Benzenesulfonic acid anti-biofilm preparation as well as preparation method and application thereof

Abstract

The invention relates to a benzenesulfonic acid anti-biofilm preparation which comprises the following components in percentage by mass: 10-75% of benzenesulfonic acid, 10-40% of propylene glycol, 5-25% of a gel matrix and a stabilizer, 0.001-0.2% of a color indicator and the balance of water, the gel matrix and the stabilizer are colloidal silicon dioxide, and the color indicator is a pharmaceutically or food-grade acceptable dye; the invention also relates to a preparation method of the benzenesulfonic acid anti-biofilm preparation and application of the benzenesulfonic acid anti-biofilm preparation in removal of bacterial biofilms. In the benzenesulfonic acid anti-biofilm preparation, benzenesulfonic acid shows anti-biofilm activity superior to that of other traditional alkyl sulfonic acid under relatively low mass concentration; propylene glycol is adopted as an organic solvent and a penetration enhancer, is proved to have a better effect in a plurality of biological membrane models, has biological safety and is free of unpleasant odor; the preparation has the broad-spectrum antibacterial biofilm characteristic, and the application of the preparation covers multiple fields of medical treatment, sanitation, industry and the like.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials technology, and particularly relates to a benzenesulfonic acid antibiofilm preparation, its preparation method and application. Background Technology

[0002] Bacterial biofilms are membrane-like complexes formed by bacteria adhering to the surface of biological materials or organismal tissues during their growth process to adapt to their living environment. They encapsulate themselves within an extracellular matrix containing polysaccharides, proteins, and nucleic acids. Biofilm formation significantly enhances bacterial resistance to antibiotics and the host's immune system, contributing significantly to chronic infections, medical device-related infections, and biocontamination of industrial pipelines, posing substantial challenges to healthcare, industrial production, and other fields.

[0003] To address the challenges posed by biofilms, various intervention strategies have been developed, including physical debridement, enzymatic hydrolysis, combined use of antimicrobial drugs, and chemical debridement. Among these, chemical debridement, with its advantages of ease of operation, rapid action, and wide applicability, has found some application in clinical and industrial settings. Its core principle is to use chemical reagents to disrupt the extracellular polymeric structure of the biofilm, kill bacteria within the membrane, or inhibit bacterial adhesion, thereby achieving the removal and detachment of the biofilm.

[0004] Existing technologies include chemical debridement using sulfonic acids. For example, DEBX (WO2021148124A1) uses ethanesulfonic acid or 1-propanesulfonic acid in combination with dimethyl sulfoxide (DMSO) as a proton acceptor. While effective, this method utilizes highly acidic short-chain sulfonic acids (pKa approximately -1.3 to -0.86), posing a significant potential irritant to normal tissues. It is particularly unsuitable for debridement of biofilms in sensitive areas such as chronic wounds and mucous membranes. Furthermore, while DMSO, a potent transdermal absorption enhancer, can promote the penetration of sulfonic acid compounds into biofilms, it may also accelerate the absorption of other potentially harmful substances. Its long-term biosafety is controversial, and it has an unpleasant garlic odor, severely impacting the user experience.

[0005] Therefore, there is an urgent need to develop a bacterial biofilm removal solution that can circumvent the above-mentioned defects and has high removal efficiency, good biosafety, and wide applicability. Summary of the Invention

[0006] To address the shortcomings of existing anti-biofilm agents, such as insufficient efficacy, poor solvent compatibility, and narrow applicability, this invention provides a benzenesulfonic acid anti-biofilm agent containing a combination of benzenesulfonic acid and propylene glycol, along with its preparation method and application. It clarifies the highly effective active ingredient and optimal preparation conditions, achieving highly efficient removal of biofilms from various bacteria. While ensuring highly effective wound cleaning capabilities, it significantly improves the safety and reliability of the formulation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is to provide a benzenesulfonic acid anti-biofilm formulation comprising, by weight percentage: 10%-75% benzenesulfonic acid and 10%-40% propylene glycol.

[0008] The aforementioned benzenesulfonic acid, as an active ingredient, can achieve excellent biofilm clearance effects within a limited concentration range. Its effect is superior to that of higher concentrations of methanesulfonic acid or ethanesulfonic acid, and its irritation to normal tissues is significantly reduced.

