Biological long-acting bactericide as well as preparation method and application thereof

Abstract

The invention relates to a biological long-acting bactericide and a preparation method and application thereof.The biological long-acting bactericide can release a quaternary ammonium salt type sophorolipid sterilization unit with the sterilization effect according to needs, the sterilization time is effectively prolonged, the recycling performance of the bactericide is improved, and the biotoxicity of the bactericide is reduced; the excessive use of the bactericide is avoided to a great extent; in addition, the biological long-acting bactericide also has excellent substrate adhesion and washing resistance, and is beneficial to inhibition of adhesion of bacteria on the surface of a material, so that the surface of the material washed by water still keeps relatively high antibacterial ability.

Description

Technical Field

[0001] The present disclosure relates to a bactericide, and specifically, to a biological long-acting bactericide, a preparation method thereof, and an application thereof. Background Art

[0002] Problems such as infections caused by bacteria and metal corrosion have caused huge losses to global public health and economic development. For example, serious corrosion caused by bacteria can be found in production and processing equipment such as industrial water transportation, oil transportation, and gas transportation, and even cause perforation and leakage. According to the results of a corrosion survey, the total corrosion cost in China in 2014 was approximately 2,127.8 billion yuan, and the loss caused by bacterial corrosion accounted for ~20%. Quaternary ammonium salt surfactants are the most commonly used type of bactericides in daily life and industrial production. However, during their use, they are often released explosively and ultimately discharged into water bodies. Due to the lack of long-acting bactericidal properties, the application frequency of such bactericides is very high, but their biodegradability is poor. The large accumulation of bactericides in the environment will not only impose a heavy burden on the ecological environment, but also further accelerate the triggering of bacteria to develop drug resistance.

[0003] In view of the many drawbacks exposed by short-acting bactericides, it is necessary to develop materials with long-acting bactericidal properties. At present, the preparation methods for long-acting bactericidal materials are very limited, and usually involve problems such as complex chemical synthesis conditions, high production costs, difficulty in large-scale preparation, and poor biodegradability. For example, cationic quaternary ammonium salt fragments are grafted onto non-leaching polymer molecular brushes or polymer emulsions through chemical synthesis methods to extend the bactericidal time (Biomacromolecules, 2022, 23, 1, 424–430; ACS Appl. Mater. Interfaces 2018, 10, 6124-6136). However, the chemical surface grafting method generally requires a specific substrate and has high selectivity for applicable substrates. Moreover, the cumbersome and delicate synthesis process is difficult to achieve large-scale preparation, which greatly limits its practical application in actual life and production. Summary of the Invention

[0004] The purpose of the present disclosure is to provide a biological long-acting bactericide, a preparation method thereof, and an application thereof. The biological long-acting bactericide can release quaternary ammonium salt-type sophorolipid bactericidal units with bactericidal effects as needed, effectively extending the bactericidal time, improving the recyclability, reducing the biological toxicity of the bactericide, and greatly avoiding the overuse of the bactericide.

[0005] To achieve the above purpose, in the first aspect of the present disclosure, a biological long-acting bactericide is provided. The biological long-acting bactericide comprises a quaternary ammonium salt-type sophorolipid and a natural polyphenol compound. The quaternary ammonium salt-type sophorolipid has a structure shown in the following formula (1):

[0006]

[0007] wherein X in formula (1) - is selected from F - , Cl - , Br - or I - ;

[0008] R1 and R2 are each independently selected from alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, substituted or unsubstituted phenyl groups having 6 to 14 carbon atoms, and substituted or unsubstituted cycloalkyl groups having 3 to 6 carbon atoms;

[0009] said R3 is selected from alkyl groups having 8 to 18 carbon atoms, alkoxy groups having 8 to 18 carbon atoms, substituted or unsubstituted phenyl groups having 6 to 24 carbon atoms, and substituted or unsubstituted cycloalkyl groups having 8 to 18 carbon atoms;

[0010] The substituents in R1 to R3 are each independently selected from one or more of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0011] Optionally, the natural polyphenol compound is selected from one or more of flavonoid compounds, anthocyanin compounds, ellagic acid compounds, and catechin compounds; the flavonoid compounds are selected from one or more of rutin, quercetin, and kaempferol; the anthocyanin compounds are selected from one or more of pelargonidin, cyanidin, and delphinidin; the ellagic acid compounds are selected from one or more of methoxy ellagic acid, methylenedioxy ellagic acid, and 3,3'-dimethoxy ellagic acid; the catechin compounds are selected from one or more of tea polyphenols, tannic acid, gallic acid, catechin, epigallocatechin, epigallocatechin gallate, and catechin hydrate; preferably, the natural polyphenol compound is selected from one or more of tea polyphenols, tannic acid, gallic acid, catechin, epigallocatechin, epigallocatechin gallate, quercetin, ellagic acid, and catechin hydrate.

[0012] Optionally, there is a non-covalent interaction between the quaternary ammonium salt type sophorolipid and the natural polyphenol compound, and the non-covalent interaction includes one or more of electrostatic interaction, hydrophobic interaction, hydrogen bond, π-π stacking interaction, and van der Waals force.

[0013] The second aspect of the present disclosure provides a preparation method of a biological long-acting bactericide, and the preparation method includes: contacting a quaternary ammonium salt type sophorolipid, a natural polyphenol compound with water to obtain a mixture containing the biological long-acting bactericide.

