An environmentally friendly antimicrobial adherent self-film forming spray and coating integrating polylactic acid and equalin II

Abstract

The present application belongs to the technical field of metal material protection, and specifically discloses an environment-friendly self-film-forming spray and coating with antimicrobial adhesion integrated with polylactic acid and palytoxin II, wherein the spray is obtained by completely dissolving palytoxin II into a dichloromethane solution of polylactic acid; and wherein 20-40 mL of the dichloromethane solution of polylactic acid contains 0.5-1.5 g of polylactic acid, and 10-30 mg of palytoxin II is added accordingly. By introducing palytoxin II into the polylactic acid solution dissolved in dichloromethane, the polylactic acid and palytoxin II are co-dissolved, and the obtained solution system can self-form a film in air, and the obtained coating can resist bacteria and prevent fouling, and is especially suitable for corrosion-resistant treatment of metal pipes. Compared with the related prior art of pure polylactic acid coating, the present application can effectively improve the environmental resistance, anti-bioadhesion property, hydrophobicity and protection of the substrate material of the polylactic acid coating.

Description

Technical Field

[0001] This invention belongs to the field of metal material protection technology, and more specifically, relates to an environmentally friendly self-forming film spray and coating that integrates polylactic acid and isoflavone toxin II, which has both environmental friendliness and antibacterial and antifouling capabilities, and has a huge application market in microbial corrosive environments. Background Technology

[0002] Due to the complex industrial environment, corrosion caused by microbial growth and metabolism accounts for 20% of the overall corrosion of existing structures. This is particularly evident in the oil and gas and marine industries, where microbial corrosion of metal structures (such as metal pipelines) reaches 50% and 70%, respectively. These microorganisms have evolved over long periods and developed strong adaptability to their surroundings, making them prone to adhering to the surfaces of metal structures. To avoid direct contact between metal structures and environmental microorganisms, coatings have become an important protective measure in the field of microbial corrosion protection. Coatings are favored by industry and academia due to their wide range of applications (high-temperature coatings, high-salt coatings, high-humidity coatings), diverse film-forming methods, unique spraying processes, and advantages such as strong electron transfer inhibition and environmental stability. Currently, most coating applications are based on organic coatings such as epoxy resins and polyurethanes. However, epoxy coatings cannot repel the adhesion of corrosive sulfate-reducing bacteria. For example, Progress in Organic Coatings (176, 2023, 107401) reported that epoxy coatings could not remain stable for 14 days in an environment of sulfate-reducing bacteria, and even after adding PHGM, which can kill bacteria, to the epoxy coating, its integrity was still compromised.

[0003] Polylactic acid (PLA), a polymer based on lactic acid (primarily starch), possesses excellent comprehensive mechanical properties, good transparency and processability, as well as eco-friendliness. It can be manufactured into various products using traditional processing techniques such as injection molding, spinning, film extrusion, and film formation. Therefore, increasing research is exploring the application of PLA in the coating field. For example, the journal *Progress in Organic Coatings* (145, 2020, 105682) investigated and explored the possibility of using bio-based PLA as a barrier coating material on natural kraft paper to manufacture coated and polished products, replacing traditional, non-biodegradable, synthetic polymer-based barrier coating materials. Data from various studies show that an ideal PLA layer thickness provides superior overall barrier performance, which is essential for manufacturing coated products. The journal *Electrical Discharge & Mold* (S1, 2023, 50-54) reported the preparation of polylactic acid (PLA) @CS coatings on the surface of 3D-printed calcium silicate scaffolds using the dip-coating method. This coating exhibited excellent hydrophobicity, which is unfavorable for biofilm adhesion, and the peak compressive stress reached 45 MPa, demonstrating considerable mechanical properties. The journal *Surface and Coatings Technology* (479, 2024, 130462) reported the construction of PLA coating systems on magnesium alloy surfaces using a novel FDM method. Results showed that a 240 μm thick PLA coating could withstand 12 days of immersion in Ringer's solution without corroding or dissolving the magnesium alloy substrate. Furthermore, normal human dermal fibroblasts could grow and proliferate directly on the coating surface, demonstrating the excellent beneficial cell compatibility of PLA coatings. These studies indicate that PLA and its composite coatings can be applied in different fields through various processing techniques, exhibiting good mechanical properties, hydrophobicity, and solution barrier characteristics.

[0004] On the other hand, by incorporating various bactericidal groups or antibacterial substances into polylactic acid (PLA) coatings through methods such as mutual dissolution and grafting, PLA coatings can acquire antibacterial and bactericidal properties. For example, the journal *European Urology* (85, Supplement 1, 2024, S1752) reported that grafting ciprofloxacin onto a PLA composite coating matrix can inhibit the proliferation of *Escherichia coli* and the formation of bacterial biofilms, without cytotoxicity, and the samples showed minimal deposition of shells (calcium and magnesium salts) in both in vivo and in vitro experiments. *Progress in Organic Coatings* (123, 2018, 282-291) reported the preparation of a biodegradable polylactic acid (PLA) nanocomposite coating with antibacterial properties using an active gas phase generated by low-energy electron beam dispersion of a powder mixture of PLA and antibacterial components (norfloxacin and silver nitrate, respectively). Both PLA-based coatings exhibited excellent antibacterial activity against Gram-negative Escherichia coli ATCC 25922 and Gram-positive ATCC 12600. *Materials Letters* (341, 2023, 134293) reported a PLA-povidone-iodine coating with antibacterial properties fabricated using a simple suspended flame spraying method. This coating kills bacteria by releasing iodine and exhibits significant antibacterial activity against both *Escherichia coli* and *Staphylococcus aureus*, with a bactericidal rate exceeding 99.99%. These research cases demonstrate that it is feasible and promising to form antimicrobial composite coatings by incorporating polylactic acid with bactericidal and antimicrobial substances. However, it is important to note that, on the one hand, these reports are all for implants and are not applicable to the corrosion problems of structures in industrial environments; on the other hand, and more importantly, these newly introduced bactericidal groups or antimicrobial substances often dissolve, resulting in insufficient long-term protection (as illustrated in Comparative Example 8 below).