[0009] The aforementioned propylene glycol, as an organic solvent and penetration enhancer, can not only effectively dissolve benzenesulfonic acid, but its own osmotic pressure effect can also synergize with benzenesulfonic acid, ultimately making the combined use of the two more effective in removing biofilm than formulations using DMSO as a solvent.

[0010] Furthermore, the benzenesulfonic acid anti-biofilm preparation also includes: 5%-25% gel matrix and stabilizer, and 0.001%-0.2% colorimetric indicator.

[0011] Furthermore, the gel matrix and stabilizer are selected from polyethyleneimine (PEI) crosslinked gel, polydiallyldimethylammonium chloride (PDADMAC) type strong cationic polyelectrolytes, porous crosslinked polystyrene (Amberlite, Dowex), ultra-high molecular weight polyethylene (UHMWPE) porous gel network, polyacrylamide (PAM) crosslinked gel, and anion exchange resin (Amberlite IRA-400 or IRA-900).

[0012] Furthermore, the gel matrix and stabilizer are colloidal silica.

[0013] Further, the colloidal silica is selected from at least one of the LUDEX series (such as LUDEX SM-30, LUDEX TM-50) or the AEROSIL series colloidal silica. Preferably, the colloidal silica is LUDEX SM-30. The colloidal silica is added using a commercially available colloidal silica aqueous dispersion, for example, LUDEX SM-30 is a commercially available 30 wt% colloidal silica aqueous dispersion. In the above formulations, the mass percentage of colloidal silica refers to the mass percentage of the colloidal silica aqueous dispersion.

[0014] The above-mentioned colloidal silica material can form a stable gel with good thixotropic properties under acidic conditions, making the formulation easy to apply and preventing it from dripping.

[0015] Furthermore, the colorimetric indicator is a pharmaceutically or food-grade acceptable dye.

[0016] Further, the colorimetric indicator is selected from one of methylene blue, Patent Blue V, food coloring, methyl violet, bromocresol violet, bromophenol blue, and carmine; preferably, the colorimetric indicator is selected from one of methylene blue, bromophenol blue, and bromocresol violet. The above-mentioned colorimetric indicator is added using a 1 wt% stock solution of the colorimetric indicator, for example, bromocresol violet is a 1 wt% bromocresol violet ethanol stock solution. In the formulation, the mass percentage of the colorimetric indicator is always a mass percentage of the 1 wt% stock solution of the colorimetric indicator.

[0017] The use of the aforementioned colorimetric indicator can ensure that the operator can clearly see the coverage area of ​​the gel during application, avoiding any omissions.

[0018] Furthermore, the colloidal silica is a LUDEX SM-30 dispersion, and the colorimetric indicator is a bromocresol purple solution or a bromophenol blue solution.

[0019] Furthermore, the benzenesulfonic acid antibiofilm formulation has a pH of 2.0 to 5.0. This pH range achieves an excellent balance between ensuring activity and reducing irritation, eliminating the need for additional pH adjusters.

[0020] Furthermore, the benzenesulfonic acid anti-biofilm formulation comprises 10%-75% benzenesulfonic acid, 10%-40% propylene glycol, 5%-25% gel matrix and stabilizer, 0.001%-0.2% colorimetric indicator, and the balance being water; wherein the gel matrix and stabilizer are colloidal silica, selected from at least one of the LUDEX series or AEROSIL series colloidal silica; and the colorimetric indicator is a pharmaceutically or food-grade acceptable dye, selected from bromocresol purple and bromophenol blue.

[0021] Furthermore, the benzenesulfonic acid anti-biofilm preparation is composed of 30%-60% benzenesulfonic acid, 15%-30% propylene glycol, 10%-20% colloidal silica, 0.05%-0.2% colorimetric indicator, and the balance being water; the pH of the benzenesulfonic acid anti-biofilm preparation is 2.0~4.0.

[0022] Further, the benzenesulfonic acid antibiofilm preparation comprises 50%-60% benzenesulfonic acid, 18%-25% propylene glycol, 10%-15% colloidal silica, 0.05%-0.1% colorimetric indicator, and the balance being water; wherein the colloidal silica is a LUDEXSM-30 dispersion, the colorimetric indicator is a bromocresol purple solution, and the pH of the benzenesulfonic acid antibiofilm preparation is 2.0-2.4.

