Anti-gram-negative bacterial compound

Abstract

To provide antimicrobial compounds having a broad antimicrobial spectrum.SOLUTION: The present invention provides an anti-Gram-negative bacterial compound having a sequence represented by the following chemical formula. The present invention provides Bacillus subtilis capable of producing a compound having a sequence represented by the following chemical formula. The present invention provides Bacillus subtilis MT2 strain (accession number: NITE BP-02767).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to anti-gram-negative bacterial compounds. [Background technology]

[0002] Peptides and proteins with antibacterial activity produced by bacteria are known as bacteriocins. Since bacteriocins are naturally occurring substances, they are considered to have less impact on the human body compared to antibacterial agents made of synthetic substances, and nisin, a bacteriocin produced by the lactic acid bacterium Lactococcus lactis, is approved for addition to foods as a preservative in many countries around the world (see, for example, Patent Documents 1 and 2).

[0003] Bacteriocins are known to have antibacterial effects against bacteria related to the producing bacteria, and therefore, it is generally believed that bacteriocins derived from gram-positive bacteria have antibacterial effects against bacteria related to the producing bacteria, which are gram-positive bacteria, but have no antibacterial effects against gram-negative bacteria.

[0004] The above-mentioned nisin also has an antibacterial effect against gram-positive bacteria, but not against gram-negative bacteria. Therefore, nisin has no antibacterial effect against, for example, Vibrio parahaemolyticus, a bacterium that causes food poisoning from seafood. Bacteriocins other than nisin, such as subtilin and entianin, have antibacterial effects against gram-positive bacteria, but not against gram-negative bacteria. This is thought to be because the outer membrane of gram-negative bacteria forms a barrier against bacteriocins derived from gram-positive bacteria (see, for example, Non-Patent Documents 1 to 3).

[0005] Moreover, bacteriocins derived from gram-positive bacteria are thought to have no antibacterial activity against fungi. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4111489 [Patent Document 2] Patent No. 5738561 [Non-patent literature]

[0007] [Non-Patent Document 1] J. Barbosa, et al., “Class I and Class II Lanthipeptides Produced by Bacillus spp.”, J. Nat. Prod. 2015, 78, 2850-2866 [Non-Patent Document 2] CV Prudencio, et al., “Strategies for the use of bacteriocins in Gram-negative bacteria: relevance in food microbiology”, J Food Sci Technol (September 2015), 52(9):5408-5417 [Non-Patent Document 3] Takeshi Zento et al., "Discovery and Use of Lactic Acid Bacterial Bacteriocins," Japanese Journal of Lactic Acid Bacteria, Vol. 25, No. 1, 2014, Japan Society for Lactic Acid Bacteria Summary of the Invention [Problem to be solved by the invention]

[0008] As described above, conventional bacteriocins have an antibacterial activity only against bacteria related to the bacterium that produces them, and therefore have a narrow antibacterial spectrum. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an antibacterial compound with a broad antibacterial spectrum. [Means for solving the problem]

[0009] An anti-Gram-negative bacterial compound according to one aspect of the present invention has an amino acid sequence shown in Chemical Formula 1 below. [ka]

[0010] Furthermore, an anti-Gram-negative bacteria drug according to one aspect of the present invention includes an anti-Gram-negative bacteria compound having the amino acid sequence shown in Chemical Formula 1 above.

[0011] Furthermore, a beverage or food according to one aspect of the present invention comprises an anti-Gram-negative bacteria compound having the amino acid sequence shown in Chemical Formula 1 above.

[0012] Furthermore, Bacillus subtilis according to one aspect of the present invention is a Bacillus subtilis capable of producing a compound having the amino acid sequence represented by Chemical Formula 1 above.

[0013] Furthermore, an anti-Gram-negative bacterial agent according to one aspect of the present invention includes a Bacillus subtilis capable of producing a compound having the amino acid sequence represented by Chemical Formula 1 above.

[0014] Furthermore, a beverage or food according to one aspect of the present invention comprises a Bacillus subtilis capable of producing a compound having the amino acid sequence shown in Chemical Formula 1 above.

[0015] Moreover, a Bacillus subtilis according to one aspect of the present invention is the Bacillus subtilis MT2 strain (accession number: NITE BP-02767).

[0016] Moreover, an anti-Gram-negative bacterial agent according to one aspect of the present invention includes Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767).

[0017] Furthermore, a beverage or food according to one aspect of the present invention contains Bacillus subtilis strain MT2 (accession number: NITE BP-02767).

[0018] Furthermore, a method for producing an anti-Gram-negative bacterial compound according to one aspect of the present invention includes culturing Bacillus subtilis and collecting a compound having the amino acid sequence shown in Chemical Formula 1 produced by Bacillus subtilis.

