JPH05310594A - Antibacterial agent and method for treating article with the agent - Google Patents

Abstract

PURPOSE:To provide an antibacterial agent having further improved antibacterial property of the decomposition product of lactoferrins and/or a lactoferrin- relating antibacterial peptide. CONSTITUTION:An antibacterial agent containing (A) a compound selected from the decomposition product of lactoferrins, a lactoferrinrelating antibacterial peptide and an arbitrary mixture of the product with (B) an antibiotic substance as active components. As an alternative, an antibacterial agent containing (A) a compound selected from the decomposition product of lactoferrins, a lactoferrin-relating antibacterial peptide and an arbitrary mixture of product with (B) an antibiotic substance and (C) a compound selected from a metal- chelating protein, a lysozyme, a decomposed casein, tocopherol, cyclodextrins, glycerol fatty acid esters, alcohols, EDTA, an EDTA salt, ascorbic acid, an ascorbic acid salt, citric acid, a citric acid salt, a polyphosphoric acid, a polyphosphoric acid salt, chitosan, cysteine, cholic acid and their arbitrary mixture as active components.

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 a new antibacterial agent having an excellent antibacterial action against a wide range of microorganisms, and a method for safely antibacterial treatment of articles such as foods and pharmaceuticals 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 Clostridia. 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] Further, research on the combined effect of lactoferrin and antibiotics has been conducted so far, and as an antibiotic for enhancing antibacterial activity by combined use with lactoferrin, a cephem antibiotic [Shuichi Miyazaki et al .; Chemotherapy ( chemotherapy); Volume 39; Page 829, 1
991] and beta-lactam antibiotics (Japanese Patent Publication 1
No. 3,194,63) is known.

[0008]

However, no studies have been conducted on the combined use of the above-mentioned lactoferrin degradation products or lactoferrin-related antibacterial peptides and antibiotics, and therefore antibacterial agents containing them as active ingredients. Did not exist either. Furthermore, the above-mentioned lactoferrin degradation product, or an antibacterial agent containing lactoferrin-related antibacterial peptides and antibiotics as active ingredients, by adding a specific compound, there is no attempt to further enhance its antibacterial activity. It wasn't done.

The present invention has been made in view of the above circumstances, and the degradation product of lactoferrin and / or the lactoferrin-related antibacterial peptide already invented by the inventors of the present invention is used as an antibiotic. For the purpose of providing an antibacterial agent having further enhanced antibacterial properties, by using in combination, or by using the above-mentioned lactoferrin degradation product and / or lactoferrin-related antibacterial peptide, an antibiotic and a specific compound in combination. There is.

[0010]

[Means for Solving the Problems] In order to solve the above problems, the present invention provides a compound selected from the group consisting of a degradation product of lactoferrins, a lactoferrin-related antibacterial peptide, and an arbitrary mixture thereof, and an antibiotic. An antibacterial agent containing a substance as an active ingredient and a method for treating an article using the antibacterial agent are provided.

The present invention also provides a compound selected from the group consisting of a decomposed product of lactoferrins, an antibacterial peptide related to lactoferrin, and an arbitrary mixture thereof; an antibacterial agent; , Casein degradation products, tocopherols, cyclodextrins,
Glycerin fatty acid esters, alcohols, EDT
A, EDTA salt, ascorbic acid, ascorbate,
Provided is an antibacterial agent containing citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, or cholic acid as active ingredients, and a method for treating an article using the antibacterial agent.

Next, the present invention will be described in detail. In the following description,% is a value by weight excluding the degree of decomposition of lactoferrins. In the present invention, lactoferrins include commercially available lactoferrin, colostrum of mammals (for example, humans, cows, sheep, goats, horses, etc.), transitional milk, normal milk,
Lactoferrin separated from terminal milk, etc., or skim milk, whey, etc., which is a processed product of these milks by a conventional method (for example, ion exchange chromatography), apolactoferrin deaerated with hydrochloric acid, citric acid, etc., apo Lactoferrin is chelated with metals such as iron, copper, zinc and manganese.
It is a metal-saturated or partially-saturated lactoferrin prepared by commercialization, and a commercially available product or a preparation produced by a known method can be used.

