A freeze-drying method for improving the storage stability of collagen fibers

Abstract

The present invention relates to a freeze-drying method for improving the storage stability of collagen fibers, and belongs to the field of biotechnology. The present invention uses NaIO4 to perform oxidation treatment on hydroxypropyl methylcellulose to obtain oxidized hydroxypropyl methylcellulose with a certain degree of oxidation, and mixes the oxidized hydroxypropyl methylcellulose with a collagen fiber solution, performs modification treatment on the collagen fibers to obtain a modified collagen fiber solution, and then mixes the modified collagen fiber solution with a freeze-drying stabilizer for freeze-drying treatment to obtain freeze-dried collagen fibers; the freeze-drying stabilizer contains aconite polysaccharide, salvia miltiorrhiza polysaccharide and scallop mantle polypeptide. The freeze-drying process of the collagen fibers provided by the present invention can effectively improve the storage stability of freeze-dried collagen fibers; at the same time, the freeze-dried collagen fibers have good anti-skin aging ability when acting on the skin as a cosmetic ingredient.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a freeze-drying method for improving the storage stability of collagen fibers. Background Art

[0002] With the development of science and technology, people are paying more and more attention to their health, especially skin health. Skin health is closely related to collagen fibers, which are important components for skin to maintain elasticity and firmness. However, due to various reasons, the stability of collagen fibers is often not guaranteed, which leads to skin aging and damage.

[0003] In order to solve this problem, researchers have conducted a lot of research, trying to find a method to improve the stability of collagen fibers. In this process, freeze-drying technology has become an important research direction. Freeze-drying technology is a technology that can quickly dry substances at low temperatures. It can effectively maintain the activity of substances and is therefore widely used in the biomedical field. However, existing freeze-drying technology cannot fully meet the needs of improving the stability of collagen fibers. First of all, existing freeze-drying technology often cannot achieve uniform freeze-drying of collagen fibers, resulting in changes in the structure of the fibers, affecting their stability. Secondly, existing freeze-drying technology cannot effectively prevent the oxidation of collagen fibers, further reducing their stability.

[0004] Therefore, developing a freeze-drying process that can improve the storage stability of collagen fibers is of great significance for improving skin health.

[0005] Invention CN114805548B discloses a recombinant collagen freeze-dried fiber and a preparation method thereof, belonging to the technical field of biomedical materials; the invention dissolves the pretreated recombinant collagen and performs chromatography to remove impurities, and then uses hydrophobic and molecular sieve chromatography to purify, desalt, concentrate and freeze-dry the collagen with a stable structure to obtain the recombinant collagen freeze-dried fiber, so that the obtained recombinant collagen freeze-dried fiber has a dense structure, strong stability, high temperature resistance, and maintains the original collagen activity.

[0006] Invention CN117230143A discloses a collagen peptide and a preparation method and application thereof. Giant salamander skin collagen is obtained from giant salamander skin, and the giant salamander skin collagen is enzymatically hydrolyzed to obtain the collagen peptide. The enzymatic hydrolysis comprises the following steps: dissolving the giant salamander skin collagen in ddH2O at a solid-liquid ratio of 1:20-30 to obtain a collagen solution; inactivating the enzyme in the collagen solution, and adjusting the pH value to 8.5-9.5 after cooling to room temperature; adding 2400-3200 U / g protease, reacting at 55-65°C for 4-6 hours, taking out, inactivating the enzyme in a boiling water bath, and centrifuging after cooling; taking the supernatant for dialysis and freeze-drying to obtain the collagen peptide, which has good antioxidant properties and is used to scavenge free radicals.

[0007] Invention CN114010528B discloses a recombinant human collagen freeze-dried powder and a preparation method thereof, wherein the recombinant human collagen freeze-dried powder comprises recombinant human collagen, a stabilizer, an adjuvant and a buffer, wherein the buffer comprises EDTA-2Na, bovine serum albumin, phosphate, sodium chloride and Tween 80. The stabilizer, adjuvant and buffer play a role in maintaining the stability of the recombinant human collagen, and the components of EDTA-2Na, bovine serum albumin, phosphate, sodium chloride and Tween 80 cooperate with each other to improve the stability of the recombinant human collagen, so that its biological activity can be maintained for a long time. It has the effect of promoting cell proliferation and migration, improving skin redness, reducing transepidermal water loss of the skin, and repairing damaged skin barriers. Summary of the invention

