Calf thymus peptide for improving immunity, and preparation method and application thereof

Abstract

The application discloses calf thymus peptide for improving immunity and a preparation method and application thereof, and relates to the technical field of functional food.The preparation method comprises the following steps: mixing calf thymus raw materials, an additive and water, adjusting the pH to 4.5-6, performing ball milling treatment, and obtaining raw material 1; mixing the raw material 1 and water, performing cooking, and filtering to obtain raw material 2; mixing the raw material 2 and a first composite enzyme for enzyme hydrolysis to obtain enzyme hydrolysis liquid 1; mixing the enzyme hydrolysis liquid 1 and a second composite enzyme for enzyme hydrolysis to obtain enzyme hydrolysis liquid 2; performing ultrafiltration concentration on the enzyme hydrolysis liquid 2, drying, and obtaining the calf thymus peptide.The calf thymus peptide product with high content, high activity and better quality is obtained by optimizing the extraction process of the calf thymus peptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of functional food technology, and in particular to a calf thymus peptide for improving immunity and a preparation method and application thereof. BACKGROUND

[0002] The immunity of the human body can also be called resistance in general, which is the ability of the body to resist external invasion when the body is invaded by bacteria and viruses. When the body is tired or the body function is not coordinated, the immunity of the human body will decrease.

[0003] Clinical studies have shown that the occurrence of diseases such as influenza, rhinitis, cough, allergic dermatitis, asthma and peptic ulcer is related to the decrease of immunity. Since the early symptoms of the decrease of immunity are not obvious, it is difficult for people to realize it. Once the body has obvious diseases, it means that the immunity has decreased significantly, which will bring great difficulties to the treatment of diseases and the recovery of patients. Age is an important reason for the decrease of the immunity of the human body. In addition to the decrease of immunity caused by age, bad living habits such as staying up late, smoking, alcoholism and lack of exercise will also harm the immunity of the human body. Therefore, whether for people of different age groups, improving immunity is the key to maintaining health.

[0004] The thymus is one of the important immune organs of the body. Thymus peptide is a group of polypeptide hormones secreted by the thymus, which mainly includes two types of proteins with molecular weights of about 9600 and 7000. It has therapeutic effects on various diseases of the body, such as various types of severe hepatitis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and viral pneumonia. It also has good adjuvant therapeutic effect on tumors and is currently mainly used in products for improving immunity. For example, Chinese patent CN115299545A discloses a solid beverage for improving human immunity, which comprises the following components in parts by weight: 80-120 parts of complex polypeptide, 10-25 parts of tremella polysaccharide, 10-25 parts of yeast beta-glucan and 30-40 parts of bovine colostrum powder; the complex polypeptide comprises one or more of tremella peptide powder, bovine bone collagen peptide, ginseng peptide powder and calf thymus peptide powder; the preparation method comprises the following steps: mixing the complex polypeptide, the tremella polysaccharide, the yeast beta-glucan and the bovine colostrum powder, drying, sterilizing to obtain the solid beverage for improving human immunity. The solid beverage for improving human immunity has the advantages of promoting human metabolism, enhancing the efficacy of protein, promoting material absorption, improving human immunity and anti-fatigue capacity.

[0005] At present, the calf thymus peptide preparations produced in China are all extracted from calf thymus to obtain high-activity thymus peptides. The extraction process includes acid extraction method, neutral extraction method and neutral homogenate liquid freeze-thaw extraction method.

[0006] Chinese patent CN 112450316 A discloses a method for preparing calf thymus peptide powder, which comprises the following steps: grinding 4 times, homogenizing 5 times, freezing and thawing 3 times, sterilizing, enzymatic hydrolysis, filtering, concentrating, filtering and spray drying. Through grinding, homogenizing, freezing and thawing and enzymatic hydrolysis processes, small molecular peptides with small molecular weight and high peptide content are fully extracted and retained, and high-quality calf thymus peptide powder with small molecular weight, high clarity and good taste is obtained through centrifugation and ceramic membrane filtration. The final product has a peptide content of up to 98.7% and a molecular weight distribution of 73.7% below 1000 daltons.

