Bacteriostatic mouthwash as well as preparation method and application thereof

Abstract

The invention discloses antibacterial mouthwash as well as a preparation method and application thereof, and belongs to the technical field of daily chemical products. According to the invention, egg white lysozyme, ferulic acid and grape seed procyanidine are taken as core components, and the mouthwash with an efficient bacteriostatic effect is developed. And the antibacterial rates of streptococcus mutans and porphyromonas gingivalis are 96.88% and 96.30% respectively. Researches show that bacterial biofilms can be destroyed by using the mouthwash, and the biofilm quantity is reduced by 53.7% after the bacterial biofilms are treated for 24 hours; extracellular polysaccharide is inhibited, the content of water-insoluble glucan is reduced by 62.3%, and synthesis of a key cariogenic matrix is hindered; the acid production capacity is regulated and controlled, the pH change of 24-hour culture is only 1.50, and xylitol competitively inhibits glycolysis and cooperates with ferulic acid to block bacterial metabolism. The mouthwash prepared by the invention avoids the problem of enzyme activity inhibition of the ionic surfactant of the traditional mouthwash, takes effect more quickly, and provides a basis for developing natural, safe and efficient functional mouthwash.

Description

Technical Field

[0001] This invention relates to the field of daily chemical products technology, and in particular to an antibacterial mouthwash, its preparation method, and its application. Background Technology

[0002] Oral health is an important component of overall health, and dental caries and periodontal disease are the most common oral diseases worldwide. These diseases are closely related to the excessive proliferation of oral pathogens. *Streptococcus mutans* (s. mutans) is considered a major cause of dental caries, producing acid through carbohydrate metabolism and forming biofilms, leading to demineralization of tooth hard tissues. *Porphyromonas gingivalis* (p. gingivalis) is a key pathogen of periodontal disease, causing gingivitis and periodontal tissue destruction. Most commercially available mouthwashes contain chemical antibacterial agents such as chlorhexidine and cetylpyridinium chloride. While these ingredients have some antibacterial effects, long-term use may lead to side effects such as oral flora imbalance, tooth discoloration, and altered taste. Developing mouthwashes based on natural ingredients that are safe and effective has become an important direction in oral care product research.

[0003] Lysozyme, as a natural immune protein, targets and dissolves Gram-positive bacteria by hydrolyzing the β-1,4 glycosidic bonds in the peptidoglycan of bacterial cell walls, and also has a permeation-sensitizing effect on the outer membranes of Gram-negative bacteria. Its high safety and low resistance characteristics align with the needs of maintaining the oral microecology. However, single lysozyme has limitations in the oral environment, including easy inactivation, short duration of action, and insufficient permeability to biofilms. Recent research has focused on overcoming these application bottlenecks through compounding and carrier technology. In existing mouthwash formulations, anionic surfactants easily denature and inactivate lysozyme, and while mineral components such as hydroxyapatite can immobilize enzyme molecules, they weaken its antibacterial efficacy.

[0004] This invention innovatively optimizes the above-mentioned defects by using multi-component spatial cooperation and stability enhancement, and is expected to achieve synergistic inhibition of two bacteria under low concentration lysozyme (LZM) conditions, providing a case for the development of bio-based mouthwash. Summary of the Invention

[0005] The purpose of this invention is to provide an antibacterial mouthwash, its preparation method, and its application, thereby addressing the problems existing in the prior art. This invention, through multi-component spatial cooperation and stability enhancement, is expected to achieve synergistic inhibition of two bacteria under low-concentration LYZ conditions, providing a case study for the development of bio-based mouthwashes.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In a first aspect, the present invention provides an antibacterial mouthwash, wherein each 100 mL of the antibacterial mouthwash comprises the following components: 0.5-1.5 g lysozyme, 0.5-0.9 g ferulic acid, 0.01-0.05 g grape seed proanthocyanidins, 6-14 mL glycerin, 0.01-0.05 g menthol, and 0.15-0.35 g xylitol.

[0008] Preferably, the antibacterial mouthwash also contains 0.3-0.5g sodium citrate and 0.03-0.05g ascorbic acid.

[0009] Secondly, the present invention also provides a method for preparing the aforementioned antibacterial mouthwash, comprising the following steps:

[0010] Menthol, ferulic acid, and grape seed proanthocyanidins were dissolved in propylene glycol to obtain the propylene glycol phase.

[0011] Sodium citrate was mixed with water and sterilized, and xylitol and ascorbic acid were added to dissolve it to obtain the aqueous phase.

[0012] The glycerol was sterilized to obtain the oil phase;

[0013] The propylene glycol phase was added to the aqueous phase and stirred, then the oil phase was added and stirred until the volume was adjusted.

[0014] Add lysozyme to the solution after it has been brought to a certain volume, stir, filter, and fill to obtain the antibacterial mouthwash.

[0015] Preferably, the amount of menthol used is 0.03g, the amount of ferulic acid used is 0.8g, and the amount of grape seed proanthocyanidins used is 0.02g.

[0016] Preferably, the amount of sodium citrate is 0.3g, the amount of xylitol is 0.2g, and the amount of ascorbic acid is 0.03g.

[0017] Preferably, the amount of glycerol used is 12 mL, and the amount of propylene glycol used is 10 times the amount of ferulic acid used.

[0018] Thirdly, the present invention also provides the application of the aforementioned antibacterial mouthwash in the preparation of oral care products.

[0019] Fourthly, the present invention also provides an oral care product, wherein the oral care product includes the antibacterial mouthwash.

[0020] Preferably, the oral care product also includes a pharmaceutically acceptable carrier or excipient.

