JP7211730B2 - Biofilm formation inhibitor and method for inhibiting biofilm formation - Google Patents

Description

The present invention relates to a biofilm formation inhibitor and a method for inhibiting biofilm formation.

A biofilm is a structure formed by colonies of microorganisms such as bacteria and fungi adhering to the surfaces of solids and liquids together with secretions and the like. Biofilms generated in household or factory drainage facilities, water circulation systems, etc., cause sliminess and clogging of pipes and bad odors, or cause deterioration of facilities. For example, if a biofilm adheres to a water-cooled cooling tower, it will lead to clogging of the circulation path and a decrease in heat exchange efficiency, resulting in a significant decrease in its capacity. When microorganisms form biofilms, they are difficult to eliminate because they become resistant to normal cleaning and sterilization methods.

Generally, in drainage facilities and water circulation systems, periodic water replacement and cleaning are performed to prevent biofilm formation. However, these works increase the maintenance cost of the equipment and system. Conventionally, disinfectants such as chlorine-based agents and organic nitrogen-sulfur-based agents have been used to prevent biofilms in drainage facilities, water circulation systems, and the like. However, chlorine-based agents have problems such as damage to pipes and equipment, metal corrosion, and stability, while organic nitrogen-sulfur-based agents have problems such as skin damage and stability. Therefore, none of the agents can be used at concentrations sufficient for sterilization, and biofilm formation cannot be suppressed for a long period of time.

Patent Document 1 describes a method for suppressing biofilm formation on hard surfaces using a polyoxyalkylene alkyl (or alkenyl) ether compound. In Patent Document 2, a hard surface using a polymer compound having a structural unit having one or more groups selected from an amino group and a quaternary ammonium group and a structural unit derived from a vinyl monomer having an anionic group A method of inhibiting biofilm formation is described.

JP 2009-078986 A JP 2010-163429 A

The present invention provides a biofilm formation inhibitor and a method for inhibiting biofilm formation.

The present inventors have found that a copolymer having an alkylene glycol group and a cationic group and / or a hydrophobic group in the side chain can suppress biofilm formation without depending on bactericidal or antibacterial action. .

Accordingly, the present invention includes (A) monomeric units having cationic and/or hydrophobic groups and (B) water-soluble monomeric units having polyalkylene glycol groups terminated with —CH or —OH _, Provided is a biofilm formation inhibitor comprising, as an active ingredient, a copolymer having a content of (A) of 7 to 25% by mass and a content of (B) of 75 to 93% by mass.
The present invention also provides a method for inhibiting biofilm formation, which comprises adding the biofilm formation inhibitor to water contained in an environment where inhibition of biofilm formation is desired.

The biofilm formation inhibitor of the present invention suppresses adhesion of microorganisms to drainage facilities, water circulation systems, and the like, thereby suppressing biofilm formation. INDUSTRIAL APPLICABILITY The biofilm formation inhibitor of the present invention is safer for the human body because it can inhibit biofilm formation without depending on bactericidal or antibacterial action. In addition, the biofilm formation inhibitor of the present invention can prevent the reduction of the effect due to the acquisition of resistance of bacteria as in the case of using a fungicide or an antibacterial agent, so even if it is used for a long time, it is effective for biofilm formation. can be suppressed. Furthermore, the biofilm formation inhibitor of the present invention is less likely to damage pipes and equipment than conventional disinfectants, and is therefore more economical.

The biofilm formation inhibitor of the present invention includes (A) a monomer unit having a cationic group and / or a hydrophobic group, and (B) a water _- soluble monomer having a polyalkylene glycol group having -CH or -OH at the end. unit, as an active ingredient.