[0007] At present, there is a great demand for functional foods that can improve immunity, and there is an urgent need for an extraction method for calf thymus peptide with high content, high activity and stability. SUMMARY

[0008] Therefore, the purpose of the present application is to provide a calf thymus peptide for improving immunity and a preparation method and application thereof. By optimizing the extraction process of calf thymus peptide, a calf thymus peptide product with high content, high activity and better quality is obtained.

[0009] To achieve the above-mentioned purpose of the application, the technical solutions of the present application are as follows:

[0010] In one aspect, the present application provides a preparation method of a calf thymus peptide for improving immunity, comprising the following steps:

[0011] (1) Ball milling: mixing calf thymus raw materials, an additive and water, adjusting the pH to 4.5-6, and ball milling to obtain raw material 1; the additive is a mixture of calcium chloride and colloidal silicon dioxide;

[0012] (2) Cooking: mixing raw material 1 obtained in step (1) with water, cooking, and filtering to obtain raw material 2;

[0013] (3) First step of enzymatic hydrolysis: mixing raw material 2 obtained in step (2) with a first composite enzyme for enzymatic hydrolysis to obtain enzymatic hydrolysate 1; the first composite enzyme is a mixture of lipase and alkaline protease;

[0014] (4) Second step of enzymatic hydrolysis: mixing enzymatic hydrolysate 1 obtained in step (3) with a second composite enzyme for enzymatic hydrolysis to obtain enzymatic hydrolysate 2; the second composite enzyme is a mixture of papain, bromelain and carboxypeptidase;

[0015] (5) Concentration: ultrafiltration and concentration of enzymatic hydrolysate 2, and drying to obtain the calf thymus peptide.

[0016] Preferably, in step (1), the mass ratio of calcium chloride to colloidal silicon dioxide is 1-2:1-2, and further preferably 1:1.

[0017] Further preferably, the particle size of the colloidal silicon dioxide is <200 mesh.

[0018] Preferably, in step (1), the mass ratio of the calf thymus raw material, the auxiliary agent and water is 5-15:1-5:15-25, and further preferably 10:2:20.

[0019] Preferably, in step (1), the pH is adjusted to 5-5.5, and more preferably 5.

[0020] Preferably, in step (1), the ball milling is performed under the following conditions: a filling rate of 70-90%, a rotation speed of 1500-3000 r / min, and a ball milling time of 30-60 min; and further preferably, a filling rate of 80%, a rotation speed of 2000 r / min, and a ball milling time of 40 min.

[0021] Preferably, in step (2), the cooking is performed under the following conditions: a pressure of 0.25±2 Mpa, a temperature of 120±5℃, and a cooking time of 3-8 h; and further preferably, a pressure of 0.25±2 Mpa, a temperature of 120±5℃, and a cooking time of 4 h.

[0022] Preferably, in step (3), the mass ratio of the lipase and the alkaline protease is 1:2-5, and further preferably 1:3.

[0023] Preferably, in step (3), the first enzymolysis is performed under the following conditions: an enzymolysis temperature of 45-55℃, and an enzymolysis time of 0.5-1.5 h; and further preferably, an enzymolysis temperature of 50℃, and an enzymolysis time of 1 h.

[0024] Preferably, in step (3), the first compound enzyme is added in an amount of 1-3% of the mass of the raw material 2, and further preferably 2%.

[0025] Preferably, in step (4), the mass ratio of the papain, the bromelain and the carboxypeptidase is 1:0.5-2:1-5, and further preferably 1:1:2.

[0026] Preferably, in step (4), the second enzymolysis is performed under the following conditions: an enzymolysis temperature of 40-50℃, and an enzymolysis time of 30-60 min; and further preferably, an enzymolysis temperature of 45℃, and an enzymolysis time of 40 min.

