Mussel mucoprotein physical barrier protective gel and preparation method thereof

Abstract

The invention belongs to the technical field of gel preparation, and particularly relates to mussel mucoprotein physical barrier protective gel and a preparation method thereof. The mussel mucin polymer disclosed by the invention can quickly form a compact and flexible isolating membrane with self-repairing capability on the surface of vaginal mucosa. The isolating membrane can be firmly attached to the surface of a mucous membrane based on the strong biological adhesiveness of mussel mucin, can directly block sperm penetration and limit sperm swimming, and also can effectively block pathogenic microorganisms such as viruses and bacteria from contacting the mucous membrane. The acid environment provided by the lactic acid-citric acid buffer system not only can directly inhibit sperm activity, but also can enhance the stability and adhesiveness of the mussel mucin polymer, so that the mussel mucin polymer can be better attached to vaginal mucosa, and the action time of a physical barrier is prolonged. The chitosan quaternary ammonium salt has the effects of killing bacteria and viruses and the like, and the hydroxypropyl methyl cellulose serving as a thickening agent can improve the viscosity and the stability of the gel, so that the gel can be better retained in the vagina, and the action time is prolonged.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gel preparation, and particularly relates to a mussel mucin physical barrier protection gel and a preparation method thereof. BACKGROUND

[0002] Unintended pregnancy and sexually transmitted diseases (STDs) are serious global public health challenges. Viral infections such as human papillomavirus (HPV), whose persistent infection of high-risk types is directly related to major diseases such as cervical cancer, pose a long-term threat to women's reproductive health. Bacterial infections such as gonorrhea and chlamydia, which are prone to cause pelvic inflammatory disease and tubal adhesion, significantly increase the risk of infertility. More importantly, whether it is the invasion of pathogens themselves or the widespread use of some chemical spermicides, it is easy to destroy the healthy vaginal microecology dominated by lactic acid bacteria, with a pH value maintained at 3.8-4.5. Once the microecology is imbalanced and the pH value is increased, the physical barrier function will be weakened, forming a vicious cycle of "easy infection -> deteriorating microenvironment -> more susceptible to infection", making it particularly urgent to develop a product that can effectively protect and maintain the health of the microecology.

[0003] Existing protection means have obvious limitations. Hormonal contraception cannot prevent STDs. Traditional physical barriers such as condoms are effective but their use compliance is affected by multiple factors. Female physical barrier products (such as female condoms) generally have the problems of short retention time, easy displacement and shedding, resulting in unreliable protection. Chemical spermicides (such as nonoxynol-9) have the defects of strong irritation, destruction of microecological balance and inability to effectively block viruses.

[0004] Therefore, we propose a mussel mucin physical barrier protection gel and a preparation method thereof, which can effectively solve the deficiencies in the prior art. The physical barrier can inhibit sperm motility and reduce the risk of accidental pregnancy, and can also prevent the risk of cross-infection between the sexes and block and inhibit the spread of viruses and sexually transmitted diseases. SUMMARY

[0005] The present application aims to solve the existing problems and provides a mussel mucin physical barrier protection gel and a preparation method thereof.

[0006] The present application is achieved by the following technical solutions: A mussel mucin physical barrier protection gel is made of the following raw materials by weight percentage: 1.0-1.8% of mussel mucin polymer, 0.1-0.5% of chitosan quaternary ammonium salt, 0.5-0.7% of hydroxypropyl methyl cellulose, and the balance of purified water, and the overall pH is adjusted to 3.8-4.2 by a lactic acid-citric acid buffer system.

