Biofilm remover

Abstract

To elucidate the action of CI on an already formed biofilm, since it is predictable to a person skilled in the art that CI can inhibit formation of a biofilm through inhibition of GTF activity, whereas it is completely unpredictable to the person skilled in the art what kind of action CI exerts on the biofilm already formed.SOLUTION: A biofilm remover contains one or more selected from cyclodextran and derivatives thereof.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to a biofilm removing agent. [Background technology]

[0002] Streptococcus mutans is , a gram-positive facultative anaerobic bacterium, is a caries-causing bacterium present in the human oral cavity, and its The layer contains glucosyltransferase (GT) F), high molecular weight protein antigen c, glucan-binding protein There are many proteins involved in pathogenicity, such as Gbp proteins. The function of protein leads to the formation of a strong biofilm, which causes dental caries.

[0003] One of the methods for preventing dental caries is the use of sugar substitutes, one of which is cyclic isomaltulose. The oligosaccharide cyclodextran (Cycloisomaltoligosacc) CI) is a cyclic structure consisting of 7 to 12 glucose units linked together by a-1,6 bonds. As an example of CI, seven glucose units are connected in a ring with a-1,6 bonds. The resulting structure is shown in Figure 1.

[0004] Previously, the applicants have reported that CI inhibits the activity of GTF in the presence of sucrose. It has been revealed that this suppresses the occurrence of dental caries (Patent Document 1, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5770845 [Non-patent literature]

[0006] [Non-Patent Document 1] FUNANE Kazumi, et al., Finding of cyclodextrans and attempts of their industrialization for cariostatic oligosaccharides, J. Appl. Glycosci., 54, 103-107 (2007). Summary of the Invention [Problem to be solved by the invention]

[0007] However, CI can inhibit biofilm formation by inhibiting the activity of GTF. Although it can be expected by those skilled in the art that the biofilms already formed are Since it is completely impossible for a person skilled in the art to predict what kind of action will be exhibited by the compound, it is This was the challenge. [Means for solving the problem]

[0008] As a result of intensive research by the present inventors to solve the above problems, it was found that CI was already formed. The present invention was completed based on the discovery that even biofilms that have been grown on the surface of the substrate can be removed.

[0009] In addition, the removal of the biofilm was achieved by inhibiting the activity of GTFs in CI, which has been known to date. Furthermore, we have newly discovered that the inhibition of the activity of glucan-binding proteins is also involved, and have developed the present invention. I finished it.

[0010] That is, the present invention provides a method for producing a cyclodextran-based polymer comprising: The biofilm remover is characterized by containing

[0011] The present invention also provides a composition containing one or more selected from cyclodextran and its derivatives. The glucan-binding protein activity inhibitor is characterized by: [Effects of the Invention]

[0012] The biofilm removing agent of the present invention can remove biofilms that have already formed. This can be done.

[0013] Furthermore, the glucan-binding protein activity inhibitor of the present invention inhibits the degradation of glucan-binding proteins. etc. can be done. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the structural formula of cyclodextran, in which seven glucose units are linked in a ring via α-1,6 bonds. [Figure 2] Figure 2 shows the effect of CI on the amount of biofilm formed by Streptococcus mutans strain 8148. [Figure 3] Figure 3 shows the effect of CI on the biofilm structure of Streptococcus mutans strain 8148. [Figure 4] Figure 4 shows the effect of CI on the density of Streptococcus mutans strain 8148 biofilm. [Figure 5] Figure 5 shows the effect of CI on the intercellular binding strength of Streptococcus mutans strain 8148 biofilm. [Figure 6] FIG. 6 shows the effect of CI on the amount of biofilm formed by Streptococcus mutans strain 8148. [Figure 7] Figure 7 shows the effect of CI on the structure of biofilms formed by Streptococcus mutans strain 8148. [Figure 8]Figure 8 shows the effect of CI on the density of biofilms formed by Streptococcus mutans strain 8148. [Figure 9] FIG. 1 illustrates how CI affects biofilms. DETAILED DESCRIPTION OF THE INVENTION

[0015] The biofilm removing agent of the present invention is selected from cyclodextran and its derivatives. It contains one or more of the following:

[0016] The above-mentioned cyclodextran may be, for example, a dextran having 4 to 33 glucose units in the α-1,6 glucose group. It is a known cyclic isomaltooligosaccharide in which cyclic bonds are bonded together. For example, The culture medium and circular It is possible to use a product obtained from the reaction mixture of isomaltose synthase or a commercially available product. In addition, when cyclodextran is obtained using microorganisms or enzymes, unbranched saccharin is usually obtained. Microdextran (normal cyclodextran) and branched cyclodextran It is obtained as a mixture of unbranched cyclodextran and branched cyclodextran. Xanthran can be separated using an ODS column or the like (see JP 2012-14052 (See Publication No. 1).

