Preparation method of calf thymosin peptide

Abstract

The invention provides a calf thymus protein peptide preparation method, and belongs to the technical field of bioactive peptides, and the calf thymus protein peptide preparation method comprises the following steps: S1, cleaning calf thymus, removing connective tissue, crushing, adding water, stirring at a constant temperature, cooling and centrifuging to obtain a liquid-phase supernatant and solid-phase meat residues, collecting the supernatant, and filtering to obtain a calf thymus natural protein peptide solution; s2, water is added into the solid-phase meat residues, the pH is adjusted, an enzyme system is added for enzymolysis, enzymatic hydrolysate is subjected to centrifugal filtration, and a calf thymosin peptide solution is obtained; s3, the calf thymosin protein peptide solution is sequentially adsorbed by neutral activated carbon and acidic activated carbon, then ultrafiltration is conducted, liquid obtained after ultrafiltration is concentrated and freeze-dried, and the calf thymosin protein peptide is obtained. The invention provides a preparation method of calf thymosin peptide. The calf thymosin peptide which is free of peculiar smell, low in ash content and high in small molecule peptide content is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a method for preparing calf thymus protein peptide. Background Art

[0002] The calf's thymus is a core immune organ in young cattle, located near the heart. It is responsible for the production of T lymphocytes and the training of the immune system. Its core functions include promoting lymphocyte differentiation and maturation, regulating immune mechanisms, and possessing antiviral and anti-tumor activities. As cattle reach adulthood, the thymus gradually atrophies and eventually degenerates.

[0003] Calf thymus is used as a raw material for immunomodulators such as thymosin and thymopentin, which are used to treat hepatitis, tumors, and immunodeficiency disorders. Market demand for food-grade calf thymosin complex peptide continues to grow due to its safety and convenience (it can be taken orally). Despite the expansion of global beef cattle farming, a single cow only produces 150-500 grams of thymus, and the total annual output is insufficient to meet the needs of the pharmaceutical and food industries. End products such as thymosin complex peptide remain expensive. Only a few companies in China have mastered the production technology for oral thymosin. Raw material extraction requires precise enzymatic digestion and aseptic workshop support, resulting in high barriers to entry.

[0004] Bioactive peptides, also known as bioactive peptides, play a vital role in human health and disease prevention. These peptides are protein fragments with specialized physiological functions, typically containing 2 to 20 amino acid residues. In-depth research has uncovered numerous bioactive peptides with antioxidant, antibacterial, antihypertensive, anti-hypercholesterol, anti-aging, and immunomodulatory properties, demonstrating beneficial health benefits. Bovine thymus, a seasonal ingredient, is at its purest and odorless after birth, before weaning, and before the calf has eaten grass. This ingredient must be consumed immediately after slaughter, as it can easily develop a fishy odor, making it particularly precious and fragile. As the cattle mature, the thymus gradually shrinks, becoming rough and hard, and losing its original value.

[0005] Therefore, it is particularly important to rationally utilize calf thymus and give full play to its value. Deep processing of calf thymus protein can obtain a series of thymosin peptides that are beneficial to human life activities. At present, thymosin peptides are mostly prepared from calf thymus by enzymolysis, separation and other technologies. In recent years, there have been relevant reports disclosing methods for preparing thymosin, but most of them are direct enzymolysis methods, such as announcement number CN112143768B, a Chinese patent entitled "A Method for Co-preparing DNA and Thymosin from Calf Thymus", which discloses that calf thymus is enzymolyzed with proteinase K to obtain an enzymolysis solution, the supernatant of the enzymolysis solution is separated by ultrafiltration, the permeate of the ultrafiltration is used to separate thymosin, and the retentate of the ultrafiltration is used to separate DNA. However, the polypeptide products obtained in the above technical solutions and the prior art often have low content of small molecule peptides, high ash content, and disadvantages such as irritating odor. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing calf thymus protein peptide, which can obtain calf thymus protein peptide with no odor, low ash content and high content of small molecule peptides.