Examples of monomers having a cationic group forming the monomer unit (A) include (meth)acrylic acid esters having a dialkylamino group, and salts thereof or quaternary salts obtained by quaternizing them ( hereinafter simply referred to as "their quaternary salts"); (meth)acrylamides having a dialkylamino group, and salts or quaternary salts thereof; and diallylamine compounds, and salts or quaternary salts thereof, and the like. . Examples of preferred acids for obtaining the above salts include inorganic acids such as hydrochloric, sulfuric, nitric, phosphoric; and acetic, formic, maleic, fumaric, citric, tartaric, adipic, sulfamic, organic acids having 1 to 22 carbon atoms in total, such as toluenesulfonic acid, lactic acid, pyrrolidone-2-carboxylic acid, succinic acid, propionic acid, and glycolic acid; Examples of preferred quaternizing agents for obtaining the above quaternary salts include alkyl halides having 1 to 8 carbon atoms such as methyl chloride, ethyl chloride, methyl bromide and methyl iodide; general alkylating agents such as diethyl and di-n-propyl sulfate; Examples of (meth)acrylic acid esters having a dialkylamino group include dimethylaminoethyl (meth)acrylate and diethylaminoethyl (meth)acrylate. Examples of (meth)acrylamides having a dialkylamino group include dimethylaminopropyl(meth)acrylamide and diethylaminopropyl(meth)acrylamide. Examples of the diallylamine compounds include diallylmethylamine and diallylamine. Among these, a salt of dimethylaminoethyl (meth)acrylate is preferable as an example of the monomer having the cationic group.

Examples of the monomer having a hydrophobic group forming the monomer unit (A) include (meth)acrylic acid esters having an alkyl group or an alkenyl group, (meth)acrylic acid esters having an arylalkyl group, and the like. be done. Examples of (meth)acrylic acid esters having alkyl or alkenyl groups include methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, t-butyl (meth)acrylate. , cyclohexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, isooctyl (meth)acrylate, isononyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isostearyl (meth)acrylate, behenyl (meth)acrylate Acrylate, oleyl (meth)acrylate and the like. The alkyl group or alkenyl group is preferably an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms. Examples of (meth)acrylic acid esters having an arylalkyl group include benzyl (meth)acrylate. Among these, benzyl (meth)acrylate is preferable as an example of the monomer having the hydrophobic group.

The monomer units of (A) contained in the copolymer used in the present invention may each independently consist of a monomer having a cationic group and/or a hydrophobic group, preferably each independently and (ii) any one selected from the group consisting of the above-exemplified monomers having a cationic group and monomers having a hydrophobic group. Preferably, the monomer units of (A) contained in the copolymer are each independently a monomer unit consisting of a salt of dimethylaminoethyl (meth)acrylate or benzyl (meth)acrylate.

The polyalkylene glycol group in the monomer unit of (B) is preferably a polyalkylene glycol group consisting of repeating 3 to 120, more preferably 9 to 90, repeating alkylene glycol groups. The alkylene glycol group is preferably an alkylene glycol group having 2 or 3 carbon atoms, more preferably an ethylene glycol group. The polyalkylene glycol groups are terminated with --CH ₃ or --OH.

As the monomer forming the monomer unit of (B), vinyl-based monomers are preferred. Examples of the vinyl-based monomer include acrylic acid or its salts, methacrylic acid or its salts, (meth)acrylamide or its salts, etc. Among them, acrylic acid or its salts and methacrylic acid or its salts are preferable. The monomers forming the monomer units of (B) may be each independently any one of the vinyl-based monomers listed above.

As used herein, a water-soluble monomer refers to a monomer whose homopolymer has a water solubility of 1 g/L or more at 0 to 50°C. In this specification, the water-insoluble monomer refers to a monomer whose homopolymer has a water solubility of less than 1 g/L at 0 to 50°C.

Preferably, (A) and (B) are structural units represented by the following formulas (1) and (2), respectively.