[0008] The object of the present invention is to provide a freeze-drying method for improving the storage stability of collagen fibers. The freeze-dried collagen fibers prepared by the freeze-drying method have the characteristics of high storage stability and can effectively delay skin oxidative aging.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] A freeze-drying method for improving the storage stability of collagen fibers, calculated by weight, comprises the following steps:

[0011] W1. Dissolving collagen fibers in acetic acid-sodium acetate buffer to prepare a collagen fiber solution;

[0012] W2. Mix 2-4 parts of the collagen fiber solution with 1-3 parts of oxidized hydroxypropyl methylcellulose and continue stirring for 30-50 min to obtain a modified collagen fiber solution;

[0013] W3. 3-5 parts of aconite polysaccharide, 1-3 parts of salvia miltiorrhiza polysaccharide, 2-4 parts of scallop mantle polypeptide and 15-20 parts of deionized water were mixed to obtain a freeze-dried stabilizer;

[0014] W4. Mix 20-30 parts of the modified collagen fiber solution with 5-7 parts of the freeze-drying stabilizer, continue stirring for 3-5 minutes, and then freeze-dry to obtain freeze-dried collagen fibers.

[0015] As a preferred technical solution of the present invention, the molar concentration of the acetic acid-sodium acetate buffer described in step W1 is 0.4-0.6 mol / L.

[0016] As a preferred technical solution of the present invention, the mass volume concentration of the collagen fiber solution described in step W1 is 5-7 mg / mL.

[0017] As a preferred technical solution of the present invention, the method for preparing oxidized hydroxypropyl methylcellulose described in step W2 comprises the following steps:

[0018] X1. Dissolve 20-30 parts of hydroxypropyl methylcellulose in 100-120 parts of deionized water, stir until completely dissolved, add 2-4 parts of NaIO4, mix and continue stirring for 5-10 minutes, then adjust the pH value with H2SO4 solution, react at 25°C in the dark for 3-5 hours, then perform light-shielding dialysis treatment, and finally freeze-dry to obtain oxidized hydroxypropyl methylcellulose.

[0019] As a preferred technical solution of the present invention, the molar concentration of the H2SO4 solution described in step X1 is 0.8-1.2 mol / L.

[0020] As a preferred technical solution of the present invention, the pH value described in step X1 is 2.0-3.0.

[0021] As a preferred technical solution of the present invention, the preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps:

[0022] Y1. The scallop mantle was washed with deionized water, desalted and crushed to obtain a scallop mantle homogenate;

[0023] Y2. After enzymatic hydrolysis of the Chlamys farreri mantle homogenate, ultrafiltration is performed, and the filtrate is inactivated and dried to obtain the Chlamys farreri mantle polypeptide.

[0024] As a preferred technical solution of the present invention, calculated by weight, the enzymatic hydrolysis treatment described in step Y2 refers to mixing 100-120 parts of Chlamys farreri mantle homogenate with 1-2 parts of papain, and enzymolyzing for 80-100 minutes at a temperature of 50-60°C.

[0025] As a preferred technical solution of the present invention, the freeze-drying process described in step W4 comprises the following steps:

[0026] Z1. Pre-freeze at a temperature of ≤-50°C for 1-3 hours;

[0027] Z2. Sublime for 12-15h at a vacuum degree of 8-12Pa and a temperature of ≤-40°C;

[0028] Z3. Continue to dry for 1-3 hours at a pressure of 12-15 Pa and a temperature of 22-28°C to obtain freeze-dried collagen fibers.

[0029] A freeze-dried collagen fiber prepared according to the freeze-drying method.

[0030] The present invention uses oxidized hydroxypropyl methylcellulose with a certain degree of oxidation to modify collagen fibers, so that some groups on the collagen fibers are aldehyde-modified to improve the stability of the collagen fibers; at the same time, the polysaccharides and polypeptides added by the present invention are coated on the surface of the collagen fibers to improve the structural stability and antioxidant capacity of the collagen fibers.