[0021] The present invention discloses the following technical effects:

[0022] This invention uses lysozyme (LZM) as the core antibacterial component, combined with plant-derived ferulic acid (FA) to disrupt the integrity of the bacterial membrane through phenolic hydroxyl groups, enhancing penetration into the outer membrane of *P. gingivalis*. A biphasic moisturizing system of glycerol and propylene glycol is introduced to replace traditional anionic surfactants, maintaining penetrating cleansing power while preventing enzyme protein denaturation. The addition of glycosylated lysozyme stabilizer grape seed proanthocyanidin (GSPE), combined with ascorbic acid and sodium citrate, antagonizes the effects of salivary oxidative stress on enzyme activity, further promoting oral flora balance. Xylitol inhibits the glycolysis pathway of *S. mutans*, blocking extracellular polysaccharide synthesis to synergize with the biofilm dispersion effect of LYZ.

[0023] This invention successfully developed a multifunctional compound antibacterial mouthwash with egg white-derived lysozyme, FA, and GSPE as its core natural active ingredients. The significant synergistic antibacterial effect among lysozyme, FA, and GSPE was verified. This compound strategy effectively overcomes the limitations of single-component action, achieving multi-target inhibition of the growth, biofilm formation, and acid production of key oral pathogens *S. mutans* and *P. gingivalis*. The mouthwash exhibits an excellent antibacterial effect of over 96% against both *S. mutans* and *P. gingivalis*, stemming from a multi-component synergistic mechanism: lysozyme directly enzymatically breaks down bacterial cell walls; FA enhances cell membrane permeability and inhibits glucosyltransferase activity; GSPE effectively interferes with extracellular polymer polymerization; and xylitol further reduces the supply of polysaccharide substrates through metabolic competition. This "combination punch" attack enables rapid and sustained destruction of cariogenic biofilm structures and exhibits a stronger inhibitory effect on difficult-to-remove water-insoluble polysaccharides, directly targeting the most critical virulence factors in caries development. This formula effectively neutralizes the acidic environment through the sodium citrate-ascorbic acid system and significantly reduces the ΔpH value by dually inhibiting bacterial glycolysis and biofilm formation, thus demonstrating strong efficacy in the key process of anti-acid production, with an inhibition rate of 55.3% in 24 hours, weakening the cariogenic ability of pathogenic bacteria.

[0024] In summary, this invention provides a multifunctional compound antibacterial mouthwash with high sensory acceptance and excellent overall rating. It elucidates the scientific basis of its highly effective antibacterial properties at the molecular mechanism level, providing a solid theoretical basis and a highly feasible technical solution for developing new oral health care products based on natural ingredients, avoiding the side effects of chemical preservatives, and possessing highly effective multi-target functions. It has broad application prospects in future oral health care products. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Plate inhibition results of antibacterial mouthwashes S. mutans and P. gingivalis;

[0027] Figure 2 The graph shows the MIC values ​​of antibacterial mouthwash against S. mutans.

[0028] Figure 3 The figure shows the results of the determination of the effect of antibacterial mouthwash on the biofilm content of S. mutans.

[0029] Figure 4 The graph shows the results of the determination of the effect of antibacterial mouthwash on the EPS content of S. mutans.

[0030] Figure 5 The graph shows the results of the determination of the effect of antibacterial mouthwash on the acid-producing ability of S. mutans.

[0031] Figure 6 For comparison of the antibacterial effect of Comparative Example 1 and the antibacterial mouthwash of the present invention;

[0032] Figure 7 For comparison of the antibacterial effect of Comparative Example 2 and the antibacterial mouthwash of the present invention;

[0033] Figure 8 This is a comparison of the antibacterial effect of Comparative Example 3 with the antibacterial mouthwash of the present invention. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1: Preparation of antibacterial mouthwash

[0040] 1. Materials and Methods

[0041] 1.1 Materials and Reagents

[0042] Lysozyme: Egg white was extracted and ovomucoid was recovered using 0.1 mol / L NaCl solution. The supernatant sample was obtained and subjected to fractional ultrafiltration. First, it was ultrafiltered through a 50 kDa membrane to obtain the primary retentate, which was recovered. The primary filtrate was used as the raw material for the secondary ultrafiltration. It was then ultrafiltered through a 20 kDa membrane. The secondary filtrate was dialyzed, desalted, and freeze-dried to obtain lysozyme powder.

[0043] Ferulic acid (FA) was purchased from Shaanxi Ruimao Biotechnology Co., Ltd.; grape seed proanthocyanidins (GSPE) were purchased from Shandong Jinsheng Biotechnology Co., Ltd.; *S. mutans* (strain number ATCC 700610) and *P. gingivalis* (strain number ATCC BAA-308) were purchased from Wuhan Yiousai Biotechnology Co., Ltd.; BHI nutrient agar medium, BHI nutrient broth medium, vitamin K, heme chloride, and defatted cellulose horse blood were purchased from Yanshen Technology Co., Ltd.; menthol, xylitol, sodium citrate, and ascorbic acid were purchased from Weifang Yingxuan Industrial Co., Ltd.; propylene glycol and glycerol were purchased from Zhengzhou Lvjian Biotechnology Co., Ltd.

[0044] 1.2 Instruments and Equipment

[0045] Vertical automatic pressure steam sterilizer: Zhiwei Instruments Co., Ltd.; QYC-200 constant temperature incubator: Shanghai Xinmiao Medical Instrument Manufacturing Co., Ltd.; Round bottom vertical anaerobic bag, gas generator, 96-well plate: Yanshen Technology Co., Ltd.

[0046] 1.3 Experimental Methods

[0047] Heat propylene glycol to 70-75℃ and hold for 20-30 minutes. After cooling, dissolve menthol, FA, and GSPE in sterilized propylene glycol in a clean bench. Cover with sterile aluminum foil and set aside. Mix distilled water and sodium citrate, heat to 60-65℃, and hold for 20-30 minutes for sterilization. After sterilization, cool to below 60℃, add xylitol, and stir until clear. Continue cooling to room temperature (25-30℃), add ascorbic acid, and stir to dissolve. Sterilize glycerol at 70-75℃ for 20-30 minutes and set aside. In a sterile workbench, slowly add the sterilized propylene glycol phase to the sterilized aqueous phase and stir until homogeneous. Add sterilized glycerol and stir until homogeneous. Make up to 100 mL with sterile distilled water and stir until homogeneous. In a sterile workbench, add lysozyme to the homogeneous solution and stir slowly and gently until homogeneous. Filter the mixed solution. Dispense the filtered sterile solution directly into pre-sterilized containers under aseptic conditions. Dispense 12mL into each container. Seal immediately and label to obtain the antibacterial mouthwash.

