Preparation method and application of an anti-aging collagen peptide

Abstract

The present invention relates to the field of collagen peptides, and specifically to a preparation method and application of an anti-aging collagen peptide. First, fish scales are defatted and demineralized, then protease is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. After inactivating the enzyme, centrifugation is carried out, and the supernatant is separated by an ionic liquid / PVDF composite membrane. After nanofiltration of the obtained filtrate, the retentate is collected and dried to obtain the anti-aging collagen peptide. The method provided by the present invention has a high yield of collagen peptide, and can remove most of the trimethylamine oxide, improving the quality of the collagen peptide.

Description

Technical Field

[0001] The present invention relates to the field of collagen peptides, and specifically to a preparation method and application of anti-aging collagen peptides. Background Art

[0002] Aging is the most basic natural law in nature and also the most complex biological phenomenon. With the aggravation of the aging problem in our country, the anti-aging problem has become a research hotspot. During the aging process, the skin on the surface of the body is the most obvious organ, and delaying skin aging has become the top priority in anti-aging research. During the aging process, the loss of collagen will directly cause the skin to lose moisture and elasticity, making the skin age rapidly. In addition, after collagen is phagocytosed by melanin substances, the activated melanin cannot be discharged in time, which will also cause problems such as age spots, uneven skin color or dullness. The loss of collagen is also likely to cause a series of serious problems such as joint pain, osteoporosis, vision decline, and serious decline in gastrointestinal function.

[0003] Collagen peptides are small molecule products obtained by using biological or chemical methods with collagen in various organisms as raw materials to further improve the water solubility and biological activity of collagen. Research at home and abroad shows that collagen peptides have good antioxidant, anti-aging, immunomodulatory, promoting tissue wound repair, inhibiting angiotensin-I converting enzyme, anti-tumor, anticoagulant, antibacterial and other activities. Compared with collagen, collagen peptides have better antioxidant effects.

[0004] At present, most of the collagen peptides derived from fish are extracted from the fish skins of deep-sea fish. However, the cost of the fish skins of deep-sea fish is relatively high, and they are easily affected by marine pollution. In order to eliminate odors, activated carbon treatment is often used, but the adsorption effect of activated carbon will greatly reduce the yield of collagen peptides. Summary of the Invention

[0005] Object of the Invention: Aiming at the above technical problems, the present invention provides a preparation method and application of anti-aging collagen peptides.

[0006] The technical solution adopted is as follows:

[0007] A preparation method of anti-aging collagen peptides is as follows:

[0008] After degreasing and demineralizing fish scales, add protease for enzymatic hydrolysis to obtain an enzymatic hydrolysate. After inactivating the enzyme, centrifuge, separate the supernatant with an ionic liquid / PVDF composite membrane, nanofiltrate the obtained filtrate, and collect the retentate and dry it to obtain anti-aging collagen peptides.

[0009] Furthermore, the preparation method of the ionic liquid / PVDF composite membrane is as follows:

[0010] Mix triethyl phosphate, polyethylene glycol and ionic liquid evenly, heat up to 160 - 180 °C, add PVDF, stir to form a homogeneous casting solution, and cast a film after standing for degassing.

[0011] Furthermore, the mass ratio of PVDF to ionic liquid is 100:1 - 5.

[0012] Furthermore, the ionic liquid is an amino acid ionic liquid.

[0013] Furthermore, the amino acid ionic liquid is any one or a combination of arginine ionic liquid, aspartic acid ionic liquid, glutamic acid ionic liquid, glycine ionic liquid, histidine ionic liquid, isoleucine ionic liquid, leucine ionic liquid, lysine ionic liquid, methionine ionic liquid, phenylalanine ionic liquid, proline ionic liquid;

[0014] The amino acid ionic liquid is preferably aspartic acid ionic liquid.

[0015] Furthermore, the preparation method of the aspartic acid ionic liquid is as follows:

[0016] Dilute 1 - hexyl - 3 - methylimidazolium bromide ionic liquid with water and add it to hydroxide - type anion resin. After ion exchange, 1 - hexyl - 3 - methylimidazolium hydroxide ionic liquid is generated, and finally react with aspartic acid to obtain aspartic acid ionic liquid.

[0017] Furthermore, the defatting and calcium - removing operation is specifically as follows:

[0018] Crush fish scales, add them to the defatting solution, stir for 5 - 10 h and then filter. Wash the filtrate with deionized water until neutral, then add it to the calcium - removing solution to obtain a mixed solution. Place the mixed solution in an ultrasonic device at 40 - 60 °C for ultrasonic treatment for 1 - 10 h, filter again, wash the filtrate with deionized water until neutral, and finally soak it in sodium chloride solution for 8 - 36 h, filter, and dry the filtrate after washing it thoroughly with deionized water.