[0031] Beneficial effects of the present invention:

[0032] (1) The modification process of collagen fibers provided by the present invention can effectively improve the stability of collagen fibers. At the same time, freeze-drying treatment with a freeze-drying stabilizer composed of aconite polysaccharide, salvia miltiorrhiza polysaccharide and scallop mantle polypeptide can effectively improve the storage stability of freeze-dried collagen fibers.

[0033] (2) The freeze-dried collagen fibers provided by the present invention have good anti-skin aging ability when used as a cosmetic ingredient on the skin. DETAILED DESCRIPTION

[0034] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0035] Aconite polysaccharide: Sichuan Weikeqi Biotechnology Co., Ltd.

[0036] Danshen polysaccharide: Shaanxi Lanhe Biotechnology Co., Ltd.

[0037] Scallop mantle: Dalian Coral Island Seafood Development Co., Ltd.

[0038] Collagen fiber: Shaanxi Haibo Biotechnology Co., Ltd.

[0039] Hericium erinaceus polysaccharide: Sichuan Weikeqi Biotechnology Co., Ltd.

[0040] Tremella polysaccharide: Sichuan Weikeqi Biotechnology Co., Ltd.

[0041] Mantle of the Japanese scallop: Dalian Coral Island Seafood Development Co., Ltd.

[0042] Example 1

[0043] A freeze-drying method for improving the storage stability of collagen fibers, calculated by weight, comprises the following steps:

[0044] W1. The collagen fibers were dissolved in an acetic acid-sodium acetate buffer solution having a molar concentration of 0.4 mol / L to prepare a collagen fiber solution having a mass volume concentration of 5 mg / mL;

[0045] W2. Mix 2 parts of collagen fiber solution with 1 part of oxidized hydroxypropyl methylcellulose and continue stirring for 30 minutes to obtain a modified collagen fiber solution;

[0046] The preparation method of oxidized hydroxypropyl methylcellulose comprises the following steps:

[0047] X1. Dissolve 20 parts of hydroxypropyl methylcellulose in 100 parts of deionized water, stir until completely dissolved, add 2 parts of NaIO4, mix and continue stirring for 5 minutes, then adjust the pH value to 2.0 with a H2SO4 solution with a molar concentration of 0.8 mol / L, react at 25°C in the dark for 3 hours, then perform light-shielding dialysis treatment, and finally freeze-dry to obtain oxidized hydroxypropyl methylcellulose.

[0048] W3. Mix 3 parts of aconite polysaccharide, 1 part of salvia miltiorrhiza polysaccharide, 2 parts of scallop mantle polypeptide and 15 parts of deionized water to obtain a freeze-dried stabilizer;

[0049] Calculated by mass, the preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps:

[0050] Y1. After washing 100 parts of the mantle of Chlamys farreri with deionized water, soaking them in 10% n-butanol, extracting them at a temperature of 4 ° C for 60 min to remove fatty substances, washing them with distilled water until neutral, mixing them with 500 parts of deionized water and mashing them to obtain a homogenate of the mantle of Chlamys farreri;

[0051] Y2. Add 1 part of papain to 100 parts of Chlamys farreri mantle homogenate, perform enzymatic hydrolysis at 50°C for 80 minutes, and then perform ultrafiltration, inactivate the enzyme and dry the filtrate to obtain Chlamys farreri mantle polypeptide.

[0052] W4. Mix 20 parts of the modified collagen fiber solution with 5 parts of the freeze-drying stabilizer, continue stirring for 3 minutes, and then freeze-dry to obtain freeze-dried collagen fibers.

[0053] The freeze-drying process comprises the following steps:

[0054] Z1. Prefreeze at -50°C for 1h;

[0055] Z2. Sublimation for 12h at a vacuum of 8Pa and a temperature of -40°C;

[0056] Z3. Continue the desorption and drying for 1 hour under the conditions of pressure of 12 Pa and temperature of 22°C to obtain freeze-dried collagen fibers.

