Preparation method and application of triple helical collagen and Antarctic glycoprotein composition

Abstract

The present invention provides a preparation method and application of a triple helical collagen and Antarctic glycoprotein composition, belonging to the field of biotechnology and cosmetology. The present invention provides a preparation method of a triple helical collagen and Antarctic glycoprotein composition, comprising mixing MaAFP antifreeze protein, Antarctic krill glycoprotein and triple helical collagen. The triple helical collagen and Antarctic glycoprotein composition prepared by the preparation method provided by the present invention has a protective effect on UVB-induced Hacat cells, can effectively inhibit inflammatory cytokines and cell oxidative stress levels, and has a good application prospect in the preparation of anti-aging products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and cosmetology, and specifically relates to a preparation method and application of a triple helical collagen and Antarctic glycoprotein composition. Background Art

[0002] Skin aging, also known as skin aging, is an inevitable stage in the process of biological life activities. It is a gradual, genetically influenced, irreversible decline of the complex morphological structure and physiological function of the whole body that occurs with age after the organism matures. Skin aging refers to the process of skin external morphology, structure, and function decline under the influence of exogenous or endogenous factors. Endogenous aging refers to the natural aging of the human body, and exogenous aging mainly refers to photoaging. The endogenous aging and exogenous aging of the skin are interrelated and affect each other. The clinical manifestations of skin aging are dry, rough, thinning, reduced elasticity, loss of luster, loose skin, atrophy, wrinkles of varying degrees, pigmentation, capillary dilation, and other manifestations, as well as the disorder of the skin's physiological structure and functional degradation. Therefore, how to alleviate the aging process of the skin has become a hot topic in the field of anti-aging research.

[0003] The triple helix structure of collagen is a right-handed superhelical rod-shaped structure formed by three independent, left-handed helical α-peptide chains around the same central axis, connected and entangled by hydrogen bonds formed between the chains. This structure is quite stable, and glycine, proline and hydroxyproline play an important role in maintaining its structural stability. Among them, there is a glycine every two amino acids, and the bend formed by glycine fills the inside of the helix. The proline and hydroxyproline chains form smooth bends around the helix, thus forming a tight and stable three-dimensional helical structure. In the skin, collagen is the main component of the dermis and helps maintain the elasticity and water retention of the skin. With age, the content of collagen gradually decreases, causing the skin to lose elasticity and luster, and wrinkles and sagging appear. As an active polymer material, collagen is very easy to degrade in normal and high temperature environments, and its shelf life is shortened. Therefore, how to maximize the activity of collagen in an environment with elevated temperatures has become a hot topic in the field of biotechnology and anti-aging research.

[0004] In the extremely cold Antarctic glacial land, various prokaryotes live in the barren soil, rocky terrain, and countless lakes. This extreme low temperature environment does not become a survival obstacle for microbial communities. Psychrophiles and psychrotrophs are two extreme microorganisms that love cold. Similarly, in the extremely cold and oxygen-rich Antarctic waters, Antarctic krill has become a specialized species of the unique Antarctic ecosystem. Among them, antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are collectively referred to as Antarctic glycoproteins, which are one of the most important characteristics of extreme microorganisms and Antarctic fish and shrimp to adapt to the extreme cold environment. Antarctic glycoprotein components have been found in the extracellular products of Pseudoalteromonas and Antarctic krill extracts. Antarctic glycoproteins, also known as ice binding proteins (IBPs), can bind to ice crystals or low-temperature sensitive substances such as cell membranes and enzymes at low temperatures, inhibiting the formation and growth of ice crystals, while stabilizing low-temperature sensitive substances. Its activity of inhibiting the growth of ice crystals is called recrystallization inhibition activity (RI). Antarctic glycoprotein is a rare ingredient that can protect the skin in severe cold environments. It can prevent freezing, resist drying, and protect the skin from damage. In addition, Antarctic glycoprotein can lock the moisture on the surface of the skin and absorb moisture from the air; at the same time, it can increase the amount of collagen and elastin in the skin, stimulate the healthy growth of the skin from the inside out, and has the effect of promoting wound healing. In addition, Antarctic protein can stimulate cell vitality and form a silky protective film on the surface of the skin to prevent and repair skin damage caused by heavy water loss. Make the skin firm, elastic and shiny.

[0005] Although triple helical collagen and Antarctic glycoprotein have shown good application prospects in the field of anti-aging technology, the existing preparation processes for obtaining triple helical collagen and Antarctic glycoprotein cannot meet the actual application needs. In addition, there is currently no study on combining triple helical collagen with Antarctic glycoprotein for anti-aging research. Therefore, a preparation method of a triple helical collagen and Antarctic glycoprotein composition is provided, which has a good reference value for the research of anti-aging products. Summary of the invention

[0006] In view of the above-mentioned deficiencies, the present invention provides a preparation method and application of a triple-helical collagen and Antarctic glycoprotein composition. The present invention provides a preparation method of a triple-helical collagen and Antarctic glycoprotein composition, comprising mixing MaAFP antifreeze protein, Antarctic krill glycoprotein and triple-helical collagen. The triple-helical collagen and Antarctic glycoprotein composition prepared by the preparation method provided by the present invention has a protective effect on UVB-induced Hacat cells, can effectively inhibit inflammatory cytokines and cell oxidative stress levels, and has a good application prospect in the preparation of anti-aging products.

[0007] The technical solution of the present invention includes:

[0008] The preparation method of the triple helical collagen and Antarctic glycoprotein composition is characterized in that the preparation method comprises:

[0009] S1. Add freeze-dried Antarctic krill powder to distilled water, adjust the pH, add enzyme mixture, inactivate after enzymolysis, and collect the supernatant;

[0010] S2, the supernatant was dialyzed and concentrated, ethanol was added, and the mixture was allowed to stand overnight, and the precipitate was collected and freeze-dried to obtain the crude product of Antarctic krill glycoprotein;

[0011] S3, dissolving the crude Antarctic krill glycoprotein in distilled water to obtain a crude Antarctic krill glycoprotein solution, adding MaAFP antifreeze protein, adsorbing collagen with a cation exchange medium, and then eluting and separating, collecting the eluate, which is the Antarctic glycoprotein solution;

[0012] S4. The Antarctic glycoprotein solution and the triple-helical collagen hydrogel solution are uniformly mixed in a volume ratio of 0.4-1.5:1, and freeze-dried to obtain a triple-helical collagen and Antarctic glycoprotein composition.

[0013] Specifically, the preparation method of the Antarctic krill freeze-dried powder described in step S1 includes: washing the Antarctic krill, freeze-drying, and sieving to obtain uniform Antarctic krill freeze-dried powder.

[0014] Preferably, the sieving is through a 10-20 mesh sieve.

[0015] Specifically, the ratio of the Antarctic krill freeze-dried powder to distilled water in step S1 is 1:40-60 g / mL.

[0016] Preferably, the ratio of the Antarctic krill freeze-dried powder to distilled water in step S1 is 1:50 g / mL.

[0017] Preferably, the pH in step S1 is 6.0-7.0.

[0018] Specifically, the enzyme mixture in step S1 consists of cellulase, papain and neutral protease.

[0019] Preferably, the weight ratio of cellulase, papain and neutral protease in step S1 is 2:1.5:1.

[0020] Specifically, the temperature of the enzymatic hydrolysis in step S1 is 50-60°C.

[0021] Preferably, the temperature of the enzymatic hydrolysis in step S1 is 50-51°C, 51-52°C, 52-53°C, 53-54°C or 54-55°C.

