JP6560891B2 - Biofilm dispersion method - Google Patents

Description

  The present invention relates to a biofilm dispersion method.

  Biofilms are also called biofilms or slimes, and are generally formed of macromolecules such as polysaccharides, proteins, and nucleic acids produced by microbial cells when microorganisms such as bacteria adhere to the surface of substances in the aqueous system and grow. , Refers to a structure that encloses microorganisms.

When a biofilm is formed, various problems due to microorganisms occur, which is a problem in various industrial fields.
For example, when a biofilm is formed in the piping of a food plant, the biofilm may be peeled off and mixed into the product as a foreign substance, or food poisoning may be caused by toxins derived from microorganisms.
Also, biofilm formation on the metal surface causes metal corrosion and promotes aging of the equipment.
In the medical field, it is known that nosocomial infections are caused when microorganisms remaining in narrow gaps or holes in medical devices such as endoscopes form biofilms.
In addition, it is known that when a biofilm called dental plaque (dental plaque) is formed on a tooth in a human oral cavity, it causes caries and periodontal disease.

  In order to prevent various problems caused by biofilms, the idea of preventing bacteria from growing by giving bactericidal and bacteriostatic effects to microorganisms, particularly bacteria, has been generally studied. However, microbial aggregates that have formed biofilms are more resistant to microbial control agents such as bactericides and bacteriostatic agents, and the penetration of drugs into the biofilms, compared to suspended microorganisms. Is known to be inadequate. For this reason, the method of preventing the harm by a biofilm is examined.

Until now, methods using proteases have been proposed as techniques for removing biofilms (see Patent Documents 1 and 2). However, the biofilm removal performance is not yet satisfactory. Furthermore, many proteases are irritating to the living body, and safer and more effective technical development is required.
On the other hand, it is known that natto extract or carrageenan is blended in an oral composition for removing plaque in the human oral cavity or inhibiting and removing caries fungi (for example, Patent Document 3). And 4). However, the synergistic effect of biofilm dispersion by using natto extract and carrageenan in combination, and the blending amount of each component for showing the synergistic effect of biofilm dispersion by using these in combination are not known at all.

JP 2010-6720 A JP-A-6-262165 Japanese Patent Application Laid-Open No. 2011-126819 JP-A-5-139799

  An object of the present invention is to provide a biofilm dispersion method capable of effectively dispersing a biofilm.

  The present inventor conducted various studies in order to realize a method for removing a biofilm that can effectively disperse the biofilm. As a result, it has been found that a biofilm can be effectively removed by using nattokinase together with a predetermined amount of ι-carrageenan or κ-carrageenan. The present invention has been completed based on this finding.

The present invention relates to a biofilm dispersion method in which a biofilm dispersion solution containing nattokinase and carrageenan is applied to a biofilm under the following conditions (1) or (2).
(1) A biofilm dispersion solution containing nattokinase in an amount of 0.0001% by mass to 0.0005% by mass and ι-carrageenan in an amount of 0.01% by mass to 0.10% by mass. (Except for a dispersion containing 0005% by mass and 0.10% by mass of iota-carrageenan) is applied to the biofilm.
(2) A biofilm dispersion solution containing nattokinase in an amount of 0.001% to 0.010% by mass and κ-carrageenan in an amount of 0.5% to 1.0% by mass (provided that nattokinase is 0.1% by mass). 001% by weight, except for the dispersion containing 1.0% by weight of κ-carrageenan) is applied to the biofilm.

  According to the biofilm dispersion method of the present invention, the biofilm can be effectively dispersed. Furthermore, it is possible to prevent harm caused by biofilms in various fields involving microorganisms.

  In the method for dispersing a biofilm according to the present invention, a biofilm dispersion solution containing a predetermined amount of nattokinase and carrageenan is applied to the biofilm under the conditions (1) or (2). Thereby, a biofilm can be disperse | distributed effectively and synergistically rather than a mere additive effect of the biofilm dispersion | distribution effect when nattokinase and carrageenan are used individually, respectively.