[0017] Furthermore, the derivatives of cyclodextran include the above-mentioned cyclodextran mixture. Branched cyclodextran obtained by separating it using an ODS column or other column, and cyclodextran Among the known derivatives of dextrin, cyclodextran and branched cyclodextran are Examples include those substituted with dextran.

[0018] Among the derivatives of cyclodextran, one to several glucose units are linked by α-1,3-bonds. α-1,3-branched cyclodextran conjugated to cyclodextran is preferred. In branched cyclodextran, the degree of polymerization of branched glucose is up to 9, In particular, one glucose molecule is branched to cyclodextran via an α-1,3-bond. α-1,3-branched cyclodextran is preferably used. The number of glucose molecules constituting the cyclic portion of the to-oligosaccharide is 4 to 33, preferably 4 to 33. 17, preferably 4 to 12, and the preferred cyclic branched isomaltooligosaccharides The total number of glucose molecules in the molecule is 5 to 20, preferably 5 to 13.

[0019] Preferred examples of the above-mentioned cyclodextran and its derivatives are as follows: Examples include: (a) Cyclodextran (b) α-1,3-linked cyclodextran containing one to several glucose molecules -1,3-branched cyclodextran (c) Unbranched cyclodextran and one to several glucose molecules α-1,3- A mixture of α-1,3-branched cyclodextrans bound to cyclodextran via bonds. (d) Cyclodes consisting of 7 to 12 glucose units linked together in a ring by α-1,6 glycosidic bonds Containing cystran

[0020] Among the above, (b) is a cyclodextrin in which one or several glucose units are linked by an α-1,3-bond. (d) α-1,3-branched cyclodextran or (e) 7-12 glycosaminoglycans Preferably, the material contains cyclodextran cyclically linked by α-1,6 glycosidic bonds. It is a cyclode consisting of 7 to 12 glucose units linked together in a ring by α-1,6 glycosidic bonds. The (d) is, for example, commercially available from Nisshin Sugar Co., Ltd. I-Dextran mix (7-12 glucose units in a ring with α-1,6 glycosidic bonds) It is commercially available under the trade name of "Cyclodextran containing 13% or more by mass of cyclodextran linked to the polymer." You can use this in.

[0021] The biofilm removing agent of the present invention is made of cyclodextran and its derivatives. The content of the selected one or more species is not particularly limited. For example, 7 to 12 glucose units are α -1.6 Cyclodextran cyclically linked by glycosidic bonds, 10 to 25% by mass (hereinafter The content of the material containing the component (hereinafter simply referred to as "%) is 0.01% to 50%, preferably 0 It is preferably 0.01 to 10%, and more preferably 0.25 to 3%.

[0022] The biofilm removing agent of the present invention has the effect of cyclodextran and its derivatives. Furthermore, sugars that can be assimilated by the cariogenic bacteria described below can be contained. Examples of such sugars include fructose, glucose, and sucrose. One or more of these sugars can be used.

[0023] Furthermore, the biofilm removing agent of the present invention contains cyclodextran and its derivatives. As long as the effect is not impaired, for example, polyphenols such as hop extracts, oral Used in care foods, confectionery, general foods, beverages, pet foods, oral care products, detergents, etc. It is possible to contain ingredients that

[0024] The form of the biofilm removing agent of the present invention is not particularly limited, and may be, for example, a gum, a tablet, or the like. Oral care foods such as candy, gummy candy, jelly, chocolate, ice cream, biscuits, etc. Sweets such as pound cake, bread, udon, soba, yogurt, beverages, seafood paste products, etc. Oral care products such as general foods, toothpaste, mouthwash, gargle, denture cleaner, denture adhesive, etc. These include household items, pet food, bath and toilet detergents, etc. Oral care foods and oral care products are particularly preferred. except that cyclodextran and its derivatives are used in place of part or all of the sugars. In the above embodiment, the composition can be produced by a conventionally known production method.

