JP5363697B2 - Biofilm inhibitor / removal agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biofilm inhibitor/remover for eliminating biofilms in the environment. <P>SOLUTION: The biofilm inhibitor/remover contains a compound represented by formula (A) (wherein R is a 6-18C alkyl group which may be substituted; G is a residue derived from a 5-6C reducing sugar; m is a number of 0 to 200; and n is a number of 1 to 30). <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a biofilm inhibitor / removal agent that removes a biofilm from the environment.

  A biofilm is a community of microorganisms formed by microorganisms, such as bacteria and mold, adhering to the surface of solids and liquids and forming secretions. If microorganisms inhabit the environment, they must be in a biofilm state. There are very many. Biofilms can cause serious problems in the living environment and industry. For example, in a living environment, drainage outlets such as kitchens, bathrooms, and toilets become slimy, clogged, and cause bad odors, resulting in discomfort. Infectious diseases may develop due to bacteria in the biofilm formed in a circulating bath in a hot spring facility or the like. On the other hand, in the industry, it is known to cause corrosion by being formed on a sewer pipe or a ship bottom. Biofilms on factory production lines can cause microbial contamination. In the medical field, biofilms formed on medical devices such as dialysis tubes, endoscopes, and contact lenses can be the source of infection, and can also cause disease due to biofilm formation in human tissues such as skin. . In the food industry, fresh food products such as vegetables, raw materials for processed foods, and biofilms formed on cooking utensils cause rot and food poisoning.

  Furthermore, it is known that microorganisms form a biofilm, which makes it much stronger than when it is in a floating state against stress such as cleaning removal, chemicals, and heat. In many cases, the effect cannot be fully exhibited, and it is a serious problem because it makes it difficult to thoroughly eliminate microorganisms.

  As a method for removing or sterilizing biofilms, in addition to using general detergents and sterilizers, combinations of surfactants such as monoglyceride polycarboxylic acid esters and enzymes such as dextranase, peptidase, and glucanase are combined. It has been proposed to use them and chemically remove them with alkaline electrolyzed water (see Patent Documents 1 to 4). In addition, a method of physically removing the biofilm by irradiating it with a pulse laser, bubbling, or the like while the attached portion of the biofilm is in contact with a liquid has been proposed (see Patent Documents 5 to 7).

  However, once a biofilm is formed, it is not easy to sufficiently remove viscous substances such as polysaccharides produced from bacteria simply by flowing a cleaning solution or rubbing them. Therefore, in order to exclude the biofilm from the environment, it is effective to suppress the formation of the biofilm. As these methods, a method of suppressing biofilm formation using farnesol or xylitol (see Patent Document 8), or a method of suppressing biofilm formation containing a lactone derivative and / or a furan derivative as an active ingredient (Patent Document 9). Etc.) have been proposed, but there are problems such as the effect is not sufficient and the target bacterial species is limited. Therefore, there is a demand for an agent that has a higher effect and has the ability to suppress biofilm formation of many bacterial species.

Alkyl glycosides such as lauryl glucoside and octyl galactoside are widely known as hypoallergenic nonionic surfactants. However, nothing is known about the effect of eliminating microbial biofilms from the environment.
Japanese Patent Laid-Open No. 10-234294 Japanese Patent Laid-Open No. 10-234595 Japanese Patent Laid-Open No. 3-193 JP 2003-250436 A Japanese Patent Application Laid-Open No. 2004-275979 JP 2004-65612 A JP 2002-284604 A JP 2004-324045 A JP 2004-155681 A

  The present invention relates to providing a biofilm inhibiting / removing agent for eliminating biofilm in the environment.

  The present inventors examined a compound that is highly safe and can be widely used in a living space and the like. As a result, the alkylglycoside represented by the following formula (A) has excellent biofilm formation suppressing action and biofilm removing action. It was found to have

  That is, the present invention provides the following formula (A):

[In the formula, R represents an optionally substituted alkyl group having 6 to 18 carbon atoms, G represents a residue derived from a reducing sugar having 5 to 6 carbon atoms, m represents a number of 0 to 200, n Indicates a number from 1 to 30. ]
The biofilm inhibitor / removal agent containing the compound represented by this is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the adhesion | attachment of the biofilm formed in various environments on a residence and industry can be suppressed and removed simply and efficiently.