[0027] Preferably, in step (4), the second compound enzyme is added in an amount of 0.5-2% of the mass of the raw material 2, and further preferably 1%.

[0028] Preferably, in step (5), the ultrafiltration membrane has a molecular weight of 10000.

[0029] As a specific example of the present application, the preparation method comprises the following steps:

[0030] (1) Pretreatment: the calf thymus raw material is washed, minced, mixed with an additive (a mixture of calcium chloride and colloidal silicon dioxide (particle size < 200 mesh) at a mass ratio of 1:1), and water, the mass ratio of the calf thymus raw material, the additive, and water is 10:2:20, the pH is adjusted to 5, ball milling is performed by adding a ball mill, the filling rate is 80%, the rotation speed is 2000 r / min, and the ball milling time is 40 min to obtain a raw material 1;

[0031] (2) The raw material 1 obtained in step (1) is mixed with 3 times the weight of water, added to a cooking pot, the pressure is maintained at 0.25±2 Mpa, the temperature is maintained at 120±5℃, and cooking is performed for 4 h, then filtration is performed, the cooking liquid is collected, and cooling is performed to obtain a raw material 2.

[0032] (3) A first composite enzyme is added to the raw material 2 obtained in step (2), the mass of the first composite enzyme added is 2% of the raw material 2, the pH is adjusted to 8.0, and mixed enzyme hydrolysis is performed to obtain an enzyme hydrolysis liquid 1; wherein the first composite enzyme is a mixture of lipase and alkaline protease at a mass ratio of 1:3, the enzyme hydrolysis temperature is 50℃, and the time is 1 h.

[0033] (4) Second step enzyme hydrolysis: the pH of the enzyme hydrolysis liquid 1 obtained in step (3) is adjusted to 7.0, a second composite enzyme is added, the mass of the second composite enzyme added is 1% of the raw material 2, and mixed enzyme hydrolysis is performed to obtain an enzyme hydrolysis liquid 2; wherein the second composite enzyme is a mixture of papain, bromelain, and carboxypeptidase at a mass ratio of 1:1:2, the enzyme hydrolysis temperature is 45℃, and the time is 40 min.

[0034] (5) Concentration: the enzyme hydrolysis liquid 2 is ultrafiltrated and concentrated (the ultrafiltration membrane molecular weight is 10000), sterilized, and spray dried to obtain the calf thymus peptide.

[0035] On the other hand, the present application provides the calf thymus peptide prepared by the preparation method.

[0036] Finally, the present application provides the application of the calf thymus peptide prepared by the preparation method in the preparation of an immunity improving product.

[0037] The present application has the following beneficial effects:

[0038] (1) The present application optimizes the extraction process of the calf thymus peptide, and obtains a calf thymus peptide product with high content, high activity, and high stability by ball milling and two-step enzyme hydrolysis.

[0039] (2) The present application selects specific ball milling additives, conditions, and composite enzyme types for the extraction of the calf thymus peptide, which significantly improves the product quality. DETAILED DESCRIPTION

[0040] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following specific examples are further illustrated, but the following examples are only preferred embodiments of the present application, not all. Based on the examples in the embodiments, other examples obtained by those skilled in the art without creative labor are within the scope of the present application. In the following examples, if not otherwise specified, the operation method used is the conventional operation method, the equipment used is the conventional equipment, and the equipment materials used in each example are the same.

[0041] In the following examples, the lipase enzyme activity is 100,000 U / g, the papain enzyme activity is 100,000 U / g, the alkaline protease enzyme activity is 100,000 U / g, the papain enzyme activity is 100,000 U / g, the bromelain enzyme activity is 100,000 U / g, the carboxypeptidase activity is 200,000 U / g, the neutral protease enzyme activity is 200,000 U / g, and the colloidal silicon dioxide is Hupooxin colloidal silicon dioxide 200PHARM.

