Elastin peptides and processes for their preparation

Abstract

The application discloses an elastin peptide and a preparation process thereof, and relates to the technical field of deep processing. The elastin peptide and the preparation process thereof are designed in the application. Through specific pretreatment degreasing process and two-stage enzymolysis process, the influence of fat substances in the elastin peptide on the use effect of the elastin peptide is greatly reduced, the biological activity of the elastin peptide is retained, the contents of desmosine and isodesmosine are improved, the elastin peptide is applied to skin anti-wrinkle, the skin elasticity can be obviously enhanced, and good anti-wrinkle effect can be achieved, and the elastin peptide has good application value.

Description

Technical Field

[0001] This invention relates to the field of animal raw material deep processing technology, and in particular to an elastin peptide and its preparation process. Background Technology

[0002] Elastin peptides are the main components of elastic fibers. They are abundant in mammalian lungs, aortas, skin, ligaments, and fish bulbos, tissues and organs that are frequently subjected to stress and deformation. Elastin contains special cross-linked structures—demodex and isodemodex—and its soluble precursor, elastin, forms a dense protein structure through cross-linking with demodex and isodemodex.

[0003] Elastin tissue is often rich in lipids, which severely affects its effectiveness. Degreasing treatment improves the enzymatic hydrolysis efficiency and purity of elastin. Secondly, in the prevention and treatment of skin aging, desmokine and isodesmokine in elastin peptides have significant effects on improving skin, especially skin elasticity and anti-wrinkle properties. Therefore, increasing the content of desmokine and isodesmokine in elastin peptides is of great significance for their application in skincare and cosmetic fields. How to further reduce the fat content of elastin peptide products while preserving their activity, 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 objective of this invention is to develop an elastin peptide with high bioactivity and good anti-wrinkle effect on the skin, as well as its preparation process.

[0005] To achieve the above objectives, this invention proposes a preparation process for elastin peptides, comprising the following steps:

[0006] S1. Take animal tissue raw materials, crush them into uniformly sized blocks, wash them, soak them in physiological saline, and let them stand for 2 to 3 hours.

[0007] S2. Soak the block raw material after soaking in step S1 in a degreasing solution, and use microwave to intermittently heat the degreasing solution;

[0008] S3. Place the heated degreasing liquid from step S2 into an ice bath and let it stand. The degreasing liquid separates into a solidified oil phase, a liquefied oil phase, and an aqueous phase from top to bottom. The blocky raw material sinks into the aqueous phase. After removing the solidified oil phase, the aqueous phase is obtained by liquid separation.

[0009] S4. The aqueous phase obtained in step S3 is subjected to homogenization and sterilization treatment in sequence to obtain a homogenized liquid.

[0010] S5. Pre-treat the homogenate obtained in step S4, and then perform a two-stage enzymatic hydrolysis: add trypsin and elastase in sequence; inactivate the enzymes, centrifuge, and take the supernatant to obtain crude elastin peptide hydrolysate.

[0011] S6. The crude enzymatic hydrolysate of elastin peptide obtained in step S5 is subjected to ion exchange resin chromatography, ultrafiltration, concentration, and drying to obtain elastin peptide.

[0012] Specifically, after the block-shaped raw material is soaked in physiological saline to remove blood contamination, it undergoes periodic microwave heating in a composite degreasing solution. A 3cm diameter is achieved by controlling the blade spacing. 3 ~5cm 3 The regular block shape ensures uniform heating and reaction of the material during subsequent processing. The instantaneous high temperature generated by microwaves dissolves the oil, and the settling stage allows the dissolved oil to aggregate in the degreasing solution. Ice bath treatment induces the liquid oil to crystallize and solidify, forming an easily separable solid oil layer. Through the above technical solution, this application achieves the low-fat preparation of elastin peptides, effectively retaining cosmetic active ingredients such as desmosin and isodesmosin. The temperature gradient formed during the degreasing stage promotes oil separation 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 cosmetic efficacy of elastin peptides.

[0013] In one embodiment, the animal tissue material includes at least one of bovine heart tube and skipjack tuna heart bulb.