[0019] Furthermore, the defatting solution is any one of sodium bicarbonate solution, sodium hydroxide solution or potassium hydroxide solution;

[0020] The calcium - removing solution is any one of lactic acid solution, acetic acid solution, hydrochloric acid solution, citric acid solution.

[0021] Furthermore, the protease is any one or a combination of neutral protease, trypsin, papain, alkaline protease.

[0022] The present invention also provides the application of the anti - aging collagen peptide prepared by the above method in cosmetics and / or health foods.

[0023] The present invention provides a preparation method of anti-aging collagen peptide, which has the following beneficial effects:

[0024] (1) Fish scales do not participate in blood circulation, so the possibility of being polluted by the environment is very small, and the raw materials are abundant, so the cost is relatively low. Compared with fish skin, it has certain cost and safety advantages;

[0025] (2) Modifying PVDF with amino acid-based ionic liquids can effectively improve the hydrophilicity of the PVDF membrane, reduce the resistance of water passing through the membrane, improve the filtration performance, and eliminate fishy smell without passing through activated carbon treatment, thereby improving the yield of collagen peptide. The reason may be that there is a relatively strong C-H···π and C-H···N hydrogen bond interaction between the trimethylamine oxide molecule and the imidazole ring of the ionic liquid, or the ionic liquid adsorbs and intercepts most of the trimethylamine oxide molecules through electrostatic adsorption, thereby eliminating fishy smell and improving the quality of collagen peptide;

[0026] (3) The collagen peptide prepared by the present invention has in vitro antioxidant activity and can be applied in anti-aging cosmetics and health foods, which is of great significance for improving the added value of fish scales, comprehensive utilization, industrial application, reducing resource waste and environmental pollution. Specific embodiments

[0027] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase. The technologies not mentioned in the present invention refer to the prior art. Unless otherwise specified, the following examples and comparative examples are parallel tests and adopt the same treatment steps and parameters.

[0028] Example 1:

[0029] A preparation method of anti-aging collagen peptide:

[0030] First, wash the tilapia fish scales thoroughly with deionized water and then dry them. Crush the dried scales with a pulverizer and pass them through an 80-mesh sieve to obtain fish scale powder. Add the fish scale powder to a 0.8 mol / L sodium bicarbonate solution at a solid-liquid ratio of 1:25 (g:ml), stir at room temperature for 8 h, and then filter. Wash the filtrate with deionized water until it is neutral and then dry it. Then, add it to a 0.8 mol / L lactic acid solution at a solid-liquid ratio of 1:25 (g:ml) to obtain a mixed solution. Place the mixed solution in an ultrasonic device at 45 °C with a power of 100 W and perform ultrasonic treatment for 2 h. Filter again, wash the filtrate with deionized water until it is neutral and then dry it. Finally, add it to a 0.5 mol / L sodium chloride solution at a solid-liquid ratio of 1:25 (g:ml), soak for 12 h, filter, and wash the filtrate thoroughly with deionized water and then dry it to obtain defatted and demineralized fish scale powder. Add it to deionized water at a solid-liquid ratio of 1:25 (g:mL), adjust the pH of the system to 11 with 0.1 mol / L sodium hydroxide solution, preheat it in a 50 °C constant temperature water bath for 30 min, add alkaline protease, mix well, and then enzymatically hydrolyze for 5 h. The addition amount of alkaline protease is 9000 U / g. Inactivate the enzyme solution at 100 °C for 15 min. After the enzyme solution cools to room temperature, centrifuge it under the conditions of 4000 r / min and 3 min. Collect the supernatant, separate it with an ionic liquid / PVDF composite membrane, collect the obtained filtrate, perform nanofiltration desalination through a nanofiltration membrane system, and collect the retentate and freeze-dry it at low temperature to obtain anti-aging collagen peptides.

