Oral gargle based on synthetic biological material

Abstract

The invention relates to the technical field of gargle preparation, in particular to an oral gargle based on synthetic biological materials. Comprising the following steps: preparing a pre-crosslinked mussel protein adhesion network precursor solution, preparing a temperature-sensitive gel matrix, preparing a liquid main agent, preparing a nano-emulsion and preparing the oral gargle based on the synthetic biological material. Through sequential combination of tea polyphenol pretreatment and immobilized tyrosinase catalysis, the recombinant mussel byssus protein is subjected to directional chemical modification, in a weakly alkaline buffer solution, a catechol structure of tea polyphenol can be combined with a protein chain to play a role in pre-organization and stabilization, and the tea polyphenol plays a role in improving the stability of the mussel byssus protein while participating in network construction. The inherent antibacterial and antioxidant functions are integrated into a material matrix in situ, a mechanical stability foundation is created for the subsequent compounding of a protein network and temperature-sensitive hydrogel, the uniform coexistence of a protein phase and a hydrogel phase is ensured, and the wet adhesion strength of the protein material is essentially improved.

Description

Technical Field

[0001] This invention relates to the field of mouthwash preparation technology, and specifically to an oral mouthwash based on synthetic biomaterials. Background Technology

[0002] Oral mouthwashes, as an important class of oral care and adjunctive treatment products, utilize the fluid dynamics of rinsing to help flush away food debris and some plaque from hard-to-reach areas such as the gingival sulcus and interdental spaces, effectively supplementing daily mechanical cleaning. Simultaneously, the antibacterial ingredients in mouthwashes, such as chlorhexidine, cetylpyridinium chloride, and essential oils, can kill or inhibit airborne bacteria, interfering with plaque formation and thus chemically supporting plaque control, reducing the risk of cavities and gingivitis. For patients with gingivitis, oral ulcers, dry mouth, or post-operative conditions, specially formulated mouthwashes can soothe inflammation, relieve pain, promote healing, and moisturize the mouth, improving patient comfort and quality of life. Therefore, oral mouthwashes, through their liquid formulation, provide convenient and comprehensive contact and treatment of the entire oral cavity. They play an indispensable role in maintaining oral health and preventing and adjunctive treating diseases.

[0003] Most existing mouthwashes are based on simple solutions of water, alcohol, or surfactants, which have weak adhesion to the moist oral mucosa and tooth enamel. The active ingredients are quickly diluted and washed away by saliva and expelled with rinsing, resulting in an effective duration often measured in minutes. This makes it difficult to maintain their antibacterial and restorative effects, failing to provide long-term protection. Users need to rinse frequently to maintain the effect. Furthermore, many promising natural antibacterial ingredients, such as plant essential oils or bioactive molecules like peptides and enzymes, have poor water solubility, are chemically unstable, and are volatile. They are prone to precipitation, degradation, or rapid loss in traditional solutions, making it difficult to maintain activity within their shelf life. The oral environment requires long-term, precise, and multifunctional intervention, but existing products generally suffer from short retention time, unstable ingredients, limited functionality, and a lack of intelligent response. Therefore, this invention provides a mouthwash based on synthetic biomaterials to solve the problems of the existing technology. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide an oral rinse based on synthetic biomaterials.

[0005] A mouthwash based on synthetic biomaterials, the preparation process of which specifically includes the following steps: S1: Preparation of pre-crosslinked mussel protein adhesion network precursor solution The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer, tea polyphenols were added, and the mixture was stirred in an ice bath. Immobilized tyrosinase was added and reacted for 50-60 min. The reaction system was then transferred to an ice-water bath to cool to 0-4℃ to stop enzyme activity. The solids were then removed by filtration, and the supernatant was retained. The supernatant was filtered through a sterile filter membrane to obtain the pre-crosslinked mussel protein adhesion network precursor solution. S2: Preparation of temperature-sensitive gel matrix Glycerin and deionized water were mixed and cooled in an ice-water bath. The mixture was stirred and methylcellulose powder was added to obtain methylcellulose sol. Calcium chloride and disodium hydrogen phosphate were added to glycerin and stirred to obtain glycerin suspension. Then, β-glycerophosphate sodium is mixed with deionized water pre-cooled to 0-4℃ to obtain β-glycerophosphate sodium solution. The mixture is stirred, and the prepared glycerol suspension and methylcellulose sol are added to the stirred β-glycerophosphate sodium solution in sequence to obtain a thermosensitive gel matrix. S3: Preparation of liquid main agent Stir 30-40 parts by weight of the pre-crosslinked mussel protein adhesion network precursor solution at a speed of 200-250 r / min, then slowly add 70-80 parts by weight of the thermosensitive gel matrix over a time of 15-20 min. After the addition is complete, increase the speed to 300-350 r / min and stir for 30-40 min to obtain the liquid main agent. S4: Preparation of nanoemulsions Poloxamer 188 was added to purified water and stirred until clear. After cooling, an aqueous phase was obtained. Thymol and eucalyptus oil were mixed evenly to obtain an oil phase. Under high-speed magnetic stirring, the oil phase was added dropwise to the aqueous phase and stirred continuously to obtain a crude emulsion. The crude emulsion was emulsified using a probe-type ultrasonic disruptor to obtain a nanoemulsion, which was stored in the dark at 4°C. S5: Preparation of oral rinse solution based on synthetic biomaterials The nanoemulsion was slowly added dropwise to the liquid main agent, followed by the addition of menthol propylene glycol solution and citric acid solution. The mixture was stirred, the pH was adjusted, and the solution was filtered and sterilized in a sterile operating table to obtain an oral rinse solution based on synthetic biomaterials.