The compound represented by the formula (A) used in the present invention includes one or more reducing sugars having 5 to 6 carbon atoms, directly or via one or more oxyethylene groups to an alkyl group having 6 to 18 carbon atoms. It is a compound in which the residue derived is glycosidically bonded in the α-configuration or β-configuration.
As the “alkyl group having 6 to 18 carbon atoms”, either a straight chain or a branched chain may be used. For example, n-hexyl group, n-heptyl group, n-octyl group, n-decyl group, n-dodecyl group, n -Tetradecyl group, n-hexadecyl group, n-octadecyl group, isostearyl group (isooctadecyl group), 2-ethylhexyl group, etc. are mentioned. Among these, an alkyl group having 8 to 16 carbon atoms is preferable and 8 to 14 carbon atoms are more preferable in terms of blending and efficacy.

  The hydrogen atom of the alkyl group may be substituted with a substituent. Examples of the substituent include an alkoxy group having 1 to 6 carbon atoms, a halogen atom (eg, fluorine, chlorine, bromine, iodine, etc.), a nitro group, a haloalkyl group having 1 to 6 carbon atoms, and a haloalkoxy group having 1 to 6 carbon atoms. Groups.

  Examples of the “reducing sugar having 5 to 6 carbon atoms” include glucose, fructose, galactose, xylose, mannose, arabinose, and the like, and a combination thereof may be used. Among these, glucose and galactose are preferable from the viewpoint of the biofilm formation inhibitory effect. In addition, these reducing sugars include any of pyranose type, furanose type or a mixture thereof.

  The “residue derived from reducing sugar” means a sugar residue remaining after removing hydrogen atoms of all non-glycosidic hydroxyl groups and hydrogen atoms of glycosidic hydroxyl groups in reducing sugars or condensates thereof.

  Although m shows the number of 0-200, 0-12 are preferable from the surface of biofilm formation suppression effect, and 0-3 are more preferable. Although n which shows the condensation degree of a reducing sugar is a number of 1-30, 1-6 are preferable and 1-3 are more preferable. When n is larger than 1, that is, when G is composed of two or more sugar chains, the sugar chain binding mode is 1-2, 1-3, 1-4, 1-6 bond, and these are mixed It may be a binding mode.

  Among the compounds represented by the formula (A), particularly preferable examples include β-octyl glucoside, α-octyl galactoside, β-octyl galactoside, laryl galactoside, trioxyethylene lauryl galactoside, lauryl glucoside, C8 to C16. A glucoside mixture, a C8-C16 galactoside mixture, etc. are mentioned.

  The compound represented by the formula (A) can be produced by the method of Hori et al. (Pharmaceutical Journal, Vol. 79, No. 1, p80-83) and the production examples described later. Commercial products such as mydol (C8-C16 glucoside mixture, manufactured by Kao Corporation) can also be used.

  Such compounds (A) can be used in various forms such as gram-positive bacteria such as Pseudomonas aeruginosa, Sephacia, Escherichia coli and Serratia, Gram-positive bacteria such as Staphylococcus aureus, and fungi such as yeast. It has the action of suppressing the formation and removal of biofilms by microorganisms present in various environments. Therefore, the compound (A) is widely useful as a biofilm inhibitor / removal agent in various environments. Here, suppression / removal of the biofilm in the present invention refers to inhibiting or suppressing the formation of a community (biofilm) formed by microorganisms (bacteria and fungi) together with secretions and deposits, and / or formed bio. This refers to removing the film, and the types of microorganisms involved in formation and the number of types are not particularly limited.

  The biofilm inhibitor / removal agent of the present invention is appropriately added with an emulsifier, a fixing agent, a dispersant, a wetting agent, a stabilizer, a propellant, etc. It can be formulated as aerosols, smoke agents, coating agents, cleaning agents, powders and granules, and can be used as pharmaceuticals such as cosmetics and external preparations for skin and quasi drugs. Such cosmetics, quasi-drugs, detergents and the like contain the compound (A) of the present invention, have a biofilm formation inhibitory and / or removal action, and are labeled as such. Can be.

Here, examples of the cleaning agent include residential cleaning agents, kitchen detergents, industrial cleaning agents, and medical cleaning agents; cosmetics include makeup cosmetics, basic cosmetics, hair cosmetics, and the like. Examples of pharmaceuticals include wounds and the like; quasi drugs (Article 2, Paragraph 2 of the Pharmaceutical Affairs Law) include, for example, bath preparations, medicinal cosmetics, contact lens disinfectants, skin disinfectants, scratches Examples include disinfectant protective agents and cracking / redening agents.
These are various environments in which biofilms are formed, for example, living environments such as floors, tatami mats, bathtubs, walls, toilets, etc., ship bottoms, factory piping, etc., human bodies such as skin, wounds, contact lenses, endoscopes, etc. The effect can be exhibited by spraying, spraying, applying, or evaporating to other medical devices.