[0025] The biofilm removing agent of the present invention described above can remove biofilms. When removing a biofilm using the biofilm removing agent of the present invention, for example, For oral care foods, sweets, general foods, and pet foods, take them 1 to 3 times a day. Oral care products can be used appropriately, for example, before and after eating and drinking, before and after going to bed, etc. Furthermore, in the case of a detergent, it may be used as needed, for example, before cleaning.

[0026] Furthermore, there are no particular limitations on the type of biofilm that can be removed by the biofilm removing agent of the present invention. However, for example, Streptococcus mutans and Porphyromonas gingivalis (P orphyromonas gingivalis), Prevotella intermedia ( Prevotella intermedia), Aggregatibacter actinomycete Aggregatibacter actinomycetemcoi tans), Streptococcus sobrinus Biofilms formed on the tooth surface by cariogenic bacteria such as Rhodotorula inus, (Rhodotorula), Methylobacterium (Methylobacteriu) m), which is formed in bathtubs, etc. by bacteria belonging to the Legionella genus, etc. Biofilm, Pseudomonas aeruginosa, Escherichia coli ( Escherichia coli, Staphylococcus aureus aureus, Staphylococcus epidermidis Among these biofilms, biofilms formed by bacteria such as cephalosporins and cephalosporins are also important. Biofilms that are already formed on the surface of teeth are preferred, and biofilms that are formed on the surface of teeth are preferred. It is more preferable, especially for the bacteria formed on the surface of teeth by Streptococcus mutans. Ophiophyllum is preferred.

[0027] The biofilm removing agent of the present invention has a GTF function that has been known to be cyclodextran. In addition to inhibiting the activity of glucan-binding proteins, Therefore, the biofilm removing agent of the present invention is a biofilm removing agent containing a glucan-binding protein. It is possible to remove a wide range of biofilms that have the desired properties. Inhibiting the activity of the protein means breaking down the glucan-binding protein, This includes weakening the activity of glucan-binding proteins through its action.

[0028] Therefore, one or more selected from cyclodextran and its derivatives are By having it, it becomes an inhibitor of the activity of glucan-binding protein.

[0029] Glucan-binding proteins are sugar-binding proteins that are components of cell walls, etc. The glucan-binding protein activity inhibitor of the present invention is useful for the development of useful substances such as antibiotics ( (breaking down the cell walls of bacteria and fungi to extract useful substances, etc.), food processing (softening fibers etc.), bioenergy production (producing sugar from biomass, etc.), soil environmental conservation (soil In addition, glucan-binding proteins can be used for various purposes, such as decomposing organic matter in soil. Since glucan-binding protein activity inhibitors of the present invention are related to various diseases, they are useful for treating inflammatory diseases. treatment of inflammatory diseases (because glucan-binding proteins are involved in the development of inflammatory diseases), cancer treatment (glucan Glucan-binding proteins are involved in the growth and metastasis of cancer cells), and in the treatment of infectious diseases (glucan Glucan-binding proteins are present in the cell walls of bacteria and fungi), diabetes treatment (glucan-binding proteins) Proteins can also be used to treat conditions such as diabetes (as proteins can interfere with the action of insulin).

[0030] The inhibitor of glucan-binding protein activity of the present invention is cyclodextran and its derivatives. The same as the biofilm remover as described above, except that the active ingredient is one or more selected from the group consisting of conductors. The same composition, form, manufacturing method, or composition, form as those of known techniques used for the above-mentioned applications. , and manufacturing method. [Example]

[0031] The present invention will be described in detail below with reference to examples of the present invention. However, it is not limited to the above.

