Antioxidant for improving stability of mussel mucin and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to an antioxidant for improving the stability of mussel mucin and application of the antioxidant. The antioxidant provided by the invention is a combination of an antioxidant A and an antioxidant B, or a combination of the antioxidant A, the antioxidant B and an antioxidant C. The antioxidant A is composed of Ectoine, mussel mucin type 6 (Mfp-6), glycerin and polyvinylpyrrolidone (PVP), the antioxidant B is an antioxidant containing reduced sulfur, and the antioxidant C is a hydrogen supply type antioxidant. The mussel mucin is protected by the components in the antioxidant through multiple mechanisms, so that the oxidation degree and the oxidation rate of the mussel mucin in the mussel mucin preparation are obviously slowed down.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and specifically relates to an antioxidant that improves the stability of mussel adhesive protein and its application. Background Technology

[0002] Mussel adhesive protein (MAP) is a protein extracted from the byssal glands of mussels or obtained through bio-fermentation. MAP contains abundant 3,4-dihydroxyphenylalanine (DOPA) groups, enabling it to form a strong, water-resistant, network-like micro-biofilm scaffold on the mussel's surface while also exhibiting excellent free radical scavenging capabilities. This results in significant anti-inflammatory, antioxidant, and wound-healing effects, making it promising for applications in biomedicine, medical devices, and cosmetics. However, during storage, the DOPA groups in MAP are easily oxidized to dopaquinone, which not only weakens its anti-inflammatory, antioxidant, and wound-healing effects but also causes discoloration in MAP preparations, negatively impacting users. Therefore, avoiding or slowing down the oxidation rate and degree of MAP is a pressing issue that needs to be addressed.

[0003] To address the oxidation and discoloration of dopa groups in mussel adhesive protein during long-term storage, Chinese patents CN118121504B and CN118121744B respectively slow down the oxidation rate of mussel adhesive protein by adding ergothioneine and specific antioxidant components (butylated hydroxytoluene, sodium metabisulfite, etc.). However, both of these methods protect mussel adhesive protein by adding a single system of antioxidants, which has limited protective effect.

[0004] Therefore, it is necessary to develop an antioxidant composition that can effectively slow down the oxidation of mussel adhesive protein and solve the problem that mussel adhesive protein is easily oxidized during long-term storage and use. Summary of the Invention

[0005] The purpose of this invention is to provide an antioxidant that improves the stability of mussel adhesive protein and its application. This antioxidant effectively solves the problem of easy oxidation of mussel adhesive protein during long-term storage.

[0006] In a first aspect, the present invention provides an antioxidant for improving the stability of mussel adhesive protein, the antioxidant comprising antioxidant A and antioxidant B, wherein antioxidant A is composed of ectoine, mussel adhesive protein type 6 (Mfp-6), glycerol and polyvinylpyrrolidone (PVP).

[0007] In this invention, on the one hand, the four components in antioxidant A work synergistically to enhance the antioxidant capacity of mussel adhesive protein. The active hydrogen atoms and amino groups of ectoine in antioxidant A, along with the thiol groups of cysteine ​​rich in Mfp-6, can bind with hydroxyl radicals and peroxy radicals generated during the oxidation of mussel adhesive protein, thereby halting the oxidation reaction of mussel adhesive protein. On the other hand, the hydroxyl and carbonyl groups of glycerol and polyvinylpyrrolidone (PVP) in antioxidant A can form a wide range of hydrogen bond networks with the amino and imino groups on the lysine residues in mussel adhesive protein through hydrogen bonding. This network structure dynamically covers the surface of mussel adhesive protein molecules at the microscopic level and fills the spaces between protein molecules, forming a physical barrier (i.e., a "polymer-protein composite protective layer") that effectively blocks the diffusion of oxygen molecules. This barrier effectively isolates oxidizing substances (such as oxygen) from direct contact with mussel adhesive protein, thereby reducing the probability of mussel adhesive protein being oxidized. Meanwhile, glycerol and PVP also play a "supporting and protective" role in the composition system. The hydrogen bond protection between glycerol and mussel adhesive protein, combined with the film-forming effect of PVP, further improves the long-term storage stability of mussel adhesive protein and prolongs its efficacy duration. In addition, the moisturizing and lubricating properties of glycerol and the adhesiveness of PVP make the prepared composition easier to adhere to the surface of organisms (such as wounds and skin), thus maximizing its effectiveness.

