A kind of bonito elastin peptide and preparation method thereof

Abstract

The invention discloses a bonito elastin peptide and a preparation method thereof, a bonito elastin peptide preparation method, comprising the following steps: S1, cleaning, removing impurities, cutting into pieces after controlling water, adding anhydrous ethanol, stirring, and rinsing with clean water after 2h; S2, adding pure water ultrasonic homogenization after controlling water, adjusting pH to 12, shaking reaction for 2h; S3, centrifugation, adding pure water to the precipitate, adjusting pH to 1 with HCl, shaking reaction and centrifugation; S4, adding the precipitate to NaCl solution, shaking reaction and centrifugation; S5, adding pure water to the precipitate, adjusting pH to 7.8, adding elastase, centrifugation, and crushing the precipitate after freeze drying to obtain bonito elastin peptide. The bonito elastin peptide preparation method provided by the present invention has mild conditions and high yield, and the obtained bonito elastin peptide does not have safety hazards, can be eaten / used with confidence, and the low molecular weight can better cross the epidermal barrier to reach the target site, thereby better exerting its biological effect.

Description

Technical Field

[0001] The present invention relates to the technical field of protein peptides, in particular to a bonito elastin peptide and a preparation method thereof. Background Art

[0002] Many people believe that the key to retaining youthful skin is to replenish collagen. In fact, elastin also plays an important role in maintaining skin elasticity. Elastin and collagen coexist in the body, giving skin tissue elasticity and tensile strength. The relationship between elastin and collagen is like the ribs and canopy of an umbrella. Elastin is the rib, responsible for support, while collagen is the canopy, responsible for filling. The two are tied together and coexist, and neither is indispensable.

[0003] Elastin is particularly important for tissues and organs that are often deformed by stress, such as the lungs, heart, aorta, elastic cartilage, skin, ligaments and bladder. The main function of elastin is to provide tissues and organs with the ability to resist repeated compression and deformation. Like a rubber band, its length can be stretched to several times its normal length, and when it shrinks, it can return to its original length, thus giving these tissues elasticity and tensile strength.

[0004] The amino acids unique to elastin are desmosin and isodesmosin, which covalently cross-link with lysine residues to form a network structure. Through the change of the configuration of this network structure, elastin has strong elasticity. Studies have shown that the stretchability of elastin is at least 5 times greater than that of a rubber strip with the same cross-sectional area.

[0005] The synthesis of elastin in the human body begins at birth and gradually increases, reaching a peak during puberty. However, the body stops producing elastin after the age of 25. The damage caused by various internal and external factors gradually accumulates and becomes difficult to repair, and degradation increases, causing the elastin in the body to be continuously degraded, fragmented, and its content to decrease until it disappears. The skin begins to show aging phenomena such as decreased elasticity, loose / sagging, and fine lines. The elasticity of elastic organs decreases until they disappear and their functions decrease.

[0006] "Bonito elastin peptide" is the only edible elastin raw material currently approved for use by my country's National Health Commission. Bonito heart arterial bulb is a high-quality source of elastin peptides, which contains the iconic amino acids of elastin - desmosin and isodesmosin, which can replenish the lost elastin in the body and have a good effect on maintaining the elasticity, appearance and metabolism of the skin and blood vessels.

[0007] At present, the commonly used methods for extracting and purifying bonito elastin peptides include hot alkali method, hot acid method, salt extraction, enzyme digestion and the like. The hot alkali method and hot acid method have strong reactions, severe degradation of elastin and low yield. The salt used in the salt extraction method is mostly harmful to the human body, such as guanidine phenol hydrochloride. Summary of the invention

[0008] The purpose of the present invention is to provide a bonito elastin peptide and a preparation method thereof, so as to solve the problems of serious elastin degradation, low yield and potential safety hazards of the extraction reagents used to human health in the current bonito elastin peptide extraction process.

[0009] To achieve the above object, the present invention provides a bonito elastin peptide and a preparation method thereof. A bonito elastin peptide preparation method comprises the following steps:

[0010] S1. Wash the purchased bonito heart artery bulb with clean water to remove visible impurities, cut the bonito heart artery bulb into pieces after controlling the water, add it to anhydrous ethanol, and stir it continuously. After 2 hours, remove it from the anhydrous ethanol and rinse it with clean water until there is no obvious ethanol smell;

[0011] S2. After controlling the water, add 3-4 times the mass of pure water of the arterial bulb, adjust the pH to 12 after ultrasonic homogenization, and shake the reaction for 2 hours;

[0012] S3, centrifugation, adding purified water to the precipitate and adjusting the pH to 1 with HCl, shaking the reaction and centrifuging;

[0013] S4, add the precipitate to 1 mol / L NaCl solution, shake the reaction, and then centrifuge;

[0014] S5. Add purified water to the precipitate, adjust the pH to 7.8, add elastase, centrifuge, freeze-dry the precipitate, and grind it through an 80-mesh sieve to obtain bonito elastin peptide.

