JP2013075927A - Biofilm formation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new biofilm formation inhibitor which can inhibit the formation of a biofilm.SOLUTION: The biofilm formation inhibitor comprises lactoferrin as an active ingredient.

Description

  The present invention relates to a biofilm formation inhibitor for suppressing the formation of a biofilm.

  Biofilm is a film that wraps organisms, as its name suggests, and is also called a biofilm or slime. The component consists of the exopolysaccharide which bacteria produce | generate around a microbial cell (nonpatent literature 1). The mucus layer formed by the biofilm exists around the cell wall so as to surround the fungus, and plays an important role in the bacterial growth process. That is, (1) Adhering to a place to be a growth scaffold, (2) Adhering so that the proliferated bacterial cells are not separated from each other, (3) Covering the formed bacterial mass and preventing attacks from the external environment It is. For this reason, even when the bacterium is exposed to an infection defense mechanism or a drug in the living body, it is considered that the mucus layer increases adhesion and prevents the attack by firmly covering the microbial cell.

  The bacteria that form biofilms are widespread and occur in various places where bacteria exist. For example, the slime that can be formed in the triangular corner of the sink and the moss attached to the wall of the water pipe or the water tank are biofilms. As for what occurs in the human body, dental plaque (plaque) in the oral cavity is a biofilm. In addition, biofilms may be formed on medical materials such as dentures, contact lenses, and catheters.

  Biofilms are not preferred in many cases because of the propagation of bacteria, and it is desirable that they be completely removed. For example, if a biofilm propagates in a home kitchen or a food factory piping, there is a risk of contamination due to bacterial contamination of the food. Moreover, dental plaque becomes a cause of periodontal disease when it gets worse. The generation of biofilms on contact lenses and catheters is a high risk because they are likely to suffer from diseases such as infections because they directly contact the human body.

The following reports are known regarding the suppression of biofilm formation.
For example, an antibacterial composition in which hygromycin and / or epihygromycin having a biofilm formation inhibitory action and a destructive action is blended with an antibacterial agent such as pyridonecarboxylic acid is disclosed (Patent Document 1). Moreover, the method of suppressing or removing the biofilm using proteases is disclosed (patent document 2). In addition, a biofilm control agent composition containing one or more compounds selected from thiols having a specific structure, carboxylic acid esters, and amines and specific enzymes is disclosed (Patent Document 3).

The following reports are known as focusing on the suppression of the formation of biofilms generated in the oral cavity.
For example, a composition for inhibiting biofilm formation (Patent Document 4) containing a sugar alcohol and / or an amino acid is disclosed. The sugar alcohol content is particularly preferably 10 to 50% by mass, and the amino acid content is 0. It is described that .1 to 2% by mass is particularly preferable. Moreover, dentifrice is mentioned as an Example.

  Moreover, the water-containing oral gel composition (patent document 5) containing orthophosphoric acid, pyrophosphoric acid, sorbitol, and a calcium salt is disclosed, and about 0.25-3 mass% as A: orthophosphoric acid about an active ingredient. Give orthophosphoric acid or a salt thereof, B: give 0.3 to 2% by mass as pyrophosphoric acid, give pyrophosphoric acid or a salt thereof, C: 20 to 60% by mass sorbitol, D: 0.15 to 1% by mass as calcium It is characterized by immediately neutralizing the acid produced in dental plaque. Examples include dentifrices and coating agents.

  Furthermore, a biofilm inhibitor containing epigallocatechin-3-gallate as an active ingredient is disclosed (Patent Document 6). It is described that this inhibitor is preferably used in an effective amount of at least 0.4 mM with respect to the inhibitory effect of the biofilm formed by the oral bacteria Eikenella corrodens. Examples also include dental drugs.

JP-A-11-246419 JP-A-6-262165 JP2008-137717 JP 2005-29484 A JP 2006-182667 A JP2008-13458

New Ophthalmology, Medical Sakai Publishing, Volume 17 (Special Issue), 2000, p. 15-17

  Thus, a means for suppressing the formation of a biofilm has been demanded. Accordingly, an object of the present invention is to provide a novel biofilm formation inhibitor.

  Furthermore, as described above, as a method for suppressing the formation of a biofilm, a method of physically scraping and removing, and a method of killing bacteria by applying or spraying a strong bactericidal agent are general. For example, many of the known inhibitors for the formation of oral bacteria-producing biofilms exhibit a formation-inhibiting effect based on bacterial growth-inhibiting action and killing (bactericidal) action. Physical removal is limited to the cases where it is possible, and there are many parts that are difficult to reach, such as in the oral cavity, where they are complicated and physically scraped, and the surface is easily damaged. Is not necessarily valid. In addition, the use of a strong disinfectant remains uneasy for daily use in areas such as the oral cavity. Furthermore, the use of such a strong disinfectant can be applied to a variety of foods, pharmaceuticals, feeds, agricultural chemicals, etc. Many problems still remain to be applied to new products.

  Accordingly, an object of the present invention is also to provide a novel biofilm formation inhibitor capable of suppressing the formation of a biofilm without relying on a strong bactericidal action.

  Furthermore, this invention is also providing the biofilm formation inhibitor suitable for the biofilm which Porphyromonas gingivalis (Porphyromonas gingivalis) which is typical oral bacteria.

  The present inventors have found that low-concentration lactoferrin, which has not been known for its effect, suppresses the formation of biofilms, and has completed the present invention.

The invention that solves this problem is a biofilm formation inhibitor containing lactoferrin as an active ingredient.
The present invention preferably contains lactoferrin at a concentration of 8 to 2000 μg / ml. In the present invention, the lactoferrin is preferably apolactoferrin, natural lactoferrin or hololactoferrin.
Furthermore, the biofilm is preferably a biofilm produced by Porphyromonas gingivalis of oral bacteria, and preferably in the form of a troche.

Therefore, the present invention includes the following [1] to [7].
[1] A biofilm formation inhibitor containing lactoferrin as an active ingredient.
[2] The biofilm formation inhibitor according to [1], which contains lactoferrin at a concentration of 8 to 2000 μg / ml.
[3] The biofilm formation inhibitor according to [1] or [2], wherein the biofilm is a biofilm produced by Porphyromonas gingivalis.
[4] The biofilm formation inhibitor according to any one of [1] to [3], wherein the suppression of biofilm formation is suppression of an increase in the amount of biofilm or a decrease in the amount of biofilm.
[5] The biofilm formation inhibitor according to [1], which is in the form of a troche.
[6] The biofilm formation inhibitor according to [5], wherein the content of lactoferrin with respect to the total mass of the lozenge is 10 to 30%.
[7] An antibacterial auxiliary agent for antibiotics containing the biofilm formation inhibitor according to any one of [1] to [6].

Furthermore, the present invention also includes the following [8] to [13].
[8] A pharmaceutical composition for inhibiting biofilm formation, comprising lactoferrin as an active ingredient.
[9] A pharmaceutical composition for inhibiting biofilm formation, comprising lactoferrin as an active ingredient and a pharmaceutically acceptable carrier.
[10] A biofilm formation inhibitor comprising lactoferrin as an active ingredient.
[11] A method for suppressing the formation of a biofilm by administering lactoferrin.
[12] Use of lactoferrin for inhibiting biofilm formation.
[13] Use of lactoferrin for producing a biofilm formation inhibitor.