The decomposed product of lactoferrin used in the present invention is obtained by hydrolyzing the above-mentioned lactoferrin with an acid or an enzyme, for example, Japanese Patent Application No. 3-171736.
Can be obtained by the method described in No. That is, in the case of performing hydrolysis with an acid, lactoferrins are dissolved in water, an inorganic acid or an organic acid is added thereto, and the mixture is heated at a predetermined temperature for a predetermined time to perform hydrolysis. When the hydrolysis is carried out with an enzyme, an aqueous solution of lactoferrin is adjusted to an optimum pH of the enzyme to be used, and an enzyme such as pepsin or trypsin is added thereto, and the mixture is kept at a predetermined temperature for a predetermined time and then hydrolyzed. Inactivate the enzyme by the method. The hydrolyzate obtained by acid or enzymatic hydrolysis 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 degree of decomposition was calculated from the following formula by measuring the total nitrogen of the sample by the Kjeldahl method and the formal nitrogen of the sample by the formol titration method.

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 as a concentrated liquid, or a powder dried after concentration In the form of proteins, proteins that chelate antibiotics or antibiotics and metals, lysozyme, casein degradation products, tocopherols, cyclodextrins, glycerin fatty acid esters, alcohols, EDTA, ED
Mix with a compound selected from the group consisting of TA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and any mixture thereof.

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
It can be obtained by the method described in each invention of Japanese Patent Application No. 94492, Japanese Patent Application No. 3-94493, and Japanese Patent Application No. 3-94494. That is, lactoferrins are hydrolyzed with an acid or an enzyme, and a method for obtaining a fraction containing an antimicrobial peptide by a separation means such as liquid chromatography from the obtained peptide mixture, or obtained as described above The amino acid sequence of the peptide having antibacterial properties is determined by a known method (for example, a method using a gas phase sequencer), and the peptide having the amino acid sequence is known by a known method (for example, an automatic peptide synthesizer is used). Method) to obtain the desired peptide. These lactoferrin-related antibacterial peptides are, for example, the following peptides having the following amino acid sequences. : Antibacterial peptides having the amino acid sequences of SEQ ID NOS: 1, 2 and 27 or derivatives thereof (Japanese Patent Application No. 3-48196 invention); Derivative (Japanese Patent Application No. 3-94
492 invention); antibacterial peptides having the amino acid sequences of SEQ ID NOs: 7, 8, 9 and 31 or derivatives thereof (Japanese Patent Application No. 3-94449 invention); SEQ ID NOs: 10, 11, 1
2, 13, 14, 15, 16, 17, 18, 19, 20
And an antibacterial peptide having the amino acid sequence of 21 or its derivative (the invention of Japanese Patent Application No. 3-94494); and SEQ ID NOs: 22, 23, 24, 25, 26, 28, 2
An antibacterial peptide having 9 and 30 amino acid sequences or a derivative thereof (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 then drying it, and a protein, a lysozyme, a casein degradation product, and a tocopherol which chelate an antibiotic or a metal with an antibiotic. , Cyclodextrins, glycerin fatty acid esters, alcohols, EDTA, EDTA salts, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid and any of these Mix with a compound selected from the group consisting of mixtures.

The antibiotics used in the present invention include penicillin, semi-synthetic penicillin, cephem antibiotics, carbapenem antibiotics, monobactam antibiotics, aminoglycoside antibiotics, peptide antibiotics, tetracycline antibiotics, chloram. Examples thereof include phenicol, macrolide antibiotics, rifamycin, vancomycin, fosfomycin, chemically synthesized antibacterial agents, antifungal agents, antituberculous agents, and polymyxin B. As these antibiotics, commercially available products or those prepared by known methods can be used.

Proteins that chelate metals, lysozyme,
Casein degradation products, tocopherols, cyclodextrins, glycerin fatty acid esters, alcohols, ED
TA, EDTA salt, ascorbic acid, ascorbic acid, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid, etc. may be commercially available products, or prepared by known methods. May be used. For example, a metal chelating protein is a protein capable of forming a chelate compound by coordinating with a metal ion, and examples thereof include lactoferrins, transferrin, conalbumin, and casein phosphopeptide. The casein degradation product is a mixture of degradation products obtained by hydrolyzing casein such as milk with a proteolytic enzyme, an acid or an alkali by a conventional method, or a product obtained by fractionating a specific component from the degradation product. Especially, the number average molecular weight is 380
Preference is given to using a mixture of peptides (and amino acids) with a molecular weight distribution of more than 75 and less than about 10,000 in order. Examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin, and their alkyl derivatives (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 monohydric, dihydric, trihydric or polyhydric alcohols, such as ethyl alcohol, propylene glycol and glycerol.