[0019] Furthermore, a method for producing an anti-Gram-negative bacterial compound according to one aspect of the present invention includes culturing Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767) and collecting a compound having the amino acid sequence shown in Chemical Formula 1 produced by the Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767).

[0020] Furthermore, one aspect of the present invention is the use of an anti-Gram-negative bacteria compound having the amino acid sequence shown in Chemical Formula 1 in the manufacture of an anti-Gram-negative bacteria drug.

[0021] Furthermore, one aspect of the present invention is the use of Bacillus subtilis capable of producing a compound having the amino acid sequence shown in Chemical Formula 1 in the production of an anti-Gram-negative bacterial drug.

[0022] Moreover, one aspect of the present invention is the use of the Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767) in the production of an anti-Gram-negative bacterial agent.

[0023] Furthermore, a method for treating enteritis according to one aspect of the present invention comprises administering to a patient an anti-Gram-negative bacterial compound having the amino acid sequence represented by Chemical Formula 1 above.

[0024] Furthermore, a method for treating enteritis according to one aspect of the present invention comprises administering to a patient a Bacillus subtilis capable of producing a compound having the amino acid sequence represented by Chemical Formula 1 above.

[0025] Furthermore, a method for treating enteritis according to one aspect of the present invention comprises administering Bacillus subtilis MT2 strain (accession number: NITE BP-02767) to a patient. Effect of the Invention

[0026] According to the present invention, it is possible to provide an antibacterial compound with a broad antibacterial spectrum. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1(a) shows the bacteriocin production gene, FIG. 1(b) shows the bacteriocin prepeptide, and FIG. 1(c) shows the bacteriocin (active peptide). [Diagram 2] 1 is a graph showing the results of homology analysis between the entire genome sequence of Bacillus subtilis strain MT2 and the entire genome sequence of Bacillus subtilis subsp. subtilis 168 by the ANI method. [Diagram 3] 1 is a graph showing the results of homology analysis between the entire genome sequence of Bacillus subtilis strain MT2 and the entire genome sequence of Bacillus subtilis subsp. subtilis 6051-HGW by the ANI method. [Figure 4] 1 is a graph showing the results of homology analysis between the entire genome sequence of Bacillus subtilis strain MT2 and the entire genome sequence of Bacillus subtilis subsp. spizizenii W23 by the ANI method. [Diagram 5] 1 is a graph showing the results of homology analysis by the ANI method between the entire genome sequence of Bacillus subtilis strain MT2 and the entire genome sequence of Bacillus tequilensis FJAT-14262a. [Figure 6] 1 is a graph showing the results of mass spectrometry of bacteriocins. [Figure 7] 1 is a graph showing the antibacterial activity of bacteriocins. [Figure 8] 1 is a graph showing the antibacterial activity of bacteriocins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The following is a detailed description of the embodiments of the present invention. Note that the embodiments shown below are merely examples of devices and methods for embodying the technical idea of ​​the present invention, and the technical idea of ​​the present invention is not limited to the combination of components described below. The technical idea of ​​the present invention can be modified in various ways within the scope of the claims.

[0029] The anti-Gram-negative bacteria compound according to this embodiment has an amino acid sequence shown in the following chemical formula 2. The amino acid sequence shown in the following chemical formula 2 is 150 H 231 N 39 O 38 S 5 The anti-gram-negative bacteria compound according to this embodiment has an antibacterial effect against gram-negative bacteria, fungi, and gram-positive bacteria. [ka]

[0030] Examples of gram-negative bacteria that the anti-gram-negative bacteria compound according to the present embodiment exhibits antibacterial activity include Vibrio bacteria. Examples of Vibrio bacteria include Vibrio parahaemolyticus, Vibrio cholerae, Non O1 Vibrio cholerae, Vibrio mimicus, Vibrio fluvialis, Vibrio vulnificus, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio furnissii.

[0031] Examples of fungi against which the anti-gram-negative bacteria compound according to the present embodiment exhibits an antibacterial effect include Trichophyton spp. Examples of Trichophyton spp. include Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton spp., and Trichophyton verrucosum.

[0032] Examples of gram-positive bacteria against which the anti-gram-negative bacteria compound according to the present embodiment exhibits antibacterial activity include Bacillus genus bacteria. Examples of Bacillus genus bacteria include Bacillus subtilis, Bacillus cereus, Bacillus anthracis, and Bacillus stearothermophilus. Bacillus subtilis is harmful in the manufacturing process of soy sauce and miso paste.