[0057] Example 2

[0058] A freeze-drying method for improving the storage stability of collagen fibers, calculated by weight, comprises the following steps:

[0059] W1. The collagen fibers were dissolved in an acetic acid-sodium acetate buffer solution having a molar concentration of 0.5 mol / L to prepare a collagen fiber solution having a mass volume concentration of 6 mg / mL;

[0060] W2. Mix 3 parts of collagen fiber solution with 2 parts of oxidized hydroxypropyl methylcellulose and continue stirring for 40 minutes to obtain a modified collagen fiber solution;

[0061] The preparation method of oxidized hydroxypropyl methylcellulose comprises the following steps:

[0062] X1. Dissolve 25 parts of hydroxypropyl methylcellulose in 110 parts of deionized water, stir until completely dissolved, add 3 parts of NaIO4, mix and continue stirring for 7 minutes, then adjust the pH value to 2.5 with a H2SO4 solution with a molar concentration of 1.0 mol / L, react at 25°C in the dark for 4 hours, then perform light-shielding dialysis treatment, and finally freeze-dry to obtain oxidized hydroxypropyl methylcellulose.

[0063] W3. Mix 4 parts of aconite polysaccharide, 2 parts of salvia miltiorrhiza polysaccharide, 3 parts of scallop mantle polypeptide and 17 parts of deionized water to obtain a freeze-dried stabilizer;

[0064] Calculated by mass, the preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps:

[0065] Y1. After washing 110 parts of the mantle of Chlamys farreri with deionized water, soaking them in 10% n-butanol, extracting them at a temperature of 4 ° C for 60 min to remove fatty substances, washing them with distilled water until neutral, mixing them with 550 parts of deionized water and mashing them to obtain a homogenate of the mantle of Chlamys farreri;

[0066] Y2. Add 1 part of papain to 110 parts of Chlamys farreri mantle homogenate, perform enzymatic hydrolysis at 55°C for 90 minutes, and then perform ultrafiltration, inactivate the enzyme and dry the filtrate to obtain Chlamys farreri mantle polypeptide.

[0067] W4. Mix 25 parts of the modified collagen fiber solution with 6 parts of the freeze-drying stabilizer, continue stirring for 4 minutes, and then freeze-dry to obtain freeze-dried collagen fibers.

[0068] The freeze-drying process comprises the following steps:

[0069] Z1. Prefreeze at -60°C for 2h;

[0070] Z2. Sublimation for 13h at a vacuum degree of 10Pa and a temperature of -50°C;

[0071] Z3. Continue the desorption and drying for 2 hours under the conditions of pressure of 13Pa and temperature of 25°C to obtain freeze-dried collagen fibers.

[0072] Example 3

[0073] A freeze-drying method for improving the storage stability of collagen fibers, calculated by weight, comprises the following steps:

[0074] W1. The collagen fibers were dissolved in an acetic acid-sodium acetate buffer solution having a molar concentration of 0.6 mol / L to prepare a collagen fiber solution having a mass volume concentration of 7 mg / mL;

[0075] W2. Mix 4 parts of collagen fiber solution with 3 parts of oxidized hydroxypropyl methylcellulose and continue stirring for 50 minutes to obtain a modified collagen fiber solution;

[0076] The preparation method of oxidized hydroxypropyl methylcellulose comprises the following steps:

[0077] X1. Dissolve 30 parts of hydroxypropyl methylcellulose in 120 parts of deionized water, stir until completely dissolved, add 4 parts of NaIO4, mix and continue stirring for 10 minutes, then adjust the pH value to 3.0 with a H2SO4 solution with a molar concentration of 1.2 mol / L, react at 25°C in the dark for 5 hours, then perform light-shielding dialysis treatment, and finally freeze-dry to obtain oxidized hydroxypropyl methylcellulose.

[0078] W3. Mix 5 parts of aconite polysaccharide, 3 parts of salvia miltiorrhiza polysaccharide, 4 parts of scallop mantle polypeptide and 20 parts of deionized water to obtain a freeze-dried stabilizer;

[0079] Calculated by mass, the preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps:

[0080] Y1. After washing 120 parts of the mantle of Chlamys farreri with deionized water, soaking them in 10% n-butanol, extracting them at a temperature of 4 ° C for 60 min to remove fatty substances, washing them with distilled water until neutral, mixing them with 600 parts of deionized water and mashing them to obtain a homogenate of the mantle of Chlamys farreri;

[0081] Y2. Add 2 parts of papain to 120 parts of Chlamys farreri mantle homogenate, perform enzymatic hydrolysis at 60°C for 100 minutes, and then perform ultrafiltration, inactivate the enzyme and dry the filtrate to obtain Chlamys farreri mantle polypeptide.

