Preparation method and application of calf thymus active polypeptide
By pretreatment and multi-step extraction treatment of the calf thymus, including three centrifugation and multi-stage enzymatic lysis, the problem of low yield of active peptides in the calf thymus was solved, and efficient extraction of active peptides and stable product preparation was achieved.
Patent Information
- Application Number
- CN202510278001.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-03
AI Technical Summary
During the preparation of calf thymus active polypeptide, the problem of low yield of active polypeptide proteins is faced.
The steps of calf thymus pretreatment, defat, crushing and beating, centrifugation and extraction, enzymatic decomposition, enzyme decomposition, ultrafiltration, concentration and ultra-high temperature sterilization were adopted. The yield of active peptides was improved through three different rates of centrifugation and multi-level enzymatic decomposition, combined with the use of acid decompressants.
The yield of calves thymus active polypeptides has been significantly improved, the stability and biological activity of the product are ensured, and the problem of low yield in the original technology has been solved.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology. Specifically, it relates to a preparation method and application of calf thymus active polypeptide. Background Art
[0002] The thymus is a part of the immune system in humans and vertebrates. Its main function is to regulate and enhance the immune system, promote the differentiation of T lymphocytes, and assist in anti-tumor treatment. It is an important part of the body's immune system. In recent years, the research on calf thymus active polypeptide has gradually emerged. Due to its potential application value in immune regulation, anti-tumor and other aspects, it has attracted much attention. However, the current research on calf thymus active polypeptide is still in the development stage. During the preparation process, many challenges are faced, and the most prominent one is the problem of low yield of active polypeptide protein. Summary of the Invention
[0003] The present invention provides a preparation method and application of calf thymus active polypeptide, which solves the problem of low yield of calf thymus active polypeptide in the related art.
[0004] The technical solution of the present invention is as follows: The present invention provides a preparation method of calf thymus active polypeptide, which includes the following steps: After pretreatment of calf thymus, through degreasing, pulverizing and pulping, centrifugal extraction, enzymatic hydrolysis, enzyme inactivation, ultrafiltration, concentration, and ultra-high temperature sterilization, calf thymus active polypeptide is obtained. The centrifugation is carried out three times, and the centrifugation rates of the three times are different, and the centrifugation rate of the first centrifugation < the centrifugation rate of the second centrifugation < the centrifugation rate of the third centrifugation.
[0005] As a further technical solution, the centrifugation rate of the first centrifugation is 40000 r / min, the centrifugation rate of the second centrifugation is 44000 r / min, and the centrifugation rate of the third centrifugation is 48000 r / min.
[0006] In the present invention, a high-speed centrifugation method is used to obtain a high-yield protein solution. A higher centrifugation rate can quickly sediment large particle impurities and achieve rapid solid-liquid separation. A higher centrifugation rate helps to break the residual structure of cells and the interaction between proteins, so that the active polypeptide originally wrapped in cells or combined with other substances is more fully released into the extraction solution, thereby improving the yield of calf thymus active polypeptide.
[0007] As a further technical solution, the total centrifugation time is 30 min, and the centrifugation time for the first centrifugation, the second centrifugation, and the third centrifugation is 10 min each.
[0008] As a further technical solution, the centrifugal extraction includes the following steps: subjecting the pulverized slurry to primary centrifugation to obtain a first extract, subjecting the first protein solution to secondary centrifugation to obtain a second extract, mixing the first extract and the second extract, subjecting the mixture to tertiary centrifugation, and adding an acid hydrolyzing agent to obtain a protein solution.
[0009] As a further technical solution, the acid hydrolyzing agent includes citric acid, and the mass ratio of the acid hydrolyzing agent to the calf thymus is 1:100.
[0010] In the present invention, adding citric acid for acid hydrolysis can promote the hydrolysis of biological macromolecules, facilitate ribonucleic acid and enzymatic reactions, and improve the yield of active polypeptides from calf thymus.
[0011] As a further technical solution, the acid hydrolyzing agent further includes gallic acid, and the mass ratio of citric acid to gallic acid is 6:1 to 2.
