JP3261175B2 - Antimicrobial agent and method of treating articles using this antimicrobial agent - Google Patents

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 an article using the antibacterial agent. More specifically, the present invention relates to a novel antibacterial agent having an excellent antibacterial activity against a wide range of microorganisms, and a method for safely performing an antibacterial treatment on articles such as foods and pharmaceuticals using the antibacterial agent.

[0002]

2. Description of the Related Art In vivo, lactoferrin is a natural iron-binding protein contained in tears, saliva, peripheral blood, milk and the like, and has an antibacterial action against harmful microorganisms such as Escherichia coli, Candida and Clostridium. [Journal of Pediatrics (Journal of P.
ediatrics), Vol. 94, p. 1: 1979]. Also,
For staphylococci and enterococci, 0.5 to 30 mg /
It is known to have an antibacterial effect at a concentration of ml [Nonecke and Smith; Journal of Dairy Science (Nonnecke, BJ & Smith, KL; Journal of Dairy).
Science) 67, 606: 1984].

On the other hand, a large number of inventions have been known for peptides having antibacterial activity against various microorganisms. For example, phosphonotripeptides (JP-A-57-106689) and phosphonodipeptide derivatives (JP-A-58-135) which are effective against Gram-positive and Gram-negative bacteria
No. 94), cyclic peptide derivatives (JP-A-58-21)
No. 3744), peptides exhibiting antibacterial and antiviral activities (JP-A-59-51247), polypeptides effective for yeast (JP-A-60-130599), glycopeptide derivatives effective for gram-positive bacteria (JP-A-60-1
No. 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 Unexamined Patent Publication (Kokai) No. 63-17897) and other antimicrobial peptides extracted from blood cells of horseshoe crab produced in North America (Japanese Unexamined Patent Publication No. 2-53)
799) and an antimicrobial peptide isolated from bee's hemolymph (Japanese Patent Application Laid-Open No. 2-5000084).

[0004] The inventors of the present invention intend to inexpensively isolate a substance having no undesirable side effects (eg, antigenicity), heat resistance and strong antibacterial activity from nature, Mammalian lactoferrin, apolactoferrin and / or metal-saturated lactoferrin (hereinafter, referred to as
These compounds are sometimes referred to as lactoferrins), and it has been found that a hydrolyzate obtained by hydrolyzing the lactoferrins with an acid or an enzyme has higher heat resistance and antibacterial property than undecomposed lactoferrins, and has already filed a patent application (Japanese Patent Application Hei 3-171
736 [JP-A-5-32068] ).

Further, the inventors of the present invention have no undesirable side effects (eg, antigenicity), have heat resistance,
As a lactoferrin-related antimicrobial peptide having strong antimicrobial activity, an antimicrobial peptide having 20 amino acid residues ( Japanese Patent Application No. 3-186260 [JP-A-5-929]).
No. 94] ), an antimicrobial peptide having 11 amino acid residues ( Japanese Patent Application No. 3-48196 [JP-A-5-783 ] ).
No. 92] ), an antimicrobial peptide having 6 amino acid residues ( Japanese Patent Application No. 3-94492 [JP-A-5-14882 ] ).
No. 97] ), an antimicrobial peptide having 5 amino acid residues ( Japanese Patent Application No. 3-94493 [ JP-A-5-1482 ] ).
No. 96] ), an antimicrobial peptide having 3 to 6 amino acid residues ( Japanese Patent Application No. 3-94494 [JP-A-5-14 ] ).
No. 8295 ) and a patent application has already been filed.

Various studies have been made to enhance the antibacterial properties of lactoferrin, and lysozyme, IgA and glycopeptides are known as such physiological activity enhancing adjuvants. For example, a method of synergistically enhancing the antibacterial action by coexistence of lactoferrin and lysozyme [Japanese Patent Laid-Open No. 62-24993], a method of synergistically enhancing the antibacterial action by coexistence of lactoferrin and secretory IgA [ Stephens, etc .;
S., et al; Immunology); 41, 597: 1980.
Year] has been reported. Furthermore, Spick etc.
F. Williams and J.D. Baum (A.
F. Williams & JDBaum), “Human Milk Banking”, Nestle Nutrition Workshop Series, Vol. 5, (Ne
stle Nutrition Workshop Series Volume 5), 133
Page, Raven Press Books,
Ltd.), 1984], reported that lactoferrin has an effect of preventing bacteria from adhering to mucosal surfaces, and that this effect is enhanced by the presence of lysozyme or glycopeptide.

[0007] In addition, studies have also been made on the effect of using lactoferrin in combination with antibiotics, and cephem antibiotics [Shuichi Miyazaki et al .; Chemotherapy ( chemotherapy); volume 39; page 829,
1991] and beta-lactam antibiotics (Japanese Application flat No. 1-319463 [JP-A-3-181421 Patent Publication No.
Information] ) is known.