[0008] In some embodiments, the mass ratio of ectoin, mussel adhesive protein type 6 (Mfp-6), glycerol and polyvinylpyrrolidone (PVP) is (1~3):(0.05~0.5):(0.1~2):(0.1~1).

[0009] In some embodiments, the mass ratio of ectoin, mussel adhesive protein type 6 (Mfp-6), glycerol and polyvinylpyrrolidone (PVP) is 1:0.1:1:0.3.

[0010] In this invention, antioxidant A prepared using ectoin, mussel adhesive protein type 6 (Mfp-6), glycerol and polyvinylpyrrolidone (PVP) within the above-mentioned mass ratio range can better prevent the oxidation of mussel adhesive protein.

[0011] In some embodiments, the antioxidant B is a sulfur-containing antioxidant selected from one or more of sodium bisulfite, sodium sulfite, sodium metabisulfite, sodium thiosulfate, and lipoic acid.

[0012] In some embodiments, the mass ratio of antioxidant B to ectoine is (0.05~0.3):(1~3).

[0013] In this invention, antioxidant B not only protects mussel adhesive protein and reduces its probability of oxidation, but also repairs minor oxidative damage to mussel adhesive protein through a thiol-disulfide bond exchange reaction. Furthermore, when antioxidant A and antioxidant B are combined, the reducing sulfur groups (such as thiol groups) contained in antioxidant B can lower the redox potential of the system by providing electrons and capturing reactive oxygen species, thereby enhancing the reducing power of the system. This maintains the solution in a reducing environment with a lower oxidation potential, making it easier to maintain the active form of antioxidant A. Through the synergistic effect of antioxidant A and antioxidant B, the antioxidant properties of mussel adhesive protein can be stabilized and improved.

[0014] In some embodiments, the antioxidant is a combination of antioxidant A, antioxidant B and antioxidant C, wherein antioxidant C is a hydrogen-donating antioxidant selected from one or more of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG).

[0015] In some embodiments, the mass ratio of the antioxidant C to the ectoine is (0.01~0.1):(1~3).

[0016] In this invention, antioxidant C is a hydrophobic antioxidant. Its hydroxyl groups can form hydrogen bonds with glycerol, and based on the principle of "like dissolves like," it enters the localized polar regions formed by glycerol. Through hydrogen bonding and the principle of "like dissolves like," antioxidant C can disperse in aqueous solution, interrupting the oxidation reaction by capturing free radicals. Simultaneously, antioxidant C, along with ectoine and Mfp-6 in antioxidant A, enhances the protective effect on mussel adhesive proteins through a synergistic hydrophilic-hydrophobic effect (ectoine and Mfp-6 scavenge reactive oxygen species in the aqueous phase, while antioxidant C terminates the chain oxidation in the hydrophobic microregions).

[0017] Secondly, the present invention provides the application of the antioxidant described in the first aspect in improving the stability of mussel adhesive protein.

[0018] Thirdly, the present invention also provides a composition containing mussel adhesive protein, the composition comprising the antioxidant, mussel adhesive protein and water described in the first aspect of the present invention.

[0019] In some embodiments, the mussel agaric in the composition is 0.01% to 30% by weight, preferably 0.01% to 5%.