Combining any of the above-mentioned antibiotics with any one of a lactoferrin degradation product, a lactoferrin-related antibacterial peptide, or any mixture thereof.
Alternatively, any of them in combination with a metal chelating protein, lysozyme, casein degradation product, tocopherol, cyclodextrin, glycerin fatty acid ester, alcohol, EDTA, EDTA salt, ascorbic acid , Ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine or cholic acid 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, the purpose of use and the like. These components can be mixed in a liquid or powder form, and a known diluent or excipient may be appropriately added.

The antibacterial agent of the present invention includes various bacteria, yeasts,
It has strong antibacterial properties against mildew, and is not only used as a drug, but also as a quasi drug that is taken into the human or animal body or applied to the body surface, and generally prevents bacterial growth. Alternatively, it can be used by blending it in any product in which suppression is desired, and those products or raw materials can be treated with the antibacterial agent of the present invention. That is, the antibacterial agent of the present invention can be directly administered to humans or animals, or pharmaceuticals (for example, eye drops, mastitis therapeutic agents, antidiarrheal agents, athlete's foot medicines),
Quasi-drugs (for example, mouthwash, antiperspirant, hair nourishing agent, etc.), various cosmetics (for example, hair dressing, cream, milky lotion, etc.), various toothpaste products (for example toothpaste, toothbrush etc.), various sanitary products. , Various baby products (for example, diapers), various elderly products (for example, denture fixatives, diapers, etc.), various detergents (for example, soap, medicated soap, shampoo, rinse, laundry detergent, kitchen detergent, Household detergents, etc.), various sanitizing products (for example, kitchen sanitizing paper, toilet sanitizing paper, etc.), feed (eg livestock feed,
Pet hood, etc.), raw materials thereof, and generally any other article for which the growth or prevention of microorganisms is desired to be added, blended, sprayed, attached, coated, impregnated, etc. It can be used for the treatment of any article for which the prevention or suppression of the proliferation of spores is desired.

It should be noted that, as is apparent from the test examples, the antibacterial agent of the present invention exhibits remarkable antibacterial activity against microorganisms resistant to various antibiotics. That is, most antibiotics are ineffective and cause nosocomial infections, such as methicillin-resistant Staphylococcus aureus, even if the antibiotic alone does not show any effect, a lactoferrin degradation product, lactoferrin The combined use of the related antimicrobial peptide, or any mixture thereof, with a particular antibiotic shows a marked antimicrobial effect. Therefore, the antibacterial agent of the present invention is also extremely effective in preventing nosocomial infections, which have recently become a major social problem.

Next, the present invention will be described in more detail by showing test examples and examples, but the present invention is not limited to the following examples. First, the preparation of the sample common to the following test examples and the test method will be described collectively. 1. Preparation of sample (1) Decomposed product of lactoferrin (powder) The sample was prepared by the method described in Example 1 and used.

(2) Antibacterial peptide (powder) Prepared by the method described in Example 3 and used. (3) Antibiotics 12 kinds of antibiotics listed in Table 1 and Table 5 (commercially available products)
It was used. (4) Lactoferrin: Commercially available bovine lactoferrin (manufactured by Sigma) was used. (5) Lysozyme: Commercially available egg white lysozyme (manufactured by Seikagaku Corporation) was used.

(6) 1-Monocapryloyl-rac-glycerol: a commercially available product (manufactured by Sigma) was used. 2. Test method (1) Preparation of pre-culture liquid of test bacteria 1 platinum loop was taken from the freeze-preserved suspension of the test bacteria, and smeared on Trypticase Soy agar medium (BBL) 3
After culturing at 7 ℃ for 16 hours, the colonies grown on the surface of trypticase soy agar were scraped with a platinum loop to give 2.1%.
37 for Miura-Hinton Broth (manufactured by Difco)
Culture was carried out at a temperature of several hours for several hours, and a bacterial solution in the logarithmic phase that had grown to a bacterial concentration of 3 × 10 ⁸ / ml was used as a preculture liquid.