[0008]

[SUMMARY OF THE INVENTION However, the combined effect of the La <br/> Kutoferin related antimicrobial peptides and antibiotics have not been conventionally at all study, therefore exist antimicrobial agent to them as an active ingredient Did not. Further, with respect to the antimicrobial agent to the La Kutoferin associated antimicrobial peptide and an antibiotic and an active ingredient, by adding a specific component has not been completely performed also attempts to further enhance its antimicrobial activity.

[0009] The present invention has been made in view of the circumstances as per above, by combining the above La Kutoferin related antimicrobial peptides inventors have already invented the present invention with antibiotic or above by combination with La Kutoferin associated antimicrobial peptide and an antibiotic and a specific component, and its object is to provide an antimicrobial agent that antibacterial activity was further enhanced.

[0010]

SUMMARY OF THE INVENTION The present invention, as to solve the foregoing problems, La Kutoferin related antimicrobial peptides or with any mixture thereof, and an antimicrobial agent containing as an active ingredient the antibiotic, the A method for treating an article with an antimicrobial agent is provided.

[0011] The present invention, La Kutoferin related antimicrobial peptides or with any mixture thereof, and an antimicrobial agent, lactoferrin, transferrin, Kon'arubu
Min, casein phosphopeptide , lysozyme, casein hydrolyzate, tocopherol, cyclodextrins, glycerin fatty acid esters, alcohols, EDTA, E
DTA salts, ascorbic acid, ascorbate, citric acid, citrate, polyphosphate, polyphosphate, chitosan, cysteine, co Lumpur acid or any mixture of these,
An antibacterial agent comprising a substance as an active ingredient, 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 (eg, human, cow, sheep, goat, horse, etc.), transitional milk, normal milk,
Late milk or the like, or skim milk, which is a processed product of these milks, lactoferrin separated from whey or the like by a conventional method (for example, ion exchange chromatography), apolactoferrin or apolactoferrin obtained by removing them with hydrochloric acid, citric acid or the like Clean lactoferrin with metals such as iron, copper, zinc and manganese
Metal-saturated or partially-saturated lactoferrin, and commercially available products or preparations prepared by known methods can be used.

[0013] hydrolyzate of lactoferrin to use is obtained by hydrolyzing the above lactoferrin with an acid or an enzyme, can be obtained for example by the method described in Japanese Patent Application No. 3-171736. That is, when 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 to a predetermined temperature for a predetermined time to perform hydrolysis. When performing hydrolysis with an enzyme, an aqueous solution of lactoferrin is adjusted to the optimum pH of the enzyme to be used, enzymes such as pepsin and trypsin are added thereto, and the mixture is hydrolyzed by holding at a predetermined temperature for a predetermined time and then subjecting to hydrolysis. Deactivate the enzyme by the method. The hydrolyzate obtained by hydrolysis with an acid or an enzyme is a mixture of antimicrobial peptides having various molecular weights. Degradation degree by the above hydrolysis,
A range of 6 to 20% is desirable. The degree of decomposition was determined by measuring the total nitrogen of the sample by the Kjeldahl method and the formol nitrogen of the sample by the formol titration method, and calculating the following formula from these values.

Degree of decomposition = (formol 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 obtained by a conventional method. Cooled, neutralized, desalted, and decolored by a conventional method, if necessary, and further fractionated by a conventional method, if necessary, and the resulting solution is used as it is or in a concentrated liquid state, or a powder dried after concentration. With antibiotics, or antibiotics and lactoferrins, transferrin, conalbumin
, Casein phosphopeptide , lysozyme, casein hydrolyzate, tocopherol, cyclodextrins, glycerin fatty acid esters, alcohols, EDTA, ED
TA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid or any mixture thereof
It is mixed with.

The lactoferrin-related antimicrobial peptide used in the present invention is the same as an antimicrobial peptide obtained by a separation means from a lactoferrin degradation product and an antimicrobial peptide obtained from a lactoferrin decomposition product by a separation means. Or a peptide having a homologous chemical structure (amino acid sequence) or a chemical structure (amino acid sequence) identical to or homologous to an antimicrobial peptide obtained by a separation means from a degradation product of lactoferrins
Derivatives of peptides having a or any mixture thereof. These lactoferrin-related antimicrobial peptides are described, for example, in Japanese Patent Application Nos. 3-186260 and 3-48.
No. 196, Japanese Patent Application No. 3-94492, Japanese Patent Application No. 3-944
No. 93, and Japanese Patent Application No. 3-94494. That is, a method of hydrolyzing lactoferrins using an acid or an enzyme and obtaining a fraction containing a peptide having antibacterial properties from the resulting peptide mixture by separation means such as liquid chromatography, or obtained as described above. The amino acid sequence of a peptide having antimicrobial properties is determined by a known method (eg, a method using a gas-phase sequencer), and the peptide having those amino acid sequences is determined by a known method (eg, using an automatic peptide synthesizer). Method) to obtain the desired peptide. These lactoferrin-related antimicrobial peptides are, for example, the following peptides having the following amino acid sequences : antimicrobial peptides having the amino acid sequences of SEQ ID NOs : 1, 2, and 27 or derivatives thereof (Japanese Patent Application No. 3-48196) An antimicrobial peptide having the amino acid sequence of SEQ ID NOs: 3, 4, 5, and 6 or a derivative thereof (the invention of Japanese Patent Application No. 3-94492);
An antimicrobial peptide having the amino acid sequences of 8, 9 and 31 or a derivative thereof (Japanese Patent Application No. 3-94493);
SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16,
Antimicrobial peptides having the amino acid sequences of 17, 18, 19, 20 and 21 or derivatives thereof (Japanese Patent Application No. 3-94)
494 invention); and SEQ ID NOs: 22, 23, 24,
Antimicrobial peptides having amino acid sequences of 25, 26, 28, 29 and 30 or derivatives thereof (Japanese Patent Application No. 3-18 / 1990)
No. 6260 invention).

These antimicrobial peptides may be used as they are, in the form of a concentrated liquid, or in the form of a powder after concentration and drying, with antibiotics, or antibiotics and lactofu.
Erins, transferrin, conalbumin, casei
Phosphopeptide , lysozyme, casein hydrolyzate, tocopherol, cyclodextrin, glycerin fatty acid ester, alcohol, EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphate, polyphosphate, chitosan, cysteine, and mixed with cholic acid or any mixture thereof.

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

Lactoferrins, transferrin,
Albumin, casein phosphopeptide , lysozyme,
Casein hydrolyzate, tocopherol, cyclodextrin, glycerin fatty acid ester, alcohol, ED
TA, EDTA salt, ascorbic acid, ascorbate, citric acid, citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine, cholic acid, etc. can be used commercially available products or prepared by known methods. May be used. Lactoferrins, transferrin,
Albumin and casein phosphopeptide are proteins (metals) that can coordinate with metal ions to form chelate compounds.
Is a protein that chelate) . The casein hydrolyzate is a mixture of hydrolyzates obtained by hydrolyzing casein such as milk with a protease, an acid or an alkali, or a product obtained by fractionating a specific component from the hydrolyzate. In particular, the number average molecular weight is about 380, the molecular weight distribution is 75 or more, and about 1
The use of a mixture of peptides (and amino acids) of 0000 or less is preferred. As cyclodextrins, α-
Examples thereof include cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin, and alkyl derivatives thereof (branched cyclodextrin). Glycerin fatty acid esters are
Esters of fatty acids and glycerin or polyglycerin and derivatives thereof, glycerin fatty acid esters,
Examples thereof include polyglycerin fatty acid esters. The alcohol is an aliphatic monohydric, dihydric, trihydric or polyhydric alcohol, and examples thereof include ethyl alcohol, propylene glycol, and glycerol.

[0019] La Kutoferin related antimicrobial peptides or any whether combining, or any and lactoferrin any combination thereof either with, the antibiotic any mixture thereof, trans ferri
, Conalbumin, casein phosphopeptide , lysozyme, casein degradation product, tocopherol, cyclodextrins, glycerin fatty acid esters, alcohols, EDTA, EDTA salt, ascorbic acid, ascorbate, citric acid, Combination with citrate, polyphosphoric acid, polyphosphate, chitosan, cysteine or cholic acid can be appropriately selected depending on the purpose of use. The mixing ratio can be appropriately selected depending on the type of each component, the purpose of use, and the like. These components can be mixed in liquid or powder form, and a known diluent or excipient may be added as appropriate.

The antibacterial agent of the present invention includes various bacteria, yeasts,
It has strong antibacterial properties against mold and prevents the growth of bacteria, not only as pharmaceuticals, but also quasi-drugs, etc., which are taken into humans or animals or applied to the body surface, and in general Alternatively, the antibacterial agent of the present invention can be used to treat those products or raw materials with any of the products whose suppression is desired. That is, the antibacterial agent of the present invention can be administered to humans or animals as it is, or a drug (eg, eye drops, mastitis therapeutic agent, antidiarrheal agent, athlete's foot drug),
Quasi-drugs (for example, mouthwash, antiperspirant, hair tonic, etc.), various cosmetics (for example, hair styling, cream, milky lotion, etc.), various toothpastes (for example, toothpaste, toothbrush, etc.), various sanitary products , Various baby products (eg, diapers, etc.), various elderly products (eg, denture fixatives, diapers, etc.), various detergents (eg, soap, medicated soap, shampoo, rinse, laundry detergent, kitchen detergent, Household detergents, etc., various sanitizing articles (for example, kitchen sanitizing paper, toilet sanitizing paper, etc.), feed (for example, livestock feed,
Pet foods, etc.), their raw materials, and generally any other articles for which the prevention or suppression of microbial growth is desired, may be added, blended, sprayed, adhered, coated, impregnated, etc. It can be used for the treatment of any articles for which the prevention and suppression of the proliferation of marine products are desired.

