This international application claims the priority based on Japanese Patent Application No. 2012-055939 filed Mar. 13, 2012 in the Japan Patent Office, and the entire disclosure of Japanese Patent Application No. 2012-055939 is incorporated herein by reference.
The present invention relates to a biofilm inhibitor.
Biofilm is a structure formed with secretions or the like of one or more types of microorganisms that are gathered and adhered to the surface of various substances. Most microorganisms in the environment are present in the form of bio film.
Biofilm is known to cause serious problems in human bodies, living environments, and medical fields.
In human bodies, biofilm can cause oral diseases such as dental caries, periodontal disease, oral malodor, etc. In addition, microbial infections in bodies as well as in oral cavities are said to be caused by microorganisms growing in bio film. In the living environments, bio film can be a cause of slime, clogging, and malodor in drains, pipes, etc. in a kitchen, and so on. In some cases, biofilm formed on the surface of equipment necessary for food processing causes food poisoning or the like, due to adhesion of microorganisms to food after processed. In the medical fields, biofilm formed on the surface of a catheter may cause serious infections. A number of methods to inhibit or remove bio film have been reported. For example, Japanese Unexamined Patent Application Publication No. 2007-145876 reports that a composition for oral use incorporating sugar alcohol and/or amino acid therein suppresses adhesion, aggregation or the like of bacteria in the process of forming bio film (Patent Document 1). Japanese Unexamined Patent Application Publication No. 2004-155681 reports that an oral biofilm inhibitor including a lactone derivative and/or a furan derivative that has at least one oxygen atom-containing functional group in a molecule and having a molecular weight of 220 or less, as an active ingredient, can effectively control oral bacteria and biofilm that cause oral diseases (Patent Document 2). Japanese Unexamined Patent Application Publication No. 2007-518400 reports that a cell-free fermentation product derived from one or more types of fermentative bacteria selected from Lactobacillus species, Lactococcus species, and Pediococcus species prevents biofilm formation, reduces existing biofilm, and decreases populations of bacteria (Patent Document 3).
In the methods of removing or inhibiting a biofilm described in Patent Documents 1 to 3 described above, when the biofilm has a drug resistance, removal of microorganisms that form the biofilm becomes difficult. There is a problem in that a desired biofilm formation inhibitory effect cannot be achieved.
Therefore, in the present invention, it is desirable to provide a biofilm inhibitor excellent in bio film formation inhibitory effect.
The bio film inhibitor of the present invention is characterized in containing a lectin, at least one recognizable sugar chain of which is N-acetylglucosamine (GlcNAc).
By containing a lectin, at least one recognizable sugar chain of which is N-acetylglucosamine (GlcNAc), the biofilm inhibitor of the present invention has ability to inhibit a bio film.
The reason why the effect of the present invention (have ability to inhibit a biofilm) is produced is estimated as described below.
A film of polysaccharide is formed on the surface of a bio film. One of the components of the film of polysaccharide is N-acetylglucosamine (GlcNAc). As the lectin recognizes N-acetylglucosamine (GlcNAc) and binds to the film of polysaccharide, the film of polysaccharide is removed. Along with the removal of the film of polysaccharide, biofilm formation is inhibited. Thereby, it is possible to inhibit a biofilm.
In addition, the biofilm has acquired resistance to every drug by exchange of substances between the film of polysaccharide and microorganisms. According to the biofilm inhibitor of the present invention, since the film of polysaccharide is to be removed, it is possible to reduce resistance to drugs of the biofilm formed.
For the lectin, known lectins, at least one recognizable sugar chain of which is N-acetylglucosamine (GlcNAc), can be appropriately selected and used.
In addition, a lectin concentration in the biofilm inhibitor of the present invention is preferably 0.0001 mg/mL or above, more preferably 0.0001 mg/mL to 2 mg/mL, and further more preferably 0.0001 mg/mL to 1 mg/mL, in saturation concentration. The saturation concentration is the maximum concentration of lectin that can be contained in the biofilm inhibitor.
The biofilm inhibitor of the present invention preferably contains a lectin, at least one recognizable sugar chain of which is sialic acid (Sia).
If the biofilm inhibitor contains the lectin, at least one recognizable sugar chain of which is sialic acid (Sia), the lectin recognizes the sialic acid, and it is thereby possible to further inhibit a biofilm.
In this case, the reason why the effect of the present invention (to have ability to inhibit a biofilm) is produced is estimated as described below.