[0007] To achieve the above object, the present invention provides a method for preparing calf thymoprotein peptide, comprising the following steps: S1. Wash the calf thymus, remove the connective tissue, grind it, add water and stir it at a constant temperature, cool and centrifuge to obtain a liquid supernatant and a solid meat residue, collect the supernatant, and filter to obtain a calf thymus natural protein peptide solution; S2, taking the solid meat residue, adding water and adjusting the pH, adding an enzyme system for enzymatic hydrolysis, and centrifuging and filtering the enzymatic hydrolyzate to obtain a calf thymus protein peptide solution; S3, successively adsorbing the calf thymosin peptide solution on neutral activated carbon and acid activated carbon, and then ultrafiltration, concentrating and freeze-drying the ultrafiltered liquid to obtain calf thymosin peptide.

[0008] Optionally, the enzymatic hydrolysis of the enzyme system comprises three stages: the first stage is the addition of alkaline protease, the second stage is the addition of compound flavor protease and deamidase, and the third stage is the addition of methionine aminopeptidase.

[0009] Optionally, the added amount of the alkaline protease accounts for 1~1.5% of the mass of the solid-phase meat residue, the added amount of the composite flavor protease accounts for 0.5~1% of the mass of the solid-phase meat residue; the added amount of the deamidase accounts for 0.1~0.3% of the mass of the solid-phase meat residue; and the added amount of the methionine aminopeptidase accounts for 0.05~0.1% of the mass of the solid-phase meat residue.

[0010] Optionally, the pH value of the first stage is adjusted to 8.5~9.5, the temperature is 45~55℃, and the time is 1.5~2.5h; the pH value of the second stage is adjusted to 5~7, the temperature is 35~45℃, and the time is 2.5~3.5h; the pH value of the third stage is adjusted to 6~7, the temperature is 35~40℃, and the time is 0.5~1.5h.

[0011] Optionally, the constant temperature conditions in S1 are a temperature of 50-60° C. and a time of 5-6 hours, and the centrifugal conditions in S1 are a temperature of 4-8° C., a rotation speed of 10,000-12,000 rpm, and a time of 15-25 minutes.

[0012] Optionally, the membrane used for filtration in S1 and S2 is 0.22 μm, and the centrifugal conditions in S2 are a temperature of 4-8° C., a rotation speed of 10,000-12,000 rpm, and a time of 15-25 min.

[0013] Optionally, the pH of the neutral activated carbon is 6.8, and the pH of the acidic activated carbon is 4.5.

[0014] Optionally, the ultrafiltration in S3 is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da; and the concentration is performed using a rotary evaporator.

[0015] Optionally, the freeze drying is performed at a temperature of -30°C to -45°C and a pressure of 25 to 30 Pa for 24 to 36 hours.

[0016] Optionally, when adding water to the solid-phase meat residue in S2, L-cysteine and ethylenediaminetetraacetic acid are also added; the amount of L-cysteine added is 0.09-0.11% of the mass of the solid-phase meat residue, and the amount of ethylenediaminetetraacetic acid added is 0.04-0.06% of the mass of the solid-phase meat residue.