[0006] Furthermore, step S1 of the preparation process, which involves preparing a pre-crosslinked mussel protein adhesion network precursor solution, specifically includes the following steps: The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer pre-cooled to 0-4℃ under magnetic stirring at 300-320r / min. The solid-liquid ratio of the recombinant mussel byssal protein lyophilized powder to PBS buffer was 8g:(10-12)mL. The mixture was stirred for 10-16min to ensure complete dispersion and dissolution. Then, 10-20% of the system mass of tea polyphenols was added, and the mixture was placed in an ice bath and stirred at 300-320r / min for 10-20min. The system temperature was then raised to 26-28℃, the stirring speed was adjusted to 150-200 r / min, 4-5% of the immobilized tyrosinase was added, and the reaction was allowed to proceed for 50-60 min. The reaction system was then transferred to an ice-water bath to cool to 0-4℃ to stop enzyme activity. Subsequently, solids were removed by magnetic adsorption filtration, and the supernatant was retained. The supernatant was then filtered through a 0.22 μm sterile filter membrane to obtain the pre-crosslinked mussel protein adhesion network precursor solution.

[0007] Furthermore, step S2 of the preparation process, which involves preparing the temperature-sensitive gel matrix, specifically includes the following steps: After mixing glycerol and deionized water, the mixture is cooled to 0-4℃ in an ice-water bath. Then, stirring is started, and methylcellulose powder is added at a speed of 600-800 r / min. The mixture is then stirred at a speed of 400-500 r / min for 15-20 min. After that, the mixture is allowed to stand at 0-4℃ for 4-5 h to obtain methylcellulose sol. Add 0.1-0.3 parts by weight of calcium chloride and 0.1-0.2 parts by weight of disodium hydrogen phosphate to 5-7 parts by weight of glycerol, and stir at 400-500 r / min for 30-60 min to obtain a glycerol suspension; Next, mix 1-2 parts by weight of sodium β-glycerophosphate with 40-45 parts by weight of deionized water pre-cooled to 0-4℃ to obtain a sodium β-glycerophosphate solution. Start stirring at 300-400 r / min. Add the prepared glycerol suspension and methylcellulose sol to the sodium β-glycerophosphate solution while stirring, and increase the stirring speed to 500-600 r / min. Stir for 1-2 h to obtain a thermosensitive gel matrix.

[0008] Furthermore, step S4 of the preparation process, which involves preparing the nanoemulsion, specifically includes the following steps: Add 1-2 parts by weight of poloxamer 188 to 8-10 parts by weight of purified water, stir magnetically at 40-45°C until completely dissolved and clear, and cool to room temperature (22-24°C) to obtain the aqueous phase; Mix 0.15-0.25 parts by weight of thymol with 0.15-0.3 parts by weight of eucalyptus oil to obtain the oil phase; Under high-speed magnetic stirring at 800-1000 r / min, the oil phase was added dropwise to the aqueous phase, and stirring was continued for 20 minutes to obtain a crude emulsion. The crude emulsion was placed in an ice bath at 0-4℃ and emulsified using a probe-type ultrasonic disruptor to obtain a nano-emulsion, which was then stored at 4℃ away from light.

[0009] Furthermore, step S5 of the preparation process, which involves preparing an oral rinse solution based on synthetic biomaterials, specifically includes the following steps: 100-110 parts by weight of the liquid main agent were stirred at 200-300 rpm on a magnetic stirrer in an ice bath at 0-4℃. 10-12 parts by weight of the nanoemulsion, pre-cooled to 0-4℃, were slowly added dropwise to the liquid main agent over 1-3 minutes. Then, 3-3.2 parts by weight of menthol propylene glycol solution and 10-12 parts by weight of citric acid solution were added. The stirring speed was increased to 300-340 rpm, and stirring was continued in the ice bath for 20-30 minutes. The pH was adjusted to 6.6-7, and the solution was filtered and sterilized using a 0.22 μm PES membrane in a sterile operating room to obtain an oral gargle based on synthetic biomaterials.

[0010] Furthermore, in step S2 of the preparation process, the mass ratio of glycerol, deionized water and methylcellulose powder is 10:30:(1-2).

[0011] Furthermore, in step S1 of the preparation process, the immobilized tyrosinase is specifically prepared by first electrostatically adsorbing and immobilizing the tyrosinase solution with a magnetic metal-organic framework material Fe3O4 as a carrier, and then chemically cross-linking it to obtain a magnetically immobilized tyrosinase.

[0012] Furthermore, the parameters of the probe-type ultrasonic disruptor in step S4 of its preparation process are: power 50W, ultrasonic on for 2 seconds, ultrasonic off for 2 seconds, and total time 5-8 minutes.

[0013] Furthermore, the menthol-propylene glycol solution in step S5 of the preparation process is obtained by mixing menthol and propylene glycol in a mass ratio of 1:(40-42).

[0014] Furthermore, the citric acid solution in step S5 of the preparation process is a solution prepared by mixing citric acid and water with a citric acid content of 1-2% (w / v).

[0015] The present invention has the following advantages: 1. This invention utilizes a sequential combination of tea polyphenol pretreatment and immobilized tyrosinase catalysis to directionally chemically modify recombinant mussel byssal proteins. In a weakly alkaline buffer, the catechol structure of tea polyphenols can bind to the protein chain, playing a pre-organization and stabilizing role. Subsequently, under mild conditions, the immobilized tyrosinase specifically catalyzes the oxidation of tyrosine residues in the protein to highly active dopaquinone, driving a controllable covalent cross-linking reaction between molecules. Furthermore, the immobilized enzyme can be physically separated after the reaction, achieving immediate termination of the reaction and reuse of the enzyme, making the process controllable and economical. While participating in network construction, the inherent antibacterial and antioxidant functions of tea polyphenols are in situ integrated into the material matrix, creating a mechanical stability basis for the subsequent composite of the protein network with a thermosensitive hydrogel, ensuring the uniform coexistence of the protein phase and the hydrogel phase, and fundamentally improving the wet adhesion strength of the protein material.