Sialic acid (Sia) is one of the components of the polysaccharide exposed on the surface of microorganisms. As the lectin recognizes the sialic acid (Sia) and binds to the microbial surface, the microorganisms firmly aggregate. Along with the firm aggregation of the microorganisms, adhesion to adherent bodies of the microorganisms is inhibited. Thus, it is possible to inhibit a bio film.
In addition, the biofilm has acquired resistance to every drug by exchange of substances between the film of polysaccharide and microorganisms. According to the biofilm inhibitor of the present invention, the microorganisms firmly aggregate. Thus, it is possible to further reduce resistance to drugs of the biofilm formed.
As for the lectin, at least one recognizable sugar chain of which is sialic acid (Sia), known lectins may be appropriately selected and used.
In the biofilm inhibitor of the present invention, the lectin is preferably an extract from one or more natural products selected from the group consisting of bitter gourd, pokeweed, gorse, mushrooms, wheat, and jack bean.
In case that the lectin is an extract from one or more natural products selected from the group consisting of bitter gourd, pokeweed, gorse, mushrooms, wheat, and jack beans, the lectin recognizes N-acetylglucosamine (GlcNAc) and binds to the film of polysaccharide. Thereby, the film of polysaccharide is removed. Along with the removal of the film of polysaccharide, biofilm formation is inhibited. Thus, it is possible to further inhibit a biofilm. Further, in case that the lectin is an extract from wheat, mushrooms, or mushrooms and wheat, the lectin recognizes sialic acid (Sia) and binds to the microbial surface. Thereby, the microorganisms firmly aggregate. Along with the firm aggregation of microorganisms, adhesion to adherent bodies of microorganisms is inhibited. Thus, it is possible to further inhibit a biofilm.
In addition, the biofilm has acquired resistance to every drug by exchange of substances between the film of polysaccharide and microorganisms. According to the biofilm inhibitor of the present invention, since the film of polysaccharide is to be removed, it is possible to further lower the resistance to drugs of the biofilm formed. Further, in case that the lectin is an extract from mushrooms, wheat, or mushroom and wheat, the microorganisms firmly aggregate. Thus, it is possible to further reduce the resistance to drugs of the biofilm formed.
The lectin can be acquired by appropriately selecting and using known techniques from each of one or more natural products selected from the group consisting of bitter gourd, pokeweed, gorse, mushrooms, wheat, and jack bean. For example, it is possible to use the technique described below.
Each of the one or more natural products selected from the group consisting of bitter gourd, pokeweed, gorse, mushrooms, wheat, and jack bean is ground by a known method, using such as a coffee mill, a homogenizer, and the like, until ground to a desired size. The resulting powder is extracted using an extraction solution commonly used in extraction of protein (e.g., distilled water, Tris buffer, phosphate buffer, Tricine buffer, Hepes buffer, MOPS buffer, carbonate buffer, citrate buffer, borate buffer, MES buffer, and PIPES buffer; the extraction solution is not particularly limited to these). Ethanol is added to the acquired extract to acquire a precipitate. After the precipitate is dried, the dried precipitate is added to a physiological saline. Thereafter, the physiological saline is centrifuged to collect the supernatant. Thereby, it is possible to acquire a crude extract. The resulting crude extract is purified with an affinity column which binds sugar. Thereby, it is possible to acquire a lectin. Sugar to be bound to the affinity column is chosen depending on the sugar specificity of the lectin to be purified. For example, in the case of bitter gourd, the lectin can be purified efficiently with an affinity column which binds galactose. Further, if necessary, it is possible to add a technique such as gel filtration. Alternatively, or in addition, purification can be achieved using one or a combination of known techniques used for protein purification, for example, ammonium sulfate precipitation, sedimentation by organic solvent (ethanol, methanol, acetone, etc.), chromatography such as ion exchange chromatography, isoelectric chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption chromatography, affinity chromatography using substrates or antibodies, and reverse phase column chromatography, filtration such as microfiltration, ultrafiltration, and reverse osmosis filtration.
Further, each lectin extracted from the one or more natural products selected from the group consisting of bitter gourd, pokeweed, gorse, mushrooms, wheat, and Jack bean, which are commercially available, may be appropriately selected and used.
In the biofilm inhibitor of the present invention, the lectin preferably has at least one of the following natures (1) and (2):
(1) nature to peel off at least one of the biofilm adhering to an adherent body and the bacteria forming the biofilm, from the adherent body; and
(2) nature to aggregate microorganisms that form the biofilm.
