Elastin peptide and preparation process thereof

Abstract

The invention discloses elastin peptide and a preparation process thereof, and relates to the technical field of deep processing. According to the designed preparation process of the elastin peptide, the influence of fat substances in the elastin peptide on the use effect of the elastin peptide is reduced to a great extent through a specific pretreatment degreasing process and a two-stage enzymolysis process; meanwhile, the biological activity of the elastin peptide is reserved, the content of desmosine and isodesmosine is increased, when the elastin peptide is applied to skin wrinkle resistance, the skin elasticity can be obviously enhanced, a good wrinkle resistance effect is achieved, and good application value is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of animal raw materials, in particular to an elastin peptide and a preparation process thereof. Background Art

[0002] Elastin peptides are the primary components of elastic fibers. They are found in large quantities in tissues and organs that are frequently deformed by stress, such as the lungs, aorta, skin, and ligaments of mammals, and the bulbus arteriosus of fish. Elastin contains a unique cross-linking structure called desmosin and isodesmosin. Elastin's soluble precursor, tropoelastin, is cross-linked through desmosin and isodesmosin to form a dense protein structure.

[0003] Elastin tissue is often rich in lipids, which seriously affect the effectiveness of elastin. Degreasing can improve the enzymatic hydrolysis efficiency and purity of elastin. Secondly, in the prevention and treatment of skin aging, desmosin and isodesmosin in elastin peptides have significant effects on improving skin, especially increasing skin elasticity and anti-wrinkle. Therefore, increasing the content of desmosin and isodesmosin in elastin peptides is of great significance for the application of elastin peptides in skin care products and beauty products. How to further reduce the fat content of elastin peptide products while retaining the activity of elastin peptides during preparation, and how to improve the cosmetic effects of the resulting elastin peptides, are urgent problems to be solved in this field. Summary of the Invention

[0004] The main purpose of the present invention is to develop an elastin peptide with high biological activity and good anti-wrinkle effect on skin and a preparation process.

[0005] To achieve the above object, the present invention proposes a preparation process of elastin peptide, comprising the following steps: S1. Crush animal tissue into blocks of uniform size, wash, soak in physiological saline, and let stand for 2-3 hours; S2, soaking the block raw material after the soaking treatment in step S1 in a degreasing liquid, and intermittently heating the degreasing liquid using microwaves; S3, placing the degreasing liquid heated in step S2 in an ice bath and allowing it to stand, wherein the degreasing liquid is separated from top to bottom into a solidified oil phase, a liquefied oil phase, and an aqueous phase, and the block raw material is sunk into the aqueous phase. After removing the solidified oil phase, the aqueous phase is obtained by liquid separation; S4, sequentially homogenizing and sterilizing the aqueous phase obtained in step S3 to obtain a homogenous solution; S5, pre-treating the homogenized solution obtained in step S4, and then performing a two-stage enzymatic hydrolysis: adding trypsin and elastase in sequence; inactivating the enzymes, centrifuging, and collecting the supernatant to obtain a crude elastin peptide hydrolysate; S6. The crude elastin peptide hydrolysate obtained in step S5 is subjected to ion exchange resin chromatography, ultrafiltration, concentration, and drying to obtain elastin peptide.

[0006] Specifically, the block raw material is soaked in physiological saline to remove blood stains, and then undergoes periodic microwave heating in a composite degreasing liquid. 3 ~5cm 3 Regular blocks ensure that the material is heated and reacted evenly during subsequent processing. The instantaneous high temperature generated by microwaves dissolves the oil, and the dissolved oil is accumulated in the degreasing liquid during the static stage. Ice bath treatment induces the crystallization and solidification of liquid oil to form a solid oil layer that is easy to separate. Through the above technical solution, the present application realizes the low-fat preparation of elastin peptides, effectively retaining the beauty active ingredients such as desmosin and isodesmosin. The temperature gradient formed in the degreasing stage promotes the separation of oils and reduces the burden of subsequent purification. The entire process effectively reduces the fat content of the product, which is beneficial to improving the user experience and beauty effect of elastin peptides.

[0007] In one embodiment, the animal tissue material comprises at least one of a bovine heart tube and a bonito heart bulb.

[0008] It can be understood that the bovine cardiac tube is a specific part of the cow's heart. It is not a real blood vessel, but a muscular structure of the inner wall of the ventricle. The bonito heart bulb refers to the spherical elastic tissue connecting the arteries of the bonito heart.