[0016] 2. This invention constructs a multifunctional temperature-sensitive gel system by combining sodium β-glycerophosphate and glycerol with calcium phosphate salts and incorporating them into a gel based on methylcellulose gum. The principle is as follows: First, glycerol serves as a dispersion medium for the calcium phosphate salts, allowing calcium chloride and disodium hydrogen phosphate to form nanoscale, metastable calcium phosphate clusters. Then, sodium β-glycerophosphate acts as an ionic strength modifier, integrating into the calcium phosphate clusters to adjust their strength and improve their compatibility. Subsequently, the calcium phosphate clusters are encapsulated by methylcellulose gum. The calcium phosphate clusters, integrated and regulated by glycerol and sodium β-glycerophosphate, form a complex system. Adding them to the methylcellulose gum matrix increases the complexity of the matrix, resulting in a gel with better adhesion properties. Furthermore, the calcium phosphate clusters themselves have the function of remineralizing tooth enamel. Encapsulated in the gel, they are slowly released in the oral cavity, and combined with improved adhesion, achieve long-lasting adhesion and repair of the oral cavity by the gel components in the mouthwash.

[0017] 3. This invention combines poloxamer 188 with thymol and eucalyptus oil, enabling the oil-phase functional components of thymol and eucalyptus oil to disperse and integrate in an aqueous thermosensitive matrix. The nonionic surfactant poloxamer 188, through its amphiphilic molecular structure, effectively encapsulates and stabilizes the mixed oil droplets of thymol and eucalyptus oil, forming fine-particle-size oil-in-water nanoemulsions. Furthermore, the combined mixed oil droplets of thymol and eucalyptus oil maintain the liquid adhesion of the thermosensitive matrix and protect the growth of heat-sensitive components such as mussel protein. The activity of the polymer is enhanced, thereby improving its antibacterial properties. Furthermore, the nanoemulsion formed by the mixed oil droplets prepared from thymol and eucalyptus oil is also introduced as a homogenizing unit into the liquid main agent containing mussel protein and thermosensitive polymer, avoiding phase separation between the thermosensitive gel component and the mussel protein adhesion network, ensuring the uniformity of the final product composition, and integrating it into a homogeneous, sterile liquid formulation. This formulation is a gargling liquid at room temperature and can be transformed into an adhesive gel at oral temperature, thereby achieving long-term retention and sustained release of antibacterial components. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of the oral rinse solution based on synthetic biomaterials according to the present invention. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this invention. Example

[0020] A mouthwash based on synthetic biomaterials, the preparation process of which is as follows: Figure 1 As shown, it includes the following steps: S1: Preparation of pre-crosslinked mussel protein adhesion network precursor solution The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer pre-cooled to 0℃ under magnetic stirring at 300r / min. The solid-liquid ratio of the recombinant mussel byssal protein lyophilized powder to PBS buffer was 8g:10mL. The mixture was stirred for 10min to ensure complete dispersion and dissolution. Then, 10% of the system mass of tea polyphenols was added, and the mixture was placed in an ice bath and stirred at 300r / min for 10min. The system temperature was then raised to 26°C, the stirring speed was adjusted to 150 r / min, and 4% (by mass) of immobilized tyrosinase was added. The reaction was allowed to proceed for 50 min, and then the reaction system was transferred to an ice-water bath to cool to 0°C to stop enzyme activity. Subsequently, magnetic adsorption filtration was performed to remove solids, and the supernatant was retained. The supernatant was then filtered through a 0.22 μm sterile filter membrane to obtain a pre-crosslinked mussel protein adhesion network precursor solution. Specifically, the immobilized tyrosinase was first electrostatically adsorbed and immobilized with the tyrosinase solution using Fe3O4 as a magnetic metal-organic framework material, and then chemically crosslinked to obtain a magnetically immobilized tyrosinase.

[0021] S2: Preparation of temperature-sensitive gel matrix Glycerin and deionized water were mixed and cooled to 0°C in an ice-water bath. Stirring was then started, and methylcellulose powder was added at 600 rpm. The mixture was then stirred at 400 rpm for 15 min and allowed to stand at 0°C for 4 h to obtain methylcellulose sol. The mass ratio of glycerin, deionized water and methylcellulose powder was 10:30:1. Add 0.1 parts by mass of calcium chloride and 0.1 parts by mass of disodium hydrogen phosphate to 5 parts by mass of glycerol, and stir at 400 r / min for 30 min to obtain a glycerol suspension; Then, 1 part by mass of sodium β-glycerophosphate was mixed with 40 parts by mass of deionized water pre-cooled to 0°C to obtain a sodium β-glycerophosphate solution. The solution was stirred at 300 r / min. The prepared glycerol suspension and methylcellulose sol were added to the sodium β-glycerophosphate solution while it was being stirred. The stirring speed was increased to 500 r / min and stirred for 1 h to obtain a thermosensitive gel matrix.