In addition to the compound (A) of the present invention, an antibacterial and antifungal agent, an antiseptic / antifungal agent, an antibiotic, a penetration enhancer, a surfactant, a deodorant, a fragrance and the like are added to the above preparation. Is.
Examples of the antibacterial and antifungal agents include thiabendazole, triclosan, chlorhexidine, zinc pyrithione, chlorxylenol, and the like, and natural antibacterial components such as chitosan, catechin, thymol, hinokitiol, scorpion bamboo extract, mustard essential oil, and wasabi essential oil.

  Examples of antiseptic / antifungal agents include paraoxybenzoates (parabens), 2-phenoxyethanol, benzoic acid and its salts, salicylic acid and its salts, sorbic acid and its salts, dehydroacetic acid and its salts, p-toluenesulfonic acid And salts thereof, alcohols such as ethanol and isopropanol, quaternary ammonium salts such as benzalkonium chloride and the like, and polyhydric alcohols that are antiseptic raw materials, medium chain fatty acids and esters thereof, glycerol derivatives, and Examples include chelating agents such as EDTA.

  The compounding amount of the compound (A) in the biofilm inhibitor / removal agent of the present invention is preferably 0.01 to 10% by mass, particularly preferably 0.03 to 5% by mass.

Production Example 1 Production of α, β-laurylgalactoside D-galactose and lauryl alcohol were reacted in the presence of a catalytic amount of paratoluenesulfonic acid monohydrate while dehydrating under heating and reduced pressure conditions. The resulting mixture was purified by a silica gel column to obtain lauryl galactoside having a degree of galactose condensation of 1 to 3. As a result of analysis by gel permeation chromatography, gas chromatography and ¹ H-NMR, the average saccharide condensation degree of the obtained lauryl galactoside was 1.48, and the composition of lauryl monogalactoside in the component was pyranoside / furanoside = 83 / 17. Of these, the α / β ratio of pyranoside was 75/25.

Production Example 2 Production of β-trioxyethylene lauryl galactoside Pentaacetyl-D-galactose and trioxyethylene monolauryl ether were reacted in dichloromethane in the presence of boron trifluoride diethyl ether complex at room temperature. After distilling off the solvent under reduced pressure, β-trioxyethylene lauryl-2,3,4,6-tetraacetylgalactoside was obtained by purification with a silica gel column. This was deacetylated with sodium methoxide to obtain β-trioxyethylene lauryl galactoside.

¹ H-NMR (400MHz, CDCl ₃ ) 0.88 (t, 3H), 1.2-1.35 (m, 18H), 1.57 (m, 2H), 3.35-3.8 (overlapped, 13H), 3.84 (t, 2H), 3.97 -4.07 (overlapped, 3H), 4.17 (d, 1H), 4.29 (d, J = 7.6 Hz, 1H), 4.41 (d, 1H).

Production Example 3 Production of α- and β-octylgalactoside α, β-octylgalactoside produced using octyl alcohol as a raw material in the same manner as in Production Example 2 was purified by a column to obtain α-octylgalactoside and β-octylgalactoside.

  α-form: 0.78 (t, 3H), 1.1-1.3 (m, 10H), 1.47 (m, 2H), 3.45-3.70 (overlapped, 7H), 4.63 (d, J = 2.8Hz, 1H), β-form: 0.86 (t, 3H), 1.2-1.35 (m, 10H), 1.51 (m, 2H), 3.25-3.75 (overlapped, 7H), 4.09 (d, J = 7.6Hz, 1H).

Production Example 4 Production of C8 to C16 Galactoside Mixture In the same manner as in Production Example 1, D-galactose and C8 to C16 mixed alcohol (C8: C10: C12: C14 = <1: 60: 30: 10: <1) Reacted. Unreacted alcohol was distilled off from the obtained mixture under reduced pressure to obtain a C8-C16 galactoside mixture. As a result of analysis, the average degree of sugar condensation was 1.3.

  Α, β-lauryl galactoside, β-trioxyethylene lauryl galactoside, α-octyl galactoside and β-octyl galactoside, C8-C16 galactoside mixture and β-n-octyl glucoside (Dojin) manufactured in the above Production Examples 1 to 4 Chemical Laboratory), C8-C16 glucoside mixture (“Mydoll 12” manufactured by Kao Corporation)) were used as test substances in the examples.

