Preparation method of silk fibroin peptide

Abstract

The invention discloses a preparation method of silk fibroin protein peptide, and relates to the technical field of animal proteolysis, sand mud and impurities in cocoon husks can be effectively removed through cocoon opening, cleaning and soaking, and the biological activity and nutritional ingredients of silk fibroin are reserved to the maximum extent after pulping softening, enzymolysis and enzyme deactivation operation. The preparation method comprises the following steps: performing enzymolysis, performing 5000 Dn interception ultrafiltration by adopting ultrafiltration to prepare a silk fibroin solution, and concentrating and spraying powder on the silk fibroin solution to prepare the silk fibroin peptide powder with small molecular weight, so that the silk fibroin peptide powder not only has small molecular weight, but also can maintain excellent dissolvability and bioavailability; the silk fibroin peptide not only has good antioxidant and moisturizing properties, but also can promote skin health, and has high market requirements.

Description

Technical Field

[0001] The invention relates to the technical field of animal proteolysis, and in particular to a method for preparing silk fibroin peptide. Background Art

[0002] With the development of biotechnology, the application areas of protein peptides have continued to expand, covering industries such as food, medicine and health care products. In nutrition, protein peptides are regarded as a high-quality source of protein because they are easy to digest and absorb, help increase muscle synthesis and promote recovery.

[0003] In the silkworm industry, by-products produced after reeling or spinning, such as cocoon skins, discarded silk and silk residue, are usually used as silk spinning raw materials with low added value. In addition, these cocoon skins contain sand, mud, organic impurities and sericin, so their value has not been fully tapped. If these by-products can be decomposed and high-purity silk protein peptides can be extracted, higher added value can be achieved in the beauty industry and health food fields. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing silk fibroin peptide to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing silk fibroin peptide, comprising the following steps:

[0006] S1. Cocoon opening: Select high-quality original cocoons of mulberry silkworms, comb the original cocoons at high speed through a cocoon opening machine, and obtain partially cleaned combed cocoons;

[0007] S2, cleaning: cleaning the combed cocoon to obtain a cocoon without sand and mud;

[0008] S3, soaking: put the sand-free cocoon into a soaking machine, inject pure water at a ratio of 1:20, add NaOH with a purity of ≥98%, start the machine, soak automatically for 10 to 12 hours, then take out the cocoon and wash it to obtain softened silk fiber;

[0009] S4, beating: Put the softened silk fiber into a vertical high-speed beating machine, inject pure water at a ratio of 1:3 and start beating to form silk fibroin powder. Remove organic impurities after precipitation. After multiple beatings and resting, obtain pure ultrafine silk fibroin powder with a particle size of more than 500 meshes;

[0010] S5, enzymatic hydrolysis: put the ultrafine silk fibroin powder into an enzymatic hydrolysis tank, inject pure water at a ratio of 1:15, add 1% compound papain, and perform enzymatic hydrolysis for 4-6 hours to obtain a silk fibroin enzymatic hydrolysis mixture;

[0011] S6, inactivating enzyme: heating the silk fibroin protein hydrolyzate mixture to 85-95°C, inactivating enzyme for 10-20 minutes, and obtaining a silk fibroin protein mixture;

[0012] S7, ultrafiltration: cooling the silk fibroin mixed solution to room temperature, and performing 5000Dn cutoff ultrafiltration to obtain a silk fibroin solution;

[0013] S8, concentration: concentrating the silk fibroin solution to obtain a silk fibroin concentrated solution, the molecular weight of which is ≤5000Dn;

[0014] S9, powder spraying: spray the concentrated silk fibroin solution with powder at high temperature to obtain silk fibroin peptide powder with a molecular weight of ≤5000Dn.

[0015] Preferably, in S1, the rotation speed of the cocoon opening machine is 200-500 rpm, and the feeding speed of the original cocoon skin is 1-3 m / min.

[0016] Preferably, in S3, the concentration of NaOH added is 2%-2.2%.

[0017] Preferably, in S4, the beating of the vertical high-speed beater is set to a beating time of ≤1 minute each time, followed by a rest period of 30 minutes.

[0018] Preferably, in S5, the temperature of the enzymolysis tank is set at 50-60°C, and the stirring speed is 250 rpm.