[0009] In one embodiment, in step S2, the degreasing liquid includes the following raw materials in weight percentage: Vegetable oil: 3wt%~6wt%; animal oil: 3wt%~7wt%; sodium hexametaphosphate: 0.6wt%~1.2wt%; sodium bicarbonate: 0.5wt%~1wt%; papain: 0.1wt%~0.2wt%; and the balance is physiological saline.

[0010] In one embodiment, the vegetable oil can be selected from one or more of coconut oil and palm oil.

[0011] It should be noted that the amphiphilic system dissolves lipids of different polarities, for example, vegetable oils can dissolve non-polar lipids, and animal oils can dissolve polar lipids; the synergistic effect of sodium hexametaphosphate and papain helps expand the gaps in animal tissues and hydrolyze fat cell membrane proteins, promoting the dissolution and release of lipids during the subsequent heating process. In addition, the combination of sodium hexametaphosphate and sodium bicarbonate adjusts the pH while softening the tissue, and papain precisely breaks down lipoprotein complexes. This technology avoids the damage to desmosin and isodesmosin caused by high temperatures or strong solvents, while improving degreasing efficiency. Secondly, trace amounts of vegetable oils or animal oils in the degreasing solution that remain in the aqueous phase can enhance the flavor of the resulting elastin peptide to a certain extent, enabling the development of elastin peptide products with better taste.

[0012] Through the above technical solution, this application achieves efficient degreasing of elastin tissue, reduces the difficulty of subsequent enzymatic hydrolysis, and improves product purity. The cross-linked structure of elastin is completely preserved during the degreasing process, preventing the loss of desmosin and isodesmosin during the degreasing process. The resulting elastin peptide has superior effects in improving skin elasticity and anti-wrinkle.

[0013] In one embodiment, in step S2, the temperature of the degreasing liquid is raised to 80° C. to 90° C. using microwaves, and each heating time is maintained for 1 min to 2 min, followed by standing for 3 min to 10 min.

[0014] In one embodiment, in step S2, one microwave heating and one standing is regarded as one cycle, and a total of (8-14) cycles are performed.

[0015] It is understandable that by reasonably controlling the standing time and utilizing the temperature gradient to cause the liquefied fat to float and stratify, the standing environment can be maintained by natural cooling or auxiliary heat dissipation devices. This period can ensure that the fat phase is fully separated from the animal tissue raw material. Specifically, during the microwave heating stage, electromagnetic waves penetrate the block raw material, causing the internal fat to expand due to heat, and the cell membrane to rupture and release lipid substances. During the standing stage, the temperature gradient of the degreasing liquid causes low-density fat to float, while the elastin in the aqueous phase maintains a stable structure because the temperature does not exceed the denaturation threshold. Through 8 to 14 cycles of operation, the fat inside and outside the block raw material is released one by one, and the standing stratification process continues to remove free fat, ultimately achieving a synergistic effect of efficient degreasing and ingredient protection.

[0016] In one embodiment, in step S2, before the last three cycles begin, soda ash is added to the degreasing solution to a concentration of 0.3M to 0.5M.

[0017] In one embodiment, in step S2, before the last three cycles begin, the degreasing liquid is shaken or stirred at a rotation speed of 200 rpm to 250 rpm.

[0018] It's important to note that adding soda ash in the final stage of degreasing breaks down lipids in the oil phase into small molecules, preventing them from aggregating and reattaching to the surface of the animal tissue. It also helps further decompose any remaining oil particles in the aqueous phase. Secondly, raising the pH of the system promotes the further precipitation of incompletely separated, stubborn lipids while preventing premature introduction of an alkaline environment that could inhibit the activity of papain.

[0019] In one embodiment, in step S2, the weight ratio of the block raw material to the degreasing liquid is 1:(6-13).

[0020] In one embodiment, in step S3, the degreasing liquid is placed in an ice bath, and the temperature of the degreasing liquid is maintained at 2° C. to 8° C. during the standing process, and the degreasing liquid is allowed to stand for 3 h to 6 h.

[0021] It's important to note that within the temperature range of 2°C to 8°C, the animal and vegetable oils in the degreasing solution selectively solidify due to their melting point differences. The high-melting-point component forms a solid oil phase, while the low-melting-point component forms a liquid oil phase, while the water phase settles due to density differences. This temperature range prevents the solution from freezing and potentially destroying the structure of the active ingredients, while also enhancing the interfacial separation between the oil and water phases through phase transition.