[0015] Preferably, the area of ​​the bonito heart artery bulb after being cut in step S1 is no larger than 5×5 mm 2 ; The mass volume ratio of bonito heart arterial bulb: anhydrous ethanol is 1:1.5~1:2; the stirring speed is 200r / min.

[0016] Preferably, the ultrasonic time in step S2 is 10 to 15 minutes; the reagent used for adjusting pH is NaOH or KOH solution; the shaking reaction in steps S2 to S5 is carried out on a shaker with a shaking frequency of 60 to 70 times / min.

[0017] Preferably, the centrifugation conditions in steps S3 to S5 are 7000 to 8000 r / min, 4°C, and centrifugation for 10 to 15 min; in step S3, the supernatant is removed to obtain a precipitate, and then the precipitate is washed with a NaOH or KOH solution with a pH of 12 and then centrifuged again, and the above steps are repeated 2 to 3 times before adjusting the pH with HCl, and then the above steps are repeated 1 to 2 times with purified water.

[0018] Preferably, in step S4, before adding the precipitate to the NaCl solution, the precipitate needs to be resuspended with 1 mol / L HCl and then centrifuged, and the above steps are repeated 2 to 3 times, and then resuspended with pure water and centrifuged once; and the reaction is shaken for 2 to 3 hours.

[0019] Preferably, in step S5, the steps of resuspending the precipitate with purified water and removing the supernatant by centrifugation are repeated twice before adjusting the pH; the mass ratio of the added elastase to the precipitate is 1:200-250, and the enzymatic hydrolysis is performed for 15-20 minutes.

[0020] A bonito elastin peptide obtained by the above-mentioned method for preparing the bonito elastin peptide.

[0021] Except for elastin, the bonito heart arterial bulb mainly includes fat, acidic protein, alkaline protein, collagen, globulin, etc. The preparation method provided by the present invention removes fat from the bonito heart arterial bulb by leaching in anhydrous ethanol. At the same time, because anhydrous ethanol is a non-toxic organic solvent with strong dissolving ability, most of the fishy smell in the arterial bulb can also be removed at the same time; by adjusting the pH of the extract to alkaline and acidic, the acidic protein and alkaline protein in the arterial bulb are dissolved in the extract successively, and are removed respectively by solid-liquid separation. At the same time, most of the collagen can also be removed under alkaline conditions; finally, the globulin is removed by a salt solution.

[0022] Therefore, the bonito elastin peptide and preparation method thereof provided by the present invention have the following technical effects:

[0023] (1) The preparation method provided by the present invention does not use any reagents that pose a potential safety hazard to human health, and the prepared elastin peptide is safe and harmless to users and can be eaten / used with confidence;

[0024] (2) The preparation method provided by the present invention has mild conditions and does not involve a heating process, which greatly reduces the rate of thermal degradation and lays a foundation for increasing the yield of elastin peptides;

[0025] (3) The preparation method provided by the present invention greatly improves the yield of bonito protein peptide by reducing degradation, making ultrasonic crushing more delicate, and utilizing the characteristics of elastin being resistant to acid, alkali and organic reagents;

[0026] (4) The preparation method provided by the present invention utilizes elastase to hydrolyze the elastin, thereby greatly reducing the molecular weight of the elastin, thereby enabling it to better pass through the epidermal barrier to reach the target site and better exert its biological effect.

[0027] The technical solution of the present invention is further described in detail below through embodiments. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further illustrated by the following examples.

[0029] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and complete, the technical scheme in the present invention is clearly and completely described below through specific embodiments. The following detailed descriptions are all descriptions of embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0030] Bonito heart arterial bulb was purchased from Ningbo Jinri Food Co., Ltd.; the electric shaker was purchased from Wuhan Huicheng Technology Biological Co., Ltd., model PY45; the centrifuge was a Thermo ST16R refrigerated centrifuge; elastase was purchased from Hefei Norda Biotechnology Co., Ltd., with an enzyme activity of 100,000; the freeze dryer was a LGJ-10D freeze dryer produced by Beijing Sihuan Scientific Instrument Factory.