[0022] S3: Preparation of liquid main agent 30 parts by weight of the pre-crosslinked mussel protein adhesion network precursor solution were stirred at 200 r / min, and then 70 parts by weight of the thermosensitive gel matrix were slowly added dropwise over a period of 15 min. After the addition was completed, the stirring speed was increased to 300 r / min and stirred for 30 min to obtain the liquid main agent. S4: Preparation of nanoemulsions Add 1 part by mass of poloxamer 188 to 8 parts by mass of purified water, stir magnetically at 40°C until completely dissolved and clear, and cool to room temperature (22°C) to obtain the aqueous phase; Mix 0.15 parts by weight of thymol with 0.15 parts by weight of eucalyptus oil until homogeneous to obtain the oil phase; Under high-speed magnetic stirring at 800 r / min, the oil phase was added dropwise to the aqueous phase, and stirring was continued for 20 minutes to obtain a crude emulsion. The crude emulsion was placed in an ice bath at 0°C and emulsified using a probe-type ultrasonic disruptor to obtain a nano-emulsion, which was then stored at 4°C in the dark. The parameters of the probe-type ultrasonic disruptor were: power 50W, ultrasonic on for 2 seconds, ultrasonic off for 2 seconds, and total time 5 minutes.

[0023] S5: Preparation of oral rinse solution based on synthetic biomaterials 100 parts by weight of the liquid main agent were stirred at 200 r / min on a magnetic stirrer in an ice bath at 0℃. 10 parts by weight of the nanoemulsion pre-cooled to 0℃ were slowly added dropwise to the liquid main agent over a time of 1 min. Then, 3 parts by weight of menthol propylene glycol solution and 10 parts by weight of citric acid solution were added. The stirring speed was increased to 300 r / min, and stirring was continued in the ice bath for 20 min. The pH value was adjusted to 6.6. The solution was filtered and sterilized using a 0.22 μm PES filter membrane in a sterile operating table to obtain an oral gargle solution based on synthetic biomaterials. The menthol-propylene glycol solution is obtained by mixing menthol and propylene glycol at a mass ratio of 1:40; the citric acid solution is a solution prepared by mixing citric acid with a citric acid content of 1% (w / v) and water. Example

[0024] A mouthwash based on synthetic biomaterials, the preparation process of which is as follows: Figure 1 As shown, it includes the following steps: S1: Preparation of pre-crosslinked mussel protein adhesion network precursor solution The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer pre-cooled to 2°C under magnetic stirring at 310 r / min. The solid-liquid ratio of the recombinant mussel byssal protein lyophilized powder to PBS buffer was 8 g: 11 mL. The mixture was stirred for 14 min to ensure complete dispersion and dissolution. Then, 15% of the system mass of tea polyphenols was added, and the mixture was placed in an ice bath and stirred at 310 r / min for 15 min. The system temperature was then raised to 27°C, the stirring speed was adjusted to 175 r / min, and 4% (by mass) of immobilized tyrosinase was added. The reaction was allowed to proceed for 55 min, and the reaction system was then transferred to an ice-water bath to cool to 2°C to stop enzyme activity. Subsequently, magnetic adsorption filtration was performed to remove solids, and the supernatant was retained. The supernatant was then filtered through a 0.22 μm sterile filter membrane to obtain a pre-crosslinked mussel protein adhesion network precursor solution. Specifically, the immobilized tyrosinase was first electrostatically adsorbed and immobilized with the tyrosinase solution using Fe3O4 as a magnetic metal-organic framework material, followed by chemical crosslinking to obtain a magnetically immobilized tyrosinase.

[0025] S2: Preparation of temperature-sensitive gel matrix Glycerin and deionized water were mixed and cooled to 2°C in an ice-water bath. Stirring was then started, and methylcellulose powder was added at 700 rpm. The mixture was then stirred at 450 rpm for 17 min and allowed to stand at 2°C for 4.5 h to obtain methylcellulose sol. The mass ratio of glycerin, deionized water and methylcellulose powder was 10:30:1.5. Add 0.2 parts by mass of calcium chloride and 0.15 parts by mass of disodium hydrogen phosphate to 6 parts by mass of glycerol, and stir at 450 r / min for 45 min to obtain a glycerol suspension; 1.5 parts by mass of sodium β-glycerophosphate were mixed with 43 parts by mass of deionized water pre-cooled to 2°C to obtain a sodium β-glycerophosphate solution. The solution was stirred at 350 r / min. The prepared glycerol suspension and methylcellulose sol were added to the stirred sodium β-glycerophosphate solution in sequence, and the stirring speed was increased to 550 r / min. The mixture was stirred for 1.5 h to obtain a thermosensitive gel matrix.

[0026] S3: Preparation of liquid main agent 35 parts by weight of the pre-crosslinked mussel protein adhesion network precursor solution were stirred at 220 r / min, and then 75 parts by weight of the thermosensitive gel matrix were slowly added dropwise over a period of 17 min. After the addition was completed, the stirring speed was increased to 320 r / min and stirred for 35 min to obtain the liquid main agent. S4: Preparation of nanoemulsions Add 1.5 parts by weight of poloxamer 188 to 7 parts by weight of purified water, stir magnetically at 43°C until completely dissolved and clear, and cool to room temperature (23°C) to obtain the aqueous phase; Mix 0.2 parts by weight of thymol with 0.2 parts by weight of eucalyptus oil until homogeneous to obtain the oil phase; Under high-speed magnetic stirring at 900 r / min, the oil phase was added dropwise to the aqueous phase, and stirring was continued for 20 minutes to obtain a crude emulsion. The crude emulsion was placed in an ice bath at 0-4℃ and emulsified using a probe-type ultrasonic disruptor to obtain a nano-emulsion. It was then stored at 4℃ in the dark. The parameters of the probe-type ultrasonic disruptor were: power 50W, ultrasonic on for 2 seconds, ultrasonic off for 2 seconds, and total time 7 minutes.