[0023] Furthermore, the benzenesulfonic acid anti-biofilm preparation is composed of 50%-60% benzenesulfonic acid, 20% propylene glycol, 10% colloidal silica, 0.1% colorimetric indicator, and the balance being water.

[0024] Furthermore, the benzenesulfonic acid antibiofilm formulation is composed of 50% benzenesulfonic acid, 20% propylene glycol, 10% LUDEX SM-30 dispersion, 0.1% methylene blue solution, and 19.9% ​​water, with a pH of 2.4; or, the benzenesulfonic acid antibiofilm formulation is composed of 60% benzenesulfonic acid, 20% propylene glycol, 10% LUDEX SM-30 dispersion, 0.1% methylene blue solution, and 9.9% water, with a pH of 2.0.

[0025] In the above formulations, this invention has demonstrated that: when DMSO is used as a solvent, benzenesulfonic acid exhibits a superior scavenging effect compared to other sulfonic acid compounds, indicating the superiority of benzenesulfonic acid itself in biofilm scavenging; compared to other solvent groups, when propylene glycol is used as a solvent, benzenesulfonic acid exhibits a superior scavenging effect at the same concentration compared to other solvent groups, indicating that propylene glycol can reduce the effective concentration of benzenesulfonic acid.

[0026] In the above formulation, the core function of colloidal silica in this invention is to form a "stable, thixotropic gel." Its content is not determined independently, but rather is predicated on the presence of propylene glycol. The gelation stability of colloidal silica in an acidic system (pH 2-5, preferably pH 2-4) is highly dependent on the polarity and compatibility of the dispersion medium. Without propylene glycol, using only pure water as the medium, colloidal silica particles will aggregate due to electrostatic effects under acidic conditions. Even if the content is increased to over 30%, a uniform gel cannot be formed (layering, sedimentation, and flow after coating will occur). This is determined by the physicochemical properties of colloidal silica. This invention determines the content range of colloidal silica to be 10%-20% precisely because propylene glycol can adjust the polarity of the system, forming a good dispersion and compatibility environment with colloidal silica, inhibiting its aggregation, and enabling stable gelation even at low contents. The compatibility of propylene glycol with colloidal silica within this content range enables the formulation to achieve the practical effect of being "easy to coat and non-dripping"; if propylene glycol does not affect the content of colloidal silica, this low content range cannot meet the gelation requirements at all, which further proves that the use of propylene glycol reduces the concentration of colloidal silica to a certain extent.

[0027] In the above formulation, the addition of propylene glycol reduces the colloidal silica content from the conventional ≥25% to 10%-20% (specifically 10%). This reduces raw material costs and optimizes the coating performance of the formulation, avoiding a thick texture and difficulty in application due to excessive colloidal content. More importantly, this compatible system of "low colloidal content + propylene glycol + high concentration benzenesulfonic acid" has not been disclosed in existing technologies. Its stability and performance cannot be derived through conventional formulation design, fully demonstrating the unpredictability of the ratio of the three components.

[0028] A second aspect of the present invention is to provide a method for preparing a benzenesulfonic acid anti-biofilm agent according to any of the first aspects of the present invention, comprising the steps of: S1. Dissolve benzenesulfonic acid in propylene glycol under stirring conditions to form a mixed solution; S2. Slowly add the gel matrix and stabilizer to the mixed solution and continuously stir at high speed to form a uniform gel. S3. Add a display indicator to the gel and make up the amount with purified water, stir evenly, and obtain the benzenesulfonic acid anti-biofilm preparation.

[0029] Further, in step S1, the operating conditions are as follows: at room temperature (20~30 ℃), continuously stir at a speed of 300~800 rpm for 5~20 min; preferably, continuously stir at a speed of 400~600 rpm for 5~15 min; more preferably, continuously stir at a speed of 500 rpm for 10 min.

[0030] Further, in step S2, the operating conditions are as follows: homogenizing and stirring at a speed of 3000~8000 rpm for 10~30 min; preferably, homogenizing and stirring at a speed of 4000~6000 rpm for 15~20 min; more preferably, homogenizing and stirring at a speed of 6000 rpm for 20 min.

[0031] Further, in step S3, the operating conditions are: stirring at a speed of 200~600 rpm for 5~20 min; preferably stirring at a speed of 400~600 rpm for 5~15 min; more preferably stirring at a speed of 500 rpm for 10 min.