[0048] Example 2: Optimization of the dosage of different components in antibacterial mouthwash

[0049] 1. Single-factor experiment

[0050] Single-factor experiments were conducted to optimize the formulation by adjusting the amounts of lysozyme, fatty acid (FA), GSPE, glycerol, menthol, and xylitol. Five gradients were set for each factor: lysozyme dosages of 0.5, 0.75, 1, 1.25, and 1.5 g; FA dosages of 0.5, 0.6, 0.7, 0.8, and 0.9 g; GSPE dosages of 0.01, 0.02, 0.03, 0.04, and 0.05 g; glycerol dosages of 6, 8, 10, 12, and 14 mL; menthol dosages of 0.01, 0.02, 0.03, 0.04, and 0.05 g; and xylitol dosages of 0.15, 0.2, 0.25, 0.3, and 0.35 g. 0.3g sodium citrate was added as a pH buffer to maintain a slightly acidic environment; 0.03g ascorbic acid enhanced antioxidant activity, resulting in a synergistic effect; propylene glycol was added at 10 times the amount of FA as a solubilizer and stabilizer. The effects of different factor levels on the antibacterial effects and sensory evaluation of *S. mutans* and *P. gingivalis* were assessed.

[0051] 2. Orthogonal experiment

[0052] Based on the antibacterial effects and sensory evaluation of each group of experiments on *S. mutans* and *P. gingivalis*, three appropriate levels were selected to conduct a six-factor, three-level orthogonal experiment to screen the optimal formulation. The orthogonal experimental design is shown in Table 1.

[0053] Table 1 Orthogonal Experimental Design Table

[0054]

[0055] 3. Antibacterial experiments on *S. mutans* and *P. gingivalis*

[0056] The antibacterial effect was determined according to the experimental method of GB / T 43543-2023 "Mouthwash". The test bacterial suspension was appropriately diluted with PBS, and the recovered bacterial count of S. mutans and P. gingivalis was 1×10⁻⁶. 5 CFU•mL -1 Take 5.0 mL of the original sample solution and place it in a sterile test tube. Incubate at 20°C for 5 min. Take 0.1 mL of the test bacterial solution and add it to the test tube containing 5.0 mL of the sample. Mix quickly and start timing immediately. After 5 min of incubation, dilute the mixture of test bacteria and sample with PBS 10 times and mix thoroughly in a sterile test tube. Take 1 mL of the sample solution and place it in a sterile Petri dish. Pour in 15 mL of BHI nutrient agar medium cooled to 45°C. Rotate the Petri dish to ensure thorough mixing. After the agar solidifies, invert the Petri dish and incubate at (36±1)°C for (48±2) h. Count the viable colonies. For p. gingivalis, pour in 15 mL of freshly prepared BHI blood nutrient agar medium. Use PBS instead of the test sample and follow the above steps as a control sample. Repeat the experiment 3 times and calculate the arithmetic mean. The inhibition rate is calculated using the following formula. The result is expressed as a percentage (%) and retained to two decimal places.

[0057] ;

[0058] In the formula: Y, inhibition rate, %; I, average colony count of control sample, CFU·mL -1 II. Average colony count of test samples, CFU·mL -1 .

[0059] 4. Sensory rating

[0060] Referring to the QB / T 2945-2012 standard for oral hygiene solutions, a sensory evaluation standard for antibacterial mouthwash was established, as shown in Table 2. The sensory evaluation indicators mainly consist of four aspects: color, odor, taste, and post-use sensation. A fuzzy comprehensive evaluation method was used to assess the sensory quality of the antibacterial mouthwash, reducing subjective factors in the sensory evaluation. The main purpose was to provide a more scientific and effective method for the sensory evaluation of antibacterial mouthwash.

[0061] Table 2 Sensory Evaluation Table for Antibacterial Mouthwash

[0062]

[0063] 5. Data Processing

[0064] Data statistical analysis was performed using Origin 2021 software for graphing. SPSSPRO software was used to perform dimensionless processing and analysis on the antibacterial rate and sensory score data, and objective weights were assigned. The antibacterial effect on S. mutans accounted for 30%, the antibacterial effect on P. gingivalis accounted for 30%, and the sensory score accounted for 40%, and a comprehensive score was obtained.

[0065] 6. Experimental Results

[0066] 6.1 Single-factor experiment

[0067] 6.1.1 Effects of Lysozyme Dosage on the Antibacterial Effects and Sensory Scores of *S. mutans* and *P. gingivalis*

[0068] The results are shown in Table 3.

[0069] Table 3. Effects of lysozyme dosage on the antibacterial effect and sensory score of *S. mutans* and *P. gingivalis*.

[0070]

[0071] Note: Different letters indicate significant differences (P < 0.05), as shown in the table below.