(2) Preparation of test medium An aqueous solution of the lactoferrin degradation product or the antimicrobial peptide prepared in (1) or (2) of the preparation of the sample described in 1 above and (3) an aqueous solution of an antibiotic are sterilized with a filter. Bacteria were sterilized by (Advantech) and added to a basic medium (Mueller-Hinton Broth) prepared to have a final concentration of 2.1%, and adjusted to the specified concentrations to prepare test mediums.

(3) Antibacterial effect test 100 μl of the preculture liquid of the test bacteria prepared in the above (1) was diluted with 2.1% Mueller-Hinton broth at a ratio of 2 × 10 ⁶ / ml, and added to the above (2). ) Test medium 100 prepared in
It was added to μl and cultured at 37 ° C. for 16 hours, and the antibacterial effect was examined by the presence or absence of turbidity of the medium.

(4) Viability test 20 μl of the preculture liquid of the test bacteria prepared in (1) above was added to the above
After adding 2 ml of the test medium prepared in (2) and culturing at 37 ° C. for 1 hour, 200 μl was taken from the culture and diluted 10 ⁿ times with a 1% peptone aqueous solution.
Then, 110 μl of the solution was applied on the plate and cultured at 37 ° C. for 24 hours to measure the number of bacteria (test bacteria).

Separately, as a control, 20 μl of the preculture liquid of the test bacteria prepared in (1) above was added to 2.1% Miura-Hinton
Broth was added to 2 ml, and the number of bacteria (the number of control bacteria) was measured in the same manner as in the measurement of the number of test bacteria. The survival rate was calculated by the following formula. Survival rate = (number of test bacteria / number of control bacteria) × 100 Test Example 1 The final concentration of the lactoferrin hydrolyzate described in (1) of the sample preparation was 0 mg / ml, 0.4 mg / 1.6 m.
g and 6.4 mg, or the final concentration of the antimicrobial peptide described in (2) of the sample preparation was 0 μg per ml, 16
μg, 64 μg, and 256 μg, the final concentration of the antibiotic described in (3) was 0 μg, 0.01 μg per ml,
Adjusted to 0.1 μg, 1 μg and 10 μg respectively,
Escherichia coli O-111 and Staphylococcus
The antibacterial effect against Aureus (JCM2151) was examined, and the growth inhibitory concentration of the antibiotic was determined when each concentration of antibacterial peptide coexisted.

The test results are shown in Tables 1 to 4. As is clear from Tables 1 to 4, it was confirmed that the antibacterial peptides enhance the antibacterial effect of antibiotics. In addition, no bactericidal effect was observed when the antimicrobial peptide was added and no antibiotic was added. Therefore, it is clear that the above-mentioned enhancement of the bactericidal effect is a synergistic effect of the antibacterial peptide and the antibiotic. Similar tests were conducted on antibacterial peptides other than the above antibacterial peptides and lactoferrin degradation products, and it was confirmed that the antibacterial effects of the antibiotics were also enhanced in this case.

[0030]

[Table 1]

[0031]

[Table 2]

[0032]

[Table 3]

[0033]

[Table 4]

Test Example 2 The final concentration of the antimicrobial peptide described in (2) of the sample preparation was 0 μg, 10 μg, 100 μg, and 1000 μg per ml, and the final concentration of the antibiotic described in (3) was 1
It was adjusted to 0 μg, 10 μg, and 50 μg per ml, respectively, and the survival rate test of the antibiotic-resistant microorganism [methicillin-resistant Staphylococcus aureus (wild type)] was performed.

The results of this test are shown in Table 5. As is clear from Table 5, it was confirmed that the antibacterial peptide enhances the bactericidal effect of the antibiotic. Also, if the antimicrobial peptide was added and no antibiotic 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 the antibiotic. 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 of the antibiotics was also enhanced in this case.

[0036]

[Table 5]

Test Example 3 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was 0 μg / ml, or the antimicrobial peptide and the sample preparation (4), (5), (6) The described lactoferrin, lysozyme or 1-monocapryloyl-rac-
The final concentration of glycerol was 10 μg per ml and 100 μg each, and the final concentration of the antibiotic described in (3) was 1 μm.
The viability test of Staphylococcus aureus (JCM-2151) was performed after adjusting to 0 μg and 1 μg per ml, respectively.

