Composition for inhibiting adhesion of escherichia coli and use thereof

Abstract

To provide an alternative technique capable of inhibiting infection caused by Escherichia coli.SOLUTION: A composition for inhibiting adhesion of Escherichia coli contains a branched saturated fatty acid having 14 or more and 17 or less carbon atoms and / or a salt thereof.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present disclosure relates to a composition for inhibiting adhesion of Escherichia coli. [Background technology]

[0002] It has been known that pathogenic bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli form biofilms. For example, Non-Patent Document 1 discloses a compound that acts on pathogenic bacteria to inhibit the formation of a biofilm. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] J Bacteriol. 2009 Mar; 191(5): 1393-1403. Summary of the Invention [Problem to be solved by the invention]

[0004] Infection by pathogenic E. coli such as E. coli O157 is caused by the attachment of pathogenic E. coli to various items such as food ingredients, cooking utensils, medical instruments, and daily necessities. However, the compound described in Non-Patent Document 1 is effective against normal bacteria other than E. coli, and as a result, there is a risk of destroying the bacterial flora. For this reason, there has been a demand for other technologies capable of suppressing infection by E. coli. [Means for solving the problem]

[0005] The present invention can be realized in the following forms.

[0006] (1) According to one aspect of the present invention, there is provided a composition for inhibiting adhesion of E. coli. This composition for inhibiting adhesion of E. coli contains a branched-chain saturated fatty acid having 14 to 17 carbon atoms and / or a salt thereof. This aspect of the composition for inhibiting adhesion of E. coli can specifically inhibit adhesion of E. coli.

[0007] (2) In the composition for inhibiting adhesion of E. coli described in (1) above, the branched-chain saturated fatty acid may have a carbon number of 15. According to this form of the composition for inhibiting adhesion of E. coli, adhesion of E. coli can be specifically inhibited.

[0008] (3) In the composition for inhibiting adhesion of E. coli described in (1) above, the branched-chain saturated fatty acid may be at least one selected from the group consisting of the following chemical formulas (1) to (6). The composition for inhibiting adhesion of E. coli in this form can specifically inhibit adhesion of E. coli. [ka] [ka] [ka] [ka] [ka] [ka]

[0009] (4) According to another aspect of the present disclosure, there is provided an agent for inhibiting adhesion of E. coli, comprising as an active ingredient the composition for inhibiting adhesion of E. coli according to any one of (1) to (3) above. The agent for inhibiting adhesion of E. coli in this aspect can specifically inhibit adhesion of E. coli.

[0010] (5) According to another aspect of the present disclosure, there is provided an article to which the composition for inhibiting adhesion of E. coli according to any one of (1) to (3) above is added or applied. The article of this aspect can specifically inhibit adhesion of E. coli.

[0011] (6) According to another aspect of the present disclosure, there is provided a method for inhibiting adhesion of E. coli, the method comprising the step of adding or applying a branched-chain saturated fatty acid having 14 to 17 carbon atoms or a salt thereof to an article. According to this aspect of the method for inhibiting adhesion of E. coli, adhesion of E. coli can be specifically inhibited.

[0012] (7) According to another aspect of the present disclosure, there is provided a method for producing a composition for inhibiting adhesion of E. coli, the method comprising a step of collecting a component secreted from Bacillus bacteria designated by accession number NITE P-03755. According to this aspect of the method for producing a composition for inhibiting adhesion of E. coli, a composition capable of specifically inhibiting adhesion of E. coli can be produced.

[0013] The present invention can be realized in various forms, for example, as an inhibitor of biofilm formation of Escherichia coli. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is an explanatory diagram showing the results of co-culturing the NB4 strain and E. coli. [Diagram 2] FIG. 1 is an explanatory diagram showing the results of co-culturing the NB4 strain with other test bacteria. [Diagram 3] FIG. 1 is an explanatory diagram showing changes in colony morphology caused by a culture extract of the NB4 strain. [Figure 4] FIG. 1 is an explanatory diagram showing the evaluation results of the adhesion ability of E. coli. [Diagram 5] FIG. 1 is an explanatory diagram showing the evaluation results of the cell surface hydrophobicity of E. coli. [Figure 6] FIG. 1 is an explanatory diagram showing the results of GCMS analysis of active fractions. [Figure 7] FIG. 1 is an explanatory diagram showing the difference in colony morphology change depending on the presence or absence of a branched structure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] As shown in the examples described below, the inventors of the present application co-cultured Bacillus sp. and Escherichia coli (E. coli) with the accession number NITE P-03755 (contracted institution: National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depository Center, date of receipt: September 20, 2022). They then revealed that compounds secreted by Bacillus sp. change the colony morphology of E. coli and weaken adhesion to the surface of a material, and completed the present invention based on this finding.