[0031] Among them, the preparation method of the ionic liquid / PVDF composite membrane is as follows:

[0032] Dilute 1-hexyl-3-methylimidazolium bromide ionic liquid with water at a solid-liquid ratio of 1:5 (g:mL), add it to 201×7 hydroxide-form anion exchange resin, and generate 1-hexyl-3-methylimidazolium hydroxide ionic liquid aqueous solution through ion exchange. Then, add aspartic acid to the 1-hexyl-3-methylimidazolium hydroxide ionic liquid aqueous solution. The molar ratio of 1-hexyl-3-methylimidazolium hydroxide ionic liquid to aspartic acid is 1:1.2. Stir and react at room temperature for 24 h, then add a large amount of a mixed solution composed of methanol and acetonitrile with a volume ratio of 1:9, and let it stand at low temperature of 5 °C for 5 h. After filtering out the unreacted aspartic acid that precipitates, distill the filtrate under reduced pressure and dry it to obtain aspartic acid ionic liquid. Mix 20 parts by weight of triethyl phosphate, 55 parts by weight of polyethylene glycol-400, and 1 part by weight of aspartic acid ionic liquid, heat up to 180 °C, add 25 parts by weight of PVDF, stir to form a uniform casting solution, let it stand and defoam, and then cast a film on a glassware. Place the glassware in a vacuum oven and dry it to obtain an ionic liquid / PVDF composite membrane with a thickness of about 100 μm.

[0033] The excessive accumulation of free radicals, manifested at the skin level, is the skin aging phenomenon that everyone is extremely concerned about, such as the decline of skin elasticity, the denaturation of collagen, the formation of wrinkles, and the relaxation of the skin, as well as damaged barriers, increased inflammatory responses, and pigmentation. All these are closely related to the damaging effects of free radicals. Therefore, scavenging free radicals is one of the important means of anti-aging. DPPH is a very stable nitrogen-centered free radical, which appears dark purple in solution and has a strong absorption at 517 nm. In the antioxidant test, antioxidants can directly scavenge DPPH. As this free radical decreases, the color of the solution gradually changes from dark purple to colorless. Therefore, the antioxidant capacity of the antioxidant can be calculated from the change value of the absorbance.

[0034] Preparation of the DPPH stock solution: Accurately weigh 7.2 mg of DPPH and dissolve it in 25 ml of methanol. Store it in the dark at -20 °C. Before use, take a small amount of the stock solution and dilute it 5 times to obtain the DPPH working solution, and measure the absorbance value (A1) at 517 nm.

[0035] Dissolve the anti-aging collagen peptide prepared in this example in methanol to prepare a working solution with a concentration of 5 mg·mL -1 Take 100 μL of the working solution and add it dropwise to 100 μL of the DPPH working solution, then mix well by shaking. Perform light shielding treatment and react at room temperature for 20 min, and measure the absorbance value (A2) at 517 nm.

[0036] The calculation formula for the DPPH free radical scavenging rate G is as follows:

[0037]

[0038] After calculation, the DPPH free radical scavenging rate of the anti-aging collagen peptide prepared in this example is 90.6%.

[0039] Take another commercially available collagen peptide (Dezhou Lanli Biotechnology Co., Ltd.) and test it using the same method as above. After calculation, the DPPH free radical scavenging rate of the commercially available collagen peptide is 81.1%.

[0040] From the above comparison, it can be seen that the anti-free radical oxidation performance of the anti-aging collagen peptide prepared in this example is better than that of the commercially available collagen peptide.

[0041] Example 2:

[0042] A preparation method of an anti-aging collagen peptide:

[0043] First, wash the Tilapia scales thoroughly with deionized water, then dry them. Crush them with a pulverizer and pass through an 80-mesh sieve to obtain scale powder. Add the scale powder to a 0.8 mol / L sodium bicarbonate solution at a solid-liquid ratio of 1:25 (g:ml), stir at room temperature for 8 h, and then filter. Wash the filtrate with deionized water until neutral, and then dry it. Then add it to a 0.8 mol / L lactic acid solution at a solid-liquid ratio of 1:25 (g:ml) to obtain a mixed solution. Place the mixed solution in an ultrasonic device at 45 °C with a power of 100 W and perform ultrasonic treatment for 2 h. Filter again, wash the filtrate with deionized water until neutral, and then dry it. Finally, add it to a 0.5 mol / L sodium chloride solution at a solid-liquid ratio of 1:25 (g:ml), soak for 12 h, filter, wash the filtrate thoroughly with deionized water, and dry it to obtain defatted and decalcified scale powder. Add it to deionized water at a solid-to-liquid ratio of 1:25 (g:mL), preheat it in a 50 °C constant temperature water bath for 30 min, add papain, mix well, and then enzymatically hydrolyze for 5 h. The addition amount of papain is 9000 U / g. Inactivate the enzyme solution at 100 °C for 15 min. After the enzyme solution cools to room temperature, centrifuge it under the conditions of 4000 r / min and 3 min. Collect the supernatant, separate it with an ionic liquid / PVDF composite membrane, collect the obtained filtrate, perform nanofiltration desalination through a Pura membrane nanofiltration membrane system, and collect the retentate and freeze-dry it at low temperature to obtain anti-aging collagen peptides.