[0033] The anti-gram-negative bacteria compound according to the present embodiment is produced, for example, from Bacillus subtilis. The anti-gram-negative bacteria compound according to the present embodiment is derived from Bacillus subtilis, which is a gram-positive bacterium, and has an antibacterial effect against gram-negative bacteria and fungi equivalent to that against gram-positive bacteria. Therefore, the anti-gram-negative bacteria compound according to the present embodiment does not need to be used in combination with a treatment that supports the antibacterial effect against gram-negative bacteria.

[0034] Treatments to aid in antibacterial action against gram-negative bacteria include the addition of preservatives, such as food preservatives. Preservatives include chelating agents, surfactants, acids and salts. An example of a chelating agent is ethylenediaminetetraacetic acid (EDTA). An example of a surfactant is Tween 80. Examples of acids and salts include lactic acid, citric acid, pyrophosphoric acid, gluconic acid, acetic acid, nitrilotriacetic acid, hypochlorous acid, sorbic acid, benzoic acid and polyphosphoric acid and their salts, as well as tri-sodium phosphate.

[0035] Preservatives are believed to destabilize the outer membrane of gram-negative bacteria. Conventionally, it has been believed that in order to kill gram-negative bacteria with bacteriocins derived from gram-positive bacteria, it is necessary to use such preservatives in combination. In contrast, the anti-gram-negative bacteria compound according to the present embodiment can exhibit an effective antibacterial effect against gram-negative bacteria even without using a preservative in combination.

[0036] In addition, the treatment of supporting the antibacterial action against gram-negative bacteria includes the addition of plant essential oil. Examples of plant essential oil include oregano essential oil and thyme essential oil. It is believed that thymol and carvacrol contained in plant essential oil destabilize the outer membrane of gram-negative bacteria. However, the anti-gram-negative bacteria compound according to the present embodiment can exhibit effective antibacterial action against gram-negative bacteria even without using plant essential oil.

[0037] In addition, a treatment that supports the antibacterial action against gram-negative bacteria includes high-pressure treatment. High-pressure treatment is, for example, a treatment that applies a pressure of 30 MPa or more. It is believed that high-pressure treatment destabilizes the outer membrane of gram-negative bacteria. However, the anti-gram-negative bacteria compound according to this embodiment can exhibit an effective antibacterial action against gram-negative bacteria even without being combined with high-pressure treatment.

[0038] In addition, examples of treatments that support the antibacterial action against gram-negative bacteria include high-temperature and low-temperature treatments. High-temperature and low-temperature treatments are thought to destabilize the outer membrane of gram-negative bacteria. In addition, examples of treatments that support the antibacterial action against gram-negative bacteria include pulsed electric field treatments. Pulsed electric field treatments are thought to have the same effect as high-temperature treatments because they increase the temperature of gram-negative bacteria. However, the anti-gram-negative bacteria compound according to this embodiment can exhibit effective antibacterial action against gram-negative bacteria even without being used in combination with high-temperature treatment, low-temperature treatment, or pulsed electric field treatment.

[0039] Bacillus subtilis capable of producing the anti-Gram-negative bacteria compound according to this embodiment has, for example, a bacteriocin production gene as shown in FIG. 1(a) and SEQ ID NO: 1. In SEQ ID NO: 1, positions 1 to 72 encode a leader peptide. The gene sequence excluding the portion encoding the leader peptide is shown in SEQ ID NO: 2. Bacillus subtilis capable of producing the anti-Gram-negative bacteria compound according to this embodiment may have the gene sequence as shown in SEQ ID NO: 2.

[0040] The production gene shown in Fig. 1(a) and SEQ ID NO: 1 is transcribed and translated to produce the bacteriocin prepeptide shown in Fig. 1(b) and SEQ ID NO: 3. In SEQ ID NO: 3, positions 1 to 24 represent the leader peptide. The amino acid sequence excluding the leader peptide is shown in SEQ ID NO: 4.

[0041] The bacteriocin prepeptide shown in FIG. 1(b) and SEQ ID NO:3 is post-translationally modified and the leader peptide is cleaved to produce an active peptide having the sequence shown in FIG. 1(c) and chemical formula 2 above.

[0042] The serine at position 3 of the prepeptide shown in SEQ ID NO: 4 is replaced with alanine in the active peptide. The serine at position 5 of the prepeptide shown in SEQ ID NO: 4 is replaced with dehydroalanine in the active peptide. The cysteine ​​at position 7 of the prepeptide shown in SEQ ID NO: 4 is replaced with alanine in the active peptide. The threonine at position 8 of the prepeptide shown in SEQ ID NO: 4 is replaced with aminobutyric acid in the active peptide.