[0020] In this invention, the mussel adhesive proteins are classified into naturally extracted and recombinant types according to their source. Naturally extracted mussel adhesive proteins include, but are not limited to, Mfp-1, Mfp-2, Mfp-3, Mfp-4, Mfp-5, Mfp-6 or mixtures thereof. Recombinant mussel adhesive proteins include, but are not limited to, Mfp-1, Mfp-2, Mfp-3, Mfp-4, Mfp-5, Mfp-6, Mfp-131, Mfp-151, Mfp-353, and fusion recombinant mussel adhesive proteins with ECM functional peptides introduced thereon, such as Mfp-151-RGD. The functional peptides can be selected from one or more of RGD (from fibronectin), YIGSR (from laminin), and GEFYFDLRLKGDK (from type IV collagen).

[0021] Fourthly, the present invention also provides a method for preparing the composition described in the third aspect of the present invention, wherein the preparation method is (A) or (B). The preparation method (A) includes the following steps: (1) Weigh out the required amount of water and introduce an inert gas into it; (2) Add antioxidant A and antioxidant B, mussel adhesive protein to the water and stir until completely dissolved; (3) Put the solution obtained in step (2) into a bottle, and pass in inert gas again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Then stop passing the gas and seal the bottle. Alternatively, the preparation method (B) includes the following steps: (1) Disperse the required amount of antioxidant C with glycerol in antioxidant A to obtain solution 1; (2) Weigh out the required amount of water and introduce an inert gas into it; (3) Add solution 1, the other components of antioxidant A except glycerol, antioxidant B and mussel adhesive protein to the water in sequence, and stir until completely dissolved; (4) Put the solution obtained in step (3) into a bottle, and pass in inert gas again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Stop passing gas and seal the bottle.

[0022] In some embodiments, the inert gas is nitrogen or argon, and the inert gas introduction rate is 50~200 mL / min.

[0023] In some embodiments, the preparation method further includes the step of sterilizing the above-mentioned composition containing mussel adhesive protein by moist heat sterilization or irradiation sterilization to obtain a sterile mussel adhesive protein composition.

[0024] In some embodiments, the moist heat sterilization conditions are 110℃~130℃ for 5~20 minutes; the irradiation sterilization method includes, but is not limited to, electron beam, gamma ray, X-ray, etc.

[0025] Fifthly, the present invention also provides the use of the antioxidants described in the first aspect of the present invention or the compositions described in the third aspect of the present invention in the preparation of medical devices, daily chemical products or biopharmaceutical products.

[0026] The present invention has the following technical effects compared with the prior art.

[0027] (1) The antioxidant provided by the present invention protects mussel adhesive protein through the synergistic effect of multiple mechanisms, and significantly slows down the degree and rate of oxidation of mussel adhesive protein. Firstly, ectoine in antioxidant A synergistically binds with Mfp-6 to reactive oxygen species (ROS) generated during the oxidation of mussel adhesive protein, directly halting the oxidation process. Simultaneously, glycerol and polyvinylpyrrolidone (PVP) can form a "polymer-protein complex protective layer" with the hydroxyl and amino groups in mussel adhesive protein through hydrogen bonding, isolating oxidizing substances (such as oxygen) from direct contact with the mussel adhesive protein. This reduces the risk of oxidation at the source and enhances the stability of mussel adhesive protein during long-term storage. Secondly, antioxidant B not only reduces the probability of mussel adhesive protein oxidation but also repairs minor oxidative damage through thiol-disulfide bond exchange reactions. Thirdly, when antioxidant A and antioxidant B are combined, the reducing sulfur groups (such as thiol groups) in antioxidant B can lower the redox potential of the system, creating a low-oxidation-potential reducing environment. This environment more easily maintains the active form of antioxidant A, thereby enhancing the overall antioxidant activity. Furthermore, the active hydrogen atoms in antioxidant C can enter the hydrophobic network formed by glycerol to interrupt the oxidation reaction by capturing free radicals. Simultaneously, through structural complementarity, antioxidant C, along with ectoine and Mfp-6 in antioxidant A, comprehensively covers both the hydrophilic and hydrophobic regions of mussel adhesive protein, forming a comprehensive, three-dimensional protective network against mussel adhesive protein. Ultimately, through the synergistic effect of these multiple mechanisms, the overall antioxidant protection effect against mussel adhesive protein is significantly enhanced.