[0017] The above technical solution of the present invention includes at least the following beneficial effects: In the method for preparing calf thymus protein peptides, the cleaned calf thymus is first subjected to a constant temperature extraction at 60°C. This process accelerates the enzymatic hydrolysis rate and efficiency of the enzyme system. L-cysteine and ethylenediaminetetraacetic acid used in the present invention inhibit the oxidation of sulfur-containing amino acids. The three-stage enzymatic hydrolysis system combines deamidase with a compound flavor protease to reduce the hydrophobicity of the peptide chain, simultaneously reducing bitterness and off-flavors. Furthermore, the specific removal of methionine by Met-AP reduces the source of sulfide formation, effectively addressing the disadvantage of poor taste and removing a large amount of oil and off-flavor. This is the key to obtaining a low-ash, harmonious-tasting calf thymus protein peptide powder. Furthermore, the 60°C constant temperature extraction of the cleaned calf thymus also yields a small amount of natural peptide, which has been tested to have high antioxidant activity, but its stability is poor and requires further study. Finally, the calf thymus protein peptide prepared by the present invention has the advantages of high safety, strong antioxidant activity and easy absorption. It can be used as a raw material for food, medicine and health products, so that the calf thymus resources are fully developed and utilized, providing effective protection for the development of the industry, and has long-term scientific theoretical guidance significance and high economic value significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 ABTS free radical scavenging ability of the examples and comparative examples of the present invention and commercially available thymosin peptides; Figure 2 DPPH free radical scavenging ability of the examples and comparative examples of the present invention and commercially available thymosin peptides; Figure 3 This is a graph showing the hydroxyl radical scavenging ability of the examples of the present invention, the comparative examples, and commercially available thymosin peptides; Figure 4 This is a graph showing the superoxide anion free radical scavenging ability of the examples of the present invention, the comparative examples, and commercially available thymosin peptides; Figure 5 This is a high performance liquid chromatogram of commercially available 2 thymosin peptides of the present invention; Figure 6 This is a high performance liquid chromatogram of the protein peptide of Example 1 of the present invention; Figure 7 This is a high performance liquid chromatography diagram of the protein peptide of comparative example 2 of the present invention. DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figures 1 to 7 , clearly and completely describing the technical solutions of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the described embodiments of the present invention, all other embodiments derived by ordinary technicians in this field fall within the scope of protection of the present invention.

[0020] Example 1 The present invention provides a method for preparing calf thymosin peptide, comprising the following steps: S1. Wash the calf thymus, remove the connective tissue, and grind it into minced meat in a meat grinder. Add water at a mass ratio of 1:3 between minced meat and water. Maintain a constant temperature of 60°C with stirring and extract for 6 hours. Cool to room temperature, and centrifuge at 4°C and 12,000 rpm for 20 minutes to obtain a liquid supernatant and a solid meat residue. Collect the supernatant, filter through a 0.22 μm membrane to obtain a calf thymus natural protein peptide solution, and store at -20°C. S2. Take the solid meat residue, add water in a meat residue:water ratio of 1:3, add L-cysteine and ethylenediaminetetraacetic acid, the amount of L-cysteine added is 0.1% of the weight of the solid meat residue, and the amount of ethylenediaminetetraacetic acid added is 0.05% of the weight of the solid meat residue. Adjust the pH to 8.5. In the first stage, add alkaline protease accounting for 1.2% of the weight of the solid meat residue. The enzymatic hydrolysis conditions are temperature 50°C and time for 2 hours. Adjust the pH to 6.5. In the second stage, add 0. 8% of a composite flavor protease and 0.2% of a deamidase by weight of the solid phase meat residue were hydrolyzed at 40°C for 3 hours; the pH was adjusted to 7, and in the third stage, 0.08% of a methionine aminopeptidase by weight of the solid phase meat residue was added, and the hydrolysis conditions were 37°C for 1 hour; the resulting hydrolyzate was centrifuged at 4°C and 12,000 rpm for 20 minutes, and the centrifuge was then filtered through a 0.22 μm membrane to obtain a calf thymus protein peptide solution; S3. The calf thymus protein peptide solution was successively adsorbed by neutral activated carbon (pH 6.8) and acidic activated carbon (pH 4.5), and then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The ultrafiltered liquid was concentrated in a rotary evaporator under the conditions of a temperature of 60°C and a pressure of 0.08MPa, and then freeze-dried at a temperature of -35°C and a pressure of 25Pa for 30h to obtain calf thymus protein peptide.