[0082] W4. Mix 30 parts of the modified collagen fiber solution with 7 parts of the freeze-drying stabilizer, continue stirring for 5 minutes, and then freeze-dry to obtain freeze-dried collagen fibers.

[0083] The freeze-drying process comprises the following steps:

[0084] Z1. Pre-freeze at -70°C for 3h;

[0085] Z2. Sublimation for 15h at a vacuum degree of 12Pa and a temperature of -60°C;

[0086] Z3. Continue the desorption and drying for 3 hours under the conditions of a pressure of 15 Pa and a temperature of 28°C to obtain freeze-dried collagen fibers.

[0087] Comparative Example 1

[0088] The collagen fibers were modified without adding oxidized hydroxypropyl methylcellulose, and the remaining steps were the same as those in Example 3.

[0089] Comparative Example 2

[0090] Without adding aconite polysaccharide, 8 parts of salvia miltiorrhiza polysaccharide, 4 parts of scallop mantle polypeptide and 20 parts of deionized water were mixed to obtain a freeze-dried stabilizer, and the remaining operating steps were referred to Example 3.

[0091] Comparative Example 3

[0092] Without adding Salvia miltiorrhiza polysaccharide, 8 parts of Aconitum polysaccharide, 4 parts of Chlamys farreri mantle polypeptide and 20 parts of deionized water were mixed to obtain a freeze-dried stabilizer. The remaining operating steps were referred to Example 3.

[0093] Comparative Example 4

[0094] Without adding Chlamys farreri mantle polypeptide, 7 parts of Aconitum polysaccharide, 5 parts of Salvia miltiorrhiza polysaccharide and 20 parts of deionized water were mixed to obtain a freeze-dried stabilizer, and the remaining operating steps were referred to Example 3.

[0095] Comparative Example 5

[0096] No freeze-drying stabilizer was added, and the remaining steps were referred to Example 3.

[0097] Comparative Example 6

[0098] No oxidized hydroxypropyl methylcellulose was added to modify the collagen fibers, no freeze-drying stabilizer was added, and the remaining operation steps were referred to Implementation 3.

[0099] Comparative Example 7

[0100] A freeze-drying method for improving the storage stability of collagen fibers, calculated by weight, comprises the following steps:

[0101] W1. The collagen fibers were dissolved in an acetic acid-sodium acetate buffer solution having a molar concentration of 0.6 mol / L to prepare a collagen fiber solution having a mass volume concentration of 7 mg / mL;

[0102] W2. Mix 4 parts of collagen fiber solution with 3 parts of oxidized hydroxypropyl methylcellulose and continue stirring for 50 minutes to obtain a modified collagen fiber solution;

[0103] The preparation method of oxidized hydroxypropyl methylcellulose comprises the following steps:

[0104] X1. Dissolve 30 parts of hydroxypropyl methylcellulose in 120 parts of deionized water, stir until completely dissolved, add 1 part of NaIO4, mix and continue stirring for 10 minutes, then adjust the pH value to 3.0 with a H2SO4 solution with a molar concentration of 1.2 mol / L, react at 25°C in the dark for 5 hours, then perform light-shielding dialysis treatment, and finally freeze-dry to obtain oxidized hydroxypropyl methylcellulose.

[0105] W3. Mix 5 parts of aconite polysaccharide, 3 parts of salvia miltiorrhiza polysaccharide, 4 parts of scallop mantle polypeptide and 20 parts of deionized water to obtain a freeze-dried stabilizer;

[0106] Calculated by mass, the preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps:

[0107] Y1. After washing 120 parts of the mantle of Chlamys farreri with deionized water, soaking them in 10% n-butanol, extracting them at a temperature of 4 ° C for 60 min to remove fatty substances, washing them with distilled water until neutral, mixing them with 600 parts of deionized water and mashing them to obtain a homogenate of the mantle of Chlamys farreri;

[0108] Y2. Add 2 parts of papain to 120 parts of Chlamys farreri mantle homogenate, perform enzymatic hydrolysis at 60°C for 100 minutes, and then perform ultrafiltration, inactivate the enzyme and dry the filtrate to obtain Chlamys farreri mantle polypeptide.

[0109] W4. Mix 30 parts of the modified collagen fiber solution with 7 parts of the freeze-drying stabilizer, continue stirring for 5 minutes, and then freeze-dry to obtain freeze-dried collagen fibers.

