JP2021046385A - Water-dispersible fine particle - Google Patents

Abstract

To provide a material that serves as an agent for suppressing biofilm formation and can be dispersed in water, while keeping its effects even under environments such as a wet area, and also has high safety and low environmental load.SOLUTION: The present disclosure provides a water-dispersible fine particle containing a complex of a polycarboxylic acid derivative and a ε-poly-L-lysine free base. The present disclosure also provides an antimicrobial agent and/or a biofilm removing agent containing the fine particle, and a method for producing a fine particle that includes a reaction between a polycarboxylic acid derivative and a ε-poly-L-lysine free base to occur in a reaction liquid to form a complex.SELECTED DRAWING: None

Description

The present invention relates to a method for suppressing the formation of a biofilm produced by a microorganism, and can suppress the production of a biofilm and remove the biofilm not only for Gram-negative bacilli having high biofilm productivity but also for Gram-positive cocci. Regarding the composition.

Biofilms are structures produced by microorganisms that have settled on the surface of materials, and are known as so-called slimes, and are involved in the generation of pathogens, corrosion of materials, etc. in all environments such as medical care, daily life, and industry. It causes many hygiene problems.

For example, in medical settings such as hospitals and nursing care, the outbreak of pathogenic bacteria (drug-resistant bacteria) that have acquired resistance to drugs such as antibacterial agents and antibiotics has become a problem. Drug-resistant bacteria are said to create an environment in which resistance to drugs and external stress can be easily acquired by forming biofilms. In addition, periodontal biofilms that occur in the oral cavity of people requiring long-term care may lead to serious diseases such as aspiration pneumonia. Furthermore, biofilms cause unpleasant odors, food poisoning, and corrosion of materials in familiar water areas such as kitchens, bathrooms, and toilets.

After the microorganisms have settled on the surface of the material, the biofilm is formed and grown in the process of growth of the microorganisms, and is released to the outside as colonies containing the microorganisms and pathogenic substances. Once the biofilm is formed, it exhibits resistance to environmental stress, antibacterial agents, etc., and it becomes difficult to remove microorganisms in the biofilm.

As a method for removing the biofilm, a cleaning treatment with an oxidizing disinfectant such as hypochlorous acid, ozone, or chlorine dioxide may be performed. However, since the oxidizing fungicide has high corrosiveness, its application range is limited from the viewpoint of environment and safety.

In order to solve the above problems, non-oxidizing biofilm sterilizers are known instead of highly corrosive oxidative sterilization methods. For example, isopropylmethylphenol (IPMP) in Patent Document 1 is known to permeate periodontal pathogenic biofilms and exhibit bactericidal properties. Since IPMP is water-insoluble, when used as a liquid, it is necessary to use it in combination with a solubilizer such as an organic solvent or a surfactant, and a water-based formulation may not be applicable.

Patent Document 2 discloses a biofilm remover using ε-poly-L-lysine (ε-PL). The invention describes that when an aqueous solution containing ε-PL is immersed in a biofilm, the biofilm is removed. ε-PL is known as a highly safe antibacterial polyamino acid, has high solubility in water, and is excellent in prescribability as an aqueous solution. However, when ε-PL is used as a coating agent for suppressing biofilm formation, it is difficult to retain it on the material or the surface of the biofilm because it easily elutes in water. In particular, the biofilm-suppressing effect of ε-PL may be lost depending on the usage environment such as around water where bacteria and biofilms easily propagate.

In order to solve the above problems, the inventor has developed fine particles containing ε-PL hydrochloride in a polymer for the purpose of retaining an antibacterial component in a material. It has been confirmed that the polymer fine particles have an inhibitory effect on a biofilm derived from Gram-negative bacilli (P. bluerescens) having high biofilm productivity. On the other hand, it has become clear that the biofilm-suppressing and / or removing effect may not be sufficient for Gram-positive cocci (S. aureus), which have recently become a problem of adhesion to medical devices such as catheters. ..

As described above, techniques for effectively suppressing and / or removing biofilms are required in many industries, and in particular, measures against biofilms derived from the genus Staphylococus, which is a problem in the medical field, are an urgent issue. is there.

Japanese Patent No. 5573111 Japanese Patent No. 5982088

Antibacterial and antifungal magazine, 1995, 23, 6. Nanoscale Research Letters, 2014, 9, 373.

The present invention, as an agent for suppressing and / or removing the formation of gram-positive cocci, particularly Staphylococcus aureus-derived biofilm, is dispersible in water but has a long-lasting effect even in an environment such as around water, and is further safe. Is to provide materials with high and low environmental load.