[0021] Example 2 The present invention provides a method for preparing calf thymosin peptide, comprising the following steps: S1. Wash the calf thymus, remove the connective tissue, and grind it into minced meat in a meat grinder. Add water in a mass ratio of 1:3, maintain a constant temperature of 50°C and stir for 5.5 hours, cool to room temperature, and centrifuge at 8°C and 10,000 rpm for 25 minutes to obtain a liquid supernatant and a solid meat residue. Collect the supernatant, filter through a 0.22 μm membrane to obtain a calf thymus natural protein peptide solution, and store at -20°C. S2, take the solid phase meat residue, add water according to the meat residue: water mass ratio of 1:3, also add L-cysteine and ethylenediaminetetraacetic acid, the addition amount of L-cysteine is 0.11% of the mass of the solid phase meat residue, the addition amount of the ethylenediaminetetraacetic acid is 0.04% of the mass of the solid phase meat residue, adjust the pH to 9.5, add alkaline protease accounting for 1% of the mass of the solid phase meat residue in the first stage, the enzymatic hydrolysis conditions are temperature 45 ° C, time is 2.5h; adjust the pH to 7, add complex 1% of the mass of the solid phase meat residue in the second stage Flavor protease and deamidase accounting for 0.3% by weight of the solid phase meat residue are combined, and the enzymatic hydrolysis conditions are a temperature of 45°C and a time of 2.5 hours; the pH is adjusted to 7, and in the third stage, methionine aminopeptidase accounting for 0.05% by weight of the solid phase meat residue is added, and the enzymatic hydrolysis conditions are a temperature of 40°C and a time of 0.5 hour; the enzymatic hydrolyzate is obtained, and the enzymatic hydrolyzate is centrifuged at a temperature of 8°C and a speed of 10,000 rpm for 15 minutes, and the centrifuge is then filtered through a 0.22 μm membrane to obtain a calf thymus protein peptide solution; S3. The calf thymus protein peptide solution was successively adsorbed by neutral activated carbon (pH 6.8) and acidic activated carbon (pH 4.5), and then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The ultrafiltered liquid was concentrated in a rotary evaporator under the conditions of temperature 60°C and pressure 0.08MPa, and then freeze-dried at a temperature of -45°C and a pressure of 27Pa for 24 hours to obtain calf thymus protein peptide.

[0022] Example 3 S1. Wash the calf thymus, remove the connective tissue, and grind it into minced meat in a meat grinder. Add water in a meat mince ratio of 1:3, maintain a constant temperature of 55°C with stirring and extract for 5 hours, cool to room temperature, and centrifuge at 4°C and 11,000 rpm for 15 minutes to obtain a liquid supernatant and a solid meat residue. Collect the supernatant, filter through a 0.22 μm membrane to obtain a calf thymus natural protein peptide solution, and store at -20°C. S2, take the solid phase meat residue, add water according to the meat residue: water mass ratio of 1:3, also add L-cysteine and ethylenediaminetetraacetic acid, the addition amount of L-cysteine is 0.09% of the mass of the solid phase meat residue, the addition amount of the ethylenediaminetetraacetic acid is 0.06% of the mass of the solid phase meat residue, adjust the pH to 8.5, add alkaline protease accounting for 1.5% of the mass of the solid phase meat residue in the first stage, the enzymatic hydrolysis conditions are temperature 55 ° C, time is 1.5h; adjust the pH to 5, add 0.5% of the mass of the solid phase meat residue in the second stage % composite flavor protease and 0.1% deamidase by weight of the solid phase meat residue, the enzymatic hydrolysis conditions are a temperature of 35°C and a time of 3.5 hours; the pH is adjusted to 6, and in the third stage, 0.1% methionine aminopeptidase by weight of the solid phase meat residue is added, the enzymatic hydrolysis conditions are a temperature of 35°C and a time of 1.5 hours; the enzymatic hydrolyzate is obtained, and the enzymatic hydrolyzate is centrifuged at a temperature of 6°C and a speed of 11000 rpm for 25 minutes, and then the centrifuge is filtered through a 0.22 μm membrane to obtain a calf thymus protein peptide solution; S3. The calf thymus protein peptide solution was successively adsorbed by neutral activated carbon (pH 6.8) and acidic activated carbon (pH 4.5), and then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The ultrafiltered liquid was concentrated in a rotary evaporator under the conditions of a temperature of 60°C and a pressure of 0.08MPa, and then freeze-dried at a temperature of -30°C and a pressure of 30Pa for 36 hours to obtain calf thymus protein peptide.

[0023] Comparative Example 1 Compared with Example 1, the only difference is that the enzyme system includes only alkaline protease, and the remaining steps and raw materials are the same as those in Example 1.