[0007] Further, the preparation of the mussel mucin polymer comprises the following steps: (1) Dissolve the mussel adhesive protein in weakly basic PBS buffer according to the mass-volume ratio of 1 g: 50-60 mL, add a small amount of dopamine oxidase according to the proportion of 0.5 U / mg mussel adhesive protein, constant temperature stirring at 25°C and 200-300 rpm for 30-40 min, filter the solution with a 0.2 μm microporous filter after the reaction is completed, and obtain a pretreated mussel adhesive protein solution; (2) Dissolve the quaternary ammonium chitosan in deionized water according to the mass-volume ratio of 1 g: 20-25 mL, stir at 200-300 rpm until dissolved at a water bath temperature of 50-55°C, then add glycidyl acrylate at a rate of 1 mL / min under N2 protection according to the molar ratio of quaternary ammonium chitosan amino group to glycidyl acrylate 1:1.5, stir at 40-45°C and 100-200 rpm for 10-12 h, transfer the reaction solution to a dialysis bag, and dialyze and purify with deionized water for 48-50 h, replace the deionized water every 6-8 h, and freeze-dry after dialysis to obtain a functional crosslinking agent; (3) Dissolve the pretreated mussel adhesive protein solution and the functional crosslinking agent in PBS buffer according to the mass ratio of 50-60:1, then add a photo initiator and a catechol-Fe 3+ complex solution, stir and mix uniformly, then stand at room temperature for 5-7 min, form a mussel adhesive protein quinone-HACC amino dynamic Schiff base bond, then transfer the solution to a quartz reactor, and irradiate under 365 nm UV for 5-7 min while maintaining magnetic stirring at 100-200 rpm; (4) After the reaction is completed, transfer the polymer solution to a dialysis bag, dialyze with PBS buffer containing 0.1 mM EDTA for 24 h, replace the liquid every 6-8 h, and then freeze-dry and grind into powder.

[0008] Further, the pH of the PBS buffer in step (1) is 8.0; The total enzyme amount of dopamine oxidase is 2500 U.

[0009] Further, the pH of the PBS buffer in step (3) is 7.4; The photo initiator is photo initiator 12959, and the final concentration is 0.05% w / v.

[0010] Further, the volume ratio of the catechol-Fe 3+ complex solution to the pretreated mussel adhesive protein solution in step (3) is 5:1; The catechol-Fe 3+Preparation method of complex solution: FeCl3·6H2O and catechol are added into deionized water according to a mass ratio of 27:34, and stirred at room temperature to form a 0.1mM catechol-Fe 3+ complex solution.

[0011] Further, the power of the UV light irradiation in step (3) is 300W, and the irradiation distance is 10-12cm.

[0012] Further, the preparation method of the lactic acid-citric acid buffer system is as follows: anhydrous citric acid is dissolved in purified water, and stirred until completely dissolved to obtain a citric acid solution with a concentration of 0.055-0.0625mol / L; under the condition of stirring at 200-300rpm, a pipette is used to add 85% lactic acid solution to the citric acid solution until the pH is 3.8-4.2.

[0013] A preparation method of a mussel mucin physical barrier protection gel, comprising the following steps: S1, according to the formula amount, prepare each raw material, add purified water to the reaction kettle, heat to 60-65℃, and add hydroxypropyl methyl cellulose under the condition of stirring at 200-300rpm, after stirring for 15-20min, add chitosan quaternary ammonium salt, and continue to stir for 10-15min; S2, cool to 40-43℃, under the condition of stirring at 200-300rpm, add mussel mucin polymer powder, after stirring and mixing, adjust the pH of the whole solution to 3.8-4.2 with a lactic acid-citric acid buffer system, and then add purified water to constant volume, then transfer the solution to a homogenizing emulsifier for homogenizing emulsification, and then perform sterilization and preservation.

[0014] Further, the homogenizing emulsification treatment condition in step S3 is 2000rpm homogenizing emulsification for 5-7min.