[0042] Example 1

[0043] (1) Pretreatment: The calf thymus raw material was washed, chopped, and mixed with an additive (calcium chloride and colloidal silicon dioxide (particle size <200 mesh) in a mass ratio of 1:1), and water. The mass ratio of the calf thymus raw material, the additive, and water was 10:2:20. The pH was adjusted to 5.0 with citric acid. Ball milling was performed using a ball mill. The ball milling conditions were: filling rate 80%, rotation speed 2000 r / min, and ball milling time 40 min. Raw material 1 was obtained.

[0044] (2) The raw material 1 obtained in step (1) was mixed with 3 times its weight of water, and added to a cooking pot. The pressure was maintained at 0.25±2 Mpa, and the temperature was maintained at 120±5℃. Cooking was performed for 4 h. After filtration, the cooking liquid was collected and cooled to obtain raw material 2.

[0045] (3) A first composite enzyme was added to the raw material 2 obtained in step (2). The mass of the first composite enzyme added was 2% of the mass of the raw material 2. The pH was adjusted to 8.0. Mixed enzymolysis was performed to obtain enzyme solution 1. The first composite enzyme was a mixture of lipase and alkaline protease in a mass ratio of 1:3. The enzymolysis temperature was 50℃, and the time was 1 h.

[0046] (4) Second step enzymolysis: The enzyme solution 1 obtained in step (3) was subjected to high-temperature enzyme inactivation. After cooling, the pH was adjusted to 7.0. A second composite enzyme was added for mixed enzymolysis. The mass of the second composite enzyme added was 1% of the mass of the raw material 2. Enzyme solution 2 was obtained. The second composite enzyme was a mixture of papain, bromelain, and carboxypeptidase in a mass ratio of 1:1:2. The enzymolysis temperature was 45℃, and the time was 40 min.

[0047] (5) Concentration: the enzyme solution 2 was concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray dried to obtain the calf thymus peptide.

[0048] Example 2

[0049] (1) Pretreatment: the calf thymus raw material was washed, shredded, mixed with an auxiliary agent (a mixture of calcium chloride and colloidal silicon dioxide (particle size <200 mesh) in a mass ratio of 2:1), and water in a mass ratio of 10:1:20. The pH was adjusted to 4.2 with citric acid, and a ball mill was added for grinding treatment. The grinding conditions were: filling rate 80%, rotation speed 2000 r / min, and grinding time 40 min to obtain raw material 1.

[0050] (2) The raw material 1 obtained in step (1) was mixed with 3 times its weight of water, added to a cooking pot, and maintained at a pressure of 0.25±2 Mpa and a temperature of 120±5℃ for 4 h. After filtration, the cooking liquid was collected and cooled to obtain raw material 2.

[0051] (3) A first complex enzyme was added to the raw material 2 obtained in step (2) in an amount of 1% of the mass of the raw material 2, and the pH was adjusted to 8.0. The enzyme solution 1 was obtained by mixing and enzymatic hydrolysis. The first complex enzyme was a mixture of lipase and alkaline protease in a mass ratio of 1:2, the enzymatic hydrolysis temperature was 50℃, and the time was 1 h.

[0052] (4) Second-step enzymatic hydrolysis: the enzyme solution 1 obtained in step (3) was high-temperature inactivated, and after cooling, the pH was adjusted to 7.0. A second complex enzyme was added for mixing and enzymatic hydrolysis in an amount of 2% of the mass of the raw material 2. The second complex enzyme was a mixture of papain, bromelain, and carboxypeptidase in a mass ratio of 1:2:5, the enzymatic hydrolysis temperature was 45℃, and the time was 40 min.

[0053] (5) Concentration: the enzyme solution 2 was concentrated by ultrafiltration (ultrafiltration membrane molecular weight 10000), sterilized, and spray dried to obtain the calf thymus peptide.