[0014] Understandably, the bovine heart tube is a specific part of the bovine heart, not a true blood vessel, but a muscular structure of the ventricular wall. The skipjack tuna heart arterial bulb refers to the spherical elastic tissue connecting the arteries to the heart of a skipjack tuna.

[0015] In one embodiment, in step S2, the degreasing solution comprises the following raw materials by weight percentage:

[0016] 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 remainder being physiological saline.

[0017] In one embodiment, the vegetable oil may be one or more of coconut oil and palm oil.

[0018] It should be noted that an amphiphilic system dissolves lipids of different polarities; for example, vegetable oils dissolve nonpolar lipids, while animal fats dissolve polar lipids. The synergistic effect of sodium hexametaphosphate and papain helps to expand the interstitial spaces of animal tissues and hydrolyze adipocyte membrane proteins, promoting the dissolution and release of lipids during subsequent heating. Furthermore, the combination of sodium hexametaphosphate and sodium bicarbonate regulates pH while softening tissue, and papain precisely breaks down lipoprotein complexes. This technology avoids the damage to desmolysin and isodesmolysin caused by high temperatures or strong solvents, while simultaneously improving defatting efficiency. Secondly, the trace amounts of vegetable or animal fat remaining in the aqueous phase of the defatting solution can, to some extent, improve the flavor of the resulting elastin peptides, enabling the development of better-tasting elastin peptide products.

[0019] Through the above technical solution, this application achieves efficient degreasing of elastin tissue, reducing the difficulty of subsequent enzymatic hydrolysis and improving product purity. The cross-linked structure of elastin is completely preserved during the degreasing process, preventing the loss of desmokinin and isodesmokinin. The resulting elastin peptides exhibit superior effects in improving skin elasticity and reducing wrinkles.

[0020] In one embodiment, in step S2, the temperature of the degreasing solution is raised to 80°C to 90°C using microwaves, with each heating time lasting 1 to 2 minutes, followed by standing for 3 to 10 minutes.

[0021] In one embodiment, step S2 consists of one microwave heating and one resting period as one cycle, and is performed for a total of (8~14) cycles.

[0022] Understandably, by rationally controlling the settling time and utilizing the temperature gradient to induce the liquefied oil to float and separate into layers, the settling environment can be maintained through natural cooling or auxiliary heat dissipation devices. This period ensures the complete separation of the fat phase from the animal tissue raw material. Specifically, during the microwave heating stage, electromagnetic waves penetrate the blocky raw material, causing the internal oil to expand due to heat, and the cell membranes to rupture, releasing lipid substances. During the settling stage, the temperature gradient of the degreasing solution causes the low-density oil to float, while the elastin in the aqueous phase remains structurally stable because the temperature does not exceed the denaturation threshold. Through 8 to 14 cycles, the oil inside and outside the blocky raw material is released successively, while the settling and stratification process continuously removes free oil, ultimately achieving a synergistic effect of efficient degreasing and component protection.

[0023] 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.

[0024] In one embodiment, in step S2, before the last three cycles begin, the degreasing solution is oscillated or stirred at a speed of 200 rpm to 250 rpm.

[0025] It should be noted that adding soda ash in the final stage of defatting breaks down lipids in the oil phase into smaller molecules, preventing oil particles from agglomerating and re-adhering to the surface of the animal tissue raw material. It also promotes further decomposition of residual oil particles in the aqueous phase. Secondly, increasing the pH of the system encourages the further precipitation of incompletely separated, stubborn lipids, while avoiding premature introduction of an alkaline environment that could inhibit the activity of papain in the early stages.

[0026] In one embodiment, in step S2, the weight ratio of the block raw material to the degreasing liquid is 1:(6~13).

[0027] In one embodiment, in step S3, the degreasing solution is placed in an ice bath, and the temperature of the degreasing solution is maintained at 2°C to 8°C during the settling process for 3 to 6 hours.

[0028] It should be noted that within the temperature range of 2℃ to 8℃, animal and vegetable oils in the degreasing solution undergo selective solidification due to their differences in melting points. The high-melting-point component forms a solid oil phase, while the low-melting-point component forms a liquid oil phase, and the aqueous phase settles due to density differences. This temperature range not only prevents the solution from freezing and thus destroying the structure of the effective components, but also enhances the interfacial separation between the oil and aqueous phases through phase change.