In formula (1), R ¹ represents a hydrogen atom or a methyl group, preferably a methyl group. R ² represents a cationic group and/or a hydrophobic group. Preferred examples of R ² include dimethylaminoethyl group, diethylaminoethyl group, dimethylaminopropyl group, diethylaminopropyl group, diallylmethyl group, methyl group, ethyl group, butyl group, isobutyl group, t-butyl group and cyclohexyl group. , 2-ethylhexyl group, isooctyl group, isononyl group, lauryl group, stearyl group, isostearyl group, behenyl group, oleyl group, benzyl group, and salts and quaternary salts thereof. Examples of preferred acids for obtaining the above salts include inorganic acids such as hydrochloric, sulfuric, nitric, phosphoric; and acetic, formic, maleic, fumaric, citric, tartaric, adipic, sulfamic, organic acids having 1 to 22 carbon atoms in total, such as toluenesulfonic acid, lactic acid, pyrrolidone-2-carboxylic acid, succinic acid, propionic acid, and glycolic acid; Preferred examples of quaternizing agents for obtaining the above quaternary salts include alkyl halides having 1 to 8 carbon atoms such as methyl chloride, ethyl chloride, methyl bromide and methyl iodide; general alkylating agents such as diethyl and di-n-propyl sulfate;

In formula (2), R ³ and R ⁴ each independently represent a hydrogen atom or a methyl group, preferably a methyl group. AG ₁ represents an alkylene glycol group having 2 to 3 carbon atoms, preferably an ethylene glycol group. m represents an integer of 3-120, preferably 9-90.

Among the constitutional units of formula (1) or (2) contained in the copolymer used in the present invention, R ¹ to R ⁴ and AG ₁ are different groups even if they are the same group. It may be In addition, m may be the same or different between the structural units of formula (2) contained in the copolymer used in the present invention.

More preferred examples of (A) include at least one selected from the group consisting of monomer units consisting of 2-(dimethylamino)ethyldiethyl methacrylate sulfate and monomer units consisting of benzyl methacrylate. A more preferable example of (B) is a monomer unit composed of methacrylate of methoxypolyethylene glycol (the number of moles of EO added is 9 to 90).

In the copolymer used in the present invention, the content of (A) may be 7 to 25% by mass, preferably 8 to 20% by mass. The content of (B) in the copolymer may be 75 to 93% by mass, preferably 80 to 92% by mass. In a preferred embodiment, the copolymer used in the present invention is a copolymer consisting of (A) and (B) above, and the content of (A) in the copolymer is 7 to 25% by mass, The content of (B) is 75 to 93 mass %, preferably the content of (A) is 8 to 20 mass % and the content of (B) is 80 to 92 mass %. The equivalent ratio of (A) and (B) in the copolymer is preferably (A)/(B) = 7/93 to 25/75, more preferably (A)/(B) = 8/92 to 20/80. In the present specification, the contents of (A) and (B) in the copolymer are calculated from the blended amounts of the monomers before polymerization. Further, the copolymer preferably has a weight average molecular weight of 30,000 or more and 1,000,000 or less. The weight average molecular weight of the copolymer in this specification is the weight average molecular weight measured by gel permeation chromatography (GPC).

In the copolymer used in the present invention, the above (A) may have the function of adsorbing the copolymer to the surface of equipment or microorganisms where biofilm formation is desired to be suppressed. On the other hand, in the copolymer used in the present invention, the above (B) may have the function of suppressing adhesion of microorganisms to the surface of the equipment.

The biofilm formation inhibitor of the present invention may consist of the copolymer described above, or may be a composition containing the copolymer and other components. Such other components include bactericides, anionic copolymers, and the like.