Furthermore, the present invention also includes the following [14] to [25].
[14] A pharmaceutical composition for enhancing antibacterial action, comprising lactoferrin as an active ingredient.
[15] A pharmaceutical composition for enhancing antibacterial action, comprising lactoferrin as an active ingredient and a pharmaceutically acceptable carrier.
[16] An antibacterial action enhancer comprising lactoferrin as an active ingredient.
[17] An additive for enhancing antibacterial action, comprising lactoferrin as an active ingredient.
[18] A method for enhancing the antibacterial action of an antibacterial agent by administering lactoferrin together with the antibacterial agent.
[19] Use of lactoferrin for enhancing antibacterial action.
[20] Use of lactoferrin for producing an antibacterial action enhancer.
[21] An antibacterial pharmaceutical composition comprising lactoferrin as an active ingredient for enhancing antibacterial action and an antibacterial ingredient.
[22] An antibacterial pharmaceutical composition comprising lactoferrin as an active ingredient for enhancing antibacterial action, an antibacterial ingredient, and a pharmaceutically acceptable carrier.
[23] An antibacterial agent comprising lactoferrin as an active ingredient for enhancing antibacterial action and an antibacterial ingredient.
[24] Use of lactoferrin for producing an antibacterial agent with enhanced antibacterial action.
[25] A method for producing an antibacterial agent, comprising a step of adding lactoferrin to the antibacterial component.

Furthermore, the present invention also includes the following [26] to [29].
[26] An antibacterial action enhancer comprising the biofilm formation inhibitor according to any one of [1] to [6].
[27] An antibacterial agent comprising the biofilm formation inhibitor according to any one of [1] to [6] and an antibacterial component.
[28] A method for producing an antibacterial agent, comprising a step of adding the biofilm formation inhibitor according to any one of [1] to [6] to an antibacterial component.
[29] An antibacterial agent for biofilm-forming bacteria comprising lactoferrin and an antibacterial component as active ingredients.

According to the present invention, lactoferrin exhibits an effective effect as a biofilm formation inhibitor and an antibacterial action enhancer at a low concentration not conventionally known. The concentration of lactoferrin at the time of its action is, for example, in the range of 8 to 2000 μg / ml (0.008 to 2 mg / ml), preferably in the range of 31 to 2000 μg / ml, more preferably in the range of 50 to 2000 μg / ml, Alternatively, for example, 8 to 1000 μg / ml, preferably 31 to 1000 μg / ml, more preferably 130 to 1000 μg / ml, or for example, 8 to 500 μg / ml, preferably 31 to 500 μg / ml, more preferably 130 to A concentration in the range of 500 μg / ml can be mentioned.
Here, the density | concentration at the time of action means the density | concentration at which the effect of the biofilm formation inhibitor of this invention and an antibacterial action enhancer is exhibited.
That is, when acting on the oral biofilm, it corresponds to the concentration at the time of administration or the concentration at the time of ingestion, and it is applied to biofilms such as slime that can be formed in the triangular corner of the sink and moss attached to the wall surface of the water pipe or water tank. When acting, it corresponds to the concentration at the time of use.
That is, the present invention also relates to a pharmaceutical composition, antibacterial adjuvant, biofilm formation inhibitor, antibacterial action enhancer, antibacterial action enhancing additive, and antibacterial agent to which lactoferrin is administered in such a concentration range. There are also pharmaceutical compositions, antibacterial adjuvants, biofilm formation inhibitors, antibacterial action enhancers, antibacterial action enhancing additives, and antibacterial agents containing lactoferrin at concentrations in such a range, such as There is also the use of lactoferrin to administer lactoferrin at a range of concentrations, and there are also methods of administering lactoferrin at such a range of concentrations. In addition, since administration at such a low concentration is suitable for the oral cavity of humans, the present invention relates to a pharmaceutical composition, antibacterial adjuvant, biofilm formation inhibitor, antibacterial action enhancement for oral cavity. There are also agents, additives for enhancing antibacterial action, and antibacterial agents, and there are also methods for administration into the oral cavity and use of lactoferrin to be administered into the oral cavity. Regarding the unit of concentration, since the specific gravity of water is 1 g / ml, for example, a lactoferrin concentration of 8 μg / ml can be rephrased as 8 μg / g.
In addition, since the antibacterial action enhancement according to the present invention suitably enhances the antibacterial action against bacteria that form biofilms, the present invention provides pharmaceutical compositions, antibacterial adjuvants, and biofilm formation inhibitors for biofilm-forming bacteria. And antibacterial action enhancers, additives for enhancing antibacterial action, and antibacterial agents, and there are also methods for administration to biofilm-forming bacteria and use of lactoferrin to be administered to biofilm-forming bacteria.

In a preferred embodiment of the present invention, in addition to lactoferrin as an active ingredient, a lactoperoxidase system component (lactoperoxidase, glucose oxidase, glucose) is added to sufficiently exert a biofilm formation inhibitory effect. Can be made. Accordingly, the present invention also includes the following [30] to [36].
[30] A biofilm formation inhibitor containing lactoferrin, lactoperoxidase, glucose oxidase, and glucose as active ingredients.
[31] The biofilm formation inhibitor according to [30], wherein the biofilm is a Porphyromonas gingivalis production biofilm.
[32] An antibacterial adjuvant for antibiotics containing the biofilm formation inhibitor according to any one of [30] to [31].
[33] A pharmaceutical composition for suppressing biofilm formation, comprising lactoferrin, lactoperoxidase, glucose oxidase, glucose, and a pharmaceutically acceptable carrier as active ingredients.
[34] A method for inhibiting the formation of a biofilm by administering lactoferrin, lactoperoxidase, glucose oxidase, and glucose.
[35] An antibacterial agent comprising lactoferrin, lactoperoxidase, glucose oxidase and glucose as active ingredients.
[36] An antibacterial agent for biofilm-forming bacteria comprising lactoferrin, lactoperoxidase, glucose oxidase and glucose as active ingredients.

Furthermore, the present invention also includes the following [37] to [57].
[37] A biofilm formation inhibitor containing lactoferrin as an active ingredient.
[38] The biofilm formation inhibitor according to [37], which contains lactoferrin at a concentration of 8-2000 μg / ml.
[39] The biofilm formation inhibitor according to any of [37] to [38], wherein the lactoferrin is one or more selected from the group consisting of apolactoferrin, natural lactoferrin, or hololactoferrin. .
[40] The biofilm formation inhibitor according to any of [37] to [39], wherein the biofilm is a Porphyromonas gingivalis producing biofilm.
[41] The biofilm formation inhibitor according to [37], which is in the form of a troche.
[42] The biofilm formation inhibitor according to [41], wherein the content of lactoferrin with respect to the total mass of the lozenge is 10 to 30%.
[43] The biofilm formation inhibitor according to any of [37] to [42], which contains lactoferrin as an active ingredient, and further lactoperoxidase, glucose oxidase, and glucose.
[44] A method for suppressing the formation of a biofilm by administering lactoferrin.
[45] Use of lactoferrin for inhibiting biofilm formation.
[46] Use of lactoferrin for producing a biofilm formation inhibitor.
[47] An antibacterial agent for biofilm-forming bacteria comprising lactoferrin and an antibacterial component as active ingredients.
[48] The antibacterial agent for biofilm-forming bacteria according to [47], wherein the lactoferrin is one or more selected from the group consisting of apolactoferrin, natural lactoferrin, or hololactoferrin.
[49] A method for sterilizing biofilm-forming bacteria by administering the antibacterial agent for biofilm-forming bacteria according to any one of [47] to [48].
[50] a step of containing an antibacterial component,
A step of containing lactoferrin,
A method for producing an antibacterial agent for biofilm-forming bacteria.
[51] An antibacterial action enhancer comprising lactoferrin as an active ingredient.
[52] The antibacterial action enhancer according to [51], wherein the lactoferrin is one or more selected from the group consisting of apolactoferrin, natural lactoferrin, or hololactoferrin.
[53] The antibacterial action enhancer according to any of [51] to [52], wherein the antibacterial action is an antibacterial action against biofilm-forming bacteria.
[54] A method for enhancing the antibacterial action of an antibacterial component, comprising the step of administering lactoferrin simultaneously with or before and after administration of the antibacterial component.
[55] The antibacterial activity enhancer according to [54], wherein the antibacterial component is an antibacterial component that acts on biofilm-forming bacteria.
[56] Use of lactoferrin to enhance the antibacterial action of the antibacterial component.
[57] Use of lactoferrin for producing an antibacterial action enhancer.