[0031] Embodiment 1

[0032] The method for preparing bonito elastin peptide comprises the following steps:

[0033] S1. Wash the purchased bonito heart artery bulb with clean water to remove visible impurities. After draining the water, cut the bonito heart artery bulb into pieces so that the area of ​​the artery bulb fragments is no larger than 5×5mm. 2 , weigh 1kg of arterial bulb fragments, add them to 1.5L of anhydrous ethanol, stir them continuously at a speed of 200r / min, remove them from the anhydrous ethanol after 2h, drain the anhydrous ethanol slightly, add them to 2L of clean water, stir and wash, and there is no obvious ethanol smell and fishy smell after rinsing 3 times;

[0034] S2. Drain the cleaned arterial bulb fragments, add them to 3 kg of pure water, homogenize them by ultrasonic for 10 min, add 1 mol / L NaOH while stirring, and continuously check the pH of the homogenate. Stop adding NaOH when the pH of each part of the homogenate is 12. Adjust the shaking frequency of the electric shaker to 60 times / min, and place the homogenate on the shaker for 2 h;

[0035] S3. Divide the homogenate into 6 1L centrifuge bottles in equal amounts, centrifuge at 7000r / min for 10min at 4°C, remove the supernatant, add 200mL of NaOH solution with a pH of 12, mix by pipetting, repeat the centrifugation, remove the supernatant, and add NaOH solution 3 times. Add 200mL of pure water to the obtained precipitate, mix by pipetting, centrifuge under the above centrifugal conditions, remove the supernatant, add 300mL of pure water, adjust the pH to 1 with 1mol / L HCl solution, shake the reaction on an electric shaker with a shaking frequency of 70 times / min for 2h, and centrifuge at 7000r / min for 10min at 4°C;

[0036] S4, remove the supernatant, add 200mL 1mol / L HCl to the precipitate to resuspend, then centrifuge at 7000r / min at 4°C for 10min, repeat the operation of adding HCl to resuspend and centrifuge 3 times, then add purified water to resuspend and centrifuge, add 300mL 1mol / L NaCl solution to the obtained precipitate, shake at a frequency of 70 times / min for 3h, then centrifuge at 7000r / min at 4°C for 15min;

[0037] S5. Add 200 mL of purified water to the precipitate, resuspend the precipitate, mix it by pipetting, and then centrifuge it at a speed of 7000 r / min and 4°C for 15 min. Repeat the steps of adding purified water, mixing, centrifuging, and retaining the precipitate twice. Add 300 mL of purified water to the obtained precipitate, adjust the pH of the precipitate to 7.8 with NaOH and HCl, add 1 mg of elastase to each centrifuge bottle, hydrolyze it for 15 min, centrifuge it at 8000 r / min and 4°C for 10 min, remove the supernatant, place the precipitate in a freeze dryer for freeze drying, and crush the dried precipitate through an 80-mesh sieve to obtain bonito elastin peptide.

[0038] Embodiment 2

[0039] The preparation of bonito elastin peptide includes the following steps which are exactly the same as those in Example 1, except for the following differences:

[0040] The amount of anhydrous ethanol added in step S1 is 1.8 L;

[0041] The ultrasonic time in step S2 is 12 min, and the shaking frequency of the shaking table is 65 times / min;

[0042] The centrifugal conditions in step S3 are a rotation speed of 7500 r / min, centrifugation at 4°C for 15 min, washing the precipitate twice with pure water, and shaking at a frequency of 65 times / min;

[0043] The centrifugation conditions in step S4 are 7500 r / min, centrifugation at 4°C for 15 min, shaking frequency of 65 times / min, shaking reaction for 3 h, HCl resuspension and centrifugation operations are repeated twice, and then pure water is added for resuspension and centrifugation twice;

[0044] The centrifugal conditions in step S5 are a rotation speed of 7500 r / min, centrifugation at 4° C. for 15 min, addition of 0.8 mg of elastase, and enzymatic hydrolysis for 20 min.