[0012] In the present invention, adding both citric acid and gallic acid as the acid hydrolyzing agent for enzymatic hydrolysis can exert their synergistic effect, strengthen the decomposition of biological macromolecules, and improve the yield of active polypeptides from calf thymus.
[0013] As a further technical solution, the enzymatic hydrolysis is carried out in six stages. Different proteases are used in the six-stage enzymatic hydrolysis. 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 alkaline protease and papain, and the protease used in the sixth-stage enzymatic hydrolysis is composed of neutral protease and papain.
[0014] In the present invention, during the six-stage enzymatic hydrolysis process, different proteases are used in each stage of enzymatic hydrolysis to achieve deep and precise hydrolysis of proteins. Single enzymes are used in the first to third stages, which can first perform preliminary and targeted cleavage of proteins, open the overall structure of proteins, and make them more loose. Composite enzymes are used in the fourth to sixth stages. Different proteases in the composite enzyme can act synergistically to further perform more refined cleavage on the peptide segments after preliminary hydrolysis, decompose them into smaller active polypeptide fragments, and achieve deep hydrolysis of proteins to improve the yield of active polypeptides from calf thymus.
[0015] As a further technical solution, the temperature of the enzymatic hydrolysis is 40 - 60 °C, and the time for each stage of enzymatic hydrolysis is independently 10 - 50 min.
[0016] As a further technical solution, the total amount of protease used in each stage of enzymatic hydrolysis is 0.1% of the mass of the calf thymus.
[0017] In the present invention, a reasonable enzymatic hydrolysis temperature and the time of each stage of enzymatic hydrolysis are set to promote the protease to maintain a high activity, control the enzymatic hydrolysis rate, and improve the yield of calf thymus active polypeptide.
[0018] As a further technical solution, the mass ratio of alkaline protease to neutral protease in the fourth-stage enzymatic hydrolysis is 1:1, the mass ratio of alkaline protease to papain in the fifth-stage enzymatic hydrolysis is 1:1, and the mass ratio of neutral protease to papain in the sixth-stage enzymatic hydrolysis is 1:1.
[0019] In the present invention, the synergistic effect of the compound protease is fully exerted to enhance the enzymatic hydrolysis efficiency and improve the yield of calf thymus active polypeptide.
[0020] 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.
[0021] As a further technical solution, the solution used for defatting is an aqueous sodium hydroxide solution with a mass concentration of 0.1%. The defatting is carried out twice, and the defatting solution is replaced every 2 h.
[0022] In the present invention, the defatting is carried out twice to ensure the cumulative effect of the defatting effect and achieve the purpose of deep defatting.
[0023] As a further technical solution, the temperature of the defatting is 0 - 25°C, and the mass ratio of the aqueous sodium hydroxide solution with a mass concentration of 0.1% used for each defatting to the calf thymus is 100:1.
[0024] As a further technical solution, the pretreatment includes the following steps: cutting the calf thymus into small segments of 3 cm.
[0025] As a further technical solution, the temperature for inactivating the enzyme is 95 - 100°C, and the time is 30 min.
[0026] As a further technical solution, the ultrafiltration membrane specification for ultrafiltration is 10KD.
[0027] The present invention also provides a composition containing calf thymus active polypeptide, including the calf thymus active polypeptide obtained by the preparation method of the calf thymus active polypeptide.
[0028] The present invention also provides a composition containing calf thymus active polypeptide, which comprises the following components in parts by weight: 80 - 90 parts of calf thymus active polypeptide, 1 - 5 parts of bovine bone collagen peptide, 3 - 5 parts of lemon fruit powder, 1 - 3 parts of polygonatum sibiricum, 1 - 2 parts of ganoderma lucidum, 0.5 - 1.5 parts of astragalus membranaceus, 0.5 - 1 part of black sesame, 0.5 - 1 part of safflower, 0.01 - 0.03 parts of konjac, 0.01 - 0.03 parts of xanthan gum, and 0.01 - 0.03 parts of sodium carboxymethyl cellulose.
[0029] The present invention also provides a preparation method of a composition containing calf thymus active polypeptide, which comprises the following steps: mixing calf thymus active polypeptide, bovine bone collagen peptide, lemon fruit powder, polygonatum sibiricum extract, ganoderma lucidum extract, astragalus membranaceus extract, black sesame extract, safflower extract, konjac extract, xanthan gum and sodium carboxymethyl cellulose evenly, and then granulating and drying to obtain the composition containing calf thymus active polypeptide.