[0022] Specifically, the enzymatic hydrolysis time in step S1 is 2-4 hours.

[0023] Preferably, the enzymatic hydrolysis time in step S1 is 2-3 h or 3-4 h.

[0024] Specifically, the inactivation temperature in step S1 is 90-100°C.

[0025] Preferably, the inactivation temperature in step S1 is 90-91°C, 91-92°C, 92-93°C, 93-94°C, 94-95°C, 95-96°C, 96-97°C, 97-98°C, 97-98°C, 98-99°C, 99-100°C.

[0026] Specifically, the inactivation time in step S1 is 15-30 minutes.

[0027] Preferably, the inactivation time described in step S1 is 15-16min, 16-17min, 17-18min, 18-19min, 19-20min, 20-21min, 21-22min, 22-23min, 23-24min, 24-25min.

[0028] Specifically, the method for collecting the supernatant in step S1 is: collecting the supernatant by centrifugation.

[0029] Preferably, the centrifugation condition is: 3000-8000 rpm for 10-30 min.

[0030] Specifically, the dialysis described in step S2 includes the following steps: putting the supernatant into a dialysis bag and performing dialysis using distilled water as the dialysis medium.

[0031] Preferably, the molecular cut-off of the dialysis bag is 10-15 kDa, which may be 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa or 15 kDa.

[0032] Preferably, the concentration in step S2 is to concentrate the dialysate to 1 / 5 of its volume.

[0033] Specifically, the final volume concentration of ethanol described in step S2 is 70%-80% v / v.

[0034] Preferably, the final volume concentration of ethanol described in step S2 is 75% v / v.

[0035] Specifically, the method for collecting the precipitate in step S2 is: collecting the precipitate by centrifugation.

[0036] Preferably, the centrifugation condition is 3000-8000 rpm for 10-30 min.

[0037] Specifically, the amino acid sequence of the MaAFP antifreeze protein described in step S3 is shown in SEQ ID NO.1.

[0038] SEQ ID NO.1:

[0039] GSGNDQIVIDNDVEKWLDAGDGDNSIYVGDDVNRNNSAGIKTGSGDDDIFVKDNVDSTIQTGGGNDRVQIGYDLGNGYHSAHINLGDGDNNLIIKNDVNNYSTVHSGSG DDVVSIGDDVRYDADIQLGDGNDRLTIGDKIEKEVSINLGSGNDVLVVGGKVSDEAWVDGGSGSDSVWFESYSRSDYNSDKDGIKSRFANFENFKFSDGTVIGNASAF.

[0040] Specifically, the method for preparing the MaAFP antifreeze protein in step S3 comprises:

[0041] ① Inoculate 3-5% of Pseudoalteromonas into a culture medium containing glutamic acid, proline, vitamin B1 and sucrose, and culture to obtain a bacterial solution;

[0042] ② After centrifugation of the bacterial solution, collect the supernatant, filter, and collect the filtrate;

[0043] ③ The filtrate is ultrafiltered and dialyzed, the retentate is collected, and the powder is obtained after freeze-drying;

[0044] ④ After the powder is dissolved, the cation exchange medium adsorbs the protein and then elutes and separates it, and the eluate is collected to obtain the MaAFP antifreeze protein.

[0045] Preferably, the culture medium described in step S3① is a culture medium containing final concentrations of 0.2-0.6 g / L glutamic acid, 0.15-0.45 g / L proline, 15-30 mg / L vitamin B1, and 1-3 g / L sucrose.

[0046] Preferably, the culture medium described in step S3① is 2216E liquid culture medium.

[0047] Preferably, the Pseudoalteromonas described in step S3① is SHMCC JCM18779.

[0048] Preferably, the inoculation amount of Pseudoalteromonas described in step S3① includes 3%-4% or 4%-5%.

[0049] Preferably, the culture conditions in step S3① are 15-20°C and 200-500 r / min for 5-7 days.

[0050] Preferably, the centrifugation in step S3② is performed at 3000-8000 rpm for 10-15 min.

[0051] Preferably, the filtration in step S3② is performed by filtering with a 0.22 μm filter membrane.

[0052] Preferably, the ultrafiltration in step S3③ is: using an ultrafiltration membrane of 15-30 kDa and performing ultrafiltration at a pressure of 5-8 bar.

[0053] Preferably, the dialysis described in step S3③ comprises the following steps: placing the ultrafiltrate into a dialysis bag and performing dialysis using PBS with a pH of 7.4 as a dialysis medium.

[0054] Further preferably, the molecular cutoff of the dialysis bag used for dialysis in step S3③ is 10-15 kDa, which can be 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa or 15 kDa.

[0055] Further preferably, the dialysis condition in step S3③ is dialysis at 20-25°C for 24-48h.

[0056] Preferably, the solution used for dissolving in step S3④ is PBS with a pH of 7.4.

[0057] Preferably, the cation exchange medium described in step S3④ is selected from one or more of DEAE (N,N-diethylaminoethyl) Sepharose Fast Flow, Q (N,N-diethylamino-2-hydroxypropyl), SP (sulfopropyl)-Sephadex dextran gel, and Sephadex G-100; more preferably Sephadex G-100.

[0058] Preferably, the solution used for elution in step S3④ is PBS with a pH of 7.4.

[0059] Specifically, the cation exchange medium in step S3 is selected from one or more of DEAE (N, N-diethylaminoethyl) Sepharose Fast Flow, Q (N, N-diethylamino-2-hydroxypropyl), and SP (sulfopropyl)-Sephadex dextran gel;

[0060] Preferably, the cation exchange medium in step S3 is SP (sulfopropyl)-Sephadex dextran gel.

[0061] Preferably, the solution used for elution in step S3 is a 10 mM Tris-HCl aqueous solution containing 0.5-0.9 wt % sodium salt.

[0062] Specifically, the volume ratio of the MaAFP antifreeze protein to the crude Antarctic krill glycoprotein solution in step S3 is 0.3-1.0:1.

[0063] More specifically, the volume ratio of the MaAFP antifreeze protein to the crude Antarctic krill glycoprotein solution in step S3 is 0.3-0.4:1, 0.4-0.5:1, 0.5-0.6:1, 0.6-0.7:1, 0.7-0.8:1, 0.8-0.9:1 or 0.9-1.0:1.

[0064] Preferably, the volume ratio of the MaAFP antifreeze protein to the crude Antarctic krill glycoprotein solution in step S3 is 0.4-0.5:1, 0.5-0.6:1 or 0.6-0.7:1.

[0065] Further specifically, the Antarctic glycoprotein solution and the triple-helix collagen hydrogel solution described in step S4 are evenly mixed according to the volume ratio of 0.4-0.5:1, 0.5-0.6:1, 0.6-0.7:1, 0.7-0.8:1, 0.8-0.9:1, 0.9-1.0:1, 1.0-1.1:1, 1.1-1.2:1, 1.2-1.3:1, 1.3-1.4:1, and 1.4-1.5:1.

[0066] Preferably, the Antarctic glycoprotein solution and the triple-helix collagen hydrogel solution in step S4 are uniformly mixed in a volume ratio of 1.2-1.3:1.