[0014] Optionally, the preparation method further includes:

[0015] (1) Dissolve quaternary ammonium salt type sophorolipid in water to obtain a quaternary ammonium salt type sophorolipid solution, and dissolve natural polyphenolic compounds in water to obtain a polyphenol solution;

[0016] (2) Mix and stir the quaternary ammonium salt type sophorolipid solution and the polyphenol solution to obtain a mixture; perform solid-liquid separation on the mixture to obtain a solid containing the biological long-acting bactericide; optionally, the solid-liquid separation includes one or more of centrifugation, precipitation, and filtration, preferably centrifugation.

[0017] Optionally, the concentration of the quaternary ammonium salt type sophorolipid solution is 0.02 - 10 mmol / L, preferably 0.1 - 1 mmol / L; the concentration of the polyphenol solution is 5 - 60 mmol / L, preferably 5 - 30 mmol / L; the volume ratio of the quaternary ammonium salt type sophorolipid solution to the polyphenol solution is (3 - 15):(1 - 15), preferably (3 - 8):(1 - 7); the temperature of the mixing is 10 - 40 °C, preferably 25 - 40 °C; the stirring time is 10 - 60 min, preferably 20 - 40 min; the rotation speed of the centrifugation is 4000 - 10000 rpm, preferably 5000 - 8000 rpm.

[0018] The third aspect of the present disclosure provides a biological long-acting bactericide prepared by using the preparation method described in the second aspect of the present disclosure.

[0019] The fourth aspect of the present disclosure provides an application of the biological long-acting bactericide described in the first aspect and the third aspect of the present disclosure in the field of antibacterial.

[0020] Optionally, the application includes: dissolving the biological long-acting bactericide in a solvent to obtain a solution containing the biological long-acting bactericide; coating or spraying the solution onto the surface of a solid, and forming a bactericidal coating on the surface of the solid after solvent evaporation induction; bringing the bactericidal coating into contact with bacteria to play a bactericidal role; or, putting the biological long-acting bactericide into the water body containing bacteria to be treated and bringing it into contact with bacteria to play a bactericidal role.

[0021] Optionally, the solvent is selected from one or more of methanol, ethanol, propylene glycol, isopropanol, methanol / water, ethanol / water, and propylene glycol / water; the coating method includes spin coating and / or blade coating; the content of the biological long-acting bactericide contained in the bactericidal coating is 5 - 110 μg / cm 2 , preferably 50 - 60 μg / cm 2 .

[0022] Optionally, the dosage of the biological long-acting bactericide is 10 - 1000 μg / mL, preferably 100 - 500 μg / mL.

[0023] Through the above technical solutions, the present disclosure provides a biotype long-acting bactericide and its preparation method and application. The biotype long-acting bactericide comprises quaternary ammonium salt type sophorolipid and natural polyphenolic compounds. The biotype long-acting bactericide can exist in the form of supramolecular assemblies in an aqueous system, wherein the quaternary ammonium salt type sophorolipid and natural polyphenolic compounds will spontaneously assemble together through non-covalent interactions in the aqueous system to form the above supramolecular assemblies. Compared with the one-time explosive release of conventional quaternary ammonium salt type sophorolipids, the biotype long-acting bactericide of the present disclosure can release the bactericidal unit of quaternary ammonium salt type sophorolipid with bactericidal effect as needed, effectively extending the bactericidal time limit, improving its recyclability, reducing the biological toxicity of the bactericide, and greatly avoiding the overuse of the bactericide; in addition, the biotype long-acting bactericide also has excellent substrate adhesion and water washing resistance, which is beneficial to inhibiting the adhesion of bacteria on the material surface and enabling the material surface after water washing to still maintain a high antibacterial ability. The preparation method of the biotype long-acting bactericide of the present disclosure uses non-petroleum-based green biomass as raw materials, and forms a biotype long-acting bactericide by simply modifying quaternary ammonium salt type sophorolipid with natural polyphenolic compounds. This method has mild reaction conditions, low energy consumption, high biodegradability, is easy to realize industrial batch preparation, and has good industrial application prospects.

[0024] Other features and advantages of the present disclosure will be described in detail in the subsequent specific implementation part. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings are used to provide a further understanding of the present disclosure, and constitute a part of the specification. Together with the following specific implementation, they are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the drawings:

[0026] Figure 1 is the surface contact angle of QSL-C used in Example 1 of the present disclosure 12 and the biotype long-acting bactericide 1 prepared and water;

[0027] Figure 2 is the SEM image of QSL-C used in Example 2 of the present disclosure 14 and the biotype long-acting bactericide 2 prepared;

[0028] Figure 3 is the X-ray photoelectron spectroscopy of the biotype long-acting bactericide 2 prepared in Example 2 of the present disclosure;

[0029] Figure 4 is the mildness test diagram of QSL-C used in Example 2 of the present disclosure 14 and the biotype long-acting bactericide 2 prepared;

[0030] Figure 5 is the quaternary ammonium salt type sophorolipid QSL-C prepared in Example 1 of the present disclosure12 ESI mass spectrum Specific embodiments

[0031] The following further describes the specific embodiments of the present disclosure with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present disclosure, and are not intended to limit the present disclosure.