[0030] As a further technical solution, the binder for granulation is purified water, and the drying temperature is 135 °C.
[0031] The present invention also provides an application of a composition containing calf thymus active polypeptide or a composition containing calf thymus active polypeptide obtained by the preparation method thereof in the field of health products or medicine.
[0032] The working principle and beneficial effects of the present invention are as follows: In the present invention, taking calf thymus as the raw material, performing pretreatment and defatting operations on it can effectively remove fats, impurities and possible microorganisms in the raw material. This can not only avoid the interference of fats on the extraction of active polypeptide in the subsequent preparation process, prevent it from wrapping the active ingredients and affecting the extraction efficiency, but also reduce the adverse effects of impurities on key steps such as enzymatic hydrolysis and ultrafiltration, providing a pure and high-quality raw material basis for the subsequent preparation process, thereby helping to improve the yield of the final product; three centrifugations at different rates, and the centrifugation rate gradually increases. The first centrifugation uses a relatively low centrifugation rate, which can separate and remove large particle impurities in the raw material, playing a role in initially purifying the extract and avoiding the influence of these large particle substances on subsequent fine operations. The second centrifugation increases the centrifugation rate, which can further separate smaller impurity particles, making the extract more pure. The third centrifugation uses the highest centrifugation rate to ensure a high yield of the finally obtained active polypeptide protein; subsequent processes of enzymatic hydrolysis, enzyme inactivation, concentration and ultra-high temperature sterilization ensure the stability and biological activity of the active polypeptide, and ensure the safety and stability of the product. Specific embodiments
[0033] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. 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 creative efforts fall within the scope of protection of the present invention.
[0034] In the following examples and comparative examples: Alkaline protease: Bacillus licheniformis protease, with an enzyme activity of 200,000 U / g; Neutral protease: Bacillus subtilis protease, with an enzyme activity of 100,000 U / g; Papain: with an enzyme activity of 100,000 U / g; Chondroprotease: chondroprotein hydrolase, with an enzyme activity of 300,000 U / g.
[0035] Example 1 A method for preparing calf thymus active polypeptide includes the following steps: Cut 80 g of calf thymus into strips with a length of 3 cm, add it to 800 g of 1% sodium hydroxide aqueous solution, stir at 0 °C for 2 h for defatting, replace the defatting solution, which is still 800 g of 1% sodium hydroxide aqueous solution, and then stir at 0 °C for 2 h for defatting again. After defatting, crush and make a slurry. The crushed slurry is centrifuged at high speed by a centrifuge to remove the precipitate, add 0.8 g of acid hydrolyzing agent for acid hydrolysis, the acid hydrolyzing agent is citric acid, separate to obtain the protein solution, and the protein solution is subjected to hierarchical enzymatic hydrolysis. After inactivating the enzyme at 95 °C for 30 min, the active polypeptide is separated and purified by a 10 KD ultrafiltration membrane, collect the filtrate, and after concentration and ultra-high temperature sterilization, 18.9 g of calf thymus active polypeptide is obtained; The centrifugation includes the following steps: Centrifuge the crushed slurry once to obtain the first extraction solution, centrifuge the first protein solution twice to obtain the second extraction solution, mix the first extraction solution and the second extraction solution, and perform three centrifugations; among them, the centrifugation rate for the first centrifugation is 40,000 r / min, and the centrifugation time is 10 min; the centrifugation rate for the second centrifugation is 44,000 r / min, and the centrifugation time is 10 min; the centrifugation rate for the third centrifugation is 48,000 r / min, and the centrifugation time is 10 min; Enzymatic hydrolysis process: The protease used for the first-stage enzymatic hydrolysis is alkaline protease, the temperature of enzymatic hydrolysis is 50 °C, the time of enzymatic hydrolysis is 50 min, the dosage is 0.08 g, and the enzymatic hydrolysis pH is 8; The protease used for the second-stage enzymatic hydrolysis is neutral protease, the temperature of enzymatic hydrolysis is 45 °C, the time of enzymatic hydrolysis is 30 min, the dosage is 0.08 g, and the enzymatic hydrolysis pH is 6; The protease used in the three-stage enzymatic hydrolysis is papain. The temperature of enzymatic hydrolysis is 60 °C, the time is 60 min, the dosage is 0.08 g, and the pH of enzymatic hydrolysis is 6; The protease used in the four-stage enzymatic hydrolysis consists of alkaline protease and neutral protease (mass ratio 1:1). The temperature of enzymatic hydrolysis is 45 °C, the time is 40 min, the total dosage is 0.08 g, and the pH of enzymatic hydrolysis is 6; The protease used in the five-stage enzymatic hydrolysis consists of alkaline protease and papain (mass ratio 1:1). The temperature of enzymatic hydrolysis is 55 °C, the time is 60 min, the total dosage is 0.08 g, and the pH of enzymatic hydrolysis is 6; The protease used in the six-stage enzymatic hydrolysis consists of neutral protease and papain (mass ratio 1:1). The temperature of enzymatic hydrolysis is 50 °C, the time is 60 min, the total dosage is 0.08 g, and the pH of enzymatic hydrolysis is 6; A preparation method of a composition containing calf thymus active polypeptide, comprising the following steps: Mix 80 parts of calf thymus active polypeptide, 1 part of bovine bone collagen peptide, 3 parts of lemon fruit powder, 1 part of polygonatum sibiricum extract, 1 part of ganoderma lucidum extract, 0.5 part of astragalus membranaceus extract, 0.5 part of black sesame extract, 0.5 part of safflower extract, 0.01 part of konjac extract, 0.01 part of xanthan gum, and 0.01 part of sodium carboxymethyl cellulose evenly, add purified water as a binder for granulation, and dry at 135 °C to obtain the composition containing calf thymus active polypeptide.
[0036] Example 2 A preparation method of calf thymus active polypeptide, comprising the following steps: Cut 85 g of calf thymus into strips with a length of 3 cm, add it to 850 g of 1% sodium hydroxide aqueous solution, replace the degreasing solution, and the degreasing solution is still 850 g of 1% sodium hydroxide aqueous solution. Then stir and degrease at 15 °C for 2 h. After degreasing, crush and beat into a pulp. Centrifuge the crushed pulp at high speed with a horizontal centrifuge, add 0.8 g of acid hydrolyzing agent for acid hydrolysis. The acid hydrolyzing agent is citric acid. Separate to obtain a protein solution. The protein solution is subjected to fractional enzymatic hydrolysis. After inactivating the enzyme at 98 °C for 30 min, separate and purify the active polypeptide through a 10 KD ultrafiltration membrane, collect the filtrate, and obtain 19.7 g of calf thymus active polypeptide after concentration and ultra-high temperature sterilization; The centrifugation includes the following steps: Centrifuge the crushed pulp once to obtain a first extract, centrifuge the first protein solution twice to obtain a second extract, and mix the first extract and the second extract and centrifuge three times; Among them, the centrifugation rate for the first centrifugation is 40000 r / min, and the centrifugation time is 10 min; The centrifugation rate for the second centrifugation is 44000 r / min, and the centrifugation time is 10 min; The centrifugation rate for the third centrifugation is 48000 r / min, and the centrifugation time is 10 min; Enzymatic hydrolysis process: The protease used in the first-stage enzymatic hydrolysis is alkaline protease. The temperature of enzymatic hydrolysis is 50 °C, the time of enzymatic hydrolysis is 50 min, the dosage is 0.085 g, and the pH of enzymatic hydrolysis is 9; The protease used in the second-stage enzymatic hydrolysis is neutral protease. The temperature of enzymatic hydrolysis is 45 °C, the time of enzymatic hydrolysis is 30 min, the dosage is 0.085 g, and the pH of enzymatic hydrolysis is 7; The protease used in the third-stage enzymatic hydrolysis is papain. The temperature of enzymatic hydrolysis is 60 °C, the time of enzymatic hydrolysis is 60 min, the dosage is 0.085 g, and the pH of enzymatic hydrolysis is 7; The protease used in the fourth-stage enzymatic hydrolysis consists of alkaline protease and neutral protease (mass ratio 1:1). The temperature of enzymatic hydrolysis is 45 °C, the time of enzymatic hydrolysis is 40 min, the