[0022] Through the above technical solution, the present application achieves efficient removal of oil components during the degreasing process, significantly reducing the lipid residues in the final elastin peptide product. At the same time, the integrity of the cross-linked structure of desmosin and isodesmosin is maintained through a low-temperature protection mechanism, providing raw material guarantee for the subsequent enzymatic hydrolysis preparation of highly active elastin peptides.

[0023] In one embodiment, in step S4, the homogenization treatment is to place the aqueous phase obtained in step S3 in a grinder and homogenize it into a paste.

[0024] In one embodiment, in step S4, during the sterilization process, the temperature is raised to 85° C. to 95° C. and kept warm for 20 min to 30 min.

[0025] In one embodiment, in step S5, the pretreatment of the homogenized liquid includes: adding oxalic acid to the homogenized liquid to a concentration of 0.2M to 0.4M, heating the homogenized liquid to 80°C to 90°C, and keeping the temperature for 0.5h to 1.5h.

[0026] In one embodiment, in step S5, the first enzymatic hydrolysis is performed using trypsin, the pH of the system is adjusted to 6.5-7.5, the hydrolysis is performed at 45°C-55°C for 1-3 hours, and then the enzyme is inactivated at 85°C-95°C for 20-30 minutes to obtain a first enzymatic hydrolyzate; wherein the amount of trypsin used is 0.1wt%-0.3wt% of the weight of the dry material of the first enzymatic hydrolysis; In step S5, the second enzymatic hydrolysis is performed using elastase, the pH of the system is adjusted to 7.5-8.5, the hydrolysis is performed at 40°C-50°C for 3h-8h, and then the enzyme is inactivated at 85°C-95°C for 20min-30min to obtain a second enzymatic hydrolyzate; wherein the amount of elastase used is 0.3wt%-0.8wt% of the weight of the dry material of the second enzymatic hydrolysis.

[0027] It should be noted that oxalic acid pretreatment helps further hydrolyze and untie the cross-links of small organic molecules such as lysine, releasing more organic compounds that are easily absorbed by the human body. In the first enzymatic hydrolysis, trypsin preferentially acts on the cross-links of myofibrillar protein under weakly acidic to neutral conditions. In the second enzymatic hydrolysis, elastase acts on the peptide bonds near the cross-linked regions of elastin in a weakly alkaline environment, releasing active peptides. The lipids removed during the pretreatment stage can reduce interference with the subsequent enzymatic hydrolysis process, and the division of labor and cooperation between the two enzymatic hydrolysis stages ensure the efficient release of active peptides. The elastin peptides obtained by this method are more suitable for applications in beauty fields such as skin anti-wrinkle while maintaining their biological activity.

[0028] The present invention also provides an elastin peptide, which is prepared by applying the preparation process of the elastin peptide.

[0029] The preparation process of the elastin peptide designed in the present application, through a specific pre-treatment degreasing process and a two-stage enzymatic hydrolysis process, greatly reduces the impact of fatty substances in the elastin peptide on the use effect of the elastin peptide, while retaining the biological activity of the elastin peptide and increasing the content of desmosin and isodesmosin. Its application in skin anti-wrinkle treatment can significantly enhance skin elasticity and achieve a good anti-wrinkle effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0031] Figure 1 is the chromatogram of desmosin standard with different concentrations; Figure 2 Chromatograms of elastin peptide samples and extracted ion chromatograms of desmosin; Figure 3 is the chromatogram of isodesmosin standard at different concentrations; Figure 4 Chromatograms of elastin peptide samples and extracted ion chromatograms of isodesmosin; The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0033] It should be noted that if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of such features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or a scheme in which A and B are satisfied at the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0034] The present invention is further described below by means of specific examples: The raw materials used in the embodiments of the present invention are all commercially available, and the present invention does not impose any restrictions on the sources of the raw materials.

[0035] Trypsin: purchased from Shanghai MacLean Reagent, product number P917424, active ingredient content ≥250 U / mg protein, CAS number: 9002-07-7.

[0036] Elastase: purchased from Shanghai McLean Reagent, model E6156, active ingredient content ≥4 units / mg protein, CAS number: 39445-21-1.