[0029] Through the above technical solution, this application achieves efficient removal of oil components during the defatting process, significantly reduces lipid residues in the final elastin peptide product, and maintains the integrity of the cross-linked structure of desmolysin and isodesmolysin through a low-temperature protection mechanism, providing raw material guarantee for the subsequent enzymatic hydrolysis preparation of highly active elastin peptides.

[0030] In one embodiment, in step S4, the homogenization process involves placing the aqueous phase obtained in step S3 into a grinder and homogenizing it into a paste.

[0031] In one embodiment, during step S4, the sterilization process involves raising the temperature to 85°C to 95°C and holding it at that temperature for 20 to 30 minutes.

[0032] In one embodiment, the pretreatment of the homogenate in step S5 includes: adding oxalic acid to the homogenate to 0.2M~0.4M, heating to 80℃~90℃ and holding at that temperature for 0.5h~1.5h.

[0033] 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, hydrolysis is carried out at 45℃-55℃ for 1-3 hours, and then enzyme inactivation treatment is performed at 85℃-95℃ for 20-30 minutes to obtain the first enzymatic hydrolysate; wherein, the amount of trypsin used is 0.1wt%-0.3wt% of the weight of the dry material from the first enzymatic hydrolysis.

[0034] In step S5, the second enzymatic hydrolysis is performed using elastase. The pH of the system is adjusted to 7.5-8.5, and hydrolysis is carried out at 40℃-50℃ for 3-8 hours. Then, the enzyme is inactivated at 85℃-95℃ for 20-30 minutes to obtain the second enzymatic hydrolysate. The amount of elastase used is 0.3wt%-0.8wt% of the weight of the dry material from the second enzymatic hydrolysis.

[0035] It should be noted that oxalic acid pretreatment facilitates further hydrolysis of the cross-linking bonds of small organic molecules such as chain cleavage enzymes, releasing more organic compounds that are easily absorbed by the human body. In the first enzymatic hydrolysis stage, trypsin preferentially acts on the cross-linking bonds of myofibril proteins under weakly acidic to neutral conditions. In the second enzymatic hydrolysis stage, elastase acts on peptide bonds near the cross-linking regions of elastin in a weakly alkaline environment, releasing active peptides. The lipids removed during the pretreatment stage reduce interference in subsequent enzymatic hydrolysis processes, and the division of labor and cooperation between the two enzymatic hydrolysis stages ensures the efficient release of active peptides. The elastin peptides obtained by this method, while maintaining biological activity, are more suitable for applications in cosmetic fields such as anti-wrinkle skincare.

[0036] The present invention also proposes an elastin peptide, which is prepared using the same process as the elastin peptide.

[0037] The preparation process of the elastin peptide designed in this application, through a specific pretreatment degreasing process and a two-stage enzymatic hydrolysis process, greatly reduces the impact of fatty substances in the elastin peptide on its effectiveness, while retaining the bioactivity of the elastin peptide and increasing the content of desmopressin and isodesmopressin. When applied to skin anti-wrinkle treatment, it can significantly enhance skin elasticity and achieve a good anti-wrinkle effect. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0039] Figure 1 Chromatograms of desmodium standards at different concentrations;

[0040] Figure 2 Chromatogram of elastin peptide sample and ion chromatogram of desmokinin extraction;

[0041] Figure 3 Chromatograms of isodesin standards at different concentrations;

[0042] Figure 4 Chromatogram of elastin peptide sample and ion chromatogram of isodesin extraction;

[0043] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] It should be noted that if the embodiments of the present invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Additionally, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0046] The present invention will be further illustrated below through specific embodiments:

[0047] All raw materials used in the embodiments of this invention are commercially available, and this invention does not impose any restrictions on the source of raw materials.

[0048] Trypsin: Purchased from Shanghai Maclean Reagent, catalog number P917424, active ingredient content ≥250 U / mg protein, CAS number: 9002-07-7.