[0016] According to one embodiment of the present disclosure, there is provided a composition for inhibiting adhesion of E. coli, the composition for inhibiting adhesion of E. coli containing a branched-chain saturated fatty acid having 14 to 17 carbon atoms and / or a salt thereof.

[0017] The branched chain saturated fatty acid having 14 to 17 carbon atoms is not particularly limited, but from the viewpoint of enhancing the adhesion inhibitory effect of E. coli, the number of carbon atoms is preferably 15 or 17, and the number of carbon atoms is more preferably 15. The branched position in the branched chain saturated fatty acid is not particularly limited, and may be branched at any of the carbons 2 to 15 counted from the carboxy group side, but is preferably branched at the terminal side counted from the carboxy group side. The branched position in the branched chain saturated fatty acid is preferably iso type (iso) or anteiso type (anteiso), and more preferably anteiso type, from the viewpoint of enhancing the adhesion inhibitory effect of E. coli. The length of the branched chain in the branched chain saturated fatty acid is not particularly limited, but from the viewpoint of enhancing the adhesion inhibitory effect of E. coli, the branched chain is preferably 4 or less carbon atoms, more preferably 3 or less carbon atoms, even more preferably 2 or less carbon atoms, and particularly preferably 1 carbon atom branched chain.

[0018] From the viewpoint of further enhancing the effect of suppressing adhesion of Escherichia coli, the branched-chain saturated fatty acid is preferably at least one selected from the group consisting of the following chemical formulas (1) to (6) or a salt thereof. The compound represented by the following chemical formula (1) is anteiso-C 15:0The compound represented by the following chemical formula (2) is iso-C 15:0 The compound represented by the following chemical formula (3) is anteiso-C 17:0 The compound represented by the following chemical formula (4) is iso-C 17:0 The compound represented by the following chemical formula (5) is iso-C 16:0 The compound represented by the following chemical formula (6) is iso-C 14:0 It is.

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[0025] The salt of the branched-chain saturated fatty acid having 14 to 17 carbon atoms is not particularly limited, but examples thereof include sodium salt, potassium salt, calcium salt, magnesium salt, ethanolamine salt, triethanolamine salt, and ammonium salt.

[0026] The composition for inhibiting adhesion of E. coli according to the present disclosure may contain two or more types of branched-chain saturated fatty acids and / or salts thereof having 14 to 17 carbon atoms. In other words, the composition for inhibiting adhesion of E. coli according to the present disclosure contains at least one type of branched-chain saturated fatty acid and / or salt thereof having 14 to 17 carbon atoms. The branched-chain saturated fatty acid and / or salt thereof having 14 to 17 carbon atoms may be produced by chemical synthesis.

[0027] The composition for inhibiting adhesion of E. coli according to the present disclosure can specifically inhibit adhesion of E. coli to the surface of an article. Although the mechanism behind this is unclear, it is presumed that the branched-chain saturated fatty acid having 14 to 17 carbon atoms penetrates from the cell surface of E. coli into the interior of the cell, increasing the hydrophilicity of the cell surface of E. coli, which results in a weakening of the adhesive performance of E. coli.

[0028] According to the composition for suppressing adhesion of E. coli in the present disclosure, the adhesion of E. coli to various articles can be specifically suppressed, and therefore infection caused by pathogenic E. coli can be suppressed. Examples of pathogenic E. coli include, but are not limited to, enterohemorrhagic E. coli (EHEC: enterohemorrhagic E. coli) such as O157, enteropathogenic E. coli (EPEC: enteropathogenic E. coli), enteroinvasive E. coli (EIEC: enteroinvasive E. coli), enterotoxigenic E. coli (ETEC: enterotoxigenic E. coli), enterodiffuse-adherent E. coli (EAEC: enteroroadhesive E. coli), enteroaggregative E. coli (EAggEC: enteroaggrigative E. coli), and extraintestinal pathogenic E. coli. According to the composition for inhibiting adhesion of E. coli of the present disclosure, the adhesive ability of E. coli can be specifically reduced, and therefore, the sterilization of other types of bacteria such as indigenous bacteria other than E. coli can be suppressed, and the destruction of bacterial flora can be suppressed. Furthermore, while the use of general antibacterial agents and bactericides poses problems such as the emergence of resistant bacteria and the destruction of ecosystems due to discharge into the environment, according to the composition for inhibiting adhesion of E. coli of the present disclosure, the decrease in effectiveness caused by the emergence of resistant bacteria can be suppressed, and the burden on the environment can be reduced.