[0028] (2) The antioxidant provided by this invention can effectively slow down the oxidation rate of mussel adhesive protein through the synergistic effect of multiple antioxidant mechanisms. At the same time, glycerol and polyvinylpyrrolidone (PVP) in the antioxidant can further improve the long-term storage stability of mussel adhesive protein and prolong its efficacy duration. In addition, the moisturizing and lubricating effect of glycerol and the adhesiveness of PVP make the prepared composition easier to adhere to the skin surface or wound, thereby fully exerting the anti-inflammatory and repair effects of mussel adhesive protein.

[0029] (3) The composition for improving the stability of mussel adhesive protein provided by the present invention has a wide range of applications. After being placed under accelerated testing conditions at 40°C for 3 months, the composition retains its color and the core functions (such as anti-oxidation, anti-inflammation, and repair) and physicochemical properties of the mussel adhesive protein remain stable. Furthermore, this composition can be widely used in medical devices, biomedicine, and daily chemical products, and can be directly added as a raw material to related products in the aforementioned fields. Attached Figure Description

[0030] Figure 1 The results are the stability test results of the antioxidants prepared in Examples 1-6 and Comparative Examples 1-16.

[0031] Figure 2 The stability test results are for the compositions prepared in Examples 7-8 and Comparative Examples 17-18.

[0032] Figure 3 Facial VISIA images of subjects in the experimental cases before and one month after use using Examples 7, 8, 17, and 18. Specific Implementation

[0033] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. All reagents and materials used in the embodiments are commercially available.

[0034] Examples 1-3

[0035] (1) Weigh the raw materials and prepare a product system with a total mass of 20g according to the mass percentage of all components in Table 1.

[0036] (2) Disperse antioxidant C with all the glycerol in antioxidant A to form solution 1.

[0037] (3) Nitrogen gas is introduced into the purified water for 5 minutes at a rate of 50 mL / min.

[0038] (4) Add solution 1, the remaining components of antioxidant A, antioxidant B, and mussel adhesive protein to the water in sequence, and stir until completely dissolved.

[0039] (5) The obtained solution is placed in a sample bottle, and nitrogen gas is introduced again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Then, the gas is stopped, the bottle is sealed, and the mussel adhesive protein composition is obtained.

[0040] Table 1. Mass percentage of each substance in Examples 1-3

[0041] Examples 4-6

[0042] (1) Weigh the raw materials and prepare a product system with a total mass of 20g according to the mass percentage of all components in Table 2.

[0043] (2) Introduce argon gas into the purified water for 5 minutes at a rate of 50 mL / min.

[0044] (3) Continue to add antioxidant A and antioxidant B, mussel adhesive protein to the purified water and stir until completely dissolved.

[0045] (4) The obtained solution is placed in a sample bottle, and argon gas is introduced again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Then, the gas is stopped, the bottle is sealed, and the mussel adhesive protein composition is obtained.

[0046] Table 2. Mass percentage of each substance in Examples 4-6

[0047] Comparative Example

[0048] Compared to Example 1, Comparative Examples 1-16 have different amounts of different components in the antioxidants (see underlined parts for details), and the specific component contents are shown in Table 3.

[0049] Table 3. Ingredients of Example 1 and Comparative Examples 1-16

[0050]

[0051] Example 7

[0052] A spray dressing containing the mussel adhesive protein composition was prepared, and the formulation is shown in Table 4.

[0053] Preparation process:

[0054] (1) Weigh the raw materials and prepare a product system with a total mass of 50g by weight percentage of all components.

[0055] (2) Disperse BHA with glycerol and pentylene glycol to form solution 1.