[0049] Elastase: Purchased from Shanghai Maclean's Reagent Center, model E6156, active ingredient content ≥4 units / mg protein, CAS number: 39445-21-1.

[0050] Example 1

[0051] The preparation process of the elastin peptide in Example 1 includes the following steps:

[0052] S1. Take animal tissue raw materials, crush them into blocks of approximately 5mm*5mm*5mm in size, wash them, soak them in physiological saline, and let them stand for 2 hours.

[0053] S2. Soak the blocky raw material after soaking in step S1 in a defatting solution. The weight ratio of the blocky raw material to the defatting solution is 1:9. The defatting solution includes the following raw materials by weight percentage: coconut oil: 4wt%; edible butter: 6wt%; sodium hexametaphosphate: 0.8wt%; sodium bicarbonate: 0.6wt%; papain: 0.2wt%; and the balance is physiological saline. The defatting solution is intermittently heated with a microwave at a power of 500W. The temperature is raised to 80°C each time, and the heating time is maintained for 2 minutes each time. Then, it is left to stand for 8 minutes. This process is repeated 10 times.

[0054] Before the last three cycles begin, add soda ash to the degreasing solution to a concentration of approximately 0.3M and stir at a speed of 200 rpm.

[0055] S3. Place the heated degreasing liquid in an ice bath, control the temperature at about 5°C, and let it stand for 3 hours. The degreasing liquid will separate into solidified oil phase, liquefied oil phase and water phase from top to bottom. After removing the solidified oil phase, the water phase is obtained by separation.

[0056] S4. The aqueous phase obtained in step S3 is placed in a grinder and homogenized until it becomes a paste; then it is sterilized at 90°C for 20 minutes to obtain a homogenized liquid.

[0057] S5. Pretreatment of the homogenized solution: Add oxalic acid to a concentration of approximately 0.3M and heat to 80℃ for 0.5 hours; then perform a two-stage enzymatic hydrolysis: The first stage uses trypsin to hydrolyze the system, adjusting the pH to 6.5-7.5, hydrolyzing at 50℃ for 1 hour, and then inactivating the enzyme at 90℃ for 30 minutes to obtain the first hydrolysate; the amount of trypsin used is 0.1 wt% of the weight of the dry material from the first stage hydrolysis; The second stage uses elastase to hydrolyze the system, adjusting the pH to 7.5-8.5, hydrolyzing at 45℃ for 3 hours, and then inactivating the enzyme at 90℃ for 30 minutes to obtain the second hydrolysate; the amount of elastase used is 0.3 wt% of the weight of the dry material from the second stage hydrolysis; after enzyme inactivation, centrifuge and collect the supernatant to obtain crude elastin peptide hydrolysate;

[0058] S6. Dissolve the crude enzymatic hydrolysate of elastin peptide obtained in step S5 in deionized water to prepare a solution with a concentration of 25 mg / mL. Pass the solution 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. After ultrafiltration, molecules with a molecular weight of less than 5000 Da are collected, concentrated, and dried to obtain elastin peptide.

[0059] Example 2

[0060] The preparation process of the elastin peptide in Example 2 includes the following steps:

[0061] S1. Take animal tissue raw materials, crush them into blocks of approximately 5mm*5mm*5mm in size, wash them, soak them in physiological saline, and let them stand for 2 hours.

[0062] S2. Soak the blocky raw material after soaking in step S1 in a defatting solution. The weight ratio of the blocky raw material to the defatting solution is 1:6. The defatting solution includes the following raw materials by weight percentage: coconut oil: 5wt%; edible pork fat: 7wt%; sodium hexametaphosphate: 1wt%; sodium bicarbonate: 1wt%; papain: 0.2wt%; and the balance is physiological saline. The defatting solution is intermittently heated using a microwave. The heating power is controlled at 500W. The temperature is raised to 80°C each time, and the heating time is maintained for 2 minutes each time. Then, it is left to stand for 8 minutes. This process is repeated 10 times.

[0063] Before the last three cycles begin, add soda ash to the degreasing solution to a concentration of approximately 0.5M and stir at a speed of 200 rpm.