[0072] When the amount of lysozyme added was 0.75g, the inhibition rate against *S. mutans* (G+) reached a peak of 90.62%, which decreased significantly with increasing addition (P<0.05), dropping to 77.73% at 1.5g. For *P. gingivalis* (G-), the inhibition rate continuously increased with increasing lysozyme addition, reaching a peak of 91.72% at 1.25g and then stabilizing. The sensory score reached a maximum of 90.88 points at 1g, then dropped sharply to only 69.85 points at 1.5g. This difference may be due to the characteristic of lysozyme to target and hydrolyze the peptidoglycan of *S. mutans* (G+), but high concentrations of lysozyme (≥1g / 100mL) may lead to enzyme molecule aggregation and interaction with Na+ in the formulation. + The high ionic strength of the enzyme shields charge repulsion, promotes hydrophobic interactions between molecules, accelerates lysozyme aggregation, and leads to decreased enzyme activity due to these interactions. For *P. gingivalis* (G-), low concentrations have limited effectiveness against the outer membrane barrier, requiring higher concentrations to penetrate and exert their effect. Sensory degradation may occur because high concentrations of lysozyme easily induce a bitter taste and a sticky feeling, disrupting the synergistic effect with xylitol and menthol in flavor enhancement. The high score at 1g indicates that at this dosage, the combination of the enzyme with flavor enhancers xylitol and menthol, and humectants glycerin and propylene glycol, balances a refreshing taste with mildness. In conclusion, to achieve the optimal balance between antibacterial effect and sensory acceptance, subsequent experiments were conducted using lysozyme dosages of 0.75g, 1g, and 1.25g, which had higher overall scores.

[0073] 6.1.2 Effects of FA Addition Amount on Antibacterial Efficacy and Sensory Score of *S. mutans* and *P. gingivalis*

[0074] The results are shown in Table 4.

[0075] Table 4. Effects of FA addition amount on the antibacterial effect and sensory score of *S. mutans* and *P. gingivalis*.

[0076]

[0077] The amount of FA (acetaminophen) added showed a non-linear effect on the inhibition rate, sensory score, and overall score of two oral pathogens, *S. mutans* (G+) and *P. gingivalis* (G-), with significant differences between species. The inhibition rate of *S. mutans* (G+) initially increased and then decreased with increasing FA addition, peaking at 90.53% at 0.7g, but significantly decreasing to 75.93% at 0.9g. This is mainly due to the phenolic hydroxyl groups of FA disrupting the integrity of the bacterial cell membrane, leading to leakage of intracellular solutes. However, high concentrations may induce molecular aggregation or competitive binding with lysozyme, thus reducing bioavailability. The inhibition rate of *P. gingivalis* (G-) continuously increased with addition, reaching a maximum of 96.11% at 0.9g. The outer membrane of Gram-negative bacteria contains lipopolysaccharides; the increased hydrophobicity of FA may promote its penetration of the outer membrane, inhibit N-acetyltransferase, or disrupt biofilm formation. Sensory scores were best at 0.6-0.7g, scoring 89.2 and 88.25 respectively, while significantly decreasing to 72.2 at 0.9g. The bitterness and astringency introduced by high concentrations of FA are due to the binding of its phenolic hydroxyl groups to oral mucosal proteins, producing astringency, and the increased free acid content exacerbates the bitterness. Furthermore, excessive addition may lead to turbidity, affecting transparency and taste. The highest overall score of 89.39 was achieved at 0.7g FA, balancing broad-spectrum antibacterial efficacy with good taste. In conclusion, to achieve the optimal balance between antibacterial effect and sensory acceptance, subsequent experiments selected FA addition amounts with higher overall scores: 0.6g, 0.7g, and 0.8g.

[0078] 6.1.3 Effects of GSPE Addition Amount on Antibacterial Efficacy and Sensory Score of *S. mutans* and *P. gingivalis*

[0079] The results are shown in Table 5.

[0080] Table 5. Effects of GSPE dosage on the antibacterial effects and sensory scores of *S. mutans* and *P. gingivalis*.

[0081]

[0082] With increasing GSPE dosage, the antibacterial rate of *S. mutans* (G+) increased from 83% to 95.63%, with the increase slowing down after 0.04g, from 94.88% to 95.63%. GSPE exerts its antibacterial effect by disrupting bacterial cell membrane permeability, inhibiting biofilm formation, and suppressing intracellular protein synthesis; its effect saturates at high concentrations. The antibacterial rate against *P. gingivalis* (G-) peaked at 89.13% at 0.03g, slightly increasing to 89.88% at 0.05g, but declining to 87.36% at 0.04g. GSPE can inhibit the expression of inflammatory factors TNF-α and IL-1β in periodontal pathogens, but excessively high concentrations may lead to decreased solubility due to increased polyphenol polymerization, affecting the release of active ingredients. The sensory score peaked at 89.88 at 0.03g, then plummeted to 69.85 at 0.05g. This may be due to the astringent taste and reddish-brown color of GSPE. At low concentrations (≤0.03g), menthol and xylitol can mask the odor; however, at high concentrations, the bitterness intensifies, and the pigment deepens, affecting the appearance and reducing palatability. Proanthocyanidin mouthwash needs to balance efficacy and taste; concentrations exceeding 30mg / mL are not recommended. -1 This can easily lead to decreased acceptability. The peak overall score occurred at 0.03g (90.12 points), balancing the antibacterial rates of 91.45% and 89.13% for *S. mutans* and *P. gingivalis*, respectively, with sensory experience. Higher addition amounts resulted in a decrease in the overall score due to sensory degradation. In summary, to achieve the optimal balance between antibacterial effect and sensory acceptability, subsequent experiments were conducted using GSPE addition amounts with higher overall scores of 0.02g, 0.03g, and 0.04g.

[0083] 6.1.4 Effect of Glycerin Addition Amount on Antibacterial Efficacy and Sensory Score of *S. mutans* and *P. gingivalis*

[0084] The results are shown in Table 6.

[0085] Table 6. Effects of glycerol addition amount on the antibacterial effect and sensory score of *S. mutans* and *P. gingivalis*.