[0037] Example 1 The preparation process of the elastin peptide in Example 1 comprises the following steps: S1. Crush animal tissue into blocks of approximately 5 mm * 5 mm * 5 mm in size, wash, soak in physiological saline, and let stand for 2 hours; S2. Soaking the bulk raw material after the soaking treatment in step S1 in a degreasing liquid, wherein the weight ratio of the bulk raw material to the degreasing liquid is 1:9, wherein the degreasing liquid comprises the following raw materials in weight percentage: coconut oil: 4wt%; edible butter: 6wt%; sodium hexametaphosphate: 0.8wt%; sodium bicarbonate: 0.6wt%; papain: 0.2wt%; and the balance is normal saline; intermittently heating the degreasing liquid using microwaves, with the heating power controlled at 500W, each time heating to 80°C, each heating time maintained for 2 minutes, and then standing for 8 minutes, for a total of 10 cycles; Before the last three cycles, soda ash was added to the degreasing solution to a concentration of approximately 0.3 M and stirred at 200 rpm; S3, placing the heated degreasing liquid in an ice bath, controlling the temperature at about 5°C, and letting it stand for 3 hours. The degreasing liquid is separated from top to bottom into a solidified oil phase, a liquefied oil phase, and an aqueous phase. After removing the solidified oil phase, the aqueous phase is obtained by liquid separation; S4. The aqueous phase obtained in step S3 is placed in a grinder and homogenized until it becomes a paste; then sterilized at 90° C. for 20 min to obtain a homogenous solution; S5. Pretreating the homogenized liquid: adding oxalic acid to a concentration of about 0.3 M and heating to 80° C. for 0.5 h; then performing a two-stage enzymatic hydrolysis: in the first stage, using trypsin for enzymatic hydrolysis, adjusting the system pH to 6.5-7.5, hydrolyzing at 50° C. for 1 h, and then inactivating the enzyme at 90° C. for 30 min to obtain a first enzymatic hydrolyzate; the amount of trypsin used is 0.1 wt% of the weight of the dry material of the first enzymatic hydrolysis; in the second stage, using elastase for enzymatic hydrolysis, adjusting the system pH to 7.5-8.5, hydrolyzing at 45° C. for 3 h, and then inactivating the enzyme at 90° C. for 30 min to obtain a second enzymatic hydrolyzate; the amount of elastase used is 0.3 wt% of the weight of the dry material of the second enzymatic hydrolysis; inactivating the enzyme, centrifuging, and taking the supernatant to obtain a crude elastin peptide hydrolyzate; S6. The crude elastin peptide hydrolysate obtained in step S5 was dissolved in deionized water to form a solution with a concentration of 25 mg / mL. The solution was passed through a D113 weakly acidic cation exchange resin at a flow rate of 25 m / h, and then through a D301 weakly basic anion exchange resin at a flow rate of 15 m / h. The molecules with a molecular weight of less than 5000 Da were ultrafiltrated, concentrated, and dried to obtain the elastin peptide.