It should be noted that the antibacterial agent of the present invention has remarkable antibacterial properties against microorganisms having resistance to various antibiotics, as apparent from the test examples. In other words, microorganisms that are causing nosocomial infections without twist is most antibiotics for example methicillin resistant Staphylococcus aureus and the like, the antibiotic alone, even if no effect, La Kutoferin related antimicrobial peptides, Alternatively, a combination of an arbitrary mixture thereof and a specific antibiotic exhibits a remarkable antibacterial effect. Therefore, the antibacterial agent of the present invention is 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 by the following examples. First, the preparation of a sample and the test method common to each of the following test examples are collectively described. 1. Preparation of sample (1) Lactoferrin hydrolyzate (powder) was prepared and used according to the method described in Reference Example 1. (2) Antimicrobial peptide (powder) It was prepared and used according to the method described in Example 1 . (3) Antibiotics Twelve antibiotics listed in Tables 1 and 5 (commercially available)
It was used. (4) Lactoferrin: Commercially available bovine lactoferrin (manufactured by Sigma) was used. (5) Lysozyme: A 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 solution of test bacteria One loop of platinum was collected from the freeze-preserved suspension of the test bacteria, and spread on Trypticase Soy agar medium (manufactured by BBL).
After culturing at 7 ° C. for 16 hours, the colony grown on the surface of Trypticase soy agar medium was scraped off with a platinum loop and 2.1%
37 for Miura Hinton Bros (Difco)
A logarithmic phase bacterial solution grown at a temperature of 3 ° C. for several hours and grown to a bacterial concentration of 3 × 10 ⁸ / ml was used as a preculture solution. (2) Preparation of test medium The aqueous solution of the lactoferrin hydrolyzate or the antimicrobial peptide prepared in (1) or (2) of the preparation of the sample described in 1 above, and (3) the aqueous solution of the antibiotic were sterilized with a sterile filter (Advantech Co., Ltd.). Was added to a basic medium (Muura-Hinton Broth) prepared to a final concentration of 2.1%, and adjusted to the specified concentrations to prepare a test medium. (3) Antibacterial effect test 100 μl of the pre-cultured solution of the test bacterium prepared in the above (1) diluted with 2.1% Miura Hinton broth at a rate of 2 × 10 ⁶ / ml was prepared in the above (2). Test medium 100
μl, and cultured at 37 ° C. for 16 hours. The antibacterial effect was examined based on the presence or absence of turbidity of the medium. (4) Survival rate test 20 μl of the pre-culture solution of the test bacterium prepared in (1) above was
Add 2 ml of the test medium prepared in (2), culture at 37 ° C. for 1 hour, collect 200 μl from the culture, dilute 10 ^n- fold with 1% peptone aqueous solution,
110 μl was spread on the plate and cultured at 37 ° C. for 24 hours to measure the number of bacteria (the number of test bacteria).

As a separate control, 20 μl of the pre-cultured solution of the test bacterium prepared in the above (1) was added to 2.1% Miura Hinton
It was added to 2 ml of broth, and the number of cells (control cell number) was measured in the same manner as in the measurement of the number of test cells.

The survival rate was calculated by the following equation. 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, 0.4 mg, and 1.6 m per ml.
g and 6.4 mg or the final concentration of the antimicrobial peptide described in (2) of the sample preparation was 0 μg / ml, 16 μg / ml.
μg, 64 μg, and 256 μg, the final concentration of the antibiotic described in (3) was set to 0 μg, 0.01 μg,
Adjusted to 0.1 μg, 1 μg and 10 μg respectively,
Escherichia coli O-111 and Staphylococcus
The antibacterial effect on aureus (JCM2151) was examined, and the growth inhibitory concentration of the antibiotic in the presence of each concentration of antimicrobial peptide was determined.

The test results are as shown in Tables 1 to 4. As is clear from Table 1 to Table 4, it was confirmed that the antimicrobial peptide enhanced the antimicrobial effect of the antibiotic. When the antibacterial peptide was added and the antibiotic was not added, no bactericidal effect was observed. Therefore, it is clear that the above enhancement of the bactericidal effect is a synergistic effect of the antimicrobial peptide and the antibiotic. A similar test was conducted for antibacterial peptides other than the above antibacterial peptides and lactoferrin hydrolyzate. In this case, however, it was confirmed that the antibacterial effect of the antibiotic was enhanced.