[0024] Comparative Example 2 The only difference from Example 1 is that in step S1, the minced meat is not subjected to subsequent operations such as constant temperature stirring and extraction. Specifically, S1 involves washing the calf thymus, removing connective tissue, and grinding the minced meat in a meat grinder. The remaining steps and raw materials are the same as in Example 1.

[0025] 1. Ash content determination Ash content of the calf thymus natural protein peptide obtained by freeze-drying the calf thymus natural protein peptide solution in Example 1, and the calf thymus protein peptide obtained in Example 1 and Comparative Examples 1-2 were measured.

[0026] The total ash content was determined according to the ash content determination method in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 1.

[0027] Table 1 Results of calf thymus protein peptide yield and ash content

[0028] The results showed that the yield of calf thymus protein peptide in Example 1 was higher and the ash content was lower. Although the yield of calf thymus protein peptide in Comparative Example 2 was only slightly lower than that in Example 1, the ash content of Comparative Example 2 was significantly higher than that in Example 1.

[0029] 2. Antioxidant activity determination The antioxidant activity of the finished product was determined by measuring the ABTS free radical scavenging ability, DPPH free radical scavenging ability, hydroxyl free radical scavenging ability, and superoxide anion free radical scavenging ability.

[0030] The antioxidant activity of the calf thymus natural protein peptide obtained by freeze-drying the calf thymus natural protein peptide solution in Example 1 (i.e., the natural peptide in Example 1), the calf thymus protein peptides obtained in Example 1 and Comparative Example 2 (i.e., the oligopeptide in Example 1 and the oligopeptide in Comparative Example 2), and the calf thymus protein peptides purchased on the market (i.e., Commercially Purchased 1 and Commercially Purchased 2) were determined.

[0031] The ABTS free radical scavenging ability and DPPH free radical scavenging ability were determined using a kit purchased from Nanjing Jiancheng Bioengineering Institute. Figure 1 and Figure 2 ; The hydroxyl radical scavenging ability was determined by salicylic acid method, and the results are shown in Figure 3 ; The superoxide anion free radical scavenging ability was determined by pyrogallol method, and the results are shown in Figure 4 .

[0032] Depend on Figures 1 to 4 It can be seen that the overall antioxidant activity of calf thymoprotein peptide is ranked as follows: natural peptide of Example 1 > protein peptide of Example 1 > protein peptide of Comparative Example 2 > commercially available peptide 1 > commercially available peptide 2. This shows that the thymoprotein peptide prepared by the method of Example 1 not only solves the problems of poor taste and irritating odor of thymoprotein peptide, but also has higher antioxidant activity than the protein peptide obtained in Comparative Example 2 and commercially available thymoprotein peptide. Figure 1 It can also be seen that although the natural peptide of Example 1 has high antioxidant activity, its superoxide anion radical scavenging ability is too low. In combination with Table 1, it can be seen that its yield is low.

[0033] 3. Determination of amino acid composition and content The amino acid composition and content of the commercially available thymosin 2, the protein peptide of Example 1 and the protein peptide of Comparative Example 2 were detected by HPLC. The results are shown in Table 2. The chromatogram of the commercially available thymosin 2 is shown in Figure 5 The chromatogram of the protein peptide in Example 1 is shown in Figure 6 The chromatogram of the protein peptide of comparative example 2 is shown in Figure 7 .

[0034] The HPLC method was used for determination. The HPLC conditions were as follows: The HPLC column used was Thermo BDS HYPERSIL C18, the column temperature was 40°C, the injection volume was 10 μl, the detection wavelength was 254 nm, the flow rate was 1.0 ml / min, and the gradient elution conditions were as follows:

[0035] Mobile phase A: 0.1 mol / L sodium acetate solution (dissolve 13.6 g of sodium acetate trihydrate in 1000 ml of water and adjust the pH to 6.5 with glacial acetic acid) - 75 ml of acetonitrile. Mobile phase B: Mix 200 ml of water and 800 ml of acetonitrile to obtain an 80% acetonitrile solution.