[0043] The cysteine ​​at position 11 of the prepeptide shown in SEQ ID NO: 4 is replaced with alanine in the active peptide. The threonine at position 13 of the prepeptide shown in SEQ ID NO: 4 is replaced with aminobutyric acid in the active peptide. The threonine at position 18 of the prepeptide shown in SEQ ID NO: 4 is replaced with dehydrobutyrine in the active peptide. The cysteine ​​at position 19 of the prepeptide shown in SEQ ID NO: 4 is replaced with alanine in the active peptide.

[0044] The threonine at position 23 of the prepeptide shown in SEQ ID NO: 4 is replaced with aminobutyric acid in the active peptide. The threonine at position 25 of the prepeptide shown in SEQ ID NO: 4 is replaced with aminobutyric acid in the active peptide. The cysteine ​​at position 26 of the prepeptide shown in SEQ ID NO: 4 is replaced with alanine in the active peptide. The cysteine ​​at position 28 of the prepeptide shown in SEQ ID NO: 4 is replaced with alanine in the active peptide. The serine at position 31 of the prepeptide shown in SEQ ID NO: 4 is replaced with dehydroalanine in the active peptide.

[0045] In the active peptide, the alanine corresponding to the serine at position 3 of the prepeptide shown in SEQ ID NO: 4 and the alanine corresponding to the cysteine ​​at position 7 of SEQ ID NO: 4 are bonded via a thioether bond to form a lanthionine.

[0046] In the active peptide, aminobutyric acid corresponding to the threonine at position 8 of the prepeptide shown in SEQ ID NO: 4 and alanine corresponding to the cysteine ​​at position 11 of SEQ ID NO: 4 are bonded via a thioether bond to form 3-methyllanthionine.

[0047] In the active peptide, aminobutyric acid corresponding to the threonine at position 13 of the prepeptide shown in SEQ ID NO: 4 and alanine corresponding to the cysteine ​​at position 19 of SEQ ID NO: 4 are bonded via a thioether bond to form 3-methyllanthionine.

[0048] In the active peptide, aminobutyric acid corresponding to the threonine at position 23 of the prepeptide shown in SEQ ID NO: 4 and alanine corresponding to the cysteine ​​at position 26 of SEQ ID NO: 4 are bonded via a thioether bond to form 3-methyllanthionine.

[0049] In the active peptide, aminobutyric acid corresponding to the threonine at position 25 of the prepeptide shown in SEQ ID NO: 4 and alanine corresponding to the cysteine ​​at position 28 of SEQ ID NO: 4 are bonded via a thioether bond to form 3-methyllanthionine.

[0050] The Bacillus subtilis MT2 strain, which is one of the Bacillus subtilis strains capable of producing the anti-gram-negative bacteria compound according to this embodiment, has been internationally deposited at the National Institute of Technology and Evaluation (Japan, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Room 122), and has the deposit number NITE BP-02767. The Bacillus subtilis MT2 strain was domestically deposited at the National Institute of Technology and Evaluation (Japan) on August 22, 2018 (domestic deposit date), and a request for transfer to an international deposit based on the Budapest Treaty was made to the National Institute of Technology and Evaluation (Japan) on October 4, 2018.

[0051] The anti-gram-negative bacteria compound according to the present embodiment may be contained in a medicine. The medicine containing the anti-gram-negative bacteria compound according to the present embodiment can be used as a disinfectant for gram-negative bacteria, gram-positive bacteria, and fungi. In addition, the medicine containing the anti-gram-negative bacteria compound according to the present embodiment can be used as a treatment for pneumonia, food poisoning, and enteritis. Furthermore, the medicine containing the anti-gram-negative bacteria compound according to the present embodiment can be used as a treatment for mycoses such as tinea, candidiasis, cryptococcosis, and aspergillosis.

[0052] The pharmaceutical product contains an effective amount of the anti-Gram-negative bacteria compound according to the present embodiment. The effective amount is the amount necessary to exert an antibacterial effect, and is appropriately determined depending on the target bacteria, symptoms, age, weight, and sex of the patient, etc.

[0053] The pharmaceutical containing the anti-gram-negative bacteria compound according to the present embodiment may be, for example, a tablet, capsule, liquid, cream, ointment, paste, gel, wax, and spray. The pharmaceutical containing the anti-gram-negative bacteria compound according to the present embodiment may be, for example, a disinfectant, an oral drug, a skin topical drug such as a topical treatment, and an eye drop. The pharmaceutical containing the anti-gram-negative bacteria compound according to the present embodiment may be applied to, for example, internal organs such as the intestines, the skin of the human body including the fingers and toes, hair, the oral cavity, and the eyeballs. The disinfectant containing the anti-gram-negative bacteria compound according to the present embodiment may be applied to, for example, food, cooking utensils, walls and floors of buildings such as hospitals, and furniture such as desks.