[0029] According to another embodiment of the present disclosure, there is provided an adhesion inhibitor for E. coli, which contains the above-mentioned composition for inhibiting adhesion of E. coli as an active ingredient. The concentration of the above-mentioned branched chain saturated fatty acid in the adhesion inhibitor for E. coli is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and even more preferably 0.1% by mass or more, from the viewpoint of enhancing the cell adhesion inhibitory effect. In addition, the concentration of the above-mentioned branched chain saturated fatty acid in the adhesion inhibitor for E. coli is preferably 30% by mass or less, more preferably 20% by mass or less, and even more preferably 10% by mass or less, from the viewpoint of inhibiting aggregation of components. The form of the adhesion inhibitor for E. coli is not particularly limited, but may be, for example, liquid, gel, paste, powder, granule, etc. The adhesion inhibitor for E. coli may contain any component other than the above-mentioned branched chain saturated fatty acid. The optional component is not particularly limited, but may be, for example, a buffer, a solvent, a stabilizer, a thickener, a preservative, an antioxidant, an emulsifier, a surfactant, an aroma, a colorant, etc. The optional components may be appropriately selected depending on the application or form, and one type may be used alone, or two or more types may be used in combination.

[0030] According to another aspect of the present disclosure, an article is provided to which the above-mentioned composition for inhibiting adhesion of E. coli is added or applied. The article is not particularly limited, but may be, for example, various articles such as food ingredients, cooking utensils, medical instruments, daily necessities, medicines, etc. More specifically, examples of articles to which the composition for inhibiting adhesion of E. coli is added include cleaning agents, toothpaste, mouthwash, gum, etc. Examples of articles to which the composition for inhibiting adhesion of E. coli is applied include catheters, implants, food packaging, kitchenware, etc.

[0031] According to another embodiment of the present disclosure, there is provided a method for inhibiting adhesion of E. coli. This method includes a step of adding or applying a branched-chain saturated fatty acid having 14 to 17 carbon atoms and / or a salt thereof to an article. The specific method of adding to the article is not particularly limited, but may be, for example, performed by mixing in advance with the material constituting the article. In addition, the specific method of applying to the article is not particularly limited, but may be, for example, performed by spraying on the surface of the article or spreading droplets. The method for inhibiting adhesion of E. coli may include, prior to the above step, a step of preparing a branched-chain saturated fatty acid having 14 to 17 carbon atoms and / or a salt thereof.

[0032] According to another embodiment of the present disclosure, there is provided a method for producing a composition for inhibiting adhesion of Escherichia coli. This production method includes a step of collecting a component secreted from a Bacillus bacterium designated by accession number NITE P-03755. The Bacillus bacterium designated by accession number NITE P-03755 may be cultured, for example, at 25°C to 37°C for 2 to 30 days using any medium suitable for the growth of Bacillus bacteria, to cause the secretion of metabolic products. In the step of collecting a component secreted from a Bacillus bacterium, for example, the culture may be centrifuged and precipitated with acetone to recover the supernatant. In addition, the component may be purified as necessary. The purification may be performed, for example, by subjecting the recovered supernatant to HP-20 column chromatography to recover a 100% MeOH elution fraction. EXAMPLES

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0034] (1) Co-culture with NB4 strain As the bacillus bacteria, the bacillus bacteria isolated from human epidermis (Bacillus sp. NB4 strain (hereinafter also referred to as "NB4 strain") designated by the accession number NITE P-03755) was used. The NB4 strain and E. coli JM109 strain bacterial liquids adjusted to a turbidity of 0.1 were dropped onto a YNB agar medium supplemented with 1% by mass of casamino acid and 0.1% by mass of glucose. After that, they were co-cultured at 30°C for 2 days, and the colony morphological changes of E. coli were visually observed. Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, Candida albicans, and Cryptococcus neoformans were also co-cultured with the NB4 strain under the same conditions, and the colony morphological changes were visually observed.

