JP5545870B2 - Biofilm remover - Google Patents

Description

  The present invention relates to a biofilm remover. More specifically, the present invention relates to a biofilm remover for removing biofilms composed of microorganisms and microorganism-producing substances from a solid surface, and preventing harm caused by biofilms in various fields involving microorganisms.

  A biofilm is also called a biofilm or slime, and it is generally an aqueous system in which microorganisms adhere to and grow on the surface of substances to produce macromolecular substances such as polysaccharides, proteins, and nucleic acids from inside the microbial cells to form structures. Point to. When a biofilm is formed, harms caused by microorganisms occur and cause problems in various industrial fields. For example, when a biofilm is formed in the piping of a food plant, the biofilm is peeled off and not only leads to contamination of foreign substances in the product, but also causes food poisoning due to microorganism-derived toxins. Furthermore, biofilm formation on the metal surface causes metal corrosion and promotes aging of the equipment.

  Furthermore, the effects of microbial control agents such as bactericides and bacteriostatic agents are not sufficiently exerted on microbial aggregates that have formed biofilms, compared to microorganisms that are dispersed and suspended in water. There are also many. For example, in the medical field, many cases of nosocomial infections have been reported in recent years due to microorganisms remaining in narrow gaps and holes in medical devices to form biofilms. In the human oral cavity, it is well known that biofilms formed on teeth, so-called dental plaque, cause dental caries and periodontal disease, and these problems have been studied for a long time.

  It is known that proteins form a network structure between molecules due to changes in the environment, and form, for example, strong structures and aggregates called amyloid and inclusion bodies (Non-Patent Documents 1 and 2). Biofilms are known to be very difficult to remove once they are formed and mature, and this is thought to be due to the existence of such strong protein structures.

  As technologies for removing biofilms, enzymes (Patent Documents 1 and 2), bactericides containing cationic surfactants (Patent Document 3), hypohalous acid and hydrogen peroxide (Patent Document 4) A method of decomposing or removing a biofilm attached to a solid surface using a drug such as the above has been proposed.

JP-T-2006-507850 Special table 2010-511623 gazette JP 2007-297318 A JP 2008-184516 A

Hasegawa et al., Cell Engineering, vol. 20, p. 1495-1501 (2001) M. M. Carrio et al., J. Biotech., Vol. 96, p. 3-12 (2002)

  Biofilms that have already matured are covered in a film with a high-molecular substance produced by microorganisms, so it is difficult to cause drugs such as enzymes and bactericides to act deep in the biofilm. In particular, it is difficult to efficiently remove the biofilm formed with the above-described strong protein structure even by the technique disclosed in Patent Documents 1-4. Further, with respect to the prior art using an enzyme, the enzyme activity largely depends on the temperature, so that there is a limitation that a strict temperature adjustment is required to exert a sufficient effect.

  An object of the present invention is to efficiently produce a biofilm formed on a solid surface, in particular, a biofilm having a protein composition ratio of 30% by mass or more in terms of BSA (bovine serum albumin), which is particularly difficult to remove in the prior art. It is to provide a biofilm remover that can be removed.

  As a result of intensive studies on the above problems, the present applicant has found that a compound having a structure represented by the following formula (1) has a protein composition ratio formed on a solid surface of 30% by mass or more in terms of BSA (bovine serum albumin). The inventors have found that the removal effect is expressed specifically for the biofilm, and have completed the present invention.

  That is, the present invention comprises a biofilm containing one or more selected from the compound represented by formula (1) and having a protein composition ratio formed on a solid surface of 30% by mass or more in terms of BSA (bovine serum albumin). A biofilm remover for removal is provided.

_{R-CONH- (CH 2) n} -COOM (1)
[In formula, R shows the C10-22 hydrocarbon group which may be substituted by the hydroxy group, n shows the integer of 1-6, M shows a hydrogen atom or a salt-forming cation. ]

  The biofilm remover of the present invention can efficiently remove a biofilm having a protein composition ratio formed on a solid surface of 30% by mass or more in terms of BSA (bovine serum albumin).

[Biofilm remover]
The biofilm remover of the present invention contains one or more compounds represented by the formula (1) [hereinafter referred to as compound (1)] as active ingredients.