[0110] The freeze-drying process comprises the following steps:

[0111] Z1. Pre-freeze at -70°C for 3h;

[0112] Z2. Sublimation for 15h at a vacuum degree of 12Pa and a temperature of -60°C;

[0113] Z3. Continue the desorption and drying for 3 hours under the conditions of a pressure of 15 Pa and a temperature of 28°C to obtain freeze-dried collagen fibers.

[0114] Comparative Example 8

[0115] A freeze-drying method for improving the storage stability of collagen fibers, calculated by weight, comprises the following steps:

[0116] W1. The collagen fibers were dissolved in an acetic acid-sodium acetate buffer solution having a molar concentration of 0.6 mol / L to prepare a collagen fiber solution having a mass volume concentration of 7 mg / mL;

[0117] W2. Mix 4 parts of collagen fiber solution with 3 parts of oxidized hydroxypropyl methylcellulose and continue stirring for 50 minutes to obtain a modified collagen fiber solution;

[0118] The preparation method of oxidized hydroxypropyl methylcellulose comprises the following steps:

[0119] X1. Dissolve 30 parts of hydroxypropyl methylcellulose in 120 parts of deionized water, stir until completely dissolved, add 5 parts of NaIO4, mix and continue stirring for 10 minutes, then adjust the pH value to 3.0 with a H2SO4 solution with a molar concentration of 1.2 mol / L, react at 25°C in the dark for 5 hours, then perform light-shielding dialysis treatment, and finally freeze-dry to obtain oxidized hydroxypropyl methylcellulose.

[0120] W3. Mix 5 parts of aconite polysaccharide, 3 parts of salvia miltiorrhiza polysaccharide, 4 parts of scallop mantle polypeptide and 20 parts of deionized water to obtain a freeze-dried stabilizer;

[0121] Calculated by mass, the preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps:

[0122] Y1. After washing 120 parts of the mantle of Chlamys farreri with deionized water, soaking them in 10% n-butanol, extracting them at a temperature of 4 ° C for 60 min to remove fatty substances, washing them with distilled water until neutral, mixing them with 600 parts of deionized water and mashing them to obtain a homogenate of the mantle of Chlamys farreri;

[0123] Y2. Add 2 parts of papain to 120 parts of Chlamys farreri mantle homogenate, perform enzymatic hydrolysis at 60°C for 100 minutes, and then perform ultrafiltration, inactivate the enzyme and dry the filtrate to obtain Chlamys farreri mantle polypeptide.

[0124] W4. Mix 30 parts of the modified collagen fiber solution with 7 parts of the freeze-drying stabilizer, continue stirring for 5 minutes, and then freeze-dry to obtain freeze-dried collagen fibers.

[0125] The freeze-drying process comprises the following steps:

[0126] Z1. Pre-freeze at -70°C for 3h;

[0127] Z2. Sublimation for 15h at a vacuum degree of 12Pa and a temperature of -60°C;

[0128] Z3. Continue the desorption and drying for 3 hours under the conditions of a pressure of 15 Pa and a temperature of 28°C to obtain freeze-dried collagen fibers.

[0129] Comparative Example 9

[0130] The polysaccharide from Aconitum carmichaelii was replaced with polysaccharide from Hericium erinaceus, and the remaining steps were referred to Example 3.

[0131] Comparative Example 10

[0132] Replace Salvia miltiorrhiza polysaccharide with Tremella fuciformis polysaccharide, and refer to Example 3 for the remaining steps.

[0133] Comparative Example 11

[0134] The Chlamys farreri mantle is replaced with the Yesso scallop mantle, that is, the obtained polypeptide is the Yesso scallop mantle polypeptide, and the remaining operating steps refer to Example 3.

[0135] Performance Testing

[0136] 1. Test on freeze-dried stability of collagen fibers

[0137] The collagen fibers prepared in Examples 1-3 and Comparative Examples 1-8 were freeze-dried and mixed with deionized water to prepare a 5% collagen fiber solution. 10 ml of the collagen fiber solution was mixed with an equal volume of water, incubated at 92° C. for 0.5 h, and then stored at 25° C. for 18 h. The particle size change and PDI value of the collagen fiber solution were measured; freeze-thaw stability: 10 ml of the collagen solution was mixed with an equal volume of water, incubated at -25° C. for 12 h, and then thawed at 35° C. for 3 h. The particle size change of the collagen solution was measured. The measurement was repeated 3 times, and the average value was taken and recorded as shown in Table 1 below;

[0138] After the collagen fibers prepared in Examples 1-3 and Comparative Examples 1-8 were freeze-dried and stored for 12 months, the above test was repeated, and the records are shown in Table 1.