The biofilm formation inhibitor of the present invention can be used for various environments where inhibition of biofilm formation is desired. Environments in which suppression of biofilm formation is desired include environments in which biofilms may form, such as in homes or factory facilities. Preferably, the biofilm formation inhibitor of the present invention is used in facilities in which water is flowed or stored, such as water flowing pipes, tanks and pools for storing water, water circulation systems, and equipment attached thereto. used to inhibit biofilm formation in Among these facilities, the biofilm formation inhibitor of the present invention is preferably used in facilities where at least a part thereof reaches a temperature at which biofilms are likely to form, for example, 20° C. or higher, preferably 25° C. or higher. Suitable examples of facilities through which the water is flowed or stored include water-cooled cooling towers, factory facilities and building cooling systems using the same, water circulation systems such as hot water heating systems, and industrial cooling systems. Examples include swimming pools, industrial water supplies, water storage or drainage channels, wastewater treatment plants, reverse osmosis membranes used in seawater desalination plants, paper mill plumbing, household sinks, and the like. More preferably, the biofilm formation inhibitor of the present invention is used to inhibit biofilm formation in water cooled cooling towers.

The biofilm formation inhibitor of the present invention has the effect of inhibiting the adhesion of microorganisms to the environment to which it is applied (for example, the facility where the water is drained or stored as described above). For example, the biofilm formation inhibitor of the present invention has the action of suppressing the adhesion of microorganisms and the formation of biofilms on the surface of the facility. More specifically, the biofilm formation inhibitor of the present invention can adsorb to the surface of the facility and/or microorganisms to suppress the adhesion of microorganisms to the surface of the facility. Suitable examples of surfaces of the equipment include surfaces of the equipment that come into contact with water, such as pipe walls, pool walls, heat exchangers, filters, and the like. Preferably, the surface may be a surface made of fibers or mixed materials thereof, such as metal, concrete, mortar, plastic, resin, wood, glass, asphalt, ore.

Preferably, the biofilm formation inhibitor of the present invention is applied to water contained in the environment where inhibition of biofilm formation is desired. Preferably, the biofilm formation inhibitor of the present invention is added to the water contained in the above-described equipment through which water is flowed or stored (e.g., water flowing, circulating, or stored in the equipment) be done. More preferred examples of water to which the biofilm formation inhibitor of the present invention is added include water used in water-cooled cooling towers or factory equipment and building cooling systems using the same, and water used in flowing hot water heating systems. water circulating in water circulation systems such as hot water, industrial cooling pool water, industrial water supply, water stored or in the drainage path, wastewater from wastewater treatment facilities, reverse osmosis membranes used in seawater desalination plants Water passing through pipes, water in piping equipment of paper mills, and the like.

Biofilms generated in such facilities contain Gram-negative microorganisms such as the genus Pseudomonas such as Pseudomonas aeruginosa, the genus Sphingomonas, and the genus Sphingopyxis. Furthermore, Gram-negative microorganisms of the genera Klebsiella, Flavobacterium and Roseomonas have been reported as microorganisms present in biofilms in water-cooled cooling towers (Biofouling 2011, 27(4): 393-402, Biocontrol Science 2013, 18 (4):205-209). Preferably, the biofilm formation inhibitor of the present invention prevents biofilms formed by the aforementioned microorganisms.

The amount of the biofilm formation inhibitor of the present invention added to the water contained in the environment where the suppression of biofilm formation is desired is converted to the concentration of the above copolymer in the water to which the biofilm formation inhibitor is added. , preferably 1 ppm (mass ppm, hereinafter the same in this specification) or more, more preferably 5 ppm or more, still more preferably 10 ppm or more, and preferably 10000 ppm or less, more preferably 5000 ppm or less, further preferably 1000 ppm or less Alternatively, it is preferably 1 to 10000 ppm, more preferably 5 to 5000 ppm, still more preferably 10 to 1000 ppm.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

<Measurement of weight average molecular weight>
The weight average molecular weight (Mw) of the polymer was measured by gel permeation chromatography (GPC) under the following conditions.
(Examples 1 and 3 and Comparative Examples 1 to 3)
Column: α-M × 2 (manufactured by Tosoh Corporation)
Eluent: (50 mmol/L lithium bromide, 1% acetic acid/ethanol): water = 3:7 (vol%)
Flow rate: 0.6mL/min
Column temperature: 40°C
Sample: 5 mg/mL
Detection: RI
Reduced molecular weight: polyethylene glycol (Example 2)
Column: K-804L × 2 (manufactured by Tosoh Corporation)
Eluent: chloroform containing 1 mmol/L Pharmin DM20 Flow rate: 1.0 mL/min
Column temperature: 40°C
Sample: 5 mg/mL
Detection: RI
Reduced molecular weight: Polystyrene

<Production of copolymer>
(Example 1)
75 g of ethanol in a 500 mL four-necked flask, 5.6 g of a 90% by mass aqueous solution of 2-(dimethylamino)ethyldiethyl methacrylate (MOEDES; manufactured by Kao Corporation), and methoxypolyethylene glycol (EO added mol Formula 9) A mixture of 45.0 g of methacrylate (PEG(9)MA; manufactured by Shin-Nakamura Chemical Co., Ltd., NK-Ester M-90G) was bubbled with nitrogen for 30 minutes. Thereafter, the solution was heated to 65° C., and a mixture of 0.1 g of 2,2′-azobis(2,4-dimethylvaleronitrile) (V-65B; manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and 3 g of ethanol was added. was added all at once to initiate polymerization. After stirring the reaction solution for 3 hours, the temperature was raised to 70° C., aged for 2 hours, and then cooled to obtain a polymer solution. After adding 208 g of ion-exchanged water to the resulting polymer solution, the solution was dialyzed using a dialysis tube, and then concentrated with a rotary evaporator to produce the final target polymer (Mw 290,000, Table 1).

(Example 2)
The final target polymer (Mw 265,000, Table 1) was manufactured.

(Example 3)
The same as in Example 1 except that 45 g of methoxypolyethylene glycol (EO addition mole number 90) methacrylate (PEG (90) MA; manufactured by NOF Corporation, Blenmer PME-4000) was used instead of PEG (9) MA. The procedure produced the final target polymer (Mw 182,000, Table 1).

(Comparative example 1)
A liquid mixture of 200 g of ethanol, 5.6 g of 90 mass % MOEDES aqueous solution, and 45 g of PEG(9)MA in a 500 mL four-necked flask was bubbled with nitrogen for 30 minutes. Thereafter, the temperature of the solution was raised to 65° C., and a mixture of 0.5 g of V-65B and 3 g of ethanol was added all at once to initiate polymerization. After stirring the reaction solution for 3 hours, the temperature was raised to 70° C., aged for 2 hours, and then cooled to obtain a polymer solution. After adding 80 g of ion-exchanged water to the obtained polymer solution, it was dialyzed using a dialysis tube, and then concentrated by a rotary evaporator to produce the final target polymer (Mw 27,000, Table 1).

(Comparative Examples 2-3)
A polymer was obtained in the same manner as in Example 1, except that the ratio of PEG(9)MA and MOEDES in the mixture was changed as shown in Table 1.

<Test 1 Evaluation of biofilm formation suppression ability>
One colony of the genus Sphingopyxis was cultured with shaking in 10 mL of R2A medium (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) at 30° C. for 12 hours. The resulting bacterial solution is centrifuged at 5000 rpm for 5 minutes, the supernatant is removed, 10 mL of DPBS (no calcium, no magnesium, pH = 7.0-7.3) is added, and the precipitate is redispersed with a touch mixer. The operation was performed twice. After that, the solution was passed through a 5 μm filter, and the bacterial solution was diluted with DPBS so that the OD600 was 0.1 to obtain a diluted bacterial solution.