According to such biofilm inhibitor having at least one of the natures of (1) and (2), it possible to further inhibit a biofilm.
The adherent body can be any body as long as the biofilm can be adhered, such as, for example, teeth, dentures, dental plaque, drainage ditches of basin, bathroom, and toilet, or a catheter for medical use, and the like.
In the biofilm inhibitor of the present invention, the biofilm inhibitor preferably inhibits a biofilm formed by one or more microorganisms selected from the group consisting of Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, and E. coli.
In the case of inhibiting a biofilm formed by one or more microorganisms selected from the group consisting of Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, and E. coli, it is possible to suppress formation of fields where the microorganisms grow. Therefore, according to the biofilm inhibitor of the present invention, it is possible to eradicate these microorganisms and suppress infections caused by the microorganisms. Further, it is possible to suppress contamination of an adherent body caused by adhesion of these microorganisms.
Hereinafter, a description will be given, by way of example, for embodiments of the present invention. The present invention, however, should not to be limited to these embodiments.
(Biofilm Inhibitor 1 which Contains Bitter Gourd Lectin)
The biofilm inhibitor 1 which contains a bitter gourd lectin was produced as described below, based on the production methods described in Japanese Patent No. 3,849,945, and in Japanese Unexamined Patent Application Publication No. 2011-241192.
Bitter gourd seeds were pulverized into fine powder by a coffee mill. The fine powder acquired was placed in a container, to which distilled water was added, and the acquired solution was stirred with a stirrer. Thereafter, the solution was left for a while and seed components of bitter gourd were extracted. The extract was filtered, and the filtrate was centrifuged to collect the supernatant. Ethanol was added to the supernatant and stirred. The mixture was incubated for about 12 hours at 4° C., to acquire a bitter gourd crude extract.
The bitter gourd crude extract was purified with an affinity column which binds galactose, thereby to acquire a biofilm inhibitor 1.
(Biofilm Inhibitor 2 which Contains Mushroom Lectin)
A biofilm inhibitor 2 was prepared by adjusting the concentration of a mushroom lectin to a desired concentration. As the mushroom lectin, the model number J102 produced by Cosmo Bio Co., Ltd. was used.
(Bio Film Inhibitor 3 which Contains Pokeweed Lectin)
A biofilm inhibitor 2 was prepared by adjusting the concentration of a pokeweed lectin to a desired concentration. As the pokeweed lectin, the model number J116 produced by Cosmo Bio Co., Ltd. was used.
(Biofilm Inhibitor 4 which Contains Wheat Germ Lectin)
A biofilm inhibitor 4 was prepared by adjusting the concentration of a wheat germ lectin to a desired concentration. As the wheat germ lectin, the model number J120 produced by Cosmo Bio Co., Ltd. was used.
(Biofilm Inhibitor 5 which Contains Needle Broom Lectin)
A biofilm inhibitor 5 was prepared by adjusting the concentration of a furze lectin to a desired concentration. As the furze lectin, the model number J119 produced by Cosmo Bio Co., Ltd. was used.
(Biofilm Inhibitor 6 which Contains Jack Bean Lectin)
A biofilm inhibitor 6 was prepared by adjusting the concentration of a jack bean lectin to a desired concentration. As the jack bean lectin, the model number J103 produced by Cosmo Bio Co., Ltd. was used.
A biofilm inhibitor was prepared by adjusting the concentration of a soybean lectin to a desired concentration. As the soybean lectin, the model number J117 produced by Cosmo Bio Co., Ltd. was used.
A biofilm inhibitor was prepared by adjusting the concentration of a pea lectin to a desired concentration. As the pea lectin, the model number J115 produced by Cosmo Bio Co., Ltd. was used.
A biofilm inhibitor was prepared by adjusting the concentration of a lentil lectin to a desired concentration. As the lentil lectin, the model number J107 produced by Cosmo Bio Co., Ltd. was used.
A biofilm inhibitor was prepared by adjusting the concentration of a peanut lectin to a desired concentration. As the peanut lectin, the model number J114 produced by Cosmo Bio Co., Ltd. was used.
A biofilm inhibitor was prepared by adjusting the concentration of a lotus lectin to a desired concentration. As the lotus lectin, the model number J109 produced by Cosmo Bio Co., Ltd. was used.
A biofilm inhibitor was prepared by adjusting the concentration of a Japanese elder lectin to a desired concentration. As the Japanese elder lectin, the model number J118 produced by Cosmo Bio Co., Ltd. was used.