[0015] Compared with the prior art, the present application has the following advantages: The mussel mucus protein in the mussel mucus protein physical barrier protection gel of the present application can form a tight, flexible and self-repairing isolation film on the surface of the vaginal mucosa through the synergistic effect of dynamic Schiff base bond and covalent cross-linking network. The isolation film can be firmly attached to the mucosa surface relying on the strong biological adhesion of mussel mucus, which can not only directly block the penetration of sperm and limit the movement of sperm, but also effectively block the contact of pathogenic microorganisms such as viruses (human papilloma virus HPV) and bacteria with the mucosa, and limit the colonization of pathogens on the mucosa surface through the physical barrier. The acidic environment provided by the lactic acid-citric acid buffer system not only directly inhibits the activity of sperm, but also enhances the stability and adhesion of mussel mucus protein, so that it can better adhere to the vaginal mucosa and prolong the action time of the physical barrier. Chitosan quaternary ammonium salt has the effect of killing bacteria and viruses, and hydroxypropyl methyl cellulose as a thickening agent can improve the viscosity and stability of the gel, so that the gel can better stay in the vagina and prolong the action time. DETAILED DESCRIPTION

[0016] In order to further explain the present application, the following specific examples are combined for illustration.

[0017] Example 1 A preparation method of a mussel mucus protein physical barrier protection gel, comprising the following steps: S1, according to the formula, prepare each raw material: mussel mucus protein 1.0%, chitosan quaternary ammonium salt 0.1%, hydroxypropyl methyl cellulose 0.5%, and the rest is purified water; S2, add purified water to the reaction kettle, heat to 60℃, add hydroxypropyl methyl cellulose under the condition of 200rpm stirring, stir for 15min, then add chitosan quaternary ammonium salt, continue to stir for 10min; S3, cool to 40℃, under the condition of 200rpm stirring, add mussel mucus protein powder, stir evenly, then adjust the pH of the whole solution to 3.8 with lactic acid-citric acid buffer system, and then add purified water to constant volume, then transfer the solution to a homogenizer at 2000rpm for 5min, and then sterilize and store; The preparation method of the lactic acid-citric acid buffer system is as follows: dissolve anhydrous citric acid in purified water, stir until completely dissolved to obtain a citric acid solution with a concentration of 0.0625mol / L, and then add 85% lactic acid solution dropwise to the citric acid solution under the condition of 300rpm stirring until the pH is 3.8; The preparation of the mussel mucus protein includes the following steps: (1) Dissolve the mussel myoglobin in weakly basic PBS buffer (pH 8.0) according to the mass-volume ratio of 1 g:50 mL, add a small amount of dopamine oxidase (total enzyme amount is 2500 U) according to the proportion of 0.5 U / mg mussel myoglobin, constant temperature stirring at 25℃ and 200 rpm for 30 min, filter the solution with 0.2 μm microporous membrane after the reaction is completed, and obtain a pretreated mussel myoglobin solution; (2) Dissolve the quaternary ammonium chitosan in deionized water according to the mass-volume ratio of 1 g:20 mL, stir at 200 rpm until dissolved at a water bath temperature of 50℃, then drop glycidyl acrylate at a rate of 1 mL / min under N2 protection according to the molar ratio of quaternary ammonium chitosan amino group to glycidyl acrylate 1:1.5, stir at 40℃ and 100 rpm for 10 h, transfer the reaction solution to a dialysis bag, and dialyze and purify with deionized water for 48 h, replace the deionized water every 6 h, and freeze-dry after dialysis to obtain a functional crosslinking agent; (3) Dissolve the pretreated mussel myoglobin solution and the functional crosslinking agent in PBS buffer (pH 7.4) according to the mass ratio of 50:1, then add a photoinitiator 12959 (final concentration is 0.05% w / v) and a catechol-Fe 3+ complex solution, the volume ratio of the catechol-Fe 3+ complex solution to the pretreated mussel myoglobin solution is 5:1, stir and mix uniformly, then stand for 5 min at room temperature to form a mussel myoglobin quinone-HACC amino dynamic Schiff base bond, then transfer the solution to a quartz reactor, and irradiate under 365 nm UV for 5 min, the power is 300 W, the irradiation distance is 10 cm, and the magnetic stirring is kept at 100 rpm at the same time; The preparation method of the catechol-Fe 3+ complex solution is as follows: add FeCl3·6H2O and catechol into deionized water according to the mass ratio of 27:34, stir at room temperature to form a 0.1 mM catechol-Fe 3+ complex solution; (4) After the reaction is completed, transfer the polymer solution to a dialysis bag, dialyze with PBS buffer containing 0.1 mM EDTA for 24 h, replace the liquid every 6 h, remove free Fe 3+ , and freeze-dry after dialysis, then grind into powder.