[0064] S3. Place the heated degreasing liquid from step S2 into an ice bath, control the temperature at about 3°C, and let it stand for 3 hours. The degreasing liquid will separate into solidified oil phase, liquefied oil phase and water phase from top to bottom. After removing the solidified oil phase, the water phase will be obtained by separation.

[0065] S4. The aqueous phase obtained in step S3 is placed in a grinder and homogenized until it becomes a paste; then it is sterilized at 90°C for 20 minutes to obtain a homogenized liquid.

[0066] S5. Pretreatment is performed on the homogenized solution obtained in step S4 by adding oxalic acid to a concentration of approximately 0.5M and heating to 80℃ for 0.5 hours. Then, a two-stage enzymatic hydrolysis is performed: The first stage uses trypsin, adjusting the pH to 6.5-7.5, hydrolyzing at 50℃ for 1 hour, followed by enzyme inactivation at 90℃ for 30 minutes to obtain the first hydrolysate; the amount of trypsin used is 0.3 wt% of the weight of the dry material from the first stage hydrolysis. The second stage uses elastase, adjusting the pH to 7.5-8.5, hydrolyzing at 45℃ for 3 hours, followed by enzyme inactivation at 90℃ for 30 minutes to obtain the second hydrolysate; the amount of elastase used is 0.6 wt% of the weight of the dry material from the second stage hydrolysis. After enzyme inactivation, centrifugation is performed, and the supernatant is collected to obtain the crude elastin peptide hydrolysate.

[0067] S6. Dissolve the crude enzymatic hydrolysate of elastin peptide obtained in step S5 in deionized water to prepare a solution with a concentration of 25 mg / mL. Pass the solution 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. After ultrafiltration, molecules with a molecular weight of less than 5000 Da are collected, concentrated, and dried to obtain elastin peptide.

[0068] Example 3

[0069] The preparation process of the elastin peptide in Example 3 includes the following steps:

[0070] S1. Take animal tissue raw materials, crush them into blocks of approximately 3mm*3mm*3mm in size, wash them, soak them in physiological saline, and let them stand for 2 hours.

[0071] S2. The blocky raw material after soaking in step S1 is soaked in a defatting solution with a weight ratio of 1:13. The defatting solution includes the following raw materials by weight percentage: coconut oil: 6wt%; edible tallow: 3wt%; sodium hexametaphosphate: 0.6wt%; sodium bicarbonate: 0.5wt%; papain: 0.1wt%; and the balance being physiological saline. The defatting solution is intermittently heated using a microwave with a heating power controlled at 500W. The temperature is raised to 90°C each time, and the heating time is maintained for 2 minutes each time. After that, it is left to stand for 3 minutes. This process is repeated 10 times.

[0072] Before the last three cycles begin, add soda ash to the degreasing solution to a concentration of approximately 0.3M and stir at a speed of 200 rpm.

[0073] S3. Place the heated degreasing liquid from step S2 into an ice bath, control the temperature at about 5°C, and let it stand for 3 hours. The degreasing liquid will separate into solidified oil phase, liquefied oil phase and water phase from top to bottom. After removing the solidified oil phase, the water phase will be obtained by separation.

[0074] S4. The aqueous phase obtained in step S3 is placed in a grinder and homogenized until it becomes a paste; then it is sterilized at 90°C for 20 minutes to obtain a homogenized liquid.

[0075] S5. Pretreatment is performed on the homogenized solution obtained in step S4 by adding oxalic acid to a concentration of approximately 0.3M and heating to 80℃ for 0.5 hours. Then, a two-stage enzymatic hydrolysis is performed: The first stage uses trypsin, adjusting the pH to 6.5-7.5, hydrolyzing at 50℃ for 1 hour, followed by enzyme inactivation at 90℃ for 30 minutes to obtain the first hydrolysate; the amount of trypsin used is 0.1 wt% of the weight of the dry material from the first stage hydrolysis. The second stage uses elastase, adjusting the pH to 7.5-8.5, hydrolyzing at 45℃ for 3 hours, followed by enzyme inactivation at 90℃ for 30 minutes to obtain the second hydrolysate; the amount of elastase used is 0.3 wt% of the weight of the dry material from the second stage hydrolysis. After enzyme inactivation, centrifugation is performed, and the supernatant is collected to obtain crude elastin peptide hydrolysate.