[0005] Furthermore, current research primarily involves grafting soluble bactericidal substances onto modified polylactic acid (PLA) coatings. This process is cumbersome and only applicable to a limited range of antibacterial and bactericidal effects, thus revealing insufficient long-term protective properties of the coatings. Therefore, a simple, cost-effective coating strategy that enhances the long-term protection of PLA is needed. This strategy should align with the strongly advocated environmentally friendly and degradable approach for engineering applications while also considering the sustainable development strategy of providing long-term protection. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide an environmentally friendly, self-forming film-forming spray and coating integrating polylactic acid (PLA) and isoflavone toxin II. By introducing isoflavone toxin II into a PLA solution dissolved in dichloromethane, the PLA and isoflavone toxin II are co-dissolved, resulting in a solution system that self-forms a film in the air. The resulting coating exhibits antibacterial and antifouling properties, making it particularly suitable for corrosion-resistant treatment of metal pipes. Compared to existing technologies involving pure PLA coatings, the present invention effectively improves the environmental resistance, anti-bioadhesion properties, hydrophobicity, and protection of the substrate material of PLA coatings.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a self-forming spray combining polylactic acid and isoflavone toxin II, which integrates environmental friendliness, antibacterial properties, and antifouling effects, is provided. The spray is characterized by being obtained by completely dissolving isoflavone toxin II in a dichloromethane solution of polylactic acid; wherein each 20 mL to 40 mL of the dichloromethane solution of polylactic acid contains 0.5 g to 1.5 g of polylactic acid, and each 20 mL to 40 mL of the dichloromethane solution of polylactic acid contains 10 mg to 30 mg of isoflavone toxin II.

[0008] According to a second aspect of the present invention, a method for using the above-mentioned self-forming spray of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness and antibacterial and antifouling properties, is provided, characterized in that the liquid film-forming spray is sprayed onto the metal surface to be protected and then dried, thereby forming a protective coating on the metal surface.

[0009] As a further preferred embodiment of the present invention, the drying is natural air drying.

[0010] According to a third aspect of the present invention, a method for preparing the above-described self-forming spray of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness, antibacterial and antifouling properties, is provided, characterized by comprising the following steps:

[0011] (1) Polylactic acid particles are added to dichloromethane and the polylactic acid particles are completely dissolved under sealed conditions to obtain a dichloromethane solution of polylactic acid; wherein the ratio of the mass of the polylactic acid particles added to the volume of the dichloromethane is (0.5g~1.5g):(20mL~40mL);

[0012] (2) Add isoflavone toxin II to the polylactic acid dichloromethane solution obtained in step (1) until it is completely dissolved to obtain an environmentally friendly antibacterial and antifouling polylactic acid and isoflavone toxin II composite liquid film-forming spray; wherein, the ratio of the mass of isoflavone toxin II added to the volume of the polylactic acid dichloromethane solution is (10mg~30mg): (20mL~40mL).

[0013] According to a fourth aspect of the present invention, a self-forming coating of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness, antibacterial and antifouling properties, is provided. The coating is characterized in that it is obtained by completely dissolving isoflavone toxin II in a dichloromethane solution of polylactic acid; wherein each 20 mL to 40 mL of the dichloromethane solution of polylactic acid contains 1 g to 3 g of polylactic acid; and each 20 mL to 40 mL of the dichloromethane solution of polylactic acid contains 10 mg to 30 mg of isoflavone toxin II.

[0014] According to a fifth aspect of the present invention, a method for using the above-mentioned self-forming film coating composed of polylactic acid and isoflavone toxin II, which integrates environmental friendliness and antibacterial and antifouling properties, is provided, characterized in that the liquid film-forming coating is applied to the metal surface to be protected and then dried, thereby forming a protective coating on the metal surface.

[0015] As a further preferred embodiment of the present invention, the coating is specifically applied by spin coating or brush coating;

[0016] The drying process involves natural air drying.

[0017] According to a sixth aspect of the present invention, a method for preparing the above-mentioned self-film-forming coating composed of polylactic acid and isoflavone toxin II, which integrates environmental friendliness, antibacterial and antifouling properties, is provided, characterized by comprising the following steps:

[0018] (1) Polylactic acid particles are added to dichloromethane and the polylactic acid particles are completely dissolved under sealed conditions to obtain a dichloromethane solution of polylactic acid; wherein the ratio of the mass of the polylactic acid particles added to the volume of the dichloromethane is (1g~3g):(20mL~40mL);

[0019] (2) Add isoflavone toxin II to the polylactic acid dichloromethane solution obtained in step (1) until it is completely dissolved to obtain an environmentally friendly antibacterial and antifouling liquid film-forming coating composed of polylactic acid and isoflavone toxin II; wherein, the ratio of the mass of isoflavone toxin II added to the volume of the polylactic acid dichloromethane solution is (10mg~30mg): (20mL~40mL).