total dosage is 0.085 g, and the pH of enzymatic hydrolysis is 8; The protease used in the fifth-stage enzymatic hydrolysis consists of alkaline protease and papain (mass ratio 1:1). The temperature of enzymatic hydrolysis is 55 °C, the time of enzymatic hydrolysis is 60 min, the total dosage is 0.085 g, and the pH of enzymatic hydrolysis is 7; The protease used in the sixth-stage enzymatic hydrolysis consists of neutral protease and papain (mass ratio 1:1). The temperature of enzymatic hydrolysis is 50 °C, the time of enzymatic hydrolysis is 60 min, the total dosage is 0.085 g, and the pH of enzymatic hydrolysis is 7; A preparation method of a composition containing calf thymus active polypeptide, comprising the following steps: mixing 85 parts of calf thymus active polypeptide, 3 parts of bovine bone collagen peptide, 4 parts of lemon fruit powder, 2 parts of polygonatum sibiricum extract, 1.5 parts of ganoderma lucidum extract, 1 part of astragalus membranaceus extract, 0.8 part of black sesame extract, 0.8 part of safflower extract, 0.02 part of konjac extract, 0.02 part of xanthan gum, and 0.02 part of sodium carboxymethyl cellulose obtained by the above preparation method evenly, adding purified water as a binder for granulation, and drying at 135 °C to obtain the composition containing calf thymus active polypeptide.
[0037] Example 3 A preparation method of calf thymus active polypeptide, comprising the following steps: cutting 90 g of calf thymus into strips with a length of 3 cm, adding it to 900 g of 1% sodium hydroxide aqueous solution, stirring and degreasing at 25 °C for 2 h, replacing the degreasing solution, which is still 900 g of 1% sodium hydroxide aqueous solution, and then stirring and degreasing at 25 °C for 2 h. After degreasing, it is crushed and pulped. The crushed slurry is centrifuged at high speed by a horizontal centrifuge, 0.8 g of acid hydrolyzing agent is added for acid hydrolysis, the acid hydrolyzing agent is citric acid, the protein solution is separated, the protein solution is subjected to fractional enzymatic hydrolysis, after inactivating the enzyme at 100 °C for 30 min, the active polypeptide is separated and purified by a 10 KD ultrafiltration membrane, the filtrate is collected, and after concentration and ultra-high temperature sterilization, 19.6 g of calf thymus active polypeptide is obtained; Centrifugation includes the following steps: subjecting the pulverized slurry to primary centrifugation to obtain a first extract, subjecting the first protein solution to secondary centrifugation to obtain a second extract, and mixing the first extract and the second extract and subjecting the mixture to tertiary centrifugation. The centrifugation rate for the primary centrifugation is 40,000 r / min and the centrifugation time is 10 min; the centrifugation rate for the secondary centrifugation is 44,000 r / min and the centrifugation time is 10 min; the centrifugation rate for the tertiary centrifugation is 48,000 r / min and the centrifugation time is 10 min; Enzymolysis process: For the primary enzymolysis, the protease used is alkaline protease, the enzymolysis temperature is 50 °C, the enzymolysis time is 50 min, the dosage is 0.09 g, and the enzymolysis pH is 12; For the secondary 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 tertiary 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 quaternary 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 quinary enzymolysis, the protease used is composed of alkaline protease and papain (mass ratio 1:1), the enzymolysis temperature is 55 °C, the enzymolysis time is 60 min, the total dosage is 0.09 g, and the enzymolysis pH is 10; For the senary enzymolysis, the protease used is composed of neutral 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 method for preparing a composition containing calf thymus active polypeptide includes the following steps: mixing 90 parts of calf thymus active polypeptide, 5 parts of bovine bone collagen peptide, 5 parts of lemon fruit powder, 3 parts of polygonatum sibiricum extract, 2 parts of ganoderma lucidum extract, 2 parts of astragalus membranaceus extract, 1 part of black sesame extract, 1 part of safflower extract, 0.03 part of konjac extract, 0.03 part of xanthan gum, and 0.03 part of sodium carboxymethylcellulose obtained by the above preparation method evenly, adding purified water as a binder for granulation, and drying at 135 °C to obtain the composition containing calf thymus active polypeptide.