As a result of diligent studies, the present inventor has found that the above problems can be solved by the means shown below, and has arrived at the present invention.

（式中のＲは、水素原子または、炭素数１〜８の炭化水素基を示す。また、前記炭化水素基において、複数の水素原子が窒素原子、酸素原子または硫黄原子に置換されたものであってもよい。また、式中のｎは、１〜３の整数を示す。）
［４］ 前記ポリカルボン酸は、ポリグルタミン酸である、［１］〜［３］のいずれかに記載の微粒子。
［５］ 前記微粒子の平均粒子径は、０．０１μｍ〜０．５μｍである、［１］〜［４］のいずれかに記載の微粒子。
［６］ ［１］〜［５］のいずれかに記載の微粒子を含有する、抗微生物剤および／またはバイオフィルム除去剤。
［７］ 前記ポリカルボン酸誘導体と前記ε−ポリ−Ｌ−リジン遊離塩基とを反応液中で反応させて複合体を形成させる、［１］〜［５］のいずれかに記載の微粒子の製造方法。
［８］ 前記反応液のｐＨが７．０〜１０．０である、［７］に記載の微粒子の製造方法。 That is, the present invention has the following configuration.
[1] Water-dispersible fine particles containing a complex composed of a polycarboxylic acid derivative and an ε-poly-L-lysine free base.
[2] The fine particles according to [1], wherein the fine particles form an aggregate of the complex.
[3] The fine particles according to [1] or [2], wherein the polycarboxylic acid derivative is a compound obtained by condensing a polycarboxylic acid with a propargyl amino acid of the following general formula (1).

(R in the formula indicates a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms. In the hydrocarbon group, a plurality of hydrogen atoms are replaced with nitrogen atoms, oxygen atoms or sulfur atoms. In addition, n in the formula indicates an integer of 1 to 3).
[4] The fine particles according to any one of [1] to [3], wherein the polycarboxylic acid is polyglutamic acid.
[5] The fine particles according to any one of [1] to [4], wherein the average particle size of the fine particles is 0.01 μm to 0.5 μm.
[6] An antimicrobial agent and / or a biofilm remover containing the fine particles according to any one of [1] to [5].
[7] Production of the fine particles according to any one of [1] to [5], wherein the polycarboxylic acid derivative and the ε-poly-L-lysine free base are reacted in a reaction solution to form a complex. Method.
[8] The method for producing fine particles according to [7], wherein the pH of the reaction solution is 7.0 to 10.0.

The fine particles of the present invention have excellent water dispersibility, have adhesiveness to the surface of the material, and can retain antibacterial components for a long time. The formation of the biofilm to be produced can be suppressed. In addition, since raw materials derived from natural products are used, the burden on the environment is small.

It is a schematic diagram which shows the formation process of fine particles. A photograph of bromophenol blue staining before and after immersion of the fine particle dispersion coat in water is shown. The photograph of the halo test before and after the immersion in water of the fine particle dispersion coating is shown.

The present invention is a water-dispersible polymer fine particle containing a complex consisting of a polycarboxylic acid derivative and a ε-poly-L-lysine (ε-PL) free base. Further, the polycarboxylic acid derivative is a graft polymer in which a substituent having an interaction with the ε-PL free base is introduced into the polycarboxylic acid. By forming a strong bond such as a hydrogen bond or a covalent bond between the polycarboxylic acid derivative and the ε-PL free base, fine particles having excellent dispersibility can be formed. Furthermore, not only can the fine particles stay on the surface of the material for a long period of time, but also the permeability into the biofilm is improved, so that the persistence of the contained drug and the sustainability of the effect can be expected.

In the present invention, the complex composed of the polycarboxylic acid and the ε-PL free base can self-assemble the polycarboxylic acid derivative and the drug in water to form a stable aggregate by having the above-mentioned interaction. it can.