Nattokinase is a protease produced by Bacillus natto, and can be produced, for example, by the method described in JP-A-6-153777. The amino acid sequence of nattokinase is registered as NCBI Accession: AHZ12722.1, Accession: AHW57451.1, Accession: AEV91244.1, Accession: AHY94992.1, and the like. Furthermore, the base sequence is registered as Accession: KJ174339.1, Accession: KJ174338.1, Accession: KJ495732.1, etc. Based on this information, nattokinase can also be produced using a conventional genetic recombination technique.
Although it does not restrict | limit especially as a nattokinase used by this invention, For example, commercially available things, such as a nattokinase by a Wako Pure Chemical Industries Ltd., can be used.

Carrageenan is a kind of sulfur-containing polysaccharide obtained from red algae, and is a compound mainly composed of D-galactose and sulfuric acid.
ι-Carrageenan is a carrageenan obtained from Eucheuma spinosum , and κ -carrageenan is a carrageenan obtained from Kappaphycus cottonii . These carrageenans can be produced, for example, by the methods described in JP-A-58-98046 and JP-A-58-98047.
The ι-carrageenan and κ-carrageenan used in the present invention are not particularly limited. For example, commercially available products such as ι-carrageenan and κ-carrageenan manufactured by Tokyo Chemical Industry Co., Ltd. can be used.

In the present invention, the nattokinase and carrageenan are dissolved or dispersed in a solvent, and the obtained dispersion solution is applied to an object to which the biofilm is attached to disperse the biofilm.
Although there is no restriction | limiting in particular as a solvent which can be used by this invention, Water, ethanol, propylene glycol, dimethylsulfoxide, etc. are mentioned. Of these, water and ethanol are preferred.
In addition, as long as the nattokinase and carrageenan come into a solution state when the nattokinase and carrageenan come into contact with the biofilm, the pre-application dosage form may be a tablet, granule, powder or the like. Therefore, pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, dilutions An appropriate combination of agents, other medicinal ingredients, and the like can be used.

The contents of the nattokinase and carrageenan in the dispersion solution used in the present invention can be appropriately determined so as to achieve the above-mentioned predetermined concentration.
For example, in the case of combined use of nattokinase and ι-carrageenan, the total concentration of both components contained in the dispersion solution is preferably 0.0101% by mass or more and 0.1001% by mass or less, and in the case of combined use of nattokinase and κ-carrageenan, The total concentration of both components contained in the solution is preferably 0.501% by mass or more and 1.01% by mass or less.

The biofilm dispersion solution used in the present invention can be prepared by dissolving the nattokinase and carrageenan in a solvent so as to have a predetermined concentration.
Alternatively, a dilution composition containing nattokinase and carrageenan at a high concentration was prepared, and diluted with a solvent so that nattokinase and carrageenan had a predetermined concentration before being applied to a biofilm. Diluents can also be used. In this case, the dilution ratio of each component is preferably 2 times or more, more preferably 5 times or more, more preferably 10 times or more, and preferably 99 times or less, more preferably 75 times or less, More preferably, it is 50 mass times or less.