_{R-CONH- (CH 2) n} -COOM (1)
[In formula, R shows the C10-22 hydrocarbon group which may be substituted by the hydroxy group, n shows the integer of 1-6, M shows a hydrogen atom or a salt-forming cation. ]

  In the compound (1), examples of the hydrocarbon group for R include an alkyl group, an alkenyl group, and a hydroxyalkyl group, and an alkyl group and an alkenyl group are preferable. From this point, it is a C10-22 thing, a C12-18 thing is preferable, and a C12-14 saturated alkyl group and a C12-18 unsaturated alkyl group are still more preferable especially. R may be a hydrocarbon group of a single composition or a mixed composition, and a mixed alkyl group of natural origin (for example, derived from palm oil, palm kernel oil, soybean, rapeseed) is also suitable. Can be used.

  In the compound (1), n is preferably 1 or 2, more preferably 1, from the viewpoint of the biofilm removing effect.

  M is a hydrogen atom or a salt-forming cation, and examples of the salt-forming cation include alkali metal, alkaline earth metal, alkanolamine, and basic amino acid cations. From the viewpoint of the biofilm removal effect, M is preferably a hydrogen atom, an alkali metal, an alkanolamine, or a basic amino acid, more preferably a hydrogen atom or an alkali metal, and particularly preferably an alkali metal. Here, the alkali metal salt is preferably a sodium salt or a potassium salt, and the alkaline earth metal is preferably a calcium salt or a magnesium salt. Further, as the alkanolamine salt, triethanolamine salt and aminomethylpropanol salt are preferable, and as the basic amino acid salt, arginine salt, histidine salt and lysine salt are preferable.

  Preferable examples of compound (1) include N-acylglycine and N-acyl-β-alanine, or alkali metal salts, alkanolamine salts, and basic amino acid salts thereof. Of these, sodium salts and potassium salts of N-acylglycine are preferable because they exhibit high biofilm removal performance.

  Compound (1) can be obtained by reacting a (hydroxy) fatty acid with an amino acid, but the production method is not particularly limited. Moreover, the compound (1) may contain unreacted substances and by-products as long as the biofilm removal effect is not inhibited.

  The biofilm remover of the present invention is usually used in the form of an aqueous solution in a scene where it acts on a biofilm. The concentration of the compound (1) in the aqueous solution is preferably 0.3 to 30% by mass, more preferably 1.0 to 10% by mass, and still more preferably 1.0 to 7.0% by mass. The pH of the aqueous solution (25 ° C.) is preferably in the range of 6 to 12.5, more preferably in the range of 7 to 12, and still more preferably in the range of 7.5 to 11.5, from the viewpoint of the biofilm removal effect. The range of 8 to 11 is particularly preferable. Here, pH (25 degreeC) of aqueous solution is the value measured with the undiluted | stock solution using HORIBA pH Meter F-21 (made by Horiba, Ltd.).

  The biofilm removing agent of the present invention preferably contains a metal ion scavenger (chelating agent) from the viewpoint of capturing polyvalent metal ions such as calcium and iron and further enhancing the biofilm removing effect.

  Specific examples of suitable metal ion scavengers (chelating agents) include nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), hydroxyethylidene diphosphone. Examples include acid (HEDP), aminotrimethylene phosphonic acid (ATMP), and salts thereof. Among them, EDTA or a salt thereof is preferable because it provides a high biofilm removal effect.

  When these metal ion scavengers (chelating agents) are used, the concentration of the metal ion scavenger (chelating agent) in the aqueous solution can be changed as appropriate depending on the polyvalent metal ion concentration to be captured. The range of -2.5 mass% is preferable, and the range of 0.5-2 mass% is still more preferable.

Examples of the method for removing the biofilm using the biofilm remover of the present invention include a method in which the biofilm remover is brought into contact with the solid surface on which the biofilm is formed by dipping, coating or spraying. At this time, a physical force such as a sponge, towel, brush, or water flow may be further applied. The time for which the biofilm remover is kept in contact varies depending on the amount of biofilm attached, the concentration of the biofilm remover, the temperature at the time of contact, and the presence or absence of physical force. In view of workability, it is preferably 10 seconds or longer, more preferably 10 seconds to 1 hour, still more preferably 10 seconds to 30 minutes, and particularly preferably 10 seconds to 10 minutes. After the contact treatment, it is desirable to quickly rinse away the removed biofilm with running water or the like.
Further, the temperature at which the biofilm remover is kept in contact does not need to be strictly controlled, but is preferably in the range of 0 to 100 ° C, more preferably in the range of 10 to 80 ° C, and more preferably 20 to 20 ° C. 50 ° C. is more preferable.