[0067] Specifically, the preparation method of the triple-helix collagen hydrogel solution described in step S4 includes:

[0068] (1) taking pig skin, soaking it in a trypsin solution containing EDTA, and rinsing it; soaking it in a detergent solution, shaking it, and rinsing it; soaking it in a neutral protease solution, and rinsing it; soaking it in a detergent solution, shaking it, and rinsing it; repeating the above steps 1-3 times to obtain a pig skin free of immunogenicity;

[0069] (2) Soaking the de-immunized pig skin in an α-1,3-galactosidase solution and washing to obtain a precipitate A; adding a filter aid solution to the precipitate A, stirring, filtering, and collecting the supernatant; concentrating the supernatant, adsorbing collagen through a cation exchange medium, and then eluting and separating to obtain an eluate;

[0070] (3) adding a modified polysaccharide at a concentration of 10-40 mg / mL to the eluate of step (2), wherein the mass ratio of the eluate to the modified polysaccharide is 5:1-1:3, to perform modification and obtain a hydrogel;

[0071] (4) sterilizing and filtering the hydrogel obtained in step (3) using a 0.22 μm filter membrane to obtain a triple-helix collagen hydrogel solution;

[0072] More specifically, the descaling agent solution in step S4 (1) is a sodium phosphate buffer solution with a pH of 6.0-8.0 containing 0.05-0.15 wt % of an ionic descaling agent; the ionic descaling agent is a mixture of sodium dodecyl sulfate and Triton X-200 in a mass ratio of 4:6;

[0073] Preferably, the sodium phosphate buffer is a sodium phosphate buffer with a pH of 7.2.

[0074] More specifically, the concentration of EDTA in the trypsin solution containing EDTA in step S4 (1) is 0.01wt%-0.04wt%; the concentration of trypsin is 0.25wt%-0.35wt%.

[0075] Preferably, the concentration of EDTA in the trypsin solution containing EDTA in step S4 (1) is 0.025 wt %; the concentration of trypsin is 0.3 wt %.

[0076] More specifically, the soaking temperature of the trypsin solution containing EDTA in step S4 (1) is 30-37° C., preferably 37° C., and the soaking time is 12-24 h, preferably 24 h.

[0077] Preferably, the temperature of soaking in the descaling solution in step S4 (1) is 24-32°C, preferably 30°C; and the time is 12-24h, preferably 20h.

[0078] More specifically, the concentration of the neutral protease solution in step S4 (1) is 500-700 U / L.

[0079] More specifically, the soaking temperature of the neutral protease solution in step S4 (1) is 28-35°C, preferably 35°C, and the soaking time is 12-24h, preferably 24h.

[0080] More specifically, the concentration of the α-1,3-galactosidase solution in step S4 (2) is 10-20 μg / mL.

[0081] Preferably, the soaking temperature of the α-1,3-galactosidase solution in step S4 (2) is 24-37° C., preferably 37° C., and the soaking time is 1-4 h, preferably 3 h.

[0082] More specifically, the filter aid in step S4 (2) is selected from one or more of diatomaceous earth, perlite, cellulose, asbestos, magnesium oxide and activated carbon; preferably diatomaceous earth.

[0083] More specifically, the mass ratio of the filter aid to the precipitate A in step S4 (2) is 0.5-3:1.

[0084] More specifically, the cation exchange medium in step S4 (2) is selected from one or more of DEAE (N, N-diethylaminoethyl) Sepharose Fast Flow, Q (N, N-diethylamino 2-hydroxypropyl), and SP (sulfopropyl)-Sephadex dextran gel; preferably, SP (sulfopropyl)-Sephadex dextran gel.

[0085] More specifically, the elution solution for elution separation in step S4 (2) is a 10 mM Tris-HCl aqueous solution containing 0.5-0.9 wt % sodium salt.

[0086] The modified polysaccharide described in step S4 (3) is methacrylic anhydride-modified hyaluronic acid, oat β-glucan and aldehyde-modified hyaluronic acid in a mass ratio of 2:5:3.

[0087] The preparation method of the methacrylic anhydride (MA) modified hyaluronic acid is as follows:

[0088] Weigh 1g of hyaluronic acid (HA) powder and dissolve it in 100mL of deionized water, stir thoroughly to make a 1% HA solution. Then, slowly add 0.64mL of MA to the HA solution, adjust and maintain the pH value of the reaction solution at 8.0 while adding, and react for 4h. During the reaction, the pH value of the reaction solution is maintained at 8.0. After the reaction, the reaction solution is repeatedly washed with ethanol to obtain a flocculent precipitate, centrifuged, and then the white precipitate obtained by centrifugation is redissolved in deionized water and dialyzed with a dialysis bag (molecular weight cutoff 8000-14000kDa) for 5 days. After the end, it is placed in a vacuum freeze dryer to dry to obtain a modified sponge-like HAMA.

[0089] The preparation method of the aldehyde-modified hyaluronic acid is as follows:

[0090] Hyaluronic acid (HA) with a molecular weight of 60KDa and sodium periodate were dissolved in deionized water respectively. Then the sodium periodate solution was slowly dripped into the HA solution and reacted for 3 hours at room temperature and in the dark. After the reaction, it was placed in a dialysis bag (molecular weight cutoff 8000-14000kDa) and dialyzed for 5 days. After the reaction, it was placed in a vacuum freeze dryer to dry to obtain spongy aldehyde-modified hyaluronic acid.

[0091] The hyaluronic acid is moistuHA produced by Dongying Foster Bioengineering Co., Ltd., and the molecular weight of hyaluronic acid is 20KDa-2000KDa; oat β-glucan (1%) is produced by Guangzhou Beta Health Biotechnology Co., Ltd., with batch number BT2212101005B.

[0092] In a second aspect, the present invention provides a triple-helical collagen and Antarctic glycoprotein composition prepared by the above preparation method.

[0093] In a third aspect, the present invention provides the use of the above-mentioned Antarctic glycoprotein composition in the preparation of anti-aging products; the products include medical devices, medicines or cosmetics.

[0094] In a fourth aspect, the present invention provides an anti-aging cosmetic, comprising the triple-helical collagen and Antarctic glycoprotein composition prepared by the above preparation method or the triple-helical collagen and Antarctic glycoprotein composition.

[0095] Specifically, the anti-aging cosmetics include but are not limited to facial cleansers, cleansing milk, toners, lotions, creams, essences, eye creams, facial masks, foundations, concealers, sunscreens, sunscreen sprays, shampoos, conditioners, shower gels or body lotions.

[0096] Specifically, the cosmetic also includes cosmetically acceptable additives.

[0097] Preferably, the additives include, but are not limited to, one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, and buffers.

[0098] In a fifth aspect, the present invention provides an anti-aging drug, which comprises the triple-helical collagen and Antarctic glycoprotein composition prepared by the above preparation method or the above triple-helical collagen and Antarctic glycoprotein composition.

[0099] Specifically, the anti-aging drug also includes pharmaceutically acceptable excipients.

[0100] Preferably, the excipients include but are not limited to solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0101] In a sixth aspect, the present invention provides an anti-aging medical device, comprising the triple-helical collagen and Antarctic glycoprotein composition prepared by the above-mentioned preparation method or the above-mentioned triple-helical collagen and Antarctic glycoprotein composition.

[0102] Specifically, the medical devices include medical equipment and medical consumables.

[0103] Preferably, the medical devices include but are not limited to: medical sanitary materials and dressings, medical polymer materials products, medical metal materials products, medical optical instruments, instruments and endoscopic equipment.

[0104] The beneficial effects of the present invention are:

[0105] The triple-helix collagen and Antarctic glycoprotein composition prepared by the preparation method provided by the present invention has a protective effect on UVB-induced Hacat cells, can effectively inhibit inflammatory cytokines and cell oxidative stress levels, and is an anti-aging product. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The results of cell survival rate determination; ** in the figure represents a significant difference compared with the blank group, and p < 0.01; *** represents a significant difference compared with the blank group, and p < 0.001; ### represents a significant difference compared with the model group (p < 0.001).

[0107] Figure 2Inflammation and oxidative stress measurement results; A is the expression level of IFN-γ; B is the expression level of IL-6; C is the expression level of MDA; ### represents a significant difference compared with the model group (p < 0.001). DETAILED DESCRIPTION

[0108] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0109] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, it is understood that the reaction is carried out at room temperature.

[0110] The purchase information of all materials of the present invention is shown in Table 1:

[0111] Table 1 Materials used in the present invention

[0112]

[0113] Basic Experimental Example 1 Preparation of Methacrylic Anhydride (MA) Modified Hyaluronic Acid

[0114] Weigh 1g of hyaluronic acid (HA) powder and dissolve it in 100mL of deionized water, stir thoroughly to make a 1% HA solution. Then, slowly add 0.64mL of MA to the HA solution, adjust and maintain the pH value of the reaction solution at 8.0 while adding, and react for 4h. During the reaction, the pH value of the reaction solution is maintained at 8.0. After the reaction, the reaction solution is repeatedly washed with ethanol to obtain a flocculent precipitate, centrifuged, and then the white precipitate obtained by centrifugation is redissolved in deionized water and dialyzed with a dialysis bag (molecular weight cutoff 8000-14000kDa) for 5 days. After the end, it is placed in a vacuum freeze dryer to dry to obtain a modified sponge-like HAMA.

[0115] Basic Experimental Example 2 Preparation of Aldehyde Hyaluronic Acid

[0116] Hyaluronic acid (HA) with a molecular weight of 60KDa and sodium periodate were dissolved in deionized water respectively. Then the sodium periodate solution was slowly dripped into the HA solution and reacted for 3 hours at room temperature and in the dark. After the reaction, it was placed in a dialysis bag (molecular weight cutoff 8000-14000kDa) and dialyzed for 5 days. After the reaction, it was placed in a vacuum freeze dryer to dry to obtain spongy aldehyde-modified hyaluronic acid.

[0117] Example 1 Preparation of triple helical collagen and Antarctic glycoprotein composition

[0118] 1. Preparation of triple-helical collagen hydrogel

[0119] The triple helical collagen hydrogel was prepared according to the method described in patent CN115944545B. The specific method is as follows:

[0120] (1) Pretreatment of pigskin:

[0121] A certain amount of animal pig skin is added to a 0.25wt% trypsin solution containing 0.01wt% EDTA, soaked at 30°C for 24 hours, and rinsed; then soaked in a phosphate buffer (pH 6.0) containing 0.05wt% ion detergent at 24°C for 24 hours and shaken, rinsed, and the pre-treated pig skin is obtained;

[0122] (2) De-immunization of pig skin:

[0123] The pigskin pretreated in step (1) is added to a neutral protease solution with a concentration of 500 U / L and soaked at 28° C. for 24 hours, and then rinsed; then soaked in a phosphate buffer (pH 6.0) containing 0.05 wt% ion detergent at 24° C. for 24 hours and shaken, and rinsed; after repeating step (1) and step (2) 1-3 times, the immunogenic pigskin is obtained;

[0124] The de-immunized pig skin obtained after the treatment was soaked in a 10 μg / mL α-1,3-galactosidase solution at 24°C for 4 hours, and then rinsed to obtain a precipitate A;

[0125] (3) Filter adsorption:

[0126] Add the diatomaceous earth solution to the precipitate A obtained in step (2), stir, filter, and collect the supernatant for later use;

[0127] (4) Separation and purification:

[0128] The supernatant collected in step (3) is concentrated, and then the collagen is adsorbed on SP (sulfopropyl)-Sephadex gel and then eluted and separated using a 10 mM Tris-HCl aqueous solution containing 0.5 wt % sodium salt to obtain an eluate;

[0129] (5) Hydrogel preparation:

[0130] Adding a modified polysaccharide at a concentration of 10 mg / mL to the eluate in step (4), wherein the mass ratio of the eluate to the modified polysaccharide is 1:3, to perform modification and obtain a hydrogel;

[0131] (6) Sterile filtration:

[0132] The hydrogel obtained in step (5) is sterilized and filtered using a 0.22 μm filter membrane to obtain a sterile soluble collagen hydrogel solution.

[0133] The ionic descaling agent in step (1) and step (2) is a mixture of sodium dodecyl sulfate and TritonX-200 in a mass ratio of 4:6;

[0134] The mass ratio of the filter aid to the precipitate A in step (3) is 0.5:1;

[0135] The modified polysaccharide described in step (5) is methacrylic anhydride-modified hyaluronic acid, oat β-glucan and aldehyde-modified hyaluronic acid in a mass ratio of 2:5:3.

[0136] 2. Preparation of MaAFP antifreeze protein

[0137] 1) Preparation of culture medium:

[0138] Glutamic acid, proline, vitamin B1, and sucrose were added to the 2216E liquid culture medium to obtain a 2216E liquid culture medium containing a final concentration of 0.4 g / L glutamate, 0.3 g / L proline, 20 mg / L vitamin B1, and 2 g / L sucrose.

[0139] 2) Strain culture:

[0140] Pseudoalteromonas was inoculated into the above culture medium at a 5% inoculation rate and cultured at 20°C and 200 r / min for 7 days. The bacterial solution was centrifuged at 5000 rpm for 10 min, the supernatant was collected, and the filtrate was filtered using a 0.22 μm filter membrane.

[0141] 3) Separation:

[0142] An ultrafiltration membrane with a molecular cutoff of 30 kDa was selected, and ultrafiltration was performed at a pressure of 8 bar to collect the filtrate; the ultrafiltered sample was placed in a dialysis bag with a molecular cutoff of 10 kDa, and PBS with a pH of 7.4 was used as the dialysis medium, and dialyzed at 25°C for 24 hours, during which the buffer solution was replaced every 8 hours; the dialyzate was collected, and after freeze-drying, a powder was obtained.

[0143] 4) Purification:

[0144] The powder was dissolved in PBS at pH 7.4, passed through a Sephadex G-100 column, eluted with PBS at pH 7.4, and the eluate was collected, which was the MaAFP antifreeze protein.

[0145] 3. Preparation of triple helical collagen and Antarctic glycoprotein composition

[0146] S1. Antarctic krill pretreatment

[0147] Fresh Antarctic krill was selected as raw material, washed, and placed in a vacuum freeze dryer for freeze drying. The freeze-dried Antarctic krill was crushed and passed through a 10-mesh sieve to obtain uniform Antarctic krill freeze-dried powder.

[0148] S2, Enzymatic Hydrolysis:

[0149] Antarctic krill freeze-dried powder was added to distilled water (liquid-to-solid ratio 1:50 g / mL), the pH was adjusted to 6.0-7.0, and the enzyme mixture was added according to 0.75 wt% of Antarctic krill freeze-dried powder. The enzyme was hydrolyzed at 55 °C for 3 h to obtain the hydrolyzate, which was heated at 90 °C for 20 min. After cooling, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected for later use.

[0150] The enzyme mixture consists of cellulase, papain and neutral protease, and the weight ratio of the cellulase, papain and neutral protease is 2:1.5:1.

[0151] S3. Dialysis:

[0152] The collected supernatant was placed in a dialysis bag with a molecular cutoff of 10 kDa, and distilled water was used as the dialysis medium for 48 hours. The dialysate was concentrated to 1 / 5 of the volume, and ethanol was added to make the final ethanol volume concentration of 75% (v / v). After standing overnight, the supernatant was centrifuged at 8000 rpm for 10 minutes, the precipitate was collected, and freeze-dried to obtain the crude Antarctic krill glycoprotein.

[0153] S4, Refining:

[0154] The crude Antarctic krill glycoprotein was dissolved in distilled water to obtain a crude Antarctic krill glycoprotein solution. MaAFP antifreeze protein was added according to a volume ratio of MaAFP antifreeze protein to crude Antarctic krill glycoprotein solution of 0.65:1, collagen was adsorbed by SP (sulfopropyl)-Sephadex dextran gel, and eluted and separated using a 10mM Tris-HCl aqueous solution containing 0.5wt% sodium salt, and the eluate was collected to obtain the Antarctic glycoprotein solution.

[0155] S5. Mixed freeze-drying:

[0156] The Antarctic glycoprotein solution and the triple-helical collagen hydrogel solution are uniformly mixed in a volume ratio of 1.25:1, and freeze-dried to obtain a triple-helical collagen and Antarctic glycoprotein composition.

[0157] Example 2 Preparation of triple helical collagen and Antarctic glycoprotein composition

[0158] 1. Preparation of triple-helical collagen hydrogel

[0159] The triple helical collagen hydrogel was prepared according to the method described in patent CN115944545B. The specific method is as follows:

[0160] (1) Pretreatment of pigskin:

[0161] A certain amount of animal pig skin was added to a 0.35wt% trypsin solution containing 0.04wt% EDTA, soaked at 37°C for 12 hours, and rinsed; then soaked in a phosphate buffer (pH 8.0) containing 0.15wt% ion detergent at 32°C for 12 hours and shaken, rinsed, and obtained the pre-treated pig skin;

[0162] (2) De-immunization of pig skin:

[0163] The pigskin pretreated in step (1) is added to a neutral protease solution with a concentration of 600 U / L, soaked at 35° C. for 12 hours, and rinsed; then, it is soaked in a phosphate buffer (pH 8.0) containing 0.15 wt% ion detergent at 32° C. for 12 hours and shaken, and rinsed; after repeating step (1) and step (2) 1-3 times, the immunogenic pigskin is obtained;

[0164] The de-immunized pig skin obtained after the treatment was soaked in 15 μg / mL α-1,3-galactosidase solution at 37°C for 1 hour, and then rinsed to obtain precipitate A;

[0165] (3) Filter adsorption:

[0166] Add the diatomaceous earth solution to the precipitate A obtained in step (2), stir, filter, and collect the supernatant for later use;

[0167] (4) Separation and purification:

[0168] After the supernatant collected in step (3) is concentrated, collagen is adsorbed on SP (sulfopropyl)-Sephadex gel and then eluted and separated using a 10 mM Tris-HCl aqueous solution containing 0.9 wt % sodium salt to obtain an eluate;

[0169] (5) Hydrogel preparation:

[0170] Adding a modified polysaccharide at a concentration of 40 mg / mL to the eluate in step (4), wherein the mass ratio of the eluate to the modified polysaccharide is 5:1, to perform modification to obtain a hydrogel;

[0171] (6) Sterile filtration

[0172] The hydrogel obtained in step (5) is sterilized and filtered using a 0.22 μm filter membrane to obtain a sterile soluble collagen hydrogel solution.

[0173] The ionic descaling agent in step (1) and step (2) is a mixture of sodium dodecyl sulfate and TritonX-200 in a mass ratio of 4:6;

[0174] The mass ratio of the filter aid to the precipitate A in step (3) is 3:1;

[0175] The modified polysaccharide described in step (5) is methacrylic anhydride-modified hyaluronic acid, oat β-glucan and aldehyde-modified hyaluronic acid in a mass ratio of 2:5:3.

[0176] 2. Preparation of MaAFP antifreeze protein

[0177] 1) Preparation of culture medium:

[0178] Glutamic acid, proline, vitamin B1, and sucrose were added to the 2216E liquid culture medium to obtain a 2216E liquid culture medium containing a final concentration of 0.2 g / L glutamate, 0.45 g / L proline, 30 mg / L vitamin B1, and 3 g / L sucrose.

[0179] 2) Strain culture:

[0180] Pseudoalteromonas was inoculated into the above culture medium at a 4% inoculation rate and cultured at 15°C and 500 r / min for 5 days. The bacterial solution was centrifuged at 8000 rpm for 10 min, the supernatant was collected, and the filtrate was filtered using a 0.22 μm filter membrane.

[0181] 3) Separation:

[0182] An ultrafiltration membrane with a molecular cutoff of 15 kDa was selected, and ultrafiltration was performed at a pressure of 5 bar to collect the filtrate; the ultrafiltration sample was placed in a dialysis bag with a molecular cutoff of 15 kDa, and PBS with a pH of 7.4 was used as the dialysis medium, and dialyzed at 25°C for 48 hours, during which the buffer solution was replaced every 8 hours; the retentate was collected, and freeze-dried to obtain a powder.

[0183] 4) Purification:

[0184] The powder was dissolved in PBS at pH 7.4, passed through a Sephadex G-100 column, eluted with PBS at pH 7.4, and the eluate was collected, which was the MaAFP antifreeze protein.

[0185] 3. Preparation of triple helical collagen and Antarctic glycoprotein composition

[0186] S1. Antarctic krill pretreatment

[0187] Fresh Antarctic krill was selected as raw material, washed, and placed in a vacuum freeze dryer for freeze drying. The freeze-dried Antarctic krill was crushed and passed through a 10-mesh sieve to obtain uniform Antarctic krill freeze-dried powder.

[0188] S2, Enzymatic Hydrolysis:

[0189] Antarctic krill freeze-dried powder was added to distilled water (liquid-to-solid ratio 1:40 g / mL), the pH was adjusted to 6.0-7.0, and the enzyme mixture was added at 1.0 wt% of Antarctic krill freeze-dried powder. The mixture was enzymolyzed at 60 °C for 2 h to obtain an enzymolysis solution, which was heated at 100 °C for 15 min. After cooling, it was centrifuged at 5000 rpm for 20 min, and the supernatant was collected for later use.

[0190] The enzyme mixture consists of cellulase, papain and neutral protease, and the weight ratio of the cellulase, papain and neutral protease is 2:1.5:1.

[0191] S3. Dialysis:

[0192] The collected supernatant was placed in a dialysis bag with a molecular cutoff of 15 kDa, and dialyzed for 48 hours using distilled water as the dialysis medium. The dialysate was concentrated to 1 / 5 of the volume, and ethanol was added to make the final ethanol volume concentration of 70% (v / v). After standing overnight, the supernatant was centrifuged at 5000 rpm for 20 minutes, the precipitate was collected, and freeze-dried to obtain the crude Antarctic krill glycoprotein.

[0193] S4, Refining:

[0194] The crude Antarctic krill glycoprotein was dissolved in distilled water to obtain a crude Antarctic krill glycoprotein solution. MaAFP antifreeze protein was added according to a volume ratio of MaAFP antifreeze protein to crude Antarctic krill glycoprotein solution of 0.3:1, collagen was adsorbed by SP (sulfopropyl)-Sephadex dextran gel, and eluted and separated using a 10mM Tris-HCl aqueous solution containing 0.9wt% sodium salt, and the eluate was collected to obtain the Antarctic glycoprotein solution.

[0195] S5, mixed freeze-drying

[0196] The Antarctic glycoprotein solution and the triple-helical collagen hydrogel solution are uniformly mixed in a volume ratio of 0.4:1, and freeze-dried to obtain a triple-helical collagen and Antarctic glycoprotein composition.

[0197] Example 3 Preparation of triple helical collagen and Antarctic glycoprotein composition

[0198] 1. Preparation of triple-helical collagen hydrogel

[0199] The triple helical collagen hydrogel was prepared according to the method described in patent CN115944545B. The specific method is as follows:

[0200] (1) Pretreatment of pigskin:

[0201] A certain amount of animal pig skin was added to a 0.3wt% trypsin solution containing 0.025wt% EDTA, soaked at 37°C for 24 hours, and rinsed; then soaked in a phosphate buffer (pH 7.2) containing 0.10wt% ion detergent at 30°C for 20 hours and shaken, rinsed, and obtained the pre-treated pig skin;

[0202] (2) De-immunization of pig skin:

[0203] The pigskin pretreated in step (1) is added to a neutral protease solution with a concentration of 700 U / L and soaked at 35° C. for 24 hours, and then rinsed; then soaked in a phosphate buffer solution (pH 7.2) containing 0.10 wt% ion detergent at 30° C. for 20 hours and shaken, and rinsed; after repeating step (1) and step (2) 1-3 times, the immunogenic pigskin is obtained;

[0204] The de-immunized pig skin obtained after the treatment was soaked in 20 μg / mL α-1,3-galactosidase solution at 37°C for 3 hours, and then washed to obtain precipitate A;

[0205] (3) Filter adsorption:

[0206] Add the diatomaceous earth solution to the precipitate A obtained in step (2), stir, filter, and collect the supernatant for later use;

[0207] (4) Separation and purification:

[0208] After the supernatant collected in step (3) is concentrated, collagen is adsorbed on SP (sulfopropyl)-Sephadex gel and then eluted and separated using a 10 mM Tris-HCl aqueous solution containing 0.8 wt % sodium salt to obtain an eluate;

[0209] (5) Hydrogel preparation:

[0210] Adding a modified polysaccharide at a concentration of 25 mg / mL to the eluate in step (4), wherein the mass ratio of the eluate to the modified polysaccharide is 3.5:2, to perform modification and obtain a hydrogel;

[0211] (6) Sterile filtration

[0212] The hydrogel obtained in step (5) is sterilized and filtered using a 0.22 μm filter membrane to obtain a sterile soluble collagen hydrogel solution.

[0213] The ionic descaling agent in step (1) and step (2) is a mixture of sodium dodecyl sulfate and TritonX-200 in a mass ratio of 4:6;

[0214] The mass ratio of the filter aid to the precipitate A in step (3) is 1:1;

[0215] The modified polysaccharide described in step (5) is methacrylic anhydride-modified hyaluronic acid, oat β-glucan and aldehyde-modified hyaluronic acid in a mass ratio of 2:5:3.

[0216] 2. Preparation of MaAFP antifreeze protein

[0217] 1) Preparation of culture medium:

[0218] Glutamic acid, proline, vitamin B1, and sucrose were added to the 2216E liquid culture medium to obtain a 2216E liquid culture medium containing a final concentration of 0.6 g / L glutamate, 0.15 g / L proline, 15 mg / L vitamin B1, and 1 g / L sucrose.

[0219] 2) Strain culture:

[0220] Pseudoalteromonas was inoculated into the above culture medium at a 3% inoculation rate and cultured at 20°C and 200 r / min for 7 days. The bacterial solution was centrifuged at 3000 rpm for 15 min, the supernatant was collected, and the filtrate was filtered using a 0.22 μm filter membrane.

[0221] 3) Separation:

[0222] An ultrafiltration membrane with a molecular cutoff of 30 kDa was selected, and ultrafiltration was performed at a pressure of 8 bar to collect the filtrate; the ultrafiltration sample was placed in a dialysis bag with a molecular cutoff of 10 kDa, and PBS with a pH of 7.4 was used as the dialysis medium, and dialyzed at 20°C for 24 hours, during which the buffer solution was replaced every 8 hours; the retentate was collected, and freeze-dried to obtain a powder.

[0223] 4) Purification:

[0224] The powder was dissolved in PBS at pH 7.4, passed through a Sephadex G-100 column, eluted with PBS at pH 7.4, and the eluate was collected, which was the MaAFP antifreeze protein.

[0225] 3. Preparation of triple helical collagen and Antarctic glycoprotein composition

[0226] S1. Antarctic krill pretreatment

[0227] Fresh Antarctic krill was selected as raw material, washed, and placed in a vacuum freeze dryer for freeze drying. The freeze-dried Antarctic krill was crushed and passed through a 10-mesh sieve to obtain uniform Antarctic krill freeze-dried powder.

[0228] S2, Enzymatic Hydrolysis:

[0229] Antarctic krill freeze-dried powder was added to distilled water (liquid-to-solid ratio 1:60 g / mL), the pH was adjusted to 6.0-7.0, and the enzyme mixture was added according to 0.5wt% of Antarctic krill freeze-dried powder. The mixture was enzymolyzed at 50℃ for 4h to obtain the enzymolysis solution, which was heated at 90℃ for 30min. After cooling, it was centrifuged at 3000rpm for 30min, and the supernatant was collected for later use.

[0230] The enzyme mixture consists of cellulase, papain and neutral protease, and the weight ratio of the cellulase, papain and neutral protease is 2:1.5:1.

[0231] S3. Dialysis:

[0232] The collected supernatant was placed in a dialysis bag with a molecular cutoff of 14 kDa, and dialyzed for 48 hours using distilled water as the dialysis medium. The dialysate was concentrated to 1 / 5 of the volume, and ethanol was added to make the final ethanol volume concentration of 80% (v / v). After standing overnight, the supernatant was centrifuged at 3000 rpm for 30 minutes, the precipitate was collected, and freeze-dried to obtain the crude Antarctic krill glycoprotein.

[0233] S4, Refining:

[0234] The crude Antarctic krill glycoprotein was dissolved in distilled water to obtain a crude Antarctic krill glycoprotein solution. MaAFP antifreeze protein was added according to a volume ratio of 1:1 between MaAFP antifreeze protein and crude Antarctic krill glycoprotein solution, collagen was adsorbed by SP (sulfopropyl)-Sephadex dextran gel, and eluted and separated by 10mM Tris-HCl aqueous solution containing 0.75wt% sodium salt, and the eluate was collected to obtain the Antarctic glycoprotein solution.

[0235] S5, mixed freeze-drying

[0236] The Antarctic glycoprotein solution and the triple-helical collagen hydrogel solution are uniformly mixed in a volume ratio of 1.5:1, and freeze-dried to obtain a triple-helical collagen and Antarctic glycoprotein composition.

[0237] Comparative Example 1 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0238] Comparative Example 1 was set up with reference to Example 1, and the only difference between Comparative Example 1 and Example 1 was that the enzyme mixture in the enzymatic hydrolysis in step S2 was different.

[0239] The enzyme mixture described in Comparative Example 1 consists of cellulase and papain, and the weight ratio of the cellulase to the papain is 2:2.5.

[0240] Comparative Example 2 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0241] Comparative Example 2 was set up with reference to Example 1. The only difference between Comparative Example 2 and Example 1 was that the enzyme mixture in the enzymatic hydrolysis in step S2 was different.

[0242] The enzyme mixture described in Comparative Example 2 consists of cellulase and neutral protease, and the weight ratio of the cellulase to the neutral protease is 2:2.5.