[0076] S6. Dissolve the crude enzymatic hydrolysate of elastin peptide obtained in step S5 in deionized water to prepare a solution with a concentration of 25 mg / mL. Pass the solution 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. After ultrafiltration, molecules with a molecular weight of less than 5000 Da are collected, concentrated, and dried to obtain elastin peptide.

[0077] Comparative Example 1

[0078] Comparative Example 1 is based on Example 1, except that in the preparation process of elastin peptides in Comparative Example 1, the defatting solution is replaced with a 0.5M sodium hydroxide solution in step S2.

[0079] Comparative Example 2

[0080] Comparative Example 2 is based on Example 1, except that: in the preparation process of elastin peptides in Comparative Example 2, microwave intermittent heating of the degreasing solution is not used in step S2, but instead:

[0081] Heat the degreasing solution to 80°C for 20 minutes. At the 14th minute, add soda ash to the degreasing solution to a concentration of approximately 0.3M and stir at a speed of 200 rpm.

[0082] Comparative Example 3

[0083] Comparative Example 3 is based on Example 1, except that the preparation process of elastin peptides in Comparative Example 3 does not include a pretreatment process in step S5.

[0084] Performance testing:

[0085] (1) The peptide chain distribution in Examples 1-3 and Comparative Examples 1-3 was tested by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

[0086] Table 1

[0087]

[0088] (2) The contents of desmosin and isodesmosin in the elastin peptides prepared in Example 1 were determined with reference to standard T / CHC 1010-2023.

[0089] Accurately measure 10g of the elastin peptide prepared in Example 1, add 2mL of 6mol / L hydrochloric acid, hydrolyze at 110℃ for 20h, neutralize to pH 7-8, add a small amount of mobile phase to a 10mL volumetric flask to fully dissolve and mix the sample, dilute to the mark with mobile phase, and filter through a 0.22µm organic phase filter. Transfer the filtrate to a vial and analyze by high-performance liquid chromatography-mass spectrometry (HPLC-MS).

[0090] (a) Determination of desmokinin content:

[0091] Accurately weigh the desmodium standard and prepare standard working solutions of 0.25 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively, and plot the standard working curve.

[0092] Quantitative testing was performed using the external standard method: Formula: x = C * V / m × 100; where X is the percentage content (%) of desmokinin and isodesmokinin in the sample; C is the concentration of desmokinin (μg / mL); V is the final volume (mL); and m is the mass (mg) of elastin peptides weighed.

[0093] Figure 1 Chromatograms of desmodium standards at different concentrations; Figure 2 The image shows the chromatogram of the elastin peptide sample (upper half) and the ion chromatogram of desmokinin extraction (lower half).

[0094] The regression equation obtained is: y = 101528x + 2170, R0 2 =0.9997.

[0095] The content of desmokinin in the elastin peptide in Example 1 is shown in Table 2.

[0096] Table 2

[0097]

[0098] (II) Determination of isodesin content:

[0099] Accurately weigh isodesin standard and prepare standard working solutions of 0.25 μg / mL, 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively, and plot the standard working curve.

[0100] Quantitative testing was performed using the external standard method: Formula: x = C * V / m × 100; where X is the percentage content (%) of desmokinin and isodesmokinin in the sample; C is the concentration of desmokinin (μg / mL); V is the final volume (mL); and m is the mass (mg) of elastin peptides weighed.

[0101] Figure 3 Chromatograms of isodesin standards at different concentrations; Figure 4 The image shows the chromatogram of the elastin peptide sample (upper half) and the ion chromatogram of isodesin extraction (lower half).

[0102] The regression equation was obtained as: y = 118485x + 20282, R0 2 =0.9999.

[0103] The content of isodesin in the elastin peptide in Example 1 is shown in Table 3.

