JPH05320067A - Antibacterial agent and method for treating article with the antibacterial agent - Google Patents

Abstract

PURPOSE:To provide the antibacterial agent containing the decomposition prod uct of a lactoferrin compound and/or a lactoferrin-related antibacterial peptide, and having a further strengthened antibacterial property. CONSTITUTION:This antibacterial agent contains as active ingredients (A) a compound selected from a group consisting of the decomposition products of lactoferrin compounds, lactoferrin-related antibacterial peptides and their arbitrary mixtures and (B) a compound selected from a group consisting of metal- bonded proteins, tocopherol, cyclodextrin compounds, fatty acid glycerol esters, alcohols, EDTA, EDTA salts, ascorbic acid, ascorbic acid salts, citric acid, citric acid salts, polyphosphoric acid, polyphosphoric acid salts, chitosan, cysteine, cholic acid and their arbitrary mixtures.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial agent and a method for treating articles with the antibacterial agent. More specifically, the present invention relates to an antibacterial agent in which the antibacterial property of a decomposed product of lactoferrin and / or lactoferrin-related antibacterial peptide is further enhanced, and a method of treating an article using the antibacterial agent.

[0002]

BACKGROUND OF THE INVENTION Lactoferrin is a natural iron-binding protein contained in tears, saliva, peripheral blood, milk, etc. in vivo, and has an antibacterial action against harmful microorganisms such as Escherichia coli, Candida and Clostridium. Is known to indicate [Journal of Pediatrics (Journal of P
ediatrics), Vol. 94, p. 1: 1979]. Also,
0.5 to 30 mg / for Staphylococcus and Enterococcus
It is known to have an antibacterial action at a concentration of ml [Nonnecke, BJ & Smith, KL; Journal of Dairy
Science) 67, 606: 1984].

On the other hand, many inventions have been known for peptides having an antibacterial activity against various microorganisms. For example, phosphonotripeptides effective against Gram-positive bacteria and Gram-negative bacteria (JP-A-57-106689) and phosphonodipeptide derivatives (JP-A-58-135).
94), cyclic peptide derivatives (JP-A-58-21)
3744), peptides having antibacterial and antiviral effects (JP-A-59-51247), polypeptides effective in yeast (JP-A-60-130599), and glycopeptide derivatives effective in Gram-positive bacteria. (JP-A-60-1
72998, JP-A-61-251699,
JP-A-63-44598), oligopeptides effective against Gram-positive bacteria (JP-A-62-22798),
Peptide antibiotics (JP-A-62-51697,
Japanese Patent Laid-Open No. 63-17897) and other antibacterial peptides extracted from blood cells of horseshoe crabs produced in North America (Japanese Patent Laid-Open No. 2-53)
799), an antibacterial peptide isolated from the hemolymph of bees (Japanese Patent Publication No. 50504/1990), and the like.

The inventors of the present invention intend to inexpensively isolate from nature the substance which has no adverse side effects (eg, antigenicity), is heat resistant, and has a strong antibacterial action. Mammalian lactoferrin, apolactoferrin and / or metal-saturated lactoferrin (hereinafter,
We found that the hydrolyzate of these (sometimes referred to as lactoferrins) with acid or enzyme has stronger heat resistance and antibacterial properties than undecomposed lactoferrins, and already filed a patent application Flat 3-171
736).

Furthermore, the inventors of the present invention have no adverse side effects (eg, antigenicity) and have heat resistance,
As a lactoferrin-related antibacterial peptide having a strong antibacterial action, an antibacterial peptide having 20 amino acid residues (Japanese Patent Application No. 3-186260) and an antibacterial peptide having 11 amino acid residues (Japanese Patent Application No. Flat 3-48196
No.), an antimicrobial peptide having 6 amino acid residues (Japanese Patent Application No. 3-94492), an antimicrobial peptide having 5 amino acid residues (Japanese Patent Application No. 3-94493), amino acid residues Is an antimicrobial peptide having 3 to 6 residues (Japanese Patent Application No. 3-9
4494) and filed a patent application.

Various studies have been conducted so far to enhance the antibacterial activity of lactoferrin, and lysozyme, IgA and glycopeptides are known as such physiological activity enhancing auxiliary agents. For example, a method of synergistically enhancing antibacterial action by coexistence of lactoferrin and lysozyme [JP-A-62-249931], a method of synergistically enhancing antibacterial action by coexistence of lactoferrin and secretory IgA [ Immunology (Stephens,
S., et al; Immunology); 41, 597: 1980.
Year] etc. have been reported. In addition, spik etc.
F. Williams and J. D. Baum (A.
F.Williams & JDBaum, edited by "Human Milk Banking", Nestle Nutrition Workshop Series, Volume 5, (Nest
le Nutrition Workshop Series Volume 5), 133
Page, Raven Press Books,
Ltd.), 1984], lactoferrin has an action of preventing bacteria from adhering to the mucosal surface, and this action is enhanced by the presence of lysozyme or glycopeptides.

[0007]

However, a method for enhancing the antibacterial activity of the above-mentioned decomposed products of lactoferrins or lactoferrin-related antibacterial peptides, especially multi-component products and articles (for example, milk etc.), has the above-mentioned effects. There is no known method for enhancing the antibacterial activity of a lactoferrin degradation product or a lactoferrin-related antibacterial peptide.

The present invention uses the above-mentioned decomposed product of lactoferrin and / or lactoferrin-related antibacterial peptide, which the inventors of the present invention have already invented, particularly in multi-component products and articles (eg milk). To provide an antibacterial agent in which a decomposed product of lactoferrin and / or an antibacterial peptide related to lactoferrin is further enhanced, and a method for treating an article using the antibacterial agent I am trying.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies on the above problems, and as a result, by combining a degradation product of lactoferrin and / or an antibacterial peptide related to lactoferrin with a specific compound. , Compared to the case where the degradation product of lactoferrin and / or the lactoferrin-related antibacterial peptide is used alone,
They have found that they can increase antibacterial properties and completed the invention.

That is, the present invention provides a compound selected from the group consisting of (A) a lactoferrin degradation product, a lactoferrin-related antibacterial peptide, and an arbitrary mixture thereof, and (B) a metal-binding protein, tocophero-
, Cyclodextrin, glycerin fatty acid ester, alcohol, EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and these And an antibacterial agent containing a compound selected from the group consisting of an arbitrary mixture thereof as an active ingredient.

The present invention further provides (A) a compound selected from the group consisting of a lactoferrin degradation product, a lactoferrin-related antibacterial peptide, and an arbitrary mixture thereof, and (B) a metal-binding protein, tocophero- Le,
Cyclodextrins, glycerin fatty acid esters,
Selected from the group consisting of alcohols, EDTA, EDTA salts, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and any mixture thereof. Provided is a method for treating an article with an antibacterial agent containing a compound as an active ingredient.

In a preferred embodiment of the present invention, the metal-binding protein is lactoferrin, transferrin, conalbumin, or casein phosphopeptide. Next, the present invention will be described in detail. In the present invention, lactoferrins are commercially available lactoferrin, colostrum of mammals (eg, humans, cattle, sheep, goats, horses, etc.), transitional milk, normal milk, end-stage milk, etc., or defatted products of these milks. From milk, whey, etc., a conventional method (for example,
Lactoferrin separated by ion exchange chromatography), apolactoferrin deironed with hydrochloric acid, citric acid, etc., metal saturated or partially saturated lactoferrin bonded with a metal such as apolactoferrin, iron, copper, zinc, manganese, or these Is a mixture of
A commercially available product may be used, or a commercially available product may be prepared and used.

The decomposed product of lactoferrin used in the present invention is obtained, for example, by hydrolyzing the above-mentioned lactoferrin with an acid or an enzyme, for example, Japanese Patent Application No. 3-171.
It can be obtained by the method described in the invention of No. 736. That is, in the case of performing hydrolysis with an acid, lactoferrins are made into an aqueous solution, an inorganic acid or an organic acid is added thereto, and the mixture is heated to a predetermined temperature for a predetermined time for hydrolysis. When carrying out hydrolysis with an enzyme, lactoferrins are made into an aqueous solution, adjusted to an optimum pH of the enzyme to be used, pepsin, an enzyme such as trypsin is added thereto, and the mixture is kept at a predetermined temperature for a predetermined time for hydrolysis, The enzyme is inactivated by a conventional method. Degradation products obtained by hydrolysis with acid or enzyme,
It is a mixture of antimicrobial peptides with different molecular weights.
The degree of decomposition by the above hydrolysis is preferably in the range of 6 to 20%. The above-mentioned degree of decomposition was determined by measuring the total nitrogen of the sample by the Kjeldahl method and the formaldehyde of the sample by the formol titration method.
Ru nitrogen was measured and calculated from these values by the following formula.

Decomposition degree = (formal nitrogen content / total nitrogen content) × 100 The reaction solution (solution of lactoferrin-decomposed product) obtained by hydrolysis with an acid or an enzyme as described above is prepared by a conventional method. Cooled, neutralized, desalted, and decolorized by a conventional method as needed, and further fractionated by a conventional method as needed, and the obtained solution as it is or in a concentrated liquid form, or a powder dried after concentration -Like, metal-binding protein, tocophero-
, Cyclodextrin, glycerin fatty acid ester, alcohol, EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and these And a compound selected from the group consisting of any mixture of

The lactoferrin-related antibacterial peptide used in the present invention is the same as the antibacterial peptide obtained from the decomposed product of lactoferrin by the separation means and the antibacterial peptide obtained from the decomposed product of the lactoferrin by the separation means. Alternatively, a peptide having a homologous chemical structure (amino acid sequence), or a chemical structure (amino acid sequence) identical or homologous to a peptide having an antibacterial property obtained by a separation means from a degradation product of lactoferrin
Is a compound selected from the group consisting of derivatives of peptides having, and any mixtures thereof. These lactoferrin-related antibacterial peptides are disclosed, for example, in Japanese Patent Application No.
-186260, Japanese Patent Application No. 3-48196, Japanese Patent Application No. 3-48196
No. 94492, Japanese Patent Application No. 3-94493, Japanese Patent Application No. 3-9
It can be obtained by the method described in each invention of No. 4494. That is, lactoferrins are hydrolyzed with an acid or an enzyme, and a method for obtaining a fraction containing a peptide having antibacterial properties from the resulting peptide mixture by a separation means such as liquid chromatography, or the antibacterial obtained as described above Method of the amino acid sequence of a peptide having a property (for example, a method using a gas phase sequencer, etc.)
Can be prepared by a method of obtaining a desired peptide by synthesizing peptides having those amino acid sequences by a known method (for example, a method using an automatic peptide synthesizer). These lactoferrin-related antimicrobial peptides are, for example, the following peptides having the following amino acid sequences: SEQ ID NO: 1,
Antibacterial peptides having amino acid sequences of 2 and 27 (Japanese Patent Application No. 3-48196); SEQ ID NOs: 3, 4, 5
And the antibacterial peptide having the amino acid sequence of 6 (the invention of Japanese Patent Application No. 3-94492); the antibacterial peptide having the amino acid sequence of SEQ ID NOs: 7, 8, 9 and 31 (the Japanese patent application No. 3 of 4).
No. 94493 invention); SEQ ID NOs: 10, 11, 12, 1
Antibacterial peptide having the amino acid sequence of 3, 14, 15, 16, 17, 18, 19, 20, and 21 (Japanese Patent Application No.
-94494 invention); SEQ ID NOs: 22, 23, 24, 2
An antibacterial peptide having an amino acid sequence of 5, 26, 28, 29 and 30 (Japanese Patent Application No. 3-186260).

These antibacterial peptides are used in the form of a solution as it is, or in a concentrated liquid form, or in a powder form obtained by concentrating and drying, a metal-binding protein, tocopherol, cyclodextrins, glycerin fatty acid esters, alcohols. And a compound selected from the group consisting of EDTA, EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and any mixture thereof. Mix.

A protein, tocopherol, which binds a metal with any of a decomposed product of lactoferrins, an antibacterial peptide related to lactoferrin, or any mixture thereof.
Cyclodextrins, glycerin fatty acid esters,
Combine with any of alcohols, EDTA, EDTA salts, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid or any mixture thereof. It can be appropriately selected depending on the purpose of use.
Further, the mixing ratio thereof can be appropriately selected depending on the type of each component and the purpose of use. The above mixture is
As described above, they can be mixed in the form of liquid or powder, and in this case, known diluents and excipients can be appropriately added.

Proteins that bind metals, tocopherols,
Cyclodextrins, glycerin fatty acid esters,
Alcohols, EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid, etc. may be commercially available products or are known. You may prepare and use by the method of. For example, a metal-binding protein is a protein capable of forming a compound by coordinating with a metal ion, and examples thereof include lactoferrins, transferrin, conalbumin, and casein phosphopeptide. Cyclodextrins include α
-Cyclodextrin, β-cyclodextrin, γ-
Examples thereof include cyclodextrin, δ-cyclodextrin, and alkyl derivatives thereof (branched cyclodextrin). The glycerin fatty acid ester is an ester of fatty acid and glycerin or polyglycerin and a derivative thereof, and examples thereof include glycerin fatty acid ester and polyglycerin fatty acid ester. The alcohols are aliphatic monovalent, divalent, trivalent or polyvalent alcohols, such as ethyl alcohol, propylene glycol and glycerol.

The antibacterial agent of the present invention comprises various bacteria, yeasts,
It has strong antibacterial properties against fungi and is not only used as a drug but also as a food, feed, cosmetic, etc. that is taken into the human or animal body or applied to the surface of the body, and generally it does not grow bacteria. The antibacterial agent of the present invention can be used by blending it with any product for which prevention or inhibition is desired, and can treat those products or raw materials. That is, the antibacterial agent of the present invention can be directly administered to humans or animals, or food (eg, chewing gum, etc.), drug (eg, eye drops, mastitis therapeutic agent, antidiarrheal drug, athlete's foot drug, etc.), pharmacy External products (for example, mouthwash, antiperspirant, hair nourishing agent, etc.), various cosmetics (for example, hair dressing, cream, emulsion, etc.), various toothpaste products (for example, toothpaste, toothbrush, etc.), various sanitary products, various Baby
Goods (for example, diapers), various elderly goods (for example,
Denture fixative, diapers, etc.), various detergents (eg soap,
Medicated soap, shampoo, rinse, laundry detergent, kitchen detergent, household detergent, etc.), various sterilization products (for example, kitchen sterilization paper, toilet sanitization paper, etc.),
Feed (e.g., pet hood, etc.), raw materials thereof, and other generally, the growth of microorganisms can be added, compounded, sprayed, attached, coated, impregnated, etc. to any article for which inhibition or suppression of microorganisms is desired, In addition, it can be generally used for treating any article for which the prevention or inhibition of the growth of microorganisms is desired.

The present invention will be described in more detail with reference to test examples. First, the preparation of a sample common to the following test examples and the test method will be described collectively. 1. Sample Preparation (1) Lactoferrin Degradation Product (Powder) The lactoferrin degradation product prepared by the method described in Example 1 was used.

The lactoferrin degradation product 2 prepared by the method described in Example 2 was used.

(2) Antimicrobial peptide (powder) The peptide (SEQ ID NO: 26) prepared by the method described in Example 3 was used. The peptide (SEQ ID NO: 27) prepared by the method described in Example 4 was used.

(3) Lactoferrin: Commercially available bovine lactoferrin (manufactured by Sigma) was used. (4) Casein phosphopeptide: publicly known method (JP-A-59)
No. 159792 method). (5) Tocopherol: A commercially available product (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

(6) β-Cyclodextrin: A commercially available product (manufactured by Nippon Shokubai Co., Ltd.) was used. (7) 1-Monocapryloyl-rac-glycerol: a commercially available product (manufactured by Sigma) was used. (8) Ethyl alcohol: Commercially available 99.5% ethyl alcohol (manufactured by Nacalai Tesque, Inc.) was used.

(9) Glycerol: a commercially available product (manufactured by Nacalai Tesque) was used. (10) Propylene glycol: a commercially available product (manufactured by Wako Pure Chemical Industries, Ltd.) was used. (11) EDTA / sodium: a commercially available product (manufactured by Wako Pure Chemical Industries, Ltd.) was used. (12) Ascorbic acid: a commercially available product (manufactured by Kanto Chemical Co., Inc.) was used. (13) Citric acid: a commercially available product (manufactured by Nacalai Tesque, Inc.) was used.

(14) Polyphosphoric acid: A commercially available product (manufactured by Merck) was used. (15) Chitosan: A commercially available product (manufactured by Nacalai Tesque, Inc.) was used. It was dissolved in a dilute solution of acetic acid. (16) L-Cysteine: A commercially available product (manufactured by Sigma) was used. The aqueous solution was sterilized with a sterile filter.

(17) Polyethylene glycol # 2000:
A commercially available product (manufactured by Nacalai Tesque) was used. (18) Glycerin fatty acid ester 1-monolauroyl-rac-glycerol: a commercially available product (manufactured by Sigma) was used.

1-Monomyristoyl-rac-glycerol: A commercially available product (manufactured by Sigma) was used. 1-Monostearoyl-rac-glycerol: A commercially available product (manufactured by Sigma) was used. Both were used as water suspensions.

(19) Cholic acid: A commercial product (manufactured by Wako Pure Chemical Industries, Ltd.) was used as an aqueous suspension. 2. Test method (1) Preparation of staphylococcal preculture liquid One platinum loop was taken from a preserved slant of Staphylococcus aureus JCM-2151 and spread on a standard agar medium (Eiken Chemical Co., Ltd.) at 37 ° C. After culturing for 16 hours, the colony grown on the surface of standard agar medium is scraped off with a platinum loop.
% Peptone (manufactured by Difco) was cultivated at 37 ° C. for several hours, and the bacterial solution in the logarithmic phase grown to a bacterial concentration of 3 × 10 ⁸ / ml was used as a preculture liquid.

(2) Preparation of basic medium (milk medium) The above sample was added to commercially available milk, the concentration of milk was diluted to 1/2 with purified water, and sterilized at 115 ° C. for 15 minutes to obtain a basic medium. .

(3) Preparation of test medium and control medium (3-1) Preparation of test medium Aqueous solution of lactoferrin degradation product or antibacterial peptide prepared in (1) or (2) of the sample preparation described in 1 above ,
And (3), (4), (6), (11), (12), (13) or (16)
The aqueous solution of the compound (1) was sterilized with a sterilizing filter (manufactured by Advantech), added to the basic medium and adjusted to the final concentration specified for each, to prepare a test medium.

(5), (7), (8), (9), (10), (14), (1
5), (17), (18) and (19) are compound aqueous solutions ((5), (7)
In this case, the sterilization of the water suspension) with a sterilizing filter was omitted, and the test medium was prepared in the same manner as in (3) except for the above. (3-2) Preparation of Control Medium 1 Commercially available milk was diluted 2-fold with purified water and sterilized at 115 ° C. for 15 minutes to prepare Control Medium 1.

(3-3) Preparation of Control Medium 2 (3), (4), (6), (11), (1
Sterile filter the aqueous solution of the compound of 2), (13) or (16)
A control medium 2 was prepared by removing the bacteria with Advantech and then adding it to the basic medium to adjust the concentration to the same as that of the test medium.

(5), (7), (9), (10), (14), (15), (1
7), (18) and (19) are the same as (3) etc. except that the sterilization of the compound aqueous solution (water suspension in the case of (5) and (7)) by a sterile filter is omitted. Then, control medium 2 was prepared. (4) Antibacterial effect test (4-1) Survival rate test 20 μl of the preculture liquid of Staphylococcus prepared in (1) above was
Add to 2 ml of test medium prepared in (3-1) above, and keep at 37 ℃
Incubate for 1 hour at room temperature, collect 200 μl from the culture and
After diluting 10 n times with a peptone aqueous solution, 110 μl of the solution was spread on a regular agar medium plate and cultured at 37 ° C. for 24 hours to measure the number of bacteria (test bacteria).

Separately, as Control 1, 20 μl of the preculture liquid of Staphylococcus prepared in (1) above was added to 2 ml of the control medium 1 prepared in (3-2) above to measure the number of test bacteria. The bacterial count (control bacterial count 1) was measured in the same manner as in (1). Further, as a control 2, 20 μl of the preculture liquid of Staphylococcus prepared in (1) above was added to 2 ml of the control medium 2 prepared in (3-3) above.
Then, the bacterial count (control bacterial count 2) was measured in the same manner as in the case of measuring the test bacterial count.

The survival rate was calculated by the following formula. Survival rate 1 = (Number of test bacteria / Number of control bacteria 1) × 100 Survival rate 2 = (Number of control bacteria 2 / Number of control bacteria 1) × 100 (Note: In each table below, the survival rate 2 is an antimicrobial peptide or The concentration of the lactoferrin degradation product is displayed in the row of 0) Test Example 1 The final concentration of the antibacterial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 ml per ml.
The final concentration of lactoferrin described in (3) is 1 ml.
0 mg, 0.1 mg, 1 mg and 10 mg per
Each was adjusted and a survival rate test was conducted.

The test results are shown in Table 1. Table 1
As is clear from the above, it was confirmed that lactoferrin enhances the bactericidal effect of the antibacterial peptide. When lactoferrin was added and no antimicrobial peptide was added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and lactoferrin. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that lactoferrin also enhanced the bactericidal effect in this case.

[0038]

[Table 1]

Test Example 2 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 ml per ml.
The final concentration of the casein phosphopeptide described in (4) is 0 mg, 1 mg, 10 mg and 20 m / ml.
Each was adjusted to g and a survival rate test was conducted.

The results of this test are shown in Table 2. As is clear from Table 2, it was confirmed that the casein phosphopeptide enhances the bactericidal effect of the antibacterial peptide. Further, when the casein phosphopeptide was added and the antibacterial peptide was not added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and the casein phosphopeptide. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that the casein phosphopeptide also enhanced the bactericidal effect in this case.

[0041]

[Table 2]

Test Example 3 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 ml per ml.
1 mg of the final concentration of tocopherol described in (5) to mg.
0 mg, 0.1 mg, 0.5 mg and 1 mg per
, And the survival rate test was performed.

The test results are shown in Table 3. Table 3
As is clear from the above, it was confirmed that tocopherol enhances the bactericidal effect of the antibacterial peptide. When tocopherol was added and no antibacterial peptide was added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and tocopherol. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that tocopherol enhanced the bactericidal effect also in this case.

[0044]

[Table 3]

Test Example 4 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 ml per ml.
The final concentration of β-cyclodextrin described in (6) is 0 mg, 0.1 mg, 1 mg and 2.
The viability test was conducted after adjusting to 5 mg.

The test results are shown in Table 4. Table 4
As is clear from the above, it was confirmed that β-cyclodextrin enhances the bactericidal effect of the antibacterial peptide. Further, when β-cyclodextrin was added and no antimicrobial peptide was added, almost no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and β-cyclodextrin. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that β-cyclodextrin also enhanced the bactericidal effect in this case.

[0047]

[Table 4]

Test Example 5 The final concentration of the antibacterial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 ml per ml.
The final concentration of monocapryloyl glycerol described in (7) was adjusted to 0 mg, 0.5 mg, 1 mg, and 2 mg, respectively, and a viability test was conducted.

The test results are shown in Table 5. Table 5
As is clear from the above, it was confirmed that monocapryloyl glycerol enhances the bactericidal effect of the antibacterial peptide. When monocapryloyl glycerol was added and no antimicrobial peptide was added, no bactericidal effect was observed up to a concentration of 1 mg / ml. Even when the concentration of monocapryloyl glycerol is 2 mg / ml, when combined with the antibacterial peptide, monocapryloyl glycerol-
The antibacterial peptide and monocapryloyl glycerol have a synergistic effect because the bactericidal effect is remarkably enhanced as compared with the case where the antibacterial peptide is used alone (concentration 2 mg / ml) or the antibacterial peptide is used alone (each concentration). It is clear to have Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides, and lactoferrin degradation products. In this case, monocapryloyl glycero-
It was found that the bactericidal effect is enhanced by the syrup.

[0050]

[Table 5]

Test Example 6 The final concentration of the antimicrobial peptide described in (2) of the preparation of the sample was 0 mg, 0.5 mg, 1 mg and 2 m per ml.
1 g of the final concentration of ethyl alcohol described in (8)
It was adjusted to 0 mg, 1 mg, 10 mg and 20 mg, respectively, and a survival rate test was conducted.

The results of this test are shown in Table 6. As is clear from Table 6, low concentrations of ethyl alcohol
It was confirmed that the antibacterial peptide enhances the bactericidal effect of the antibacterial peptide. Further, when ethyl alcohol was added and no antimicrobial peptide was added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect between the antibacterial peptide and ethyl alcohol. Similar tests were conducted on antibacterial peptides other than the above-mentioned antibacterial peptides and lactoferrin degradation products, and it was confirmed that the bactericidal effect was enhanced by ethyl alcohol in this case as well.

[0053]

[Table 6]

Test Example 7 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 per ml.
The final concentration of glycerol described in (9) was adjusted to 0 mg, 1 mg, 10 mg, and 20 mg per ml, and a viability test was conducted.

The test results are shown in Table 7. Table 7
As is clear from the above, it was confirmed that glycerol enhances the bactericidal effect of the antibacterial peptide. When glycerol was added and no antimicrobial peptide was added,
No bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and glycerol. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that the bactericidal effect was enhanced by glycerol also in this case.

[0056]

[Table 7]

Test Example 8 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was 0 mg, 0.5 mg, 1 mg and 2 ml per ml.
The final concentration of propylene glycol described in (10) is 0 mg, 1 mg, 10 mg and 20 mg per ml.
, And the survival rate test was performed.

The test results are shown in Table 8. Table 8
As is clear from the above, it was confirmed that propylene glycol enhances the bactericidal effect of the antibacterial peptide. Also,
No bactericidal effect was observed when propylene glycol was added and no antimicrobial peptide was added. Therefore, it is clear that the enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and propylene glycol. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that the bactericidal effect was enhanced by propylene glycol in this case as well.

[0059]

[Table 8]

Test Example 9 The final concentration of the lactoferrin degradation product described in (1) of the above sample preparation was 0 mg, 10 mg, 20 mg and 40 mg per ml, and the final concentration of EDTA / sodium was 0 mg per 1 ml described in (11). , 0.1 mg, 1 mg and 5 mg, respectively, and the survival rate test was conducted.

The results of this test are shown in Table 9. As is clear from Table 9, it was confirmed that EDTA / disodium enhances the bactericidal effect of the lactoferrin degradation product. When EDTA / disodium was added and the lactoferrin degradation product was not added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and EDTA / disodium. A similar test was conducted using an antibacterial peptide instead of the lactoferrin degradation product, and it was confirmed that the bactericidal effect was enhanced by EDTA / sodium in this case as well.

[0062]

[Table 9]

Test Example 10 The final concentration of the lactoferrin degradation product described in (1) of the above sample preparation was 0 mg, 10 mg, 20 mg and 40 mg per ml, and the final concentration of ascorbic acid described in (12) was 0 mg per ml, It adjusted to 0.1 mg, 0.5 mg, and 1 mg, respectively, and the survival rate test was done.

The results of this test are shown in Table 10. As is clear from Table 10, it was confirmed that ascorbic acid enhances the bactericidal effect of the lactoferrin degradation product. Further, when ascorbic acid was added and no lactoferrin degradation product was added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and ascorbic acid. A similar test was conducted using an antibacterial peptide instead of the lactoferrin degradation product, and it was found that ascorbic acid also enhanced the bactericidal effect in this case.

[0065]

[Table 10]

Test Example 11 The final concentration of the lactoferrin degradation product described in (1) of the above sample preparation was 0 mg, 10 mg, 20 mg and 40 mg per ml, and the final concentration of citric acid described in (13) was 1 m.
0 mg, 0.1 mg, 1 mg and 5 mg per liter,
Each was adjusted and a survival rate test was conducted.

The results of this test are shown in Table 11. As is clear from Table 11, it was confirmed that citric acid enhances the bactericidal effect of the lactoferrin degradation product. Further, when citric acid was added and lactoferrin degradation product was not added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and citric acid. A similar test was conducted using an antibacterial peptide instead of the lactoferrin degradation product, and it was confirmed that citric acid also enhanced the bactericidal effect in this case.

[0068]

[Table 11]

Test Example 12 The final concentration of the lactoferrin degradation product described in (1) of the sample preparation was 0 mg, 10 mg, 20 mg and 40 mg per ml, and the final concentration of the polyphosphoric acid described in (14) was 1 mg / ml.
0 mg, 0.1 mg, 1 mg and 5 mg per ml
, And the survival rate test was performed.

The results of this test are shown in Table 12. As is clear from Table 12, it was confirmed that polyphosphoric acid enhances the bactericidal effect of the lactoferrin degradation product.
Further, when polyphosphoric acid was added and a lactoferrin decomposition product was not added, no bactericidal effect was observed.
Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and polyphosphoric acid. A similar test was conducted using an antibacterial peptide instead of the lactoferrin degradation product, and it was confirmed that the bactericidal effect was enhanced by polyphosphoric acid in this case as well.

[0071]

[Table 12]

Test Example 13 The final concentration of the antimicrobial peptide of SEQ ID NO: 27 described in (2) of the sample preparation was 0 mg / ml, 0.5 mg / ml,
For 1 mg and 2 mg, the final concentration of chitosan described in (15) was 0 mg, 0.004 mg, 0.02 mg per ml.
And 0.1 mg, respectively, and the survival rate test was conducted.

The results of this test are shown in Table 13. As is clear from Table 13, it was confirmed that chitosan enhances the bactericidal effect of the antibacterial peptide. Further, the bactericidal effect was low when chitosan was added and no antimicrobial peptide was added. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and chitosan. A similar test was conducted on a lactoferrin degradation product instead of the antibacterial peptide, and it was found that the bactericidal effect was enhanced by chitosan in this case as well.

[0074]

[Table 13]

Test Example 14 The final concentration of the antimicrobial peptide of SEQ ID NO: 27 described in (2) of the sample preparation was 0 mg / ml, 0.5 mg / ml,
For 1 mg and 2 mg, the final concentration of L-cysteine described in (16) was 0 mg, 1 mg, 5 mg and 1 ml per ml.
The survival rate test was conducted after adjusting to 0 mg.

The results of this test are shown in Table 14. As is clear from Table 14, it was confirmed that L-cysteine enhances the bactericidal effect of the antibacterial peptide. Moreover, when L-cysteine was added and no antimicrobial peptide was added, the bactericidal effect was low. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and L-cysteine. A similar test was conducted on a lactoferrin degradation product instead of the antibacterial peptide, and it was confirmed that the bactericidal effect was enhanced by L-cysteine in this case as well.

[0077]

[Table 14]

Test Example 15 Lactoferrin degradation product 2 described in (1) of the above-mentioned sample preparation
Final concentration of 0 mg, 10 mg, 20 mg per ml
And 40 mg, the final concentration of polyethylene glycol # 2000 described in (17) is 0 mg, 1 mg per ml,
It adjusted to 10 mg and 20 mg, respectively, and the survival rate test was done.

The results of this test are shown in Table 15. As is clear from Table 15, it was confirmed that polyethylene glycol # 2000 enhances the bactericidal effect of the lactoferrin degradation product 2. Further, when polyethylene glycol # 2000 was added and lactoferrin degradation product 2 was not added, almost no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the lactoferrin degradation product 2 and polyethylene glycol # 2000. A similar test was conducted using an antibacterial peptide instead of the lactoferrin degradation product 2, and in this case as well, polyethylene glycol #
It was found that 2000 increased the bactericidal effect.

[0080]

[Table 15]

Test Example 16 The final concentration of the lactoferrin degradation product described in (1) of the sample preparation was 0 mg, 10 mg, 20 mg, and 40 mg per ml, and the final concentration of cholic acid described in (19) was 1 m.
0 mg, 1 mg, 10 mg and 20 mg per liter,
Each was adjusted and a survival rate test was conducted.

The results of this test are shown in Table 16. As is clear from Table 16, it was confirmed that cholic acid enhances the bactericidal effect of the lactoferrin degradation product. Moreover, the bactericidal effect was low when the cholic acid was added and the lactoferrin degradation product was not added. Therefore, it is clear that the enhancement of the bactericidal effect is a synergistic effect of the lactoferrin degradation product and cholic acid. A similar test was conducted on the antibacterial peptide instead of the lactoferrin degradation product, and it was confirmed that the bactericidal effect was enhanced by the cholic acid in this case as well.

[0083]

[Table 16]

Test Example 17 The final concentration of the antimicrobial peptide of SEQ ID NO: 26 described in (2) of the sample preparation was 0 mg / ml, 0.5 mg / ml,
The final concentration of 1-monolauroyl-rac-glycerol described in (18) was added to 1 mg and 2 mg of 0 m / ml.
After adjusting to g, 0.5 mg, 1 mg and 2 mg respectively, a survival rate test was conducted.

The results of this test are shown in Table 17. As is apparent from Table 17, 1-monolauroyl-
It was confirmed that rac-glycerol enhances the bactericidal effect of antimicrobial peptides. Also, 1-monolauroyl-
When rac-glycerol was added and no antimicrobial peptide was added, almost no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and 1-monolauroyl-rac-glycerol. A similar test was conducted using a lactoferrin degradation product instead of the antibacterial peptide. In this case as well, 1-monolauroyl-rac was used.
-It was found that glycerol enhances the bactericidal effect.

[0086]

[Table 17]

Test Example 18 The final concentration of the antimicrobial peptide of SEQ ID NO: 26 described in (2) of the above sample preparation was 0 mg / ml, 0.5 mg / ml,
The final concentration of 1-monomyristoyl-rac-glycerol as described in (18) was added to 1 mg and 2 mg at 0 per ml.
It adjusted to mg, 0.5 mg, 1 mg, and 2 mg, respectively, and the survival rate test was done.

The results of this test are shown in Table 18. As is clear from Table 18, it was confirmed that 1-monomyristoyl-rac-glycerol enhances the bactericidal effect of the antibacterial peptide. Further, when 1-monomyristoyl-rac-glycerol was added and no antibacterial peptide was added, the bactericidal effect was low. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and 1-monomyristoyl-rac-glycerol. A similar test was conducted using a lactoferrin degradation product instead of the antibacterial peptide, and it was confirmed that 1-monomyristoyl-rac-glycerol enhanced the bactericidal effect also in this case.

[0089]

[Table 18]

Test Example 19 The final concentration of the antimicrobial peptide of SEQ ID NO: 26 described in (2) of the sample preparation was 0 mg / ml, 0.5 mg / ml,
The final concentration of 1-monostearoyl-rac-glycerol described in (18) was added to 1 mg and 2 mg at 0 per ml.
It adjusted to mg, 0.5 mg, 1 mg, and 2 mg, respectively, and the survival rate test was done.

The results of this test are shown in Table 19. As is clear from Table 19, it was confirmed that 1-monostearoyl-rac-glycerol enhances the bactericidal effect of the antibacterial peptide. In addition, when 1-monostearoyl-rac-glycerol was added and no antimicrobial peptide was added, no bactericidal effect was observed. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and 1-monostearoyl-rac-glycerol. A similar test was conducted using a lactoferrin degradation product in place of the antibacterial peptide, and in this case as well, 1-monostearoyl-r was used.
It was found that the bactericidal effect was enhanced by ac-glycerol.

[0092]

[Table 19]

Test Example 20 The final concentration of the antibacterial peptide of SEQ ID NO: 26 described in (2) of the above sample preparation was 0 mg and 1 mg per ml.
The final concentration of 1-monolauroyl-rac-glycerol according to (18) is 0 mg and 0.5 mg per ml.
Furthermore, the final concentration of bovine lactoferrin described in (3) of the sample preparation was adjusted to 0 mg and 1 mg per ml, respectively, and a viability test was conducted.

The results of this test are shown in Table 20. As is clear from Table 20, 1-monolauroyl-
It was confirmed that rac-glycerol and bovine lactoferrin further enhance the bactericidal effect of the antibacterial peptide. A similar test was conducted on a lactoferrin degradation product instead of the antibacterial peptide, and it was confirmed that the bactericidal effect was also enhanced in this case.

[0095]

[Table 20]

Test Example 21 The final concentration of the antimicrobial peptide of SEQ ID NO: 26 described in (2) of the above sample preparation was 0 mg and 1 mg per ml.
The final concentration of 1-monolauroyl-rac-glycerol according to (18) is 0 mg and 0.5 mg per ml.
In addition, the final concentration of chitosan described in (15) was adjusted to 0 mg and 0.01 mg per ml, and a viability test was conducted.

The results of this test are shown in Table 21. As is clear from Table 21, 1-monolauroyl-
It was confirmed that rac-glycerol and chitosan further enhance the bactericidal effect of the antibacterial peptide. A similar test was conducted using a lactoferrin degradation product instead of the antibacterial peptide, and it was confirmed that the bactericidal effect was also enhanced in this case.

[0098]

[Table 21]

Test Example 22 The final concentration of the antimicrobial peptide of SEQ ID NO: 26 described in (2) of the sample preparation was 0 mg and 1 mg per ml.
The final concentration of 1-monolauroyl-rac-glycerol according to (18) is 0 mg and 0.5 mg per ml.
Furthermore, the final concentration of cholic acid described in (19) was adjusted to 0 mg and 1 mg per ml, and a viability test was conducted.

The results of this test are shown in Table 22. As is clear from Table 22, 1-monolauroyl-
It was confirmed that rac-glycerol and cholic acid further enhance the bactericidal effect of the antibacterial peptide. A similar test was conducted on a lactoferrin degradation product instead of the antibacterial peptide, and it was confirmed that the bactericidal effect was also enhanced in this case.

[0101]

[Table 22]

Next, the present invention will be described more specifically and in detail by showing examples, but the present invention is not limited to the following examples.

[0103]

Example 1 50 g of commercially available lactoferrin (manufactured by Oleofina Co., Belgium) as separated from milk was dissolved in 950 g of purified water, and 1N hydrochloric acid was added to the resulting solution to adjust the pH to 2. It is adjusted, heated at 120 ° C. for 15 minutes, cooled, and then lactoferrin degradation product solution (lactoferrin degradation product concentration: 5
%) About 1000 g was obtained. The degradation degree of this lactoferrin degradation product was 9%.

The above-mentioned lactoferrin degradation product solution was concentrated under reduced pressure and lyophilized to give about 49 powders of the lactoferrin degradation product.
g was obtained. ED was added to 10 g of this lactoferrin decomposition product powder.
An antibacterial agent was prepared by uniformly mixing 1 g of TA and disodium (manufactured by Wako Pure Chemical Industries, Ltd.). Example 2 1 kg of commercially available lactoferrin (manufactured by Oleofina Co., Belgium) as separated from milk was dissolved in 9 kg of purified water, and 2 molar citric acid was added to adjust the pH to 2.5. 30 g of pork pepsin (1: 10,000, manufactured by Wako Pure Chemical Industries, Ltd.) was added and mixed uniformly, and the mixture was heated to 37 ° C. for 1 hour.
The mixture was kept for 80 minutes, heated at 85 ° C. for 10 minutes to inactivate the enzyme, and then cooled to obtain about 10 kg of a lactoferrin degradation product solution (lactoferrin degradation product concentration: 10%). The decomposition degree of this lactoferrin decomposition product was 11.3%.

The above-mentioned lactoferrin degradation product solution was concentrated under reduced pressure and lyophilized to give about 9 powders of the lactoferrin degradation product.
60 g were obtained. 100g of this lactoferrin decomposition product powder
An antibacterial agent was prepared by uniformly mixing 30 g of citric acid (manufactured by Nacalai Tesque, Inc.). Example 3 50 mg of commercially available bovine lactoferrin (manufactured by Sigma) was dissolved in 0.9 ml of purified water, and the pH was adjusted to 2. with 0.1N hydrochloric acid.
Adjusted to 5, then commercial pig pepsin (manufactured by Sigma) 1 m
g, and hydrolyzed at 37 ° C. for 6 hours. Then 0.
Adjust the pH to 7.0 with 1N sodium hydroxide and
The enzyme was inactivated by heating at 0 ° C for 10 minutes, cooled to room temperature, and centrifuged at 15,000 rpm for 30 minutes to obtain a transparent supernatant. 100 μl of this supernatant was added to TSK gel ODS-
After high-performance liquid chromatography using 120T (manufactured by Toso Co., Ltd.), 10 samples were injected at a flow rate of 0.8 ml / min.
20 minutes with 0.05% TFA (trifluoroacetic acid)
% Acetonitrile, then 0.05% T for 30 minutes
Elute with a gradient of 20-60% acetonitrile containing FA, collect fractions eluting between 24-25 minutes,
Vacuum dried. This dried product was dissolved in purified water at a concentration of 2% (W / V), and again subjected to high performance liquid chromatography using TSK gel ODS-120T (manufactured by Toso Corporation),
0.05% for 10 minutes after sample injection at a flow rate of 0.8 ml / min
Elute with 24% acetonitrile containing TFA, then 30
Fractions eluting with a gradient of 24-32% acetonitrile containing 0.05% TFA per minute and eluting between 33.5 and 35.5 minutes were collected. The above operation was repeated 25 times and dried in vacuum to obtain about 1.5 mg of antibacterial peptide.

The above antimicrobial peptide was hydrolyzed with 6N hydrochloric acid, and the amino acid composition was analyzed by an ordinary method using an amino acid analyzer. The same sample was subjected to Edman degradation 25 times using a gas phase sequencer (manufactured by Applied Biosystems) to determine the sequence of 25 amino acid residues. In addition, a disulfide bond analysis method using DTNB (5,5-dithio-bis (2-nitrobenzoic acid)) [Analytic Biochemistry (Analytic
al Biochemistry), 67, 493, 1975.
Year] confirmed the existence of disulfide bonds.

As a result, this peptide was composed of 25 amino acid residues, the 3rd and 20th cysteine residues were disulfide-bonded, and the 3rd cysteine residue resulted in N
It was confirmed to have the amino acid sequence of SEQ ID NO: 26 in which two amino acid residues were bonded to the terminal side and five amino acids were bonded to the C-terminal side from the 20th cysteine residue, respectively. ..

100 g of the above antimicrobial peptide powder was uniformly mixed with 1 g of beef lactoferrin (manufactured by Sigma),
An antibacterial agent was prepared. Example 4 Using a peptide automatic synthesizer (Pharmacia LKB Biotechnology, LKBBiolynx4170), Shepard et al. [Journal of Chemical Society Parkin I (Journal of Chemical Societ)
y Perkin I), p. 538, 1981], and an antibacterial peptide was synthesized as follows.

N, N-dicyclohexylcarbodiimide was added to an amino acid whose amine functional group was protected by a 9-fluorenylmethoxycarbonyl group to produce the desired anhydride of the amino acid, and this Fmoc-amino acid anhydride was synthesized. Using. In order to produce a peptide chain, Fmoc-lysine anhydride corresponding to the C-terminal lysine residue is used as a catalyst for ultrocin A resin (Pharmacia LKB Biotechnology-
Fixed). The resin is then washed with dimethylformamide containing piperidine to remove the amine functional protecting group of the C-terminal amino acid. Later amino acid sequence C
The penultimate corresponding Fmoc-lysine anhydride was coupled via the C-terminal amino acid residue to the deprotected amine functional group of lysine immobilized on the resin. Similarly, methionine, arginine, tryptophan,
Glutamine, tryptophan, arginine, arginine, threonine, and lysine were fixed. After completion of coupling of all amino acids and formation of a peptide chain having a desired amino acid sequence, 94% TFA, 5% pheno-
And a solvent consisting of 1% ethanediol to remove protecting groups other than acetamidomethyl and elimination of the peptide, the peptide was purified by high performance liquid chromatography, and the solution was concentrated and dried, Peptide powder was obtained.

The peptide thus obtained was analyzed for amino acid composition by an ordinary method using an amino acid analyzer, and it was confirmed to have the amino acid sequence of SEQ ID NO: 27. 100 mg of antibacterial peptide powder prepared by the above synthesis
Then, 2 g of casein phosphopeptide (the same one used in Test Example 3) was uniformly mixed to prepare an antibacterial agent. Example 5 An eye drop (aqueous solution) having the following composition was produced by a conventional method.

Boric acid 1.60 (%) Antibacterial agent of Example 4 0.15 Methylcellulose 0.50 Example 6 Chewing gum having the following composition was produced by a conventional method.

Gum base 25.00 (%) Calcium carbonate 2.00 Fragrance 1.00 Antibacterial agent of Example 2 0.02 Sorbitol powder 71.98 Example 7 A dentifrice having the following composition was produced by a conventional method. did.

Dicalcium phosphate dihydrate 36.93 (%) sorbitol 45.00 glycerin 15.00 carbooxymethylcellulose sodium 1.50 sorbitan fatty acid ester 0.50 saccharin sodium 1.00 Example Antibacterial agent 1 of 0.07 Example 8 A skin cleansing solution having the following composition was produced by a conventional method. This skin cleansing solution is diluted 50 times with water before use.

Sodium chloride 8.0 (%) Antibacterial agent of Example 3 0.8 Purified water 91.2 Example 9 An external preparation (ointment) having the following composition was produced by a conventional method.

Ethyl paraoxybenzoate 0.10 (%) Butyl paraoxybenzoate 0.10 Lauromacrogol 0.50 Cetanol 18.00 White petrolatum 40.00 Purified water 40.85 Peptide of SEQ ID NO: 27 0.15 1- Monomyristoyl-rac-glycerol 0.30 Example 10 A hand lotion having the following composition was produced by a conventional method.

Carbowax 1500 8.00 (%) Alcohol 5.00 Propylene glycol 52.00 Purified water 33.90 Fragrance 0.30 Peptide of SEQ ID NO: 26 0.20 1-Monolauroyl-rac-glycerol 0.20 Cole Acid 0.40

[0117]

The effects of the present invention are as follows. (1) An antibacterial agent having an excellent antibacterial action against a wide range of microorganisms is provided. (2) An extremely safe antibacterial agent is provided for use in foods and pharmaceuticals. (3) Since the antibacterial agent of the present invention exhibits an antibacterial effect even in a small amount, it has almost no effect on the flavor when used for treating articles such as foods.

[0118]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 2 Sequence length: 11 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 3 Sequence length: 6 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 5 Sequence length: 6 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 6 Sequence length: 6 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence feature: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 8 Sequence length: 5 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys) SEQ ID NO: 10 Sequence length: 6 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide , And a peptide containing this peptide as a fragment SEQ ID NO: 11 Sequence length: 5 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 12 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 13 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment SEQ ID NO: 18 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 19 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 21 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment SEQ ID NO: 22 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment In the following sequence, Cys No. 2 Cys 19 is disulfide-bonded.

[0119] SEQ ID NO: 23 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment Cys * is a disulfide bond in the following sequence Cysteine is a chemically modified thiol group to prevent the formation of cysteine.

[0120] SEQ ID NO: 24 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and peptide containing this peptide as a fragment In the following sequence, Cys No. 2 Cys 19 is disulfide-bonded.

[0121] SEQ ID NO: 25 Sequence length: 20 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment Cys * is a disulfide bond in the following sequence Cysteine is a chemically modified thiol group to prevent the formation of cysteine.

[0122] SEQ ID NO: 26 Sequence length: 25 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment Cys No. 3 in the following sequence Cys 20 is disulfide-bonded.

[0123] SEQ ID NO: 27 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment SEQ ID NO: 28 Sequence length: 38 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence feature: Peptide containing this peptide and this peptide as a fragment Cys of 16 in the following sequence No. 33 Cys is disulfide-bonded.

[0124] SEQ ID NO: 29 Sequence length: 32 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment In the following sequence, Cys number 10 It has a disulfide bond with Cys 27.

[0125] SEQ ID NO: 30 Sequence length: 47 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment In the following sequence, a sequence length of 36 And number 9 and 26
Nos. 9 and Cys of peptides having Cys at Nos. 35 and 35
And Cys at No. 26 are disulfide-bonded, and the Cys at No. 35 of the peptide having a sequence length of 36 and the Cys at No. 10 of the peptide having a sequence length of 11 and having Cys at 10 are disulfide-bonded. is doing.

[0126] SEQ ID NO: 31 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment (In the above sequence, Xaa represents any amino acid residue except Cys)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location // A23K 1/16 304 Z 9123-2B A23L 3/34 A61K 7/00 J 9165-4C Z 9165 -4C 7/16 7252-4C 7/32 C07K 5/10 8018-4H 7/06 ZNA Z 8318-4H 7/08 8318-4H 7/10 8318-4H C07K 99:00 (72) Inventor Mitsunori Takase Saitama 138-10 Minami-Nakamaru, Omiya-shi, Japan (72) Inventor Wayne Berami- 3-22-6 Higashihara, Zama-shi, Kanagawa 3-22-6 Villa Sagamino West C-5 (72) Instructor Tsuneharu Yamauchi 4-2 Tamawa, Kamakura-shi, Kanagawa -Garden Heights Kamakura Tamawa 405 (72) Inventor Hiroyuki Wakabayashi 118 Minami Kibogaoka, Asahi-ku, Yokohama-shi, Kanagawa Morinaga Kibogaoka Dormitory (72) Inventor Yukiko Tokita 3-6 Asahicho, Sagamihara-shi, Kanagawa Flatsto Down Sagami No. 201

Claims (4)

[Claims] 1. A compound selected from the group consisting of (A) lactoferrin degradation products, lactoferrin-related antibacterial peptides, and arbitrary mixtures thereof, and (B) a metal-binding protein, tocopherol, cyclo. Dextrins, glycerin fatty acid esters, alcohols, E
A compound selected from the group consisting of DTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and any mixture thereof, as an active ingredient Antibacterial agent. 2. The antibacterial agent according to claim 1, wherein the metal-binding protein is lactoferrin, transferrin, conalbumin, or casein phosphopeptide. 3. A compound selected from the group consisting of (A) a lactoferrin degradation product, a lactoferrin-related antibacterial peptide, and an arbitrary mixture thereof, and (B) a metal-binding protein, tocopherol, cyclo Dextrins, glycerin fatty acid esters, alcohols, E
A compound selected from the group consisting of DTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and any mixture thereof, as an active ingredient And a method of treating an article with an antibacterial agent. 4. A method for treating an article with an antibacterial agent according to claim 3, wherein the metal-binding protein is lactoferrin, transferrin, conalbumin, or casein phosphopeptide.