[0054] A pharmaceutical containing the anti-Gram-negative bacteria compound according to this embodiment may contain appropriate pharmaceutical ingredients such as liquid oils and fats, solid oils and fats, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, silicones, moisturizers, water-soluble polymers, thickeners, coating agents, sequestering agents, lower alcohols, polyhydric alcohols, sugars, amino acids, organic amines, pH adjusters, skin nutrients, vitamins, fragrances, powders, coloring materials, and water, depending on the purpose.

[0055] The anti-gram-negative bacteria compound according to the present embodiment may be contained in beverages or foods. The anti-gram-negative bacteria compound according to the present embodiment may also be contained in toothpaste, oral cleansers, and the like.

[0056] The Bacillus subtilis capable of producing the anti-gram-negative bacteria compound according to this embodiment may be contained in a medicine, beverage, or food. For example, the Bacillus subtilis contained in a medicine, beverage, or food produces the anti-gram-negative bacteria compound and exerts an antibacterial effect against gram-negative bacteria, fungi, and gram-positive bacteria. The target disease, shape, and usage of the medicine containing the Bacillus subtilis capable of producing the anti-gram-negative bacteria compound according to this embodiment are the same as those of the medicine containing the anti-gram-negative bacteria compound according to this embodiment.

[0057] The Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767) may be contained in medicines, beverages, or foods. The target disease, form, and usage of the medicine containing the Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767) are the same as those of the medicine containing the anti-Gram-negative bacteria compound according to the present embodiment.

[0058] The anti-gram-negative bacteria compound according to this embodiment may be produced, for example, by culturing Bacillus subtilis in a medium and collecting a peptide having the amino acid sequence shown in the above chemical formula 2 produced by Bacillus subtilis in the medium. There is no particular limitation on the method for collecting and purifying the peptide from the medium. The anti-gram-negative bacteria compound according to this embodiment may be chemically synthesized.

[0059] As described above, the anti-Gram-negative bacteria compound and the like according to each embodiment of the present invention have the configurations and effects according to the following examples of any one or a combination of a plurality of the above-mentioned components.

[0060] The anti-gram-negative bacteria compound according to this embodiment has an amino acid sequence shown in the above chemical formula 2. The anti-gram-negative bacteria compound according to this embodiment has an antibacterial effect against gram-negative bacteria, gram-positive bacteria, and fungi.

[0061] The anti-gram-negative bacteria compound according to the present embodiment may be produced from Bacillus subtilis. The anti-gram-negative bacteria compound according to the present embodiment may be produced from Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767). The anti-gram-negative bacteria compound according to the present embodiment has an antibacterial effect against gram-negative bacteria, even though it is derived from Bacillus subtilis, which is a gram-positive bacterium.

[0062] The gram-negative bacterium against which the anti-gram-negative bacterium compound according to this embodiment exhibits an antibacterial activity may be a bacterium belonging to the genus Vibrio. The vibrio bacterium may be Vibrio parahaemolyticus.

[0063] The fungus against which the anti-gram-negative bacteria compound according to the present embodiment exhibits an antibacterial effect may be a Trichophyton fungus. The Trichophyton fungus may be a Trichophyton fungus, a Trichophyton trichophyton, or an Interdigital Fungus.

[0064] The anti-gram-negative bacteria compound according to the present embodiment does not need to be used in combination with a treatment that supports the antibacterial action against gram-negative bacteria. The treatment that supports the antibacterial action against gram-negative bacteria may be the addition of a preservative. The preservative may be a chelating agent. The anti-gram-negative bacteria compound according to the present embodiment has an antibacterial action against gram-negative bacteria equivalent to the antibacterial action against gram-positive bacteria, even if it is not used in combination with a treatment that supports the antibacterial action against gram-negative bacteria.

[0065] The anti-gram-negative bacteria drug according to this embodiment may contain the above-mentioned anti-gram-negative bacteria compound. The beverage or food according to this embodiment may contain the above-mentioned anti-gram-negative bacteria compound.

[0066] The Bacillus subtilis according to this embodiment may be a Bacillus subtilis capable of producing a compound having the amino acid sequence shown in Chemical Formula 2 above.

[0067] The anti-Gram-negative bacterial agent according to this embodiment may contain Bacillus subtilis capable of producing a compound having the amino acid sequence shown in the above Chemical Formula 2. The beverage or food according to this embodiment may contain Bacillus subtilis capable of producing a compound having the amino acid sequence shown in the above Chemical Formula 2.