[0038] Example 4 Compared with Example 1, the difference in Example 4 is the centrifugation rate. In this example, the centrifugation rate for the primary centrifugation is 30,000 r / min, the centrifugation rate for the secondary centrifugation is 34,000 r / min, and the centrifugation rate for the tertiary centrifugation is 38,000 r / min; In this example, 18.4 g of calf thymus active polypeptide was obtained.
[0039] Example 5 Compared with Example 1, the difference in Example 5 is that the acid hydrolyzing agent is gallic acid; In this example, 19.7 g of calf thymus active polypeptide was obtained.
[0040] Example 6 Compared with Example 1, the difference in Example 6 is that the acid hydrolyzing agent is composed of citric acid and gallic acid with a mass ratio of 6:1; In this example, 24.0 g of calf thymus active polypeptide was obtained.
[0041] Example 7 Compared with Example 1, the difference in Example 7 is that the acid hydrolyzing agent is composed of citric acid and gallic acid with a mass ratio of 3:1; In this example, 25.5 g of calf thymus active polypeptide was obtained.
[0042] Comparative Example 1 Compared with Example 1, the difference in Comparative Example 1 is that the centrifugation rate is different. In this comparative example, the centrifugation rate of the first centrifugation = the centrifugation rate of the second centrifugation = the centrifugation rate of the third centrifugation, all of which are 48000 r / min; In this comparative example, 17.6 g of calf thymus active polypeptide was obtained.
[0043] Comparative Example 2 Compared with Example 1, the difference in Comparative Example 2 is that the centrifugation rate is different. In this comparative example, the centrifugation rate of the first centrifugation is 48000 r / min, the centrifugation rate of the second centrifugation is 44000 r / min, and the centrifugation rate of the third centrifugation is 40000 r / min; In this comparative example, 15.8 g of calf thymus active polypeptide was obtained.
[0044] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is that the centrifugation process is different. In this comparative example, the centrifugation process is carried out in two steps. The centrifugation includes the following steps: The pulverized slurry is centrifuged for the first time to obtain the first extraction solution, and the first extraction solution is centrifuged for the second time to obtain the protein solution. Among them, the centrifugation rate of the first centrifugation is 40000 r / min, and the centrifugation time is 15 min; the centrifugation rate of the second centrifugation is 44000 r / min, and the centrifugation time is 15 min; In this comparative example, 12.8 g of calf thymus active polypeptide was obtained.
[0045] Comparative Example 4 Compared with Example 1, the difference in Comparative Example 4 lies in the centrifugation process. In this comparative example, the centrifugation process is only carried out once, the centrifugation rate is 48000 r / min, and the centrifugation time is 30 min; 10.7 g of calf thymus active polypeptide was obtained in this comparative example.
[0046] Experimental Example 1 Calculate the yields of the calf thymus active polypeptides prepared in Examples 1-7 and Comparative Examples 1-4, where the yield = (content of calf thymus active polypeptide / theoretical content of calf thymus active polypeptide) × 100%.
[0047] The test results are shown in Table 1: Table 1 Yields of calf thymus active polypeptides prepared in Examples 1-7 and Comparative Examples 1-4
[0048] Compared with Comparative Examples 1-4, the yield of Example 1 is higher than that of Comparative Examples 1-4, indicating that when centrifugation is carried out three times and the centrifugation rates of the three centrifugations are different, with the centrifugation rate of the first centrifugation < the centrifugation rate of the second centrifugation < the centrifugation rate of the third centrifugation, the yield of calf thymus active polypeptide can be increased.
[0049] Compared with Example 4, the yield of Example 1 is higher than that of Example 4, indicating that when the centrifugation rate of the first centrifugation is 40000 r / min, the centrifugation rate of the second centrifugation is 44000 r / min, and the centrifugation rate of the third centrifugation is 48000 r / min, that is, when the centrifugation rate remains at a relatively high level, it helps to increase the yield of calf thymus active polypeptide.