[0032] Example 1 Biofilm removal test: (1) Effect on biofilm formation Todd Hewitt liquid medium (Becton Dickinson and C Company, Franklin Lakes, NJ, USA: hereinafter referred to as "TH medium" Streptococcus mutans MT8148 strain (a Japanese small After inoculation with the strain isolated from the infant (standard strain), the bacteria were cultured at 37°C for 18 hours to prepare the bacterial solution. Chemically defined medium containing 1% sucrose was prepared. edium (CDM); 58 mM K2HPO4; Nacalai Tesque, Kyoto, 15 mM KH 2PO4,10mM(NH4)2SO4,35mM NaCl,0.2%Casamino acids,100μM MnCl2·4H2O(pH7.4),20mM Nicoti nic acid,50mM Pyridoxine HCl,5mM Pantoth enic acid,0.5mM Riboflavin,0.15mM Thiami n HCl, 0.015mM D-biotin, 50mM L-glutamic a cid,12.5mM L-arginine HCl,16.25mM L-cyst eine HCl, 1.25mM L-tryptophan, 1M MgSO4·7H2 100 μl of the bacterial solution was inoculated into a tube containing 0.01% ethanol (Wako Pure Chemical Industries, Ltd., Osaka), and the tube was divided into 0.01% ethanol (Wako Pure Chemical Industries, Ltd., Osaka), and 0.1% ethanol (Wako Pure Chemical Industries, Ltd., Osaka) was inoculated into a tube containing 0.01% ethanol (Wako Pure Chemical Industries, Ltd., Osaka), and the tube was divided into 0.01% ethanol ( Cyclodextran (CI-Dextran mix, manufactured by Nisshin Sugar Co., Ltd.) was used as the CI at 0%. Cyclodextrins are cyclic molecules consisting of 7 to 12 glucose units linked together by α-1,6 glycosidic bonds. These test solutions were added to each of the plates. 100 μl of the mixture was dispensed into wells and cultured at 37°C for 48 hours under anaerobic conditions. The test solution was stained with Leviolet red and the OD 570The absorbance was measured, and the results are shown in Figure 2. Ta.

[0033] The amount of biofilm formed decreased depending on the CI concentration, and was higher than that without CI. When 10% was added, the amount of sucrose was reduced by approximately 70%. This suggests that the presence of CI does not affect the inhibitory effect of CI on bifoil formation.

[0034] (2) Effect on biofilm structure After preparing a bacterial solution of Streptococcus mutans strain 8148 in the same manner as in (1), The cells were separated by centrifugation at 3,000 × g for 15 minutes. The stained cells were then stained with 1% sucrose-containing chemical solution. OD in adult medium 570 The absorbance of the mixture was adjusted to 0.1 and inoculated. In addition, 0 to 10% cyclodextran (CI-Dextran manufactured by Nisshin Sugar Co., Ltd.) was added as CI. The test solution was prepared by adding the mixture of the test solution and the chamber slide well. 200 μl of the solution was dispensed into each well and cultured at 37°C for 24 hours under anaerobic conditions. Point scanning laser microscope LSM780 (Version 4.2, Carl Zeiss) MicroImaging Co., Oberkochen, Germany The structure and cross section (thickness) of the ion film were observed. The results are shown in Figure 3. The biofilm density was also determined from the observations, and the results are shown in Figure 4.

[0035] The biofilm structure showed a 28% decrease in density with increasing CI concentration. The thickness was reduced by 55%. The thickness decreased by 55% when the CI concentration was increased. When comparing the cases where CI is not added and where it is added at 10%, A decrease in density of approximately 65% ​​was observed.

[0036] (3) Effect on biofilm disruption Cyclodextran (Nisshin Sugar Co., Ltd.: CI-Dextran mix) was used as the CI. Streptococcus aureus was grown in TH medium containing 1% sucrose, with the final concentration of 0-1%. These were inoculated into a 6-well cell culture microtest plate. 10 ml of the mixture was dispensed into each well and cultured at 37°C for 24 hours under anaerobic conditions. The bacterial solution in the well was subjected to ultrasound to disrupt the biofilm. After washing with saline (PBS), the remaining biofilm was removed with a cell scraper. The mixture was serially diluted with saline and plated on Trypticase Soy Agar (Bect on Dickinson and company) and incubated at 37°C for 48 hours. The number of colonies was counted before and after ultrasonic treatment, and the remaining bacteria were calculated using the following formula: The percentage of the number of cases was calculated, and the results are shown in Figure 5.

[0037]

number

[0038] As the concentration of CI increased, the number of bacteria in the biofilm decreased. When comparing the case where the amount of added 1% with the case where the amount of added 1% was 100, the number of bacteria in the biofilm was approximately It was a 65% decrease.

[0039] Example 2 Biofilm removal test: (1) Effect on the amount of biofilm formed After inoculating TH liquid medium with Streptococcus mutans MT8148, The bacteria were cultured at ℃ for 18 hours under anaerobic conditions to prepare a bacterial solution. 100 μl of the bacterial solution was inoculated and dispensed into each well of the plate in 100 μl portions. The cells were cultured at ℃ for 48 hours under anaerobism to form a biofilm. Cyclodextran (CI-Dextran, manufactured by Nisshin Sugar Co., Ltd.) was used as CI at a concentration of 0-1%. After adding the α-amyloid β-amyloid mix, the cells were cultured at 37°C for 3, 6, or 12 hours under anaerobic conditions. The test solution was stained with crystal violet and the OD 570 The absorbance of the sample was measured. Shown in 6.