The results of this test are shown in Table 6. As is clear from Table 6, the antibacterial peptide and lactoferrin, lysozyme or 1-monocapryloyl-r
It was confirmed that ac-glycerol enhances the bactericidal effect of antibiotics. In addition, antibacterial peptide and lactoferrin, lysozyme or 1-monocapryloyl-rac-
When glycerol was added and no antibiotic was added, almost no bactericidal effect was observed. Therefore, the above-mentioned enhancement of the bactericidal effect is achieved by the antibacterial peptide and lactoferrin, lysozyme or 1-monocapryloyl-ra.
It is clear that there is a synergistic effect of c-glycerol and the antidote. In addition, antibacterial peptides other than the above antibacterial peptides, and proteins that chelate lactoferrin degradation products and metals, casein degradation products, tocopherols, cyclodextrins, glycerin fatty acid esters, alcohols, EDTA, EDTA salt, ascorbic acid,
Similar tests were carried out on ascorbate, citric acid, citrate, polyphosphoric acid, or polyphosphate and an antibacterial substance, and it was found that the bactericidal effect of the antibiotic was also enhanced in this case.

[0039]

[Table 6]

[0040]

【Example】

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, 120 Heat at 15 ° C. for 15 minutes, cool, 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 freeze-dried to obtain about 49 powders of the lactoferrin degradation product.
g was obtained. 2 mg of streptomycin was uniformly mixed with 40 g of this lactoferrin degradation product powder to prepare an antibacterial agent. 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 decomposition product solution was concentrated under reduced pressure and freeze-dried to obtain about 9 powders of the lactoferrin decomposition product.
60 g was obtained. 100g of this lactoferrin decomposition product powder
An antibacterial agent was prepared by uniformly mixing 1 g of polymyxin B with. Example 3 An antibacterial agent was prepared by uniformly mixing 0.1 g of oxytetracycline with 500 g of lactoferrin degradation product powder prepared by the same method as in Example 2. Example 4 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 0.1 with hydrochloric acid of 0.1 standard.
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 1 standard 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 consists of 25 amino acid residues, the 3rd and 20th cysteine residues are disulfide-bonded, and the 3rd cysteine residue leads to N
It was confirmed to have the amino acid sequence of SEQ ID NO: 26 in which two amino acid residues were bound to the -terminal side and 5 amino acids were bound to the C-terminal side from the 20th cysteine residue, respectively. .

An antibacterial agent was prepared by uniformly mixing 100 mg of the above-mentioned antibacterial peptide powder with 1 mg of the tetracycline antibiotic minocycline. Example 5 An antibacterial agent was prepared by uniformly mixing 100 mg of the antibacterial peptide powder prepared by the same method as in Example 4 with 1 mg of penicillin G. Example 6 10 mg of antibacterial peptide powder prepared by the same method as in Example 4 was added to 100 mg of lysozyme and penicillin G1m.
g was uniformly mixed to prepare an antibacterial agent. Example 7 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 antibacterial peptides were 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-asparagine anhydride corresponding to the C-terminal asparagine residue is immobilized on Ultrosin A resin (Pharmacia LKB Biotechnology) using its dimethyl group as a catalyst through its carboxyl group. To do. The resin is then washed with dimethylformamide containing piperidine to remove the amine functional protecting group of the C-terminal amino acid. Thereafter, Fmoc-arginine anhydride, which corresponds to the second position from the C-terminal of the amino acid sequence, was coupled to the deprotected amine functional group of arginine immobilized on the resin via the C-terminal amino acid residue. In the same manner, glutamine, tryptophan, glutamine, and phenylalanine were sequentially fixed in the same manner. 94% TF after coupling of all amino acids and formation of a peptide chain of the desired amino acid sequence
A, 5% phenol, and 1% ethanediol were used to remove protective groups other than acetamidomethyl and peptides to remove the peptide.
The peptide was purified by and the solution was concentrated and dried to obtain a peptide powder.

The amino acid composition of the peptide thus obtained was analyzed by an ordinary method using an amino acid analyzer, and it was confirmed that it had the amino acid sequence of SEQ ID NO: 10. 100 mg of antibacterial peptide powder prepared by the above synthesis
0.5 mg of gentamicin was uniformly mixed to prepare an antibacterial agent. Example 8 An eye drop (aqueous solution) having the following composition was produced by a conventional method.