  The biofilm to be removed by the biofilm remover of the present invention is a biofilm having a protein composition ratio formed on a solid surface of 30% by mass or more in terms of BSA (bovine serum albumin).

  As described in the Background Art section, a biofilm is a structure formed by producing a high-molecular substance such as a polysaccharide, protein, or nucleic acid from a microbial cell in the process of microorganisms adhering to a solid surface and growing. is there. In general, it is known that the composition of biofilms formed differs depending on the type of microorganisms, and the environment including the solid surface state where the microorganisms are derived or placed. Depending on the type of microorganism that adheres and proliferates, the composition of the polymer material of the biofilm formed varies greatly, and the biofilm remover that has been developed by the composition that forms the biofilm and the biofilm removability of various surfactants I found that it had a big influence. Regarding the composition of biofilms formed by microorganisms, as shown in Table 1 below, Pseudomonas aeruginosa (NBRC13275) has a nucleic acid as a main component and forms a biofilm with relatively little protein (24.8% by mass; BSA equivalent). On the other hand, Acinetobacter (environmental isolate) has a biofilm mainly composed of polysaccharide and a relatively large amount of protein (40.2% by mass; BSA equivalent), and Staphylococcus aureus (NBRC13276) has a protein (70. It was confirmed that a biofilm with an extremely large amount of 6% by mass (converted to BSA) was formed (details of the experimental method are described in the column <Measurement method of biofilm composition ratio>).

  With respect to conventional biofilm removers, although a sufficient removal effect is exhibited with respect to biofilms with a small amount of protein derived from Pseudomonas aeruginosa or the like (for example, refer to Patent Document 2 above), the amount of protein in the biofilm With the increase, the removal effect is significantly reduced. In contrast, the biofilm remover of the present invention is specific for a biofilm having a protein composition ratio of 30% by mass or more in terms of BSA (bovine serum albumin) (for example, a biofilm derived from Acinetobacter). The removal effect is expressed. Unexpectedly, the biofilm remover of the present invention is very difficult to remove with the conventional biofilm remover. The protein composition ratio reaches 50% by mass or more in terms of BSA (bovine serum albumin). It was confirmed that the biofilm (for example, a biofilm derived from Staphylococcus aureus) has a higher removal effect (see Examples). That is, the biofilm remover of the present invention exhibits particularly high removal performance for biofilms produced by Staphylococcus aureus.

  As described above, the biofilm remover of the present invention using the compound (1) as an active ingredient has a specific removal effect on a biofilm with a large amount of protein, which was very difficult to remove with a conventional biofilm remover. Is expressed.

<Measurement method of biofilm composition ratio>
Select Staphylococcus aureus (NBRC13276), Acinetobacter (environmental isolate), and Pseudomonas aeruginosa (NBRC13275), known as causative bacteria that are harmful to the human body or poor in terms of aesthetics. About the film, the ratio of the protein, polysaccharide, and nucleic acid was measured with the following method.
Staphylococcus aureus is TSB No.2 liquid medium (Sigma), Acinetobacter is MHB liquid medium (Wako Pure Chemicals), and Pseudomonas aeruginosa is LB medium (Wako Pure Chemicals) at 37 ° C. And cultured for 20 hours. After diluting the culture solution of various bacteria 100 times with sterile saline, Staphylococcus aureus is TSB No.2 agar medium, Acinetobacter is MHB agar medium, Pseudomonas aeruginosa is NAC agar medium (Eiken Chemical Co., Ltd.) 10 of each were smeared and cultured at 37 ° C. for 48 hours. Add 5 ml of an aqueous solution of pH 7.5 containing 140 mM NaCl and 10 mM EDTA (abbreviated as solution A) on each agar medium, and scrape the fungus layer with a congeal bar twice on each plate. The bacterial solution was collected. The collected bacterial solution was stirred for 30 minutes and centrifuged at 8000 rpm and 20 min at 15 ° C. The supernatant was collected and filtered through a 0.2 μm membrane filter (Nalgene filtration product). On the other hand, add 100 ml of solution A to the fungus layer, stir in the same way, collect the solution in a centrifuge tube, stir for 1 minute with vortex (Scientific Industry, G-560), centrifuge and filter. I went twice. The filtrate was collected, sealed in a dialysis cellulose tube, and dialyzed under 5 L of ion-exchanged water for 48 hours (exchanged twice with ion-exchanged water) to remove low molecular compounds.
The collected liquid was measured by the Lowry method (measurement kit manufactured by Nacalai Tesque), and the protein concentration was quantified from the calibration curve using bovine serum albumin according to the instructions of the measurement kit to obtain the amount of protein-derived biofilm. The collected liquid was quantified by the phenol sulfate method, and the concentration of polysaccharides was determined from the calibration curve with mannose for Staphylococcus aureus and Acinetobacter, and the calibration curve with alginate for Pseudomonas aeruginosa. Reference 3). Furthermore, after measuring the absorption maximum at 260 nm for the collected liquid to confirm the presence of nucleic acid, the amount of nucleic acid was obtained by subtracting the amount of protein and the amount of polysaccharide from the solid content of the dialyzed portion to obtain the amount of nucleic acid-derived biofilm.