[0243] Comparative Example 3 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0244] Comparative Example 3 was set up with reference to Example 1. The only difference between Comparative Example 3 and Example 1 is that the "enzyme mixture" in the enzymatic hydrolysis of step S2 was replaced by "cellulase".

[0245] Comparative Example 4 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0246] Comparative Example 4 was set up with reference to Example 1. The only difference between Comparative Example 4 and Example 1 was that the enzyme mixture in the enzymatic hydrolysis in step S2 was different.

[0247] The enzyme mixture described in Comparative Example 4 consists of cellulase, papain and neutral protease, and the weight ratio of the cellulase, papain and neutral protease is 1:1.5:2.

[0248] Comparative Example 5 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0249] Comparative Example 5 is set up with reference to Example 1. The only difference between Comparative Example 5 and Example 1 is that: in step S2 enzymolysis, "adding the enzyme mixture at 0.75 wt % of Antarctic krill freeze-dried powder" is replaced by "adding the enzyme mixture at 1.5 wt % of Antarctic krill freeze-dried powder".

[0250] Comparative Example 6 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0251] Comparative Example 6 is set with reference to Example 1. The only difference between Comparative Example 6 and Example 1 is that "2. Preparation of MaAFP antifreeze protein" is not included and "Step S4. Purification" is different.

[0252] Step S4 of Comparative Example 6 is:

[0253] The crude Antarctic krill glycoprotein was dissolved in distilled water to obtain a crude Antarctic krill glycoprotein solution. According to the volume ratio of physiological saline to crude Antarctic krill glycoprotein solution of 0.65:1, physiological saline was added, collagen was adsorbed by SP (sulfopropyl)-Sephadex dextran gel, and eluted and separated using a 10mM Tris-HCl aqueous solution containing 0.5wt% sodium salt, and the eluate was collected to obtain the Antarctic glycoprotein solution.

[0254] Comparative Example 7 Preparation of Antarctic glycoprotein freeze-dried powder

[0255] Comparative Example 7 is set up with reference to Example 1. The only difference between Comparative Example 7 and Example 1 is that "1. Preparation of triple-helix collagen hydrogel" is not included and "Step S5. Mixing and freeze-drying" is different.

[0256] Step S5 of Comparative Example 7 is:

[0257] The Antarctic glycoprotein solution and physiological saline were evenly mixed in a volume ratio of 1.25:1, and freeze-dried to obtain Antarctic glycoprotein freeze-dried powder.

[0258] Comparative Example 8 Preparation of triple helical collagen hydrogel freeze-dried powder

[0259] Comparative Example 8 is set up with reference to Example 1. The only difference between Comparative Example 8 and Example 1 is that "2. Preparation of triple-helical collagen hydrogel" and "3. Preparation of triple-helical collagen and Antarctic glycoprotein composition" are not included.

[0260] Physiological saline and triple-helix collagen hydrogel solution were evenly mixed in a volume ratio of 1.25:1, and freeze-dried to obtain triple-helix collagen hydrogel freeze-dried powder.

[0261] Comparative Example 9 Preparation of a composition of triple helical collagen and Antarctic glycoprotein

[0262] Comparative Example 9 was set up with reference to Example 1. The only difference between Comparative Example 9 and Example 1 was that "Steps S4 and S5" in "3. Preparation of a composition of triple-helical collagen and Antarctic glycoprotein" were different.

[0263] S4, Refining:

[0264] The crude Antarctic krill glycoprotein was dissolved in distilled water to obtain a crude Antarctic krill glycoprotein solution. MaAFP antifreeze protein was added according to a volume ratio of MaAFP antifreeze protein to crude Antarctic krill glycoprotein solution of 1.5:1, collagen was adsorbed by SP (sulfopropyl)-Sephadex dextran gel, and eluted and separated using a 10mM Tris-HCl aqueous solution containing 0.5wt% sodium salt, and the eluate was collected to obtain the Antarctic glycoprotein solution.

[0265] S5. Mixed freeze-drying:

[0266] The Antarctic glycoprotein solution and the triple-helical collagen hydrogel solution are uniformly mixed in a volume ratio of 2:1, and freeze-dried to obtain a triple-helical collagen and Antarctic glycoprotein composition.

[0267] Experimental Example 1 Cultivation and passaging of HaCaT cells

[0268] The culture conditions of HaCaT cells are: DMEM medium containing 10% FBS and 1% streptomycin solution; 37°C, 5% CO2. When the cell density reaches 80%-90%, the cells are passaged. HaCaT cells are passaged more than 2-3 times, and cells in the logarithmic growth phase are used for experiments.

[0269] Experimental Example 2 HaCaT cell survival rate experiment

[0270] 1. This experiment is divided into 14 groups, namely:

[0271] (1) Model control group reagents: DMEM culture medium containing 10% FBS and 1% streptomycin solution.

[0272] (2) Blank control group reagent: DMEM culture medium containing 10% FBS and 1% streptomycin solution.

[0273] (3) Reagents of Example 1: The triple-helical collagen and Antarctic glycoprotein composition prepared in Example 1 was prepared at a concentration of 2000 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0274] (4) Reagents of Example 2: The triple-helical collagen and Antarctic glycoprotein composition prepared in Example 2 was prepared at a concentration of 800 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0275] (5) Reagents of Example 3: The triple helical collagen and Antarctic glycoprotein composition prepared in Example 3 was prepared at a concentration of 6000 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0276] (6) Comparative Example 1 Reagents: The triple helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 1 was prepared at a concentration of 2000 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0277] (7) Reagents for Comparative Example 2: The triple-helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 2 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0278] (8) Reagents of Comparative Example 3: The triple-helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 3 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0279] (9) Reagents of Comparative Example 4: The triple helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 4 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0280] (10) Reagents for Comparative Example 5: The triple-helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 5 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0281] (11) Reagents for Comparative Example 6: The triple-helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 6 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0282] (12) Reagents for Comparative Example 7: The Antarctic glycoprotein freeze-dried powder prepared in Comparative Example 7 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0283] (13) Reagents for Comparative Example 8: The triple-helical collagen hydrogel freeze-dried powder prepared in Comparative Example 8 was prepared at a concentration of 2000 μg / mL using DMEM medium containing 10% FBS and 1% streptomycin solution.

[0284] (14) Reagents for Comparative Example 9: The triple-helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 9 was prepared at a concentration of 2000 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0285] 2. Cell viability determination:

[0286] HaCaT cells in the logarithmic growth phase were used to prepare a cell suspension, with 1×10 4 The cells were inoculated at a density of 100 μL / well in a 96-well plate and cultured in an incubator at 37°C with 5% CO2 for 24 hours, and the supernatant was discarded. 100 μL of reagent was added according to the experimental group and cultured for 2 hours. Except for the blank control group, UVB radiation was irradiated at 300 nJ / cm 2 The cells were cultured for 15 min. After culturing for 24 h, 100 µL of basal medium containing 10% CCK-8 was added and the cells were incubated in a carbon dioxide incubator for 2 h. The absorbance was measured at 450 nm using a microplate reader to calculate the cell survival rate.

[0287] The results of the test are as follows Figure 1As shown, compared with the normal group, the cell survival rate of the model group was reduced to 46.75±5.00%, proving that the cell model was successfully modeled, and the Hacat cells at this time showed a photoaging state. After intervention with the triple-helical collagen and Antarctic glycoprotein composition prepared in Comparative Example 6, the Antarctic glycoprotein freeze-dried powder prepared in Comparative Example 7, and the Antarctic glycoprotein freeze-dried powder prepared in Comparative Example 8, the cell survival rate increased to 67.47%-76.28%, which had a certain anti-aging effect. Examples 1-3 showed the best anti-aging effect, and were able to maintain the survival rate of Hacat cells to 92.78%-97.18%, which was basically the same as the blank group cell effect. Therefore, the triple-helical collagen and Antarctic glycoprotein compositions prepared in Examples 1-3 were selected for subsequent inflammation and oxidative stress determinations.