[0019] Preferably, in S6, the pH value of the silk fibroin enzymatic hydrolysis mixture is adjusted to 6-7.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention can effectively remove sand, mud and impurities in the cocoon coat by opening the cocoon, washing and soaking, and after pulping, softening, enzymatic hydrolysis and enzyme inactivation, the biological activity and nutritional components of silk fibroin are retained to the greatest extent; by adopting ultrafiltration and powder spraying processes after enzymatic hydrolysis, not only can small molecular weight silk fibroin protein peptide powder be obtained, but also its excellent solubility and bioavailability can be maintained, so that the silk fibroin protein peptide not only has good antioxidant and moisturizing properties, but also can promote skin health, and has a high market demand. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Embodiment 1

[0023] The present embodiment provides a method for preparing silk fibroin peptide, comprising the following steps: S1, opening cocoons: selecting high-quality original cocoons of mulberry silkworms, combing the original cocoons at high speed through a cocoon opening machine to obtain partially decontaminated combed cocoons, the cocoon opening machine rotating speed is 500 rpm, and the original cocoon feeding speed is 2 m / min;

[0024] S2, cleaning: cleaning the combed cocoon to obtain a cocoon without sand and mud;

[0025] S3, soaking: put the sand-free cocoon into a soaking machine, inject pure water at a ratio of 1:20, add NaOH with a purity of ≥98% and a concentration of 2%, start the machine, soak automatically for 10 hours, then take out the cocoon and wash it to obtain softened silk fiber;

[0026] S4, beating: put the softened silk fiber into a vertical high-speed beating machine, inject pure water at a ratio of 1:3 and start beating, set the beating time to ≤1 minute each time, then let it stand for 30 minutes to form silk fibroin powder, remove organic impurities after precipitation, and obtain pure ultrafine silk fibroin powder after multiple beatings and standing, with a particle size of more than 500 mesh;

[0027] S5, enzymatic hydrolysis: put the ultrafine silk fibroin powder into an enzymatic hydrolysis tank, inject pure water at a ratio of 1:15, add 1% compound papain, and perform enzymatic hydrolysis for 4 hours to obtain a silk fibroin enzymatic hydrolysis mixture. The temperature of the enzymatic hydrolysis tank is set at 60°C and the stirring speed is 250 rpm;

[0028] S6, inactivating enzyme: heating the silk fibroin protein enzymatic hydrolysis mixture to 85°C, inactivating enzyme for 20 minutes, to obtain a silk fibroin protein mixture, and adjusting the pH value of the silk fibroin protein enzymatic hydrolysis mixture to 6;

[0029] S7, ultrafiltration: cooling the silk fibroin mixed solution to room temperature, and performing 5000Dn cutoff ultrafiltration to obtain a silk fibroin solution;

[0030] S8, concentration: concentrating the silk fibroin solution to obtain a silk fibroin concentrated solution, the molecular weight of which is ≤5000Dn;

[0031] S9, powder spraying: spray the concentrated silk fibroin solution with powder at high temperature to obtain silk fibroin peptide powder with a molecular weight of ≤5000Dn.

[0032] Embodiment 2

[0033] The present embodiment provides a method for preparing silk fibroin peptide, comprising the following steps: S1, opening cocoons: selecting high-quality original cocoons of mulberry silkworms, combing the original cocoons at high speed through a cocoon opening machine to obtain partially decontaminated combed cocoons, the cocoon opening machine rotating speed is 500 rpm, and the original cocoon feeding speed is 2 m / min;

[0034] S2, cleaning: cleaning the combed cocoon to obtain a cocoon without sand and mud;

[0035] S3, soaking: put the sand-free cocoon into a soaking machine, inject pure water at a ratio of 1:20, add NaOH with a purity of ≥98% and a concentration of 2%, start the machine, automatically soak for 12 hours, then take out the cocoon and wash it to obtain softened silk fiber;

[0036] S4, beating: put the softened silk fiber into a vertical high-speed beating machine, inject pure water at a ratio of 1:3 and start beating, set the beating time to ≤1 minute each time, then let it stand for 30 minutes to form silk fibroin powder, remove organic impurities after precipitation, and obtain pure ultrafine silk fibroin powder after multiple beatings and standing, with a particle size of more than 500 mesh;

[0037] S5, enzymatic hydrolysis: put the ultrafine silk fibroin powder into an enzymatic hydrolysis tank, inject pure water at a ratio of 1:15, add 1% compound papain, and perform enzymatic hydrolysis for 6 hours to obtain a silk fibroin enzymatic hydrolysis mixture. The temperature of the enzymatic hydrolysis tank is set at 60°C and the stirring speed is 250 rpm;

[0038] S6, enzyme inactivation: heating the silk fibroin protein enzymatic hydrolysis mixture to 95°C and inactivating the enzyme for 20 minutes to obtain a silk fibroin protein mixture, and adjusting the pH value of the silk fibroin protein enzymatic hydrolysis mixture to 6;

[0039] S7, ultrafiltration: cooling the silk fibroin mixed solution to room temperature, and performing 5000Dn cutoff ultrafiltration to obtain a silk fibroin solution;

[0040] S8, concentration: concentrating the silk fibroin solution to obtain a silk fibroin concentrated solution, the molecular weight of which is ≤5000Dn;

[0041] S9, powder spraying: spray the concentrated silk fibroin solution with powder at high temperature to obtain silk fibroin peptide powder with a molecular weight of ≤5000Dn.