Associated with the formation of the biofilm, the bacteria form a biofilm dispersion by the method of the present invention, for example, Pseudomonas (Pseudomonas) bacteria, Porphyromonas (Porphyromonas) bacteria, Yersinia (Yersinia) bacteria, Escherichia (Escherichia) bacteria, Salmonella (Salmonella) bacteria, Haemophilus (Haemophilus) bacteria, Helicobacter (Helicobacter) bacteria, Bacillus (Bacillus) bacteria, Borrelia (Borrelia) bacteria, Neisseria (Neisseria) bacteria, Campylobacter ( Campylobacter) bacteria belonging to the genus, Day Roh Cox (Deinococcus) bacteria belonging to the genus Mycobacterium (Mycobacterium) bacteria belonging to the genus Enterococcus (Enterococcus) bacteria belonging to the genus Streptococcus (Streptococcus) bacteria belonging to the genus Shigella (Shigella) bacteria belonging to the genus Aeromonas (Aeromonas) bacteria belonging to the genus, Aiken La (Eikenella) bacteria belonging to the genus Clostridium (Clostridium) bacteria, Staphylococcus (Staphylococcus) bacteria belonging to the genus Lactobacillus (Lactobacillus) bacteria belonging to the genus Actinobacillus (Actinobacillus) bacteria belonging to the genus Actinomyces (Actinomyces) bacteria belonging to the genus Bacteroides ( Bacteroides) bacteria belonging to the genus, Cap Roh site file moth (Capnocytophaga) bacteria belonging to the genus Klebsiella (Klebsiella) bacteria belonging to the genus, Harobachirusu (Halobacillus) bacteria, Fusobacterium (Fusobacterium) bacteria belonging to the genus Erwinia (Erwinia) bacteria belonging to the genus, Erubenera (Elbenella) bacteria belonging to the genus , Lactobacillus (Lactobacillus) bacteria belonging to the genus Listeria (Listeria) bacteria belonging to the genus Mannheimia (Mannheimia) bacteria belonging to the genus, Peputokokkasu (Peptococcus) bacteria belonging to the genus Prevotella (Prevotella) bacteria belonging to the genus, Proteus (Proteus) bacteria belonging to the genus Serratia (Serratia) bacteria belonging to the genus as well as Such as Ironera (Veillonella) bacteria belonging to the genus, and the like.
More specifically, Vibrio harveyi (Vibrio harveyi), Vibrio fischeri (Vibrio fischeri), cholera (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio Aruginorichikasu (Vibrio alginolyticus), Pseudomonas Hosuhoreumu (Pseudomonas phosphoreum ), poly Philo Sphingomonas gingivalis (Porphyromonas gingivalis), Yersinia enterocolitica (Yersinia enterocolitica), E. (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), Haemophilus influenzae (Haemophilus influenzae), Helicobacter pylori (Helicobacter pylori), Bacillus subtilis (Bacillus subtilis), Borrelia burgdorferi (Borrelia burgfdorferi), meningococcal (Neisseria meningitidis), Neisseria gonorrhoeae (Neisseria gonorrhoeae), Yersinia pestis (Yersinia pestis), Campylobacter Jeji Two (Campylobacter jejuni), Day Roh Cox radiodurans (Deinococcus radiodurans), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Enterococcus faecalis (Enterococcus faecalis), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus mu chest (Streptococcus mutans), Staphylococcus aureus (Staphylococcus aureus), Mita Shigella (Shigella flexneri), Shigella Boidei (Shigella boydii), Bacillus cereus (Bacillus cereus), Bacillus Kubuchirisu (Bacillus cubtilis), Aeromonas hydrophila ( Aeromonas hydrophila), Salmonella typhi (Salmonella enterica), Eikenera-Korodensu (Eikenella corrodens), Helicobacter Hepatikasu (Helicobacter hepaticus), Clostridium perfringens (Clostridium perfringens), staphylococcal · Haemoritikasu (Staphylococcus haemolyticus), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus casei (Lactobacillus casei ), Streptococcus Sanginisu (Streptococcus sanguinis), Streptococcus anginosus (Streptococcus anginosus), Streptococcus oralis (Streptococcus oralis), Streptococcus bovis (Streptococcus bovis), Streptococcus Gorudoni (Streptococcus gordonii), Streptococcus mitis (Streptococcus mitis), Actinobacillus actinomycetemcomitans (Actinobacillus actinomycetemcomitans), Biburi Vulnificus (Vivrio vulnificus), Vibrio Mimikusu (Vibrio mimicus), Vibrio Anguirarumu (Vibrio anguillarum), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Erwinia Amirobora (Erwinia amylovora), Erwinia Karotobara (Erwinia carotovara), Harobachirusu - halophilus (Halabacilus halophilus), Serratia Pimuchika (Serratia pymuthica), Serratia marcescens (Serratia marcescens), Bacteroides fragilis (Bacteroides fragilis), Bacteroides vulgatus (Bacteroides vulgatus), Bacteroides Disutasonisu (Bacteroides distasonis), Listeria Genes ( Listeria monocytogenes ), Staphylococcus epidermidis , Clostridium difficile ( C lostridium difficile), Aeromonas hydrophila Philia (Aeromonas hydrophilia), Mannheimia Haemoraitika (Mannhemia haemolytica), Klebsiella pneumoniae (Klebsiella pneumoniae), Bacillus anthracis (Bacillus anthracis), Campylobacter coli (Campylobacter coli), Campylobacter rectus (Campylobacter rectus), Proteus mirabilis (Proteus mirabilis), Actinomyces Naesurandi (Actinomyces naeslundii), Peputokokkasu-Anaerobiusu (Peptococcus anaerobius), Fusobacterium nucleatum (Fusobacterium nucleatum), Beironera-Parla (Veillonella parvula), Cap Roh site file like moth-Suputigena (Capnocytophaga sputigena) and Prevotella inter media (Prevotella intermedia).