[0026]

[Table 1]

[0027]

[Table 2]

[0028]

[Table 3]

[0029]

[Table 4]

Test Example 2 The final concentration of the antimicrobial peptide described in (2) of the above sample preparation was set to 0 μg, 10 μg, 100 μg, and 1000 μg per ml, and the final concentration of the antibiotic described in (3) was set to 1
The survival rate of microorganisms having antibiotic resistance (methicillin-resistant Staphylococcus aureus (wild type)) was adjusted to 0 μg, 10 μg, and 50 μg per ml, respectively.

The results of this test are as shown in Table 5. As is clear from Table 5, it was confirmed that the antimicrobial peptide enhanced the bactericidal effect of the antibiotic. Also, if you add an antimicrobial peptide and do not add an antibiotic,
Almost no bactericidal effect was observed. Therefore, it is clear that the above enhancement of the bactericidal effect is a synergistic effect of the antimicrobial peptide and the antibiotic. Similar tests were conducted on antibacterial peptides other than the above-mentioned antibacterial peptides and lactoferrin hydrolyzate. In this case, it was confirmed that the bactericidal effect of the antibiotic was enhanced.

[0032]

[Table 5]

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

The results of this test are shown in Table 6. As is apparent from Table 6, the antimicrobial peptide and lactoferrin, lysozyme or 1-monocapryloyl-r
It was confirmed that ac-glycerol enhanced the bactericidal effect of antibiotics. In addition, an 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 combining the antimicrobial peptide with lactoferrin, lysozyme or 1-monocapryloyl-ra.
It is clear that there is a synergistic effect between c-glycerol and the anti-drug. 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,
A similar test was conducted for ascorbate, citric acid, citrate, polyphosphate, or polyphosphate and an antibacterial substance, and in this case, it was recognized that the bactericidal effect of the antibiotic was enhanced.

[0035]

[Table 6]

[0036]

Reference Example 1 50 g of commercially available lactoferrin (produced by Oreofina Co., Belgium) as separated from milk was dissolved in 950 g of purified water, and 1N hydrochloric acid was added to the obtained solution to adjust the pH to 2. The mixture was heated at 120 ° C. for 15 minutes, cooled and cooled to obtain a lactoferrin hydrolyzate solution (lactoferrin hydrolyzate concentration: 5
%) About 1000 g were obtained. The degree of decomposition of this lactoferrin hydrolyzate was 9%.

The lactoferrin hydrolyzate solution was concentrated under reduced pressure and freeze-dried to obtain a powder of lactoferrin hydrolyzate of about 49%.
g was obtained. Streptomycin (2 mg) was uniformly mixed with 40 g of the lactoferrin hydrolyzate powder to prepare an antibacterial agent. Reference Example 2 1 kg of commercially available lactoferrin (manufactured by Oreofina of Belgium) as separated from milk was dissolved in 9 kg of purified water, and the pH was adjusted to 2.5 by adding 2 molar concentration of citric acid. 30 g of pork pepsin (1: 10,000; manufactured by Wako Pure Chemical Industries, Ltd.) is added and mixed uniformly.
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 hydrolyzate solution (lactoferrin hydrolyzate concentration: 10%). The degree of decomposition of this lactoferrin hydrolyzate was 11.3%.

The lactoferrin hydrolyzate solution was concentrated under reduced pressure and freeze-dried to obtain a powder of lactoferrin hydrolyzate of about 9%.
60 g were obtained. 100 g of this lactoferrin hydrolyzate powder
Was uniformly mixed with 1 g of polymyxin B to prepare an antibacterial agent. Reference Example 3 0.1 g of oxytetracycline was uniformly mixed with 500 g of the lactoferrin hydrolyzate powder prepared by the same method as in Reference Example 2 to prepare an antibacterial agent. Example 1 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 2.0 with hydrochloric acid of 0.1 standard.
Adjust to 5, then 1m of commercially available pork pepsin (Sigma)
g was added and hydrolyzed at 37 ° C. for 6 hours. Then 0.
The pH was adjusted to 7.0 with sodium hydroxide of 1 regulation, and 8
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 clear supernatant. 100 μl of this supernatant was applied to TSK gel ODS-
After high-performance liquid chromatography using 120T (manufactured by Tosoh Corporation), the sample was injected at a flow rate of 0.8 ml / min.
20 minutes containing 0.05% TFA (trifluoroacetic acid) for 20 minutes
Eluted with 5% acetonitrile, then 0.05% T for 30 minutes
Eluting with a gradient of 20-60% acetonitrile containing FA, collecting fractions eluting between 24-25 minutes,
Vacuum dried. This dried product was dissolved in purified water at a concentration of 2% (W / V) and subjected to high-performance liquid chromatography using TSK gel ODS-120T (manufactured by Tosoh Corporation) again.
0.05% for 10 minutes after sample injection at a flow rate of 0.8 ml / min
Elution with 24% acetonitrile containing TFA followed by 30%
The fractions eluted with a gradient of 24-32% acetonitrile containing 0.05% TFA for minutes and the fractions eluting between 33.5 and 35.5 minutes were collected. The above operation was repeated 25 times and vacuum-dried to obtain about 1.5 mg of the antimicrobial peptide.