[0035] FIG. 1 is an explanatory diagram showing the results of co-cultivating the NB4 strain and E. coli. FIG. 2 is an explanatory diagram showing the results of co-cultivating the NB4 strain and other test bacteria. A photograph of the agar medium is shown. FIGS. 1 and 2 show photographs of the agar medium after co-cultivation. As shown in FIG. 1, morphological changes were observed in E. coli colonies adjacent to the NB4 strain colonies, and it was shown that the colony edges were elongated. On the other hand, as shown in FIG. 2, no morphological changes were observed in bacteria other than E. coli, even in colonies adjacent to the NB4 strain colonies. From these results, it was found that the substance secreted by the NB4 strain acts specifically on E. coli to induce colony morphological changes.

[0036] (2) Study using culture extract of NB4 strain The NB4 strain, adjusted to a turbidity of 0.1, was inoculated into a YNB agar medium supplemented with 1% by mass of casamino acid and 0.1% by mass of glucose, and cultured at 30°C for 2 days. The culture was extracted by centrifugation (10,000×G, 10 minutes), and the supernatant was filter-sterilized using a 0.22 μm sterilizing filter (Millex-GV, Merck Millipore), and then immersed in a paper disk (Advantec) with a diameter of 8 mm. The paper disk was placed on a YNB agar medium on which a bacterial solution of E. coli JM109 strain had been dropped, and the cultured at 30°C for 2 days, and the colony morphological changes of E. coli were visually observed.

[0037] After the above-mentioned cultivation, the paper disks containing the NB4 strain culture extract were removed, and the colonies were lightly washed with water. The colonies were visually observed before and after washing to evaluate the change in the adhesion ability of E. coli to the agar medium surface. Furthermore, the change in cell surface hydrophobicity of E. coli with changed colony morphology was evaluated using the MATH method. In the MATH method, E. coli colonies were suspended in PBS buffer and the turbidity was measured, and after adding hexadecane and thoroughly stirring, the turbidity of the aqueous layer was measured again. The cell surface hydrophobicity (%) was calculated using the following formula. Cell surface hydrophobicity (%) = [(turbidity of bacterial suspension - turbidity after addition of hexadecane) / turbidity of bacterial suspension] x 100

[0038] Figure 3 is an explanatory diagram showing colony morphological changes caused by the culture extract of the NB4 strain. A photograph of an agar medium is shown in Figure 3. As shown in Figure 3, a morphological change was observed in the E. coli colony adjacent to the paper disk containing the culture extract of the NB4 strain, and it was found that the edge of the colony was elongated.

[0039] Figure 4 is an explanatory diagram showing the evaluation results of the adhesive ability of E. coli. Figure 4 shows photographs of the agar medium before and after washing with water. As shown in Figure 4, colonies that showed morphological changes after lightly washing with water were easily peeled off. This result suggests that E. coli cells whose colony morphology was changed by the culture extract of the NB4 strain had a significantly reduced adhesive ability to solid surfaces (Figure 3).

[0040] Fig. 5 is an explanatory diagram showing the results of evaluating the cell surface hydrophobicity of E. coli. As shown in Fig. 5, the results of evaluating the change in cell surface hydrophobicity of E. coli by the MATH method showed that the cell surface hydrophobicity of E. coli that had undergone colony morphological changes was significantly reduced. It was suggested that E. coli cells whose colony morphology had been changed by the culture extract of the NB4 strain had increased hydrophilicity on the cell surface, which resulted in a weakening of adhesive performance.

[0041] (3) Purification and analysis of metabolites from NB4 strain that induce colony morphological changes in E. coli The NB4 strain was inoculated onto YNB agar medium supplemented with 1% by mass of casamino acids and 0.5% by mass of glucose, and cultured at 30°C for 5 days. The culture was centrifuged (10,000 x G, 10 minutes) to recover the supernatant, which was then further recovered by acetone precipitation. This fraction was subjected to HP-20 column chromatography, and a 100% MeOH elution fraction was recovered. This was then subjected to LH-20 column chromatography, and the colony morphology change activity against E. coli was evaluated by the paper disc method to obtain an active fraction. Regarding the obtained active fraction, 1 1 H NMR analysis was performed. 1 In the H NMR analysis, the sample was dissolved in deuterated methanol and measured at 600 MHz using an ECZ600 (manufactured by JEOL). The NMR results suggested that the substances contained in the active fraction were fatty acids. In addition, the obtained active fraction was subjected to GCMS analysis after methyl esterification. The GCMS analysis was performed using a QP2020NX instrument (manufactured by Shimadzu Corporation) equipped with an Equity-1 column, with the inlet and detector temperatures at 280°C and the column temperature increasing from 175°C to 275°C at a rate of 30°C / min. The fatty acid species were identified by comparing the retention time and mass spectrum with those of fatty acid standards.