[0044] Among them, the preparation method of the ionic liquid / PVDF composite membrane is the same as that in Example 1.

[0045] Example 3:

[0046] A preparation method of anti-aging collagen peptides:

[0047] First, wash tilapia fish scales thoroughly with deionized water, then dry them. Crush them with a pulverizer and pass through an 80-mesh sieve to obtain fish scale powder. Add the fish scale powder to a 0.8 mol / L sodium bicarbonate solution at a solid-liquid ratio of 1:25 (g:ml), stir at room temperature for 8 h, and then filter. Wash the filtrate with deionized water until neutral, and then dry it. Then, add it to a 0.8 mol / L lactic acid solution at a solid-liquid ratio of 1:25 (g:ml) to obtain a mixed solution. Place the mixed solution in an ultrasonic device at 45 °C with a power of 100 W for ultrasonic treatment for 2 h, filter again, wash the filtrate with deionized water until neutral, and then dry it. Finally, add it to a 0.5 mol / L sodium chloride solution at a solid-liquid ratio of 1:25 (g:ml), soak for 12 h, filter, wash the filtrate thoroughly with deionized water, and dry it to obtain defatted and demineralized fish scale powder. Add it to deionized water at a solid-to-liquid ratio of 1:25 (g:mL), preheat it in a 50 °C constant temperature water bath for 30 min, add neutral protease, mix well, and then enzymatically hydrolyze for 5 h. The addition amount of neutral protease is 9000 U / g. Inactivate the enzyme solution at 100 °C for 15 min. After the enzyme solution cools to room temperature, centrifuge it under the conditions of 4000 r / min and 3 min. Collect the supernatant, separate it with an ionic liquid / PVDF composite membrane, collect the obtained filtrate, perform nanofiltration desalination through a Pura membrane nanofiltration membrane system, and collect the retentate, and then freeze-dry it at low temperature to obtain anti-aging collagen peptides.

[0048] Among them, the preparation method of the ionic liquid / PVDF composite membrane is the same as that in Example 1.

[0049] Example 4:

[0050] A preparation method of anti-aging collagen peptides:

[0051] First, wash the tilapia fish scales thoroughly with deionized water and then dry them. Crush the dried scales with a pulverizer and pass them through an 80-mesh sieve to obtain fish scale powder. Add the fish scale powder to a 0.8 mol / L sodium bicarbonate solution at a solid-liquid ratio of 1:25 (g:ml), stir at room temperature for 8 h, and then filter. Wash the filtrate with deionized water until it is neutral and then dry it. Then, add it to a 0.8 mol / L lactic acid solution at a solid-liquid ratio of 1:25 (g:ml) to obtain a mixed solution. Place the mixed solution in an ultrasonic device at 45 °C with a power of 100 W and perform ultrasonic treatment for 2 h. Filter again, wash the filtrate with deionized water until it is neutral and then dry it. Finally, add it to a 0.5 mol / L sodium chloride solution at a solid-liquid ratio of 1:25 (g:ml), soak for 12 h, filter, wash the filtrate thoroughly with deionized water, and dry it to obtain defatted and demineralized fish scale powder. Add it to deionized water at a solid-liquid ratio of 1:25 (g:mL), preheat it in a 50 °C constant temperature water bath for 30 min, add trypsin, mix well, and enzymatically hydrolyze for 5 h. The addition amount of trypsin is 9000 U / g. Inactivate the enzyme solution at 100 °C for 15 min. After the enzyme solution is cooled to room temperature, centrifuge it under the conditions of 4000 r / min and 3 min. Collect the supernatant, separate it with an ionic liquid / PVDF composite membrane, collect the obtained filtrate, perform nanofiltration desalination through a film nanofiltration membrane system, and collect the retentate and freeze-dry it at low temperature to obtain anti-aging collagen peptides.

[0052] Among them, the preparation method of the ionic liquid / PVDF composite membrane is the same as that in Example 1.

[0053] Comparative Example 1:

[0054] It is basically the same as Example 1, except that a commercially available PVDF ultrafiltration membrane (Shandong Bona) is used to separate the supernatant instead of the ionic liquid / PVDF composite membrane.