[0020] According to a seventh aspect of the present invention, a protective coating is provided formed by a self-forming spray using the above-described integrated environmentally friendly and antibacterial / antifouling polylactic acid and isoflavone toxin II composite, or by a self-forming coating using the above-described integrated environmentally friendly and antibacterial / antifouling polylactic acid and isoflavone toxin II composite.

[0021] According to the eighth aspect of the present invention, the application of the above-described self-forming spray of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness and antibacterial and antifouling properties, or the above-described self-forming coating of polylactic acid and isoflavone toxin II composite, in antibacterial and antifouling applications is provided.

[0022] Compared with existing technologies, the technical solution conceived in this invention, through the dissolution of polylactic acid in dichloromethane followed by the miscibility of isoflavone toxin II, yields a composite solution system of polylactic acid and isoflavone toxin II. This system can be sprayed (or coated) onto a metal surface to form a self-film-forming polylactic acid composite coating containing isoflavone toxin II, achieving excellent protection, anti-biofilm adhesion properties, and antibacterial properties under field working conditions. The spray (or coating) and the corresponding coating obtained in this invention do not contain heavy metal elements and are environmentally friendly.

[0023] This invention improves the antibacterial and antifouling properties of a membrane by adding antibacterial and hydrophobic isoflavone toxin II to an environmentally friendly polylactic acid (PLA) liquid matrix. The synergistic effect of PLA and isoflavone toxin II enhances the hydrophobicity of the PLA coating, while PLA is completely miscible with isoflavone toxin II. This allows for highly uniform film formation in the liquid system, resulting in a coating with fewer bacterial attachment sites, preventing performance inhomogeneity and porosity. The resulting membrane combines environmental friendliness with antibacterial and antifouling properties, achieving a bacterial desorption rate exceeding 99.9%, demonstrating excellent effectiveness and significant application potential in the protection and maintenance of metal materials.

[0024] The polylactic acid / isodextrin II composite solution system obtained based on the present invention can be used as a spray. Compared with the traditional polylactic acid and its composite coating film formation process, the present invention simplifies the on-site operation process to the greatest extent by spray drying film formation method and has no safety hazards. For example: (1) The suspension flame deposition technology used in Materials Letters (341, 2023, 134293) to prepare polylactic acid coatings on stainless steel surfaces requires specially designed flame spray deposition equipment, which not only puts forward corresponding requirements on the shape and size of the sample, but also requires the use of combustible gas as a guide, which not only poses safety hazards, but also requires customization of the shape and size of the material. (2) Surface and Coatings Technology (388, 2020, 125593) reported that multilayer polylactic acid coatings were obtained by dip-coating after several soakings. This method is prone to causing the accumulation of polylactic acid solution at the bottom of the sample, and in the subsequent dip-coating process, the polylactic acid coating formed in the previous step may be dissolved and destroyed, resulting in uneven surface coating. On the other hand, considering that many equipment and facilities need to be installed before coating is applied in actual working conditions, it is more convenient to use this spray film formation at some bends and joints.

[0025] The preparation method of this invention is simple and easy to implement, has excellent protective performance, is easy to mass-produce and scale up, and has a good industrial production foundation and broad application prospects.

[0026] Specifically, the present invention can achieve the following beneficial effects:

[0027] (1) The polylactic acid / isodactylyotoxin II composite coating obtained by the liquid film-forming spray (and coating) of the present invention, which is environmentally friendly, has antimicrobial adhesion properties and antimicrobial and antifouling effects. This invention is the first to utilize the antibacterial polypeptide isoflavone II as a co-solubilizer added to a polylactic acid (PLA) system. Dichloromethane is used as a dispersant (on the one hand, dichloromethane can simultaneously dissolve both PLA and isoflavone II; on the other hand, dichloromethane evaporates rapidly, facilitating practical use). Taking spray formation as an example, the concentration of PLA in the spray is controlled at 0.5g–1.5g per 20mL–40mL, and the concentration of isoflavone II is controlled at 10mg–30mg per 20mL–40mL. This ensures that the spray is neither too dry (excessive dispersion concentration), affecting spraying, nor too dilute (excessive dispersion concentration), affecting film formation. Furthermore, PLA and isoflavone II hardly dissolve. For coatings, the concentration of PLA can be increased to 1g–3g per 20mL–40mL, effectively guaranteeing the coating effect.

[0028] (2) The preparation and application process of the spray (or coating) of the present invention is simple, greatly simplifying the cumbersome sample preparation steps, and sample preparation can be completed without any other procedures. The resulting coating can be used in almost all working conditions, especially for components that are prone to corrosion but have not yet reached the service life of the equipment.

[0029] (3) The protective coating obtained based on this invention has long-lasting protective properties. As described below. Figures 2 to 4 As exemplified, the polylactic acid and isoflavone toxin II composite membrane obtained by the present invention, after being immersed in a solution of sulfate-reducing bacteria Desulfovibrioferrophilus for 60 days, showed almost no bacterial cell coverage, and the composite membrane remained intact and pore-free, exhibiting long-lasting protection. Attached Figure Description

[0030] Figure 1 The images show the X-ray diffraction patterns of the polylactic acid / isodactylyanthropoietin II composite membrane and its individual components, prepared by co-solution.