[0139] 2. Anti-skin aging test of freeze-dried collagen fibers

[0140] Anti-aging detection method: Skin aging is mainly related to elastic fibers, which are composed of elastin and microfibrils. Elastic fibers will be decomposed by elastase, causing collagen loss and thus leading to skin aging. Therefore, the purpose of anti-aging effect detection can be achieved by measuring the inhibition rate of elastase by freeze-dried collagen fibers.

[0141] The specific operation is to mix 100 μL of 0.2 mol / L tris-HCl buffer, 25 μL of 10 mmol / L N-methoxysuccinyl-alanine-proline-valine-4-nitroaniline and 30 mg of collagen fiber freeze-dried, let it stand at 25°C for 10 minutes, add 25 μL of 0.3 U / mL elastase and continue to stand for 10 minutes, and finally detect the OD value at 410 nm, and use 0.3 mL of deionized water instead of collagen fiber freeze-dried as a blank control. The results are recorded in Table 1 below.

[0142] The inhibition rate of elastase by freeze-dried collagen fibers (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group × 100%.

[0143] 3. Antioxidant properties test of freeze-dried collagen fibers

[0144] 30 mg of lyophilized collagen fibers were placed in a test tube, 5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol was added, and the mixture was shaken for 10 seconds, and then reacted at room temperature for 20 minutes. After the reaction, the OD value of the reaction mixture was measured at 517 nm, and 0.3 mL of deionized water was used instead of lyophilized collagen fibers as a blank control. The results are recorded in Table 1 below.

[0145] The scavenging rate of DPPH free radicals by freeze-dried collagen fibers (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group × 100%.

[0146] Table 1 Performance test results

[0147]

[0148] As can be seen from Table 1, the particle size change values ​​of the freeze-dried collagen fibers prepared in Examples 1-3 are smaller than those in Comparative Examples 1-11, and the particle size change values ​​of the freeze-dried collagen fibers prepared in Examples 1-3 after storage for 12 months are also smaller than those in Comparative Examples 1-11. In addition, the particle size change values ​​of the freeze-dried collagen fibers prepared in Examples 1-3 after storage for 12 months are not much different from those before storage, while the particle size change values ​​of the freeze-dried collagen fibers prepared in Comparative Examples 1-11 after storage for 12 months are larger than those before storage, indicating that the collagen fibers in Examples 1-3 have a smaller change range, indicating that the freeze-dried collagen fibers prepared in Examples 1-3 have better storage stability.

[0149] The particle size change values ​​of the freeze-dried collagen fibers prepared in Comparative Example 1 and the particle size change values ​​after storage for 12 months are larger than those in Example 3. The reason is that the collagen fibers are not modified by using oxidized hydroxypropyl methylcellulose, resulting in the structure of the freeze-dried collagen fibers being less stable than the structure of the modified freeze-dried collagen fibers prepared in Example 3.

[0150] The particle size change values ​​of the freeze-dried collagen fibers prepared in Comparative Examples 2-4 and Comparative Examples 9-11 and the particle size change values ​​after storage for 12 months are larger than those in Example 3. The reason is that a freeze-dried stabilizer composed of specified components was not used, resulting in the freeze-dried stabilizer being unable to form a good coating on the collagen fibers.

[0151] The particle size change values ​​of the freeze-dried collagen fibers obtained in Comparative Example 5 and the particle size change values ​​after storage for 12 months are larger than those in Example 3. The reason is that the structure of the collagen fibers during the freeze-drying process and subsequent storage is not as stable as the freeze-dried collagen fibers obtained in Example 3 without the use of a freeze-drying stabilizer.

[0152] The particle size change values ​​of the freeze-dried collagen fibers obtained in Comparative Example 6 and the particle size change values ​​after storage for 12 months are larger than those in Example 3. The reason is that no freeze-dried stabilizer is used and no oxidized hydroxypropyl methylcellulose is used to modify the collagen fibers, resulting in its structure being less stable than the freeze-dried collagen fibers obtained in Example 3.