[Non-Patent Document 3] Hodge, J.E. and Hofreiter, B.T.1962.Methods in Carbohydrate Chemistry vol.1, pp.380-394

Each compounding component used in Examples and Comparative Examples is shown below.
<Compound (1)>
Compound 1-1: N-coconut oil fatty acid acylglycine potassium salt (Ajinomoto Co. Amilite GCK-12K, 30% aqueous solution)
Compound 1-2: N-coconut oil fatty acid acylglycine (prepared by neutralizing amylite GCK-12K with 100 mM HCl (manufactured by Wako Pure Chemical Industries, Ltd.))
Compound 1-3: N-coconut oil fatty acid acylglycine sodium salt (Ajinomoto Amylite GCS)
Compound 1-4: N-lauroyl-β-alanine potassium salt (prepared by equimolar neutralization of N-lauroyl-β-alanine (LBA, manufactured by Mitsui Chemicals, Inc.) with KOH (Wako Pure Chemical Industries, Ltd.))
<Compound (1 ′): Comparative Component of Compound (1)>
Compound 1′-1: polyoxyethylene (4) potassium lauryl ether sulfate (Emal 4.0K, manufactured by Kao Corporation)
Compound 1′-2: Sodium octanoate (manufactured by Kanto Chemical Co., Inc.)
Compound 1′-3: Sodium octyl sulfate (manufactured by Wako Pure Chemical Industries)
Compound 1′-4: alkylamidopropyl betaine (Ahitole 20AB, 30% aqueous solution manufactured by Kao Corporation)
Compound 1′-5: Alkylsulfohydroxybetaine (Ahitole 20HD, 30% aqueous solution manufactured by Kao Corporation)
Compound 1′-6: Palm oil fatty acid sorbitan (Reodol Super SP-L10 manufactured by Kao)
Compound 1′-7: polyoxyethylene (5) lauryl ether (Emulgen 106 manufactured by Kao Corporation)
-Compound 1'-8: Cetyltrimethylammonium chloride (Coatamine 60W, 30% aqueous solution manufactured by Kao Corporation)
Compound 1′-9: alkyl glucoside (Myo Dole 12 manufactured by Kao Corporation)
Compound 1′-10: Lauroylmethyl taurine sodium salt (Nikko Chemical Nikkor LMT)
Compound 1′-11: N-acyl aspartic acid sodium salt (Aminoformer FLMS-P1 manufactured by Asahi Kasei Chemicals Corporation)
-Compound 1'-12: N-acyl glutamic acid potassium salt (Ajinomoto Co. Amisoft CK-11F)
Compound 1'-13: Calcium chloride (Wako Pure Chemical Industries)
Compound 1′-14: α-amylase (Termamyl 120L manufactured by Novozymes)
<Metal ion scavenger (chelating agent)>
Chelating agent 1: ethylenediaminetetraacetic acid tetrasodium salt (manufactured by Wako Pure Chemical Industries, Ltd.)
Chelating agent 2: nitrilotriacetic acid trisodium salt (manufactured by Akzo Nobel, Dissolvin A-92)

Example 1-22 and Comparative Example 1-15
Using the components shown in Table 2, biofilm removers having the compositions shown in Table 2 were prepared by the method shown below (Here, Examples 1-22 and Comparative Example 1-14 are <Preparation of Biofilm Remover>1>, and Comparative Example 15 was prepared in accordance with <Preparation 2 of Biofilm Remover> below). The biofilm removal performance of the obtained biofilm remover was evaluated as follows. The results are shown in Table 2.
In addition, the density | concentration in Table 2 is shown by the effective part density | concentration (mass%) with respect to biofilm removal agent whole quantity. Further, in the following description, “%” means “mass%” unless otherwise specified.