[0031] Figure 2 The images show surface scanning and fluorescence images of the polylactic acid / isodactylyotoxin II composite membrane prepared in Example 1 and the polylactic acid membrane prepared in Comparative Example 1 after immersion in the marine sulfate-reducing bacterium *Desulfovibrio ferrophilus* for 60 days; wherein, Figure 2 (a) in the image corresponds to the surface scan of the polylactic acid membrane. Figure 2 (b) in the image corresponds to the surface scan of the polylactic acid / isotropic anemone toxin II composite coating. Figure 2 (c) in the image corresponds to the fluorescence image of the polylactic acid membrane. Figure 2 (d) in the image corresponds to the fluorescence image of the polylactic acid / isotropic anemone toxin II composite coating.

[0032] Figure 3 The images show surface scanning and fluorescence images of the polylactic acid / isodactylyotoxin II composite membrane prepared in Example 2 and the polylactic acid membrane prepared in Comparative Example 2 after immersion in marine sulfate-reducing bacteria *Desulfovibrio ferrophilus* for 60 days; wherein, Figure 3 (a) in the image corresponds to the surface scan of the polylactic acid membrane. Figure 3 (b) in the image corresponds to the surface scan of the polylactic acid / isotropic anemone toxin II composite coating. Figure 3 (c) in the image corresponds to the fluorescence image of the polylactic acid membrane. Figure 3 (d) in the image corresponds to the fluorescence image of the polylactic acid / isotropic anemone toxin II composite coating.

[0033] Figure 4The images show surface scanning and fluorescence images of the polylactic acid / isodactylyotoxin II composite membrane prepared in Example 3 and the polylactic acid membrane prepared in Comparative Example 3 after immersion in the marine sulfate-reducing bacterium *Desulfovibrio ferrophilus* for 60 days; wherein, Figure 4 (a) in the image corresponds to the surface scan of the polylactic acid membrane. Figure 4 (b) in the image corresponds to the surface scan of the polylactic acid / isotropic anemone toxin II composite coating. Figure 4 (c) in the image corresponds to the fluorescence image of the polylactic acid membrane. Figure 4 (d) in the image corresponds to the fluorescence image of the polylactic acid / isotropic anemone toxin II composite coating.

[0034] Figure 5 The images show surface scanning and fluorescence images of the polylactic acid / isodactylyotoxin II composite membrane prepared in Example 4 and the polylactic acid membrane prepared in Comparative Example 4 after immersion in the marine sulfate-reducing bacterium *Desulfovibrio ferrophilus* for 60 days; wherein, Figure 5 (a) in the image corresponds to the surface scan of the polylactic acid membrane. Figure 5 (b) in the image corresponds to the surface scan of the polylactic acid / isotropic anemone toxin II composite coating. Figure 5 (c) in the image corresponds to the fluorescence image of the polylactic acid membrane. Figure 5 (d) in the image corresponds to the fluorescence image of the polylactic acid / isotropic anemone toxin II composite coating.

[0035] Figure 6 The images show surface scanning and fluorescence images of the polylactic acid / isodactylyanthropoietin II composite membrane prepared in Example 5 and the polylactic acid membrane prepared in Comparative Example 5, respectively, after being immersed in marine sulfate-reducing bacteria *Desulfovibrio ferrophilus* for 60 days. Figure 6 (a) in the image corresponds to the surface scan of the polylactic acid membrane. Figure 6 (b) in the image corresponds to the surface scan of the polylactic acid / isotropic anemone toxin II composite coating. Figure 6 (c) in the image corresponds to the fluorescence image of the polylactic acid membrane. Figure 6 (d) in the image corresponds to the fluorescence image of the polylactic acid / isotropic anemone toxin II composite coating.

[0036] Figure 7 This is a comparison of the contact angle detection results of water droplets between the polylactic acid / isodactylyotropic anemone toxin II composite membrane prepared in Example 1 and the polylactic acid membrane prepared in Comparative Example 1; wherein, Figure 7 In the figure, (a) corresponds to the contact angle between the polylactic acid film and the water droplet. Figure 7 (b) in the figure corresponds to the contact angle between the polylactic acid / isotropic anemone toxin II composite membrane and the water droplet.

[0037] Figure 8This is a comparison of the weight loss of the metal substrate and the bacterial adsorption / desorption on the coating surface after the polylactic acid / isodactyly anemone toxin II composite membrane prepared in Example 1 and the polylactic acid membrane prepared in Comparative Example 1 were immersed in marine sulfate-reducing bacteria Desulfovibrio ferrophilus for 14 days.

[0038] Figure 9 These are physical images of the coatings obtained in Comparative Examples 6 and 7; among them, Figure 9 (a) corresponds to a ratio of 6. Figure 9 The corresponding ratio for (b) in the text is 7.

[0039] Figure 10 This is a comparison of the number of attached cells on the surface of polylactic acid (PLA) membranes immersed in sulfate-reducing bacteria *Desulfovibrio ferrophilus* medium for 14 days in Comparative Example 8, and PLA membranes with added amoxicillin. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0041] In general, the polylactic acid and isoflavone toxin II composite liquid film-forming spray (coating) of the present invention, which is both environmentally friendly and antibacterial and antifouling, can be prepared and used according to the following steps: (1) Prepare a polylactic acid liquid matrix; (2) Embed isoflavone toxin II into the polylactic acid liquid matrix to form a composite liquid; (3) After the composite liquid is filled into a spray bottle and sprayed out, it adheres to the surface of the facility to be protected (maintained) and evaporates in the air to form a film, thus obtaining a protective film.