In addition to the free base (hereinafter, may be referred to as ε-PL free base), the ε-PL is an inorganic acid salt formed by ε-PL and an inorganic acid such as hydrochloric acid, sulfuric acid and phosphoric acid, and acetic acid. , Propionic acid, fumaric acid, malic acid, citric acid, maleic acid, adipic acid, gluconic acid and lactic acid, and organic acid salts formed by ε-PL, caproic acid, lauric acid and stearic acid, etc. Saturated fatty acid salts formed by medium- and long-chain saturated fatty acids and ε-PL, non-saturated fatty acids formed by medium- and long-chain unsaturated fatty acids such as oleic acid, linoleic acid and arachidonic acid and ε-PL. Examples include saturated fatty acid salts. As the ε-PL free base, for example, those manufactured by JNC Corporation or Ichimaru Falcos Co., Ltd. are available. Further, as the hydrochloride type ε-PL, for example, one manufactured by Toronto Research Chemicals Co., Ltd. is available. When ε-PL forms an amine salt, the adsorptivity between the amine and the cell membrane of the genus Staphylococus may be weakened due to the electrostatic interaction of counterions, and the antibacterial property may be lowered. Therefore, it is preferable to use an ε-PL free base having no counterion.

The genus Staphylococcus (Staphylococcus genus) includes Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Staphylococcus saprophyticus, etc. Has microbial, microbial killing, disinfecting or sterilizing effects.

In the present invention, the polycarboxylic acid derivative is a derivative obtained by modifying at least one propargyl amino acid with respect to a carboxylic acid having two or more carboxyl groups in the molecule. Examples of the carboxylic acid having two or more carboxyl groups in the molecule include low molecular weight carboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, citric acid and tartaric acid, polyacrylic acid and polymethacrylic acid. Examples thereof include high molecular weight synthetic polymers, natural polysaccharides such as pectin, alginic acid, hyaluronic acid and carboxymethyl cellulose, and polypeptides such as poly-γ-glutamic acid, poly-α-glutamic acid and polyaspartic acid. Among these, poly-γ-glutamic acid and salts thereof are preferable because they are polymers having a plurality of cross-linking points in the molecule, can effectively utilize biological resources, and have high biodegradability and safety.

In the present invention, the propargyl amino acid is an amino acid derivative represented by the general formula (1). R in the formula is a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms. Further, in the hydrocarbon group, a plurality of hydrogen atoms may be substituted with a nitrogen atom, an oxygen atom or a sulfur atom. Further, R in the formula may be the same or different. Further, n in the formula represents an integer of 1 to 3.

The amino acids constituting the propargyl amino acid of the general formula (1) are not limited regardless of which one is used, and any commercially available amino acid may be used, but from the viewpoint of safety, it constitutes a biological protein. It is preferable to use amino acids. Aspartic acids, aspartic acid, alanine, arginine, cysteine cystine, glutamine, glutamic acid, glycine, proline, serine, tyrosine, isoleucine, leucine, valine, histidine, lysine (lysine), methionine, etc. Examples thereof include phenylalanine, threonine (threonine), and tryptophan, and it is preferable to use the L form for all of them. Among these, it is preferable to use glycine, which is easily available, has high safety, and has the simplest structure.

The propargyl amino acid of the general formula (1) can be obtained by condensing the amino acid with propargyl alcohol, propargyl halide or the like as shown in the following reaction formula (2). More specifically, a propargyl amino acid can be easily produced by mixing and dissolving an amino acid and propargyl alcohol or the like, and adding a condensing agent such as thionyl chloride to cause a reaction. In addition, X in the formula represents a hydroxyl group or a halogen group. Further, R in the formula represents a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms. Further, in the hydrocarbon group, a plurality of hydrogen atoms may be substituted with a nitrogen atom, an oxygen atom or a sulfur atom. Further, R in the formula may be the same or different. Further, n in the formula represents an integer of 1 to 3.

In the present invention, the polycarboxylic acid derivative can be obtained by condensing the propargyl amino acid of the general formula (1) with the carboxyl group of the polycarboxylic acid as shown in the reaction formula (3) below. As a method for condensing the polycarboxylic acid and the propargyl amino acid, it can be prepared by reacting the polycarboxylic acid and the propargyl amino acid at room temperature (1 to 30 ° C.) in the presence of a dehydration condensing agent. More specifically, it can be prepared by dissolving the polycarboxylic acid in a solvent, adding the prepared propargyl amino acid, and adding a condensing agent and, if necessary, a reaction accelerator to react. At this time, the propargyl amino acid, which is a carboxylic acid active group, is preferably 0.25 to 1.0 equivalent with respect to the carboxylic acid unit constituting the polycarboxylic acid. If the molar ratio of propargyl amino acid to the carboxylic acid unit is too small, the reaction may be insufficient and the modification rate may decrease. On the other hand, if the molar ratio of propargyl amino acid to the carboxylic acid unit is too large, the amounts of unreacted raw materials and by-products may increase.