[0056] (3) Nitrogen gas is introduced into the purified water for 5 minutes at a rate of 50 mL / min.

[0057] (4) Add solution 1, ectoine, Mfp-6, PVP, sodium bisulfite, trehalose and mussel adhesive protein to the water in sequence, and stir until completely dissolved.

[0058] (5) Add phenoxyethanol / ethylhexylglycerin to the above solution and stir until completely dissolved.

[0059] (6) Place the obtained solution in a sample bottle, and purge with nitrogen again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Stop purging, seal, and obtain the spray dressing.

[0060] Table 4. Formulation of Spray Dressing Products

[0061] Example 8

[0062] A face mask containing the mussel adhesive protein composition was prepared, and the formula of the face mask liquid is shown in Table 5.

[0063] Preparation process:

[0064] (1) Weigh the raw materials and prepare a product system with a total mass of 50g by weight percentage of all components.

[0065] (2) Disperse PG, hydroxyethyl cellulose and carbomer with glycerol and propylene glycol to form solution 1.

[0066] (3) Nitrogen gas is introduced into the purified water for 5 minutes at a rate of 50 mL / min.

[0067] (4) Add solution 1, ectoine, Mfp-6, PVP, thioctic acid, trehalose, β-glucan and mussel adhesive protein to the water in sequence, and stir until completely dissolved.

[0068] (5) Add phenoxyethanol / ethylhexylglycerin and triethanolamine to the above solution and stir until completely dissolved.

[0069] (6) Place the obtained solution in a sample bottle, and purge with nitrogen again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Stop purging, seal, and obtain the mask product.

[0070] Table 5 Facial Mask Product Formula

[0071] Comparative Example 17

[0072] The formula and preparation process are the same as in Example 7, except that no antioxidant is added (only mussel adhesive protein is added).

[0073] Comparative Example 18

[0074] The formula and preparation process are the same as in Example 8, except that no antioxidant is added (only mussel adhesive protein is added).

[0075] Stability testing method: The sample to be tested was placed at 40℃ for 3 months to examine its stability and observe its properties.

[0076] Methods for detecting dopa content

[0077] 1. Experimental principle: Substances containing the 3,4-dihydroxyphenylalanine ("DOPA" or "DOPA") structure are yellow under acidic conditions, and turn deep orange-red when an excess of alkali is added.

[0078] 2. Reagents and Instruments

[0079] 2.1 Hydrochloric acid solution: Take 0.1 mL of hydrochloric acid and dilute it with pure water to a final volume of 100 mL.

[0080] 2.2 DOPA standard solution: Weigh 20 mg of DOPA standard and dilute to 100 mL with hydrochloric acid solution. Prepare fresh before use.

[0081] 2.3 Reagent A: Take 4.3 mL of hydrochloric acid and dilute it with pure water to a final volume of 100 mL.

[0082] 2.4 Reagent B: Take 10g of sodium molybdate dihydrate and 10g of sodium nitrite, dissolve them in pure water and bring the volume to 100mL.

[0083] 2.5 C Reagent: Take 4.0g of sodium hydroxide and dilute it with pure water to a final volume of 100mL.

[0084] 2.6 Instruments and equipment: Ultraviolet spectrophotometer (UV-265).

[0085] 3 steps

[0086] 3.1 Take 0 mL, 0.1 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL and 3.0 mL of DOPA standard solution respectively, dilute with hydrochloric acid solution (2.1) to 10 mL, shake well and set aside.

[0087] 3.2 Take 1 mL of the above standard solution (3.1), add 0.5 mL of reagent A to each test tube, and then add 1.5 mL of reagent B and 2 mL of reagent C to each test tube in sequence (reagent C should be added within 5 min after reagent B is added), and shake well.

[0088] 3.3 Take 1 mL of the sample to be tested into a 10 mL test tube and operate according to the above method. Use tube 0 as a blank and use a 1 cm cuvette to measure the absorbance of the prepared standard solution and sample solution at a wavelength of 500 nm.