<Preparation 1 of biofilm remover>
In a 100 ml glass beaker, insert a stirrer piece (diameter 8mm x 30mm, manufactured by Masuda Rika Kogyo Co., Ltd.) Of ingredients were added. The temperature was raised to 80 ° C. in a water bath, and the mixture was stirred at 100 rpm for 30 minutes. After confirming that the appearance of the aqueous solution became uniform, it was cooled to room temperature (25 ° C.).

<Preparation 2 of biofilm remover>
In a 100 ml glass beaker, insert a stirrer piece (diameter 8mm x 30mm, manufactured by Masuda Rika Kogyo Co., Ltd.) Of ingredients were added. Stirring was performed at 100 rpm for 5 minutes at room temperature (25 ° C.), and it was confirmed that the appearance of the aqueous solution was uniform.

<Evaluation of biofilm removal performance>
One strain of Staphylococcus aureus (NBRC13276) was cultured with shaking in 25 ml of TBS No. 2 medium (manufactured by Sigma) at 37 ° C. for 22 hours. The turbidity at a wavelength of 600 nm was measured (turbidimeter U-2800 manufactured by HITACHI), diluted with TBS No. 2 medium so that the turbidity was 0.1, and then a sterile 96-well plate (Falcon with a flat bottom) (0.15 ml) was added to each well and incubated at 37 ° C. for 24 or 48 hours. As a positive control, a sample was prepared by adding 0.15 ml of TSB No. 2 medium containing no bacteria to different wells of the same plate. In addition, Acinetobacter (environmentally isolated bacteria) uses MHB liquid medium (manufactured by Wako Pure Chemical Industries, Ltd.) and Pseudomonas aeruginosa (NBRC13275) uses LB medium (manufactured by Wako Pure Chemical Industries, Ltd.). Each control was prepared. After discarding the supernatant, 0.2 ml of sterilized ion-exchanged water (hereinafter referred to as “sterilized water”) was added to each well, and the operation of discarding the supernatant (hereinafter referred to as “wash”) was performed twice. Next, 0.2 ml of sterilized water was added to each well and held until just before each biofilm remover shown in Table 2 was added.
Immediately after discarding the supernatant, unless otherwise stated, each biofilm remover shown in Table 2 stored at room temperature (25 ° C.) was added to each well and left at room temperature (25 ° C.) for 1 minute. For samples other than room temperature, the samples were stored overnight in advance in each temperature incubator and immediately added to each well immediately after discarding the supernatant so that the temperature of the samples did not change.
Thereafter, the supernatant was discarded and washed twice. Moreover, what performed the same operation using sterilized water instead of the biofilm removal agent was made into negative control, and positive control performed the same operation with sterilized water.
Thereafter, 0.2 ml of a 0.1% aqueous solution of crystal violet (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well and left at room temperature for 10 minutes. After washing twice with sterilized water, 0.2 ml of 95% ethanol solution (manufactured by Sigma) was added to each well and left overnight at 4 ° C. The absorbance at 570 nm was then measured for each well. The obtained absorbance value was substituted into the following formula to calculate the biofilm removal rate.
All the above steps were carried out under aseptic conditions.

  Biofilm removal rate (%) = 100 × [(An−Ap) − (As−Ap)] / (An−Ap)

Where
As: Sample absorbance An: Negative control absorbance Ap: Positive control absorbance

Claims (5)

A biofilm remover that contains one or more compounds represented by formula (1) and removes a biofilm having a protein composition ratio of 30% by mass or more in terms of BSA (bovine serum albumin) formed on a solid surface.
_{R-CONH- (CH 2) n} -COOM (1)
[In formula, R shows the C10-22 hydrocarbon group which may be substituted by the hydroxy group, n shows the integer of 1-6, M shows a hydrogen atom or a salt-forming cation. ]   The biofilm removing agent according to claim 1, further comprising a metal ion scavenger (chelating agent).   The biofilm remover according to claim 1 or 2, wherein R in the formula (1) is a saturated alkyl group having 12 to 14 carbon atoms.   The biofilm removing agent according to any one of claims 1 to 3, wherein n in the formula (1) is 1 or 2. A method for removing a biofilm having a protein composition ratio formed on a solid surface of 30% by mass or more in terms of BSA (bovine serum albumin) using the biofilm remover according to any one of claims 1 to 4.