The effects produced by the present invention are as follows.
1. According to the present invention, biofilm formation can be suppressed (biofilm formation suppression). Suppression of biofilm formation refers to inhibiting the new formation of biofilm to suppress the increase in the amount of biofilm, and promoting the removal of biofilm once formed to reduce the amount of biofilm. Including both. As a result, so-called bacterial nests can be destroyed to suppress bacterial adhesion and aggregation.
2. For a biofilm formed in the oral cavity, it is a simple method that can be effective only by ingestion.
3. Since lactoferrin can be used at a low concentration, there is no problem of adhesion to the oral cavity and flavor at the time of oral intake, and it is economical. Further, since lactoferrin can be used at a low concentration, the degree of freedom of product composition is high, and the product of the present invention can be provided in various forms.
4). Compared with attempts to destroy biofilms with powerful disinfectants, problems such as side effects caused by the administration of antibiotics and disinfectants, killing resistant bacteria and microorganisms beneficial to the host when ingested by the human body Can be avoided.
5. The antibacterial effect of the antibacterial agent can be enhanced by using it as an antibacterial agent, particularly an antibacterial auxiliary agent (antibacterial action enhancer) for antibiotics. Thereby, since an equivalent bactericidal effect can be obtained with a small amount of antibacterial agent, the amount of antibacterial agent (particularly antibiotics) used can be reduced.
6). By using it as an antibacterial agent for biofilm-forming bacteria using lactoferrin and antibiotics, the amount of antibiotics used can be reduced. As a result, side effects due to the use of antibiotics are less likely to occur, and the appearance of resistant bacteria is suppressed.
7). Lactoferrin is a milk-derived component and is also contained in human breast milk and has been ingested by humans for historical years as livestock animal milk, making it extremely safe for humans. high. Therefore, the biofilm formation inhibitor and antibacterial adjuvant (antibacterial action enhancer) according to the present invention can be used with confidence in the oral cavity of humans and can be administered daily for a long time. There is no worry at all. It can also be used with peace of mind for instruments used in the human oral cavity, such as dentures.

FIG. 1 is a diagram showing the biofilm formation inhibitory effect of lactoferrin. FIG. 2 is a diagram showing a decrease in the amount of biofilm by lactoferrin. FIG. 3 shows that antibacterial activity by lactoferrin is not observed. FIG. 4 is a diagram showing the effect of enhancing the antibacterial action of minocycline when used together with lactoferrin. FIG. 5 is a diagram showing the biofilm formation inhibitory effect of lactoferrin and lactoperoxidase system.

  Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.

[Biofilm]
Bacteria secrete substances around the cells to create their own living environment, forming so-called bacterial nests. The biofilm of the present invention is a substance referred to as an exopolysaccharide substance since a polysaccharide substance produced by bacteria around the microbial cell is a major component. For this reason, in this specification, a biofilm can be paraphrased as an exopolysaccharide.

  The biofilm of the present invention is a slime formed at the triangular corner of the sink, moss adhering to the wall of the water pipe or water tank, dental plaque (plaque) in the oral cavity, between teeth and gums (plaque in periodontal pocket) and tongue It includes things that occur on the surface (tongue moss), slime that occurs in medical materials such as dentures, contact lenses, and catheters.

[Bacteria]
The bacterium in the present invention is not particularly limited as long as it is a bacterium that forms a biofilm, but a gram-negative anaerobic gonococcus that forms a biofilm in the oral cavity is preferable. Among them, Porphyromonas gingivalis, Prevotera intermedia, Prevotera nigrescens, Tannerella forgience, and Tannerella forgience. actinomycetemcomitans) and Fusobacterium nucleatum are preferred, and Porphyromonas gingivalis is particularly preferred.

[Biofilm formation inhibitor]
According to the biofilm formation inhibitor of the present invention, by preventing the formation of biofilms on tooth surfaces, periodontal pockets, tongues and cheeks, the adhesion and aggregation of periodontopathic bacteria on these sites can be prevented. Can do.

  The suppression of biofilm formation in the present invention specifically means suppression of an increase in the amount (especially mass) of extracellular polysaccharides called biofilms, or a decrease in the amount (especially mass) of extracellular polysaccharides. It means to make it.

  The biofilm formation inhibitor of the present invention is effective at a very low concentration, and effectively exhibits a biofilm formation inhibitory effect at a low concentration at which no effect of lactoferrin including a bactericidal action is known. As the lactoferrin concentration at the time of administration of the biofilm formation inhibitor, lactoferrin is preferably contained at a concentration of 8-2000 μg / ml (0.008-2 mg / ml), and preferably at a concentration of 31-2000 μg / ml. More preferably, it is more preferably contained at a concentration of 50 to 2000 μg / ml. In a preferred embodiment, it can be used at a concentration in the range of, for example, 8 to 1000 μg / ml, preferably 31 to 1000 μg / ml, more preferably 130 to 1000 μg / ml, or, for example, 8 to 500 μg / ml. ml, preferably 31 to 500 μg / ml, more preferably 130 to 500 μg / ml.

  For this reason, the biofilm formation inhibitor of the present invention has a very low concentration when an effective amount is administered, and in addition to the safety of lactoferrin, there is no risk of side effects from the viewpoint of this low concentration, and the daily routine over a long period of time. There is no concern about safety for humans even if it is administered regularly. Therefore, the inhibitor of the present invention can be used regardless of age or sex when administered to humans, does not adhere to the oral cavity, and has no problem with flavor. In addition, the inhibitor of the present invention can be used very inexpensively, and if it is administered into the oral cavity of a human, the treatment cost in the dental clinic can be reduced.

  The biofilm formation inhibitor of the present invention effectively exerts a biofilm formation inhibitory effect at a low concentration that does not have any known effect of lactoferrin including a bactericidal action. That is, from the conventional knowledge, the biofilm formation suppression effect of the present invention is not based on the bactericidal action of lactoferrin. In addition, as described later, according to the present invention, the biofilm formation suppression effect of the present invention is also effectively exhibited by hololactoferrin that is saturated with metal and does not have chelating ability. This effect is not based on the bactericidal action of lactoferrin. That is, the biofilm formation inhibitor of the present invention is obtained by discovering an unknown property of lactoferrin.

[Lactoferrin]
In addition to commercially available products, lactoferrin, which is an active ingredient of the present invention, is a processed product such as colostrum, transitional milk, normal milk, end milk, etc. of mammals (eg, humans, cows, buffalos, horses, goats, sheep). Lactoferrin separated from conventional skim milk, whey, etc. by conventional methods such as ion exchange chromatography, apolactoferrin from which iron has been removed from lactoferrin by conventional methods, and apolactoferrin partially chelated with metals such as iron, copper, zinc and manganese Metal-bound lactoferrin, or metal saturated lactoferrin fully chelated with the metal can be used.
In particular, bovine-derived lactoferrin is preferable.

In addition, recombinant fungi obtained by recombinant DNA technology, human lactoferrin produced by recombinant dairy cows (transgenic cows) and the like can also be used in the present invention.
The lactoferrin used in the present invention is preferably apolactoferrin, natural lactoferrin or hololactoferrin, more preferably natural lactoferrin or hololactoferrin, and further preferably hololactoferrin.

  The biofilm formation inhibitor of the present invention can be formulated and administered in various modes by known methods, and can be administered orally in a preferred embodiment.

  The lactoferrin contained in the biofilm formation inhibitor of the present invention is effective at a very low concentration, and the biofilm formation inhibitory effect at such a low concentration that no action or effect including bactericidal action is known for lactoferrin. To effectively demonstrate. As the lactoferrin concentration at the time of administration of the biofilm formation inhibitor, lactoferrin is preferably contained at a concentration of 8 to 2000 μg / ml (0.008 to 2 mg / ml), and contained at a content of 31 to 2000 μg / ml. Is more preferable, and it is further more preferable to contain by content of 50-2000 microgram / ml. In a preferred embodiment, it can be used at a content in the range of, for example, 8-1000 μg / ml, preferably 31-1000 μg / ml, more preferably 130-1000 μg / ml, or, for example, 8-500 μg. / Ml, preferably 31 to 500 μg / ml, more preferably 130 to 500 μg / ml.