[0027] S5: Preparation of oral rinse solution based on synthetic biomaterials 105 parts by weight of the liquid main agent were stirred at 250 r / min on a magnetic stirrer in an ice bath at 2℃. 11 parts by weight of the nanoemulsion, pre-cooled to 2℃, were slowly added dropwise to the liquid main agent over a period of 2 min. Then, 3.1 parts by weight of menthol propylene glycol solution and 11 parts by weight of citric acid solution were added. The stirring speed was increased to 320 r / min, and stirring was continued in the ice bath for 25 min. The pH value was adjusted to 6.8. The solution was filtered and sterilized using a 0.22 μm PES filter membrane in a sterile operating table to obtain an oral gargle based on synthetic biomaterials. The menthol-propylene glycol solution is prepared by mixing menthol and propylene glycol at a mass ratio of 1:41; the citric acid solution is prepared by mixing citric acid with a citric acid content of 1.5% (w / v) and water. Example

[0028] A mouthwash based on synthetic biomaterials, the preparation process of which is as follows: Figure 1 As shown, it includes the following steps: S1: Preparation of pre-crosslinked mussel protein adhesion network precursor solution The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer pre-cooled to 4°C under magnetic stirring at 320 r / min. The solid-liquid ratio of the recombinant mussel byssal protein lyophilized powder to PBS buffer was 8 g: 12 mL. The mixture was stirred for 16 min to ensure complete dispersion and dissolution. Then, 20% of the system mass of tea polyphenols was added, and the mixture was placed in an ice bath and stirred at 320 r / min for 20 min. The system temperature was then raised to 28°C, the stirring speed was adjusted to 200 r / min, and 5% (by mass) of immobilized tyrosinase was added. The reaction was allowed to proceed for 60 min, and the reaction system was then transferred to an ice-water bath to cool to 4°C to stop enzyme activity. Subsequently, magnetic adsorption filtration was performed to remove solids, and the supernatant was retained. The supernatant was then filtered through a 0.22 μm sterile filter membrane to obtain a pre-crosslinked mussel protein adhesion network precursor solution. Specifically, the immobilized tyrosinase was first electrostatically adsorbed and immobilized with the tyrosinase solution using Fe3O4 as a magnetic metal-organic framework material, followed by chemical crosslinking to obtain a magnetically immobilized tyrosinase.

[0029] S2: Preparation of temperature-sensitive gel matrix Glycerin and deionized water were mixed and cooled to 4°C in an ice-water bath. Stirring was then started, and methylcellulose powder was added at 800 rpm. The mixture was then stirred at 500 rpm for 20 minutes and allowed to stand at 4°C for 5 hours to obtain methylcellulose sol. The mass ratio of glycerin, deionized water and methylcellulose powder was 10:30:2. Add 0.3 parts by mass of calcium chloride and 0.2 parts by mass of disodium hydrogen phosphate to 7 parts by mass of glycerol, and stir at 500 r / min for 60 min to obtain a glycerol suspension; Two parts by mass of sodium β-glycerophosphate were mixed with 45 parts by mass of deionized water pre-cooled to 4°C to obtain a sodium β-glycerophosphate solution. The solution was stirred at 400 r / min. The prepared glycerol suspension and methylcellulose sol were added to the sodium β-glycerophosphate solution while it was being stirred. The stirring speed was increased to 600 r / min and stirred for 2 h to obtain a thermosensitive gel matrix.

[0030] S3: Preparation of liquid main agent 40 parts by weight of the pre-crosslinked mussel protein adhesion network precursor solution were stirred at 250 r / min, and then 80 parts by weight of the thermosensitive gel matrix were slowly added dropwise over a period of 20 min. After the addition was completed, the stirring speed was increased to 350 r / min and stirred for 40 min to obtain the liquid main agent. S4: Preparation of nanoemulsions Add 2 parts by mass of poloxamer 188 to 10 parts by mass of purified water, stir magnetically at 45°C until completely dissolved and clear, and cool to room temperature (24°C) to obtain the aqueous phase; Mix 0.25 parts by weight of thymol with 0.3 parts by weight of eucalyptus oil to obtain the oil phase; Under high-speed magnetic stirring at 1000 r / min, the oil phase was added dropwise to the aqueous phase, and stirring was continued for 20 minutes to obtain a crude emulsion; The crude emulsion was placed in an ice bath at 4°C and emulsified using a probe-type ultrasonic disruptor to obtain a nano-emulsion, which was then protected from light and stored at 4°C. The parameters of the probe-type ultrasonic disruptor were: power 50W, ultrasonic on for 2 seconds, ultrasonic off for 2 seconds, and total time 8 minutes.

[0031] S5: Preparation of oral rinse solution based on synthetic biomaterials 110 parts by weight of the liquid main agent were stirred at 300 r / min on a magnetic stirrer in an ice bath at 4°C. 12 parts by weight of the nanoemulsion, pre-cooled to 4°C, were slowly added dropwise to the liquid main agent over a period of 3 min. Then, 3.2 parts by weight of menthol propylene glycol solution and 12 parts by weight of citric acid solution were added. The stirring speed was increased to 340 r / min, and stirring was continued in the ice bath for 30 min. The pH value was adjusted to 7. The solution was filtered and sterilized using a 0.22 μm PES filter membrane in a sterile operating table to obtain an oral gargle solution based on synthetic biomaterials. The menthol-propylene glycol solution is prepared by mixing menthol and propylene glycol at a mass ratio of 1:42; the citric acid solution is prepared by mixing citric acid with a citric acid content of 2% (w / v) and water.