[0042] FIG. 6 is an explanatory diagram showing the results of GCMS analysis of the active fraction. As shown in FIG. 6, the main fatty acid contained in the active fraction is anteiso-C. 15:0 and iso-C 15:0 In addition, it was found that iso-C, a branched saturated fatty acid, 14:0 , iso-C 16:0 , iso-C 17:0 , anteiso-C 17:0 It was also found that palmitic acid (C 16:0 ) was also identified.

[0043] (4) Study on branched structures of fatty acids The main fatty acid in the active fraction was anteiso-C. 15:0 and iso-C 15:0 Therefore, the commercially available anteiso-C 15:0 and iso-C 15:0 The colony morphological changes of E. coli were observed using the reagents listed above. More specifically, the commercially available anteiso-C 15:0 and iso-C 15:0 The paper disk was soaked in a concentration of 0.1% by mass, placed on a YNB agar medium on which a bacterial solution of E. coli JM109 strain had been dropped, and cultured at 30°C for 2 days. The morphological changes of the E. coli colonies were visually observed. 15:0 ) and palmitic acid (C 16:0 ) and observed the morphological changes in the E. coli colonies.

[0044] Figure 7 is an explanatory diagram showing the difference in colony morphology depending on whether or not there is a branched structure. In Figure 7, a photograph of the agar medium is shown together with the chemical formulas of the reagents used. As shown in Figure 7, anteiso-C 15:0 and iso-C 15:0 It was observed that the E. coli colony morphological change was significantly induced, with the colony margins elongating. It was also suggested that the adhesive ability of the colonies that showed the morphological change was significantly reduced. On the other hand, pentadecanoic acid (C 15:0 ) and palmitic acid (C 16:0 ) did not induce colony morphological changes in E. coli. These results suggest that the branched structure of the fatty acids is necessary for activity.

[0045] These results suggest that the branched fatty acids secreted by Bacillus sp. NB4 strain change the colony morphology of E. coli, and that the hydrophobicity of the cell surface of E. coli with altered colony morphology is significantly reduced, resulting in a decreased ability to adhere to solid surfaces. [Industrial Applicability]

[0046] Pathogenic E. coli that adheres to the surfaces of medical instruments and food processing instruments form biofilms and exhibit high drug resistance, becoming the starting point of infection. In addition, the overuse of common antibacterial drugs and disinfectants also destroys normal microflora and promotes the emergence of resistant bacteria. The composition disclosed herein can specifically reduce the adhesive ability of E. coli without killing other types of bacteria, leading to the development of new control technologies for pathogenic E. coli infections. The composition disclosed herein is expected to be applied to new medical and food materials (catheters, implants, food packaging, surface treatment of kitchen utensils, etc.) and drugs (cleaning agents, toothpaste, mouthwash, gum, etc.) that inhibit the adhesion of pathogenic E. coli.

[0047] The present invention is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present invention. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the Summary of the Invention column can be appropriately replaced or combined in order to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

Claims

1. Contains a branched-chain saturated fatty acid having 14 to 17 carbon atoms and / or a salt thereof, A composition for inhibiting adhesion of Escherichia coli.

2. The composition for inhibiting adhesion of Escherichia coli according to claim 1, The branched chain saturated fatty acid has 15 carbon atoms. A composition for inhibiting adhesion of Escherichia coli.

3. The composition for inhibiting adhesion of Escherichia coli according to claim 1, The branched chain saturated fatty acid is at least one selected from the group consisting of the following chemical formulas (1) to (6): A composition for inhibiting adhesion of Escherichia coli. 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】

4. The composition for inhibiting adhesion of Escherichia coli according to claim 1 or 2 is contained as an active ingredient. An adhesion inhibitor for E. coli.

5. A substrate to which the composition for inhibiting adhesion of E. coli according to claim 1 or 2 has been added or applied. Goods.

6. The method includes adding or applying a branched-chain saturated fatty acid having 14 to 17 carbon atoms and / or a salt thereof to an article. Method for inhibiting adhesion of E. coli.

7. The method includes a step of collecting a component secreted from a bacillus bacterium having the accession number NITE P-03755, A method for producing a composition for inhibiting adhesion of Escherichia coli.

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