[0054] Example 3

[0055] (1) Pretreatment: the calf thymus raw material was washed, shredded, mixed with an auxiliary agent (a mixture of calcium chloride and colloidal silicon dioxide (particle size <200 mesh) in a mass ratio of 1:2), and water in a mass ratio of 10:5:20. The pH was adjusted to 6.0 with citric acid, and a ball mill was added for grinding treatment. The grinding conditions were: filling rate 80%, rotation speed 2000 r / min, and grinding time 40 min to obtain raw material 1.

[0056] (2) The raw material 1 obtained in step (1) is mixed with 3 times its weight of water, added to a cooking pot, the pressure is maintained at 0.25±2Mpa, the temperature is maintained at 120±5℃, and cooked for 4h, filtered, the cooking liquid is collected, cooled, and the raw material 2 is obtained;

[0057] (3) A first composite enzyme is added to the raw material 2 obtained in step (2), the mass of the first composite enzyme added is 3% of the raw material 2, the pH is adjusted to 8.0, and mixed enzymolysis is carried out to obtain an enzymolysis liquid 1; wherein the first composite enzyme is a mixture of lipase and alkaline protease in a mass ratio of 1:2, the enzymolysis temperature is 50℃, and the time is 1h.

[0058] (4) Second-step enzymolysis: the enzymolysis liquid 1 obtained in step (3) is high-temperature inactivated, after cooling, the pH is adjusted to 7.0, a second composite enzyme is added for mixed enzymolysis, the mass of the second composite enzyme added is 0.5% of the raw material 2, and the enzymolysis liquid 2 is obtained; wherein the second composite enzyme is a mixture of papain, bromelain and carboxypeptidase in a mass ratio of 1:0.5:1, the enzymolysis temperature is 45℃, and the time is 40min.

[0059] (5) Concentration: the enzymolysis liquid 2 is ultrafiltration concentrated (ultrafiltration membrane molecular weight 10000), sterilized, and spray dried to obtain the calf thymus peptide.

[0060] Example 4

[0061] The difference from Example 1 is that the ball milling time is 30min, and the rest is the same.

[0062] Example 5

[0063] The difference from Example 1 is that the ball milling time is 60min, and the rest is the same.

[0064] Comparative Example 1

[0065] The difference from Example 1 is that the ball milling time is 20min, and the rest is the same.

[0066] Comparative Example 2

[0067] The difference from Example 1 is that the ball milling time is 80min, and the rest is the same.

[0068] Comparative Example 3

[0069] (1) Pretreatment: the raw material of calf thymus is washed and shredded to obtain the raw material 1;

[0070] (2) The raw material 1 obtained in step (1) is mixed with 3 times its weight of water, added to a cooking pot, the pressure is maintained at 0.25±2Mpa, the temperature is maintained at 120±5℃, and cooked for 4h, filtered, the cooking liquid is collected, cooled, and the raw material 2 is obtained;

[0071] (3) adding a first complex enzyme to the raw material 2 obtained in step (2), the first complex enzyme being added in an amount of 2% of the raw material 2, adjusting the pH to 8.0, and mixing and enzymatically hydrolyzing to obtain an enzymatic hydrolysate 1; wherein the first complex enzyme is a mixture of lipase and alkaline protease in a mass ratio of 1:3, and the enzymatic hydrolysis is carried out at a temperature of 50°C for 1 hour.

[0072] (4) second-step enzymatic hydrolysis: high-temperature enzyme inactivation of the enzymatic hydrolysate 1 obtained in step (3), adjusting the pH to 7.0 after cooling, adding a second complex enzyme for mixing and enzymatically hydrolyzing, the second complex enzyme being added in an amount of 1% of the raw material 2, to obtain an enzymatic hydrolysate 2; wherein the second complex enzyme is a mixture of papain, bromelain and carboxypeptidase in a mass ratio of 1:1:2, and the enzymatic hydrolysis is carried out at a temperature of 45°C for 40 minutes.

[0073] (5) concentration: ultrafiltration concentration (ultrafiltration membrane molecular weight 10000) of the enzymatic hydrolysate 2, sterilization, and spray drying to obtain the calf thymus peptide.

[0074] Comparative Example 4

[0075] Different from Example 1, the ball milling is replaced by split-type colloid milling for 40 minutes at a speed of 2000 r / min, and the rest are the same.

[0076] Comparative Example 5

[0077] Different from Example 1, in step (1), the pH is not adjusted, and the rest are the same.

[0078] Comparative Example 6

[0079] Different from Example 1, no auxiliary agent is added in the ball milling, and the rest are the same.

[0080] Comparative Example 7

[0081] Different from Example 1, no colloidal silicon dioxide is added in the ball milling, and the rest are the same.

[0082] Comparative Example 8

[0083] Different from Example 1, in step (4), the second complex enzyme is a mixture of papain, bromelain and neutral protease in a mass ratio of 1:1:2, and the rest are the same.

[0084] Comparative Example 9

[0085] Different from Example 1, in step (3), the mass ratio of lipase and alkaline protease is 1:1, and the rest are the same.

[0086] Results detection

[0087] 1. Molecular weight distribution detection

[0088] According to GB 31645-2018 Appendix A GPC / UV, the proportion of peptide segments with a relative molecular weight of not more than 2000 was calculated, and the results of the examples and comparative examples are as follows:

[0089]

[0090] 2. Protein and peptide content detection

[0091] According to GB 5009.5-2016 first method for protein content detection; according to GB22492-2008 Appendix B for peptide content detection.

[0092] The results are as follows:

[0093]

[0094]

[0095] 3. Biological activity detection

[0096] The T lymphocyte-E rose test was used for activity detection (reference: Cai Hongjie, "Improvement of calf thymus peptide production process and analysis of preparation", No. 1672-0369 (2011) 19-2414-04), and the results are as follows:

[0097] Group Ea-RFC(%) Control 35 Example 1 73 Example 2 70 Example 3 72 Example 4 74 Example 5 72 Comparative Example 1 68 Comparative Example 2 65 Comparative Example 3 64 Comparative Example 4 66 Comparative Example 5 71 Comparative Example 6 68 Comparative Example 7 64 Comparative Example 8 67 Comparative Example 9 68

[0098] The detection results of molecular weight distribution detection, protein and peptide content detection, and biological activity detection show that the calf thymus peptide prepared in examples 1-5 has improved short peptide chain content, higher peptide content, and higher activity. Examples 1, 4, 5, comparative examples 1 and 2 screened the effect of ball milling time on product performance, and the results showed that when the ball milling time was 30-60 min, the comprehensive performance of the calf thymus peptide was better, the short peptide chain content and the peptide content were reduced when the ball milling time was too short, and the activity was significantly reduced when the ball milling time was too long. The results of comparative examples 3 and 4 showed that ball milling pretreatment could greatly promote the subsequent enzymolysis process and obtain products with smaller molecular weight and higher peptide content. Comparative examples 5-7 screened the ball milling environmental parameters, and it was found that under the action of certain acidic conditions and complex additives, the comprehensive performance of the calf thymus peptide could be significantly improved. The results of comparative examples 8-9 showed that the specific proportion of complex enzymes had a key influence on the quality of the calf thymus peptide.

[0099] 4. Improve immunity

[0100] (1) Experimental reagents

[0101] Example 1, comparative example 3 prepared calf thymus peptide;

[0102] Spleen amino acid peptide (Guojiazhunzi H10970214).