[0089] 3.4 Plot a standard curve with DOPA concentration on the x-axis and absorbance on the y-axis, and then calculate the DOPA content of the sample based on the measured absorbance of the sample solution.

[0090] Example 1

[0091] The antioxidants prepared in Examples 1-6 and Comparative Examples 1-16 were subjected to stability testing and dopamine content testing using the above-described stability testing and dopamine testing methods, wherein the initial dopamine content was set as the dopamine content baseline value (i.e., 100%).

[0092] The formula for calculating the percentage of dopa content in samples after 1 month / 3 months is: Dopa content percentage = (detected mussel content / initial mussel content) * 100%. The results are as follows: Figure 1 As shown in Table 6.

[0093] Table 6. Stability and dopamine content detection results of Examples 1-6 and Comparative Examples 1-16

[0094] From Table 6 and Figure 1 It can be seen that after 3 months of storage, the appearance color of the samples prepared in Examples 1-3 was not significantly different from the initial state, and the dopa content retention rate was about 99%. Compared with Examples 4-6 (only antioxidant A and antioxidant B) and Comparative Examples 1-16, the color change of the samples in Examples 1-3 was more slight and the decrease in dopa content was significantly reduced. The above results indicate that the multi-mechanism synergistic antioxidant strategy adopted in Examples 1-3 can effectively reduce the oxidation degree of mussel adhesive protein and slow down its oxidation rate by complementary coverage of the hydrophilic-hydrophobic interface of the entire system through different mechanisms.

[0095] The test results from Examples 1, Comparative Examples 1-4, and Comparative Example 16 show that the protective effect on mussel adhesive protein was reduced to a certain extent when antioxidant A, antioxidant C, antioxidant B, or antioxidant B and C were used alone, or when the content of antioxidants B and C in the antioxidant composition was reduced.

[0096] At 40°C, the mussel adhesive protein compositions of Comparative Examples 1, 2, and 16 turned pale yellow after one month and yellow after three months, with the dopamine content decreasing from 100% to 58.6%, 57.3%, and 58.1%, respectively. The composition of Comparative Example 3 (with reduced antioxidant B content) turned slightly yellow after one month at 40°C and also turned yellow after three months, with the dopamine content decreasing to 59.2% during the same period. The composition of Comparative Example 4 (with reduced antioxidant C content), although less oxidized than the mussel adhesive protein of Comparative Examples 1-3, still had a dopamine content that decreased to 90.2% after three months compared to Examples 1-3.

[0097] This demonstrates that the antioxidant composition of the present invention achieves excellent protective effects through a multi-mechanism synergy: the reducing sulfur groups (such as thiol groups) in antioxidant B can maintain the active form of antioxidant A for a long time, thereby enhancing the overall antioxidant activity; at the same time, the hydrophobic antioxidant C complements antioxidant A through structural complementarity, fully covering the hydrophilic and hydrophobic regions of mussel adhesive protein, further enhancing the protective effect.

[0098] The test results from Examples 1 and Comparative Examples 5-8 show that the antioxidant protective effect on mussel adhesive protein is significantly reduced when any one component of antioxidant A (containing ectoin, glycerol, polyvinylpyrrolidone, and mussel adhesive protein Mfp-6) is missing (compared to the complete component of antioxidant A). It should be noted that the absence of ectoin and mussel adhesive protein Mfp-6 has a more significant impact on the oxidation of mussel adhesive protein than the absence of glycerol and polyvinylpyrrolidone. The above results indicate that ectoin binds to reactive oxygen species with Mfp-6 to stop the oxidation process, thereby slowing down the oxidation rate and degree of mussel adhesive protein from the source. Although glycerol and polyvinylpyrrolidone have a slightly weaker direct inhibitory effect on oxidation, they enhance the antioxidant effect by constructing a stable microenvironment. The four components together constitute a multi-component synergistic network of antioxidant A.