In the biofilm dispersion method of the present invention, the biofilm can be dispersed and removed by immersing an object with the biofilm attached to the dispersion solution and allowing it to stand. Moreover, a biofilm can be disperse | distributed and removed by apply | coating or spraying the said dispersion solution on a biofilm, and leaving still.
In order to promote the dispersion and removal of the biofilm, the surface on which the biofilm is present may be rubbed after contacting the biofilm and the dispersion solution as described above.

  The time for contacting the biofilm and the dispersion solution can be set as appropriate, but is preferably 30 seconds or longer, more preferably 1 minute or longer.

The biofilm dispersion method of the present invention also includes a mode in which the biofilm adhering to teeth is dispersed or removed therapeutically or non-therapeutically by applying the dispersion solution in the oral cavity of a human or non-human animal. In this case, the application amount of the dispersion solution may be an amount that can disperse the biofilm, and can be appropriately determined depending on the administration method, age, symptoms, and the like. For example, when nattokinase and ι-carrageenan are used in combination, the total mass of the active ingredient is usually 0.01 to 1 g, preferably 0.1 to 1 g. In the case of combined use of nattokinase and κ-carrageenan, the amount is usually 0.1 to 10 g, preferably 5 to 10 g.
In addition, although the time which applies the said dispersion solution in an oral cavity can be set suitably, Preferably it is 30 second or more, More preferably, it is 1 minute or more.
Further, in this specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

The present invention also provides a biofilm dispersion solution comprising the following solution (3) or (4).

(3) A solution containing 0.0001% by mass or more and 0.0005% by mass or less of nattokinase and 0.01% by mass or more and 0.10% by mass or less of ι-carrageenan (however, 0.0005% by mass of nattokinase And a solution containing 0.10% by mass of iota-carrageenan).
(4) A solution containing 0.001% by mass to 0.010% by mass of nattokinase and 0.5% by mass to 1.0% by mass of κ-carrageenan (however, 0.001% by mass of nattokinase) And a solution containing 1.0% by mass of κ-carrageenan).

Here, the total content of both components in the dispersion solution (3) is preferably 0.01% by mass or more and 1% by mass or less, and more preferably 0.01% by mass or more and 0.1% by mass or less. The total content of both components in the dispersion solution (4) is preferably 0.1% by mass or more and 1% by mass or less, and more preferably 0.5% by mass or more and 1% by mass or less.

  In relation to the above-described embodiments, the present invention further discloses the following biofilm dispersion method, biofilm dispersion composition, use and method.

<1> A biofilm dispersion method in which a biofilm dispersion solution containing nattokinase and carrageenan is applied to a biofilm under the following conditions (1) or (2).
(1) A biofilm dispersion solution containing nattokinase in an amount of 0.0001% by mass to 0.0005% by mass and ι-carrageenan in an amount of 0.01% by mass to 0.10% by mass. (Except for a dispersion containing 0005% by mass and 0.10% by mass of iota-carrageenan) is applied to the biofilm.
(2) A biofilm dispersion solution containing nattokinase in an amount of 0.001% to 0.010% by mass and κ-carrageenan in an amount of 0.5% to 1.0% by mass (provided that nattokinase is 0.1% by mass). 001% by weight, except for the dispersion containing 1.0% by weight of κ-carrageenan) is applied to the biofilm.