[0086]

[0087] Experiments showed that the amount of glycerol added had a non-linear effect on the antibacterial rate of two oral pathogens, *S. mutans* and *P. gingivalis*. The antibacterial rate against *S. mutans* (G+) peaked at 92.5% with 10 mL of glycerol, but decreased significantly to 85.73% after increasing to 12 mL, and slightly rebounded to 91.08% at 14 mL. The antibacterial rate against *P. gingivalis* (G-) showed a trend of first increasing and then decreasing with increasing glycerol addition, reaching a maximum of 89.3% at 10 mL and decreasing to 74.47% at 14 mL. This phenomenon may be related to the fact that moderate addition of glycerol enhances antibacterial activity. Glycerol, as a solvent and penetration enhancer, can enhance the solubility and cell membrane permeability of active ingredients such as lysozyme and ferulic acid, thereby improving the efficiency of bacterial biofilm disruption. Excessive addition weakens the antibacterial effect. High concentrations of glycerin may increase the viscosity of the system, hindering the free diffusion of antibacterial components and reducing their contact efficiency with bacteria. Simultaneously, while glycerin itself has mild antibacterial properties, excessively high concentrations may interfere with the synergistic effects of other potent ingredients such as menthol and ferulic acid (FA). Sensory scores peaked at 87.88 points when 10 mL of glycerin was added, while both excessively low (6 mL) and excessively high (14 mL) additions led to a decrease in scores. Low additions resulted in insufficient moisturizing and lubrication, leading to a rough mouthfeel, with the lowest score being only 64.95 points. Appropriate addition of glycerin as a moisturizer and thickener improves the smoothness of the antibacterial mouthwash, alleviates the irritation of menthol, and enhances overall palatability. Excessive addition (14 mL) resulted in excessively high viscosity, potentially creating a cloying sweetness, and the unique slightly sweet taste of glycerin may mask the refreshing flavor of sodium citrate, leading to an unbalanced mouthfeel and a score dropping to 81.7 points. In summary, to achieve the optimal balance between antibacterial effect and sensory acceptance, the glycerol addition amounts with higher comprehensive scores were selected as 10 mL, 12 mL, and 14 mL for subsequent experiments.

[0088] 6.1.5 Effect of Menthol Addition on Antibacterial Efficacy and Sensory Score of *S. mutans* and *P. gingivalis*

[0089] The results are shown in Table 7.

[0090] Table 7. Effects of menthol dosage on the antibacterial effects and sensory scores of *S. mutans* and *P. gingivalis*.

[0091]

[0092] The amount of menthol added showed a non-linear effect on the antibacterial rate, sensory evaluation, and overall score of two major oral pathogens (S. mutans and P. gingivalis), with significant differences. When the amount of menthol added increased from 0.01g to 0.03g, the antibacterial rate against S. mutans (G+) increased from 90.65% to 92.18%, but when the amount was further increased to 0.05g, the antibacterial rate decreased significantly to 70.20%. This indicates that low to medium concentrations of menthol can effectively inhibit this bacterium, while high concentrations weaken the effect. The antibacterial rate against P. gingivalis (G-) fluctuated more with increasing concentration, reaching a peak of 90.17% at 0.03g, but decreasing to 78.07% at 0.05g. This bacterium is more sensitive to menthol concentration, which may be related to differences in its cell membrane structure. High concentrations of menthol may disrupt the lipopolysaccharide layer, but excessive amounts may lead to the inactivation of effective antibacterial components due to solubility limitations or component interactions. Menthol enhances the inhibitory effect on *P. gingivalis* by disrupting bacterial cell membrane permeability and increasing the efficiency of lysozyme in breaking down peptidoglycan. Sensory scores peaked at 87.93 points at a concentration of 0.03g, with significant decreases at concentrations below or above this level. Menthol provides a suitable cooling sensation at 0.03g, but excessive amounts can cause mucosal burning or bitterness, reducing acceptability. High concentrations of menthol may physically interact with lysozyme and ferulic acid (FA) in the formulation, reducing their bioavailability and thus affecting antibacterial efficacy and taste. Therefore, to achieve the optimal balance between antibacterial effect and sensory acceptability, subsequent experiments were conducted using menthol concentrations of 0.02g, 0.03g, and 0.04g, which had the highest overall scores.

[0093] 6.1.6 Effect of Xylitol Addition Amount on Antibacterial Effects and Sensory Scores of *S. mutans* and *P. gingivalis*

[0094] The results are shown in Table 8.

[0095] Table 8. Effects of xylitol addition on the antibacterial effects and sensory scores of *S. mutans* and *P. gingivalis*.

[0096]

[0097] The antibacterial rate against *S. mutans* (G+) increased continuously with increasing xylitol dosage. The antibacterial rate against *P. gingivalis* (G-) peaked at 91.03% at 0.25g, decreasing above 0.3g. This may be because the outer membrane lipopolysaccharide of this Gram-negative bacterium is sensitive to low concentrations of xylitol, but high concentrations induce stress-induced biofilm thickening, similar to the adaptive response of *Pseudomonas aeruginosa*. 0.25g of xylitol provides approximately 60% of the sweetness of sucrose, effectively masking the bitterness of lysozyme and FA, but above 0.3g, the sweetness intensifies and creates sensory antagonism with the cooling sensation of menthol. Xylitol above 0.3g may increase solution viscosity, leading to enhanced oral residue. In summary, to achieve the optimal balance between antibacterial effect and sensory acceptability, xylitol dosages with higher overall scores (0.2g, 0.25g, and 0.3g) were selected for subsequent experiments.

[0098] 6.2 Orthogonal Experiment

[0099] An orthogonal experiment was conducted based on the results of the single-factor experiment, and the results are shown in Table 9.

[0100] Table 9. Results of Orthogonal Analysis

[0101]

[0102] The antibacterial mouthwash formula was optimized using orthogonal experimental results. The effects of six factors—lysozyme (A), fatty acid (FA) (B), GSPE (C), glycerin (D), menthol (E), and xylitol (F)—were investigated. The orthogonal analysis range R-values ​​showed that the order of influence of each factor on the overall score of the antibacterial mouthwash was A > B > C > D > F > E. Based on the k-values ​​of each factor, the optimal process combination was determined to be A2B3C1D2E3F1, namely 1g lysozyme, 0.8g FA, 0.02g GSPE, 12mL glycerin, 0.03g menthol, and 0.2g xylitol, the same as the 11th group formula. This combination achieved the highest overall score of 92.84, validating the effectiveness of the formula. As the core antibacterial agent, the amount of lysozyme added must be precise; insufficient addition results in a weak antibacterial effect, while excessive addition may cause a bitter taste. The synergistic effect of FA and GSPE was particularly pronounced at B3C1, associated with enhanced antioxidant and biofilm-disrupting capabilities. Glycerin's moisturizing effect balanced viscosity and mouthfeel at 12 mL; excessive amounts may reduce the fluidity of the antibacterial mouthwash. The cooling agent menthol and the sweetener xylitol had a weak impact on the overall score (R≤0.63), indicating that their dosage can be adjusted within a reasonable range, prioritizing sensory experience.