[0099] Comparative Example 9 had an ectoine concentration below the optimal range; Comparative Example 10 had an Mfp-6 concentration below the optimal range; Comparative Example 11 had an glycerol concentration below the optimal range; and Comparative Example 12 had a PVP concentration below the optimal range. In these four comparative examples, due to insufficient concentrations of key components, the components could not synergistically construct an effective antioxidant system, resulting in their antioxidant function not being fully realized, and consequently, failing to effectively protect the mussel adhesive protein.

[0100] In Comparative Example 13, the amount of ectoine added exceeded the suitable range. After being stored at room temperature for 3 months, the dopa content of the product prepared with ectoine decreased by 9.7% compared to Example 1. This is because ectoine, as a water-soluble component, significantly increases the viscosity of the formulation system when added at high concentrations. When antioxidants containing reduced sulfur are also present in the system, the high viscosity environment hinders the uniform dispersion of these antioxidants, causing some areas of mussel adhesive protein to fail to come into contact with sufficient antioxidants, thus losing effective protection and ultimately resulting in a decrease in antioxidant effect. In Comparative Example 14, the amount of mussel adhesive protein type 6 (Mfp-6) added exceeded the suitable range. After being stored at room temperature for 3 months, the dopa content of the product prepared with ectoine decreased by 25.3% compared to Example 1. The core reason is that the Mfp-6 molecule contains a dopa (DOPA) group, which has strong reducing properties and readily reacts with oxidizing factors in the system. When the amount of Mfp-6 added is too high, its own dopa group will preferentially bind to oxidizing factors, that is, it consumes the antioxidant components in the system through self-oxidation, disrupting the concentration balance of 'antioxidant components-mussel adhesive protein' in the system. This results in some mussel adhesive protein not being able to fully contact the core antioxidant, ultimately leading to a decrease in the retention rate of the dopa group. In Comparative Example 15, the amount of glycerol added exceeded the appropriate range. After the product prepared was placed at room temperature for 3 months, the dopa content was not significantly different from that of Example 1. However, the excessively high proportion of glycerol would lead to a significant increase in the viscosity of the entire system, which is not conducive to subsequent compounding with other skin care ingredients or processing into final skin care products (such as lotions, serums, etc.), and would adversely affect the production process and the skin feel of the product.

[0101] In summary, antioxidant A in the composition effectively protects mussel adhesive protein and significantly slows down the oxidation degree and rate of mussel adhesive protein preparations. In antioxidant A, the active hydrogen atoms and amino groups of ectoine and the sulfhydryl groups (-SH) in the cysteine ​​abundant in Mfp-6 can bind with hydroxyl radicals (・OH) and peroxy radicals (ROO・) generated during the oxidation of mussel adhesive protein, thereby halting the oxidation reaction. Simultaneously, the hydroxyl groups (-OH) and carbonyl groups (C=O) in glycerol and polyvinylpyrrolidone (PVP) in antioxidant A can form hydrogen bonds with the hydroxyl and amino groups on the lysine residues in mussel adhesive protein, forming a "polymer-protein complex protective layer." This layer acts as a shield, preventing direct contact between oxidizing substances (oxygen) and mussel adhesive protein, thus avoiding its oxidation.

[0102] Example 2

[0103] The compositions prepared in Examples 7-8 and Comparative Examples 17-18 were subjected to stability testing and dopamine content testing using the methods described above. The results are as follows: Figure 2 As shown in Table 7.

[0104] Table 7. Stability records of Examples 7-8 and Comparative Examples 17-18

[0105]

[0106] Experimental results show that the antioxidant provided by this invention can effectively slow down the oxidation and discoloration rate of mussel adhesive protein in aqueous products, significantly improve its stability in products, and achieve long-term stable preservation of mussel adhesive protein in aqueous products without discoloration.