[0018] Example 2 A preparation method of a mussel myoglobin physical barrier protection gel, comprising the following steps: S1, prepare each raw material according to the formula amount: mussel myoglobin polymer 1.4%, chitosan quaternary ammonium salt 0.3%, hydroxypropyl methyl cellulose 0.6%, and the rest is purified water; S2. Add purified water to the reactor, heat to 62°C, add hydroxypropyl methylcellulose while stirring at 250 rpm, add chitosan quaternary ammonium salt after stirring for 17 min, and continue stirring for 12 min. S3. Cool down to 41℃, add mussel adhesive protein polymer powder under stirring at 250rpm, stir and mix well, adjust the pH of the whole solution to 4 with lactic acid-citric acid buffer system, make up to volume with purified water, transfer the solution to a homogenizer and emulsify at 2000rpm for 6min, and then sterilize and store. The preparation method of the lactic acid-citric acid buffer system is as follows: anhydrous citric acid is dissolved in purified water and stirred until completely dissolved to obtain a citric acid solution with a concentration of 0.06 mol / L. Under the stirring state of 250 rpm, a lactic acid solution with a mass fraction of 85% is added dropwise to the citric acid solution with a pipette until the pH reaches 4.0. The preparation of the mussel adhesive protein polymer includes the following steps: (1) Dissolve mussel adhesive protein in weakly alkaline PBS buffer (pH 8.0) at a mass-to-volume ratio of 1g:55mL, add a small amount of dopamine oxidase (total enzyme amount of 2500U) at a ratio of 0.5U / mg mussel adhesive protein, stir at 25℃ and 250rpm for 35min, and after the reaction, filter the solution through a 0.2μm microporous membrane to obtain the pretreated mussel adhesive protein solution; (2) Quaternized chitosan was dissolved in deionized water at a mass-volume ratio of 1g:22mL. The mixture was stirred at 250rpm in a water bath at 52℃ until dissolved. Then, under N2 protection, glycidyl acrylate was added dropwise at a rate of 1mL / min at a molar ratio of 1:1.5 between the amino group of quaternized chitosan and glycidyl acrylate. The mixture was stirred at 150rpm in a water bath at 43℃ for 11h. The reaction solution was transferred to a dialysis bag and purified by dialysis with deionized water for 49h. The deionized water was replaced every 7h. After dialysis, the functionalized crosslinking agent was obtained by freeze drying. (3) Dissolve the pretreated mussel adhesive protein solution and the functionalized cross-linking agent in PBS buffer (pH 7.4) at a mass ratio of 55:1, and then add photoinitiator 12959 (final concentration 0.05% w / v) and catechol-Fe 3+ Complex solution, catechol-Fe 3+ The volume ratio of the complex solution to the pretreated mussel adhesive protein solution was 5:1. After stirring and mixing, the mixture was allowed to stand at room temperature for 6 minutes to form mussel adhesive protein quinone-HACC amino dynamic Schiff base bonds. The solution was then transferred to a quartz reactor and irradiated under 365nm UV light for 6 minutes at a power of 300W and an irradiation distance of 11cm, while maintaining magnetic stirring at 150rpm. The catechol-Fe3+ Preparation method of complex solution: FeCl3·6H2O and catechol were added to deionized water at a mass ratio of 27:34, and stirred at room temperature to form 0.1 mM catechol-FeCl3·6H2O. 3+ Complex solution; (4) After the reaction is complete, transfer the polymer solution to a dialysis bag and dialyze with PBS buffer containing 0.1 mM EDTA for 24 h, changing the buffer every 7 h to remove free Fe. 3+ After dialysis, the solution is freeze-dried and then ground into powder.