[0103] Therefore, the optimal formula for the antibacterial mouthwash of this invention is determined to be: per 100mL of antibacterial mouthwash, containing 1g of lysozyme, 0.8g of FA, 0.02g of GSPE, 12mL of glycerin, 0.03g of menthol, and 0.2g of xylitol, which is the same as the formula in group 11 of the orthogonal experiment. The above formula also includes 0.3g of sodium citrate and 0.03g of ascorbic acid.

[0104] The lysozyme mouthwash prepared using the optimal formula was tested for antibacterial activity on agar plates, and the results are as follows: Figure 1 As shown in the figure, the optimal formulation achieved antibacterial rates of 96.88% and 96.30% against *S. mutans* and *P. gingivalis*, respectively. Lysozyme, as the core antibacterial agent, requires precise dosage; insufficient dosage results in weak antibacterial effect, while excessive dosage may cause a bitter taste. The synergistic effect of FA and GSPE was particularly pronounced at B3C1, related to enhanced antioxidant and biofilm-disrupting capabilities. Glycerin's moisturizing effect balanced viscosity and mouthfeel at 12 mL; excessive dosage may reduce mouthwash fluidity. The cooling agent menthol and the sweetener xylitol had a weak impact on the overall score (R≤0.63), indicating that their dosage can be adjusted within a certain range, prioritizing sensory experience.

[0105] Example 3: Determination of the inhibitory effect of antibacterial mouthwash on *S. mutans*

[0106] 1. Determination of the MIC value of antibacterial mouthwash against S. mutans

[0107] The method described in the reference "Experimental Study on the In Vitro Effect of Honeysuckle on Streptococcus mutans UA159" was slightly modified. The highest concentration of the antibacterial mouthwash stock solution (100%) was used, and a series of gradient stock solutions were prepared by serially diluting the solution twofold (100%, 50%, 25%, 12.5%, 6.25%, 3.125%). Micro-dispersion was performed using a 96-well plate. 100 μL of each concentration of antibacterial mouthwash was added to the corresponding well, followed by adding 100 μL to each well to achieve a final concentration of 10... 5 CFU / mL bacterial suspension was cultured and diluted in BHI liquid medium containing 1% sucrose. A negative control (no bacterial suspension added) and a positive control (no antibacterial substances) were used. After incubating in 96-well plates at 37°C for 24 hours, OD was measured using a microplate reader. 600 The minimum inhibitory concentration (MIC) was determined by setting the value. Three independent experiments were conducted.

[0108] 2. Determination of the effect of antibacterial mouthwash on biofilm mass of *S. mutans*

[0109] Sucrose significantly enhances the accumulation and structural stability of *S. mutans* biofilm by catalyzing EPS synthesis, thereby increasing its cariogenic potential. This invention uses BHI medium containing 1% w(sucrose) to simulate a high-cariogenic oral environment, constructing a mature biofilm model. Based on this, the antibacterial efficacy of 1% lysozyme solution (w / v) and antibacterial mouthwash was evaluated. The biofilm was quantified using crystal violet staining. The effects of 1% lysozyme solution (w / v) and antibacterial mouthwash on *S. mutans* biofilm were assessed at incubation times of 5 min, 10 min, 30 min, 6 h, 12 h, and 24 h. A model without any added substances served as a control group.

[0110] 3. Determination of the effect of antibacterial mouthwash on the extracellular polysaccharide (EPS) content of *S. mutans*

[0111] *S. mutans* utilizes sucrose to generate two types of extracellular polysaccharides (EPS): soluble and insoluble EPS. The water-soluble polysaccharides are released in the culture supernatant, providing energy for the bacteria; while the water-insoluble polysaccharides adhere to the tooth surface and participate in plaque matrix formation. Since EPS content is directly related to the cariogenic ability of the bacteria, the amount of EPS produced can serve as an important indicator of the cariogenic performance of *S. mutans*. The experiment used crystal violet staining to quantify the biofilm, evaluating the effects of 1% lysozyme solution (w / v) and antibacterial mouthwash on the EPS content of *S. mutans* at culture times of 5 min, 10 min, 30 min, 6 h, 12 h, and 24 h.

[0112] 4. Determination of the effect of antibacterial mouthwash on the acid-producing capacity of *S. mutans*

[0113] Add 1% lysozyme solution (w / v) and antibacterial mouthwash to BHI medium. After inoculating S. mutans, measure the change in pH value (ΔpH) before and after culture at 5 min, 10 min, 30 min, 6 h, 12 h and 24 h.

[0114] 5. Experimental Results

[0115] 5.1 Determination of the MIC value of antibacterial mouthwash against S. mutans

[0116] See results Figure 2Experimental data showed that the antibacterial mouthwash achieved an antibacterial rate of approximately 97% at a 100% concentration. The antibacterial rate decreased in a concentration-dependent manner with decreasing concentrations; at 50%, 25%, and 12.5% ​​concentrations, the antibacterial rates were 95.62%, 90.83%, and 85.39%, respectively, indicating strong antibacterial activity at high concentrations. When the concentration dropped to 6.25%, the antibacterial rate plummeted to 35.48%, and at 3.125%, there was almost no antibacterial effect, further supporting the efficacy of MIC. 90 The concentration is between 25% and 12.5%. This potent antibacterial effect may stem from the lysozyme in the formula directly disrupting the β-1,4 glycosidic bonds of the bacterial cell wall; FA interferes with cell membrane integrity and reduces extracellular protein synthesis. This invention achieves an antibacterial rate of 90.83% at a concentration of 25%, confirming the advantages of the compound formula; GSPE inhibits biofilm formation and acid production. The addition of ascorbic acid and sodium citrate may enhance the stability of active ingredients by adjusting pH. Combined with substances such as lysozyme, it better reflects the actual application effect.