[0032] In a first aspect of the present disclosure, a biotype long-acting fungicide is provided. The biotype long-acting fungicide comprises quaternary ammonium salt type sophorolipid and natural polyphenolic compounds. The quaternary ammonium salt type sophorolipid has a structure shown in the following formula (1):

[0033]

[0034] Wherein X in formula (1) - is selected from F - 、Cl - 、Br - or I - ;

[0035] R1 and R2 are each independently selected from alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, substituted or unsubstituted phenyl groups having 6 to 14 carbon atoms, and substituted or unsubstituted cycloalkyl groups having 3 to 6 carbon atoms; preferably, R1 and R2 are each independently selected from alkyl groups having 1 to 4 carbon atoms, alkoxy groups having 1 to 4 carbon atoms, substituted or unsubstituted phenyl groups having 6 to 10 carbon atoms, and substituted or unsubstituted cycloalkyl groups having 3 to 5 carbon atoms;

[0036] The R3 is selected from alkyl groups having 8 to 18 carbon atoms, alkoxy groups having 8 to 18 carbon atoms, substituted or unsubstituted phenyl groups having 6 to 24 carbon atoms, and substituted or unsubstituted cycloalkyl groups having 8 to 18 carbon atoms; preferably, R3 is selected from alkyl groups having 10 to 16 carbon atoms, alkoxy groups having 10 to 16 carbon atoms, substituted or unsubstituted phenyl groups having 6 to 18 carbon atoms, and substituted or unsubstituted cycloalkyl groups having 10 to 16 carbon atoms;

[0037] The substituents in R1 to R3 are each independently selected from one or more of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0038] In the present disclosure, the substituents in R1 to R3 include the substituents on the substituted phenyl and the substituents on the substituted cycloalkyl. Among them, "substituted or unsubstituted cycloalkyl" refers to a cycloalkyl with substituents or an unsubstituted cycloalkyl, and the substituted cycloalkyl may be one or more hydrogen atoms in the cycloalkyl replaced by substituents; "substituted or unsubstituted phenyl" refers to a phenyl with substituents or an unsubstituted phenyl, and the substituted phenyl may be one or more hydrogen atoms in the phenyl replaced by substituents. The number of carbon atoms of the substituted or unsubstituted group refers to all the carbon atoms. For example, if the phenyl is selected from a substituted phenyl with 10 carbon atoms, then the total number of carbon atoms of the phenyl and its substituents is 10.

[0039] The biotype long-acting bactericide provided by the present disclosure comprises a quaternary ammonium salt type sophorolipid and a natural polyphenol compound. The biotype long-acting bactericide can exist in the form of a supramolecular assembly in an aqueous system, wherein the quaternary ammonium salt type sophorolipid and the natural polyphenol compound will spontaneously assemble together through non-covalent interactions in the aqueous system to form the above-mentioned supramolecular assembly. Compared with the one-time explosive release of the conventional quaternary ammonium salt type sophorolipid, the biotype long-acting bactericide of the present disclosure can release the quaternary ammonium salt type sophorolipid bactericidal unit with bactericidal effect as needed, effectively prolonging the bactericidal time limit, improving its recyclability, reducing the biological toxicity of the bactericide, and greatly avoiding the overuse of the bactericide; in addition, the biotype long-acting bactericide also has excellent substrate adhesion and water resistance, which is beneficial to inhibiting the adhesion of bacteria on the material surface and enabling the material surface to still maintain a high antibacterial ability after water washing.

[0040] In one embodiment of the present disclosure, the natural polyphenol compound is selected from one or more of flavonoid compounds, anthocyanin compounds, ellagic acid compounds, and catechin compounds; the flavonoid compounds are selected from one or more of rutin, quercetin, and kaempferol; the anthocyanin compounds are selected from one or more of pelargonidin, cyanidin, and delphinidin; the ellagic acid compounds are selected from one or more of methoxy ellagic acid, methylenedioxy ellagic acid, and 3,3'-dimethoxy ellagic acid; the catechin compounds are selected from one or more of tea polyphenols, tannic acid, gallic acid, catechin, epigallocatechin, epigallocatechin gallate, and catechin hydrate. In a preferred embodiment, the natural polyphenol compound is selected from one or more of tea polyphenols, tannic acid, gallic acid, catechin, epigallocatechin, epigallocatechin gallate, quercetin, ellagic acid, and catechin hydrate. In the above embodiments, the natural polyphenol compound has a polyhydroxy structure, which is beneficial to the modification of the quaternary ammonium salt type sophorolipid.

[0041] In the present disclosure, there is a non-covalent interaction between the quaternary ammonium salt type sophorolipid and the natural polyphenolic compound, and the non-covalent interaction includes one or more of electrostatic interaction, hydrophobic interaction, hydrogen bond, π-π stacking interaction and van der Waals force. In the above embodiments, the quaternary ammonium salt type sophorolipid and the natural polyphenolic compound can spontaneously assemble together through one or more non-covalent interactions in the presence of water to form a biological long-acting bactericide in the form of a supramolecular assembly.

[0042] The second aspect of the present disclosure provides a preparation method of a biological long-acting bactericide, and the preparation method includes: contacting a quaternary ammonium salt type sophorolipid, a natural polyphenolic compound with water to obtain a mixture containing the biological long-acting bactericide.

[0043] The preparation method provided by the present disclosure uses non-petroleum-based green biomass as a raw material, and through simple polyphenol modification of the quaternary ammonium salt type sophorolipid by using a natural polyphenol compound, a mixture containing the biological long-acting bactericide is formed. The preparation method of the present disclosure has mild conditions, low energy consumption, high biodegradability, is easy to realize industrial batch preparation, and has good industrial application prospects.

[0044] In one embodiment of the present disclosure, the preparation method further includes:

[0045] (1) Dissolving the quaternary ammonium salt type sophorolipid in water to obtain a quaternary ammonium salt type sophorolipid solution, and dissolving the natural polyphenolic compound in water to obtain a polyphenol solution;

[0046] (2) Mixing and stirring the quaternary ammonium salt type sophorolipid solution and the polyphenol solution to obtain a mixture; performing solid-liquid separation on the mixture to obtain a solid containing the biological long-acting bactericide;

[0047] Optionally, the solid-liquid separation includes one or more of centrifugation, precipitation and filtration, and preferably centrifugation.