[0032] A third aspect of the present invention is to provide a method for removing bacterial biofilms, comprising the steps of: contacting a benzenesulfonic acid antibiofilm agent with a bacterial biofilm for 10 s to 10 min; wherein the benzenesulfonic acid antibiofilm agent is prepared as described in any of the first aspects of the present invention or by any of the preparation methods described in the second aspect of the present invention.

[0033] Furthermore, the contact time is 1 min to 5 min; preferably 30 s, 1 min, 2 min, etc.

[0034] A fourth aspect of the present invention is to provide the application of a benzylsulfonic acid antibiofilm preparation or a method for removing bacterial biofilms in the removal of bacterial biofilms, wherein the benzylsulfonic acid antibiofilm preparation is as described in any of the first aspects of the present invention or is prepared by any of the preparation methods described in any of the second aspects of the present invention; and the method for removing bacterial biofilms is as described in any of the third aspects of the present invention.

[0035] The fifth aspect of the present invention is to provide the use of a benzyl sulfonic acid antibiofilm agent in the preparation of a medicament for treating diseases caused by bacterial biofilms, said benzyl sulfonic acid antibiofilm agent being prepared as described in any of the first aspects of the present invention or by any of the preparation methods described in any of the second aspects of the present invention.

[0036] The sixth aspect of the present invention is to provide the application of a benzenesulfonic acid antibiofilm preparation or a method for removing bacterial biofilms in the prevention and treatment of medical device-related infections and industrial pipeline biocontamination caused by bacterial biofilms, wherein the benzenesulfonic acid antibiofilm preparation is as described in any of the first aspects of the present invention or is prepared by any of the preparation methods described in any of the second aspects of the present invention; and the method for removing bacterial biofilms is as described in any of the third aspects of the present invention.

[0037] Furthermore, in the above-described methods and applications for removing bacterial biofilms, the bacterial biofilm includes biofilms formed by Gram-positive bacteria and Gram-negative bacteria.

[0038] Furthermore, the bacterial biofilm includes biofilms formed by Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0039] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects: (1) High selectivity of active ingredients: This invention clearly shows that benzenesulfonic acid has a significantly better anti-biofilm effect than other sulfonic acid compounds such as methanesulfonic acid and ethanesulfonic acid, and has a stronger ability to clear Escherichia coli biofilm, providing a clear direction for the selection of active ingredients in anti-biofilm preparations; (2) Solvent safety and efficacy are both taken into account: Propylene glycol is chosen as the solvent, which not only makes the anti-biofilm efficacy of benzenesulfonic acid better than that of the formulation with DMSO as the solvent, but also comparable to that of the formulation with DEGME as the solvent. At the same time, propylene glycol is safer and has a wider range of applications, solving the problem of high toxicity and insufficient efficacy of some solvents. (3) Reasonable concentration optimization: The optimal mass concentration of benzenesulfonic acid is determined to be 50%~60%. Within this concentration range, the formulation has excellent anti-biofilm efficacy, and there is no need to pursue excessively high concentrations, which can reduce raw material costs and reduce the irritation that high concentration components may cause. (4) Wide range of applications: The benzenesulfonic acid preparation of the present invention can not only effectively remove biofilms formed by Gram-negative bacteria, but also remove biofilms formed by Gram-positive bacteria. It can be applied to biofilm removal scenarios in multiple fields such as medical, health and industrial fields. Attached Figure Description