[0117] 5.2 Determination of the effect of antibacterial mouthwash on the biofilm content of *S. mutans*

[0118] See results Figure 3 The antibacterial mouthwash group showed an OD of 5 minutes. 600 The inhibition rate was significantly lower than that of the control group and the pure lysozyme group (P<0.05), reaching 19.7%, indicating that FA and GSPE in the formula can synergistically disrupt the biofilm structure with lysozyme and enhance the penetration of EPS. The OD of the antibacterial mouthwash group at 24h was significantly lower than that of the control group and the pure lysozyme group (P<0.05), with an inhibition rate of 19.7%. 600 The concentration was lower than that of the control group and lower than that of the pure lysozyme group. This long-lasting effect is attributed to xylitol interfering with bacterial sugar metabolism to reduce EPS synthesis, and sodium citrate-ascorbic acid maintaining a weakly acidic environment, delaying biofilm regeneration and enhancing lysozyme activity. Although the pure lysozyme group showed an inhibitory effect, its standard deviation was high, indicating that the penetration of a single component into mature biofilms is unstable; while the antibacterial mouthwash group had a lower standard deviation, confirming that the lysozyme + FA + GSPE multi-component combination enhances stability through a triple mechanism of cell wall disruption, EPS degradation, and metabolic inhibition. This invention uses low-dose lysozyme combined with FA and xylitol to reduce the effective concentration and minimize oral microecological disturbance through FA physical penetration, xylitol metabolic inhibition, and lysozyme enzymatic hydrolysis of the cell wall.

[0119] 5.3 Determination of the effect of antibacterial mouthwash on EPS content of S. mutans

[0120] See results Figure 4With prolonged treatment time, the contents of both water-soluble and water-insoluble polysaccharides in the control group increased significantly, indicating that *S. mutans* continuously synthesizes EPS under sucrose induction, promoting biofilm maturation. However, the increase in polysaccharide content in the 1% lysozyme solution and antibacterial mouthwash groups slowed significantly, indicating that both could inhibit EPS synthesis. The inhibitory effect of the antibacterial mouthwash was consistently superior to that of lysozyme alone, suggesting a synergistic effect between the FA, GSPE, and other components in the formula and lysozyme. The inhibition of water-insoluble dextran was most significant; at 24 hours, the water-insoluble dextran in the antibacterial mouthwash group decreased by 62.3% compared to the control group, a greater decrease than the 52.5% decrease in water-soluble dextran. This is directly related to the cariogenic mechanism of *S. mutans* biofilms. Water-insoluble dextran is a major component of the biofilm scaffold, giving EPS structural rigidity and enhancing bacterial adhesion. Lysozyme hydrolyzes peptidoglycan, disrupting bacterial integrity, while FA may interfere with activity, inhibiting dextran polymerization, thereby targeting and degrading water-insoluble polysaccharides. This experiment uses dynamic EPS quantification to simultaneously analyze the mass concentrations of water-soluble polysaccharides, water-insoluble polysaccharides, and dextran, which can more accurately reflect changes in the composition of the biofilm matrix and confirm that the antibacterial mouthwash prepared in this invention can inhibit the synthesis of S. mutans biofilm matrix through multiple targets.

[0121] 5.4 Determination of the effect of antibacterial mouthwash on the acid-producing ability of *S. mutans*

[0122] See results Figure 5 The ΔpH of the short-term (5-30 min) antibacterial mouthwash group was significantly lower than that of the control group and the single lysozyme group (P<0.05), indicating that substances such as FA and xylitol in the antibacterial mouthwash can rapidly inhibit bacterial acid production in the early stages of metabolism. With prolonged exposure, the acid-inhibiting advantage of the antibacterial mouthwash group continued to expand. At 24 h, the ΔpH was significantly lower than that of the lysozyme group and the control group (P<0.01), indicating that the antibacterial mouthwash delayed the bacterial acid production process through multi-target inhibition, interfering with glycolysis and blocking biofilm formation. Although the ΔpH of the single lysozyme group was lower than that of the control, it was significantly higher than that of the antibacterial mouthwash group, indicating that the effect of lysozyme alone was limited. In the antibacterial mouthwash group, FA could disrupt bacterial membrane integrity and enhance the penetration of lysozyme into the cell wall; xylitol competitively inhibited sugar transport, reducing the supply of lactic acid substrate. Sodium citrate and ascorbic acid in the formula constituted a pH buffer system, partially neutralizing the produced organic acids (such as lactic acid), thus lowering the ΔpH of the experimental group. The antibacterial mouthwash prepared in this invention achieves biphasic inhibition of acid production by lysozyme combined with plant-derived components FA and xylitol, along with a buffer system and stabilizers. It rapidly neutralizes the acidic environment in the short term and blocks sugar metabolism and biofilm formation in the long term.

[0123] Comparative Example 1: Preparation of Antibacterial Mouthwash

[0124] Unlike Example 1, this comparative example removes the lysozyme component, while other experimental conditions and preparation methods are the same as in Example 1.

[0125] Comparative Example 2: Preparation of Antibacterial Mouthwash

[0126] Unlike Example 1, this comparative example removes the ferulic acid component, while other experimental conditions and preparation methods are the same as in Example 1.

[0127] Comparative Example 3: Preparation of Antibacterial Mouthwash

[0128] Unlike Example 1, this comparative example removes the grape seed proanthocyanidin component, while other experimental conditions and preparation methods are the same as in Example 1.