  The biofilm formation inhibitor of the present invention can be produced by formulating lactoferrin using any additive such as a pharmaceutically acceptable excipient. In formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and solvents for injections can be used. Specific preparations include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules and soft capsules), granules (including those coated), pills, Illustrative examples include troches, encapsulated liposomes, solutions, and pharmaceutically acceptable sustained release formulations.

Since the biofilm formation inhibitor of this invention exhibits an effect by hold | maintaining a fixed concentration lactoferrin in an oral cavity, the form of a troche agent is especially preferable.
When the biofilm formation inhibitor of the present invention is a troche, lactoferrin preferably remains in the oral cavity at a concentration of 8-2000 μg / ml for 1 hour or more, and lactoferrin has a concentration of 31-2000 μg / ml in the oral cavity. It is more preferable that it remains for 1 hour or longer, and it is further preferable that lactoferrin remains in the oral cavity at a concentration of 130 to 2000 μg / ml for 1 hour or longer. Therefore, physical properties such as the size, mass, and hardness of the lozenge agent, the blending amount of lactoferrin, the type and blending amount of the additive used, and the like can be adjusted so as to satisfy the conditions.
The content of lactoferrin in the troche is preferably 10-30% and more preferably 15-25% with respect to the total mass of the troche.

  Carriers and excipients used in the formulation include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder, and binders such as starch, Examples include gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like.

  Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.

Furthermore, as a lubricant, magnesium stearate, hydrogenated vegetable oil, and macrogol, etc., as a colorant, red No. 2, yellow No. 4, and blue No. 1, etc. that are allowed to be added to pharmaceuticals, Each can be illustrated.

  Tablets and granules include sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, It can also be coated with a methacrylic acid polymer or the like.

Although the chemical | medical agent of this invention may be used independently, you may use together with the pharmaceutical composition which has an effect in other biofilm formation suppression. It is because a biofilm formation inhibitory effect can be heightened by using together.
The pharmaceutical composition to be used in combination may be contained as an active ingredient in the drug of the present invention, or may be commercialized as a separate drug without being contained in the drug of the present invention.

  In a preferred embodiment of the present invention, in addition to lactoferrin as an active ingredient, a lactoperoxidase system component (lactoperoxidase, glucose oxidase, glucose) is added to sufficiently exert a biofilm formation inhibitory effect. Can be made.

  Lactoperoxidase can be obtained from mammalian milk or the like, and can be obtained from milk such as humans, cows, horses, sheep or goats, for example. For example, as in the method disclosed in Japanese Patent Laid-Open No. 5-41981 (title of invention: liquid composition containing viable bacteria), conventional methods (for example, ion exchange chromatography, etc.) are used from unheated whey or skim milk such as milk. In addition, commercially available lactoperoxidase (for example, manufactured by DMV) or recombinant lactoperoxidase [for example, the method of Shin et al. [Biochemical and Biochemical and Biophysical Research Communications, Vol. 271, 2000, p. 831-836] or a recombinant lactoperoxidase commercially available or commercially available recombinant lactoperoxidase]. As lactoperoxidase, those derived from mammalian milk can be preferably used. When used in food additives and pharmaceutical compositions, lactoperoxidase derived from milk such as cows, sheep, goats, etc., whose milk is traditionally used for food and drink is preferred, especially those derived from milk. preferable. This is because these have been used for human consumption during historical years, and thus have a very high level of safety for humans. Unheated whey derived from milk is particularly suitable as a raw material for lactoperoxidase used in the present invention because it can be obtained stably and in large quantities as a by-product of dairy production.

As the glucose oxidase, for example, commercially available glucose oxidase (for example, manufactured by Shin Nippon Chemical Industry Co., Ltd.) which is an enzyme produced by microorganisms such as Aspergillus niger and Penicillium chrysogenum is used. be able to. As glucose, for example, glucose for commercial food additives (for example, manufactured by Nippon Shokuhin Kako Co., Ltd.) can be used. These glucose oxidase and glucose are both widely used as food additives and are commercially available and can be easily obtained.

  In addition to lactoferrin as an active ingredient, a biofilm formation inhibitor comprising components of the lactoperoxidase system (lactoperoxidase, glucose oxidase, glucose) is also formulated and manufactured as described above. Can be used.

[Antimicrobial agent for biofilm-forming bacteria]
The antibacterial agent for biofilm-forming bacteria of the present invention contains at least lactoferrin and an antibacterial component (antibacterial agent). Antibiotics are preferred as the antibacterial component.
The antibacterial agent for a biofilm-forming bacterium of the present invention is a microbial cell that forms a biofilm, and can be suitably used for sterilizing the microbial cell covered with the biofilm.
Usually, since the microbial cells exist in an integrated state with the biofilm, the biofilm-producing bacteria can also be regarded as bacteria and a biofilm complex. Therefore, the antibacterial agent for biofilm-forming bacteria of the present invention can be rephrased as “a pharmaceutical composition for inhibiting the formation of bacteria and biofilm complexes”.
The antibacterial agent for biofilm-forming bacteria of the present invention can effectively antibacterize biofilm-producing bacteria (biofilm-forming bacteria) by different functions of lactoferrin and antibacterial components, preferably antibiotics.

  The antibacterial component (antibacterial agent) that can be used as the antibacterial agent for biofilm-forming bacteria of the present invention is not particularly limited as long as it is an antibacterial component (antibacterial agent) having an antibacterial effect against the target fungus, Antibacterial components (antibacterial agents) that can be suitably used include, for example, antibiotics, and examples of antibiotics include ampicillin, ciprofloxacin, clarithromycin, minocycline, and minocycline is particularly preferable. .

The lactoferrin in the antibacterial agent for biofilm-forming bacteria of the present invention exerts an effect at a low concentration such that no effect of lactoferrin including bactericidal action is known as described above in the biofilm formation inhibitor. The present inventor believes that this effect is exhibited through the action of inhibiting biofilm formation.
Specifically, the lactoferrin concentration in the antibacterial agent for biofilm-forming bacteria is preferably 8 to 2000 μg / ml (0.008 to 2 mg / ml), preferably 31 to 2000 μg / ml. More preferably, it is contained in an amount of 50 to 2000 μg / ml. In a preferred embodiment, it can be used at a content in the range of, for example, 8-1000 μg / ml, preferably 31-1000 μg / ml, more preferably 130-1000 μg / ml, or, for example, 8-500 μg. / Ml, preferably 31 to 500 μg / ml, more preferably 130 to 500 μg / ml.
Therefore, it is as having mentioned above in the biofilm formation inhibitor that the effect of the antibacterial action enhancement of the present invention is exhibited without going through the bactericidal action of lactoferrin, and that it has high safety for humans. Furthermore, the lactoferrin concentration at the time of administration of the antibacterial adjuvant and the antibacterial action enhancer is as described above for the biofilm formation inhibitor, and can be formulated as described above for the biofilm formation inhibitor.

The antibacterial agent for biofilm-forming bacteria of the present invention is obtained by adding lactoferrin, which is an active ingredient of a biofilm formation inhibitor, to an antibacterial component (antibacterial agent) and formulating it by the above and known methods.
In addition, it is preferable that the density | concentration of the lactoferrin in the antibacterial agent for biofilm formation bacteria of this invention contains a density | concentration required for a biofilm formation inhibitory effect as a lactoferrin.
Moreover, it is preferable that the mixing ratio (mass ratio) of the lactoferrin and the antibacterial component in the antibacterial agent for biofilm-forming bacteria of the present invention is lactoferrin: antibacterial component = 5000: 1 to 1: 1.
That is, even if the amount of the antibacterial component is as small as 1/5000 of lactoferrin, the antibacterial component in the present invention sufficiently exhibits antibacterial activity against the biofilm-forming bacteria, so that an antibiotic as an antibacterial component It is also possible to reduce the amount of the antimicrobial agent, and to effectively suppress and reduce the emergence of antibiotic-resistant bacteria.
In the antibacterial agent for biofilm-forming bacteria of the present invention, since lactoferrin is considered to act first, for example, in the case of a troche or tablet, if lactoferrin is dispersed in the vicinity of the surface layer of the drug, the oral cavity precedes the antibacterial component. It is preferable that it disperses within and easily acts on the biofilm.