[0040] The effect of CI on already formed biofilms was observed. The amount of CI formation decreased in a concentration-dependent manner compared to when CI was not added. When added, the balance was about 40% after 3 hours, about 30% after 6 hours, and about 40% after 12 hours. A decrease in the amount of iofilm formation was observed.

[0041] (2) Effects on the structure of the formed biofilm After preparing a bacterial solution of Streptococcus mutans strain 8148 in the same manner as in (1), The cells were separated by centrifugation at 3,000 × g for 15 minutes. The stained cells were then stained with 1% sucrose-containing chemical solution. OD in adult medium 570 The absorbance of the solution was adjusted to 0.1 and the solution was inoculated. 200 μl of the mixture was dispensed into each well of the plate and cultured at 37°C for 24 hours under anaerobic conditions. Biofilms were formed. Each well was treated with cyclodextran at 0-1% CI. After adding Dextran (Nisshin Sugar Co., Ltd.: CI-Dextran mix), the mixture was incubated at 37°C for 24 hours. After the cultivation, the structure and cross section (thickness) of the biofilm were examined using a confocal scanning laser microscope. The results are shown in Figure 7. The density of the biofilm was also determined from this observation. The results are shown in Figure 8.

[0042] The structure of the biofilm after 3 hours of CI reaction showed a decrease in thickness and a decrease in density. Furthermore, when comparing the case where CI was not added with the case where CI was added at 10%, the thickness was about It was reduced by 50% and its density by about 55%.

[0043] (3) Examination of the effect of CI on the formed biofilm Based on the results of Examples 1 and 2, we investigated how CI affects the formed biofilm. The reason for this is as shown in Figure 9. In other words, CI is an enzyme that produces insoluble glucan. It inhibits the activity of GTF, which is involved in the formation of glutamic acid, and also inhibits the activity of glucan-binding proteins, thereby It weakens the bonds of the biofilm and destroys it.

[0044] Example 3 Biofilm remover (toothpaste): The following components were mixed to prepare a biofilm remover. CI * 0.2% Propylene glycol 3.0% Sodium alginate 0.6% Xanthan gum 0.7% Sorbitol solution (70%) 45.0% Silica anhydride 20.0% Titanium dioxide 0.4% Sodium fluoride 0.21% Sodium saccharin 0.15% Sodium lauryl sulfate 0.8% Fragrance (appropriate amount) water remainder *: Cyclodextran (Nisshin Sugar Co., Ltd.: CI-Dextran mix)

[0045] Example 4 Biofilm remover (mouthwash): The following components were mixed to prepare a biofilm remover. CI * 3.0% Benzalkonium chloride 0.2% water remainder *: Cyclodextran (Nisshin Sugar Co., Ltd.: CI-Dextran mix)

[0046] Example 5 Biofilm remover (drinkable): The following components were mixed to prepare a biofilm remover. CI * 0.12% Vitamin Mix 0.25% Citric acid 0.125% Sodium L-ascorbate 0.05% Lemon juice 0.50% water remainder *: Cyclodextran (Nisshin Sugar Co., Ltd.: CI-Dextran mix)

[0047] Example 6 Biofilm remover (tablets): The following components were mixed to prepare a biofilm remover. CI * 2.00% Sorbitol 92.05% Peppermint flavor 4.95% Sucrose fatty acid ester 1.00% *: Cyclodextran (Nisshin Sugar Co., Ltd.: CI-Dextran mix)

[0048] Example 7 Glucan-binding protein activity inhibitors: Inhibition of glucan-binding protein activity in the same manner as in the biofilm removal agents of Examples 3 to 6 The agent was manufactured. [Industrial Applicability]

[0049] The biofilm removing agent of the present invention can be used to remove biofilms.

[0050] Furthermore, the glucan-binding protein activity inhibitor of the present invention inhibits the breakdown of glucan-binding proteins. It can be used for solutions etc.

Claims

1. It is characterized by containing one or more selected from cyclodextran and its derivatives. A biofilm remover.

2. The biofilm removal method according to claim 1, wherein the biofilm is formed on the surface of a tooth. Agent.

3. The bacon according to claim 1 or 2, further comprising sugars that can be assimilated by cariogenic bacteria. Iofilm remover.

4. It is characterized by containing one or more selected from cyclodextran and its derivatives. Inhibitor of glucan-binding protein activity.

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