[0129] The antibacterial effect of the antibacterial mouthwash prepared using the optimal process of this invention was compared with that of the mouthwashes prepared in Comparative Examples 1-3. The results are shown below. Figures 6-8 .

[0130] After removing lysozyme from antibacterial mouthwash ( Figure 6 The antibacterial rate against Streptococcus mutans dropped sharply from approximately 94% in the antibacterial mouthwash to approximately 60%, a decrease of 34%, which was the most significant among all single-component deficiency groups. This indicates that lysozyme is the most effective antibacterial component against Streptococcus mutans in this antibacterial mouthwash formula, playing an irreplaceable core role in the entire compound system. This significant decrease in the antibacterial rate directly reflects the unique ability of lysozyme to disrupt bacterial cell walls. The antibacterial mechanism of lysozyme mainly lies in its ability to specifically hydrolyze the β-1,4 glycosidic bonds of peptidoglycan in bacterial cell walls, especially against Gram-positive bacteria such as Streptococcus mutans, leading to cell wall rupture and bacterial death. This mechanism is particularly important in the oral environment because lysozyme itself is one of the natural antibacterial components in human saliva, participating in maintaining the homeostasis of the oral microenvironment. In addition to its direct lysis effect, lysozyme also has multiple functions such as antibacterial activity, bacterial aggregation, and interference with bacterial metabolism. It can regulate plaque composition and reduce the production of acidic substances within plaque, thus playing a dual role in preventing dental caries.

[0131] When ferulic acid is removed from antibacterial mouthwash ( Figure 7The inhibition rate against *Streptococcus mutans* decreased from approximately 94% in the antibacterial mouthwash to approximately 79%. This indicates that ferulic acid made a significant contribution to the overall antibacterial effect of the system. Its mechanism of action primarily stems from the antibacterial activity of ferulic acid itself and its synergistic effect with other components. As a natural phenolic acid, ferulic acid's antibacterial mechanism mainly lies in its ability to disrupt the cell membrane structure of microorganisms, interfere with cellular energy metabolism, and inhibit the formation of various bacterial biofilms. Crucially, ferulic acid exhibits a synergistic effect with lysozyme. Studies have shown that by chemically modifying ferulic acid to covalently bind to lysozyme, the resulting ferulic acid-lysozyme modified complex not only retains the original activity of lysozyme but also significantly expands its antibacterial spectrum, enhancing its inhibitory effect against some Gram-negative bacteria. In antibacterial mouthwash, ferulic acid may work synergistically with lysozyme to enhance lysozyme's ability to disrupt the cell wall of *Streptococcus mutans*, or improve overall antibacterial efficiency by influencing other bacterial metabolic pathways.

[0132] After removing grape seed proanthocyanidins from antibacterial mouthwash ( Figure 8 The antibacterial rate against Streptococcus mutans decreased from approximately 94% in the antibacterial mouthwash to around 75%. This indicates that although the direct bactericidal ability of grape seed proanthocyanidins is weaker compared to core components such as lysozyme, it remains an indispensable part of this compound system. Its importance lies more in its long-term effects of interfering with bacterial adhesion and inhibiting biofilm formation. As a type of natural plant polyphenol, grape seed proanthocyanidins' main mechanism of action against Streptococcus mutans is not direct killing, but rather interference with the bacterial colonization process on the tooth surface. The value of grape seed proanthocyanidins also lies in its multiple beneficial mechanisms. In addition to directly inhibiting biofilm, it can promote remineralization of tooth tissue and enhance the mechanical properties of dentin by inducing collagen cross-linking, which is of great significance in the prevention of caries and the treatment and restoration of deep caries.

[0133] In summary, in antibacterial mouthwash formulations, lysozyme is the main antibacterial component, while FA and GSPE serve as important auxiliary components. Through direct antibacterial activity and the mechanism of inhibiting bacterial adhesion, they together constitute a multi-level, synergistic compound antibacterial system.

[0134] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antibacterial mouthwash, characterized in that, Each 100 mL of the antibacterial mouthwash contains the following components: 0.5-1.5 g lysozyme, 0.5-0.9 g ferulic acid, 0.01-0.05 g grape seed proanthocyanidins, 6-14 mL glycerin, 0.01-0.05 g menthol, and 0.15-0.35 g xylitol.

2. The antibacterial mouthwash according to claim 1, characterized in that, The antibacterial mouthwash also contains 0.3-0.5g of sodium citrate and 0.03-0.05g of ascorbic acid.

3. A method for preparing an antibacterial mouthwash according to any one of claims 1-2, characterized in that, Includes the following steps: Menthol, ferulic acid, and grape seed proanthocyanidins were dissolved in propylene glycol to obtain the propylene glycol phase. Sodium citrate was mixed with water and sterilized, and xylitol and ascorbic acid were added to dissolve it to obtain the aqueous phase. The glycerol was sterilized to obtain the oil phase; The propylene glycol phase was added to the aqueous phase and stirred, then the oil phase was added and stirred until the volume was adjusted. Add lysozyme to the solution after it has been brought to a certain volume, stir, filter, and fill to obtain the antibacterial mouthwash.

4. The preparation method according to claim 3, characterized in that, The amount of menthol used is 0.03g, the amount of ferulic acid used is 0.8g, and the amount of grape seed proanthocyanidins used is 0.02g.

5. The preparation method according to claim 3, characterized in that, The amount of sodium citrate used is 0.3g, the amount of xylitol used is 0.2g, and the amount of ascorbic acid used is 0.03g.

6. The preparation method according to claim 3, characterized in that, The amount of glycerol used is 12 mL, and the amount of propylene glycol used is 10 times the amount of ferulic acid used.

7. The use of an antibacterial mouthwash according to any one of claims 1-2 in the preparation of oral care products.

8. An oral care product, characterized in that, The oral care products include the antibacterial mouthwash according to any one of claims 1-2.

9. The oral care product according to claim 8, characterized in that, The oral care products also include pharmaceutically acceptable carriers or excipients.

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