[0038] Example 2 The preparation process of the elastin peptide in Example 2 comprises the following steps: S1. Crush animal tissue into blocks of approximately 5 mm * 5 mm * 5 mm in size, wash, soak in physiological saline, and let stand for 2 hours; S2. Soaking the block raw material after the soaking treatment in step S1 in a degreasing liquid, wherein the weight ratio of the block raw material to the degreasing liquid is 1:6, wherein the degreasing liquid comprises the following raw materials in weight percentage: coconut oil: 5wt%; edible pig fat: 7wt%; sodium hexametaphosphate: 1wt%; sodium bicarbonate: 1wt%; papain: 0.2wt%; and the balance is normal saline; using microwaves to intermittently heat the degreasing liquid, with the heating power controlled at 500W, each time the temperature is raised to 80°C, each heating time is maintained for 2 minutes, and then allowed to stand for 8 minutes, for a total of 10 cycles; Before the last three cycles, soda ash was added to the degreasing solution to a concentration of approximately 0.5 M and stirred at 200 rpm; S3, placing the degreasing liquid heated in step S2 in an ice bath, controlling the temperature at about 3°C, and letting it stand for 3 hours. The degreasing liquid is separated from top to bottom into a solidified oil phase, a liquefied oil phase, and an aqueous phase. After removing the solidified oil phase, the aqueous phase is obtained by liquid separation; S4. The aqueous phase obtained in step S3 is placed in a grinder and homogenized until it becomes a paste; then sterilized at 90° C. for 20 min to obtain a homogenous solution; S5. Pre-treating the homogenized solution obtained in step S4 by adding oxalic acid to a concentration of about 0.5 M and heating to 80° C. for 0.5 h, followed by two-stage enzymatic hydrolysis: in the first stage, trypsin is used for enzymatic hydrolysis, the pH of the system is adjusted to 6.5-7.5, hydrolysis is carried out at 50° C. for 1 h, and then the enzyme is inactivated at 90° C. for 30 min to obtain a first enzymatic hydrolyzate; the amount of trypsin used is 0.3 wt % of the weight of the dry material of the first enzymatic hydrolysis; in the second stage, elastase is used for enzymatic hydrolysis, the pH of the system is adjusted to 7.5-8.5, hydrolysis is carried out at 45° C. for 3 h, and then the enzyme is inactivated at 90° C. for 30 min to obtain a second enzymatic hydrolyzate; the amount of elastase used is 0.6 wt % of the weight of the dry material of the second enzymatic hydrolysis; inactivating the enzyme, centrifuging, and taking the supernatant to obtain a crude elastin peptide hydrolyzate; S6. The crude elastin peptide hydrolysate obtained in step S5 was dissolved in deionized water to form a solution with a concentration of 25 mg / mL. The solution was passed through a D113 weakly acidic cation exchange resin at a flow rate of 25 m / h, and then through a D301 weakly basic anion exchange resin at a flow rate of 15 m / h. The molecules with a molecular weight of less than 5000 Da were ultrafiltrated, concentrated, and dried to obtain the elastin peptide.

[0039] Example 3 The preparation process of the elastin peptide in Example 3 comprises the following steps: S1. Crush animal tissue into blocks of approximately 3 mm*3 mm*3 mm in size, wash, soak in physiological saline, and let stand for 2 h; S2. Soaking the bulk raw material after the soaking treatment in step S1 in a degreasing liquid, wherein the weight ratio of the bulk raw material to the degreasing liquid is 1:13, wherein the degreasing liquid comprises the following raw materials in weight percentage: coconut oil: 6wt%; edible butter: 3wt%; sodium hexametaphosphate: 0.6wt%; sodium bicarbonate: 0.5wt%; papain: 0.1wt%; and the balance is normal saline; intermittently heating the degreasing liquid using microwaves, with the heating power controlled at 500W, each time heating to 90°C, each heating time maintained for 2 minutes, and then standing for 3 minutes, for a total of 10 cycles; Before the last three cycles, soda ash was added to the degreasing solution to a concentration of approximately 0.3 M and stirred at 200 rpm; S3, placing the degreasing liquid heated in step S2 in an ice bath, controlling the temperature at about 5° C., and standing for 3 hours. The degreasing liquid is separated from top to bottom into a solidified oil phase, a liquefied oil phase, and an aqueous phase. After removing the solidified oil phase, the aqueous phase is obtained by liquid separation; S4. The aqueous phase obtained in step S3 is placed in a grinder and homogenized until it becomes a paste; then sterilized at 90° C. for 20 min to obtain a homogenous solution; S5. Pre-treating the homogenized solution obtained in step S4 by adding oxalic acid to a concentration of about 0.3 M and heating to 80° C. for 0.5 h, followed by two-stage enzymatic hydrolysis: in the first stage, trypsin is used for enzymatic hydrolysis, the pH of the system is adjusted to 6.5-7.5, hydrolysis is carried out at 50° C. for 1 h, and then the enzyme is inactivated at 90° C. for 30 min to obtain a first enzymatic hydrolyzate; the amount of trypsin used is 0.1 wt % of the weight of the dry material of the first enzymatic hydrolysis; in the second stage, elastase is used for enzymatic hydrolysis, the pH of the system is adjusted to 7.5-8.5, hydrolysis is carried out at 45° C. for 3 h, and then the enzyme is inactivated at 90° C. for 30 min to obtain a second enzymatic hydrolyzate; the amount of elastase used is 0.3 wt % of the weight of the dry material of the second enzymatic hydrolysis; inactivating the enzyme, centrifuging, and taking the supernatant to obtain a crude elastin peptide hydrolyzate; S6. The crude elastin peptide hydrolysate obtained in step S5 was dissolved in deionized water to form a solution with a concentration of 25 mg / mL. The solution was passed through a D113 weakly acidic cation exchange resin at a flow rate of 25 m / h, and then through a D301 weakly basic anion exchange resin at a flow rate of 15 m / h. The molecules with a molecular weight of less than 5000 Da were ultrafiltrated, concentrated, and dried to obtain the elastin peptide.