[0048] In the above embodiments, mixing and stirring the quaternary ammonium salt type sophorolipid solution and the polyphenol solution can enable the quaternary ammonium salt type sophorolipid and the natural polyphenolic compound to spontaneously assemble together through one or more non-covalent interactions, achieve the purpose of simple polyphenol modification of the quaternary ammonium salt type sophorolipid, and form a mixture containing a supramolecular composition. Performing solid-liquid separation on the obtained mixture can obtain a solid containing the biological long-acting bactericide, and the biological long-acting bactericide exists in the solid in the form of a supramolecular assembly.

[0049] In one embodiment of the present disclosure, the concentration of the quaternary ammonium salt type sophorolipid solution is 0.02 - 10 mmol / L, preferably 0.1 - 1 mmol / L; the concentration of the polyphenol solution is 5 - 60 mmol / L, preferably 5 - 30 mmol / L; the volume ratio of the quaternary ammonium salt type sophorolipid solution to the polyphenol solution is (3 - 15):(1 - 15), preferably (3 - 8):(1 - 7); the temperature of the mixing is 10 - 40 °C, preferably 25 - 40 °C; the stirring time is 10 - 60 min, preferably 20 - 40 min; the centrifugation speed is 4000 - 10000 rpm, preferably 5000 - 8000 rpm. In the above embodiment, selecting the quaternary ammonium salt type sophorolipid solution and the polyphenol solution with the preferred concentration and volume ratio is beneficial to form a biotype long-acting bactericide with a certain composition.

[0050] In one embodiment, the preparation method further includes, before the mixing in step (2), adjusting the pH value of the quaternary ammonium salt type sophorolipid solution to 6 - 9, preferably 7 - 9, with a pH regulator to make the quaternary ammonium salt type sophorolipid solution colorless and transparent; adjusting the pH value of the polyphenol solution to 5 - 10, preferably 5 - 8, with a pH regulator to make the polyphenol solution clear and transparent; wherein, the pH regulator includes one or more of sodium hydroxide solution, potassium hydroxide solution, lithium hydroxide solution, sodium carbonate solution, sodium bicarbonate solution and ammonia water, preferably sodium hydroxide solution.

[0051] In another embodiment, the method further includes washing the solid obtained after solid-liquid separation multiple times, and performing vacuum drying or freeze drying after the solid turns light brown to obtain a dry sample of the biotype long-acting bactericide powder.

[0052] The third aspect of the present disclosure provides a biotype long-acting bactericide prepared by using the preparation method described in the second aspect of the present disclosure.

[0053] The fourth aspect of the present disclosure provides the application of the biotype long-acting bactericide described in the first aspect and the third aspect of the present disclosure in the antibacterial field.

[0054] In one embodiment, the application includes: dissolving the biotype long-acting bactericide in a solvent to obtain a solution containing the biotype long-acting bactericide; coating or spraying the solution onto the surface of a solid, and forming a bactericidal coating on the surface of the solid after being induced by solvent evaporation; making the bactericidal coating contact with bacteria to play a bactericidal role. In the above embodiment, the biotype long-acting bactericide is applied to the surface of a solid in the form of a coating, so that the surface of the solid has a high bactericidal activity to inhibit the content of bacteria on the surface of the solid, wherein the bacteria on the surface of the solid include one or more of Gram-positive bacteria, Gram-negative bacteria, fungi and molds.

[0055] In a preferred embodiment, the solvent is selected from one or more of methanol, ethanol, propylene glycol, isopropanol, methanol / water, ethanol / water, and propylene glycol / water; the coating method includes spin coating and / or blade coating; the content of the biotype long-acting bactericide contained in the bactericidal coating is 5-110 μg / cm 2 , preferably 50-60 μg / cm 2 ; the thickness of the bactericidal coating is 100-1000 nm, preferably 500-800 nm.

[0056] In a specific embodiment, the dry biotype long-acting bactericide powder is fully dissolved in a methanol solvent, and a stainless steel sheet is immersed in the above solution for 3 min. Then, the stainless steel sheet is taken out and the surface of the stainless steel is dried with nitrogen or air-dried naturally to obtain a bactericidal stainless steel product. Alternatively, the dry biotype long-acting bactericide powder is fully dissolved in an ethanol solvent, and the above solution is spin-coated on the surface of a plastic sheet, and the surface of the plastic is dried with nitrogen or air-dried naturally to obtain a bactericidal plastic product. Or, the dry biotype long-acting bactericide powder is fully dissolved in a methanol solvent, and a cotton cloth is immersed in the above solution for 5 min, and the surface of the cotton cloth is dried with nitrogen or air-dried naturally to obtain a bactericidal cotton cloth product. When the obtained bactericidal stainless steel product, bactericidal plastic product, and bactericidal cotton cloth product are in contact with bacteria, they can effectively inhibit the adhesion of bacteria on the material surface, and the surface of the material after water washing still maintains a high antibacterial ability.

[0057] In another embodiment, the application includes: putting the biotype long-acting bactericide into the water body containing bacteria to be treated and contacting with bacteria to play a bactericidal role. In the above embodiment, the biotype long-acting bactericide is put into the water body containing bacteria to be treated in the form of powder, and the bacteria in the water body containing bacteria to be treated include one or more of Gram-positive bacteria, Gram-negative bacteria, fungi, and molds.

[0058] In a preferred embodiment, the dosage of the biotype long-acting bactericide is 10-1000 μg / mL, preferably 100-500 μg / mL.

[0059] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereby. Unless otherwise specified, the raw materials used in the examples and comparative examples of the present disclosure are purchased through commercial channels and are all pure reagents.