[0032] Comparative Example 1: Compared with Example 1, the difference of Comparative Example 1 is that tea polyphenols are not added in step S1, but tea polyphenols are replaced by immobilized tyrosinase by mass, and the rest of the steps remain unchanged. This is called Comparative Example 1.

[0033] Comparative Example 2: Compared with Example 1, the difference of Comparative Example 2 is that immobilized tyrosinase is not added in step S1, but tea polyphenols are replaced with an equal mass of immobilized tyrosinase. The other steps remain unchanged, and it is referred to as Comparative Example 2.

[0034] Comparative Example 3: Compared with Example 1, the difference of Comparative Example 3 is that sodium β-glycerophosphate is not added in step S2, but glycerol is replaced by an equal mass of sodium β-glycerophosphate. The other steps remain unchanged, and it is referred to as Comparative Example 3.

[0035] Comparative Example 4: Compared with Example 1, the difference of Comparative Example 4 is that in step S2, instead of preparing a suspension with glycerol, calcium chloride and disodium hydrogen phosphate, glycerol is replaced by sodium β-glycerophosphate in equal mass. Specifically, 0.1 parts by mass of calcium chloride and 0.1 parts by mass of disodium hydrogen phosphate are added to 5 parts by mass of sodium β-glycerophosphate and stirred at 400 r / min for 30 min to obtain a sodium β-glycerophosphate suspension. Next, 1 part by mass of sodium β-glycerophosphate was mixed with 40 parts by mass of deionized water pre-cooled to 0°C to obtain a sodium β-glycerophosphate solution. The mixture was stirred at 300 r / min, and the prepared sodium β-glycerophosphate suspension and methylcellulose sol were added sequentially to the stirred sodium β-glycerophosphate solution. The stirring speed was then increased to 500 r / min, and the mixture was stirred for 1 h to obtain a thermosensitive gel matrix. The remaining steps remained unchanged, and this was designated as Comparative Example 4.

[0036] Comparative Example 5: Compared with Example 1, Comparative Example 5 differs in that thymol is not added in step S4, but thymol is replaced by eucalyptus oil by mass, while the other steps remain unchanged. It is referred to as Comparative Example 5.

[0037] Comparative Example 6: Compared with Example 1, the difference of Comparative Example 6 is that eucalyptus oil is not added in step S4, but eucalyptus oil is replaced by thymol by mass, and the other steps remain unchanged. It is referred to as Comparative Example 6.

[0038] Comparative Example 7: Compared with Example 1, the difference of Comparative Example 7 is that poloxamer 188 is not added in step S4. Instead, thymol and eucalyptus oil are directly mixed to obtain the oil phase. The nanoemulsion in step S5 is replaced by the oil phase by an equal mass and added dropwise to the liquid main agent. The other steps remain unchanged. This is called Comparative Example 7.

[0039] Test the adhesive mechanical properties of Examples 1-3, Comparative Examples 1-4, and Comparative Example 7 in a simulated moist oral environment: Select freshly slaughtered pig oral mucosa, trim the oral mucosa into samples with a thickness of 2-3 mm and a smooth surface, fix them in the fixture of the lower platform of the testing machine with the mucosa side facing up, use a moist PBS cotton pad to keep the surface moist but without excess liquid accumulation, clean and dry the glass disc, apply double-sided tape to one side and firmly stick it to the fixture of the upper platform of the testing machine, and record the surface area of ​​the glass disc as A; Using a pipette, 10 μL of the oral gargle solution based on synthetic biomaterials prepared in Examples 1-3, Comparative Examples 1-4 and Comparative Example 7 was accurately pipetted and vertically dropped onto the central region of the porcine oral mucosa sample. The testing machine is manipulated to make the glass disc above descend at a constant speed of 1 mm / s, making contact with the droplets of oral gargle liquid based on synthetic biomaterials; A preload of 0.5 N was applied to the droplet and this contact state was maintained for 300 seconds while keeping the ambient humidity constant, so that the solutions of Examples 1-3, Comparative Examples 1-4 and Comparative Example 7 could spread fully at the interface, and the water and matrix could interact to form adhesion. After the contact time ended, the upper platform clamp moved vertically upwards at a speed of 10 mm / min to peel the disc from the mucosal surface. Five experiments were performed for each group. In each experiment, the testing machine software recorded the average value of the maximum force required for the peeling process in real time, which was denoted as F. max And calculate the adhesion strength, adhesion strength = F max / A, and the results are shown in Table 1.

[0040] Antibacterial tests were conducted on Examples 1-3, Comparative Examples 1-2, and Comparative Examples 5-7 using the quantitative suspension antibacterial test method in WS / T 650-2019 "Evaluation Methods for Antibacterial and Antimicrobial Effects". The selected strains were Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus mutans, and Porphyromonas gingivalis. The antibacterial rates were obtained, and the results are shown in Table 2.

[0041] Table 1 (Unit: kPa) Adhesion strength Example 1 27.8 Example 2 28.9 Example 3 29.4 Comparative Example 1 21.3 Comparative Example 2 22.4 Comparative Example 3 17.2 Comparative Example 4 17.8 Comparative Example 7 15.5 Table 2 (Unit: %) Antibacterial rate Staphylococcus aureus E. coli Candida albicans Streptococcus mutans Porphyromonas gingivalis Example 1 98.2 97.2 95.4 98.4 98.8 Example 2 98.3 97.5 95.5 98.6 98.8 Example 3 98.5 97.6 95.7 98.9 99.0 Comparative Example 1 96.4 95.8 93.1 92.1 95.2 Comparative Example 2 96.7 96.0 93.7 92.6 95.4 Comparative Example 5 96.5 95.2 92.7 91.6 94.7 Comparative Example 6 96.8 95.4 92.9 91.8 94.8 Comparative Example 7 95.2 94.1 91.4 90.5 93.3 As shown in Table 1, Examples 1-3 exhibited the highest adhesion strength, reaching 27.8-29.4 kPa. Their design effectively allows for adhesion to the mucosa in the moist environment of the oral cavity. In contrast, Comparative Examples 1-2 showed adhesion strengths of only 21.3-22.4 kPa. This indicates that in step S1, tea polyphenols are not only an antibacterial agent but also a key cross-linking agent for the adhesion network; while immobilized tyrosinase catalyzes this cross-linking reaction. Both are indispensable, jointly constructing a robust mussel protein adhesion network.