<2> The method according to <1>, wherein the total concentration of nattokinase and iota-carrageenan contained in the dispersion solution is 0.0101% by mass or more and 0.1001% by mass or less in the condition (1).
<3> The method according to <1>, wherein the total concentration of nattokinase and κ-carrageenan contained in the dispersion solution is 0.501% by mass or more and 1.01% by mass or less in the condition (2).
<4> Pseudomonas bacteria, Porphyromonas bacteria, Yersinia bacteria, Escherichia bacteria, Salmonella bacteria, Hemophilus bacteria, Helicobacter bacteria, Bacillus bacteria, Borrelia bacteria, Neisseria bacteria, Campylobacter bacteria, Deino Cox bacteria, Mycobacterium bacteria, Enterococcus bacteria, Streptococcus bacteria, Shigella bacteria, Aeromonas bacteria, Eikenella bacteria, Clostridium bacteria, Staphylococcus bacteria, Lactobacillus bacteria, Actinobacillus bacteria, Actinobacillus bacteria, Actinomyces, Bacteroides, Capnocytofaga, Klebsiella, Halobacillus, Fusobacterium, Erwinia, Ervenera, Lactobacillus, Squirrel Disperse a biofilm formed by one or more bacteria selected from the group consisting of a rear bacterium, a manheimia bacterium, a peptococcus bacterium, a prebotella bacterium, a proteus bacterium, a serratia bacterium, and a beironella bacterium, The method according to any one of <1> to <3>.
<5> Vibrio Harvey, Vibrio fischeri, Vibrio cholera, Vibrio parahaemolyticus, Vibrio arginolyticus, Pseudomonas phospholeum, Polyphyromonas gingivalis, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus Bacteria, Borrelia burgdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Plague, Campylobacter jejuni, Deinocox radiodurance, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Yellow grape Staphylococcus, Shigella Hamada, Shigella Boyday, Bacillus cereus, Bacillus subtilis, Aeromonas hydrophila, Salmonella typhi, Aikenella chordens, Helicobacter hepaticas, Well Fungus, Staphylococcus haemolyticus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivaius, Lactobacillus casei, Streptococcus sanguinis, Streptococcus anginosus, Streptococcus botris Streptococcus gordonii, Streptococcus mittis, Actinobacillus actinomycetemcomitans, Vibrio brunicius, Vibrio micix, Vibrio anguillarum, Lactobacillus ramnosus, Erwinia amyrobora, Erwinia carotobara Serratia Marcesence, Bacteroides fragilis Bacteroides bulgatas, Bacteroides disstasonis, Listeria monocytogenes, Staphylococcus epidermidis, Clostridium difficile, Aeromonas hydrophilia, Manheimia haemolytica, Klebsiella pneumoniae, Bacillus anthracis, Campylobacter proteus, Lecco・ Formation of one or more bacteria selected from the group consisting of Mirabilis, Actinomyces naesrandi, Peptococcus anaerobius, Fusobacterium nucleatum, Beironella parla, Capnocytofaga sputigena and Prevotella intermedia The method according to any one of <1> to <4>, wherein the biofilm to be dispersed is dispersed.
<6> The method according to any one of <1> to <5>, wherein an object having a biofilm attached thereto is immersed in the dispersion solution to disperse the biofilm.
<7> The method according to any one of <1> to <5>, wherein the biofilm is dispersed by applying or spraying the dispersion solution on the biofilm and allowing to stand.
<8> The method according to any one of <1> to <5>, wherein after the dispersion solution and the biofilm are brought into contact with each other, the surface on which the biofilm is present is rubbed to disperse the biofilm. .
<9> The method according to any one of <1> to <5>, wherein the dispersion solution is applied to a human oral cavity to disperse a biofilm attached to teeth.

<10> A biofilm dispersion solution comprising the following solution (3) or (4).