[0040] Comparative Example 1 Comparative Example 1 is based on Example 1, except that in the preparation process of elastin peptide in Comparative Example 1, the degreasing liquid is replaced with a sodium hydroxide solution with a concentration of 0.5 M in step S2.

[0041] Comparative Example 2 Comparative Example 2 is based on Example 1, except that: in the preparation process of elastin peptide in Comparative Example 2, microwaves are not used to intermittently heat the degreasing liquid in step S2, but instead: The degreasing liquid was heated to 80° C. for 20 minutes. At the 14th minute, soda ash was added to the degreasing liquid to a concentration of about 0.3 M, and the mixture was stirred at a speed of 200 rpm.

[0042] Comparative Example 3 Comparative Example 3 is based on Example 1, except that the preparation process of the elastin peptide in Comparative Example 3 does not include a pretreatment process in step S5.

[0043] Performance testing: (1) The peptide chain distribution in Examples 1-3 and Comparative Examples 1-3 was tested by HPLC. The test results are shown in Table 1 below.

[0044] Table 1

[0045] (2) The contents of desmosin and isodesmosin in the elastin peptide prepared in Example 1 were determined according to standard T / CHC 1010-2023.

[0046] 10 g of the elastin peptide prepared in Example 1 was taken and hydrolyzed at 110°C for 20 h with 2 mL of 6 mol / L hydrochloric acid. After neutralization, the pH was adjusted to 7-8. A small amount of mobile phase was added to a 10 mL volumetric flask to fully dissolve the sample and mix thoroughly. The sample was diluted to volume with mobile phase and filtered through a 0.22 μm pore size organic phase filter. The filtrate was placed in an injection vial and analyzed by liquid chromatography-mass spectrometry (HPLC-MS).

[0047] (1) Determination of desmosin content: Accurately weigh the desmosin standard and prepare 0.25 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, 25 μg / mL, and 50 μg / mL standard working solutions, respectively, and draw a standard working curve.

[0048] The external standard method was used for quantitative testing: formula: x=C*V / m×100; where X is the percentage of desmosin and isodesmosin in the sample (%); C is the concentration of desmosin (μg / mL); V is the fixed volume (mL); and m is the weight of the elastin peptide weighed (mg).

[0049] Figure 1 is the chromatogram of desmosin standard with different concentrations; Figure 2 The chromatogram of the elastin peptide sample (upper part) and the extracted ion current chromatogram of desmosin (lower part) are shown.

[0050] The regression equation was measured: y=101528x+2170, R 2 =0.9997.

[0051] The content of desmosin in the elastin peptide in Example 1 is shown in Table 2.

[0052] Table 2

[0053] (II) Determination of the content of isodesmosin: Accurately weigh the isodesmosin standard and prepare 0.25 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, 25 μg / mL, and 50 μg / mL standard working solutions, respectively, and draw a standard working curve.

[0054] The external standard method was used for quantitative testing: formula: x=C*V / m×100; where X is the percentage of desmosin and isodesmosin in the sample (%); C is the concentration of desmosin (μg / mL); V is the fixed volume (mL); and m is the weight of the elastin peptide weighed (mg).

[0055] Figure 3 is the chromatogram of isodesmosin standard at different concentrations; Figure 4 The chromatogram of the elastin peptide sample (upper part) and the extracted ion current chromatogram of isodesmosin (lower part) are shown.

[0056] The regression equation was measured: y=118485x+20282, R 2 =0.9999.

[0057] The content of isodesmosin in the elastin peptide in Example 1 is shown in Table 3.