[0019] Example 3 A method for preparing a mussel adhesive protein physical barrier protective gel includes the following steps: S1. Prepare the raw materials according to the formula: 1.8% mussel adhesive protein polymer, 0.5% chitosan quaternary ammonium salt, 0.7% hydroxypropyl methylcellulose, and the remainder is purified water; S2. Add purified water to the reactor, heat to 65°C, add hydroxypropyl methylcellulose while stirring at 300 rpm, add chitosan quaternary ammonium salt after stirring for 20 min, and continue stirring for 15 min. S3. Cool down to 43℃, add mussel adhesive protein polymer powder while stirring at 300 rpm, stir and mix well, adjust the pH of the overall solution to 4.2 with lactic acid-citric acid buffer system, make up to volume with purified water, transfer the solution to a homogenizer and emulsify at 2000 rpm for 7 minutes, and then sterilize and store. The preparation method of the lactic acid-citric acid buffer system is as follows: anhydrous citric acid is dissolved in purified water and stirred until completely dissolved to obtain a citric acid solution with a concentration of 0.0625 mol / L. Under stirring at 300 rpm, a lactic acid solution with a mass fraction of 85% is added dropwise to the citric acid solution using a pipette until the pH reaches 4.2. The preparation of the mussel adhesive protein polymer includes the following steps: (1) Dissolve mussel adhesive protein in weakly alkaline PBS buffer (pH 8.0) at a mass-volume ratio of 1g:60mL, add a small amount of dopamine oxidase (total enzyme amount of 2500U) at a ratio of 0.5U / mg mussel adhesive protein, stir at 25℃ and 300rpm for 40min, and after the reaction is completed, filter the solution through a 0.2μm microporous membrane to obtain the pretreated mussel adhesive protein solution; (2) Quaternized chitosan was dissolved in deionized water at a mass-volume ratio of 1g:25mL. The mixture was stirred at 300rpm in a water bath at 55℃ until dissolved. Then, under N2 protection, glycidyl acrylate was added dropwise at a rate of 1mL / min at a molar ratio of 1:1.5 between the amino group of quaternized chitosan and glycidyl acrylate. The mixture was stirred at 200rpm in a water bath at 45℃ for 12h. The reaction solution was transferred to a dialysis bag and purified by dialysis with deionized water for 50h. The deionized water was replaced every 8h. After dialysis, the functionalized crosslinking agent was obtained by freeze drying. (3) Dissolve the pretreated mussel adhesive protein solution and the functionalized cross-linking agent in PBS buffer (pH 7.4) at a mass ratio of 60:1, and then add photoinitiator 12959 (final concentration 0.05% w / v) and catechol-Fe 3+ Complex solution, catechol-Fe 3+ The volume ratio of the complex solution to the pretreated mussel adhesive protein solution was 5:1. After stirring and mixing, the mixture was allowed to stand at room temperature for 7 minutes to form mussel adhesive protein quinone-HACC amino dynamic Schiff base bonds. The solution was then transferred to a quartz reactor and irradiated under 365nm UV light for 7 minutes at a power of 300W and an irradiation distance of 12cm, while maintaining magnetic stirring at 200rpm. The catechol-Fe 3+ Preparation method of complex solution: FeCl3·6H2O and catechol were added to deionized water at a mass ratio of 27:34, and stirred at room temperature to form 0.1 mM catechol-FeCl3·6H2O. 3+ Complex solution; (4) After the reaction is complete, transfer the polymer solution to a dialysis bag and dialyze it with PBS buffer containing 0.1 mM EDTA for 24 h, changing the buffer every 8 h to remove free Fe3+. After dialysis, freeze dry and then grind into powder.