[0040] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a diagram illustrating the effect of different sulfonic acid compounds on the removal of Escherichia coli biofilm in one embodiment of the present invention; Figure 2 This is a graph showing the effect of different solvents on the biofilm removal efficiency of benzenesulfonic acid in one embodiment of the present invention. Figure 3 This is a diagram illustrating the effect of different mass concentrations of benzenesulfonic acid on the removal of Escherichia coli biofilm in one embodiment of the present invention; Figure 4 This is a diagram illustrating the effect of propylene glycol synergistically with benzenesulfonic acid on the removal of Escherichia coli biofilm in one embodiment of the present invention. Figure 5 This is a graph showing the effect of benzenesulfonic acid preparation on the removal of Escherichia coli biofilm at different action times in one embodiment of the present invention; Figure 6 This is a diagram illustrating the effect of benzenesulfonic acid preparation on the removal of biofilms formed by different bacteria in one embodiment of the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0042] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0043] The reagents and instruments used in the following examples include: (1) Reagents: benzenesulfonic acid (Shanghai Sangon Biotech, A502113-0250), methanesulfonic acid (Maclean, M813392), ethanesulfonic acid (Maclean, E809106); dimethyl sulfoxide (DMSO, Maclean, D6258), diethylene glycol monoethyl ether (DEGME, Bide, BD124874), propylene glycol (PG, Maclean, P967315), colloidal silica (LUDEX® SM-30 (abbreviated as LUDEX SM-30, LUDEX 30), Sigma, 420794); standard strains of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 43300), Pseudomonas aeruginosa (ATCC 47085); biofilm detection kit (crystal violet method, Solarbio, C8470). (2) Instruments: Microplate reader, constant temperature incubator, clean bench, vortex shaker, electronic balance. All other reagents not specified are commercially available. In the following preparations, LUDEX 30 is a commercially available 30wt% colloidal silica aqueous dispersion, and all mass percentages are the mass percentages of the colloidal silica aqueous dispersion; the colorimetric indicator is added using a 1wt% stock solution of the colorimetric indicator, for example, bromocresol purple is a 1wt% bromocresol purple ethanol stock solution, and all mass percentages of the colorimetric indicator are the mass percentages of the 1wt% stock solution of the colorimetric indicator.

[0044] In the following examples, the preparation methods for each formulation are as follows: sulfonic acid compounds are dissolved in a solvent under stirring to obtain a mixture; a gel matrix and a stabilizer (preferably colloidal silica, such as LUDEX SM-30 dispersion) are slowly added to the mixture, and high-speed homogenization is continuously carried out to form a uniform gel; a colorimetric indicator (preferably bromocresol purple solution) is added, and the remaining purified water is added to make up the difference, and the mixture is stirred evenly to obtain an anti-biofilm formulation.

[0045] In the following examples, the construction of the *E. coli* biofilm model using the crystal violet method is typically performed in a 96-well plate. *E. coli* culture in the logarithmic growth phase is diluted to a suitable concentration with fresh culture medium and added to the wells. The plates are then incubated at 37°C for 24–48 h to allow bacteria to adhere to the well walls and form a biofilm. After incubation, the supernatant is discarded, and the plates are gently washed with PBS to remove airborne bacteria. Crystal violet solution is then added for staining for 15–30 min. After staining, the plates are washed again to remove unbound dye. Once dried, the bound crystal violet is dissolved in ethanol or an ethanol-acetone solution. The degree of biofilm formation is quantitatively reflected by measuring the absorbance value.

[0046] The technical solution of the present invention will be described by way of example below in conjunction with the embodiments and accompanying drawings.

[0047] Example 1: Comparative Experiment on the Anti-Biofilm Efficacy of Different Sulfonic Acid Compounds This embodiment comparatively verifies the anti-biofilm efficacy of methanesulfonic acid, ethanesulfonic acid, and benzenesulfonic acid, specifically including the following experimental steps: (1) The composition of the preparations in the control group and the experimental group (by mass percentage); (2) Experimental methods: DMSO was used as the solvent to prepare benzenesulfonic acid, methanesulfonic acid and ethanesulfonic acid preparations with a mass concentration of 60% respectively; Escherichia coli (ATCC 25922) was selected to construct an E. coli biofilm model by crystal violet method. The solvent control group and the experimental group (four preparations) were respectively contacted with the E. coli biofilm for 30 seconds. A blank control group (Control, no solvent drug group) was also set up. Then, the biofilm detection kit was operated according to the instructions. The absorbance value at 570nm was detected by microplate reader. The lower the absorbance value, the stronger the biofilm removal ability. The pH of benzenesulfonic acid preparation was 2.0.

[0048] (3) Experimental results: such as Figure 1 As shown, compared with the solvent control group, the methanesulfonic acid preparation group, the ethanesulfonic acid preparation group, and the benzenesulfonic acid preparation group were all able to significantly remove the biofilm that Escherichia coli had formed. However, benzenesulfonic acid had a significantly better ability to remove Escherichia coli biofilm than methanesulfonic acid and ethanesulfonic acid.