[Antimicrobial adjuvant / antibacterial action enhancer]
The antibacterial auxiliary agent and the antibacterial action enhancer of the present invention can be used together with an antibacterial component (antibacterial agent), for example, an antibiotic to sterilize cells covered with a biofilm. Antibacterial effect of antibiotics can be enhanced. The antibacterial adjuvant and antibacterial action enhancer of the present invention have been obtained by discovering the unknown property of lactoferrin, which is the enhancement of the antibacterial action of the antibacterial component (antibacterial agent). Therefore, the antibacterial adjuvant and antibacterial action enhancer of the present invention can be suitably used against bacteria (bacteria) covered with a biofilm in which the biofilm formation inhibitor can be used. The target bacteria are as described above for the biofilm formation inhibitor.

  The antibacterial component (antibacterial agent) that has an enhanced antibacterial action according to the present invention is not particularly limited as long as it is an antibacterial component (antibacterial agent) having an antibacterial effect against the target bacteria, but can be suitably used. Examples of such antibacterial components (antibacterial agents) include antibiotics, and examples of antibiotics include ampicillin, ciprofloxacin, clarithromycin, minocycline, and minocycline is particularly preferable.

  As described above in the biofilm formation inhibitor, the antibacterial adjuvant and antibacterial action enhancer of the present invention have an antibacterial action enhancing effect at a low concentration such that any action effect including lactoferrin is not known. Demonstrate effectively. The present inventor believes that the effect of enhancing the antibacterial action of the present invention is exhibited through the action of inhibiting the formation of biofilm. Therefore, it is as having mentioned above in the biofilm formation inhibitor that the effect of the antibacterial action enhancement of the present invention is exhibited without going through the bactericidal action of lactoferrin, and that it has high safety for humans. Furthermore, the lactoferrin concentration at the time of administration of the antibacterial adjuvant and the antibacterial action enhancer is as described above for the biofilm formation inhibitor, and can be formulated as described above for the biofilm formation inhibitor.

  In addition, the present invention is also in an antibacterial adjuvant or antibacterial action enhancer formulated with lactoferrin, which is an active ingredient for enhancing antibacterial action, as described above. There is also an antibacterial agent with enhanced antibacterial action obtained by adding lactoferrin, which is an active ingredient, and preparing it by the above and known methods.

  Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Production Example 1]
Lactoferrin (Morinaga Milk Industry Co., Ltd., iron saturation 15%) 300 g, reduced maltose syrup (Towa Kasei Co., Ltd.) 810 g, powdered candy (Showa Sangyo Co., Ltd.) 172.5 g, corn starch (manufactured by Saneigen FFI Co., Ltd.) 1.5 g, 6 g of yogurt fragrance (manufactured by Hasegawa Fragrance Co., Ltd.), and 45 g of glycerin fatty acid ester (manufactured by Saneigen FFI Co., Ltd.) were uniformly mixed to obtain a mixed powder.
Using the tableting machine (manufactured by Hata Iron Works Co., Ltd.), this mixed powder was tableted continuously at a tableting speed of 1.5 g per tablet, 12 tablets / minute, and a troche having a hardness of 7.3 kg. 900 tablets (about 1350 g) were produced. The lactoferrin content in one obtained troche is 0.3 g.

[Production Example 2]
Lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., iron saturation 20%) 250 g, malt dextrin (manufactured by Matsutani Chemical Industry Co., Ltd.) 635 g, skim milk powder (manufactured by Morinaga Milk Industry Co., Ltd.) 85 g, stevia sweetener (manufactured by Saneigen FFI Co., Ltd.) 1 g, 5 g of yogurt flavor (manufactured by San-Ei Gen FFI Co., Ltd.) and 24 g of glycerin fatty acid ester (manufactured by Riken Vitamin Co., Ltd.) were uniformly mixed to obtain a mixed powder.
Using the tableting machine (manufactured by Hata Iron Works Co., Ltd.), this mixed powder is continuously tableted at a tableting speed of 1.5 g / tablet, 12 tablets / min, and a troche having a hardness of 8.5 kg. 600 tablets (about 900 g) were produced. The content of lactoferrin in one tablet of the obtained troche is 0.3 g.

[Production Example 3]
683.7 g of lactoferrin (Morinaga Milk Industry, iron saturation 20%), 2214.5 g of reduced maltose liquid sugar (Towa Kasei Kogyo), 461.5 g of xylitol (Towa Kasei Kogyo), erythritol fine powder (Mitsubishi Chemical Foods) 1846.0 g, trisodium citrate fine powder (manufactured by San-Ei Gen FFI Co., Ltd.) 213.3 g, citric acid (anhydrous) (manufactured by San-Ei Gen FFI Co., Ltd.) 96.4 g, anhydrous crystalline glucose 184.6 g (manufactured by Nippon Shokuhin Kako Co., Ltd.) was uniformly mixed to obtain 5700.0 g of a primary mixed powder.
1389.5 g of primary mixed powder, 3.0 g of lactoperoxidase (manufactured by DMV), 40.0 g of glucose oxidase (manufactured by Shin Nippon Chemical Industry Co., Ltd.), 4.5 g of lemon flavor MN-42135-H (manufactured by Hasegawa Fragrance Co., Ltd.), Menthol (Hasegawa Fragrance Co., Ltd.) 3.0 g, Ryoto Sugar Ester S-1170F (Mitsubishi Chemical Foods Co., Ltd.) 30.0 g, Poem RJ-38 (Riken Vitamin Co., Ltd.) 30.0 g were uniformly mixed to obtain lactoferrin. And the lactoperoxidase system containing composition 1500.0g was obtained.

[Production Example 4]
683.7 g of lactoferrin (Morinaga Milk Industry, iron saturation 20%), 2214.5 g of reduced maltose liquid sugar (Towa Kasei Kogyo), 461.5 g of xylitol (Towa Kasei Kogyo), erythritol fine powder (Mitsubishi Chemical Foods) 1846.0 g, trisodium citrate fine powder (manufactured by San-Ei Gen FFI Co., Ltd.) 213.3 g, citric acid (anhydrous) (manufactured by San-Ei Gen FFI Co., Ltd.) 96.4 g, anhydrous crystalline glucose 184.6 g (manufactured by Nippon Shokuhin Kako Co., Ltd.) was uniformly mixed to obtain 5700.0 g of a primary mixed powder.
1389.5 g of primary mixed powder, reduced maltose liquid sugar (manufactured by Towa Kasei Kogyo Co., Ltd.) 17.0 g, lactoperoxidase (manufactured by DMV) 2.0 g, glucose oxidase (manufactured by Shin Nippon Chemical Industry Co., Ltd.) 24.0 g, lemon flavor MN-42135-H (Hasegawa Fragrance Co., Ltd.) 4.5 g, Menthol (Hasegawa Fragrance Co., Ltd.) 3.0 g, Ryoto Sugar Ester S-1170F (Mitsubishi Chemical Foods Co., Ltd.) 30.0 g, Poem RJ-38 (RIKEN) 30.0 g (manufactured by Vitamin Co., Ltd.) was uniformly mixed to obtain 1500.0 g of a composition containing lactoferrin and a lactoperoxidase system having a low lactoperoxidase and glucose oxidase content compared to Production Example 3.