[0060] Example 1

[0061] In this example, quaternary ammonium salt type sophorolipid QSL-C was first prepared 12 , and the specific steps are as follows:

[0062] S1. Weigh 12.4 g (10 mmol) of an acidic sophorolipid aqueous solution (purchased from Shandong Qilu Biotechnology Co., Ltd., with a water content of 50 wt%) into a round-bottom flask. Add 0.028 g (base, 0.2 mmol) of disodium hydrogen phosphate and heat to 90 °C, then stir for 1 h. Then add 1.02 g (epihalohydrin, 11 mmol) of epichlorohydrin and 0.060 g (phase transfer catalyst, 0.186 mmol) of tetrabutylammonium bromide to obtain a mixture. The first solvent in the mixture is water. The molar ratio of epihalohydrin to sophorolipid is 1.1:1, the molar ratio of base to sophorolipid is 0.02:1, and the molar ratio of phase transfer catalyst to sophorolipid is 0.0186:1. Heat the obtained mixture under stirring for 12 h (the temperature of the heating reaction is 90 °C), stop the reaction, and rotary evaporate to remove the solvent to obtain 6.56 g of an intermediate with a yield of 92.1%.

[0063] S2. Weigh 3.57 g (5 mmol) of the intermediate, add 1.17 g (tertiary amine, where R1 and R2 are methyl groups and R3 is a dodecyl group, 5.5 mmol) of N,N-dimethyldodecylamine and 20 g of isopropanol (the second solvent), and carry out a quaternization reflux reaction at 75 °C for 24 h, then stop the reaction. The molar ratio of the intermediate to the tertiary amine is 0.9:1, and the weight ratio of the second solvent to the intermediate is 5.6:1. Rotary evaporate to remove the solvent, and after vacuum drying, 4.3 g of the product is obtained with a yield of 92.8%, obtaining a quaternary ammonium salt type sophorolipid QSL-C with the structure shown in the following formula (1) where n is 12, R1 and R2 are methyl groups, and R3 is a dodecyl group. 12

[0064] The specific reaction process is as follows:[[]]END]]

[0065]

[0066] For the quaternary ammonium salt type sophorolipid QSL-C 12 perform mass spectrometry analysis. The mass spectrum is as shown in Figure 5 where the mass spectrometry peak with a mass-to-charge ratio of 892.6345 corresponds to the molecular ion peak ([M - Cl - ) of the quaternary ammonium salt type sophorolipid after removing the chloride ion. + The mass spectrometry results indicate that the product with the above structure has been synthesized.

[0067] The method for preparing a biological long-acting fungicide using the above quaternary ammonium salt type sophorolipid QSL-C 12 is as follows:[[]]END]]

[0068] (1) The above quaternary ammonium salt type sophorolipid QSL-C 12 ​Dissolve it in water to obtain a quaternary ammonium salt type sophorolipid solution with a concentration of 0.1 mmol / L; dissolve tannic acid (a natural polyphenol compound) in water to obtain a polyphenol solution with a concentration of 15 mmol / L;

[0069] (2) Mix the quaternary ammonium salt type sophorolipid solution and the polyphenol solution in a volume ratio of 5:3, stir at 25 °C for 30 min, and obtain a milky white mixed turbid liquid mixture; place this mixture in a centrifuge, perform high-speed centrifugation at a rotation speed of 8000 rpm for 5 min, and discard the upper clear liquid. Wash it repeatedly with pure water 3 times, and place it in an oven at 50 °C to dry to obtain a powdery biological long-acting fungicide 1.

[0070] Example 2

[0071] The preparation method of the biological long-acting fungicide in this example is as follows:

[0072] Same as Example 1, the difference is only that: in step S1, 1.17 g of N,N-dimethyldodecylamine is replaced with 1.27 g of N,N-dimethyltetradecylamine to obtain a quaternary ammonium salt type sophorolipid QSL-C with the structure shown in formula (1) 14 , where R1 and R2 are methyl groups, and R3 is a tetradecyl group; in step (1), the quaternary ammonium salt type sophorolipid QSL-C 12 is replaced with QSL-C 14 , and the natural polyphenol compound tannic acid is replaced with gallic acid; in step (2), the quaternary ammonium salt type sophorolipid solution and the polyphenol solution are mixed in a volume ratio of 4:2, and finally a powdery biological long-acting fungicide 2 is obtained.

[0073] Example 3

[0074] The preparation method of the biological long-acting fungicide in this example is as follows:

[0075] Same as Example 1, the difference is only that: in step S1, 1.17 g of N,N-dimethyldodecylamine is replaced with 1.62 g of N,N-dimethylhexadecylamine to obtain a quaternary ammonium salt type sophorolipid QSL-C with the structure shown in formula (1) 16 , where R1 and R2 are methyl groups, and R3 is a hexadecyl group; in step (1), the quaternary ammonium salt type sophorolipid QSL-C 12 is replaced with QSL-C 16 , the concentration of the quaternary ammonium salt type sophorolipid solution is 0.5 mmol / L; the natural polyphenol compound tannic acid is replaced with epigallocatechin gallate, and the concentration of the polyphenol solution is 30 mmol / L; in step (2), the quaternary ammonium salt type sophorolipid solution and the polyphenol solution are mixed in a volume ratio of 1:1, and finally a powdery biological long-acting fungicide 3 is obtained.

[0076] Example 4

[0077] In this example, the preparation method of the biotype long-acting bactericide is as follows:

[0078] Same as Example 2, the difference is only that: in step (1), the concentration of quaternary ammonium salt type sophorolipid QSL-C 14 is 2 mmol / L, and the powdery biotype long-acting bactericide 4 is obtained.