(3) A biofilm dispersion containing 0.0001% by mass or more and 0.0005% by mass or less of nattokinase and 0.01% by mass or more and 0.10% by mass or less of ι-carrageenan (provided that nattokinase is 0.00%). (Excluding solutions containing 0005% by mass and 0.10% by mass of iota-carrageenan).
(4) A biofilm dispersion solution containing nattokinase in an amount of 0.001% by mass to 0.010% by mass and κ-carrageenan in an amount of 0.5% by mass to 1.0% by mass. (Excluding solutions containing 001% by weight and 1.0% by weight of κ-carrageenan).

<11> Use of the solution (3) or (4) as a biofilm dispersion solution.
<12> Use of the solution (3) or (4) for the production of a biofilm dispersion solution.
<13> A method of using the solution (3) or (4) as a biofilm dispersion solution.
<14> The solution (3) or (4) used for a method for removing a biofilm in the oral cavity.
<15> Non-therapeutic use of the solution (3) or (4) for a method for removing a biofilm in the oral cavity.
<16> Use of the solution (3) or (4) for producing a biofilm remover in the oral cavity.

<17> The <10> to <16>, wherein the total concentration of nattokinase and ι-carrageenan contained in the solution (3) is 0.0101% by mass or more and 0.1001% by mass or less. Solutions, uses or methods.
<18> The <10> to <16>, wherein the total concentration of nattokinase and κ-carrageenan contained in the solution (4) is 0.501% by mass or more and 1.01% by mass or less. Biofilm dispersion solution, use or method.
<19> The biofilm is a Pseudomonas bacterium, a Porphyromonas bacterium, a Yersinia bacterium, an Escherichia bacterium, a Salmonella bacterium, a Hemophilus bacterium, a Helicobacter bacterium, a Bacillus bacterium, a Borrelia bacterium, a Neisseria bacterium, Campylobacter bacteria, Deinocox bacteria, Mycobacterium bacteria, Enterococcus bacteria, Streptococcus bacteria, Shigella bacteria, Aeromonas bacteria, Eikenella bacteria, Clostridium bacteria, Staphylococcus bacteria, Lactobacillus bacteria, Actinobacillus bacteria, Actinomyces bacteria, Bacteroides bacteria, Capnocytofaga bacteria, Klebsiella bacteria, Halobacillus bacteria, Fusobacterium bacteria, Erwinia bacteria, Ervenella bacteria, La Biofilm formed by one or more bacteria selected from the group consisting of Tobacillus bacteria, Listeria bacteria, Manheimia bacteria, Peptococcus bacteria, Prevotella bacteria, Proteus bacteria, Serratia bacteria, and Beironella bacteria The solution, use or method according to any one of <10> to <18>, wherein
<20> The biofilm is Vibrio Harvey, Vibrio Fischeri, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio arginolyticus, Pseudomonas phospholeum, Polyphyromonas gingivalis, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Pest fungus, Campylobacter jejuni, Deinocox radiodurance, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus Mutans, Staphylococcus aureus, Shigella Hamada, Shigella Boyday, Bacillus cereus, Bacillus subtilis, Aeromonas hydrophila, Salmonella typhi, Eikenella collodens, Helicobacter・ Hepaticas, Clostridium perfringens, Staphylococcus haemolyticus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivaius, Lactobacillus casei, Streptococcus sanguinis, Streptococcus anginasus streptococcus streptococcus Bovis, Streptococcus gordonii, Streptococcus mittis, Actinobacillus actinomycetemcomitans, Vibrio Bruni ficus, Vibrio mimics, Vibrio anguillarum, Lactobacillus rhamnosus, Erwinia amyrobora, Erwinia ceratophylla・ Pimchika, Serratia Marcesence, Ba Teleoides fragilis, Bacteroides bulgatas, Bacteroides disstasonis, Listeria monocytogenes, Staphylococcus epidermidis, Clostridium difficile, Aeromonas hydrophilia, Manheimia haemolytica, Klebsiella pneumoniae, Bacillus anthracis One or more selected from the group consisting of Lectus, Proteus Mirabilis, Actinomyces naesrandi, Peptococcus anaerobius, Fusobacterium nucleatum, Beironella parla, Capnocytofaga sputigena and Prevotella intermedia Any one of the above <10> to <19> The solution, use or method described in 1.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
In the examples, “%” represents “% by mass”.