[Production Example 5]
683.7 g of lactoferrin (Morinaga Milk Industry, iron saturation 20%), 2214.5 g of reduced maltose liquid sugar (Towa Kasei Kogyo), 461.5 g of xylitol (Towa Kasei Kogyo), erythritol fine powder (Mitsubishi Chemical Foods) 1846.0 g, trisodium citrate fine powder (manufactured by San-Ei Gen FFI Co., Ltd.) 213.3 g, citric acid (anhydrous) (manufactured by San-Ei Gen FFI Co., Ltd.) 96.4 g, anhydrous crystalline glucose 184.6 g (manufactured by Nippon Shokuhin Kako Co., Ltd.) was uniformly mixed to obtain 5700.0 g of a primary mixed powder.
Primary mixed powder 277.9 g, Lemon flavor MN-42135-H (Hasegawa Fragrance Co., Ltd.) 0.9 g, Menthol (Hasegawa Fragrance Co., Ltd.) 0.6 g, Ryoto Sugar Ester S-1170F (Mitsubishi Chemical Foods Co., Ltd.) 6 0.0 g, Poem RJ-38 (manufactured by Riken Vitamin Co., Ltd.) 6.0 g, and a lactoferrin-containing composition 291. in which lactoperoxidase and glucose oxidase are not added to the composition of Production Example 3. 4 g was obtained.

Next, the present invention will be described in detail with reference to test examples.
[Test Example 1]
This test was performed for the purpose of confirming the effect of suppressing the increase of biofilm among the biofilm formation suppression effects by lactoferrin. That is, the purpose was to confirm the effect of suppressing the increase in the amount of biofilm produced and accumulated by the bacterial cells (the effect of inhibiting new formation of biofilm).

(1) Preparation of sample As lactoferrin sample, natural type lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) bound with 10% saturation amount of iron, apolactoferrin without binding iron at all, hololactoferrin bound with saturation amount of iron were used. . Natural lactoferrin used was purified from milk. Apolactoferrin and hololactoferrin use natural lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) as a raw material, and follow the method of Inorganica Chimica Acta (Inorganica Chimica Acta, Switzerland, 1979, Vol. 33, p.149-153). Each was manufactured.

  Porphyromonas gingivalis (ATCC 33277) was obtained from the American Type Culture Collection as a biofilm-forming bacterium. 5% sheep blood, hemin (5 μg / ml, manufactured by Sigma-Aldrich Japan), vitamin K1 (0.5 μg / ml, manufactured by Sigma-Aldrich Japan), yeast extract (10 mg / ml, Japan) On a Trypticase soy agar medium (Nippon Becton Dickinson) supplemented with Becton Dickinson), in a carp model 2000 incubator (manufactured by Koy Laboratory Products), anaerobic atmosphere (10% carbon dioxide, Cultured and maintained in 5% hydrogen, 85% nitrogen).

  The test bacterial solution was Trypticase soy liquid medium (Nippon Becton Dickinson) supplemented with Porphyromonas gingivalis with hemin (5 μg / ml), vitamin K1 (5 μg / ml), and yeast extract (1 mg / ml). ) And cultured at 37 ° C., and used for biofilm formation test and growth inhibition test.

(2) Test method Measurement of biofilm formation is described in Current Protocols in Microbiology (USA, 2005, p. 1B.1.1-1B.1.17). According to the above method, it was measured by a microtiter plate biofilm assay. In a well of a 96-well microtiter plate made of polyvinyl chloride (manufactured by Nippon Becton Dickinson), 0 mg / ml (control), 0.008 mg / ml, 0.031 mg / ml, 0.13 mg / ml, 0.5 mg 100 ml of trypticase soy liquid medium supplemented with hemin (5 μg / ml), vitamin K1 (5 μg / ml) and yeast extract (1 mg / ml) Then, about 10 ⁷ / ml of the test bacteria was cultured for 24 hours. As a test sample, apolactoferrin, natural lactoferrin, hololactoferrin was used.

  After 24 hours of culture, airborne bacteria were washed away with water, and each well was incubated with 125 μl of 0.1% crystal violet solution for 10 minutes to adsorb the dye to the biofilm. After washing with water and drying in air, each well was incubated with 150 μl of 95% ethanol for 10 minutes to extract the dye. 125 μl of the dye-extracted solution was transferred to a polystyrene 96-well microtiter plate (Nippon Becton Dickinson), and the absorbance at 550 nm was measured with a microplate reader MTP-32 (Corona Denki). Biofilm The amount of formation. The biofilm formation amount was measured for each of the three specimens, and the average value was obtained.

(3) Test result The result of the biofilm formation amount of Porphyromonas gingivalis by lactoferrin is shown in FIG. The vertical axis represents the amount of biofilm formed after 24 hours, and the horizontal axis represents the concentration of each lactoferrin (LF) sample. Statistical analysis was compared with controls according to t test. In the table, “*” indicates a significant difference p <0.05, and “**” indicates a significant difference p <0.001. Only the sample to which Holo LF was added at 0.008 mg / ml had a significant difference of p <0.05. In other conditions, the significant difference was p <0.001.

  That is, it was revealed that any lactoferrin effectively inhibits formation of a biofilm at a concentration of 0.008 mg / ml to 2 mg / ml (8 μg / ml to 2000 μg / ml). In addition, this biofilm formation inhibitory effect was sufficiently significant even when lactoferrin was administered at the minimum concentration, and the effect became more remarkable as the concentration of lactoferrin was increased. In addition, since hololactoferrin had a sufficiently significant biofilm formation inhibitory effect as with other lactoferrins, this biofilm formation inhibitory effect was not an effect through the bactericidal action of lactoferrin. all right.

[Test Example 2]
The purpose of this test was to confirm the effect of reducing the amount of biofilm among the biofilm formation inhibitory effects of lactoferrin. That is, the purpose was to confirm the effect of reducing the amount of biofilm once produced and accumulated by the cells (effect of reducing the amount of biofilm already formed).

(1) Preparation of sample As a lactoferrin sample, natural lactoferrin bound by 10% saturation amount of iron (manufactured by Morinaga Milk Industry), apolactoferrin not binding iron at all, hololactoferrin bound to saturation amount of iron were used. . Natural lactoferrin used was purified from milk. Apolactoferrin and hololactoferrin use natural lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) as a raw material, and follow the method of Inorganica Chimica Acta (Inorganica Chimica Acta, Switzerland, 1979, Vol. 33, p.149-153). Each was manufactured.

  Porphyromonas gingivalis (ATCC 33277) was obtained from the American Type Culture Collection as a biofilm-forming bacterium. 5% sheep blood, hemin (manufactured by Sigma-Aldrich Japan) 5 μg / ml, vitamin K1 (manufactured by Sigma-Aldrich Japan) 0.5 μg / ml, yeast extract (manufactured by Nippon Becton Dickinson) On trypticase soy agar medium (Nippon Becton Dickinson) supplemented with 10 mg / ml, in an koi model 2000 incubator (manufactured by Koi Laboratory Products), anaerobic atmosphere (10% carbon dioxide, 5% hydrogen) And maintained in nitrogen (85%).

The bacterial solution for the test was 37 ° C. in a trypticase soy liquid medium (Nippon Becton Dickinson) supplemented with Porphyromonas gingivalis with 5 μg / ml hemin, 5 μg / ml vitamin K1, and 1 mg / ml yeast extract. It was prepared by culturing in

(2) Test method The measurement of the amount of remaining biofilm is described in Current Protocols in Microbiology (USA, 2005, p. 1B.1.1-1B.1.17). According to the above method, it was measured by a microtiter plate biofilm assay. Add 100 μl of trypticase soy liquid medium containing hemin 5 μg / ml, vitamin K1 5 μg / ml, yeast extract 1 mg / ml to wells of polyvinyl chloride 96-well microtiter plate (Nippon Becton Dickinson) Then, about 10 ⁷ / ml of the test bacteria was cultured for 24 hours.

  After confirming that a biofilm was formed after culturing for 24 hours, the floating cells were removed and washed with physiological saline (0.85% NaCl (manufactured by Wako Pure Chemical Industries, Ltd.)). After stopping the growth, lactoferrin dissolved in physiological saline was added and cultured for 5 hours.