[0045] Embodiment 3

[0046] The preparation of bonito elastin peptide includes the following steps which are exactly the same as those in Example 1, except for the following differences:

[0047] The amount of anhydrous ethanol added in step S1 is 2 L;

[0048] The ultrasonic time in step S2 is 15 min, 1 mol / L KOH solution is used for pH adjustment, and the shaking frequency of the shaking table is 70 times / min;

[0049] The centrifugal condition in step S3 is a speed of 8000 r / min, centrifugation for 12 min at 4°C, the added alkali solution is a KOH solution with a pH of 12, the operation of centrifugation, removal of the supernatant, and addition of the KOH solution is repeated twice, and the mixture is washed with pure water twice, and the shaking frequency is 60 times / min;

[0050] The centrifugal conditions in step S4 are a rotation speed of 8000 r / min, centrifugation at 4°C for 12 min, a shaking frequency of 60 times / min, and a shaking reaction for 2 h;

[0051] The centrifugal conditions in step S5 are a rotation speed of 8000 r / min, centrifugation at 4° C. for 12 min, addition of 1.1 mg of elastase, and enzymatic hydrolysis for 20 min.

[0052] Results Analysis

[0053] After weighing, 1 kg of fresh bonito heart arterial bulb can obtain 30 g of freeze-dried elastin peptide using the preparation method in Example 1, 29.5 g of freeze-dried elastin peptide can be obtained using the method in Example 2, and 30.2 g of freeze-dried elastin peptide can be obtained using the method in Example 3.

[0054] 3 g of the bonito elastin peptides prepared in Examples 1, 2 and 3 were respectively sent to Pony Testing Group Co., Ltd. to detect the molecular weight of the polypeptides. The results showed that the molecular weights of the bonito elastin peptides obtained in Examples 1 to 3 were no more than 120 Da, 100 Da and 110 Da, respectively.

[0055] Therefore, the bonito elastin peptide and the preparation method thereof provided by the present invention do not use any reagents that pose a potential safety hazard to human health, and the prepared elastin peptide is safe and harmless to users and can be eaten / used with confidence; the conditions are mild and there is no heating process, which greatly reduces the rate of thermal degradation, and lays a foundation for improving the yield of elastin peptide; the yield of bonito protein peptide is greatly improved by reducing degradation, making ultrasonic crushing more delicate, and utilizing the characteristics of elastin being resistant to acid, alkali and organic reagents; and the molecular weight of elastin is greatly reduced by utilizing elastase enzymatic hydrolysis, so that it can better pass through the epidermal barrier to reach the target site and better exert its biological effect.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing bonito elastin peptide, characterized in that: The following steps are involved: S1. Wash the purchased bonito heart artery bulb with clean water to remove visible impurities, cut the bonito heart artery bulb into pieces after controlling the water, add it to anhydrous ethanol, and stir it continuously. After 2 hours, remove it from the anhydrous ethanol and rinse it with clean water until there is no obvious ethanol smell; S2. After controlling the water, add 3-4 times the mass of pure water of the arterial bulb, adjust the pH to 12 after ultrasonic homogenization, and shake the reaction for 2 hours; S3, centrifuge, remove the supernatant to obtain a precipitate, then wash the precipitate with a NaOH or KOH solution with a pH of 12 and centrifuge again, repeat the steps of washing the precipitate with aspiration and beating and centrifuging 2 to 3 times, add purified water to the precipitate, repeat the steps of washing the precipitate with aspiration and beating and centrifuging 1 to 2 times, add purified water, adjust the pH to 1 with HCl, shake the reaction and centrifuge; S4, add the precipitate to 1 mol / L NaCl solution, shake the reaction, and then centrifuge; S5, adding purified water to the precipitate, adjusting the pH to 7.8, adding elastase, centrifuging, freeze-drying the precipitate, and crushing it through an 80-mesh sieve to obtain bonito elastin peptide; In step S1, the area of ​​the bonito heart artery bulb after being cut into pieces is no larger than 5×5 mm 2 ; The mass volume ratio of bonito heart arterial bulb: anhydrous ethanol is 1:1.5~1:2; the stirring speed is 200r / min; The ultrasonic time in step S2 is 10-15 min; the reagent used for adjusting pH is NaOH or KOH solution; the shaking reaction in steps S2-S4 is carried out on a shaker with a shaking frequency of 60-70 times / min; The centrifugal conditions in steps S3 to S5 are 7000 to 8000 r / min, 4° C., and centrifugation for 10 to 15 min; In step S4, before adding the precipitate to the NaCl solution, the precipitate needs to be resuspended with 1 mol / L HCl and then centrifuged, and the above steps are repeated 2 to 3 times, and then resuspended with purified water and centrifuged once; shake the reaction for 2 to 3 hours; In step S5, the steps of resuspending the precipitate with purified water and removing the supernatant by centrifugation are repeated twice before adjusting the pH; the mass ratio of the added elastase to the precipitate is 1:200-250, and the enzymatic hydrolysis is performed for 15-20 minutes; The molecular weight of the prepared bonito elastin peptide is no more than 120Da.