[0079] Comparative Example 1

[0080] Dissolve tetradecyltrimethylammonium chloride in water to obtain a solution with a concentration of 2 mg / mL; dissolve catechin in water to obtain a solution with a concentration of 2 mg / mL. Mix the two at room temperature to obtain a water-insoluble precipitate. Place the precipitate in a centrifuge and centrifuge at a high speed of 8000 rpm for 5 min, and discard the supernatant. Wash it repeatedly with pure water 3 times and dry it in an oven at 50 °C to obtain a solid powder.

[0081] Test Example 1

[0082] Characterize the surface wettability of the quaternary ammonium salt type sophorolipid QSL-C 12 used in Example 1 and the biotype long-acting bactericide 1 prepared in Example 1:

[0083] Put equal masses of QSL-C 12 and the biotype long-acting bactericide 1 into equal volumes of methanol respectively to obtain a QSL-C 12 solution and a solution containing the biotype long-acting bactericide 1. Coat the above solutions on a silica wafer respectively. After the solvent evaporates, a substrate material with a coating is formed. The contents of QSL-C 12 and the biotype long-acting bactericide 1 contained in the coating are both 30 μg / cm 2 . Use a contact angle tester to measure the contact angle of water on their surfaces (the instrument model is KRUSS DSA100), and the results are as Figure 1 shown. It can be seen from Figure 1 that the contact angle of pure water on the surface of the coating containing QSL-C 12 is 23.1°, which indicates that QSL-C 12 has strong hydrophilicity; while the contact angle of pure water on the surface of the coating containing the biotype long-acting bactericide 1 is 81.4°, which indicates that in an aqueous system, QSL-C 12 and natural polyphenolic compounds can spontaneously assemble to form the biotype long-acting bactericide 1 in the form of a supramolecular assembly. After assembly, the surface wettability of the biotype long-acting bactericide 1 has changed significantly, showing a certain "hydrophobicity". The increase in this hydrophobicity is beneficial to inhibiting the adhesion of bacteria on the material surface.

[0084] Test Example 2

[0085] For the quaternary ammonium salt type sophorolipid QSL-C used in Example 2 14 and the biotype long-acting bactericide 2 prepared in Example 2, structural characterization was carried out:

[0086] Equal masses of QSL-C 14 and the biotype long-acting bactericide 2 were respectively put into equal volumes of methanol to obtain a QSL-C 14 solution and a solution containing the biotype long-acting bactericide 2. The above solutions were respectively dropped onto a silicon wafer substrate, and the adsorption morphology differences of the two on the substrate were compared by a Hitachi S-4800 scanning electron microscope. As Figure 2 shown, with the volatilization of the solvent, QSL-C 14 agglomerated and precipitated, presenting irregular and amorphous precipitation, while the biotype long-acting bactericide 2 assembled with natural polyphenolic compounds formed a layer of closely connected nanoscale spherical assemblies on the substrate surface. X-ray photoelectron spectroscopy analysis was carried out on the spherical assemblies, and the results were as 14 shown. The assemblies had characteristic peaks of C, O, N, and Cl elements from gallic acid and quaternary ammonium salt type sophorolipid, proving that the biotype long-acting bactericide 2 was formed by the non-covalent interaction (such as hydrogen bonds, van der Waals forces, etc.) between the quaternary ammonium salt type sophorolipid and gallic acid. Compared with the micron or millimeter-scale precipitation of the large-area aggregated QSL-C Figure 3 the nanospheres formed by the biotype long-acting bactericide 2 exposed more active sites, which could increase the effective contact area with bacteria, thereby increasing the bactericidal efficiency. 14

[0087] Test Example 3

[0088] For the quaternary ammonium salt type sophorolipid QSL-C used in Example 2 14 and the biotype long-acting bactericide 2 prepared in Example 2, the bactericidal aging time was compared:

[0089] Equal masses of QSL-C 14 and the biotype long-acting bactericide 2 were respectively put into equal volumes of methanol to obtain a QSL-C 14 solution and a solution containing the biotype long-acting bactericide 2. The above solutions were respectively coated on stainless steel, plastic, non-woven fabric, glass, and silicon wafer substrates, and after the solvent volatilized, substrate materials with bactericidal coatings were formed. The contents of QSL-C 14 and the biotype long-acting bactericide 2 contained in the bactericidal coatings were both 60 μg / cm 2 . After the above substrate materials were cleaned in pure water at a rotation speed of 300 rpm for 30 min, they were respectively combined with OD 600 ​Interacted with Staphylococcus aureus, a Gram-positive bacterium with a value of 0.2, for 30 min. The QSL-C after water washing was calculated by the plate counting method 14 and the bactericidal rates of the biotype long-acting bactericide 2. The results are shown in Table 1. It can be seen that the bactericidal activity of QSL-C 14 on different types of substrates was between 5% and 20%. This was because the water-soluble QSL-C 14 suffered significant mass loss after water washing and could not reach the effective content for killing bacteria. The bactericidal rate of the biotype long-acting bactericide 2 modified on different substrates was about 99.9%. This was mainly because the biotype long-acting bactericide 2 had a large number of hydroxyl groups, which increased its adhesion to the substrate and could still maintain high-efficiency killing of bacteria after water washing.