[0104] Table 3

[0105]

[0106] 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 transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for preparing an elastin peptide, characterized by, The preparation method of the elastin peptide comprises the following steps: S1, taking animal tissue raw materials and crushing them into uniform size block-shaped raw materials, washing, soaking in physiological saline, and standing for 2-3 hours; S2, soaking the block-shaped raw materials treated in step S1 in a degreasing solution, and using microwaves to heat the degreasing solution at intervals; using microwaves to raise the temperature of the degreasing solution to 80-90℃, maintaining each heating time for 1-2 minutes, and then standing for 3-10 minutes; In step S2, one microwave heating and one standing are taken as one cycle, and a total of 8-14 cycles are performed; before the start of the last three cycles, pure alkali is added to the degreasing solution to a concentration of 0.3-0.5M; S3, placing the heated degreasing solution in step S2 in an ice bath pool, standing, and the degreasing solution being divided into solidified oil phase, liquefied oil phase and water phase from top to bottom, and the block-shaped raw materials sinking in the water phase, and the solidified oil phase being taken out and then separated by liquid to obtain the water phase; S4, sequentially homogenizing and sterilizing the water phase obtained in step S3 to obtain a homogenate; S5, pretreating the homogenate prepared in step S4, then performing two-stage enzymolysis: sequentially adding trypsin and elastase; deactivating the enzyme, centrifuging, taking the supernatant, and obtaining an elastin peptide crude enzymatic hydrolysate; S6, ion exchange resin chromatography, ultrafiltration, concentration, and drying are performed on the elastin peptide crude enzymatic hydrolysate obtained in step S5 to prepare an elastin peptide. The degreasing solution comprises the following raw materials by weight percentage: vegetable oil and fat: 3-6wt%; animal oil and fat: 3-7wt%; sodium hexametaphosphate: 0.6-1.2wt%; sodium bicarbonate: 0.5-1wt%; papain: 0.1-0.2wt%; and the balance is physiological saline.

2. The method for preparing elastin peptides as described in claim 1, characterized in that, The animal tissue raw materials include at least one of bovine heart tube and bonito heart and arterial bulb.

3. The method for preparing elastin peptides as described in claim 1, characterized in that, In step S2, before the start of the last three cycles, the degreasing solution is oscillated or stirred at a speed of 200-250 rpm.

4. The method for preparing elastin peptides as described in claim 1, characterized in that, In step S2, the weight ratio of the block-shaped raw materials to the degreasing solution is 1:6-13.

5. The method for preparing elastin peptides as described in claim 1, characterized in that, In step S3, the degreasing solution is placed in an ice bath pool, and the temperature of the degreasing solution is maintained at 2-8℃ during the standing process for 3-6 hours.

6. The method for preparing elastin peptides as described in claim 1, characterized in that, In step S4, the homogenization treatment is to homogenize the water phase obtained in step S3 to a paste state in a grinder; In step S4, during the sterilization treatment, the temperature is raised to 85-95℃ and kept for 20-30 minutes.

7. The method for preparing elastin peptides as described in claim 1, characterized in that, In step S5, the pretreatment of the homogenate includes adding oxalic acid to the homogenate to 0.2-0.4M, raising the temperature to 80-90℃ and keeping for 0.5-1.5 hours. And / or, in the step S5, the first-stage enzymolysis uses trypsin to perform enzymolysis, the system pH is adjusted to 6.5-7.5, hydrolysis is performed at 45-55°C for 1-3h, and then enzyme inactivation treatment is performed at 85-95°C for 20-30min to obtain a first enzymolysis liquid; wherein the trypsin is used in an amount of 0.1-0.3wt% of the dry material in the first-stage enzymolysis. And / or, in the step S5, the second-stage enzymolysis uses elastase to perform enzymolysis, the system pH is adjusted to 7.5-8.5, hydrolysis is performed at 40-50°C for 3-8h, and then enzyme inactivation treatment is performed at 85-95°C for 20-30min to obtain a second enzymolysis liquid; wherein the elastase is used in an amount of 0.3-0.8wt% of the dry material in the second-stage enzymolysis.

8. An elastin peptide, characterized in that, The elastin peptide is prepared by the method of any one of claims 1-7.

Images