  The addition amount of the lactoferrin sample is a 4-fold dilution series of 0 mg / ml (control), 0.008 mg / ml, 0.031 mg / ml, 0.13 mg / ml, 0.5 mg / ml, and 2 mg / ml. The remaining amount of biofilm was confirmed after 5 hours from the addition of the lactoferrin sample.

  After 5 hours, the floating bacteria were washed away with water, and each well was incubated with 125 μl of 0.1% crystal violet solution for 10 minutes to adsorb the dye to the biofilm. After washing with water and drying in air, each well was incubated with 150 μl of 95% ethanol for 10 minutes to extract the dye. 125 μl of the dye-extracted solution was transferred to a polystyrene 96-well microtiter plate (Nippon Becton Dickinson), and the absorbance at 550 nm was measured with a microplate reader MTP-32 (Corona Denki). Biofilm The remaining amount was used. The biofilm residual amount was measured for each of the three specimens, and the average value was determined.

(3) Test result The biofilm residual amount when each lactoferrin is used is shown in FIG. The vertical axis shows the amount of biofilm remaining after 5 hours, and the horizontal axis shows each lactoferrin sample arranged in order of concentration. The unit of the horizontal axis is mg / ml. Statistical analysis was compared with the control according to t test, and the results are shown in the figure. In the table, “*” indicates a significant difference p <0.05, and “**” indicates a significant difference p <0.001.

  All the lactoferrin showed significant significance in reducing the amount of biofilm once formed. This effect was sufficiently significant even when lactoferrin was administered at a minimal concentration, and the effect became more pronounced as the concentration of lactoferrin was increased. In hololactoferrin, a sufficiently significant effect was observed as in the case of other lactoferrin. Apolactoferrin and natural lactoferrin were even more effective.

  From the results of this test, it was found that lactoferrin not only inhibits the new formation of biofilm and suppresses the increase of the amount, but also has the effect of reducing the amount of biofilm already formed. In addition, the decrease in the amount of biofilm once formed cannot be explained even if the cells can be sterilized. That is, it was found that the biofilm formation suppression according to the present invention was due to an unknown mechanism different from the bactericidal action.

[Test Example 3]
This test was conducted for the purpose of examining the antibacterial action of lactoferrin against periodontopathic bacteria at a concentration showing a biofilm formation inhibitory effect.

(1) Sample Preparation and Test Method A lactoferrin sample and periodontopathic bacteria were prepared in the same manner as in Test Example 1. In a well of a 96-well microtiter plate (manufactured by Nippon Becton Dickinson) made of polyvinyl chloride, a 4-fold diluted lactoferrin sample of 0 (control), 0.13, 0.5, 2 and 8 mg / ml, Add 150 μl trypticase soy liquid medium supplemented with hemin (5 μg / ml), vitamin K1 (5 μg / ml), yeast extract (1 mg / ml), and add about 10 ⁷ / ml periodontopathic bacteria for 8 hours or Cultured for 24 hours. 100 μl of the culture solution was transferred to a polystyrene 96-well microtiter plate (Nippon Becton Dickinson) and the absorbance at 630 nm was measured with a microplate reader MTP-32 (Corona Electric) to determine the amount of bacterial growth. .

(2) Results FIG. 3 shows the results when 0.13 to 8 mg / ml apolactoferrin, natural lactoferrin and hololactoferrin were added to Porphyromonas gingivalis and cultured for 8 hours. The results were almost the same as the growth of the control without lactoferrin, and no antibacterial activity was observed.

  That is, it has been clarified that all of apolactoferrin, natural lactoferrin, and hololactoferrin do not exhibit an antibacterial effect against Porphyromonas gingivalis in a concentration range of 0.13 to 8 mg / ml.

  Thus, lactoferrin did not inhibit the growth of bacteria involved in biofilm formation and did not exert any bactericidal action at concentrations that exhibited biofilm formation inhibitory effects. In this way, lactoferrin exerts the effect of inhibiting the formation of biofilms effectively at a concentration at which it does not exert a bactericidal action, and thus has been known to have an effect of inhibiting the growth of bacteria and the effect of killing them (bactericidal effect). It has been clarified that the effect of suppressing the formation of biofilm is exerted by a mechanism other than the mechanism based on.

[Test Example 4]
The purpose of this test was to confirm that lactoferrin has an effect as an antibacterial agent for biofilm-forming bacteria by combining with an antibiotic that is an antibacterial component.

(1) Preparation of sample Natural lactoferrin was used as a lactoferrin sample. Minocycline (manufactured by Sigma) was used as an antibiotic.

  Porphyromonas gingivalis (ATCC 33277) was obtained from the American Type Culture Collection as a biofilm-forming bacterium. 5% sheep blood, hemin (manufactured by Sigma-Aldrich Japan) 5 μg / ml, vitamin K1 (manufactured by Sigma-Aldrich Japan) 0.5 μg / ml, yeast extract (manufactured by Nippon Becton Dickinson) On trypticase soy agar medium (Nippon Becton Dickinson) supplemented with 10 mg / ml, in an koi model 2000 incubator (manufactured by Koi Laboratory Products), anaerobic atmosphere (10% carbon dioxide, 5% hydrogen) And maintained in nitrogen (85%).

(2) Test method The biofilm formation experiment is described in Current Protocols in Microbiology (USA, 2005, p. 1B.1.1-1B.1.17). Followed the method. Add 100 μl of trypticase soy liquid medium containing hemin 5 μg / ml, vitamin K1 5 μg / ml, yeast extract 1 mg / ml to wells of polyvinyl chloride 96-well microtiter plate (Nippon Becton Dickinson) Then, about 10 ⁷ / ml of the test bacteria was cultured for 24 hours.

  After confirmation of biofilm formation after 24 hours of culture, floating cells were removed and washed with physiological saline (0.85% NaCl (made by Wako Pure Chemical Industries, Ltd.)) to stop biofilm growth. Thereafter, lactoferrin and minocycline dissolved in physiological saline were added and reacted for 24 hours.

  The addition amount of lactoferrin is 0 mg / ml (control), 0.031 mg / ml, and 0.5 mg / ml, and the addition amount of minocycline is 0.1 μg / ml, 1 μg / ml, and 10 μg / ml.

After 24 hours from the addition of lactoferrin and minocycline, floating cells were removed, and 100 μl of physiological saline was further added, followed by washing with a method of removing after stirring. 100 μl of BacTiter-Glo Microbial Cell Viability Assay (Promega) was added, stirred, and allowed to stand for 15 minutes. Of this, 80 μl was transferred to another 96-well white plate (made by Greiner), the amount of luminescence was measured with a Veritas Microplate Luminometer (made by Promega), and RLU (relative luminescence unit, hereinafter abbreviated as RLU) was compared. The RLU value represents the amount of ATP. The larger the number of surviving bacteria, the higher the value, and the better the growth of the fungus, the higher the value, so it can be defined as representing the viability of the fungus. It is.

(3) Test results The results are shown in FIG. The vertical axis shows the survival amount (RLU) of Porphyromonas gingivalis in the biofilm after 24 hours, and the horizontal axis shows the combination of lactoferrin and minocycline in order of concentration. The unit of the horizontal axis is mg / ml for lactoferrin and μg / ml for minocycline. On the horizontal axis of the figure, MINO indicates minocycline, 0.1, 1, and 10 each indicate its concentration μg / ml, and No antibiotic indicates that no minocycline is added. On the horizontal axis of the figure, LF indicates lactoferrin, 0.031 and 0.5 indicate their concentrations in mg / ml, respectively, and no LF indicates no addition of lactoferrin.

When only minocycline was added without adding lactoferrin (in FIG. 4, MINO 0.1 to 10 and no LF), antibacterial activity was not observed. That is, antibacterial activity was not observed with minocycline alone against bacteria covered with biofilm.

On the other hand, even when only lactoferrin was added and minocycline was not added (in the case of No antibiotic, LF 0.031 to 0.5 in FIG. 4), antibacterial activity was not observed. That is,
Antimicrobial activity was not observed with lactoferrin alone.