[0107] Experimental Example

[0108] Twenty subjects were selected and divided into four groups of five: A, B, C, and D. Group A used the product formulated as shown in Example 7, Group B used the product formulated as shown in Example 8, Group C used the product formulated as shown in Comparative Example 17, and Group D used the product formulated as shown in Comparative Example 18. Examples 7 and Comparative Example 17 were used twice daily, while Examples 8 and Comparative Example 18 were used twice weekly. After one month of continuous use, facial images of the subjects were captured using VISIA. Figure 3 As shown, this was used to test the anti-inflammatory and soothing effects of the mussel adhesive protein composition.

[0109] The facial VISIA images of the subjects showed that after one month of use of the products from Examples 7 and 8, the area of ​​inflammation on the face was significantly reduced, indicating that Examples 7 and 8 had better anti-inflammatory and soothing effects. In contrast, the reduction in the area of ​​inflammation in subjects using the products from Comparative Examples 17 and 18 was less significant compared to those using the products from Examples 7 and 8. This indirectly proves that Comparative Examples 17 and 18 did not contain antioxidants, which prevented them from maintaining the stability of mussel adhesive protein in the product, leading to a gradual weakening of the anti-inflammatory effect.

[0110] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An antioxidant for improving the stability of mussel adhesive protein, characterized in that, The antioxidants include antioxidant A and antioxidant B. Antioxidant A is composed of ectoin, mussel adhesive protein type 6 (Mfp-6), glycerol and polyvinylpyrrolidone (PVP); antioxidant B is an antioxidant containing reduced sulfur.

2. The antioxidant according to claim 1, characterized in that, The mass ratio of ectoin, mussel adhesive protein type 6 (Mfp-6), glycerol and polyvinylpyrrolidone (PVP) is (1~3):(0.05~0.5):(0.1~2):(0.1~1).

3. The antioxidant according to claim 1, characterized in that, The antioxidant B is selected from one or more of sodium bisulfite, sodium sulfite, sodium metabisulfite, sodium thiosulfate, and lipoic acid.

4. The antioxidant according to claim 3, characterized in that, The mass ratio of antioxidant B to ectoine is (0.05~0.3):(1~3).

5. The antioxidant according to any one of claims 1 to 4, characterized in that, The antioxidant further includes antioxidant C, which is a hydrogen-donating antioxidant selected from one or more of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG); the mass ratio of antioxidant C to ectoine is (0.01~0.1):(1~3).

6. The use of the antioxidant according to any one of claims 1 to 5 in improving the stability of mussel adhesive protein.

7. A composition containing mussel adhesive protein, characterized in that, The composition comprises the antioxidant as described in any one of claims 1 to 5, mussel adhesive protein, and water.

8. The composition according to claim 7, characterized in that, The mussel adhesive protein in the composition has a mass percentage (wt) of 0.01% to 30%, preferably 0.01% to 5%.

9. The method for preparing the composition containing mussel adhesive protein according to claim 7, characterized in that, The preparation method is (A) or (B). The preparation method (A) includes the following steps: (1) Weigh out the required amount of water and introduce an inert gas into it; (2) Add antioxidant A and antioxidant B, mussel adhesive protein to the water and stir until completely dissolved; (3) Put the solution obtained in step (2) into a bottle, and pass in inert gas again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Then stop passing the gas and seal the bottle. Alternatively, the preparation method (B) includes the following steps: (1) Disperse the required amount of antioxidant C with glycerol in antioxidant A to obtain solution 1; (2) Weigh out the required amount of water and introduce an inert gas into it; (3) Add solution 1, the other components of antioxidant A except glycerol, antioxidant B and mussel adhesive protein to the water in sequence, and stir until completely dissolved; (4) Put the solution obtained in step (3) into a bottle, and pass in inert gas again until the oxygen concentration in the solution no longer decreases and tends to stabilize. Then stop passing gas, seal the bottle, and the solution is obtained.

10. The use of the composition according to any one of claims 7 to 8 in the preparation of medical devices, daily chemical products or biopharmaceutical products.

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