[0049] Example 2: Effect of different solvents on the anti-biofilm efficacy of benzenesulfonic acid This embodiment compares and verifies the effects of dimethyl sulfoxide (DMSO), diethylene glycol monoethyl ether (DEGME), and propylene glycol (PG) as solvents on the anti-biofilm efficacy of benzenesulfonic acid, specifically including the following experimental steps: (1) The composition of the preparations in the control group and the experimental group (by mass percentage); (2) Experimental methods: Equal masses of benzenesulfonic acid were dissolved in DMSO, DEGME, and PG respectively to prepare three benzenesulfonic acid preparations with a mass concentration of 60%. A biofilm model of *E. coli* was constructed using the crystal violet method. The solvent control group and the experimental group (six preparations) were respectively contacted with the *E. coli* biofilm for 30 seconds. A blank control group (control group without solvent drugs) was also set up. OD was detected using an enzyme-linked immunosorbent assay (ELISA) reader. 570 The value was used to evaluate the biofilm clearance capacity; the pH of the benzenesulfonic acid preparation was 2.0.

[0050] (3) Experimental results: such as Figure 2As shown, the benzenesulfonic acid formulation dissolved in propylene glycol has a better ability to remove E. coli biofilm than the formulation dissolved in DMSO, and is no less effective than the formulation dissolved in DEGME. Considering solvent safety and suitability, propylene glycol is determined to be the optimal solvent.

[0051] Example 3: Comparative Experiment on the Anti-Biofilm Efficacy of Different Concentrations of Benzenesulfonic Acid This embodiment compares and verifies the anti-biofilm efficacy of different concentrations of benzenesulfonic acid, specifically including the following experimental steps: (1) The composition of the preparations in the control group and the experimental group (by mass percentage); (2) Experimental methods: Benzenesulfonic acid preparations with mass concentrations of 60%, 50%, 40%, and 30% were prepared using propylene glycol as a solvent. An E. coli biofilm model was constructed using the crystal violet method. Different concentrations of the preparations were contacted with the biofilm for 30 seconds. A blank control group (control group without solvent) was also set up. OD was measured. 570 The pH value is used to evaluate the biofilm clearance capacity of benzenesulfonic acid preparations, which range from 2.0 to 4.0.

[0052] (3) Experimental results: such as Figure 3 As shown, the scavenging ability of benzenesulfonic acid at concentrations of 60% and 50% is not significantly different on biofilms, and the scavenging ability gradually weakens as the concentration decreases. Therefore, 50%–60% was determined to be the optimal concentration range. Based on the efficacy stability, safety, industrial feasibility, and component synergistic compatibility of the formulation of this invention, a 50% concentration of benzenesulfonic acid was selected as the concentration for subsequent experiments.

[0053] Example 4: Experiment on the effect of propylene glycol on the anti-biofilm efficacy of benzenesulfonic acid To determine whether propylene glycol can exert a synergistic antibacterial effect with benzenesulfonic acid, this example compares and verifies the effect of adding or not adding propylene glycol on the anti-biofilm efficacy of benzenesulfonic acid, specifically including the following experimental steps: (1) The composition of the preparations in the control group and the experimental group (by mass percentage); (2) Experimental methods: Based on the efficacy, stability, safety, industrial feasibility, and synergistic compatibility of benzenesulfonic acid preparations, a 50% concentration of benzenesulfonic acid preparation was selected as the concentration for subsequent experiments. Simultaneously, to determine whether propylene glycol could exert a synergistic antibacterial effect with benzenesulfonic acid, a ratio was added where the preparation contained only benzenesulfonic acid and no propylene glycol. An E. coli biofilm model was constructed using the crystal violet method. The preparation was brought into contact with the biofilm for 30 seconds. A blank control group (control group without solvent) was also set up. OD was measured. 570The pH value was used to evaluate the biofilm clearance capacity of the benzenesulfonic acid preparation, which was 2.4.

[0054] (3) Experimental results: such as Figure 4 As shown, benzenesulfonic acid formulations with and without propylene glycol exhibit significant differences in their biofilm removal capabilities, indicating that propylene glycol exerts a significant synergistic anti-biofilm effect with benzenesulfonic acid through osmotic pressure, resulting in a significantly improved removal effect compared to using benzenesulfonic acid alone.