(1) Evaluation of Biofilm Formation Inhibitory Effect in Case that Biofilm Inhibitor 1 was Added Before Formation of Biofilm
(1-1) Evaluation Method
Staphylococcus aureus was added to a glucose-added 804 medium and cultured until the optical density (opticaldensity: OD) of the medium at a wavelength of 600 nm is 0.1 OD. Thereafter, 500 μl of the culture solution was placed in each well of a 24-well multiwell plate. The biofilm inhibitor 1 was added to each well with the concentration of the bitter gourd lectin of 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, and 1 mg/mL each. Thereafter, the culture solutions were cultured overnight. Also, a culture solution without addition of the biofilm inhibitor was cultured in the same manner as a control.
The composition of the glucose-added 804 medium is as shown below.
Polypeptone 10 g/L, yeast extract 5 g/L, MgSO4.7H2O 1 g/L, glucose 5 g/L, distilled water 1 L
For each culture solution prepared, the degree of biofilm formation was calculated using a crystal violet method.
Degree of biofilm formation (%)=X/Y×100 (Equation 1)
where X is the absorbance for the well to which the biofilm inhibitor was added, which was acquired by measuring the decolorizing solution after the step (d) in
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
(2) Evaluation of Biofilm Formation Inhibitory Effect in Case that Each of Biofilm Inhibitor 1, Biofilm Inhibitor 5, and Biofilm Inhibitors of Comparative Examples 1 to 3 was Added Before Formation of Biofilm
(1-1) Evaluation Method
Evaluation was made in the same manner as in the evaluation method described in (1-1) of (1) above, except that each of the biofilm inhibitor 1, the biofilm inhibitor 5, and the biofilm inhibitors of Comparative Examples 1 to 3, of which lectin concentration is 0.5 mg/mL, was used as a biofilm inhibitor.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
(3) Evaluation of Biofilm Formation Inhibitory Effect in Case that Each of Biofilm Inhibitor 1 and Biofilm Inhibitor 2 was Added Before Formation of Biofilm
(1-1) Evaluation Method
Evaluation was performed in the same manner as in the evaluation method described in (1-1) of (1) above, except that each of the biofilm inhibitor 1 of which lectin concentration is 0.5 mg/mL and the biofilm inhibitor 2 of which lectin concentration is 0.5 mg/mL is used as a biofilm inhibitor.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
(4) Evaluation of Biofilm Formation Inhibitory Effect in Case that Each of Biofilm Inhibitor 1, Biofilm Inhibitor 3, Biofilm Inhibitor 4, and Biofilm Inhibitors of Comparative Examples 4 to 6 was Added Before Formation of Biofilm
(1-1) Evaluation Method
Evaluation was performed in the same manner as in the evaluation method described in (1-1) of (1) above, except that each of the biofilm inhibitor 1, the bio film inhibitor 3, the biofilm inhibitor 4, and the biofilm inhibitors of Comparative Examples 4 to 6, of which lectin concentration is 0.5 mg/mL, is used as a biofilm inhibitor.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
In case that the biofilm inhibitor 4 is added, aggregation of Staphylococcus aureus was observed as in the case that the biofilm inhibitor 2 is added.
Table 1 summarizes the above results. The higher the number of “+” is, the higher the biofilm formation inhibitory effect is. The same number of “+” indicates that the biofilm formation inhibitory effect is equivalent. In addition, sugar chains recognized by the lectin contained in each biofilm inhibitor are also shown in Table 1. It is to be noted that effect on Pseudomonas aeruginosa is shown, in regard to the biofilm inhibitor 6.
As shown in Table 1, the biofilm inhibitors 1 to 6 containing a lectin including N-acetylglucosamine (GlcNAc) in the recognizable sugar chain can achieve high biofilm formation inhibitory effect. From the results, it was shown that the lectin including N-acetylglucosamine (GlcNAc) in the recognizable sugar chain can inhibit formation of biofilm.