[0036] Table 2 Amino acid composition and content results

[0037] Combine Figures 5 to 7 As shown in Table 2, the amino acid composition of the protein peptide in Example 1 is consistent with that of the control component (commercially purchased 2), with a total content of 9.8×10 -2 mg / ml, which is higher than the content of commercially available peptide 2. However, the amino acid composition of the protein peptide in Comparative Example 2 is inconsistent with that of the control ingredient (commercially available peptide 2), with a total content of 1.73 mg / ml, which is much higher than the control. In other words, the technical solution provided by this application meets the standards for thymosin.

[0038] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing calf thymus protein peptide, characterized in that: The following steps are involved: S1. Wash the calf thymus, remove the connective tissue, grind it, add water and stir it at a constant temperature, cool and centrifuge to obtain a liquid supernatant and a solid meat residue, collect the supernatant, and filter to obtain a calf thymus natural protein peptide solution; S2, taking the solid meat residue, adding water and adjusting the pH, adding an enzyme system for enzymatic hydrolysis, and centrifuging and filtering the enzymatic hydrolyzate to obtain a calf thymus protein peptide solution; S3, successively adsorbing the calf thymosin peptide solution on neutral activated carbon and acid activated carbon, and then ultrafiltration, concentrating and freeze-drying the ultrafiltered liquid to obtain calf thymosin peptide.

2. The method for preparing calf thymus protein peptide according to claim 1, characterized in that: The enzymatic hydrolysis of the enzyme system comprises three stages: the first stage is the addition of alkaline protease, the second stage is the addition of compound flavor protease and deamidase, and the third stage is the addition of methionine aminopeptidase.

3. The method for preparing calf thymosin peptide according to claim 2, characterized in that: The added amount of the alkaline protease accounts for 1-1.5% of the mass of the solid-phase meat residue, the added amount of the composite flavor protease accounts for 0.5-1% of the mass of the solid-phase meat residue; the added amount of the deamidase accounts for 0.1-0.3% of the mass of the solid-phase meat residue; and the added amount of the methionine aminopeptidase accounts for 0.05-0.1% of the mass of the solid-phase meat residue.

4. The method for preparing calf thymus protein peptide according to claim 2, characterized in that: The pH value of the first stage is adjusted to 8.5-9.5, the temperature is 45-55°C, and the time is 1.5-2.5 hours; the pH value of the second stage is adjusted to 5-7, the temperature is 35-45°C, and the time is 2.5-3.5 hours; the pH value of the third stage is adjusted to 6-7, the temperature is 35-40°C, and the time is 0.5-1.5 hours.

5. The method for preparing calf thymosin peptide according to claim 1, characterized in that: The constant temperature conditions in S1 are 50-60° C. for 5-6 hours, and the centrifugal conditions in S1 are 4-8° C., 10,000-12,000 rpm, and 15-25 minutes.

6. The method for preparing calf thymosin peptide according to claim 1, characterized in that: The membrane used for filtration in S1 and S2 is 0.22 μm, and the centrifugal conditions in S2 are temperature 4-8° C., rotation speed 10000-12000 rpm, and time 15-25 min.

7. The method for preparing calf thymosin peptide according to claim 1, characterized in that: The pH of the neutral activated carbon is 6.8, and the pH of the acidic activated carbon is 4.

5.

8. The method for preparing calf thymosin peptide according to claim 1, characterized in that: The ultrafiltration in S3 is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da; and the concentration is performed using a rotary evaporator.

9. The method for preparing calf thymosin peptide according to claim 1, characterized in that: The freeze drying is carried out at a temperature of -30°C to -45°C and a pressure of 25 to 30 Pa for 24 to 36 hours.

10. The method for preparing calf thymosin peptide according to claim 1, wherein When the solid phase meat residue is added to water in S2, L-cysteine and ethylenediaminetetraacetic acid are also added; the amount of L-cysteine added is 0.09-0.11% of the mass of the solid phase meat residue, and the amount of ethylenediaminetetraacetic acid added is 0.04-0.06% of the mass of the solid phase meat residue.

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