[0055] Example 5: Preparation of benzenesulfonic acid formulation This embodiment describes some preferred benzenesulfonic acid anti-biofilm formulations and their preparation methods, specifically including: (1) Benzenesulfonic acid preparation formulation (by mass percentage); (2) Preparation method of benzenesulfonic acid preparations; The preparation methods for all the above formulations are the same. The dispersions and solutions in the above raw materials are prepared using conventional concentrations. Weigh each raw material according to the above formulation composition per 100 g. The preparation steps include: Slowly adding benzenesulfonic acid to propylene glycol at room temperature (20–30℃) using mechanical stirring at 500 rpm for 10 min until the benzenesulfonic acid is completely dissolved, obtaining a homogeneous transparent or semi-transparent mixed solution; Adding colloidal silica dispersion (e.g., LUDEX 30 dispersion, LUDEX 50 dispersion, AEROSIL 200 dispersion) slowly in batches to the above mixed solution under continuous stirring, followed by homogenization at 6000 rpm for 20 min using a high-speed homogenizing mixer until a homogeneous gel state is formed without obvious particles or phase separation; Adding a pre-dissolved color indicator solution (e.g., bromocresol purple solution, bromophenol blue solution, food-grade water-soluble blue pigment solution) to the obtained gel system, and adding purified water to 100 g. g, then stirred at 500 rpm for 10 min to fully disperse the colorimetric indicator and water in the gel system, to obtain a uniform and stable benzenesulfonic acid anti-biofilm formulation with a pH of 2.0-5.0.

[0056] Example 6: Effect of different treatment times on the anti-biofilm efficacy of benzenesulfonic acid This embodiment selects formulation 1 from Example 5 to verify the effect of different treatment times on the anti-biofilm efficacy of benzylsulfonic acid, specifically including the following steps: (1) The composition of the preparations in the control group and the experimental group (by mass percentage); (2) Experimental methods: A 50% benzenesulfonic acid preparation was prepared using propylene glycol as a solvent; an E. coli biofilm model was constructed using the crystal violet method, and the preparation was contacted with the biofilm for 10 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, and 10 minutes, respectively. A blank control group (without solvent drug) was also set up; OD was measured at each time point. 570 The pH value was used to evaluate the biofilm clearance capacity of the benzenesulfonic acid preparation, which was 2.4.

[0057] (3) Experimental results: such as Figure 5 As shown, compared with the control group, the biofilm removal capabilities of the formulations treated for different times were different. As the treatment time increased, the biofilm removal capability gradually increased, reaching its peak at 2 minutes.

[0058] Example 7: Experiment on the scavenging effect of benzenesulfonic acid preparation on different bacterial biofilms This embodiment selects formulation 1 from Example 5 to verify the effect of benzenesulfonic acid on different bacterial biofilms, specifically including the following steps: (1) The composition of the preparations in the control group and the experimental group (by mass percentage); (2) Experimental methods: A 50% benzenesulfonic acid preparation was prepared using propylene glycol as a solvent; Escherichia coli (E. coli) were constructed respectively. E. coli ), Pseudomonas aeruginosa ( P. aeruginosa ) and Staphylococcus aureus ( S.aureus Biofilm model; the formulation was contacted with three different biofilms for 30 seconds, and a blank control group (control group without solvent) was also set up; OD was measured. 570 The pH value was used to evaluate the scavenging ability of the benzenesulfonic acid preparation, which was 2.4.

[0059] (3) Experimental results: such as Figure 6 As shown, the 50% benzenesulfonic acid preparation has a significant ability to remove biofilms formed by two types of typical pathogenic bacteria. It can effectively act on the biofilm structure constructed by Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and can also efficiently remove the dense biofilm formed by Gram-positive bacteria (Staphylococcus aureus), demonstrating the broad-spectrum removal activity and outstanding effect of this concentration of preparation on bacterial biofilms.

[0060] As demonstrated by the above embodiments, this invention, through formulation screening, has found that benzenesulfonic acid (BSA), a structurally simple aromatic sulfonic acid, exhibits superior anti-biofilm activity compared to other traditional alkyl sulfonic acids at relatively low concentrations. Furthermore, the use of propylene glycol instead of DMSO as both an organic solvent and a penetration enhancer has been proven to be more effective in multiple biofilm models, and its biocompatibility is widely recognized, exhibiting no unpleasant odor. The formulations of this invention containing benzenesulfonic acid and propylene glycol possess broad-spectrum antibacterial biofilm properties, with applications spanning multiple fields including medical, hygiene, and industrial applications.

[0061] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A benzenesulfonic acid anti-biofilm agent, characterized in that, The benzenesulfonic acid antibiofilm preparation comprises, by weight percentage: 10%-75% benzenesulfonic acid and 10%-40% propylene glycol.

2. The benzenesulfonic acid anti-biofilm agent according to claim 1, characterized in that, The benzenesulfonic acid anti-biofilm preparation further includes: 5%-25% gel matrix and stabilizer, and 0.001%-0.2% colorimetric indicator.

3. The benzenesulfonic acid anti-biofilm agent according to claim 2, characterized in that, The gel matrix and stabilizer are selected from one of the following: polyethyleneimine crosslinked gel, polydiallyldimethylammonium chloride-based strong cationic polyelectrolytes, porous crosslinked polystyrene, ultra-high molecular weight polyethylene porous gel network, polyacrylamide crosslinked gel, anion exchange resin, and colloidal silica; and / or... The colorimetric indicator is a pharmaceutically or food-grade acceptable dye, selected from one of methylene blue, Patent Blue V, food coloring, methyl violet, bromocresol violet, bromophenol blue, and carmine; and / or The pH of the benzenesulfonic acid antibiofilm preparation is 2.0~5.

0.

4. The benzenesulfonic acid anti-biofilm agent according to claim 1, characterized in that, By weight percentage, the benzenesulfonic acid anti-biofilm formulation consists of 10%-75% benzenesulfonic acid, 10%-40% propylene glycol, 5%-25% gel matrix and stabilizer, 0.001%-0.2% colorimetric indicator and the balance being water; The gel matrix and stabilizer are colloidal silica, selected from at least one of the LUDEX series or AEROSIL series colloidal silica; the colorimetric indicator is a pharmaceutically or food-grade acceptable dye, selected from bromocresol violet or bromophenol blue.

5. The benzenesulfonic acid anti-biofilm agent according to claim 4, characterized in that, The benzenesulfonic acid antibiofilm preparation comprises 30%-60% benzenesulfonic acid, 15%-30% propylene glycol, 10%-20% colloidal silica, 0.05%-0.2% colorimetric indicator, and the balance being water; the pH of the benzenesulfonic acid antibiofilm preparation is 2.0-4.

0.

6. The benzenesulfonic acid anti-biofilm agent according to claim 4, characterized in that, The benzenesulfonic acid anti-biofilm preparation comprises 50%-60% benzenesulfonic acid, 18%-25% propylene glycol, 10%-15% colloidal silica, 0.05%-0.1% colorimetric indicator, and the balance being water; wherein the colloidal silica is LUDEX SM-30 dispersion, the colorimetric indicator is bromocresol purple solution, and the pH of the benzenesulfonic acid anti-biofilm preparation is 2.0~2.

4.

7. A method for preparing a benzenesulfonic acid anti-biofilm agent as described in any one of claims 1 to 6, characterized in that, The preparation method includes the following steps: S1. Dissolve benzenesulfonic acid in propylene glycol under stirring conditions to form a mixed solution; S2. Slowly add the gel matrix and stabilizer to the mixed solution and continuously stir at high speed to form a uniform gel. S3. Add a colorimetric indicator to the gel and make up the amount with purified water, stir evenly, and obtain the benzenesulfonic acid anti-biofilm preparation.

8. A method for removing bacterial biofilm, characterized in that, The method includes the step of contacting a benzenesulfonic acid antibiofilm agent with a bacterial biofilm for 10 s to 10 min; wherein the benzenesulfonic acid antibiofilm agent is as described in any one of claims 1 to 6, or is prepared by the preparation method described in claim 7.

9. The application of a benzenesulfonic acid anti-biofilm agent or a method for removing bacterial biofilms in the removal of bacterial biofilms, characterized in that, The benzenesulfonic acid anti-biofilm preparation is as described in any one of claims 1 to 6, or is prepared by the method described in claim 7; the method for removing bacterial biofilm is as described in claim 8.

10. The application according to claim 8, characterized in that, The bacterial biofilm includes biofilms formed by Gram-positive bacteria and Gram-negative bacteria.

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