[0068] The Bacillus subtilis according to this embodiment may be the Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767).

[0069] The anti-gram-negative bacterial agent according to this embodiment may contain the above-mentioned Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767). The beverage or food according to this embodiment may contain the above-mentioned Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767).

[0070] The method for producing an anti-Gram-negative bacterial compound according to this embodiment may include culturing Bacillus subtilis and collecting a compound having the amino acid sequence shown in the above chemical formula 2 produced by Bacillus subtilis.

[0071] The method for producing an anti-Gram-negative bacterial compound according to this embodiment may include culturing Bacillus subtilis strain MT2 (Accession Number: NITE BP-02767) and collecting a compound having the amino acid sequence shown in the above chemical formula 2, which is produced by Bacillus subtilis strain MT2 (Accession Number: NITE BP-02767).

[0072] This embodiment may be the use of an anti-gram-negative bacteriocin as described above in the manufacture of an anti-gram-negative drug.

[0073] This embodiment may also be the use of Bacillus subtilis capable of producing a compound having the amino acid sequence shown in Chemical Formula 2 above in the manufacture of an anti-Gram-negative bacterial drug.

[0074] The present embodiment may be the use of the Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767) in the production of an anti-Gram-negative bacterial agent.

[0075] The method for treating enteritis according to the present embodiment may include administering the anti-gram-negative bacteria compound to a patient. The method for treating enteritis according to the present embodiment may include administering Bacillus subtilis capable of producing a compound having the amino acid sequence shown in Chemical Formula 2 to a patient. The method for treating enteritis according to the present embodiment may include administering Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767) to a patient. The enteritis may be food poisoning.

[0076] The above-described embodiments are intended to facilitate understanding of the present invention, and are not intended to limit the present invention. The present invention may be modified / improved without departing from the spirit thereof, and equivalents are also included in the present invention. That is, those in which a person skilled in the art appropriately changes the design of each embodiment are also included in the scope of the present invention as long as they have the characteristics of the present invention. For example, the elements, materials, conditions, shapes, etc. of each embodiment are not limited to those exemplified, and can be appropriately changed. In addition, each embodiment is an example, and it goes without saying that partial replacement or combination of the configurations shown in different embodiments is possible, and these are also included in the scope of the present invention as long as they include the characteristics of the present invention. EXAMPLES

[0077] The following describes examples of the present invention, although it goes without saying that the present invention is not limited to the following examples.

[0078] (Example 1: Preparation of mugwort fermented liquid) It is said that the number of lactic acid bacteria in mugwort leaves is highest during a total of two hours, one hour before and one hour after sunrise. It is also said that outside of these times, the number of lactic acid bacteria decreases and the number of photosynthetic bacteria increases. Therefore, during these two hours, about 20 cm of the tip of the mugwort leaves was collected. The collected 6.3 kg of mugwort leaves were immediately placed in the first pickling barrel with a plastic bag inside, and 3.2 kg of molasses and 0.6 kg of coarse salt were sprinkled on the mugwort leaves, and the opening of the plastic bag was closed and sealed. A weight was placed on top of the plastic bag, and the mugwort leaves were pickled.

[0079] A few days after the pickling juice had risen above the mugwort leaves, the weight was removed. Next, 10 L of chlorine-free water for rinsing was poured into the second pickling barrel, and the mugwort leaf pickles and 10 kg of pickling juice were placed in the water. A third pickling barrel was then prepared, and a wire mesh filter was placed on the opening of the third pickling barrel. The mugwort leaves were taken out of the second pickling barrel little by little while rubbing them with their hands, and the mugwort leaves were lightly pressed with the palm of the hand onto the wire mesh filter above the opening of the third pickling barrel to squeeze out the pickling juice.

[0080] After squeezing out all the mugwort leaves, the pickling juice remaining in the second pickling juice was filtered through a wire mesh filter. Next, molasses (Hateruma brown sugar) was dissolved in the pickling juice in the third pickling barrel to a final concentration of 10% by weight, and coarse salt was dissolved in a final concentration of 3% by weight. Then, the ambient temperature of the third pickling barrel was raised to about 30°C to start fermentation. Large bubbles were observed at first, which gradually changed to fine bubbles, and finally the foaming subsided. After about one week, the pH was around 3.8 when the foaming subsided. The pickling juice at this time was used as the mugwort fermented liquid.