Boric acid 1.60 (%) Antibacterial agent of Example 1 0.15 Methylcellulose 0.50 Example 9 A skin external preparation having the following composition was produced by a conventional method.

Ethyl paraoxybenzoate 0.1 (%) Butyl paraoxybenzoate 0.1 Lauromacrogol 0.5 Cetanol 20.0 White petrolatum 40.0 Water 29.3 Antibacterial agent of Example 2 10.0 Example 10 An animal feed having the following composition was produced by a conventional method.

Fish meal 30.0 (%) Soybean meal 39.9 Wheat 30.0 Antibacterial agent of Example 3 0.1 Example 11 A skin external preparation having the following composition was produced by a conventional method.

Ethyl paraoxybenzoate 0.1 (%) Butyl paraoxybenzoate 0.1 Lauromacrogol 0.5 Cetanol 20.0 White petrolatum 40.0 Water 29.3 Antibacterial agent of Example 4 10.0 Example 12 A therapeutic agent for mastitis having the following composition was produced by a conventional method.

1,2-Hydroxystearin 1.0 (%) Glycer monostearate 0.5 Butylated hydroxyanisole 0.02 Peanut oil 93.48 Antibacterial agent of Example 5 5.0 Example 13 A skin external preparation having the following composition was produced by a conventional method.

Ethyl paraoxybenzoate 0.1 (%) Butyl paraoxybenzoate 0.1 Lauromacrogol 0.5 Cetanol 20.0 White petrolatum 40.0 Water 29.3 Antibacterial agent of Example 6 10.0 Example 14 An antibiotic preparation having the following composition was produced by a conventional method.

Antibacterial agent of Example 7 100.0 (%)

[0055]

The effects of the present invention are as follows. (1) An antibacterial agent having an excellent antibacterial action against a wide range of microorganisms. (2) It is an extremely safe antibacterial agent used in foods and pharmaceuticals. (3) Since the antibacterial agent of the present invention exhibits an antibacterial effect in a small amount, the amount of conventional antibiotics used can be greatly reduced. (4) The antibacterial agent of the present invention exhibits a remarkable antibacterial effect even on microorganisms resistant to certain antibiotics.

[0056]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: (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 sequence 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 sequence 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 sequence 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 sequence 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 characteristics: This peptide , And a peptide sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 peptide containing this peptide as a fragment Sequence: SEQ ID NO: 12 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 13 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 18 Sequence length: 6 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 19 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: SEQ ID NO: 21 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide containing this peptide as a fragment Sequence: 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.

Sequence: Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val 20 SEQ ID NO: 23 Sequence Length: 20 Sequence Type: Amino Acid Topology: Linear Type of sequence: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment In the following sequence, Cys * represents cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond. ..

Sequence: Lys Cys * Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys * Val 20 SEQ ID NO: 24 Sequence Length: 20 Sequence Type: Amino Acid Topology: Type of linear sequence: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment In the following sequence, Cys 2 and 19 are disulfide-bonded.

Sequence: Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys Ile 20 SEQ ID NO: 25 Sequence Length: 20 Sequence Type: Amino Acid Topology: Linear Type of sequence: Peptide Sequence characteristics: Peptide containing this peptide and this peptide as a fragment In the following sequence, Cys * represents cysteine in which a thiol group is chemically modified to prevent the formation of a disulfide bond. ..

Sequence: Lys Cys * Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly Pro 1 5 10 15 Pro Val Ser Cys * Ile 20 SEQ ID NO: 26 Sequence Length: 25 Sequence Type: Amino Acid Topology: Type of linear sequence: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment In the following sequence, Cys 3 and 20 are disulfide-bonded.

Sequence: Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala 1 5 10 15 Pro Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25 SEQ ID NO: 27 Sequence Length: 11 Sequence Type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and peptide containing this peptide as a fragment Sequence: 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.

Sequence: Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Glu Trp Phe Lys 1 5 10 15 Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro Ser 20 25 30 Ile Thr Cys Val Arg Arg Ala Phe 35 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 Cys number 10 in the following sequence And Cys 27 are disulfide-bonded.

Sequence: Thr Ile Ser Gln Pro Glu Trp Phe Lys Cys Arg Arg Trp Gln Trp 1 5 10 15 Arg Met Lys Lys Leu Gly Ala Pro Ser Ile Thr Cys Val Arg Arg 20 25 30 Ala Phe SEQ ID NO: 30 of the 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, the length of sequence 36 is number 9, 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.