[0153] The particle size change values ​​of the freeze-dried collagen fibers prepared in Comparative Examples 7-8 and the particle size change values ​​after storage for 12 months are larger than those in Example 3. The reason is that the collagen fibers are modified by using oxidized hydroxypropyl methylcellulose with a degree of oxidation lower or higher than the specified degree, which results in the failure to achieve the effect of improving the stability of the collagen fibers.

[0154] As can be seen from Table 1, the freeze-dried collagen fibers prepared in Examples 1-3 have good elastase inhibition rate and DPPH free radical scavenging rate, and are higher than those in Comparative Examples 1-11, indicating that the freeze-dried collagen fibers prepared by modifying the collagen fibers using a freeze-dried stabilizer composed of specified components and oxidized hydroxypropyl methylcellulose in a specified oxidation degree range have a certain anti-aging effect on the skin.

[0155] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A freeze-drying method for improving the storage stability of collagen fibers, characterized in that: Calculated by weight, it includes the following steps: W1. Dissolving collagen fibers in acetic acid-sodium acetate buffer to prepare a collagen fiber solution; W2. Mix 2-4 parts of the collagen fiber solution with 1-3 parts of oxidized hydroxypropyl methylcellulose and continue stirring for 30-50 min to obtain a modified collagen fiber solution; W3. 3-5 parts of aconite polysaccharide, 1-3 parts of salvia miltiorrhiza polysaccharide, 2-4 parts of scallop mantle polypeptide and 15-20 parts of deionized water were mixed to obtain a freeze-dried stabilizer; W4. Mix 20-30 parts of the modified collagen fiber solution with 5-7 parts of a freeze-dried stabilizer, continue stirring for 3-5 minutes, and then freeze-dry to obtain freeze-dried collagen fibers; The method for preparing oxidized hydroxypropyl methylcellulose described in step W2 comprises the following steps: X1. Dissolve 20-30 parts of hydroxypropyl methylcellulose in 100-120 parts of deionized water, stir until completely dissolved, add 2-4 parts of NaIO4, mix and continue stirring for 5-10 minutes, adjust the pH value, protect from light, dialyze in dark, and freeze-dry to obtain oxidized hydroxypropyl methylcellulose; The pH value adjustment in step X1 refers to adjusting the pH value to 2.0-3.0; The preparation of the Chlamys farreri mantle polypeptide described in step W3 comprises the following steps: Y1. After washing 100-120 parts of the mantle of Chlamys farreri with deionized water, immerse in 10% n-butanol, extract for 60 min at 4 ° C to remove fatty substances, wash with distilled water to neutrality, mix with 500-600 parts of deionized water and mash to obtain a homogenate of the mantle of Chlamys farreri; Y2. The scallop mantle homogenate was enzymatically treated and then ultrafiltered, the filtrate was inactivated and dried to obtain the scallop mantle polypeptide; Calculated by weight, the enzymatic hydrolysis treatment described in step Y2 refers to mixing 100-120 parts of Chlamys farreri mantle homogenate with 1-2 parts of papain and enzymolyzing them at a temperature of 50-60° C. for 80-100 minutes.

2. A freeze-drying method for improving the storage stability of collagen fibers according to claim 1, characterized in that: The molar concentration of the acetic acid-sodium acetate buffer described in step W1 is 0.4-0.6 mol / L.

3. A freeze-drying method for improving the storage stability of collagen fibers according to claim 1, characterized in that: The mass volume concentration of the collagen fiber solution described in step W1 is 5-7 mg / mL.

4. A freeze-drying method for improving the storage stability of collagen fibers according to claim 1, characterized in that: The freeze-drying process described in step W4 comprises the following steps: Z1. Pre-freeze at a temperature of ≤-50°C for 1-3 hours; Z2. Sublime for 12-15h at a vacuum degree of 8-12Pa and a temperature of ≤-40°C; Z3. Continue to dry for 1-3 hours at a pressure of 12-15 Pa and a temperature of 22-28°C to obtain freeze-dried collagen fibers.

5. A freeze-dried collagen fiber obtained by the freeze-drying method according to any one of claims 1 to 4.

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