[0058] Table 3

[0059] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformation made by utilizing the contents of the present invention's description and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A process for preparing elastin peptide, characterized in that: The preparation process of the elastin peptide comprises the following steps: S1. Crush animal tissue into blocks of uniform size, wash, soak in physiological saline, and let stand for 2-3 hours; S2, soaking the block raw material after the soaking treatment in step S1 in a degreasing liquid, and intermittently heating the degreasing liquid using microwaves; S3, placing the degreasing liquid heated in step S2 in an ice bath and allowing it to stand, wherein the degreasing liquid is separated from top to bottom into a solidified oil phase, a liquefied oil phase, and an aqueous phase, and the block raw material is sunk into the aqueous phase. After removing the solidified oil phase, the aqueous phase is obtained by liquid separation; S4, sequentially homogenizing and sterilizing the aqueous phase obtained in step S3 to obtain a homogenous solution; S5, pre-treating the homogenized solution obtained in step S4, and then performing a two-stage enzymatic hydrolysis: adding trypsin and elastase in sequence; inactivating the enzymes, centrifuging, and collecting the supernatant to obtain a crude elastin peptide hydrolysate; S6. The crude elastin peptide hydrolysate obtained in step S5 is subjected to ion exchange resin chromatography, ultrafiltration, concentration, and drying to obtain elastin peptide.

2. The process for preparing the elastin peptide according to claim 1, wherein: The animal tissue material includes at least one of a bovine heart tube and a bonito heart bulb.

3. The process for preparing the elastin peptide according to claim 1, wherein: In step S2, the degreasing liquid includes the following raw materials in percentage by weight: Vegetable oils: 3wt%~6wt%; Animal oils: 3wt%~7wt%; Sodium hexametaphosphate: 0.6wt%~1.2wt%; Sodium bicarbonate: 0.5wt%~1wt%; Papain: 0.1wt%~0.2wt%; and the balance is normal saline.

4. The process for preparing the elastin peptide according to claim 1, wherein: In step S2, the temperature of the degreasing liquid is raised to 80°C to 90°C using microwaves, and the heating time is maintained for 1 minute to 2 minutes each time, followed by standing for 3 minutes to 10 minutes; In the step S2, one microwave heating and one standing is regarded as one cycle, and a total of (8-14) cycles are performed.

5. The process for preparing elastin peptide according to claim 4, wherein: In step S2, before the start of the last three cycles, soda ash is added to the degreasing solution to a concentration of 0.3M to 0.5M; And / or, in step S2, before the start of the last three cycles, the degreasing liquid is oscillated or stirred at a rotation speed of 200 rpm to 250 rpm.

6. The process for preparing elastin peptide according to claim 1, wherein: In step S2, the weight ratio of the block raw material to the degreasing liquid is 1:(6-13).

7. The process for preparing elastin peptide according to claim 1, wherein: In the step S3, the degreasing liquid is placed in an ice bath, and the temperature of the degreasing liquid is maintained at 2° C. to 8° C. during the standing process, and the degreasing liquid is allowed to stand for 3 h to 6 h.

8. The process for preparing elastin peptide according to claim 1, wherein: In step S4, the homogenization treatment is to place the aqueous phase obtained in step S3 in a grinder and homogenize it into a paste; And / or, in step S4, during the sterilization process, the temperature is raised to 85° C. to 95° C. and kept warm for 20 min to 30 min.

9. The process for preparing elastin peptide according to claim 1, wherein: In step S5, the pretreatment of the homogenized liquid includes: adding oxalic acid to the homogenized liquid to a concentration of 0.2M to 0.4M, heating the homogenized liquid to 80°C to 90°C and keeping the temperature for 0.5h to 1.5h; And / or, in step S5, the first enzymatic hydrolysis is performed using trypsin, the pH of the system is adjusted to 6.5-7.5, the hydrolysis is performed at 45° C.-55° C. for 1-3 hours, and then the enzyme is inactivated at 85° C.-95° C. for 20-30 minutes to obtain a first enzymatic hydrolyzate; wherein the amount of trypsin used is 0.1 wt%-0.3 wt% of the weight of the dry material of the first enzymatic hydrolysis; And / or, in step S5, the second enzymatic hydrolysis is performed using elastase, the pH of the system is adjusted to 7.5-8.5, the hydrolysis is performed at 40°C-50°C for 3h-8h, and then the enzyme is inactivated at 85°C-95°C for 20min-30min to obtain a second enzymatic hydrolysis solution; wherein the amount of elastase used is 0.3wt%-0.8wt% of the weight of the dry material of the second enzymatic hydrolysis.

10. An elastin peptide, characterized in that The elastin peptide is prepared using the preparation process of the elastin peptide according to any one of claims 1 to 9.

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