[0081] Example 2: Preparation of Bacillus subtilis The mugwort fermentation liquid was added dropwise to Lactobacillus subtilis MRS medium (1% proteose peptone, 1% beef extract, 0.5% yeast extract, 2% glucose, 0.1% Tween 80, 0.5% ammonium citrate, 0.01% magnesium sulfate, 0.005% manganese sulfate, 0.2% dipotassium phosphate, Difco), and the bacteria contained in the mugwort fermentation liquid were cultured in the medium, and several subcultures were performed as necessary. The cultured bacteria were then smeared and cultured on Lactobacillus subtilis MRS agar medium (the above MRS medium with 3% agar added), and bacteria were collected from the resulting colonies.

[0082] Next, the strains capable of producing antibacterial substances were selected from the collected bacteria by the spot-on-lawn test using the antibacterial activity against the spore-forming lactic acid bacterium Bacillus coagulans as an index, and identified as Bacillus subtilis MT2 strain. The bacteriological properties of the selected Bacillus subtilis MT2 strain were investigated, and it was identified as Bacillus subtilis by the analysis of the homology of the 16S ribosomal DNA (rDNA) base sequence.

[0083] The average homology between the whole genome sequence of the Bacillus subtilis MT2 strain and the whole genome sequence of Bacillus subtilis subsp. subtilis 168, an existing Bacillus subtilis strain, was calculated using the Average Nucleotide Identity (ANI) method, and was found to be 98.5%, a difference of 1.5%, as shown in Figure 2.

[0084] The average homology between the whole genome sequence of the Bacillus subtilis MT2 strain and the whole genome sequence of Bacillus subtilis subsp. subtilis 6051-HGW, an existing Bacillus subtilis strain, was calculated using the ANI method to be 98.49%, as shown in Figure 3, indicating a difference of 1.51%.

[0085] The average homology value between the complete genome sequence of the Bacillus subtilis MT2 strain and the complete genome sequence of Bacillus subtilis subsp. spizizenii W23, an existing Bacillus subtilis strain, was calculated using the ANI method to be 92.03%, as shown in Figure 4, indicating a difference of 7.97%.

[0086] The average homology between the whole genome sequence of the Bacillus subtilis strain MT2 and the whole genome sequence of Bacillus tequilensis FJAT-14262a, an existing Bacillus subtilis strain, was calculated using the ANI method to be 98.49%, as shown in Figure 5, which is a difference of 1.51%.

[0087] From the comparison with the existing Bacillus subtilis strains shown above, it was considered that the Bacillus subtilis MT2 strain is a new strain of Bacillus subtilis. The Bacillus subtilis MT2 strain was internationally deposited at the National Institute of Technology and Evaluation (Japan) and was assigned the accession number: NITE BP-02767. The Bacillus subtilis MT2 strain had the gene sequences shown in SEQ ID NOs: 1 and 2.

[0088] Example 3: Preparation of bacteriocins Bacillus subtilis MT2 strain was aerobically cultured for 3 days in Lactobacillus MRS medium at 35° C. to 37° C. with shaking. The medium containing the cells was then centrifuged at 10,000 rpm for 20 minutes at 25° C. The centrifuged medium was then filtered through a 0.45 μm sterilized filter to remove the cells, and a culture supernatant was obtained.

[0089] The bacteriocin in the culture supernatant was concentrated using a reverse-phase silica gel cartridge (Sep-Pac C18, Millipore), and the active bacteriocin fraction was fractionated by reverse-phase column chromatography using Sephasil Peptide C185μST (Pharmacia).

[0090] Mass spectrometry of the fractionated active bacteriocin was performed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOFMS ultrafleX III, Bruker), and the precise molecular weight was about 3346, as shown in FIG. 6. The target plate used was MTP384 ground steel (Bruker Daltonics). After various investigations, α-cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix. The amino acid sequence of the fractionated active bacteriocin was analyzed, and it was found to have the amino acid sequence shown in Chemical Formula 2 above.

[0091] Example 4: Antibacterial activity of bacteriocins Bacillus subtilis (NBRC 3134) was used as a gram-positive bacterium, and Vibrio parahaemolyticus (12711T) was used as a gram-negative bacterium. 10 mL of either of the above bacteria was added to 10 mL of a solution containing the active bacteriocin prepared in Example 3 and having a pH of 7.8. 7 0.1 mL of a bacterial solution containing 10 mL of Vibrio parahaemolyticus (12711T) at a concentration of 1.067 mol / L was inoculated into the culture, and the viable cell count was measured after 24 hours at 25° C. As a control, 0.1 mL of a bacterial solution containing Vibrio parahaemolyticus (12711T) was inoculated into 10 mL of a phosphate buffer solution with a concentration of 0.067 mol / L and pH 7.2, and the viable cell count was measured after 24 hours at 25° C. As a result, as shown in FIG. 7, the active bacteriocin reduced both Bacillus subtilis and Vibrio parahaemolyticus to the same extent.