[0090] Table 1 Comparison results of the bactericidal time effects of QSL-C 14 and the prepared biotype long-acting bactericide 2

[0091]

[0092]

[0093] Test Example 4

[0094] Characterize the water resistance of the quaternary ammonium salt type sophorolipid QSL-C 14 used in Example 2, the biotype long-acting bactericide 2 prepared in Example 2, the biotype long-acting bactericide 4 prepared in Example 4, and the solid powder prepared in Comparative Example 1:

[0095] Put equal masses of QSL-C 14 , biotype long-acting bactericide 2, biotype long-acting bactericide 4, and solid powder into equal volumes of methanol respectively to obtain a QSL-C 14 solution, a solution containing biotype long-acting bactericide 2, a solution containing biotype long-acting bactericide 4, and a solution containing solid powder. Coat the above solutions evenly on plastic substrates respectively. After the solvent volatilizes, plastic substrates with bactericidal coatings are formed. The contents of QSL-C 14 , biotype long-acting bactericide 2, biotype long-acting bactericide 4, and solid powder on the bactericidal coatings are all 60 μg / cm 2 . After the surfaces of the above plastic substrates are dried, let them interact with Staphylococcus aureus for 30 min respectively. Take the plastic substrates after the interaction and wash them in pure water at a rotation speed of 300 rpm for 30 min. Monitor the changes in the masses of the four substances on the plastic substrates with the increase in the number of water washing times respectively. Each group of experiments has three parallels. As shown in Table 2, the mass per unit area of QSL-C 14 rapidly decreased to 4.3 μg / cm after the first water washing 2. After the first sterilizing water wash in Example 2, Example 4 and Comparative Example 1, the mass decreased to 50.1 μg / cm 2 , 43.7 μg / cm 2 and 39.8 μg / cm 2 respectively. After the second cycle of water wash, the mass decreased to 28.6 μg / cm 2 , 25.0 μg / cm 2 and 23.2 μg / cm 2 respectively. After the 5th cycle of water wash, the mass was 11.0 μg / cm 2 , 9.7 μg / cm 2 and 0 μg / cm 2 . It can be seen that the sterilization method of QSL-C 14 alone is the first explosive release, and the water wash resistance is poor. The order of water wash resistance of the other three composite materials on the plastic substrate is: Example 2 > Example 4 > Comparative Example 1. Therefore, the bio-based long-acting bactericide prepared within the preferred concentration range of the present disclosure has stronger adhesion to the substrate, less water wash loss, and the best water resistance effect.

[0096] Table 2 Comparison results of water wash resistance performance of QSL-C 14 , Example 2, Example 4 and Comparative Example 1

[0097]

[0098] Test Example 5

[0099] The quaternary ammonium salt type sophorolipid QSL-C 14 used in Example 2 and the bio-based long-acting bactericide 2 prepared in Example 2 were used to sterilize bacteria, and the changes in the bacterial membrane potential before and after sterilization were tested:

[0100] QSL-C 14 and the bio-based long-acting bactericide 2 were respectively added to the bacterial solution containing Staphylococcus aureus and allowed to act on Staphylococcus aureus. After centrifuging the inactivated bacterial solution after the action, the supernatant was discarded, and the bacterial cells were resuspended in pure water. The changes in the membrane potential of bacteria before and after the action of QSL-C 14 and the bio-based long-acting bactericide 2 were respectively tested by a Zetasizer Nano ZS instrument. The results are shown in Table 3. It can be seen that the membrane potential of Staphylococcus aureus is -19.2 mV. After the action of QSL-C 14 and the bio-based long-acting bactericide 2, the membrane potential increased to +20.9 mV and +4.6 mV respectively. This is mainly due to the positively charged QSL-C 14Inserted directly into the negatively charged bacterial membrane, resulting in a positive shift in the bacterial membrane potential, while the biotype long-acting bactericide 2 releases the quaternary ammonium salt type sophorolipid bactericidal unit into the bacterial membrane through the assembly. QSL-C 14 has a greater impact on the degree of positive shift of the bacterial membrane potential, indicating that QSL-C 14 has a higher content entering the bacterial membrane. The biotype long-acting bactericide 2 has a smaller impact on the degree of positive shift of the bacterial membrane potential, indicating that the biotype long-acting bactericide 2 has the characteristic of releasing bactericidal units on demand, which is an important factor for its long-acting bactericidal effect.

[0101] Table 3 Membrane potential of bacteria before and after sterilization by QSL-C 14 and the biotype long-acting bactericide 2

[0102]

[0103]

[0104] Test Example 6

[0105] The quaternary ammonium salt type sophorolipid QSL-C used in Test Example 2 14 and the mildness of the biotype long-acting bactericide 2 prepared in Example 2:

[0106] Select zein as the model protein to evaluate the mildness of the biotype bactericide. Equal masses of QSL-C 14 and the biotype long-acting bactericide 2 were respectively added to the 10.0 mg / mL zein protein aqueous solution. After stirring for 48 h, centrifuged at 8000 rpm for 3 min to remove the undissolved zein protein. The absorbance value of the supernatant was measured by a Thermo Fisher Evolution 201 ultraviolet-visible spectrophotometer. The test results are shown in Figure 4 as follows. QSL-C 14 has a certain solubilizing effect on zein protein. It can be seen that after QSL-C 14 is incubated with zein protein, the supernatant after centrifugation has an ultraviolet characteristic absorption belonging to zein protein near 279 nm. However, the supernatant after the biotype long-acting bactericide 2 is incubated with zein has no obvious spectral absorption, which indicates that although the biotype long-acting bactericide 2 contains quaternary ammonium salt type sophorolipid components, due to the poor water solubility of the biotype long-acting bactericide 2, the dense structure formed by it is not easily disintegrated in water to release enough quaternary ammonium salt type sophorolipid molecules to dissolve zein protein. Therefore, the biotype long-acting bactericide 2 effectively reduces the solubilization of zein protein by the quaternary ammonium salt type sophorolipid and has good mildness.