[0055] Comparative Example 2:

[0056] It is basically the same as Example 1, except that after the enzyme solution is cooled to room temperature, activated carbon is added for decolorization and deodorization. The addition amount of activated carbon is 10% of the mass of the enzyme solution. After filtration through a filter cloth, the filtrate is centrifuged under the conditions of 4000 r / min and 3 min. Then, the supernatant is collected and a commercially available PVDF ultrafiltration membrane (Shandong Bona) is used to separate the supernatant instead of the ionic liquid / PVDF composite membrane. The obtained filtrate is collected, subjected to nanofiltration desalination through a film nanofiltration membrane system, and the retentate is collected and freeze-dried at low temperature.

[0057] Performance detection:

[0058] ① Trichloroacetic acid (TCA) can dissolve small molecule proteins or peptides and react with macromolecule proteins to form white precipitates. Therefore, the TCA precipitation method was used to determine the peptide yield. 5 mL of the retentate from Examples 1-4 and Comparative Examples 1 and 2 was taken respectively, and 5 mL of TCA solution (mass fraction 30%) was added and mixed well. After standing for 10 min, it was centrifuged at 8000 r / min for 10 min to obtain the supernatant. The soluble protein content was determined by the biuret method, the amino nitrogen content in the supernatant was determined by the double indicator formaldehyde titration method, and the total protein content in tilapia fish scales was determined by the Kjeldahl method.

[0059] The calculation formula for the yield of collagen peptide is as follows:

[0060]

[0061] In the formula, W is the yield of collagen peptide, %; N1 is the protein content in the supernatant after precipitation with 30% TCA, mg; N2 is the ammonia nitrogen content in the supernatant, mg; N3 is the protein content in tilapia fish scales, mg.

[0062] ② The detection of trimethylamine oxide was carried out with reference to the reineckate spectrophotometry method. The calculation formula for the removal rate of trimethylamine oxide is as follows:

[0063]

[0064] In the formula, Y is the removal rate of trimethylamine oxide, %; M1 is the content of trimethylamine oxide in the enzymatic hydrolysate, mg; M2 is the content of trimethylamine oxide in the filtrate, mg.

[0065] The above detection results are shown in Table 1 below:

[0066] Table 1:

[0067]

[0068]

[0069] As can be seen from Table 1 above, the method provided by the present invention has a high yield of collagen peptide, and can remove most of the trimethylamine oxide, improving the quality of collagen peptide.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing anti-aging collagen peptides, characterized in that: The details are as follows: After defatting and decalcifying the fish scales, protease is added to obtain an enzymatic solution, the enzyme is inactivated and then centrifuged, the supernatant is separated by an ionic liquid / PVDF composite membrane, the obtained filtrate is nanofiltered, and the retentate is collected and dried to obtain anti-aging collagen peptides; The preparation method of the ionic liquid / PVDF composite membrane is as follows: Mix triethyl phosphate, polyethylene glycol and ionic liquid, heat to 160-180℃, add PVDF, stir to form a uniform casting solution, let stand to degas, and then cast into a film; The mass ratio of the PVDF to the ionic liquid is 100:1-5; The ionic liquid is an amino acid ionic liquid; The amino acid ionic liquid is aspartic acid ionic liquid; The preparation method of the aspartic acid ionic liquid is as follows: The bromide 1-hexyl-3-methylimidazolium ionic liquid is diluted with water and added to a hydroxide type anion resin, and ion exchange is performed to generate a hydroxide 1-hexyl-3-methylimidazolium ionic liquid, which is finally reacted with aspartic acid to obtain an aspartic acid ionic liquid.

2. The method for preparing the anti-aging collagen peptide according to claim 1, characterized in that: The degreasing and decalcification operations are as follows: The fish scales are crushed, added to the degreasing solution, stirred for 5-10 hours, and then filtered. The filtrate is washed with deionized water until it is neutral, and then added to the decalcification solution to obtain a mixed solution. The mixed solution is placed in an ultrasonic device at 40-60°C for ultrasonic treatment for 1-10 hours, filtered again, and the filtrate is washed with deionized water until it is neutral. Finally, it is added to a sodium chloride solution and soaked for 8-36 hours, filtered, and the filtrate is fully washed with deionized water and then dried.

3. The method for preparing the anti-aging collagen peptide according to claim 2, characterized in that: The degreasing solution is any one of a sodium bicarbonate solution, a sodium hydroxide solution or a potassium hydroxide solution; The decalcification solution is any one of lactic acid solution, acetic acid solution, hydrochloric acid solution and citric acid solution.

4. The method for preparing the anti-aging collagen peptide according to claim 2, characterized in that: The protease is any one or more combinations of neutral protease, trypsin, papain and alkaline protease.

5. Use of the anti-aging collagen peptide prepared by the preparation method according to claim 3 or 4 in cosmetics and / or health foods.

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