparative Example 3 Compared with Example 3, Comparative Example 3 is different in that only the quaternary enzymatic hydrolysis, quinary enzymatic hydrolysis, and senary enzymatic hydrolysis are retained. 9.4 g of bovine cartilage active polypeptide was obtained in this comparative example.

[0048] Comparative Example 4 Compared with Example 3, the difference in Comparative Example 4 is that the protease for four - stage enzymatic hydrolysis is alkaline protease and papain with a mass ratio of 1:1, and the protease for six - stage enzymatic hydrolysis is alkaline protease and neutral protease with a mass ratio of 1:1; 14.2 g of bovine cartilage active polypeptide was obtained in this comparative example.

[0049] Comparative Example 5 Compared with Example 3, the difference in Comparative Example 5 is that the five - stage enzymatic hydrolysis is removed; 11.4 g of bovine cartilage active polypeptide was obtained in this comparative example.

[0050] Experimental Example 1 Calculate the yields of the bovine cartilage active polypeptides prepared in Examples 1 - 7 and Comparative Examples 1 - 5, where the yield=(content of bovine cartilage active polypeptide / content of bovine cartilage active polypeptide)×100%.

[0051] The test results are shown in Table 1: Table 1 Performance test results of bovine cartilage active polypeptides prepared in Examples 1 - 7 and Comparative Examples 1 - 5

[0052] Compared with Comparative Examples 1 - 5, the yield of the bovine cartilage active polypeptide in Example 1 is higher than that in Comparative Examples 1 - 5, indicating that when using hierarchical enzymatic hydrolysis, and the protease used in the first - stage enzymatic hydrolysis is alkaline protease, the protease used in the second - stage enzymatic hydrolysis is neutral protease, the protease used in the third - stage enzymatic hydrolysis is papain, the protease used in the fourth - stage enzymatic hydrolysis is composed of alkaline protease and neutral protease, the protease used in the fifth - stage enzymatic hydrolysis is composed of neutral protease and chondroprotease, and the protease used in the sixth - stage enzymatic hydrolysis is composed of alkaline protease and papain, the yield of the bovine cartilage active polypeptide can be improved.

[0053] Compared with Example 1, the yields of the bovine cartilage active polypeptides in Examples 4 - 6 are higher than that in Example 1, indicating that when using a process of composite degreasing with an alkali solution and an organic solution, the yield of the bovine cartilage active polypeptide can be further improved; and when the organic solvent is petroleum ether, the effect is the best.

[0054] Experimental Example 2 The composition containing bovine cartilage active polypeptide prepared in Example 3 was packaged, with each bag containing 20 g of bovine cartilage active polypeptide, and then boxed, with 20 bags in each box. This composition has the effects of promoting the proliferation of bone cells, anti - fatigue, improving osteoporosis, enhancing immunity and promoting metabolism. The composition was given for trial use in Qiuxian Traditional Chinese Medicine Hospital. After a trial period of one cycle (10 days, 2 bags per day, 1 bag in the morning and 1 bag in the evening), the trial situation is shown in Table 2. By making a telephone follow - up of the trial users, the application effects of the composition containing bovine cartilage active polypeptide were obtained, and the application effects are shown in Table 3.

[0055] The trial situation is shown in Table 2 as follows: Table 2 Trial situation of the composition containing bovine cartilage active polypeptide prepared in Example 3

[0056] The application effect is shown in Table 3 as follows: Table 3 Application effect of the composition containing bovine cartilage active polypeptide prepared in Example 3

[0057] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing bovine cartilage active polypeptide, characterized in that: The following steps are involved: The bovine cartilage is subjected to defatting, crushing and beating, tissue homogenization, enzymolysis, enzyme inactivation, filtration, concentration and sterilization to obtain the bovine cartilage active polypeptide; the enzymolysis is graded enzymolysis, and the proteases used in the graded enzymolysis are different. The protease used in the primary enzymolysis is alkaline protease, the protease used in the secondary enzymolysis is neutral protease, the protease used in the tertiary enzymolysis is papain, the protease used in the quaternary enzymolysis is composed of alkaline protease and neutral protease, the protease used in the fifth enzymolysis is composed of neutral protease and chondroitinase, and the protease used in the sixth enzymolysis is composed of alkaline protease and papain.

2. The method for preparing a bovine cartilage active polypeptide according to claim 1, characterized in that: The temperature of each stage of enzymolysis is independently 40-60° C., and the time of each stage of enzymolysis is independently 10-50 min.

3. The method for preparing a bovine cartilage active polypeptide according to claim 1, characterized in that: The mass ratio of alkaline protease to neutral protease in the fourth-stage enzymolysis is 1:1, the mass ratio of neutral protease to chondroitinase in the fifth-stage enzymolysis is 1:1, and the mass ratio of alkaline protease to papain in the sixth-stage enzymolysis is 1:

1.

4. The method for preparing a bovine cartilage active polypeptide according to claim 1, characterized in that: The degreasing is carried out three times, and different solutions are used for the three degreasing. The solutions used for the first degreasing and the second degreasing are both sodium hydroxide solutions with a mass concentration of 0.1%, and the solutions used for the third degreasing are organic solutions.

5. The method for preparing a bovine cartilage active polypeptide according to claim 4, characterized in that: The organic solvent is petroleum ether.

6. The method for preparing a bovine cartilage active polypeptide according to claim 1, characterized in that: The degreasing temperature is 0-25° C., and the mass ratio of the solution used for each degreasing to the bovine cartilage is 100:

1.

7. A composition containing bovine cartilage active polypeptide, characterized in that: The invention relates to a bovine cartilage active polypeptide obtained by the preparation method of a bovine cartilage active polypeptide according to any one of claims 1 to 6.

8. The composition containing bovine cartilage active polypeptide according to claim 7, characterized in that: The invention comprises the following components in parts by weight: 80-90 parts of bovine cartilage active polypeptide, 2-8 parts of bovine bone collagen peptide, 1-3 parts of pear powder, 0.5-2 parts of wolfberry, 0.5-2 parts of coix seed, 1-2 parts of papaya, 0.5-1.5 parts of spiny jujube seed, 0.5-1.5 parts of polygonatum, 1-2 parts of tuckahoe, 0.5-1 parts of angelica, 0.5-1 parts of eucommia and 0.01-0.05 parts of konjac.

9. The method for preparing a composition containing bovine cartilage active polypeptide according to any one of claims 7 to 8, characterized in that: The following steps are involved: The composition containing bovine cartilage active polypeptide, bovine bone collagen peptide, pear extract, wolfberry extract, coix seed extract, papaya extract, jujube seed extract, polygonatum extract, tuckahoe extract, angelica extract, eucommia extract and konjac extract is granulated and dried to obtain the composition containing bovine cartilage active polypeptide.

10. Use of the composition containing bovine cartilage active polypeptide according to any one of claims 7 to 8 or the composition containing bovine cartilage active polypeptide obtained by the preparation method according to claim 9 in common food and health food.

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