In the present invention, the dehydration condensing agent is N, N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC), O- (benzotriazole-1-yl). -N, N, N', N'-tetramethyluronium hexafluorophosphoric acid (HBTU) and the like can be mentioned. The dehydration condensing agent is preferably added in an amount of 0.25 to 1.0 equivalent with respect to the carboxylic acid unit constituting the polycarboxylic acid.

In the present invention, the reaction solvent for the condensation reaction is not particularly limited as long as it is a solvent that dissolves the polycarboxylic acid, and examples thereof include water, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF). ..

In the present invention, when accelerating the condensation reaction, it is preferable to add a base, a catalyst, and an active ester as a reaction accelerator to the reaction solution. Bases are triethylamine _{(Et 3 N), N,} N- diisopropylethylamine amine such as, sodium carbonate, carbonates such as sodium hydrogen carbonate. Examples of the catalyst include N, N-dimethyl-4-aminopyridine (DMAP) and the like. Examples of the active ester include 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide (NHS) as the ester of the reaction intermediate. When a base is added, it is preferable to add 0.5 to 1.5 equivalents with respect to the carboxylic acid unit constituting the polycarboxylic acid. When the active ester is added, it is preferable to add 0.25 to 1.0 equivalent to the carboxylic acid unit constituting the polycarboxylic acid.

In the condensation reaction, the modification rate of propargyl amino acid with respect to polycarboxylic acid can be changed by changing the amount of each raw material such as propargyl amino acid and dehydration condensing agent. When a polycarboxylic acid is used, the modification rate of the propargyl amino acid is preferably 1 to 55%, more preferably 5 to 50%, still more preferably 10 to 50% per carboxylic acid unit. If it exceeds 55%, the solubility in a solvent may deteriorate. By adjusting the modification rate of the propargyl amino acid to the polycarboxylic acid within the above range, the size of the fine particles when the drug (ε-PL) is encapsulated can be adjusted.

In the present invention, in order to recover the polycarboxylic acid derivative after the reaction, an organic solvent (poor solvent) in which the polycarboxylic acid derivative is insoluble is added to the reaction solution to precipitate the polycarboxylic acid derivative. The poor solvent is not particularly limited as long as it is a liquid in which the polycarboxylic acid derivative is insoluble and has good dispersibility, and examples thereof include acetone, methanol, ethanol, isopropyl alcohol, and acetic acid ester. The amount of the poor solvent added is preferably 3 to 10 times the amount of the reaction solvent. If the amount of the poor solvent added is too small, the polycarboxylic acid derivative may not be sufficiently precipitated. Further, if the amount of the poor solvent added is too large, the cleaning cost increases.

In the present invention, when the polycarboxylic acid derivative is recovered and then impurities are removed, the polycarboxylic acid derivative is dissolved in a good solvent such as water, and then a poor solvent is added while stirring at room temperature to obtain the polycarboxylic acid. Examples thereof include a method of purifying the derivative using a so-called dissolution reprecipitation method in which the derivative is reprecipitated. If this is repeated 1 to 3 times as necessary, impurities can be reduced to below the detection limit. The good solvent is not particularly limited as long as it is a solvent in which the polycarboxylic acid derivative is dissolved, but water is preferable from the viewpoint of safety and environmental influence. The poor solvent is not particularly limited as long as it is a liquid in which the polycarboxylic acid derivative is difficult to dissolve, and examples thereof include acetone, methanol, and ethanol.

The amount of the good solvent can be adjusted according to the solubility of the polycarboxylic acid derivative, but a high concentration is desirable from the viewpoint of industrial productivity. For example, the amount of the good solvent is preferably in the range of 10 to 60 wt%, more preferably in the range of 10 to 30 wt%.

The amount of the poor solvent can be adjusted according to the solubility of the polycarboxylic acid derivative, but may be the minimum amount of the polycarboxylic acid derivative precipitated. For example, the amount of the poor solvent is preferably 2 to 12 times the amount of the good solvent.

By repeating the above purification, impurities can be further reduced. From the viewpoint of manufacturing cost, it may be performed once or twice.

The obtained polycarboxylic acid derivative is a compound in which a propargyl amino acid is condensed with a carboxyl group of a polycarboxylic acid, and is an amphipathic molecule composed of a hydrophilic moiety of a carboxyl group and a hydrophobic moiety of a propargyl amino group.

In the present invention, by coexisting the polycarboxylic acid derivative and the ε-PL free base in a compatible solvent (in the reaction solution), the ε-PL free base is bound to the propargyl amino acid moiety of the polycarboxylic acid derivative. The complex can be further self-assembled to form stable fine particles (FIG. 1).