[0020] Comparative Example 1 Compared with Example 2, Comparative Example 1 omits the mussel adhesive protein polymer and its preparation, while the other steps are the same as in Example 2.

[0021] Comparative Example 2 Compared with Example 2, Comparative Example 2 replaces the mussel adhesive protein polymer with untreated mussel adhesive protein and omits the preparation of the mussel adhesive protein polymer, while the other steps are the same as in Example 2.

[0022] Comparative Example 3 Compared with Example 2, Comparative Example 3 omits hydroxypropyl methylcellulose, while the other steps are the same as in Example 2.

[0023] Animals: SPF-grade sexually mature KM mice, 200 males and 180 females, weighing 30±3g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Animal Use License No. SCXK (Sichuan) 2020-030. All rats were not kept in the same cage before the experiment.

[0024] Nonoxynol-9 gel (batch number: 230201, Yaoda Pharmaceutical Co., Ltd., specification: 40mg nonoxynol-9 gel per gram); Estradiol benzoate injection (batch number: 20230201, Hefei Xinkexin Animal Pharmaceutical Co., Ltd., specification: 4mg estradiol benzoate per 2mL); Hydrocortisone butyrate cream (batch number: 23041017, Tianjin Jinyao Pharmaceutical Co., Ltd., specification: ... Each gram contains 1 mg of hydrocortisone.

[0025] 1. Experiment on the antifertility effect of gynecological gel Female mice were randomly divided into 7 groups of 10 each, namely, nonoxynol ether gel, Examples 1-3, and Comparative Examples 1-3, with 20 μL / mouse administered vaginally. Estradiol formate was injected intramuscularly into each female mouse 24 hours before mating to induce estrus. After administration, each group was mated with male mice at a 1:1 ratio, and the vaginal lavage fluid of the female mice was continuously monitored at 24-hour intervals. The presence of sperm or vaginal plugs in the vaginal lavage fluid was used as the endpoint event for terminating the mating experiment.

[0026] The pregnancy suppression rate (R) is calculated using the following formula.

[0027] In the formula, n1 represents the number of non-pregnant animals and n2 represents the number of experimental animals.

[0028] The experimental results are shown in Table 1 below.

[0029] Table 1 As can be seen from Table 1 above, the contraceptive rate of the gel of the present invention reaches 100%, which is better than the positive control nonoxynol ether gel (90%), indicating that the gel prepared by the present invention has excellent contraceptive effect.

[0030] 2. Gel vaginal irritation test Female mice were randomly divided into 7 groups of 10 mice each: nonoxynol ether gel group, Examples 1-3, and Comparative Examples 1-3. Using a 2mm diameter mouse gavage needle, physiological saline and 20μL of gel were injected into the vagina once daily. After administration, the mice were hung upside down for 10 minutes with their tails attached to prevent leakage. The mice were administered the medication continuously for 14 days. Twenty-four hours after the last administration, the mice were sacrificed, and their vaginas were longitudinally incised. The redness, swelling, bleeding, ulceration, and presence of discharge in each mouse's vagina were visually observed.

[0031] Pathological scoring criteria: According to Eckstein's irritation assessment criteria, four indicators are used for scoring: congestion, edema, inflammatory cell infiltration, and ulcer epithelium. The degree of irritation for each indicator is scored from 0 to 4 points according to its severity.

[0032] The experimental results are shown in Table 2 below.

[0033] Table 2 As can be seen from Table 2 above, the total vaginal irritation scores of Examples 1-3 were significantly lower than those of nonoxynol ether gel, indicating that the gel prepared by the method of the present invention has good biocompatibility and low irritation.