Example 1 Inhibitory effect on biofilm formation of Pseudomonas aeruginosa (1) Test strain Pseudomonas aeruginosa NBRC 13725
(2) Experimental method The test strain was diluted 100 times after shaking culture (30 ° C., 700 rpm, all day and night) in LB medium (Wako Pure Chemical Industries) (600 μL / well) using a 24-well microplate. Liquid.
Preparation of biofilm and evaluation of formation inhibitory effect were performed as follows.
1) After using a 96-well microplate (round bottom) of PVC (polyvinyl alcohol chloride), each glycolipid was added to MHB medium (Mura Hinton liquid medium, Difco) (100 μL / well) at 0.1%, 10 μL of the test bacterial solution was inoculated, and statically cultured at 30 ° C. for 24 hours.
2) After removing the culture solution, the attached cells were used as a biofilm, and 200 μL of 0.1% crystal violet solution was added to stain the biofilm.
3) After removing the crystal violet solution and washing 10 times or more with 200 μL of tap water, the remaining biofilm adhered to the plate was quantified.
4) The biofilm was quantified by dissolving the stained biofilm in 200 μL of 99.5% ethanol and measuring the absorbance (595 nm).
5) As a control, a biofilm was formed in the same manner without adding the test substance.
The results are shown in Table 1. Here, the biofilm formation amount is shown as a ratio when the control is 100.

Example 2 Inhibitory effect on biofilms formed by various microorganisms (1)
Using α, β-lauryl galactoside and the three strains shown in Table 2 as test strains, the biofilm formation inhibitory effect was evaluated in the same manner as in Example 1. The concentrations of α, β-lauryl galactoside were 0.05% and 0.2%.
The results are shown in Table 2. Here, the biofilm formation amount is shown as a ratio when the control is 100.

Example 3 Inhibitory effect on biofilms formed by various microorganisms (2)
Using the C8-16-glucoside mixture, the biofilm formation inhibitory effect was evaluated in the same manner as in Example 1, using 4 strains shown in Table 3 as test strains and 25 environmentally isolated strains. In addition, when mixing a strain, what was mixed after culture | cultivation was diluted. The concentration of C8-16-glucoside was 0.04%.
The results are shown in Table 3. Here, the biofilm formation amount is shown as a ratio when the control is 100.

Example 4 Removal effect of biofilm (1) Test strain Pseudomonas aeruginosa NBRC 13725
Serratia bacteria (Serratia marcescens NBRC 12648)
Staphylococcus aureus NBRC 13276
(2) Experimental method The test strain was diluted 100 times after shaking culture (30 ° C., 700 rpm, all day and night) in LB medium (Wako Pure Chemical Industries) (600 μL / well) using a 24-well microplate. Liquid.
The biofilm was prepared and the removal effect was evaluated as follows.
1) Using a 96-well microplate (round bottom) of PVC (polyvinyl alcohol chloride), inoculate 10 μL of the test bacterial solution into MHB medium (Mura Hinton liquid medium, Difco) (100 μL / well) at 30 ° C. for 24 hours. The culture was stationary.
2) Remove the culture solution, wash once with PBS (phosuphate-buffered saline (pH 7.4)), add 200 μL of PBS to the biofilm remaining on the plate, and add C8-16 galactoside mixture to 0.1%. After the addition, it was left at 30 ° C. for 24 hours.
3) After removing PBS, the remaining biofilm was stained and quantified in the same manner as in Example 1.
As a control, the same operation was performed without adding the C8-16 galactoside mixture.
The results are shown in Table 4. Here, the amount of biofilm is shown as a ratio when the control is 100.

Claims (1)

The following formula (A):
Represented by
Α- and / or β-lauryl galactoside, wherein R is a lauryl group, G is a residue derived from galactose, m is 0, and n is a number from 1 to 3,
Β-trioxyethylene lauryl galactoside, wherein R is a lauryl group, G is a residue derived from galactose, m is 3, and n is a number from 1 to 3,
R is an alkyl group having 8 to 16 carbon atoms, G is a residue derived from glucose, m is 0, and a C8 to C16 glucoside mixture in which n is a number from 1 to 3, and
C8-C16 galactoside mixture wherein R is an alkyl group having 8 to 16 carbon atoms, G is a residue derived from galactose, m is 0, and n is 1 to 3
A biofilm inhibiting / removing agent comprising at least one compound selected from the group consisting of: