Anti-Gram-negative compounds

Abstract

A compound for resisting Gram-negative bacteria having a sequence shown in the following chemical formula. A Bacillus subtilis capable of producing a compound having a sequence shown in the following chemical formula. A Bacillus subtilis MT2 strain (Accession No.: NITE BP‑02767).

Description

Technical Field

[0001] The present invention relates to anti-Gram-negative bacterial compounds. Background Art

[0002] Peptides and proteins with antibacterial effects produced by bacteria are known as bacteriocins. Bacteriocins are natural substances and therefore are considered to have less impact on the human body than antibacterial agents composed of synthetic substances. Nisin, a bacteriocin produced by Lactococcus lactis, a lactic acid bacterium, has been approved as a preservative for addition to food in many countries around the world (for example, see Patent Documents 1 and 2).

[0003] Bacteriocins are known to have antibacterial effects on bacteria similar to the producing bacteria. Therefore, it is generally believed that bacteriocins derived from Gram-positive bacteria have antibacterial effects on bacteria similar to the producing bacteria of Gram-positive bacteria, but have no antibacterial effects on Gram-negative bacteria.

[0004] The above-mentioned nisin also has an antibacterial effect on Gram-positive bacteria, but does not have an antibacterial effect on Gram-negative bacteria. Therefore, nisin has no antibacterial effect on Vibrio enteritidis, the pathogenic bacteria of food poisoning caused by fish and shellfish. Regarding bacteriocins other than nisin, for example, subtilin and entianin also have antibacterial effects on Gram-positive bacteria, but do not have antibacterial effects on Gram-negative bacteria. It is believed that this is because the outer membrane of Gram-negative bacteria forms a barrier to bacteriocins from Gram-positive bacteria (for example, refer to non-patent documents 1 to 3).

[0005] In addition, bacteriocins derived from Gram-positive bacteria are not believed to have antibacterial effects against fungi.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 4111489

[0009] Patent Document 2: Japanese Patent No. 5738561

[0010] Non-patent literature

[0011] Non-patent literature 1: J. Barbosa, et al., "Class I and Class II Lanthipeptides Produced by Bacillus spp.", J. Nat. Prod. 2015, 78, 2850-2866

[0012] Non-patent document 2: CVPrudencio, et al., "Strategies for the use ofbacteriocins in Gram-negative bacteria:relevance in food microbiology", J FoodSci Technol (September 2015), 52(9):5408-5417

[0013] Non-patent document 3: Takefumi Zento et al., “Exploration and application of lactic acid bacteria bacteriocins”, Japanese Journal of Lactic Acid Bacteria, Vol. 25, No. 1, 2014, Japan Society for Lactic Acid Bacteria Summary of the invention

[0014] As described above, conventional bacteriocins have an antibacterial effect only on bacteria similar to the producing bacteria, and therefore have a narrow antibacterial spectrum. The present invention has been made in view of such circumstances, and its object is to provide an antibacterial compound having a broad antibacterial spectrum.

[0015] The anti-Gram-negative bacteria compound according to one aspect of the present invention has an amino acid sequence represented by the following Chemical Formula 1.

[0016]

[0017] In addition, an anti-Gram-negative bacteria drug according to one aspect of the present invention comprises an anti-Gram-negative bacteria compound having an amino acid sequence represented by the above Chemical Formula 1.

[0018] In addition, a beverage or food according to one aspect of the present invention comprises an anti-Gram-negative bacteria compound having an amino acid sequence represented by the above Chemical Formula 1.

[0019] In addition, Bacillus subtilis according to one aspect of the present invention is a Bacillus subtilis that can produce a compound having an amino acid sequence represented by the above Chemical Formula 1.

[0020] In addition, an anti-Gram-negative bacteria drug according to one aspect of the present invention comprises Bacillus subtilis capable of producing a compound having an amino acid sequence represented by the above Chemical Formula 1.

[0021] In addition, a beverage or food according to one aspect of the present invention comprises Bacillus subtilis capable of producing a compound having an amino acid sequence represented by the above Chemical Formula 1.

[0022] In addition, the Bacillus subtilis according to one aspect of the present invention is the Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767).

[0023] In addition, an anti-Gram-negative bacteria drug according to one aspect of the present invention comprises Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767).

[0024] In addition, a beverage or food according to one aspect of the present invention contains Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767).

[0025] In addition, a method for producing an anti-Gram-negative bacteria compound according to one aspect of the present invention comprises: culturing Bacillus subtilis, and collecting a compound having an amino acid sequence shown in the above Chemical Formula 1 produced by Bacillus subtilis.

[0026] In addition, a method for producing an anti-Gram-negative bacteria compound according to one aspect of the present invention comprises: culturing Bacillus subtilis MT2 strain (accession number: NITE BP-02767), and collecting a compound having an amino acid sequence shown in the above chemical formula 1 produced by Bacillus subtilis MT2 strain (accession number: NITE BP-02767).

[0027] In addition, one aspect of the application of the present invention is the use of the anti-Gram-negative bacteria compound having the amino acid sequence represented by the above Chemical Formula 1 in the manufacture of an anti-Gram-negative bacteria drug.

[0028] In addition, one aspect of the present invention is the use of Bacillus subtilis capable of producing a compound having an amino acid sequence represented by the above Chemical Formula 1 in the manufacture of an anti-Gram-negative bacteria drug.

[0029] In addition, one aspect of the present invention is the use of Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767) in the manufacture of an anti-Gram-negative bacteria drug.

[0030] In addition, a method for treating enteritis according to one aspect of the present invention comprises administering an anti-Gram-negative bacteria compound having the amino acid sequence represented by the above Chemical Formula 1 to a patient.

[0031] In addition, a method for treating enteritis according to one aspect of the present invention comprises administering Bacillus subtilis capable of producing a compound having an amino acid sequence represented by the above Chemical Formula 1 to a patient.

[0032] In addition, a method for treating enteritis according to one aspect of the present invention comprises administering Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767) to a patient.

[0033] According to the present invention, an antibacterial compound having a broad antibacterial spectrum can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 (a) indicates the gene producing bacteriocin, Figure 1 (b) represents the propeptide of bacteriocin, Figure 1 (c) represents bacteriocin (active peptide).

[0035] Figure 2 This is a graph showing the results of homology analysis of the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of Bacillus subtilis subsp. subtilis 168 based on the ANI method.

[0036] Figure 3 This is a graph showing the results of homology analysis of the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of Bacillus subtilis subsp. subtilis 6051-HGW based on the ANI method.

[0037] Figure 4 This is a graph showing the results of homology analysis of the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of Bacillus subtilis subsp. spizizenii W23 based on the ANI method.

[0038] Figure 5 This is a graph showing the results of homology analysis of the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of Bacillus tequilensis FJAT-14262a based on the ANI method.

[0039] Figure 6 This is a graph showing the results of mass spectrometry analysis of bacteriocins.

[0040] Figure 7 It is a graph showing the antibacterial activity of bacteriocins.

[0041] Figure 8 It is a graph showing the antibacterial activity of bacteriocins. DETAILED DESCRIPTION

[0042] The following is a detailed description of the embodiments of the present invention. It should be noted that the embodiments shown below illustrate the device and method for embodying the technical idea of ​​the present invention, and the technical idea of ​​the present invention does not limit the combination of components to the following contents. The technical idea of ​​the present invention can be modified in various ways within the scope of the protection requested.

[0043] The anti-Gram-negative bacteria compound of 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 C 150H 231 N 39 O 38 S5. The anti-Gram-negative bacteria compound of this embodiment has antibacterial effects on Gram-negative bacteria, fungi and Gram-positive bacteria.

[0044]

[0045] Examples of Gram-negative bacteria on which the anti-Gram-negative bacteria compound of the present embodiment exhibits antibacterial activity include Vibrio bacteria. Examples of Vibrio bacteria include Enteritidis Vibrio, Cholera Vibrio, Non-O1 Cholera Vibrio, Vibrio mimicus, Vibrio fluvialis, Vibrio vulnificus, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio furnissii.

[0046] Examples of fungi on which the anti-Gram-negative bacteria compound of the present embodiment exhibits antibacterial activity include Trichophyton. Examples of Trichophyton include Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton spp., and Trichophyton verrucosum.

[0047] Examples of Gram-positive bacteria on which the anti-Gram-negative bacteria compound of the present embodiment exhibits antibacterial activity include Bacillus bacteria. Examples of Bacillus bacteria include Bacillus subtilis, Bacillus cereus, Bacillus anthracis, and Bacillus stearothermophilus. Bacillus subtilis is harmful in the production process of soy sauce and miso.

[0048] The anti-Gram-negative bacteria compound of the present embodiment is produced by, for example, Bacillus subtilis. The anti-Gram-negative bacteria compound of the present embodiment is derived from Bacillus subtilis, which is a Gram-positive bacterium, but has an antibacterial effect on Gram-negative bacteria and fungi that is equivalent to the antibacterial effect on Gram-positive bacteria. Therefore, the anti-Gram-negative bacteria compound of the present embodiment may not be used in combination with a treatment that assists the antibacterial effect on Gram-negative bacteria.

[0049] As a treatment to assist the antibacterial effect on Gram-negative bacteria, the addition of preservatives such as food preservatives can be cited. As preservatives, chelating agents, surfactants, acids and salts can be cited. As an example of a chelating agent, ethylenediaminetetraacetic acid (EDTA) can be cited. As an example of a surfactant, Tween 80 can be cited. As examples of acids and salts, lactic acid, citric acid, pyrophosphoric acid, gluconic acid, acetic acid, nitrilotriacetic acid, hypochlorous acid, sorbic acid, benzoic acid and polyphosphoric acid and their salts, trisodium phosphate can be cited.

[0050] It is believed that preservatives destabilize the outer membrane of Gram-negative bacteria. In the past, in order to sterilize Gram-negative bacteria with bacteriocins from Gram-positive bacteria, it was believed that such preservatives were needed. In contrast, the anti-Gram-negative bacteria compound of the present embodiment can show an effective antibacterial effect on Gram-negative bacteria even if it is not used in combination with a preservative.

[0051] In addition, as a treatment to assist the antibacterial effect on Gram-negative bacteria, the addition of plant essential oils can be cited. As examples of plant essential oils, oregano essential oil and thyme essential oil can be cited. It is believed that thymol and carvacrol contained in plant essential oils destabilize the outer membrane of Gram-negative bacteria. However, the anti-Gram-negative bacteria compound of the present embodiment can show an effective antibacterial effect on Gram-negative bacteria even if it is not used in combination with plant essential oils.

[0052] In addition, as a treatment to assist the antibacterial effect on Gram-negative bacteria, high pressure treatment can be cited. High pressure treatment refers to, for example, a treatment in which a pressure of 30 MPa or more is applied. It is believed that high pressure treatment makes the outer membrane of Gram-negative bacteria unstable. However, the anti-Gram-negative bacteria compound of the present embodiment can achieve an effective antibacterial effect on Gram-negative bacteria even if it is not used in combination with high pressure treatment.

[0053] In addition, as a treatment to assist the antibacterial effect on Gram-negative bacteria, high temperature and low temperature treatment can be cited. It is believed that high temperature and low temperature treatment make the outer membrane of Gram-negative bacteria unstable. In addition, as a treatment to assist the antibacterial effect on Gram-negative bacteria, pulsed electric field treatment can be cited. Pulsed electric field treatment increases the temperature of Gram-negative bacteria, so it is believed that it has the same effect as high temperature treatment. However, the anti-Gram-negative bacteria compound of the present embodiment can achieve an effective antibacterial effect on Gram-negative bacteria even if it is not used in combination with high temperature treatment, low temperature treatment, or pulsed electric field treatment.

[0054] The Bacillus subtilis capable of producing the anti-Gram-negative bacteria compound of the present embodiment has, for example, Figure 1(a) and the bacteriocin production gene shown in sequence number 1. In sequence number 1, positions 1 to 72 encode a leader peptide. The gene sequence excluding the portion encoding the leader peptide is shown in sequence number 2. The Bacillus subtilis capable of producing the anti-Gram-negative bacteria compound of this embodiment may have the gene sequence shown in sequence number 2.

[0055] Figure 1 (a) and the production gene shown in sequence number 1 are transcribed and translated to produce Figure 1 (b) and the propeptide of the bacteriocin shown in SEQ ID NO: 3. In SEQ ID NO: 3, positions 1 to 24 are the leader peptide. The amino acid sequence excluding the leader peptide is shown in SEQ ID NO: 4.

[0056] Figure 1 (b) and the propeptide of the bacteriocin shown in SEQ ID NO: 3 are modified after translation, and the leader peptide is cleaved to produce Figure 1 (c) and an active peptide having a sequence shown in the above chemical formula 2.

[0057] The serine at position 3 of the propeptide shown in sequence number 4 is replaced by alanine in the active peptide. The serine at position 5 of the propeptide shown in sequence number 4 is replaced by dehydroalanine in the active peptide. The cysteine ​​at position 7 of the propeptide shown in sequence number 4 is replaced by alanine in the active peptide. The threonine at position 8 of the propeptide shown in sequence number 4 is replaced by aminobutyric acid in the active peptide.

[0058] The cysteine ​​at position 11 of the propeptide shown in sequence number 4 is replaced by alanine in the active peptide. The threonine at position 13 of the propeptide shown in sequence number 4 is replaced by aminobutyric acid in the active peptide. The threonine at position 18 of the propeptide shown in sequence number 4 is replaced by dehydrotyrosine in the active peptide. The cysteine ​​at position 19 of the propeptide shown in sequence number 4 is replaced by alanine in the active peptide.

[0059] The threonine at position 23 of the propeptide shown in sequence number 4 is replaced by aminobutyric acid in the active peptide. The threonine at position 25 of the propeptide shown in sequence number 4 is replaced by aminobutyric acid in the active peptide. The cysteine ​​at position 26 of the propeptide shown in sequence number 4 is replaced by alanine in the active peptide. The cysteine ​​at position 28 of the propeptide shown in sequence number 4 is replaced by alanine in the active peptide. The serine at position 31 of the propeptide shown in sequence number 4 is replaced by dehydroalanine in the active peptide.

[0060] In the active peptide, the alanine corresponding to the serine at position 3 of the propeptide shown in SEQ ID NO: 4 and the alanine corresponding to the cysteine ​​at position 7 shown in SEQ ID NO: 4 form lanthionine via a thioether bond.

[0061] In the active peptide, aminobutyric acid corresponding to threonine at position 8 of the propeptide shown in SEQ ID NO: 4 and alanine corresponding to cysteine ​​at position 11 shown in SEQ ID NO: 4 form 3-methyllanthionine via a thioether bond.

[0062] In the active peptide, aminobutyric acid corresponding to threonine at position 13 of the propeptide shown in SEQ ID NO: 4 and alanine corresponding to cysteine ​​at position 19 shown in SEQ ID NO: 4 form 3-methyllanthionine via thioether.

[0063] In the active peptide, aminobutyric acid corresponding to threonine at position 23 of the propeptide shown in SEQ ID NO: 4 and alanine corresponding to cysteine ​​at position 26 shown in SEQ ID NO: 4 form 3-methyllanthionine via a thioether bond.

[0064] In the active peptide, aminobutyric acid corresponding to threonine at position 25 of the propeptide shown in SEQ ID NO: 4 and alanine corresponding to cysteine ​​at position 28 shown in SEQ ID NO: 4 form 3-methyllanthionine via a thioether bond.

[0065] Bacillus subtilis MT2 strain, which is one of the Bacillus subtilis strains capable of producing the anti-Gram-negative bacteria compound of the present embodiment, has been internationally deposited at the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository (Japan, postal code 292-0818, Room 122, 2-5-8, Kazusa Kamata, Kisarazu City, Chiba Prefecture), with the deposit number NITE BP-02767. Bacillus subtilis MT2 strain was domestically deposited at the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository (Japan) on August 22, 2018 (domestic deposit date), and a transfer request to the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository (Japan) was made on October 4, 2018 to the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository (Japan) for international deposit based on the Budapest Treaty.

[0066] The anti-Gram-negative bacteria compound of the present embodiment can be contained in a pharmaceutical product. The pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment can be used as a disinfectant for Gram-negative bacteria, Gram-positive bacteria and fungi. In addition, the pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment can be used as a therapeutic drug for pneumonia, food poisoning and enteritis. Furthermore, the pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment can be used as a therapeutic drug for fungal diseases such as white ringworm, candidiasis, cryptococcosis and aspergillosis.

[0067] The pharmaceutical product contains an effective amount of the anti-Gram-negative bacteria compound of the present embodiment. The effective amount refers to the amount required to exert an antibacterial effect, and can be appropriately determined according to the target bacteria, symptoms, age, weight, and sex of the patient.

[0068] The pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment can be, for example, a tablet, a capsule, a liquid, a cream, an ointment, a plaster, a gel, a wax, and a spray. In addition, the pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment is provided in the form of, for example, a disinfectant, an oral medication, a coating therapeutic drug, and other skin topical medications and eye drops. The pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment can be applied to internal organs such as intestines, human skin including fingers and toes, hair, oral cavity, and eyeballs, etc. In addition, the disinfectant containing the anti-Gram-negative bacteria compound of the present embodiment can also be applied to food, cooking utensils, walls, floors of buildings such as hospitals, and furniture such as tables.

[0069] The pharmaceutical product containing the anti-Gram-negative bacteria compound of the present embodiment may contain appropriate pharmaceutical ingredients such as liquid oils, solid oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, silicones, humectants, water-soluble polymers, thickeners, film-forming agents, metal ion chelating agents, lower alcohols, polyols, sugars, amino acids, organic amines, pH adjusters, skin nutrients, vitamins, fragrances, powders, colorants and water, depending on the purpose.

[0070] The anti-Gram-negative bacteria compound of the present embodiment may be contained in beverages or foods. In addition, the anti-Gram-negative bacteria compound of the present embodiment may be contained in toothpaste, oral cleaning agents, and the like.

[0071] The Bacillus subtilis capable of producing the anti-Gram-negative bacteria compound of the present embodiment can be contained in a pharmaceutical, beverage or food. For example, the Bacillus subtilis contained in a pharmaceutical, beverage or food produces an anti-Gram-negative bacteria compound and exerts an antibacterial effect on Gram-negative bacteria, fungi and Gram-positive bacteria. The target diseases, shape and usage of the pharmaceutical containing the Bacillus subtilis capable of producing the anti-Gram-negative bacteria compound of the present embodiment are the same as those of the pharmaceutical containing the anti-Gram-negative bacteria compound of the present embodiment.

[0072] Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767) can be contained in medicines, beverages or foods. The target diseases, form and usage of the medicine containing Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767) are the same as those of the medicine containing the anti-Gram-negative bacteria compound of this embodiment.

[0073] The anti-Gram-negative bacteria compound of this embodiment can be produced, for example, by culturing Bacillus subtilis in a culture medium and collecting a peptide having an amino acid sequence shown in the above chemical formula 2 produced by Bacillus subtilis in the culture medium. The method of collecting and purifying the peptide from the culture medium is not particularly limited. The anti-Gram-negative bacteria compound of this embodiment can also be chemically synthesized.

[0074] As described above, the anti-Gram-negative bacteria compound and the like according to each embodiment of the present invention have the following configurations and effects constituted by any one or more combinations of the above.

[0075] The anti-Gram-negative bacteria compound of this embodiment is an anti-Gram-negative bacteria compound having an amino acid sequence shown in the above Chemical Formula 2. The anti-Gram-negative bacteria compound of this embodiment has an antibacterial effect on Gram-negative bacteria, Gram-positive bacteria and fungi.

[0076] The anti-Gram-negative bacteria compound of the present embodiment can be produced by Bacillus subtilis. In addition, the anti-Gram-negative bacteria compound of the present embodiment can be produced by Bacillus subtilis MT2 strain (Deposit Number: NITE BP-02767). Although the anti-Gram-negative bacteria compound of the present embodiment is derived from Bacillus subtilis, which is a Gram-positive bacterium, it has an antibacterial effect on Gram-negative bacteria.

[0077] The Gram-negative bacteria on which the anti-Gram-negative bacteria compound of this embodiment exhibits antibacterial action may be bacteria of the genus Vibrio. The bacteria of the genus Vibrio may be Vibrio enteritidis.

[0078] The Gram-negative bacteria on which the anti-Gram-negative bacteria compound of this embodiment exhibits antibacterial activity and the fungi on which the antibacterial activity is exhibited may be Trichophyton, which may be Trichophyton, Trichophyton mentagrophytes or Trichophyton interdigitalis.

[0079] The anti-Gram-negative bacteria compound of the present embodiment may not be used in combination with a treatment that assists the antibacterial effect on Gram-negative bacteria. The treatment that assists the antibacterial effect on Gram-negative bacteria may be the addition of a preservative. The preservative may be a chelating agent. The anti-Gram-negative bacteria compound of the present embodiment has an antibacterial effect on Gram-negative bacteria equivalent to the antibacterial effect on Gram-positive bacteria even if it is not used in combination with a treatment that assists the antibacterial effect on Gram-negative bacteria.

[0080] The anti-Gram-negative bacteria drug of this embodiment may include the anti-Gram-negative bacteria compound described above. The beverage or food of this embodiment may include the anti-Gram-negative bacteria compound described above.

[0081] The Bacillus subtilis of the present embodiment may be a Bacillus subtilis that can produce a compound having an amino acid sequence represented by the above Chemical Formula 2.

[0082] The anti-Gram-negative bacteria drug of this embodiment may include Bacillus subtilis capable of producing a compound having an amino acid sequence as shown in the above Chemical Formula 2. The beverage or food of this embodiment may include Bacillus subtilis capable of producing a compound having an amino acid sequence as shown in the above Chemical Formula 2.

[0083] The Bacillus subtilis of the present embodiment may be the Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767).

[0084] The anti-Gram-negative bacteria drug of this embodiment may include the above-mentioned Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767). The beverage or food of this embodiment may include the above-mentioned Bacillus subtilis MT2 strain (Accession No.: NITE BP-02767).

[0085] The method for producing the anti-Gram-negative bacteria compound of this embodiment may include: culturing Bacillus subtilis, and collecting the compound having the amino acid sequence shown in the above Chemical Formula 2 produced by Bacillus subtilis.

[0086] The method for producing the anti-Gram-negative bacteria compound of this embodiment may include: culturing Bacillus subtilis MT2 strain (accession number: NITE BP-02767), and collecting the compound having the amino acid sequence shown in the above chemical formula 2 produced by Bacillus subtilis MT2 strain (accession number: NITE BP-02767).

[0087] This embodiment may be the use of the above-mentioned anti-Gram-negative bacteria bacteriocin in the manufacture of anti-Gram-negative bacteria drugs.

[0088] This embodiment may be the use of Bacillus subtilis capable of producing a compound having an amino acid sequence as shown in the above Chemical Formula 2 in the manufacture of an anti-Gram-negative bacteria drug.

[0089] This embodiment may be the use of Bacillus subtilis MT2 strain (accession number: NITE BP-02767) in the manufacture of anti-Gram-negative bacteria drugs.

[0090] The method for treating enteritis of this embodiment may include administering the above-mentioned anti-Gram-negative bacteria compound to the patient. The method for treating enteritis of this embodiment may include administering Bacillus subtilis capable of producing a compound having the amino acid sequence shown in the above-mentioned Chemical Formula 2 to the patient. The method for treating enteritis of this embodiment may include administering Bacillus subtilis MT2 strain (preservation number: NITE BP-02767) to the patient. Enteritis may be food poisoning.

[0091] It should be noted that the above-described embodiments are intended to facilitate understanding of the present invention rather than to restrictively interpret the present invention. The present invention can be changed / improved without departing from its purport, and the present invention also includes its equivalents. That is, as long as the embodiments obtained by those skilled in the art appropriately designing and changing the embodiments have the features of the present invention, they are also included in the scope of the present invention. For example, the various elements, materials, conditions and shapes etc. possessed by the embodiments are not limited to the illustrated contents and can be appropriately changed. In addition, the embodiments are illustrative, and of course, partial replacement or combination of the structures shown in different embodiments can be performed, and as long as they include the features of the present invention, they are also included in the scope of the present invention.

[0092] Example

[0093] Hereinafter, examples of the present invention will be described, but the present invention is of course not limited to the following examples.

[0094] (Example 1: Preparation of Artemisia selengensis fermentation broth)

[0095] It is generally believed that the number of lactic acid bacteria in Artemisia selengensis leaves is the largest during the day, 1 hour before and after sunrise, and a total of 2 hours. In addition, it is generally believed that outside these time periods, lactic acid bacteria decrease and photosynthetic bacteria increase. Therefore, during this 2-hour period, a portion of about 20 cm is collected from the front end of the Artemisia selengensis leaves. The collected 6.3 kg of Artemisia selengensis leaves are immediately placed in the first pickling barrel lined with an ethylene bag, and after sprinkling 3.2 kg of molasses and 0.6 kg of coarse salt on the Artemisia selengensis leaves, the mouth of the ethylene bag is closed and sealed. A heavy stone is placed on top of the ethylene bag to pickle the Artemisia selengensis leaves.

[0096] After a few days, the pickle rises to the top of the wormwood leaves, and the heavy stone is removed. Next, add 10L of chlorine-free water for rinsing to the second pickling barrel, and put the pickled wormwood leaves and 10kg of pickle juice in the water. Further prepare a third pickling barrel, and place a metal mesh filter on the opening of the third pickling barrel. Take out the wormwood leaves from the second pickling barrel little by little while rubbing them with your hands, and gently press the wormwood leaves on the metal mesh filter on the opening of the third pickling barrel with your palm to squeeze out the pickle juice.

[0097] After the Artemisia selengensis leaves are completely squeezed, the remaining pickle juice in the second pickle juice is filtered through a metal mesh filter. Next, molasses (Hateruma brown sugar) is dissolved in the pickle juice in the third pickling barrel to a final concentration of 10% by weight, and crude salt is dissolved to a final concentration of 3% by weight. Then, the ambient temperature of the third pickling barrel is set to about 30°C to start fermentation. Initially, large bubbles are confirmed to form, which gradually turn into small bubbles, and eventually the foaming ends. After about 1 week, the pH at the end of the foaming is about 3.8. The pickle juice at this time is used as the Artemisia selengensis fermentation liquid.

[0098] (Example 2: Preparation of Bacillus subtilis)

[0099] The Artemisia selengensis fermentation liquid was added dropwise to the lactic acid bacteria MRS medium (1% 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, manufactured by Difco), and the bacteria contained in the Artemisia selengensis fermentation liquid were cultured in the medium, and subculture was performed several times as needed. Then, the cultured bacteria were spread on the lactic acid bacteria MRS agar medium (a medium obtained by adding 3% agar to the above-mentioned MRS medium) for culture, and the bacteria were collected from the generated colonies.

[0100] Next, using the antibacterial activity against spore lactic acid bacteria, Bacillus coagulans, as an indicator, a strain with the ability to produce antibacterial substances was selected from the collected bacteria by the spot on lawn test method as the Bacillus subtilis MT2 strain. The mycological properties of the selected Bacillus subtilis MT2 strain were investigated, and the results were identified as Bacillus subtilis by analyzing the homology of the base sequence of 16S ribosomal DNA (rDNA).

[0101] The average homology value between the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of an existing Bacillus subtilis strain, namely Bacillus subtilis subsp. subtilis 168, was calculated by the ANI (Average Nucleotide Identity) method. The results are as follows: Figure 2 Shown is 98.5%, 1.5% different.

[0102] The ANI method was used to calculate the average homology value between the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of an existing Bacillus subtilis strain, namely Bacillus subtilis subsp. subtilis 6051-HGW. The results are as follows: Figure 3 Shown is 98.49%, 1.51% different.

[0103] The ANI method was used to calculate the average homology value between the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of an existing Bacillus subtilis strain, namely Bacillus subtilis subsp. Steinernema W23. The results are as follows: Figure 4 Shown is 92.03%, 7.97% different.

[0104] The ANI method was used to calculate the average homology value between the whole genome sequence of Bacillus subtilis MT2 strain and the whole genome sequence of an existing Bacillus subtilis strain, namely Bacillus tequila FJAT-14262a. The results are as follows: Figure 5Shown is 98.49%, 1.51% different.

[0105] Based on the comparison with the existing Bacillus subtilis strains shown above, it is believed that the Bacillus subtilis MT2 strain is a new strain of Bacillus subtilis. The Bacillus subtilis MT2 strain is internationally deposited in the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository (Institute for Product Evaluation and Technology Foundation) (Japan) with the deposit number NITE BP-02767. The Bacillus subtilis MT2 strain has the gene sequence shown in sequence numbers 1 and 2.

[0106] (Example 3: Preparation of bacteriocin)

[0107] The Bacillus subtilis MT2 strain was aerobically cultured in a lactic acid bacteria MRS medium at 35°C or higher and 37°C or lower for 3 days. Then, the culture medium containing the bacterial cells was centrifuged at 10,000 rpm at 25°C for 20 minutes. The centrifuged culture medium was further filtered through a sterilizing filter with a mesh size of 0.45 μm to remove the bacterial cells and obtain a culture supernatant.

[0108] The bacteriocin contained in the culture supernatant was concentrated using a reverse phase silica gel column (Sep-Pac C18, manufactured by Millipore), and the active bacteriocin fraction was further fractionated by reverse phase column chromatography using Sephasil Peptide C18 5μST (manufactured by Pharmacia).

[0109] The mass spectrometry analysis of the fractionated active bacteriocins was performed using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS ultrafleX III, manufactured by Bruker). Figure 6 As shown, the precise molecular weight is about 3346. It should be noted that the target plate uses MTP384 ground steel (Bruker daltonics). In addition, as a matrix, α-cyano-4-hydroxycinnamic acid (HCCA) was used based on various studies. The amino acid sequence of the classified active bacteriocin was analyzed, and the result showed that it had the amino acid sequence shown in the above chemical formula 2.

[0110] (Example 4: Antibacterial activity of bacteriocin)

[0111] Bacillus subtilis (NBRC 3134) as a Gram-positive bacterium and Vibrio parahaemolyticus (12711T) as a Gram-negative bacterium were prepared. 10 mL of a liquid containing the active bacteriocin prepared in Example 3 and having a pH of 7.8 was inoculated with 10 70.1 mL of a bacterial solution containing any of the above bacteria at a concentration of 100 / mL was inoculated with 0.1 mL of a bacterial solution of Vibrio parahaemolyticus (12711T) at a concentration of 0.067 mol / L and pH 7.2 as a control, and the number of viable bacteria was measured at 25°C for 24 hours. The results were as follows: Figure 7 As shown, the active bacteriocin reduced both Bacillus subtilis and Vibrio enteritidis to the same extent.

[0112] Furthermore, Trichophyton mentagrophytes (NBRC 6124) was prepared as a fungus. 10 mL of a liquid containing the active bacteriocin prepared in Example 3 and having a pH of 7.8 was inoculated with 10 7 0.1 mL of a bacterial solution containing Trichophyton at a concentration of 100 / mL was inoculated with 0.1 mL of a bacterial solution containing Trichophyton at a concentration of 100 / mL, and the number of viable bacteria was measured at 25°C for 24 hours. In addition, as a control, 0.1 mL of a bacterial solution containing Trichophyton (NBRC 6124) at a concentration of 0.067 mol / L and a pH of 7.2 was inoculated with 0.1 mL of a bacterial solution containing Trichophyton at a concentration of 100 / mL, and the number of viable bacteria was measured at 25°C for 24 hours. The results were as follows: Figure 8 As shown, active bacteriocins reduced Trichophyton. Sequence Listing <110> Murata Manufacturing Co., Ltd. <120> Anti-Gram-negative compounds <130> M18-01812 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 171 <212> DNA <213> Bacillus subtilis <400> 1 atgtcaaagt tcgatgattt cgatttggat gtagtaaaag tctctaaaca agactctaaa 60 atcactcctc aatggaaaag tgaatcactt tgtacacctg ggtgtgtaac tggtgtattg 120 caaacttgct tccttcaaac aataacttgt aactgcaaaa tctctaaata a 171 <210> 2 <211> 99 <212> DNA <213> Bacillus subtilis <400> 2 tggaaaagtg aatcactttg tacacctggg tgtgtaactg gtgtattgca aacttgcttc 60 cttcaaacaa taacttgtaa ctgcaaaatc tctaaataa 99 <210> 3 <211> 56 <212> PRT <213> Bacillus subtilis <400> 3 Met Ser Lys Phe Asp Asp Phe Asp Leu Asp Val Val Lys Val Ser Lys 1 5 10 15 Gln Asp Ser Lys Ile Thr Pro Gln Trp Lys Ser Glu Ser Leu Cys Thr 20 25 30 Pro Gly Cys Val Thr Gly Val Leu Gln Thr Cys Phe Leu Gln Thr Ile 35 40 45 Thr Cys Asn Cys Lys Ile Ser Lys 50 55 <210> 4 <211> 32 <212> PRT <213> Bacillus subtilis <400> 4 Trp Lys Ser Glu Ser Leu Cys Thr Pro Gly Cys Val Thr Gly Val Leu 1 5 10 15 Gln Thr Cys Phe Leu Gln Thr Ile Thr Cys Asn Cys Lys Ile Ser Lys 20 25 30

Claims

1. An anti-Gram-negative bacteria compound, consisting of an amino acid sequence represented by the following chemical formula:

2. An anti-Gram-negative bacteria drug comprising the anti-Gram-negative bacteria compound according to claim 1.

3. A Bacillus subtilis MT2 strain, with the deposit number being NITE BP-02767.

4. An anti-Gram-negative bacteria drug comprising Bacillus subtilis MT2 strain, wherein the deposit number of the Bacillus subtilis MT2 strain is NITE BP-02767.

5. A method for producing an anti-Gram-negative bacteria compound, comprising: Cultivation of Bacillus subtilis MT2 strain, and The compound composed of the amino acid sequence shown in the following chemical formula produced by the Bacillus subtilis MT2 strain is collected. The accession number of the Bacillus subtilis MT2 strain is NITE BP-02767.

6. Use of the anti-Gram-negative bacteria bacteriocin according to claim 1 in the manufacture of anti-Gram-negative bacteria drugs, wherein: The Gram-negative bacteria are Vibrio enteritidis.

7. Use of Bacillus subtilis MT2 strain in the manufacture of an anti-Gram-negative bacteria drug, wherein the deposit number of the Bacillus subtilis MT2 strain is NITE BP-02767, and the Gram-negative bacteria is Vibrio enteritidis.

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