<Test strain>
Streptococcus mutans (JCM5175) was cultured for 1 day under anaerobic conditions at 37 ° C. in 8 ml of BHI medium (Becton Dickinson). The obtained culture solution was diluted 20 times with BHI medium, and 200 μL of the diluted solution was dispensed into each well of a sterilized 96-well plate (Becton Dickinson), and cultured at 37 ° C. under anaerobic conditions for 48 hours. did. After discarding the supernatant, 200 μL of PBS was added to each well, and the operation of discarding the supernatant was performed three times for use in the experiment.

<Preparation of biofilm dispersion solution>
Nattokinase was obtained from Wako Pure Chemical Industries.
As κ-carrageenan and ι-carrageenan, those obtained from Tokyo Chemical Industry Co., Ltd. were used.
Nattokinase solution, κ-carrageenan solution and ι-carrageenan solution were prepared by dissolving each substance in BHI medium.

<Evaluation of biofilm dispersion performance>
100 μL of the biofilm dispersion solution dissolved in BHI medium was added to each well of the biofilm formed by Streptococcus mutans in 48 hours prepared by the above method, and cultured at 37 ° C. under anaerobic conditions for 1 hour.
After culturing for 1 hour, the plate was shaken for 1 minute at 1,000 rpm, and the absorbance of 80 μL of the culture solution was measured at a wavelength of 600 nm to evaluate biofilm dispersion performance. Absorbance was measured using a negative control prepared by adding only BHI medium instead of the biofilm dispersion solution to different wells of the same plate and performing the same operation. Further, the absorbance of the biofilm dispersion solution itself was measured as a sample blank.

Based on the absorbance of the sample (As), the absorbance of the negative control (An), the absorbance of the sample blank (Abs), and the absorbance of the negative control blank (Abn), the biofilm dispersion performance is calculated based on the following formula (1) did.

Biofilm dispersibility (OD ₆₀₀ value) = (As−Abs) − (An−Abn) Formula (1)

Furthermore, the biofilm dispersibility when nattokinase is allowed to act alone (Bn) and the biofilm dispersibility when it is acted solely by ι-carrageenan or κ-carrageenan, calculated based on the above formula (1) Based on the biofilm dispersibility (Bnc) when combined with (Bc), nattokinase, and ι-carrageenan or κ-carrageenan, the biosynthesis of the biofilm is calculated based on the following formula (2). did.

Biofilm dispersion synergy ratio = Bnc / (Bn + Bc) Formula (2)

  The dispersion evaluation results thus obtained are shown in Table 1 (dispersion synergistic effect of nattokinase and ι-carrageenan) and Table 2 (dispersion synergistic effect of nattokinase and κ-carrageenan).

  As is apparent from Tables 1 and 2, when a nattokinase and ι-carrageenan or κ-carrageenan are used in combination and a dispersion solution containing a predetermined amount of these is used, a synergistic effect of biofilm dispersion is recognized, which is excellent. Biofilm removability was demonstrated.

Claims (3)

Following (1) or condition (2), biofilm dispersion solvent solution comprising nattokinase and carrageenan.
(1) nattokinase was 0.0001 wt% 0.0005 wt% or less, and ι- carrageenan containing 0.10 wt% 0.01 wt% biofilm dispersion solution (however, nattokinase 0 containing .0005 wt%, excluding the dispersion solution containing 0.10% by weight of ι- carrageenan.).
(2) nattokinase was contained 0.010 wt% 0.001 wt%, and biofilm dispersion solution containing 1.0 wt% to 0.5 wt% of κ- carrageenan (where 0 nattokinase (Excluding dispersion solutions containing 0.001% by mass and 1.0% by mass of κ-carrageenan ).   The solution for biofilm dispersion according to claim 1, wherein the total concentration of nattokinase and iota-carrageenan is 0.0101% by mass or more and 0.1001% by mass or less in the condition (1).   2. The biofilm dispersion solution according to claim 1, wherein, under the condition (2), the total concentration of nattokinase and κ-carrageenan is 0.501% by mass or more and 1.01% by mass or less.