[0042] Embodiment 3

[0043] The present embodiment provides a method for preparing silk fibroin peptide, comprising the following steps: S1, opening cocoons: selecting high-quality original cocoons of mulberry silkworms, combing the original cocoons at high speed through a cocoon opening machine to obtain partially decontaminated combed cocoons, the cocoon opening machine rotating speed is 500 rpm, and the original cocoon feeding speed is 2 m / min;

[0044] S2, cleaning: cleaning the combed cocoon to obtain a cocoon without sand and mud;

[0045] S3, soaking: put the sand-free cocoon into a soaking machine, inject pure water at a ratio of 1:20, add NaOH with a purity of ≥98% and a concentration of 2%, start the machine, automatically soak for 11 hours, then take out the cocoon and wash it to obtain softened silk fiber;

[0046] S4, beating: put the softened silk fiber into a vertical high-speed beating machine, inject pure water at a ratio of 1:3 and start beating, set the beating time to ≤1 minute each time, then let it stand for 30 minutes to form silk fibroin powder, remove organic impurities after precipitation, and obtain pure ultrafine silk fibroin powder after multiple beatings and standing, with a particle size of more than 500 mesh;

[0047] S5, enzymatic hydrolysis: put the ultrafine silk fibroin powder into an enzymatic hydrolysis tank, inject pure water at a ratio of 1:15, add 1% compound papain, and perform enzymatic hydrolysis for 5 hours to obtain a silk fibroin enzymatic hydrolysis mixture. The temperature of the enzymatic hydrolysis tank is set at 60°C and the stirring speed is 250 rpm;

[0048] S6, inactivating enzyme: heating the silk fibroin protein enzymatic hydrolysis mixture to 90°C, inactivating enzyme for 20 minutes, to obtain a silk fibroin protein mixture, and adjusting the pH value of the silk fibroin protein enzymatic hydrolysis mixture to 6;

[0049] S7, ultrafiltration: cooling the silk fibroin mixed solution to room temperature, and performing 5000Dn cutoff ultrafiltration to obtain a silk fibroin solution;

[0050] S8, concentration: concentrating the silk fibroin solution to obtain a silk fibroin concentrated solution, the molecular weight of which is ≤5000Dn;

[0051] S9, powder spraying: spray the concentrated silk fibroin solution with powder at high temperature to obtain silk fibroin peptide powder with a molecular weight of ≤5000Dn.

[0052] Embodiment 4

[0053] The present embodiment provides a method for preparing silk fibroin peptide, comprising the following steps: S1, opening cocoons: selecting high-quality original cocoons of mulberry silkworms, combing the original cocoons at high speed through a cocoon opening machine to obtain partially decontaminated combed cocoons, the cocoon opening machine rotating speed is 500 rpm, and the original cocoon feeding speed is 2 m / min;

[0054] S2, cleaning: cleaning the combed cocoon to obtain a cocoon without sand and mud;

[0055] S3, soaking: put the sand-free cocoon into a soaking machine, inject pure water at a ratio of 1:20, add NaOH with a purity of ≥98% and a concentration of 2%, start the machine, automatically soak for 11 hours, then take out the cocoon and wash it to obtain softened silk fiber;

[0056] S4, beating: put the softened silk fiber into a vertical high-speed beating machine, inject pure water at a ratio of 1:3 and start beating, set the beating time to ≤1 minute each time, then let it stand for 30 minutes to form silk fibroin powder, remove organic impurities after precipitation, and obtain pure ultrafine silk fibroin powder after multiple beatings and standing, with a particle size of more than 500 mesh;

[0057] S5, enzymatic hydrolysis: put the ultrafine silk fibroin powder into an enzymatic hydrolysis tank, inject pure water at a ratio of 1:15, add 1% compound papain, and perform enzymatic hydrolysis for 6 hours to obtain a silk fibroin enzymatic hydrolysis mixture. The temperature of the enzymatic hydrolysis tank is set at 60°C and the stirring speed is 250 rpm;

[0058] S6, enzyme inactivation: heating the silk fibroin protein enzymatic hydrolysis mixture to 95°C and inactivating the enzyme for 20 minutes to obtain a silk fibroin protein mixture, and adjusting the pH value of the silk fibroin protein enzymatic hydrolysis mixture to 6;

[0059] S7, ultrafiltration: cooling the silk fibroin mixed solution to room temperature, and performing 5000Dn cutoff ultrafiltration to obtain a silk fibroin solution;

[0060] S8, concentration: concentrating the silk fibroin solution to obtain a silk fibroin concentrated solution, the molecular weight of which is ≤5000Dn;

[0061] S9, powder spraying: spray the concentrated silk fibroin solution with powder at high temperature to obtain silk fibroin peptide powder with a molecular weight of ≤5000Dn.