[0288] Experimental Example 3 Inflammation and oxidative stress assay

[0289] 1. This experiment is divided into 5 groups, namely:

[0290] (1) Model control group reagents: DMEM culture medium containing 10% FBS and 1% streptomycin solution.

[0291] (2) Blank control group reagent: DMEM culture medium containing 10% FBS and 1% streptomycin solution.

[0292] (3) Reagents of Example 1: The triple-helical collagen and Antarctic glycoprotein composition prepared in Example 1 was prepared at a concentration of 2000 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0293] (4) Reagents of Example 2: The triple helical collagen and Antarctic glycoprotein composition prepared in Example 1 was prepared at a concentration of 800 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0294] (5) Reagents of Example 3: The triple helical collagen and Antarctic glycoprotein composition prepared in Example 3 was prepared at a concentration of 6000 μg / mL in DMEM medium containing 10% FBS and 1% streptomycin solution.

[0295] 2. Inflammation and oxidative stress determination:

[0296] HaCaT cells in the logarithmic growth phase were used to prepare cell suspensions at 6×10 5 The cells were inoculated into a 96-well plate at a density of 100 L / well and cultured in a 7°C, 5% CO2 incubator for 24 h, then the supernatant was discarded. 100 µL of reagent was added according to the experimental group and cultured for 2 h. Except for the blank control group, UVB radiation was irradiated at 300 nJ / cm 2The culture was continued for 15 min. After culturing for 24 h, the supernatant was collected by centrifugation and the contents of IFN-γ, IL-6 and MDA were determined according to the instructions of the ELISA kit.

[0297] The results of the test are as follows Figure 2 The preparations prepared in Examples 1-3 of the present invention can effectively inhibit the expression levels of inflammatory cytokines IFN-γ and IL-6, and inhibit the production of MDA.

[0298] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A method for preparing a triple helical collagen and Antarctic glycoprotein composition, characterized in that: The preparation method comprises: S1. Add freeze-dried Antarctic krill powder to distilled water, adjust the pH, add enzyme mixture, inactivate after enzymolysis, and collect the supernatant; S2, the supernatant was dialyzed and concentrated, ethanol was added, and the mixture was allowed to stand overnight, and the precipitate was collected and freeze-dried to obtain the crude product of Antarctic krill glycoprotein; S3, dissolving the crude Antarctic krill glycoprotein in distilled water to obtain a crude Antarctic krill glycoprotein solution, adding MaAFP antifreeze protein, adsorbing the protein with a cation exchange medium, and then eluting and separating, collecting the eluate, which is the Antarctic glycoprotein solution; S4, mixing the Antarctic glycoprotein solution and the triple helical collagen hydrogel solution evenly in a volume ratio of 0.4-1.5:1, and freeze-drying to obtain a triple helical collagen and Antarctic glycoprotein composition; The enzyme mixture in step S1 is composed of cellulase, papain and neutral protease, and the weight ratio of cellulase, papain and neutral protease is 2:1.5:1; The amount of the enzyme mixture added in step S1 is 0.5wt%-1.0wt% of the Antarctic krill freeze-dried powder; The volume ratio of the MaAFP antifreeze protein to the crude Antarctic krill glycoprotein solution in step S3 is 0.3-1.0:1; The amino acid sequence of the MaAFP antifreeze protein in step S3 is shown in SEQ ID NO.1; The method for preparing the triple helical collagen hydrogel solution in step S4 comprises: (1) taking pig skin, soaking it in a trypsin solution containing EDTA, and rinsing it; soaking it in a detergent solution, shaking it, and rinsing it; soaking it in a neutral protease solution, and rinsing it; soaking it in a detergent solution, shaking it, and rinsing it; repeating the above steps 1-3 times to obtain a pig skin free of immunogenicity; (2) soaking the de-immunized pig skin in an α-1,3-galactosidase solution, and obtaining a precipitate A after rinsing; adding a filter aid solution to the precipitate A, stirring, and filtering, and collecting the supernatant; concentrating the supernatant, adsorbing collagen through a cation exchange medium, and then eluting and separating, and finally obtaining an eluate; (3) adding a modified polysaccharide at a concentration of 10-40 mg / mL to the eluate of step (2), wherein the mass ratio of the eluate to the modified polysaccharide is 5:1-1:3, to perform modification and obtain a hydrogel; (4) sterilizing and filtering the hydrogel obtained in step (3) using a 0.22 μm filter membrane to obtain a triple-helix collagen hydrogel solution; The descaling agent solution in step (1) is a sodium phosphate buffer solution with a pH of 6.0-8.0 containing 0.05-0.15 wt% of an ionic descaling agent; the ionic descaling agent is a mixture of sodium dodecyl sulfate and Triton X-200 in a mass ratio of 4:6; The modified polysaccharide described in step (3) is methacrylic anhydride-modified hyaluronic acid, oat β-glucan and aldehyde-modified hyaluronic acid in a mass ratio of 2:5:

3.

2. The preparation method according to claim 1, characterized in that: The ratio of the Antarctic krill freeze-dried powder to distilled water in step S1 is 1:40-60 g / mL; and / or, the pH in step S1 is 6.0-7.0; And / or, the enzymatic hydrolysis temperature in step S1 is 50-60° C., and the enzymatic hydrolysis time is 2-4 h.

3. The preparation method according to claim 1, characterized in that: The method for preparing the MaAFP antifreeze protein in step S3 comprises: ① Inoculate 3-5% of Pseudoalteromonas into a culture medium containing glutamic acid, proline, vitamin B1 and sucrose, and culture to obtain a bacterial solution; ② After centrifugation of the bacterial solution, collect the supernatant, filter, and collect the filtrate; ③ The filtrate is dialyzed, the retentate is collected, and the powder is obtained after freeze-drying; ④ After the powder is dissolved, the cation exchange medium adsorbs the protein and then elutes and separates it, and the eluate is collected to obtain the MaAFP antifreeze protein.

4. The preparation method according to claim 3, characterized in that: The culture medium described in step ① is a culture medium containing final concentrations of 0.2-0.6 g / L glutamic acid, 0.15-0.45 g / L proline, 15-30 mg / L vitamin B1, and 1-3 g / L sucrose.

5. The preparation method according to claim 1, characterized in that: The solution used for elution in step S3 is a 10 mM Tris-HCl aqueous solution containing 0.5-0.9 wt % sodium salt.

6. A triple-helical collagen and Antarctic glycoprotein composition prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the triple-helix collagen and Antarctic glycoprotein composition according to claim 6 in the preparation of anti-aging products; the products include dressings, medicines or cosmetics.

8. An anti-aging cosmetic, characterized in that: The anti-aging cosmetic comprises the triple helical collagen and Antarctic glycoprotein composition prepared by the preparation method according to any one of claims 1 to 5 or the triple helical collagen and Antarctic glycoprotein composition according to claim 6.

9. An anti-aging drug, characterized in that: The anti-aging drug comprises the triple helical collagen and Antarctic glycoprotein composition prepared by the preparation method according to any one of claims 1 to 5 or the triple helical collagen and Antarctic glycoprotein composition according to claim 6.

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