In the present invention, the particle size of the fine particles can be adjusted by changing the pH of the reaction solution when preparing the fine particles. The pH of the reaction solution when preparing the fine particles is preferably in the range of 5 to 10, more preferably in the range of 7 to 10. When the pH is on the acidic side, the antibacterial property may be lowered, or the fine particles may be easily aggregated due to polyion complexing. The average particle size of the fine particles is not particularly limited, but is preferably in the range of 0.01 μm to 0.5 μm.

In the present invention, the amount of ε-PL free base added to the polycarboxylic acid derivative in the reaction solution when preparing fine particles is such that the molar ratio of ε-PL free base to the propargylamino group of the polycarboxylic acid derivative is 0.01. The range of ~ 2 is preferable, and the range of 0.1 to 1.5 is more preferable. If the molar ratio is too large, it can be costly.

In the present invention, the solvent used for preparing the fine particles is not particularly limited as long as it is a solvent in which both the polycarboxylic acid derivative and the ε-PL free base are dissolved, but water is used from the viewpoint of environment and safety. preferable. If necessary, additives such as a dispersant, a surfactant, and a solubilizer may be used.

In the present invention, the concentration of ε-PL free base in the reaction solution when preparing fine particles is the value of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) when used as an antimicrobial (bacterial) agent. You can decide with reference to. For example, when ε-PL free base is used, the range is preferably 0.001 to 3 wt%, more preferably 0.01 to 1 wt%. If it exceeds 3 wt%, it may be disadvantageous in terms of raw material cost, and if it is less than 0.001 wt%, the antibacterial property may decrease.

In the present invention, the concentration of the polycarboxylic acid derivative in the reaction solution when preparing the fine particles can be determined according to the balance between the effective concentration of the ε-PL free base and the molar ratio. For example, the range of 0.1 to 1 wt% is preferable, and the range of 0.5 to 1 wt% is more preferable. If it exceeds 1 wt%, it may be disadvantageous in terms of raw material cost. If it is less than 0.1 wt%, the concentration of the drug in the fine particles is lowered, so that the antibacterial property may be lowered.

When water is used as the reaction solvent, the obtained fine particles can be used as they are as a fine particle dispersion for applications such as antimicrobial, microbial killing, disinfection and sterilization, and for removing biofilms. Examples of the fine particle dispersion liquid include a method of diluting or concentrating the biofilm already formed and spraying or spraying the fine particle dispersion liquid. After spraying or spraying the dispersion on the biofilm, it is preferable to leave it at room temperature for at least 1 minute or more. Further, if necessary, a surfactant, a proteolytic enzyme, a polysaccharide degrading enzyme, a DNA degrading enzyme, a bleaching agent and the like may be added or used in combination.

In the present invention, the fine particles are applied to the surface of the material by pre-coating the surface of the material on which the biofilm is not formed (anti-biofilm coating), in addition to spraying or spraying the fine particles on the biofilm already formed. It is possible to suppress the colonization of microorganisms and the formation of biofilms. In the coat film, the particles are immobilized on the surface of the base material by the bond interaction between the carboxylic acid group on the surface of the fine particles and the polymer on the surface of the base material, or the fine particles are physically adsorbed on the surface of the base material. The antimicrobial property is maintained without being easily spilled, and the formation of biofilm can be suppressed for a long period of time.

In the present invention, when the fine particles are used as an anti-biofilm coat, the amount of ε-PL free base attached to the surface of the material via the fine particles is preferably in the range of ^{0.04 to 0.12 μg / cm 2.} .. If the amount of the ε-PL free base attached is too small, the suppression of biofilm formation may not be sufficiently exerted. Further, if the amount of the ε-PL free base attached is too large, it is disadvantageous in terms of cost effectiveness.

When the surface of the material is coated with an anti-biofilm, examples of the base material that can be applied include ceramics, metals, metal oxides, plastics, rubbers, ores, and wood.

In the present invention, the fine particles use a material that is highly safe for the ecosystem and can be dispersed in water. Therefore, the biofilm is used not only in general households but also in medical sites such as hospitals and nursing care sites. As a countermeasure, it can be applied to many industrial applications such as around water in kitchens, toilets, bathtubs, etc.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the Examples.