[0062] The silk fibroin peptide powder prepared according to Examples 1 to 4 was used as a sample for finished product quality testing. The reagents and equipment required for the test preparation stage were NaOH solution, a particle size analyzer, and an enzyme solution. The NaOH solution was used to adjust the pH of the silk fibroin protein. The particle size analyzer was used to test the particle size distribution of the silk fibroin peptide powder. The enzyme solution was used to detect the degree of enzymatic hydrolysis and activity of the silk fibroin protein in order to understand its biological activity and potential applications.

[0063] Table 1 is the test table of silk fibroin peptide powder samples:

[0064]

[0065] In summary:

[0066] Referring to the test results in Table 1, Example 4 performs well in terms of protein content, antioxidant capacity and biological activity, and is suitable for use as a high-value nutritional supplement or functional food ingredient.

[0067] The antioxidant capacity and biological activity of Example 2 are relatively high, and the product is suitable for application in the field of daily nutrition and health care products.

[0068] Embodiment 3: The overall performance is good, and the overall indicators are slightly lower than those of embodiment 4.

[0069] Embodiment 1: The overall indicators are slightly lower, which is suitable for the low-cost nutritional supplement market. The parts not involved in the present invention are the same as the prior art or can be implemented by the prior art. Although the embodiments of the present invention have been shown and described, it can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing silk fibroin peptide, characterized in that: The following steps are involved: S1. Cocoon opening: Select high-quality original cocoons of mulberry silkworms, comb the original cocoons at high speed through a cocoon opening machine, and obtain partially cleaned combed cocoons; S2, cleaning: cleaning the combed cocoon to obtain a cocoon without sand and mud; S3, soaking: put the sand-free cocoon into a soaking machine, inject pure water at a ratio of 1:20, add NaOH with a purity of ≥98%, start the machine, soak automatically for 10 to 12 hours, then take out the cocoon and wash it to obtain softened silk fiber; S4, beating: Put the softened silk fiber into a vertical high-speed beating machine, inject pure water at a ratio of 1:3 and start beating to form silk fibroin powder. Remove organic impurities after precipitation. After multiple beatings and resting, obtain pure ultrafine silk fibroin powder with a particle size of more than 500 meshes; S5, enzymatic hydrolysis: put the ultrafine silk fibroin powder into an enzymatic hydrolysis tank, inject pure water at a ratio of 1:15, add 1% compound papain, and perform enzymatic hydrolysis for 4-6 hours to obtain a silk fibroin enzymatic hydrolysis mixture; S6, inactivating enzyme: heating the silk fibroin protein hydrolyzate mixture to 85-95°C, inactivating enzyme for 10-20 minutes, and obtaining a silk fibroin protein mixture; S7, ultrafiltration: cooling the silk fibroin mixed solution to room temperature, and performing 5000Dn cutoff ultrafiltration to obtain a silk fibroin solution; S8, concentration: concentrating the silk fibroin solution to obtain a silk fibroin concentrated solution, the molecular weight of which is ≤5000Dn; S9, powder spraying: spray the concentrated silk fibroin solution with powder at high temperature to obtain silk fibroin peptide powder with a molecular weight of ≤5000Dn.

2. The method for preparing a silk fibroin peptide according to claim 1, characterized in that: In S1, the rotation speed of the cocoon opening machine is 200-500 rpm, and the feeding speed of the original cocoon skin is 1-3 m / min.

3. The method for preparing a silk fibroin peptide according to claim 1, characterized in that: In S3, the concentration of NaOH added is 2%-2.2%.

4. The method for preparing a silk fibroin peptide according to claim 1, characterized in that: In S4, the beating of the vertical high-speed beater is set to a beating time of ≤1 minute each time, followed by a rest period of 30 minutes.

5. The method for preparing a silk fibroin peptide according to claim 1, characterized in that: In S5, the temperature of the enzymolysis tank is set at 50-60°C, and the stirring speed is 250 rpm.

6. The method for preparing a silk fibroin peptide according to claim 1, characterized in that: In S6, the pH value of the silk fibroin enzymatic hydrolysis mixture is adjusted to 6-7.

7. A silk fibroin peptide powder, wherein the silk fibroin peptide powder is prepared according to the preparation method of the silk fibroin peptide according to any one of claims 1 to 6.

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