[0092] Furthermore, tinea fungus (Trichophyton mentagrophytes, NBRC 6124) was prepared as a fungus. 10 mL of the solution containing the active bacteriocin prepared in Example 3 and having a pH of 7.8 was added to the solution. 7A 0.1 mL solution containing 10 mL of phosphate buffer with a concentration of 0.067 mol / L and pH 7.2 was inoculated with 0.1 mL of a solution of Trichophyton mentagrophytes (NBRC 6124) and the viable cell count was measured after 24 hours at 25° C. As a control, a 0.1 mL solution of Trichophyton mentagrophytes (NBRC 6124) was inoculated into 10 mL of phosphate buffer with a concentration of 0.067 mol / L and pH 7.2 and the viable cell count was measured after 24 hours at 25° C. As a result, as shown in FIG. 8, the active bacteriocin reduced the Trichophyton.

Claims

1. An anti-gram-negative bacterial compound having the amino acid sequence shown in the chemical formula below. 【Chemistry 1】

2. 2. The anti-gram-negative compound of claim 1, produced by Bacillus subtilis.

3. The anti-gram-negative bacterial compound according to claim 1, which is produced from Bacillus subtilis strain MT2 (accession number: NITE BP-02767).

4. The anti-gram-negative bacteria compound according to claim 1 , wherein the gram-negative bacteria is a Vibrio bacterium.

5. The anti-gram-negative bacteria compound according to any one of claims 1 to 3, wherein the gram-negative bacteria is Vibrio parahaemolyticus.

6. The anti-gram-negative compound according to any one of claims 1 to 5, which further has an antibacterial effect against fungi.

7. The anti-gram negative bacterial compound according to claim 6, wherein the fungus is a Trichophyton fungus.

8. The anti-gram negative compound according to claim 6, wherein the fungus is Trichophyton.

9. The anti-gram negative compound according to claim 6, wherein the fungus is Menthol or Interdigital Fungus.

10. The anti-gram-negative bacteria compound according to any one of claims 1 to 9, which further has an antibacterial activity against gram-positive bacteria.

11. 11. The anti-gram-negative compound according to any one of claims 1 to 10, not in combination with a treatment to support the antibacterial action against gram-negative bacteria.

12. The anti-gram negative bacterial compound of claim 11 , wherein the treatment is the addition of a preservative.

13. The anti-gram negative bacterial compound of claim 12 , wherein the preservative is a chelating agent.

14. An anti-gram-negative bacterial drug comprising an anti-gram-negative bacterial compound according to any one of claims 1 to 13.

15. A beverage or food comprising an anti-gram-negative bacterium compound according to any one of claims 1 to 13.

16. Bacillus subtilis capable of producing a compound having the amino acid sequence shown in the chemical formula below. 【Chemistry 2】

17. An anti-gram-negative agent comprising the Bacillus subtilis of claim 16.

18. A beverage or food comprising the Bacillus subtilis according to claim 16.

19. Bacillus subtilis strain MT2 (accession number: NITE BP-02767).

20. An anti-Gram-negative agent containing Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767).

21. Beverages or foods containing Bacillus subtilis MT2 strain (Accession Number: NITE BP-02767).

22. Cultivating Bacillus subtilis; collecting a compound having an amino acid sequence represented by the following chemical formula produced by the Bacillus subtilis; A method for producing an anti-Gram-negative bacterial compound, comprising: 【Chemistry 3】

23. Cultivating Bacillus subtilis MT2 strain (accession number: NITE BP-02767), collecting a compound having an amino acid sequence represented by the following chemical formula, which is produced by the Bacillus subtilis MT2 strain (accession number: NITE BP-02767); A method for producing an anti-Gram-negative bacterial compound, comprising: 【Chemistry 4】

24. 14. Use of an anti-gram-negative bacteriocin according to any one of claims 1 to 13 in the manufacture of an anti-gram-negative drug.

25. Use of the Bacillus subtilis strain according to claim 16 in the manufacture of an anti-Gram-negative drug.

26. Use of Bacillus subtilis MT2 strain (accession number: NITE BP-02767) in the production of an anti-Gram-negative bacterial agent.

27. 14. A method for treating enteritis comprising administering to a patient an anti-Gram-negative bacterial compound according to any one of claims 1 to 13.

28. A method for treating enteritis, comprising administering the Bacillus subtilis of claim 16 to a patient.

29. A method for treating enteritis, comprising administering Bacillus subtilis MT2 strain (accession number: NITE BP-02767) to a patient.

Images