(Preparation of glycine, 2-propyne-1-yl, ester (GPE))
A mixture of 2.1 g of glycine (Nacalai Tesque) and 30 mL of 2-propyne-1-ol (Nacalai Tesque) was prepared, and 2.4 mL of thionyl chloride (Nacalai Tesque) was added at room temperature. The reaction mixture was stirred at room temperature for 2 hours and further at 50 ° C. for 2 hours. The reaction mixture was cooled to 5 ° C. and 90 mL of ethyl acetate was added to obtain a precipitate. The precipitate was separated by filtration, washed 3 times with 30 mL of ethyl acetate, and dried (50 ° C., 12 hours) to give glycine, 2-propin-1-yl, an ester (2-propynyl aminoethaneate) (GPE). ) Was obtained.

(Measurement of propargyl amino acid modification rate)
Modification ratio of propargyl amino acid to the carboxyl group constituting the acid derivative was determined by measuring ¹ H-NMR spectrum in _{D 2 O (BRUKER, MR400)} . The modification rate was calculated by measuring the integrated intensity ratio of α-hydrogen of the carboxyl group modified with propargyl amino acid and the α-hydrogen of the carboxyl group not modified, and using the following formula.
Modification rate (%) = [modified carboxyl group α-hydrogen] / [(unmodified carboxyl group α-hydrogen) + (modified carboxyl group α-hydrogen)] × 100

(Production Example 1) Production of GPE-modified γ-PGA (40) 0.5 g of Toyobo-made sodium polyglutamate (γ-PGA) and 6 mL of water were added, and the mixture was stirred at room temperature. 0.75 equivalents of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC) and 1-hydroxybenzotriazole (HOBt) were added to the solution with respect to the carboxylic acid units constituting γ-PGA. In addition, the reaction was carried out at room temperature for 24 hours. After the reaction, 35 mL of acetone was added to precipitate the polymer. The resulting polymer was washed with acetone and dried. After drying, the crude polymer was dissolved in 5.5 mL of water, 60 mL of acetone was added, the mixture was precipitated again, and the mixture was collected by filtration. Vacuum dried at 60 ° C. for 12 hours to obtain the desired GPE-modified γ-PGA. From _{^{1 H-NMR (D 2 O}} ), modification ratio of GPE for the carboxylic acid units of gamma-PGA was confirmed to be 40%. The obtained polycarboxylic acid derivative was designated as GPE-modified γ-PGA (40).

(Production Example 2) Production of GPE-ized γ-PGA (25) The method described in Production Example 1 was used except that 0.5 equivalents of GPE, WSC, and HOBt were used as the amount of raw materials to be charged. From _{^{1 H-NMR (D 2 O}} ), modification ratio of GPE for the carboxylic acid units of gamma-PGA was confirmed to be 25%. The obtained polycarboxylic acid derivative was designated as GPE-modified γ-PGA (25).

(Production Example 3) Production of GPE-ized γ-PGA (15) The method described in Production Example 1 was used except that 0.25 equivalents of GPE, WSC, and HOBt were used as the amount of raw materials to be charged. From _{^{1 H-NMR (D 2 O}} ), modification ratio of GPE for the carboxylic acid units of gamma-PGA was confirmed to be 15%. The obtained polycarboxylic acid derivative was designated as GPE-modified γ-PGA (15).

(Preparation of fine particle dispersion of GPE-ized γ-PGA / ε-PL)
25 mL of distilled water was added to 0.05 g of GPE-modified γ-PGA obtained in Production Examples 1 to 3, dissolved at room temperature, and diluted to 0.01 to 0.1 wt% in the solution. 25 mL of an aqueous solution of base (polylysine 10) or 25 mL of an aqueous solution of ε-PL hydrochloride manufactured by Toronto Research Chemicals Co., Ltd. was added, and the mixture was stirred at room temperature for 1 minute to obtain fine particle dispersions of Examples 1 to 7 and Comparative Examples 1 to 3. The details of the obtained fine particle dispersion are shown in Table 1.

(Measurement of average particle size of fine particles in dispersion)
The average particle size of the obtained fine particle dispersion was measured with a particle size distribution measuring device (Dispersion Technology, Inc., DT1202, USA). The fine particles of Examples 1, 2 and 7 were prepared in the range of pH 9.9 to 6.5, and the average particle size of the fine particles in the obtained dispersion was measured at each pH. The pH before adjustment was 9.9, and the pH was adjusted to 6.5 with 1% hydrochloric acid. The results are shown in Table 2. In the pH range of 9.9 to 7.0, polymer fine particles having an average particle diameter of 30 to 341 nm were obtained, but below pH 7, agglutination of the particles was observed. Since the agglomerated polymer fine particles precipitate, it becomes difficult to uniformly apply the polymer fine particles to the base material, and the biofilm suppressing effect may be reduced.