Sequence: Val Ser Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn 1 5 10 15 Met Arg Lys Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp 20 25 30 Ser Pro Ile Gln Cys Ile 35 Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala 1 5 10 SEQ ID NO: 31 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and this peptide Peptide sequence included 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 indication location A61K 31/545 7252-4C 31/65 7252-4C 37/14 ADZ 8314-4C 37/16 8314- 4C A61L 2/16 Z 8718-4C // A23K 1/17 Z 9123-2B A61K 7/00 J 9165-4C Z 9165-4C 7/16 7252-4C C07K 5/10 8018-4H 7/06 Z 8318- 4H 7/08 8318-4H 7/10 ZNA 8318-4H C07K 99:00 (72) Inventor Mitsunori Takase 138-10 Minamichumaru, Omiya City, Saitama Prefecture (72) Inventor Wayne Berami- 3-22 Higashihara, Zama City, Kanagawa Prefecture -6 Villa Sagamino West C-5 (72) Inventor Tsuneharu Yamauchi 4-2-2 Tamawa, Kamakura City, Kanagawa Prefecture Garden Heights 405 Tamawaro, Kamakura City (72) Inventor Hiroyuki Wakabayashi Minami Kibogaoka, Asahi Ward, Yokohama City, Kanagawa Prefecture 118 Morinaga Hope Dormitory (72) inventor Yukiko Tokita Sagamihara, Kanagawa Prefecture Asahimachi 3-6 flat be sampled - down Sagami No. 201

Claims (8)

[Claims] 1. An antibacterial agent containing (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) an antibiotic. 2. A compound selected from the group consisting of (A) a decomposed product of lactoferrins, an antibacterial peptide related to lactoferrin, and an arbitrary mixture thereof; (B) an antibiotic; and (C) a metal. Protein, lysochi
, Casein degradation products, tocopherols, cyclodextrins, glycerin fatty acid esters, alcohols,
Effective with a compound selected from the group consisting of EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphate, polyphosphate, chitosan, cysteine, cholic acid, and any mixture thereof. Antibacterial agent as an ingredient. 3. (B) The antibiotic is penicillin, semi-synthetic penicillin, cephem antibiotics, carbapenem antibiotics, monobactam antibiotics, aminoglycoside antibiotics, peptide antibiotics, tetracycline antibiotics, chloramphenicol. The antibacterial agent according to claim 1 or 2, which is a macrolide antibiotic, rifamycin, vancomycin, fosfomycin, a chemically synthesized antibacterial agent, an antifungal agent, an antituberculous agent, or polymyxin B. 4. The antibacterial agent according to claim 2, wherein the metal-chelating protein is lactoferrin, transferrin, conalbumin, or casein phosphopeptide. 5. An antibacterial agent comprising (A) a lactoferrin degradation product, a lactoferrin-related antibacterial peptide, and a mixture selected from the group consisting of these and (B) an antibiotic as active ingredients. A method of treating an article using. 6. A compound selected from the group consisting of (A) a decomposed product of lactoferrin, a lactoferrin-related antibacterial peptide, and an arbitrary mixture thereof, (B) an antibiotic, and (C) a metal. Protein, lysochi
, Casein degradation products, tocopherols, cyclodextrins, glycerin fatty acid esters, alcohols,
Effective with a compound selected from the group consisting of EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphate, polyphosphate, chitosan, cysteine, cholic acid, and any mixture thereof. A method of treating an article with an antibacterial agent as a component. 7. (B) The antibiotics are penicillin, semisynthetic penicillin, cephem antibiotics, carbapenem antibiotics, monobactam antibiotics, aminoglycoside antibiotics, peptide antibiotics, tetracycline antibiotics, chloramphenicol. A method for treating an article with an antibacterial agent according to claims 5 and 6, which is a macrolide antibiotic, rifamycin, vancomycin, fosfomycin, a chemically synthesized antibacterial agent, an antifungal agent, an antituberculous agent, or polymyxin B. 8. A method for treating an article with an antibacterial agent according to claim 6, wherein the metal-chelating protein is lactoferrin, transferrin, conalbumin or casein phosphopeptide.