[0050] Compared with Examples 1 and 5, the yields of Examples 6 and 7 are higher than those of Examples 1 and 5, indicating that citric acid and gallic acid play a synergistic role and can further increase the yield of calf thymus active polypeptide.
[0051] Experimental Example 2 The composition containing calf thymus active polypeptide prepared in Example 3 was packaged. Each bag contained 20 g of calf thymus active polypeptide and was packed in a box with 20 bags per box. This composition has the effects of enhancing immunity, nourishing the body, and delaying aging. 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. Telephone follow-up was conducted on the trial users to obtain the application effects of the composition containing calf thymus active polypeptide, and the application effects are shown in Table 3.
[0052] The trial situation is shown in Table 2: Table 2 Trial situation of the calf thymus active polypeptide prepared in Example 3
[0053] The application effects are shown in Table 3 as follows: Table 3 Application effects of the composition containing calf thymus active polypeptide prepared in Example 3
[0054] 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 principles of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for preparing calf thymus active polypeptide, characterized in that: The following steps are involved: After the calf thymus is pretreated, it is defatted, crushed and pulped, centrifuged for extraction, enzymatically hydrolyzed, enzyme inactivated, ultrafiltered, concentrated, and sterilized at ultrahigh temperature to obtain the calf thymus active polypeptide. The centrifugation is performed three times at different centrifugal rates, and the centrifugal rate of the first centrifugation is less than the centrifugal rate of the second centrifugation and less than the centrifugal rate of the third centrifugation.
2. The method for preparing calf thymus active polypeptide according to claim 1, characterized in that: The centrifugal speed of the first centrifugation is 40000 r / min, the centrifugal speed of the second centrifugation is 44000 r / min, and the centrifugal speed of the third centrifugation is 48000 r / min.
3. The method for preparing calf thymus active polypeptide according to claim 1, characterized in that: The centrifugal extraction comprises the following steps: centrifuging the pulverized slurry once to obtain a first extract, centrifuging the first protein solution twice to obtain a second extract, mixing the first extract and the second extract, centrifuging three times, adding an acidolysis agent for acid hydrolysis, and obtaining a protein solution.
4. The method for preparing calf thymus active polypeptide according to claim 3, characterized in that: The acidolysis agent includes citric acid, and the mass ratio of the acidolysis agent to the calf thymus is 1:
100.
5. The method for preparing calf thymus active polypeptide according to claim 3, characterized in that: The acidolysis agent also includes gallic acid, and the mass ratio of the citric acid to the gallic acid is 6:1-2.
6. The method for preparing calf thymus active polypeptide according to claim 1, characterized in that: The enzymatic hydrolysis is carried out in six stages, and the proteases used in the six stages are different. 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 alkaline protease and papain, and the protease used in the sixth stage enzymatic hydrolysis is composed of neutral protease and papain.
7. A composition containing calf thymus active polypeptide, characterized in that: The invention relates to a calf thymus active polypeptide obtained by the preparation method of a calf thymus active polypeptide according to any one of claims 1 to 6.
8. The composition containing calf thymus active polypeptide according to claim 7, characterized in that: The invention comprises the following components in parts by weight: 80-90 parts of calf thymus active polypeptide, 1-5 parts of bovine collagen peptide, 3-5 parts of lemon fruit powder, 1-3 parts of polygonatum, 1-2 parts of ganoderma lucidum, 0.5-1.5 parts of astragalus, 0.5-1 parts of black sesame, 0.5-1 parts of safflower, 0.01-0.03 parts of konjac, 0.01-0.03 parts of xanthan gum and 0.01-0.03 parts of sodium hydroxymethyl cellulose.
9. The method for preparing a composition containing calf thymus active polypeptide according to any one of claims 7 to 8, characterized in that: The following steps are involved: Calf thymus active polypeptide, bovine collagen peptide, lemon fruit powder, polygonatum extract, ganoderma extract, astragalus extract, black sesame extract, safflower extract, konjac extract, xanthan gum and sodium hydroxymethylcellulose are mixed evenly, granulated and dried to obtain a composition containing calf thymus active polypeptide.
10. Use of the composition containing calf thymus active polypeptide according to any one of claims 7 to 8 or the composition containing calf thymus active polypeptide obtained by the preparation method according to claim 9 in the field of health care products or medicine.