However, when lactoferrin and minocycline were added together, the survival amount (RLU) of the bacteria was significantly reduced. When lactoferrin and minocycline are added together, the addition amount of minocycline is 0.1 μg / ml, 1 μg / ml, 10 μg / ml, regardless of whether lactoferrin is 0.031 mg / ml or 0.5 mg / ml. In any of the mls, there was a marked decrease in RLU, ie a significant antibacterial effect, compared to the case where lactoferrin or minocycline was added alone.
From this result, the mixing ratio (mass ratio) of lactoferrin and minocycline is preferably lactoferrin: minocycline = 5000: 1 to 3: 1, more preferably 5000: 1 to 31: 1, and further preferably 5000: 1 to 310: 1. It can be said that it is a preferable range. Moreover, as one mode of suitable implementation, for example, lactoferrin: minocycline = 500: 1 to 3.1: 1, preferably 500: 1 to 31: 1, more preferably 500: 1 to 310: 1 can do. Alternatively, for example, lactoferrin: minocycline = 50: 1 to 3.1: 1 is preferable, and 50: 1 to 31: 1 can be used as a more preferable range.
Thus, by using lactoferrin in combination with minocycline, which is an antibacterial component, a remarkable antibacterial effect that could not be seen when each was added alone was confirmed as a synergistic effect.

From the above results, when an antibacterial component is administered to bacteria present in a biofilm after forming a biofilm, the lactoferrin is based on the biofilm formation inhibitory effect when administered in combination with lactoferrin. It was clarified that the antibacterial component is assisted and the antibacterial action of the antibacterial component is enhanced to produce a remarkable antibacterial effect that cannot be achieved by administration of the antibacterial component alone.

[Test Example 5]
The purpose of this test was to confirm the biofilm formation inhibitory effect when the lactoperoxidase system was administered in combination with lactoferrin, that is, the biofilm formation inhibitory effect of the lactoferrin and lactoperoxidase system-containing composition.

(1) Preparation of sample The composition manufactured by the method of Production Example 3 was used as the lactoferrin and lactoperoxidase system-containing composition. Moreover, the composition manufactured by the method of Production Example 5 was used as a composition obtained by removing lactoperoxidase and glucose oxidase from the lactoferrin and lactoperoxidase system-containing composition, that is, the lactoferrin-containing composition.

  Porphyromonas gingivalis (ATCC 33277) was obtained from the American Type Culture Collection as a biofilm-forming bacterium. 5% sheep blood, hemin (5 μg / ml, manufactured by Sigma-Aldrich Japan), vitamin K1 (0.5 μg / ml, manufactured by Sigma-Aldrich Japan), yeast extract (10 mg / ml, Japan) On a Trypticase soy agar medium (Nippon Becton Dickinson) supplemented with Becton Dickinson), in a carp model 2000 incubator (manufactured by Koy Laboratory Products), anaerobic atmosphere (10% carbon dioxide, Cultured and maintained in 5% hydrogen, 85% nitrogen).

  The test bacterial solution was Trypticase soy liquid medium (Nippon Becton Dickinson) supplemented with Porphyromonas gingivalis with hemin (5 μg / ml), vitamin K1 (5 μg / ml), and yeast extract (1 mg / ml). ) And cultured at 37 ° C.

(2) Test method Measurement of biofilm formation is described in Current Protocols in Microbiology (USA, 2005, p. 1B.1.1-1B.1.17). In the well of a 96-well microtiter plate made of polyvinyl chloride (Nippon Becton Dickinson), 0% or 0.032% of the test sample, saliva component sodium thiocyanate for working the lactoperoxidase system 100 μl of trypticase soy liquid medium supplemented with 0.5 mM, hemin (5 μg / ml), vitamin K1 (5 μg / ml) and yeast extract (1 mg / ml) was added, and about 10 ⁷ / ml of test bacteria was added. Incubate for hours. As a test sample, lactoferrin, a lactoperoxidase system-containing composition, and a lactoferrin-containing composition were used.

  Airborne bacteria were washed away with water, and each well was incubated with 125 μl of 0.1% crystal violet solution for 10 minutes to adsorb the dye to the biofilm. After washing with water and drying in air, each well was incubated with 150 μl of 95% ethanol for 10 minutes to extract the dye. 125 μl of the dye-extracted solution was transferred to a polystyrene 96-well microtiter plate (Nippon Becton Dickinson), and the absorbance at 550 nm was measured with a microplate reader MTP-32 (Corona Denki). Biofilm The amount of formation. The biofilm formation amount was measured for each of the three specimens, and the average value was obtained.

(3) Test result The amount of biofilm formation of Porphyromonas gingivalis in the presence of a lactoferrin (LF) and lactoperoxidase (LPO) system-containing composition and a lactoferrin (LF) -containing composition is shown in FIG. The vertical axis represents the amount of biofilm formed after 24 hours, and the horizontal axis represents each test sample. Statistical analysis compared two groups according to the T test, and the results are shown in FIG. Both the LF-containing composition and the LF and LPO-containing composition significantly suppressed biofilm formation with p <0.01 compared to the control. Furthermore, the lactoferrin and lactoperoxidase system-containing composition showed a tendency that the formation inhibitory effect was further enhanced with p <0.10 as compared with the lactoferrin-containing composition, and the biofilm formation amount was almost zero.
That is, it was revealed that the lactoferrin-containing composition effectively suppresses formation of a biofilm, but the effect is further enhanced by using a lactoperoxidase system in combination. Thus, in addition to the lactoferrin as an active ingredient, it turned out that the biofilm formation inhibitor containing the component (a lactoperoxidase, glucose oxidase, glucose) of a lactoperoxidase system also shows sufficient inhibitory effect.

According to the biofilm formation inhibitor of this invention, the effect which suppresses biofilm formation derived from bacteria is acquired, and periodontal disease can be prevented beforehand.
In addition, the biofilm formation inhibitor of the present invention contains lactoferrin, which is one of food components, as an active ingredient, and can be used in various forms because it is effective at a low concentration. It can be widely applied to foods and drinks, agricultural chemicals, feeds, pharmaceuticals, and the like.

Claims (11)

  A Porphyromonas gingivalis-producing biofilm formation inhibitor containing 8-2000 μg / ml lactoferrin, lactoperoxidase, glucose oxidase, and glucose as active ingredients.   The biofilm formation inhibitor according to claim 1, wherein the lactoferrin is one or more selected from the group consisting of apolactoferrin, natural lactoferrin, or hololactoferrin.   The biofilm formation inhibitor according to claim 1, which is in the form of a troche.   The biofilm formation inhibitor according to claim 3, wherein the content of lactoferrin with respect to the total mass of the lozenge is 10 to 30%.   Use of 8-2000 μg / ml lactoferrin, lactoperoxidase, glucose oxidase, and glucose for producing a Porphyromonas gingivalis producing biofilm formation inhibitor.   An antibacterial agent for Porphyromonas gingivalis containing 8 to 2000 μg / ml of lactoferrin, lactoperoxidase, glucose oxidase, and glucose and an antibacterial component as active ingredients.   The antibacterial agent according to claim 6, wherein the lactoferrin is one or more selected from the group consisting of apolactoferrin, natural lactoferrin, or hololactoferrin. A step of containing an antibacterial component,
Containing 8-2000 μg / ml lactoferrin,
A step of containing lactoperoxidase, glucose oxidase, and glucose;
A method for producing an antibacterial agent for Porphyromonas gingivalis comprising:   An antibacterial activity enhancer for Porphyromonas gingivalis, comprising 8-2000 μg / ml lactoferrin, lactoperoxidase, glucose oxidase, and glucose as active ingredients.   The antibacterial action enhancer according to claim 9, wherein the lactoferrin is one or more selected from the group consisting of apolactoferrin, natural lactoferrin, or hololactoferrin.   Use of 8-2000 μg / ml lactoferrin, lactoperoxidase, glucose oxidase, and glucose for producing an antibacterial action enhancer against Porphyromonas gingivalis.

Images