[0107] The above Test Examples 1 to 6 show that the biotype long-acting bactericide of the present disclosure can not only release the quaternary ammonium salt-type sophorolipid bactericidal unit with bactericidal effect as required, effectively extend the bactericidal time limit, improve its recyclability, reduce the biological toxicity of the bactericide, and greatly avoid the overuse of the bactericide; but also has excellent substrate adhesion and water resistance, which is beneficial to inhibiting the adhesion of bacteria on the material surface, so that the material surface after water washing still maintains a high antibacterial ability.

[0108] The preferred embodiments of the present disclosure have been described in detail above in conjunction with the accompanying drawings. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0109] In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0110] In addition, any combination can be made between various different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A biotype long-acting fungicide, characterized in that, The biotype long-acting fungicide comprises quaternary ammonium salt type sophorolipid and natural polyphenolic compounds, and the quaternary ammonium salt type sophorolipid has a structure shown in the following formula (1): wherein X in formula (1) - is selected from F - 、Cl - 、Br - or I - ; R1 and R2 are each independently selected from an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a substituted or unsubstituted phenyl group having 6 to 14 carbon atoms, and a substituted or unsubstituted cycloalkyl group having 3 to 6 carbon atoms; The R3 is selected from an alkyl group having 8 to 18 carbon atoms, an alkoxy group having 8 to 18 carbon atoms, a substituted or unsubstituted phenyl group having 6 to 24 carbon atoms, and a substituted or unsubstituted cycloalkyl group having 8 to 18 carbon atoms; The substituents in R1 to R3 are each independently selected from one or more of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

2. The biotype long-acting fungicide according to claim 1, characterized in that, The natural polyphenolic compounds are selected from one or more of flavonoid compounds, anthocyanin compounds, ellagic acid compounds, and catechin compounds; The flavonoid compounds are selected from one or more of rutin, quercetin, and kaempferol; The anthocyanin compounds are selected from one or more of pelargonidin, cyanidin, and delphinidin; The ellagic acid compounds are selected from one or more of methoxy ellagic acid, methylenedioxy ellagic acid, and 3,3'-dimethoxy ellagic acid; The catechin compounds are selected from one or more of tea polyphenols, tannic acid, gallic acid, catechin, epigallocatechin, epigallocatechin gallate, and catechin hydrate; Preferably, the natural polyphenolic compounds are selected from one or more of tea polyphenols, tannic acid, gallic acid, catechin, epigallocatechin, epigallocatechin gallate, quercetin, ellagic acid, and catechin hydrate.

3. The biotype long-acting fungicide according to claim 1, characterized in that, There is a non-covalent interaction between the quaternary ammonium salt type sophorolipid and the natural polyphenolic compounds, and the non-covalent interaction includes one or more of electrostatic interaction, hydrophobic interaction, hydrogen bond, π-π stacking interaction, and van der Waals force.

4. A preparation method of a biological long-acting bactericide, characterized in that, The preparation method includes: contacting the quaternary ammonium salt type sophorolipid, the natural polyphenolic compounds, and water to obtain a mixture containing the biotype long-acting fungicide.

5. The preparation method according to claim 4, characterized in that, The preparation method further includes: (1) Dissolving the quaternary ammonium salt type sophorolipid in water to obtain a quaternary ammonium salt type sophorolipid solution, and dissolving the natural polyphenolic compounds in water to obtain a polyphenol solution; (2) Mixing and stirring the quaternary ammonium salt type sophorolipid solution and the polyphenol solution to obtain a mixture; performing solid-liquid separation on the mixture to obtain a solid containing the biotype long-acting fungicide; Optionally, the solid-liquid separation includes one or more of centrifugation, precipitation, and filtration, and preferably centrifugation.

6. The preparation method according to claim 5, characterized in that, The concentration of the quaternary ammonium salt type sophorolipid solution is 0.02 to 10 mmol / L, preferably 0.1 to 1 mmol / L; the concentration of the polyphenol solution is 5 to 60 mmol / L, preferably 5 to 30 mmol / L; the volume ratio of the quaternary ammonium salt type sophorolipid solution to the polyphenol solution is (3 to 15):(1 to 15), preferably (3 to 8):(1 to 7); The temperature of the mixture is 10 to 40 °C, preferably 25 to 40 °C; the stirring time is 10 to 60 min, preferably 20 to 40 min; the centrifugation speed is 4000 to 10000 rpm, preferably 5000 to 8000 rpm.

7. A bio-based long-acting bactericide prepared by the preparation method according to any one of claims 4 to 6.

8. Use of the bio-based long-acting bactericide according to any one of claims 1 to 3 and claim 7 in the field of antibacterial.

9. The application according to claim 8, characterized in that, The use includes: dissolving the bio-based long-acting bactericide in a solvent to obtain a solution containing the bio-based long-acting bactericide; coating or spraying the solution onto a solid surface, and forming a bactericidal coating on the solid surface after being induced by solvent evaporation; bringing the bactericidal coating into contact with bacteria to achieve a bactericidal effect; or, Putting the bio-based long-acting bactericide into the water body containing bacteria to be treated and contacting it with bacteria to achieve a bactericidal effect.

10. The application according to claim 9, wherein The solvent is selected from one or more of methanol, ethanol, propylene glycol, isopropanol, methanol / water, ethanol / water, and propylene glycol / water; the coating method includes spin coating and / or blade coating; the content of the biotype long-acting bactericide contained in the bactericidal coating is 5-110 μg / cm 2 , preferably 50-60 μg / cm 2 .

11. The application according to claim 9, characterized in that, The dosage of the bio-based long-acting bactericide is 10 to 1000 μg / mL, preferably 100 to 500 μg / mL.

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