Further, the biofilm inhibitor 2 and the biofilm inhibitor 4 can firmly aggregate bacteria and inhibit formation of biofilm. Since the biofilm inhibitor 2 and the biofilm inhibitor 4 contain a lectin including sialic acid (Sia) as well as N-acetylglucosamine (GlcNAc) in the recognizable sugar chain, it was shown that the lectin containing sialic acid (Sia) as well as N-acetylglucosamine (GlcNAc) in the recognizable sugar chain can firmly aggregate bacteria and inhibit formation of biofilm (see
(5) Evaluation of Peeling Effect of Biofilm in Case that Each of Biofilm Inhibitor 1 and Biofilm Inhibitor 2 was Added after Formation of Biofilm
(1-1) Evaluation Method
Staphylococcus aureus was added to a glucose-added 804 medium and cultured until the optical density (opticaldensity: OD) of the medium at a wavelength of 600 nm is 0.1 OD. Thereafter, 500 μl of the culture solution was placed in each well of a 24-well multiwell plate. The culture solution was cultured overnight, to form a biofilm in each well. Then, to each well, each of the biofilm inhibitor 1 of which lectin concentration is 0.5 mg/mL and the biofilm inhibitor 2 of which lectin concentration is 0.5 mg/mL was added.
The degree of biofilm formation was calculated in the same manner as described in (1-1) of (1) above.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
(6) Evaluation of Biofilm Peeling Effect in Case that Each of Biofilm Inhibitor 1, Biofilm Inhibitor 3 and Biofilm Inhibitor 4 was Added after Formation of Bio Film
(1-1) Evaluation Method
Evaluation was performed in the same manner as the evaluation method described in (1-1) of (5) above, except that each of the biofilm inhibitor 1 of which lectin concentration is 0.5 mg/mL, the biofilm inhibitor 3 of which lectin concentration is 0.5 mg/mL and the bio film inhibitor 4 of which lectin concentration is 0.5 mg/mL is used.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
Table 1 summarizes results of the above.
The biofilm inhibitor 1, the biofilm inhibitor 3 and the biofilm inhibitor 5 can produce high peeling effect on a biofilm formed. Since the biofilm inhibitor 1 and the biofilm inhibitor 5 contain a lectin including L-fucose-D-galactose-N-acetylglucosamine (Fucα1-2Galβ1-4GlcNAc) in the recognizable sugar chain, it was suggested that the lectin including L-fucose-D-galactose-N-acetylglucosamine (Fucα1-2Galβ1-4GlcNAc) in the recognizable sugar chain can peel biofilm. Further, since the biofilm inhibitor 3 contains a lectin that recognizes N-acetylglucosamine (linked (β1-4GlcNAc)n), it was suggested that the lectin that recognizes N-acetylglucosamine (linked (β1-4GlcNAc)n) has ability to peel biofilm (see
(7) Evaluation of Biofilm Formation Inhibitory Effect in Case that Each of Biofilm Inhibitor 1 and Biofilm Inhibitor 3 was Added to Biofilm Formed by Candida albicans Before Formation of Biofilm
(1-1) Evaluation Method
Evaluation was performed in the same manner as described in (1-1) of (1) above, except that candida is used as a bacteria to form a biofilm, and each of the biofilm inhibitor 1 of which lectin concentration is 0.5 mg/mL and the biofilm inhibitor 3 of which lectin concentration is 0.5 mg/mL is used as a biofilm inhibitor.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
(8) Evaluation of Biofilm Formation Inhibitory Effect in Case that Each of Biofilm Inhibitor 3, Biofilm Inhibitor 4, and Biofilm Inhibitor 6 was Added to Biofilm Formed by Pseudomonas aeruginosa Before Formation of Biofilm
(1-1) Evaluation Method
Evaluation was performed in the same manner as described in (1-1) of (1) above, except that Pseudomonas aeruginosa is used as a bacteria to form a biofilm and each of the biofilm inhibitor 3, the biofilm inhibitor 4, and the biofilm inhibitor 6, of which lectin concentration is 0.5 mg/mL, is used for a biofilm inhibitor.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
(9) Evaluation of Biofilm Formation Inhibitory Effect in Case that Biofilm Inhibitor 1 or Biofilm Inhibitor 4 was Added to Biofilm Formed by Escherichia coli Before Formation of Biofilm
(1-1) Evaluation Method
Evaluation was performed in the same manner as described in (1-1) of (1) above, except that E. coli is used as a bacteria to form a biofilm and each of the biofilm inhibitor 1 and the biofilm inhibitor 4, of which lectin concentration is 0.5 mg/mL, is used as a biofilm inhibitor.
(1-2) Results
[Results for Staining Each Well after Step (c) in
As shown in
As shown in
The present invention is not limited to the embodiments described above and can be practiced in various modes within a scope not departing from the present invention.
Number | Date | Country | Kind |
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2012-055939 | Mar 2012 | JP | national |
Filing Document | Filing Date | Country | Kind |
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PCT/JP2013/056652 | 3/11/2013 | WO | 00 |