[0042] In step (3), in addition to spraying, coating can also be done by spin coating, brushing, etc. (in this case, the composite liquid obtained in step 2 is the coating). Of course, spraying is the most convenient, especially suitable for the inner surface of small-diameter pipes that are difficult to access with tools such as brushes.

[0043] It is known in the prior art that isotropic anemone toxin II, also known as antimicrobial peptide A, has the amino acid sequence: SVPYDYNWYSNW. The isotropic anemone toxin II used in the following examples was prepared according to A. Zlotkin. Dispersion and detachment of cell aggregates. US Patent, No. 9284351B2, 2016. Additionally, the marine sulfate-reducing bacterium *Desulfovibrio ferrophilus* used in the following examples was purchased from the China Culture Collection Center (bio-136872).

[0044] The following are specific examples:

[0045] Example 1:

[0046] This embodiment provides a method for preparing a self-forming film spray coating composed of polylactic acid and isoflavone toxin II, which integrates environmental friendliness, antibacterial and antifouling properties, including the following steps:

[0047] (1) Add 0.5g of polylactic acid granules to 20mL of dichloromethane solution, seal the beaker with plastic wrap and sealing film, and obtain polylactic acid solution after the polylactic acid granules are completely dissolved.

[0048] (2) Add 10 mg of isoflavone toxin II to the polylactic acid solution obtained in step (1), and after it is completely dissolved, a composite solution of polylactic acid and isoflavone toxin II can be obtained.

[0049] (3) The composite liquid in (2) above is poured into a sprayer or spray bottle to obtain a liquid film-forming spray; the spray is applied to the surface of the metal to be protected (Q235 carbon steel is used in this embodiment, the same below) and naturally dried to obtain a composite protective coating of polylactic acid and isoflavone toxin II.

[0050] Comparative Example 1:

[0051] This comparative example is almost identical to Example 1, except that iso-finger anemone toxin II was not used, and the resulting coating sample is a polylactic acid coating.

[0052] Example 2

[0053] This embodiment provides a method for preparing a self-forming film spray coating composed of polylactic acid and isoflavone toxin II, which integrates environmental friendliness, antibacterial and antifouling properties, including the following steps:

[0054] (1) Add 1g of polylactic acid granules to 30mL of dichloromethane solution, seal the beaker with plastic wrap and sealing film, and obtain polylactic acid solution after the polylactic acid granules are completely dissolved.

[0055] (2) Add 20 mg of isoflavone toxin II to the polylactic acid solution obtained in step (1), and after it is completely dissolved, a composite solution of polylactic acid and isoflavone toxin II can be obtained.

[0056] (3) The composite liquid in (2) above can be poured into a sprayer or spray bottle to obtain a liquid film-forming spray. The spray is applied to the metal surface to be protected and allowed to dry naturally to obtain a protective coating.

[0057] Comparative Example 2:

[0058] This comparative example is almost identical to Example 2, except that isotropic anemone toxin II was not used, and the resulting coating sample is a polylactic acid coating.

[0059] Example 3

[0060] This embodiment provides a method for preparing a self-forming film spray coating composed of polylactic acid and isoflavone toxin II, which integrates environmental friendliness, antibacterial and antifouling properties, including the following steps:

[0061] (1) Add 1.5g of polylactic acid granules to 40mL of dichloromethane solution, seal the beaker with plastic wrap and sealing film, and obtain polylactic acid solution after the polylactic acid granules are completely dissolved.

[0062] (2) Add 30 mg of isoflavone toxin II to the polylactic acid solution obtained in step (1), and after it is completely dissolved, a composite solution of polylactic acid and isoflavone toxin II can be obtained.

[0063] (3) The composite liquid in (2) above can be poured into a sprayer or spray bottle to obtain a liquid film-forming spray. The spray is applied to the metal surface to be protected and allowed to dry naturally to obtain a protective coating.

[0064] Comparative Example 3:

[0065] This comparative example is almost identical to Example 3, except that iso-finger anemone toxin II is not used, and the resulting coating sample is a polylactic acid coating.

[0066] Example 4:

[0067] This embodiment provides a method for preparing a self-forming film coating of polylactic acid and isoflavone toxin II composite material that integrates environmentally friendly, antibacterial, and antifouling properties using spin coating or brush coating processes, comprising the following steps:

[0068] (1) Add 1g of polylactic acid granules to 20mL of dichloromethane solution, seal the beaker with plastic wrap and sealing film, and obtain polylactic acid solution after the polylactic acid granules are completely dissolved.

[0069] (2) Add 10 mg of isoflavone toxin II to the polylactic acid solution obtained in step (1), and after it is completely dissolved, a composite coating of polylactic acid and isoflavone toxin II can be obtained.

[0070] (3) The composite coating in (2) above is spin-coated or brushed onto the metal surface to be protected and then naturally dried to obtain a composite protective coating of polylactic acid and isotropic anemone toxin II.

[0071] Comparative Example 4:

[0072] This comparative example is almost identical to Example 4, except that isotropic anemone toxin II was not used, and the resulting coating sample is a polylactic acid coating.

[0073] Example 5:

[0074] This embodiment provides a method for preparing a self-forming film coating of polylactic acid and isoflavone toxin II composite material that integrates environmentally friendly, antibacterial, and antifouling properties using spin coating or brush coating processes, comprising the following steps:

[0075] (1) Add 3g of polylactic acid granules to 40mL of dichloromethane solution. Seal the beaker with plastic wrap and sealing film. After the polylactic acid granules are completely dissolved, a polylactic acid solution is obtained.

[0076] (2) Add 10 mg of isoflavone toxin II to the polylactic acid solution obtained in step (1), and after it is completely dissolved, a composite coating of polylactic acid and isoflavone toxin II can be obtained.

[0077] (3) The composite coating in (2) above is spin-coated or brushed onto the metal surface to be protected and then naturally dried to obtain a composite protective coating of polylactic acid and isotropic anemone toxin II.

[0078] Comparative Example 5:

[0079] This comparative example is almost identical to Example 5, except that isotropic anemone toxin II is not used, and the resulting coating sample is a polylactic acid coating.

[0080] In addition to using dichloromethane as a solvent during the research and development process of this invention, we also tried using commonly used organic solvents such as DMF and NMP, but their effects were not as good as those of dichloromethane, as detailed below:

[0081] Comparative Example 6:

[0082] This comparative example uses DMF as a solvent to prepare polylactic acid membranes. The specific operation process is as follows:

[0083] (1) Add 0.5g of polylactic acid granules to 20mL of DMF solution, seal the beaker with plastic wrap and sealing film, and obtain polylactic acid solution after the polylactic acid granules are completely dissolved.

[0084] (2) Spread the polylactic acid solution obtained in (1) on a glass plate and bake it in an oven at 80°C for 2 hours to dry it completely.

[0085] Comparative Example 7:

[0086] This comparative example uses NMP as a solvent to prepare polylactic acid membranes. The specific operation process is as follows:

[0087] (1) Add 0.5g of polylactic acid granules to 20mL of NMP solution, seal the beaker with plastic wrap and sealing film, and obtain polylactic acid solution after the polylactic acid granules are completely dissolved.

[0088] (2) Spread the polylactic acid solution obtained in (1) on a glass plate and bake it in an oven at 80°C for 2 hours to dry it completely.

[0089] The coatings obtained in Comparative Examples 6 and 7 are as follows Figure 9 As shown, it is easy to see that polylactic acid films made with DMF and NMP as solvents are powdery and cannot form a film.

[0090] Performance testing:

[0091] The composite coating of polylactic acid and isoflavone toxin II prepared by this invention does not alter the original state of its components, such as... Figure 1 As shown, the X-ray diffraction test results indicate that the polylactic acid and isoflavone toxin II composite membrane is a superposition of polylactic acid and isoflavone toxin II, indicating that the polylactic acid and isoflavone toxin II composite membrane has been successfully prepared without damaging any of its components.

[0092] The hydrophilicity and hydrophobicity of polylactic acid (PLA) coatings and PLA composite coatings containing isoflavone toxin II were measured using the water droplet method. Figure 7 The results showed that the water droplet contact angle of the polylactic acid (PLA) coating was 43.6°, while that of the PLA composite coating with added isoflavone II was 107.4°. It is clear that the water droplet contact angle of the PLA composite coating with added isoflavone II is 2.46 times that of the PLA coating, indicating a significant increase in hydrophobicity.

[0093] After anaerobic activation of the marine sulfate-reducing bacterium *Desulfovibrio ferrophilus* for three days using enriched artificial seawater (EASW) as reported in *Corrosion Science* (220, 2023, 111306), 1% (v / v) of the activated bacterial solution was injected into EASW culture medium with a composite coating (prepared in Example 1), and anaerobic cultured for 14 days and 60 days respectively in a constant temperature and humidity chamber at 37°C.

[0094] Similarly, 1% (volume ratio) of activated bacterial solution was injected into EASW culture medium coated with polylactic acid (prepared from Comparative Example 1), and anaerobic cultured for 14 days and 60 days respectively in a constant temperature and humidity chamber at 37°C.

[0095] After the 14-day bacterial immersion experiment, bacterial adsorption-desorption tests and metal matrix weight loss measurements were performed. The results are as follows: Figure 8 As shown. Using the composite membrane obtained in Example 1, the bacterial desorption rate reached 99.9%, and the weight loss of the metal substrate before and after immersion was less than 0.1 mg / cm³. 2 Using the polylactic acid coating obtained in Comparative Example 1, the bacterial desorption rate was significantly reduced, and the weight loss of the metal substrate before and after immersion reached as high as 1.7 mg / cm³. 2 .

[0096] After the 60-day bacterial immersion experiment, surface scanning and fluorescence image detection were performed, and the results were as follows: Figure 2 As shown, the polylactic acid coating surface is covered with a biofilm of sulfate-reducing bacteria *Desulfovibrio ferrophilus*, interspersed with bacterial cells, such as... Figure 2 As shown in (a); however, only a few sulfate-reducing bacteria adhered to the surface of the composite coating, and no obvious cell metabolites were found. Furthermore, the coating was intact and free of pores, as shown in (a). Figure 2 As shown in (b) of the diagram. Accordingly, from Figure 2 The surface fluorescence results in (c) and (d) show that the polylactic acid coating is almost completely covered by bacterial cells, while the composite coating surface shows almost no green fluorescence. Therefore, the bacterial desorption rate of the composite coating surface is >99.9%.

[0097] Similarly, 1% (v / v) of activated bacterial solution was injected into EASW culture media with a composite coating (prepared in Example 2) and a polylactic acid coating (prepared in Comparative Example 2), respectively, and anaerobically cultured for 60 days in a constant temperature and humidity incubator at 37°C. The results are as follows: Figure 3 As shown, after a 60-day bacterial immersion experiment, the polylactic acid coating surface was covered with a biofilm of sulfate-reducing bacteria *Desulfovibrioferrophilus*, interspersed with bacterial cells. Furthermore, the coating had extensively peeled and cracked, losing its protective effect. Figure 3As shown in (a); however, only a few sulfate-reducing bacteria adhered to the surface of the composite coating, and no obvious cell metabolites were found. Furthermore, the coating was intact and free of pores, as shown in (a). Figure 3 As shown in (b) of the diagram. Accordingly, from Figure 3 The surface fluorescence results in (c) and (d) show that the polylactic acid coating is almost completely covered by bacterial cells, while the composite coating surface shows almost no green fluorescence. Therefore, the bacterial desorption rate of the composite coating surface is >99.9%.

[0098] Similarly, 1% (v / v) of activated bacterial solution was injected into EASW culture media with a composite coating (prepared in Example 3) and a polylactic acid coating (prepared in Comparative Example 3), respectively, and anaerobically cultured for 60 days in a constant temperature and humidity incubator at 37°C. The results are as follows: Figure 4 As shown, after the 60-day bacterial immersion experiment, the polylactic acid coating surface was covered with a biofilm of sulfate-reducing bacteria *Desulfovibrioferrophilus*, interspersed with bacterial cells. The coating had also extensively peeled and cracked, revealing a significant portion of the sulfate-reducing bacteria biofilm beneath the peeled layer, rendering it ineffective as a protective agent. Figure 4 As shown in (a); however, only a few sulfate-reducing bacteria adhered to the surface of the composite coating, and no obvious cell metabolites were found. Furthermore, the coating was intact and free of pores, as shown in (a). Figure 4 As shown in (b) of the diagram. Accordingly, from Figure 4 The surface fluorescence results in (c) and (d) show that the polylactic acid coating is almost completely covered by bacterial cells, while the composite coating surface shows almost no green fluorescence. Therefore, the bacterial desorption rate of the composite coating surface is >99.9%.

[0099] Similarly, 1% (v / v) of activated bacterial solution was injected into EASW culture media with a composite coating (prepared in Example 4) and a polylactic acid coating (prepared in Comparative Example 4), respectively, and anaerobically cultured for 60 days in a constant temperature and humidity incubator at 37°C. The results are as follows: Figure 5 As shown, after the 60-day bacterial immersion experiment, a biofilm of sulfate-reducing bacteria *Desulfovibrioferrophilus* accumulated on the surface of the polylactic acid coating, interspersed with bacterial cells. Furthermore, the coating had extensively peeled and cracked, losing its protective effect. Figure 5 As shown in (a); however, only a few sulfate-reducing bacteria adhered to the surface of the composite coating, and no obvious cell metabolites were found. Furthermore, the coating was intact and free of pores, as shown in (a). Figure 5 As shown in (b) of the diagram. Accordingly, from Figure 5 The surface fluorescence results in (c) and (d) show that the polylactic acid coating is almost completely covered by bacterial cells, while the composite coating surface shows almost no green fluorescence. Therefore, the bacterial desorption rate of the composite coating surface is >99.9%.

[0100] Similarly, 1% (v / v) of activated bacterial solution was injected into EASW culture media with a composite coating (prepared in Example 5) and a polylactic acid coating (prepared in Comparative Example 5), respectively, and anaerobically cultured for 60 days in a constant temperature and humidity incubator at 37°C. The results are as follows: Figure 6 As shown, after the 60-day bacterial immersion experiment, a biofilm of sulfate-reducing bacteria *Desulfovibrioferrophilus* accumulated on the surface of the polylactic acid coating, interspersed with bacterial cells. Furthermore, the coating had extensively peeled and cracked, losing its protective effect. Figure 6 As shown in (a); however, only a few sulfate-reducing bacteria adhered to the surface of the composite coating, and no obvious cell metabolites were found. Furthermore, the coating was intact and free of pores, as shown in (a). Figure 6 As shown in (b) of the diagram. Accordingly, from Figure 6 The surface fluorescence results in (c) and (d) show that the polylactic acid coating is almost completely covered by bacterial cells, while the composite coating surface shows almost no green fluorescence. Therefore, the bacterial desorption rate of the composite coating surface is >99.9%.

[0101] In addition, to compare with traditional processes using antibacterial substances, the inventors also conducted the following comparative example 8:

[0102] Comparative Example 8

[0103] This comparative example tested the adhesion and growth of adherent cells on the surface of polylactic acid (PLA) coatings and PLA coatings containing amoxicillin after immersion in a solution of sulfate-reducing bacteria *Desulfovibrio ferrophilus* cultured in EASW medium for 14 days. Specifically, 1 g of PLA particles were added to 30 mL of dichloromethane solution. The beaker was sealed with plastic wrap and sealing film until the PLA particles were completely dissolved. Two PLA solution samples were prepared in this way. Then, 20 mg of amoxicillin powder was added to one of the PLA solution samples and mixed thoroughly to obtain a PLA / amoxicillin composite solution. The composite solution was then poured into a sprayer or spray bottle to obtain a liquid film-forming spray. The spray was applied to the metal surface to be protected and allowed to air dry to obtain the protective coating. Another polylactic acid solution without amoxicillin powder was prepared by forming a polylactic acid film using a similar procedure to that in Comparative Example 1 above.

[0104] The experimental results for the two coatings are as follows: Figure 10 As shown, the number of attached cells on the surface of the polylactic acid (PLA) coating after 14 days of culture was almost the same as that of the PLA coating with added amoxicillin, indicating that the addition of this type of water-soluble antibacterial drug cannot provide long-term inhibition of bacteria. Therefore, the drawbacks of this traditional antibacterial substance addition are obvious.

[0105] The above embodiments are merely examples. For instance, the molecular weight of polylactic acid (PLA) can be flexibly varied (e.g., 60,000, 80,000, 110,000), as long as the amount of PLA ensures complete dissolution in dichloromethane. Furthermore, dissolving PLA in dichloromethane can be achieved through stirring or without stirring, as long as the time is sufficient for complete dissolution. Besides metal substrates, this invention is also applicable to the surfaces of other protective devices requiring antibacterial and antifouling properties.

[0106] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A self-forming spray combining environmentally friendly, antibacterial, and antifouling polylactic acid and isoflavone toxin II, characterized in that, The spray is obtained by completely dissolving isoflavone toxin II in a dichloromethane solution of polylactic acid; wherein each 20 mL to 40 mL of the dichloromethane solution of polylactic acid contains 0.5 g to 1.5 g of polylactic acid, and each 20 mL to 40 mL of the dichloromethane solution of polylactic acid corresponds to the addition of 10 mg to 30 mg of isoflavone toxin II.

2. The method of using the self-forming spray of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness, antibacterial and antifouling properties, as described in claim 1, is characterized in that... The self-forming film spray is applied to the metal surface to be protected and then dried to form a protective coating on the metal surface.

3. The method of using the self-forming spray of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness, antibacterial properties, and antifouling effects as described in claim 2, is characterized in that... The drying process involves natural air drying.

4. The preparation method of the self-forming spray of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness, antibacterial and antifouling properties as described in claim 1, is characterized in that... Includes the following steps: (1) Polylactic acid particles are added to dichloromethane, and the polylactic acid particles are completely dissolved under sealed conditions to obtain a polylactic acid dichloromethane solution; wherein the ratio of the mass of the polylactic acid particles added to the volume of the dichloromethane is (0.5 g ~ 1.5 g): (20 mL ~ 40 mL); (2) Add isoflavone toxin II to the polylactic acid dichloromethane solution obtained in step (1) until it is completely dissolved to obtain an environmentally friendly antibacterial and antifouling polylactic acid and isoflavone toxin II composite self-forming spray; wherein, the ratio of the mass of isoflavone toxin II added to the volume of the polylactic acid dichloromethane solution is (10 mg ~ 30 mg): (20 mL ~ 40 mL).

5. A self-forming film coating that integrates environmentally friendly, antibacterial, and antifouling properties with polylactic acid and isoflavone toxin II, characterized in that, The coating is obtained by completely dissolving isoflavone toxin II in a dichloromethane solution of polylactic acid; wherein each 20 mL to 40 mL of the dichloromethane solution of polylactic acid contains 1 g to 3 g of polylactic acid; and each 20 mL to 40 mL of the dichloromethane solution of polylactic acid corresponds to the addition of 10 mg to 30 mg of isoflavone toxin II.

6. The method of using the self-forming film coating of polylactic acid and isoflavone toxin II composite as described in claim 5, characterized in that, The process involves applying the self-forming coating to the metal surface to be protected and then drying it to form a protective coating on the metal surface.

7. The method of using the self-forming coating of polylactic acid and isoflavone toxin II composite, which integrates environmental friendliness, antibacterial properties, and antifouling properties as described in claim 6, is characterized in that... The coating process specifically involves spin coating or brush coating; The drying process involves natural air drying.

8. The method for preparing the self-forming film coating of polylactic acid and isoflavone toxin II composite as described in claim 5, characterized in that, Includes the following steps: (1) Polylactic acid particles are added to dichloromethane and the polylactic acid particles are completely dissolved under sealed conditions to obtain a dichloromethane solution of polylactic acid; wherein the ratio of the mass of the polylactic acid particles added to the volume of the dichloromethane is (1g ~ 3g): (20 mL ~ 40 mL). (2) Add isoflavone toxin II to the polylactic acid dichloromethane solution obtained in step (1) until it is completely dissolved to obtain an environmentally friendly antibacterial and antifouling polylactic acid and isoflavone toxin II composite self-forming coating; wherein, the ratio of the mass of isoflavone toxin II added to the volume of the polylactic acid dichloromethane solution is (10 mg ~ 30 mg): (20 mL ~ 40 mL).

9. A protective coating formed by using a self-forming spray of polylactic acid and isoflavone toxin II composite as described in claim 1, which integrates environmental friendliness and antibacterial and antifouling properties, or by using a self-forming coating of polylactic acid and isoflavone toxin II composite as described in claim 5.

10. The application of the self-forming spray of polylactic acid and isoflavone toxin II composite as described in claim 1, which integrates environmental friendliness and antibacterial and antifouling properties, or the self-forming coating of polylactic acid and isoflavone toxin II composite as described in claim 5, in antibacterial and antifouling applications.

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