An aqueous polymer solution containing 0.1 wt % of the polymer of Examples 1-3 or Comparative Examples 1-3 was prepared. A SUS304 test piece (15×15×1 mm) was immersed in the polymer aqueous solution for 5 minutes and then lightly rinsed with DPBS to obtain a polymer-treated piece (n=1 for each polymer). As a control, pieces without polymer treatment were prepared (n=1). Each piece and 3 mL of the diluted bacterial solution were added to each well of a sterilized flat-bottomed 24-well plate (Falcon, polystyrene) and allowed to stand for 1 hour. The pieces were transferred to individual wells of a sterile flat-bottomed 24-well plate preloaded with 3 mL of DPBS and rinsed briefly. An aqueous SYTO ^™ 9 Green Fluorescent Nucleic Acid Stain solution adjusted to 1 ppm with sterile water was then dropped onto each piece drawn from each well. After standing for 10 minutes, each piece was transferred to a petri dish filled with sterilized water, rinsed lightly, and dried under nitrogen gas flow. The number of bacteria adhering to the piece was counted using an optical microscope (Zeiss), and the bacterial adhesion rate of the polymer-treated piece [(the number of bacteria adhering to the polymer-treated piece/the number of bacteria adhering to the control piece) × 100] (%) asked for The average value of the bacteria adhesion rate between pieces was obtained for each polymer, and was defined as the bacteria adhesion rate for the polymer. In addition, the said operation was implemented under room temperature (25 degreeC).

Table 1 shows the bacterial adhesion rates for the polymers of Examples 1-3 and Comparative Examples 1-3. The polymers of Examples 1 to 3 had a bacteria adhesion rate of less than 40%, indicating that these polymers can suppress biofilm formation by suppressing the adhesion of bacteria to the piece.

Claims (11)

(A) at least one monomer unit selected from the group consisting of a monomer unit having a cationic group, a (meth)acrylic acid ester having an alkyl group or an alkenyl group, and a (meth)acrylic acid ester having an arylalkyl group ; and (B) contains a water-soluble monomer unit having a polyalkylene glycol group having —CH ₃ or —OH at the end, the content of (A) is 7 to 25% by mass, and the content of (B) is A biofilm formation inhibitor comprising, as an active ingredient, a copolymer having an amount of 75 to 93% by mass and a weight average molecular weight of 30,000 to 1,000,000 . 2. The biofilm formation inhibitor according to claim 1, wherein (A) is a monomer unit consisting of a salt of dimethylaminoethyl (meth)acrylate or benzyl (meth)acrylate. The biofilm formation inhibitor according to claim 1 or 2, wherein the polyalkylene glycol group of (B) has 3 to 120 repeating alkylene glycol groups. (A) and (B) are represented by the following formulas (1) and (2), respectively:

(wherein R ¹ , R ³ and R ⁴ each independently represent a hydrogen atom or a methyl group, R ² represents a cationic group and/or a hydrophobic group, and AG ₁ has 2 to 3 carbon atoms). represents an alkylene glycol, m represents an integer of 3 to 120)
Represented by the biofilm formation inhibitor according to any one of claims 1 to 3. The biofilm formation according to any one of claims 1 to 4, wherein the content of (A) in the copolymer is 8 to 20% by mass and the content of (B) is 80 to 92% by mass. inhibitor. The biofilm formation inhibitor according to any one of claims 1 to 5 , wherein the biofilm is a biofilm in facilities where water is flushed or stored. The biofilm formation inhibitor according to claim 6 , which prevents adhesion of microorganisms to said equipment. A biofilm formation suppression method comprising adding the biofilm formation inhibitor according to any one of claims 1 to 7 to water contained in an environment where suppression of biofilm formation is desired. The amount of the biofilm formation inhibitor added to the water is 1 to 10,000 ppm in terms of the concentration of the copolymer in the water added with the biofilm formation inhibitor, according to claim 8 . Method. 10. The method of claim 8 or 9 , wherein the environment in which inhibition of biofilm formation is desired is a facility in which water is flushed or stored. 11. The method of claim 10 , wherein the attachment of microorganisms to said equipment is prevented.