[0034] 3. Effect of the gel on xylene-induced ear swelling in mice Male mice were randomly divided into 8 groups of 10 mice each: saline, hydrocortisone, Examples 1-3, and Comparative Examples 1-3. Each mouse in each group received a 50 μL dose of the corresponding drug applied evenly to both sides of the right ear once daily for 6 consecutive days. One hour after the last administration, 30 μL of xylene was applied evenly to both sides of the right ear. The left ear served as a control and was left untreated. Mice were sacrificed 0.5 hours later, and both ears were cut off along the auricular baseline. Circular ear pieces of equal area were punched from the same location in both ears using a 6 mm punch. The pieces were weighed, and the ear swelling degree (M) and swelling inhibition rate (Z) were calculated for each group according to the formulas to compare the differences in anti-inflammatory effects.

[0035] In the formula, m R The weight of the right ear is expressed in mg and m. L The weight of the left ear is indicated in mg.

[0036] In the formula, M0 represents the average swelling degree of the saline group; M1 represents the average swelling degree of the drug-treated group.

[0037] Table 3 As shown in Table 3 above, the swelling inhibition rate of Examples 1 to 3 all exceeded 70%, which was significantly better than that of the comparative example, indicating that the gel of the present invention has significant anti-salt and anti-allergic effects.

[0038] 4. Mussel adhesive protein physical barrier protective gel antibacterial effect Examples 1-3: Mussel adhesive protein physical barrier protective gels, comparative examples 1-3: gel samples, and negative control (sterile saline).

[0039] Test strain: Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212); Gram-negative bacteria: Escherichia coli (ATCC 25922).

[0040] Reagents and instruments: Nutrient agar medium, MH broth medium; sterile petri dishes, pipettes, constant temperature incubator (37℃), colony counter, and clean bench.

[0041] Experimental methods: Agar diffusion method (qualitative and quantitative detection of inhibition zone size) (1) Preparation of bacterial culture: Each strain was inoculated into MH broth medium and cultured at 37°C with shaking for 18-24 h. The bacterial concentration was adjusted to 1×10⁻⁶ with sterile physiological saline. 6 CFU / mL (McFarland turbidimetric assay).

[0042] (2) Culture medium plate preparation: Take the sterilized nutrient agar medium, melt it, cool it to about 50°C, add the above bacterial solution (bacterial solution: medium = 1:100, v / v), mix well and pour it into sterile petri dishes (90 mm in diameter), 20 mL per dish, and make bacterial agar plates after solidification.

[0043] (3) Sample processing and inoculation: Use a sterile punch (6 mm in diameter) to punch holes in the bacterial plate. Add 50 μL of test sample (Examples 1-3, Comparative Examples 1-3, Negative Control) to each well. Set up 3 replicates for each strain.

[0044] (4) Culture and result determination: After the plates are placed in a constant temperature incubator at 37℃ for 24 hours, the diameter (mm) of the inhibition zone around each well is measured with vernier calipers. The larger the diameter of the inhibition zone, the stronger the antibacterial activity (inhibition zone diameter ≥15mm is high sensitivity, 10~14mm is medium sensitivity, <10mm is low sensitivity or no antibacterial effect).

[0045] Table 4 As shown in Table 4 above, the gel of the present invention produces significant inhibition zones against all four types of bacteria. The antibacterial effect is significant.

[0046] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mussel adhesive protein physical barrier protective gel, characterized in that, Made from the following raw materials by weight percentage: The composition includes 1.0-1.8% mussel adhesive protein polymer, 0.1-0.5% chitosan quaternary ammonium salt, 0.5-0.7% hydroxypropyl methylcellulose, with the remainder being purified water. The overall pH is adjusted to 3.8-4.2 using a lactic acid-citric acid buffer system.