(Preparation of bacterial solution)
In order to evaluate the antibacterial property of the obtained fine particle dispersion, test bacteria were prepared. As a test bacterium, S. Aureus (Staphylococcus aureus) was obtained from the NBRC strain (NBRC13276) of the Biotechnology Center, National Institute of Technology and Evaluation. S. After reconstructing and culturing aureus, S. aureus. It was cryopreserved as an emulsion glycerol suspension. S. Thaw the aureus glycerol suspension and put the suspension (1 mL) and SCD liquid medium (12 g of SCD medium manufactured by Nippon Pharmaceutical Co., Ltd. dissolved in 400 mL of distilled water and sterilized in an autoclave) in a shaking flask. 50 mL) was added. Place in an incubator with a shaker, set the culture temperature at 35 ° C. for 19 hours, and set the stirring speed to 150 rpm. The aureus glycerol suspension was cultured. After culturing, the flask was taken out and prepared in a clean bench. 40 mL of the bacterial solution was collected from the flask after culturing and diluted with sterilized 80% glycerol (prepared from Nacalai Tesque glycerol and distilled water) (20 mL) to prepare a preservation solution. The viable cell count of the bacterial solution after culturing was measured as follows.

(Measurement of viable cell count of bacterial solution)
Bacterial solution (1 mL) was taken from the flask after culture was diluted 10 ² to 10 ⁸ times at sterile Nacalai Tesque saline. 1 mL of each diluted solution was inoculated into Petrifilm for general bacteria manufactured by 3M. Petrifilm was placed in an incubator and cultured at room temperature for 3 days. After culturing, the number of colonies observed on Petrifilm was counted to determine the viable cell count. S. cerevisiae obtained in the main culture. aureus glycerol suspension bacterial liquid viable count (before glycerol added) was 1.4 × ¹⁰ 9 cfu / mL.

(MIC measurement)
The antibacterial property of the fine particle dispersion against various bacteria was determined by measuring the minimum inhibitory concentration (MIC). The bacteria solution and SCD liquid medium at (Nippon Seiyaku of SCD medium 12g was dissolved in distilled water 400 mL, liquid medium sterilized in an autoclave), was diluted to the viable cell count becomes ¹⁰ 6 cfu / mL. A test solution prepared to have an antibacterial agent concentration of 2000 ppm was used, and diluted with a bacterial solution so that the drug (ε-PL) concentration was 800 ppm. The 800 ppm solution was further diluted with the bacterial solution to a final 25 ppm while preparing a 2-fold dilution series. A diluted solution of 800 to 25 ppm was inoculated into a 48-well microplate and cultured at 35 ° C. for 24 hours. After culturing, the minimum concentration at which the growth of the bacterium was suppressed was measured, and the MIC was determined. In addition, S. In addition to aureus, P. aureus is a gram-negative bacillus with high biofilm productivity. Fluorescence (NBRC No. 14160) was used for MIC measurement. The measurement results are shown in Table 3. The fine particle dispersions of Examples 1, 2, 4, 5, and 7 have a pH range of 6.3 to 9.8, and S.I. aureus and P. aureus Antibacterial properties against fluororescens were observed. However, at pH 6.3, the antibacterial property was slightly reduced probably because the amine of ε-PL was protonated. Further, in the case of Comparative Examples 1 to 3 using ε-PL hydrochloride, P.I. Antibacterial properties were observed against fluororescens, but S. The MIC against aureus was as high as> 800 ppm, resulting in low antibacterial activity. It is presumed that ε-PL hydrochloride has reduced antibacterial properties due to weak adsorption between amines and cell membranes of the genus Staphylococus due to the electrostatic interaction of counterions.

(Water immersion test assuming prevention of biofilm adhesion)
A water immersion test was conducted assuming the prevention of biofilm adhesion using a coated substrate of the fine particle dispersion. As the fine particle dispersion coating base material, a PVC sheet (1 cm ^{2 ) manufactured by Sumitomo Bakelite Co., Ltd. was coated with 0.04 mL / cm 2 of} the fine particle dispersions obtained in Example 2 and Comparative Example 2 and dried at room temperature. The solid content of ε-PL at this time corresponds to ^{0.08 mg / cm 2.} Assuming a biofilm forming environment, a coated base material was placed on a petri dish having a diameter of 100 mm, 20 mL of city water was added, and the mixture was allowed to stand at 30 ° C. The coated substrate was collected as appropriate, and a persistence test and an antibacterial property test of ε-PL were carried out.