The antimicrobial peptide was hydrolyzed with 6N hydrochloric acid, and the amino acid composition was analyzed by a conventional 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), and the sequence of 25 amino acid residues was determined. A disulfide bond analysis method using DTNB (5,5-dithio-bis (2-nitrobenzoic acid)) [Analytic Biochemistry (Analytic
al Biochemistry), 67, 493, 1975.
Year] confirmed that a disulfide bond was present.

As a result, this peptide was composed of 25 amino acid residues, the third and twentieth cysteine residues were disulfide-bonded, and the third cysteine residue was N-substituted.
-It has been confirmed that the amino acid sequence of SEQ ID NO: 26 has two amino acid residues on the terminal side and 5 amino acids on the C-terminal side from the cysteine residue at the 20th position bonded thereto. .

An antibacterial agent was prepared by uniformly mixing 1 mg of the tetracycline antibiotic minocycline with 100 mg of the above antibacterial peptide powder. Example 2 An antimicrobial agent was prepared by uniformly mixing 1 mg of penicillin G with 100 mg of antimicrobial peptide powder prepared by the same method as in Example 1 . Example 3 10 mg of lysozyme and 10 mg of penicillin G1m were added to 10 mg of antibacterial peptide powder prepared by the same method as in Example 1.
g was uniformly mixed to prepare an antibacterial agent. Example 4 Using an automatic peptide synthesizer (Pharmacia LKB Biotechnology, Inc .; LKBBiolynx4170), shepard et al. [Journal of Chemical Society Parkin I
y Perkin I), p. 538, 1981], an antimicrobial peptide was synthesized as follows.

N, N-Dicyclohexylcarbodiimide is added to an amino acid whose amine function is protected with a 9-fluorenylmethoxycarbonyl group to form an anhydride of the desired amino acid, and this Fmoc-amino acid anhydride is synthesized. Using. To produce a peptide chain, an Fmoc-asparagine anhydride corresponding to a C-terminal asparagine residue is immobilized on an ultrathin A resin (manufactured by Pharmacia LKB Biotechnology) via its carboxyl group using dimethylaminopyridine as a catalyst. I do. The resin is then washed with dimethylformamide containing piperidine to remove the protecting group for the amine function of the C-terminal amino acid. Thereafter, the Fmoc-arginine anhydride corresponding to the second 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. Glutamine, tryptophan, glutamine, and phenylalanine were immobilized sequentially in the same manner. After coupling of all amino acids is completed and a peptide chain having a desired amino acid sequence is formed, 94% TF
A. A solvent consisting of 5% phenol and 1% ethanediol was used to remove protecting groups other than acetamidomethyl and to remove the peptide, and to perform high performance liquid chromatography.
The solution was concentrated and dried to obtain a peptide powder.

The amino acid composition of the obtained peptide was analyzed by a conventional method using an amino acid analyzer, and it was confirmed that the peptide had the amino acid sequence of SEQ ID NO: 10. Antibacterial peptide powder 100 mg prepared by the above synthesis
Was uniformly mixed with 0.5 mg of gentamicin to prepare an antibacterial agent. Reference Example 4 An eye drop (aqueous solution) having the following composition was produced by a conventional method.

Boric acid 1.60 (%) Antibacterial agent of Reference Example 1 0.15 Methylcellulose 0.50 Reference Example 5 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 Setanol 20.0 White petrolatum 40.0 Water 29.3 Antibacterial agent of Reference Example 2 10.0 Reference Example 6 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 Reference Example 3 0.1 Example 5 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 1 10.0 Example 6 A remedy for mastitis having the following composition was produced by a conventional method.

1, 2-hydroxystearin 1.0 (%) glyceryl monostearate 0.5 butylated hydroxyanisole 0.02 peanut oil 93.48 Antibacterial agent of Example 2 5.0 Example 7 An external preparation for skin 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 3 10.0 Example 8 An antibiotic preparation having the following composition was produced by a conventional method.

The antimicrobial agent of Example 4 100.0 (%)

[0051]

The effects provided by the present invention are as follows. (1) It is an antibacterial agent that has excellent antibacterial activity 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 even in a small amount, the amount of conventional antibiotics used can be greatly reduced. (4) The antibacterial agent of the present invention has a remarkable antibacterial effect even on microorganisms having resistance to certain antibiotics.

[0052]

[Sequence List] SEQ ID NO: 1 Sequence length: 11 Sequence type: amino acid Topology :: Linear Sequence type: Peptide Sequence features: This peptide and peptides containing this peptide as a fragment Sequence: Lys Xaa Xaa Xaa Xaa Gln Xaa Xaa Met Lys Lys 1510 (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 features: This peptide and a peptide containing this peptide as a fragment Sequence: Lys Xaa Xaa Xaa Xaa Gln Xaa Xaa Met Arg Lys 1 5 10 (In the above sequence, Xaa is 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 this peptide Arg Xaa Xaa Xaa Xaa Arg 15 (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 Sequence: Lys Xaa Xaa Xaa Xaa Arg 15 (In the above sequence, Xaa is 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 peptides containing this peptide as a fragment Sequence: Lys Xaa Xaa Xaa Xaa Lys 15 (In the above sequence, Xaa represents any amino acid residue except Cys.) SEQ ID NO: 6 Sequence length: 6 Sequence type: Amino acid Topolo Di-: linear Sequence type: peptide Sequence characteristics: this peptide and a peptide containing this peptide as a fragment Sequence: Arg Xaa Xaa Xaa Xaa Lys 15 (In the above sequence, Xaa is any amino acid residue except Cys. SEQ ID NO: 7 Sequence length: 5 Sequence type: amino acid Topology: linear type of sequence: peptide Sequence characteristics: this peptide and peptides containing this peptide as a fragment Sequence: Arg Xaa Xaa Xaa Arg 15 (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 Characteristic: This peptide and a peptide containing this peptide as a fragment Sequence: Lys Xaa Xaa Xaa Arg 15 (In the above sequence, Xaa is any amino acid except Cys. SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence features: This peptide and peptides containing this peptide as a fragment Sequence: Arg Xaa Xaa Xaa Lys 15 (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 Sequence: Phe Gln Trp Gln Arg Asn 15 SEQ ID NO: 11 Sequence length: 5 Sequence type: Amino acid Topology-: Type of linear sequence : Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment Sequence: Phe Gln Trp Gln Arg 15 SEQ ID NO: 12 Sequence length: Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics: this peptide and peptides containing this peptide as a fragment Sequence: Gln Trp Gln Arg 1 SEQ ID NO: 13 Sequence length: 3 Sequence length Type: Amino acid Topology: Linear Type of sequence: Peptide Sequence characteristics: This peptide and a peptide containing this peptide as a fragment Sequence: Trp Gln Arg 1 SEQ ID NO: 14 Sequence length: 5 Sequence type: Amino acid Topology: linear Sequence type: peptide Sequence characteristics: this peptide and peptides containing this peptide as a fragment Sequence: Arg Arg Trp Gln Trp 15 SEQ ID NO: 15 Sequence length: 4 Sequence type: amino acid Topology: Linear Sequence type: Peptide Sequence features: This peptide, and fragments of this peptide Sequence containing: as Arg Arg Trp Gln 1 SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence features: Include this peptide and this peptide as a fragment Peptide sequence: Trp Gln Trp Arg 1 SEQ ID NO: 17 Sequence length: 3 Sequence type: amino acid Topology :: Linear Sequence type: Peptide Sequence characteristics: This peptide, and peptide sequence containing this peptide as a fragment : Gln Trp Arg 1 SEQ ID NO: 18 Sequence length: 6 Sequence type: amino acid Topology-: Linear Sequence type: Peptide Sequence features: This peptide and peptides containing this peptide as a fragment Sequence: Leu Arg Trp Gln Asn Asp 15 SEQ ID NO: 19 Sequence length: 5 Sequence type: amino acid Topology: linear type of sequence Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment Sequence: Leu Arg Trp Gln Asn 15 SEQ ID NO: 20 Sequence length: 4 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence characteristics: This peptide and peptides containing this peptide as a fragment Sequence: Leu Arg Trp Gln 1 SEQ ID NO: 21 Sequence length: 3 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide sequence Characteristics of: This peptide and peptides containing this peptide as a fragment Sequence: Arg Trp Gln 1 SEQ ID NO: 22 Sequence length: 20 Sequence type: Amino acid Topology :: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment In the following sequence, Cys at position 19 has a disulfide bond.

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

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: This peptide and peptides containing this peptide as a fragment In the following sequence, Cys No. 2 and Cys No. 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 Sequence type: Peptide Sequence characteristics: This peptide, and peptides containing this peptide as a fragment In the following sequence, Cys * represents cysteine in which the thiol group is chemically modified to prevent the formation of disulfide bonds. .

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 Characteristic of sequence: This peptide and peptide containing this peptide as a fragment In the following sequence, Cys No. 3 and Cys No. 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 features: This peptide and peptides containing this peptide as a fragment Sequence: Lys Thr Arg Arg Trp Gln Trp Arg Met Lys Lys 1510 SEQ ID NO: 28 Sequence length Length: 38 Sequence type: Amino acid Topology: Linear Type of sequence: Peptide Characteristic of sequence: This peptide and a peptide containing this peptide as a fragment In the following sequence, Cys at position 16 and Cys at position 33 are disulfides Are combined.

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: This peptide and peptides containing this peptide as a fragment In the following sequence, Cys No. 10 And Cys at position 27 have a disulfide bond.