[0103] (2) Experimental method

[0104] Male Kunming mice (18-22g) were selected for adaptive feeding for one week, the feeding environment temperature was 20±2℃, the relative humidity was 45%-65%, and each group was randomly divided into 10 groups, the experimental groups were respectively administered with Example 1 small cattle thymus peptide 500mg / kg / d, Comparative Example 3 small cattle thymus peptide 500mg / kg / d, spleen amino acid peptide 1.3g / kg / d, once a day by gavage (gavage volume 10mL / kg), the model group and the blank group were gavaged with the same volume of normal saline, after continuous administration for 2 weeks, the immune suppression was carried out by intraperitoneal injection of cyclophosphamide 40mg / kg for two days, and after continuous gavage for 2 weeks, the mice were sacrificed by removing the vertebrae, the thymus and spleen were taken out, and the thymus index and spleen index were calculated. The results are as follows:

[0105] Group Thymus index Spleen index Blank group 0.244±0.125 0.310±0.354 Model group 0.127±0.152** 0.224±0.266* Spleen aminopeptide group 0.263±0.201## 0.382±0.448## Example 1 0.258±0.247## 0.369±0.496## Comparative Example 3 0.218±0.138# 0.315±0.324#

[0106] In the table, the model group is compared with the blank group, *P<0.05, **P<0.01; the experimental group is compared with the model group, #P<0.05, ##P<0.01.

[0107] The results show that the small cattle thymus peptide prepared in Example 1 of the application can significantly improve the thymus index and spleen index of the immunosuppressed model mice, and the results have statistical significance, the small cattle thymus peptide prepared in Comparative Example 3 has a significant effect of improving immunity, and compared with Example 1 and spleen amino acid peptide group, there is still a gap, after process improvement, the effect of the small cattle thymus peptide in improving immunity is further improved, and it can be used for preparing an immune-enhancing product.

[0108] The above only describes the preferred embodiments of the application and is not intended to limit the application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A method for preparing calf thymosin peptides to enhance immunity, characterized in that, Including the following steps: (1) Ball milling: calf thymus raw material is mixed with adjuvant and water, pH is adjusted to 4.5-6, and ball milling is performed to obtain raw material 1; the adjuvant is a mixture of calcium chloride and colloidal silica; the ball milling conditions are: filling rate 70-90%, rotation speed 1500-3000 r / min, ball milling time 30-60 min; (2) Steaming: Mix the raw material 1 obtained in step (1) with water, steam, and filter to obtain raw material 2; (3) First step of enzymatic hydrolysis: The raw material 2 obtained in step (2) is mixed with the first complex enzyme for enzymatic hydrolysis to obtain enzymatic hydrolysate 1; the first complex enzyme is a mixture of lipase and alkaline protease; the mass ratio of the lipase and alkaline protease is 1:2-5; (4) Second step of enzymatic hydrolysis: The enzymatic hydrolysate 1 obtained in step (3) is mixed with the second complex enzyme for enzymatic hydrolysis to obtain enzymatic hydrolysate 2; the second complex enzyme is a mixture of papain, bromelain and carboxypeptidase; the mass ratio of papain, bromelain and carboxypeptidase is 1:0.5-2:1-5; (5) Concentration: The enzymatic hydrolysate 2 is concentrated by ultrafiltration and dried to obtain the calf thymopeptide.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of calcium chloride to colloidal silicon dioxide is 1-2:1-2.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of the calf thymus raw material, the auxiliary agent and water is 5-15:1-5:15-25.

4. The preparation method according to claim 1, characterized in that, In step (1), adjust the pH to 5.

5. The preparation method according to claim 1, characterized in that, In step (3), the first compound enzyme is added at a mass of 1-3% of the raw material 2.

6. The preparation method according to claim 1, characterized in that, In step (4), the second complex enzyme is added at a mass of 0.5-2% of the raw material 2.

7. The calf thymosin peptide prepared by the preparation method according to any one of claims 1-6.

8. The use of calf thymopeptide prepared by the preparation method according to any one of claims 1-6 in the preparation of products that enhance immunity.

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