2. The mussel adhesive protein physical barrier protective gel according to claim 1, characterized in that, The preparation of the mussel adhesive protein polymer includes the following steps: (1) Dissolve mussel adhesive protein in weakly alkaline PBS buffer at a mass-to-volume ratio of 1g:50~60mL, add a small amount of dopamine oxidase at a ratio of 0.5U / mg mussel adhesive protein, stir at 200~300rpm for 30~40min at 25℃, and filter the solution through a 0.2μm microporous membrane after the reaction to obtain the pretreated mussel adhesive protein solution; (2) Dissolve quaternized chitosan in deionized water at a mass-volume ratio of 1g:20~25mL. Stir at 200~300rpm in a water bath at 50~55℃ until dissolved. Then, under N2 protection, add glycidyl acrylate dropwise at a rate of 1mL / min at a molar ratio of quaternized chitosan amino to glycidyl acrylate of 1:1.

5. Stir at 40~45℃ and 100~200rpm for 10~12h. Transfer the reaction solution to a dialysis bag and dialysis with deionized water for 48~50h. Change the deionized water every 6~8h. After dialysis, freeze dry to obtain the functionalized crosslinking agent. (3) Dissolve the pretreated mussel adhesive protein solution and the functionalized cross-linking agent in PBS buffer at a mass ratio of 50~60:1, and then add the photoinitiator and catechol-Fe. 3+ After stirring and mixing the complex solution, let it stand at room temperature for 5-7 minutes to form mussel adhesive protein quinone-HACC amino dynamic Schiff base bonds. Then, transfer the solution to a quartz reactor and irradiate it under 365nm UV light for 5-7 minutes while maintaining magnetic stirring at 100-200rpm. (4) After the reaction is complete, transfer the polymer solution to a dialysis bag and dialyze it with PBS buffer containing 0.1 mM EDTA for 24 h. Change the buffer every 6-8 h. After dialysis, freeze dry the solution and grind it into powder.

3. The mussel adhesive protein physical barrier protective gel according to claim 2, characterized in that, The pH of the PBS buffer described in step (1) is 8.0; The total amount of dopamine oxidase was 2500 U.

4. The mussel adhesive protein physical barrier protective gel according to claim 2, characterized in that, The pH of the PBS buffer described in step (3) is 7.4; The photoinitiator was photoinitiator 12959, with a final concentration of 0.05% w / v.

5. The mussel adhesive protein physical barrier protective gel according to claim 2, characterized in that, The catechol-Fe described in step (3) 3+ The volume ratio of the complex solution to the pretreated mussel adhesive protein solution was 5:1; The catechol-Fe 3+ Preparation method of complex solution: FeCl3·6H2O and catechol were added to deionized water at a mass ratio of 27:34, and stirred at room temperature to form 0.1 mM catechol-FeCl3·6H2O. 3+ Complex solution.

6. The mussel adhesive protein physical barrier protective gel according to claim 2, characterized in that, The UV light power in step (3) is 300W and the light distance is 10~12cm.

7. The mussel adhesive protein physical barrier protective gel according to claim 1, characterized in that, The preparation method of the lactic acid-citric acid buffer system is as follows: dissolve anhydrous citric acid in purified water and stir until completely dissolved to obtain a citric acid solution with a concentration of 0.055~0.0625mol / L. Under stirring at 200~300rpm, add 85% lactic acid solution dropwise to the citric acid solution using a pipette until the pH reaches 3.8~4.

2.

8. A method for preparing the mussel adhesive protein physical barrier protective gel according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1. Prepare each raw material according to the formula amount, add purified water to the reaction vessel, heat to 60~65℃, add hydroxypropyl methylcellulose while stirring at 200~300rpm, add chitosan quaternary ammonium salt after stirring for 15~20min, and continue stirring for 10~15min. S2. Cool to 40~43℃, add mussel adhesive protein polymer powder while stirring at 200~300rpm, stir and mix well, adjust the pH of the overall solution to 3.8~4.2 with a lactic acid-citric acid buffer system, make up to volume with purified water, transfer the solution to a homogenizer for homogenization and emulsification, and then sterilize and store.

9. The method for preparing a mussel adhesive protein physical barrier protective gel according to claim 8, characterized in that, The homogenization emulsification conditions described in step S3 are: homogenization emulsification at 2000 rpm for 5-7 minutes.

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