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 Length: 47 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics: This peptide, and a peptide containing this peptide as a fragment In the following sequence, the sequence length is 36 and No. 9, 26
And Cys at position 9 and Cys at position 26 of the peptide having Cys at position 35 are disulfide-bonded, and Cys at position 35 of the peptide having length 36 is the sequence length 11 The peptide having Cys at position 10 has a disulfide bond with Cys at position 10.

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 1510 SEQ ID NO: 31 Sequence Length: 5 Sequence Type: Amino Acid Topology-: Linear Sequence Type: Peptide Sequence Features: This peptide and this peptide Peptide included as a fragment Sequence: Lys Xaa Xaa Xaa Lys 15 (In the above sequence, Xaa represents any amino acid residue except Cys.)

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61K 31/545 A61K 31/65 31/65 A61L 2/16 Z 38/16 A61P 31/04 38/17 A23K 1/17 Z A61L 2/16 A61K 7/00 J A61P 31/04 Z // A23K 1/17 7/16 A61K 7/00 C07K 5/10 7/06 7/16 7/08 ZNA C07K 5/10 A61K 37/18 7 / 06 37/14 7/08 ZNA 37/16 (72) Inventor Mitsunori Takase 138-10 Minaminakamaru, Omiya City, Saitama Prefecture (72) Inventor Wayne Berami-3-22-6 Higashihara, Zama City, Kanagawa Prefecture Villa Sagamino West C- 5 (72) Inventor Tsuneharu Yamauchi 4-2-2 Tamano, Kamakura, Kamakura, Kanagawa Pref. Yukiko Tokita Kanagawa 3-6 Asahimachi, Sagamihara-shi Flat Stone Sagami 201 (56) References JP-A-3-220130 (JP, A) European Patent Application Publication 438750 (EP, A1) Shuichi Miyazaki et al. Of lactoferrin in combination with various antibacterial agents in LS. ”, CAR LSSON, A. et al. , Et al. , "L actoferrin and Lys ozyme in (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/00-38/42 A61K 31/00-31/80 A61K 7/00-7/50 A61P 31 / 00-31/04 A61L 2/00-2/28 A01N 37/00-37/52 A01N 43/00-43/90 A01N 63/00-63/04 A23K 1/00-1/24 C07K 5/00 -5/12 C07K 7/00-7/66 CA (STN) MEDLINE (STN) BIOSIS (STN) BIOTECHABS (STN) EMBASE (STN) JICST file (JOIS)

Claims (6)

(57) [Claims] 1. A (A) La Kutoferin related antimicrobial peptides or with any mixture thereof, antimicrobial agent comprising as an active ingredient and (B) an antibiotic. 2. A (A) La Kutoferin related antimicrobial peptides, or any mixtures thereof, and (B) an antibiotic, (C) lactoferrin, transferrin, con
Albumin, casein phosphopeptide , lysozyme, casein degradation product, tocopherol, cyclodextrins, glycerin fatty acid esters, alcohols, ED
TA, EDTA salts, ascorbic acid, ascorbate, citric acid, citrate, polyphosphate, polyphosphate, chitosan, cysteine, cholic acid or antibacterial agents to any mixtures thereof and the active ingredient. 3. The antibiotic (B) is penicillin, semi-synthetic penicillin, cephem antibiotic, carbapenem antibiotic, monobactam antibiotic, aminoglycoside antibiotic, peptide antibiotic, tetracycline antibiotic, chloramphenicol. 3. The antibacterial agent according to claim 1, which is a macrolide antibiotic, rifamycin, vancomycin, fosfomycin, a chemically synthesized antibacterial agent, an antifungal agent, an antituberculous agent, or polymyxin B. Wherein (A) La Kutoferin related antimicrobial peptides or method of processing an article by using the mixture of any of these, an antibacterial agent comprising as an active ingredient and (B) an antibiotic. Wherein (A) La Kutoferin related antimicrobial peptides or with any mixture thereof, and (B) an antibiotic, (C) lactoferrin, transferrin, con
Albumin, casein phosphopeptide , lysozyme, casein degradation product, tocopherol, cyclodextrins, glycerin fatty acid esters, alcohols, ED
Using TA, EDTA salts, ascorbic acid, ascorbate, citric acid, citrate, polyphosphate, polyphosphate, chitosan, cysteine, an antimicrobial agent and cholic acid or active ingredient and any mixture thereof, A method of processing goods. (B) The antibiotic is penicillin, semisynthetic penicillin, cephem antibiotic, carbapenem antibiotic, monobactam antibiotic, aminoglycoside antibiotic, peptide antibiotic, tetracycline antibiotic, chloramphenicol. A method for treating an article with the antibacterial agent according to claim 4 or 5 , which is a macrolide antibiotic, rifamycin, vancomycin, fosfomycin, a chemically synthesized antibacterial agent, an antifungal agent, an antituberculous agent, or polymyxin B.