[0042] The adhesion strength of Comparative Examples 3-4 was 17.2-17.8 kPa, indicating that in step S2, sodium β-glycerophosphate as a thermosensitive trigger and glycerol as a moisturizing and plasticizing agent are indispensable for forming a thermosensitive gel matrix with suitable rheological properties and mucosal binding ability. The absence of either component would severely compromise the structural integrity of the matrix, resulting in a significant loss of overall adhesion.

[0043] Comparative Example 7 showed even lower adhesion strength, only 15.5 kPa. This indicates that the nonionic surfactant poloxamer 188, through its amphiphilic molecular structure, effectively encapsulates and stabilizes the mixed oil droplets of thymol and eucalyptus oil, forming a fine-particle-size oil-in-water nanoemulsion. This avoids phase separation between the temperature-sensitive gel components and the mussel protein adhesion network, and can transform into an adhesive gel at oral temperature. In other words, poloxamer 188 improves the adhesion performance of the temperature-sensitive gel after transforming the oil droplets into nanoemulsions.

[0044] As shown in Table 2, Examples 1-3 exhibited an antibacterial rate greater than 95.4% against the five test strains, demonstrating excellent and broad-spectrum antibacterial activity. Comparative Example 1, lacking tea polyphenols, and Comparative Example 2, lacking immobilized tyrosinase, showed a decrease in overall antibacterial rate, particularly against two key oral pathogens: *Streptococcus mutans* and *Porphyromonas gingivalis*, which decreased to 92.1%-92.6% and 95.2%-95.4%, respectively. This indicates that tea polyphenols themselves possess antibacterial activity. Furthermore, the pre-crosslinked mussel protein adhesion network formed synergistically with tea polyphenols and immobilized tyrosinase may enhance the retention and penetration of active ingredients on plaque / mucosal surfaces, thereby significantly improving the inhibitory ability against oral-specific pathogens. Comparative Examples 5 and 6 showed significantly lower antibacterial rates than the Examples 1-3, further demonstrating the synergistic antibacterial effect of thymol and eucalyptus oil in nanoemulsions. A single essential oil cannot achieve the broad-spectrum and highly effective antibacterial effect when combined with these two ingredients.

[0045] The antibacterial rates of various strains in Comparative Example 7 were lower than those in Comparative Examples 5 and 6, indicating that the nonionic surfactant poloxamer 188, through its amphiphilic molecular structure, effectively encapsulates and stabilizes the mixed oil droplets of thymol and eucalyptus oil, forming a water-in-oil nanoemulsion with fine particle size, which has a more significant effect on improving the antibacterial properties of the temperature-sensitive gel.

[0046] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims. Parts not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. An oral rinse solution based on synthetic biomaterials, characterized in that, Its preparation process specifically includes the following steps: S1: Preparation of pre-crosslinked mussel protein adhesion network precursor solution The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer, tea polyphenols were added, and the mixture was stirred in an ice bath. Immobilized tyrosinase was added and reacted for 50-60 min. The reaction system was then transferred to an ice-water bath to cool to 0-4℃ to stop enzyme activity. The solids were then removed by filtration, and the supernatant was retained. The supernatant was filtered through a sterile filter membrane to obtain the pre-crosslinked mussel protein adhesion network precursor solution. S2: Preparation of temperature-sensitive gel matrix Glycerin and deionized water were mixed and cooled in an ice-water bath. The mixture was stirred and methylcellulose powder was added to obtain methylcellulose sol. Calcium chloride and disodium hydrogen phosphate were added to glycerin and stirred to obtain glycerin suspension. Then, β-glycerophosphate sodium is mixed with deionized water pre-cooled to 0-4℃ to obtain β-glycerophosphate sodium solution. The mixture is stirred, and the prepared glycerol suspension and methylcellulose sol are added to the stirred β-glycerophosphate sodium solution in sequence to obtain a thermosensitive gel matrix. S3: Preparation of liquid main agent Stir 30-40 parts by weight of the pre-crosslinked mussel protein adhesion network precursor solution at a speed of 200-250 r / min, then slowly add 70-80 parts by weight of the thermosensitive gel matrix over a time of 15-20 min. After the addition is complete, increase the speed to 300-350 r / min and stir for 30-40 min to obtain the liquid main agent. S4: Preparation of nanoemulsions Poloxamer 188 was added to purified water and stirred until clear. After cooling, an aqueous phase was obtained. Thymol and eucalyptus oil were mixed evenly to obtain an oil phase. Under high-speed magnetic stirring, the oil phase was added dropwise to the aqueous phase and stirred continuously to obtain a crude emulsion. The crude emulsion was emulsified using a probe-type ultrasonic disruptor to obtain a nanoemulsion, which was stored in the dark at 4°C. S5: Preparation of oral rinse solution based on synthetic biomaterials The nanoemulsion was slowly added dropwise to the liquid main agent, followed by the addition of menthol propylene glycol solution and citric acid solution. The mixture was stirred, the pH was adjusted, and the solution was filtered and sterilized in a sterile operating table to obtain an oral rinse solution based on synthetic biomaterials.