(Persistent test of ε-PL)
The ε-PL persistence test was performed by staining ε-PL with bromophenol blue (BPB). A 0.1% BPB sodium aqueous solution manufactured by Tokyo Chemical Industry Co., Ltd. was applied to the coated substrate taken out in the water immersion test described above and immersed for 3 hours at room temperature to form a composite salt of BPB and ε-PL. .. After immersion, the cells were washed with a large amount of water to remove excess BPB sodium, and the persistence of ε-PL was visually observed as a BPB-stained site. As shown in FIG. 2, in the γ-PGA and ε-PL mixed solution coating (Reference Examples 1 and 2) and ε-PL alone (Reference Examples 3 and 4), ε-PL flowed out immediately after immersion in water. On the other hand, the GPE-modified γ-PGA / ε-PL fine particle dispersion showed high persistence of ε-PL even when immersed in water for 1 month.

In addition, in order to quantify the persistence of ε-PL, the BPB-stained site was extracted with 1 mL of 5% saline, and the obtained extract was measured for absorbance at 595 nm with a Biotech Japan plate reader. The residual rate of ε-PL was calculated from the following formula. In the dispersion having a pH of 9.8, the residual rate of ε-PL was the highest, and the residual rate was 31% at the time of immersion for 1 month (Table 4). On the other hand, in Comparative Example 2 using ε-PL hydrochloride, only the ε-PL residual rate up to 1 day was confirmed. Reference Examples 1 and 2 are tests using a composition composed of γ-PGA and ε-PL, and Reference Examples 3 and 4 are the results of a test using only ε-PL. In both cases, the survival rate was low. The ε-PL free base had good reactivity with GPE-modified PGA, and the result was obtained that it remained on the polymer fine particles and the surface of the substrate for a long period of time.
ε-PL residual rate (%) = 100- (absorbance immediately after immersion in water-absorbance after immersion in water) / (absorbance immediately after immersion in water) x 100

(Hello test)
In the water immersion test assuming the prevention of biofilm adhesion, the coated base material after water immersion was collected, and S.I. An antibacterial test (halo test) against aureus was carried out. In the halo test, a bacterial solution (S. aureus, 10 ⁶ cfu / mL) was inoculated on an SCD plate agar medium, and a coat base material was placed on the bacterial solution (S. aureus, 10 6 cfu / mL) to perform the halo test. The halo was statically cultured at 37 ° C. for 24 hours, and the diameter of the halo was measured. As shown in Table 5, the fine particle dispersion coating of Example 2 was observed to have halos even after being immersed for 10 days, and the antibacterial property was maintained. On the other hand, in the system using ε-PL hydrochloride (Comparative Example 2) and the system using silver nanoparticles (manufactured by Nippon Ion) (Reference Example 5), the antibacterial component was eluted or dropped into water immediately after immersion in water. Antibacterial properties were lost in a short period of time. FIG. 3 shows the results of the halo test.

The fine particles of the present invention have a wide range of application because the biofilm removing component can be dispersed in an appropriate medium such as water and applied or sprayed to effectively suppress the formation of the biofilm or remove the formed biofilm. It is effective as a biofilm countermeasure because it uses a wide range of materials that are highly safe for living organisms and the environment, and has excellent substrate adhesion.

Claims (8)

Water-dispersible fine particles containing a complex consisting of a polycarboxylic acid derivative and a ε-poly-L-lysine free base. The fine particles according to claim 1, wherein the fine particles form an aggregate of the complex. The fine particles according to claim 1 or 2, wherein the polycarboxylic acid derivative is a compound obtained by condensing a polycarboxylic acid with a propargyl amino acid of the following general formula (1).

(R in the formula indicates a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms. In the hydrocarbon group, a plurality of hydrogen atoms are replaced with nitrogen atoms, oxygen atoms or sulfur atoms. In addition, n in the formula indicates an integer of 1 to 3). The fine particles according to any one of claims 1 to 3, wherein the polycarboxylic acid is polyglutamic acid. The fine particles according to any one of claims 1 to 4, wherein the average particle size of the fine particles is 0.01 μm to 0.5 μm. An antimicrobial agent and / or a biofilm remover containing the fine particles according to any one of claims 1 to 5. The method for producing fine particles according to any one of claims 1 to 5, wherein the polycarboxylic acid derivative and the ε-poly-L-lysine free base are reacted in a reaction solution to form a complex. The method for producing fine particles according to claim 7, wherein the pH of the reaction solution is 7.0 to 10.0.

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