2. The oral rinse solution based on synthetic biomaterials according to claim 1, characterized in that, Step S1 of its preparation process involves preparing a pre-crosslinked mussel protein adhesion network precursor solution, specifically including the following steps: The recombinant mussel byssal protein lyophilized powder was poured into PBS buffer pre-cooled to 0-4℃ under magnetic stirring at 300-320r / min. The solid-liquid ratio of the recombinant mussel byssal protein lyophilized powder to PBS buffer was 8g:(10-12)mL. The mixture was stirred for 10-16min to ensure complete dispersion and dissolution. Then, 10-20% of the system mass of tea polyphenols was added, and the mixture was placed in an ice bath and stirred at 300-320r / min for 10-20min. The system temperature was then raised to 26-28℃, the stirring speed was adjusted to 150-200 r / min, 4-5% of the immobilized tyrosinase was added, and the reaction was allowed to proceed for 50-60 min. The reaction system was then transferred to an ice-water bath to cool to 0-4℃ to stop enzyme activity. Subsequently, solids were removed by magnetic adsorption filtration, and the supernatant was retained. The supernatant was then filtered through a 0.22 μm sterile filter membrane to obtain the pre-crosslinked mussel protein adhesion network precursor solution.

3. The oral rinse solution based on synthetic biomaterials according to claim 2, characterized in that, Step S2 of its preparation process involves preparing the temperature-sensitive gel matrix, specifically including the following steps: After mixing glycerol and deionized water, the mixture is cooled to 0-4℃ in an ice-water bath. Then, stirring is started, and methylcellulose powder is added at a speed of 600-800 r / min. The mixture is then stirred at a speed of 400-500 r / min for 15-20 min. After that, the mixture is allowed to stand at 0-4℃ for 4-5 h to obtain methylcellulose sol. Add 0.1-0.3 parts by weight of calcium chloride and 0.1-0.2 parts by weight of disodium hydrogen phosphate to 5-7 parts by weight of glycerol, and stir at 400-500 r / min for 30-60 min to obtain a glycerol suspension; Next, mix 1-2 parts by weight of sodium β-glycerophosphate with 40-45 parts by weight of deionized water pre-cooled to 0-4℃ to obtain a sodium β-glycerophosphate solution. Start stirring at 300-400 r / min. Add the prepared glycerol suspension and methylcellulose sol to the sodium β-glycerophosphate solution while stirring, and increase the stirring speed to 500-600 r / min. Stir for 1-2 h to obtain a thermosensitive gel matrix.

4. The oral rinse solution based on synthetic biomaterials according to claim 3, characterized in that, Step S4 of its preparation process involves preparing the nanoemulsion, specifically including the following steps: Add 1-2 parts by weight of poloxamer 188 to 8-10 parts by weight of purified water, stir magnetically at 40-45°C until completely dissolved and clear, and cool to room temperature (22-24°C) to obtain the aqueous phase; Mix 0.15-0.25 parts by weight of thymol with 0.15-0.3 parts by weight of eucalyptus oil to obtain the oil phase; Under high-speed magnetic stirring at 800-1000 r / min, the oil phase was added dropwise to the aqueous phase, and stirring was continued for 20 minutes to obtain a crude emulsion. The crude emulsion was placed in an ice bath at 0-4℃ and emulsified using a probe-type ultrasonic disruptor to obtain a nano-emulsion, which was then stored at 4℃ away from light.

5. The oral rinse solution based on synthetic biomaterials according to claim 4, characterized in that, Step S5 of the preparation process involves preparing an oral rinse solution based on synthetic biomaterials, specifically including the following steps: 100-110 parts by weight of the liquid main agent were stirred at 200-300 rpm on a magnetic stirrer in an ice bath at 0-4℃. 10-12 parts by weight of the nanoemulsion, pre-cooled to 0-4℃, were slowly added dropwise to the liquid main agent over 1-3 minutes. Then, 3-3.2 parts by weight of menthol propylene glycol solution and 10-12 parts by weight of citric acid solution were added. The stirring speed was increased to 300-340 rpm, and stirring was continued in the ice bath for 20-30 minutes. The pH was adjusted to 6.6-7, and the solution was filtered and sterilized using a 0.22 μm PES membrane in a sterile operating room to obtain an oral gargle based on synthetic biomaterials.

6. The oral rinse solution based on synthetic biomaterials according to claim 3, characterized in that, In step S2 of its preparation process, the mass ratio of glycerol, deionized water and methylcellulose powder is 10:30:(1-2).

7. The oral rinse solution based on synthetic biomaterials according to claim 2, characterized in that, The immobilized tyrosinase in step S1 of the preparation process specifically involves electrostatic adsorption and immobilization of tyrosinase solution using Fe3O4 as a magnetic metal-organic framework material as a carrier, followed by chemical cross-linking to obtain a magnetically immobilized tyrosinase.

8. The oral rinse solution based on synthetic biomaterials according to claim 4, characterized in that, The parameters of the probe-type ultrasonic disruptor in step S4 of its preparation process are: power 50W, ultrasonic on for 2 seconds, ultrasonic off for 2 seconds, and total time 5-8 minutes.

9. The oral rinse solution based on synthetic biomaterials according to claim 5, characterized in that, The menthol-propylene glycol solution in step S5 of the preparation process is obtained by mixing menthol and propylene glycol in a mass ratio of 1:(40-42).

10. The oral rinse solution based on synthetic biomaterials according to claim 5, characterized in that, The citric acid solution in step S5 of the preparation process is a solution prepared by mixing citric acid and water with a citric acid content of 1-2% (w / v).

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