JP2771068B2 - Antimicrobial peptides and antimicrobial agents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel antimicrobial peptide, an antimicrobial agent containing the peptide, an antimicrobial peptide compound containing the peptide, and a method for treating an article with the antimicrobial agent. It is. More specifically, the present invention relates to an antimicrobial agent, a peptide, or a peptide containing a novel antimicrobial peptide or a derivative thereof, a salt of the peptide, or a derivative of the peptide, or a mixture of two or more thereof as an active ingredient. And a method for treating an article with the antimicrobial agent.

[0002] In the present specification, amino acids and peptides are indicated based on the abbreviation adopted by the IUPAC-IUB Biochemical Nomenclature Committee (IUPAC-IUB CBN). For example, the following abbreviations are used. Ala-: L-alanine residue Gln-: L-glutamine residue Arg-: L-arginine residue Glu-: L-glutamic acid residue Asn-: L-asparagine residue Gly-: L-glycine residue Asp- : L-aspartic acid residue His-: L-histidine residue Cys-: L-cysteine residue Ile-: L-isoleucine residue Leu-: L-leucine residue Ser-: L-serine residue Lys-: L-lysine residue Thr-: L-threonine residue Met-: L-methionine residue Trp-: L-tryptophan residue Phe-: L-phenylalanine residue Tyr-: L-tyrosine residue Pro-: L- Proline residue Val-: L-valine residue

[0003]

2. Description of the Related Art Numerous inventions have been known for peptides having antibacterial activity against various microorganisms. For example, phosphonotripeptides (JP-A-57-106689), phosphonodipeptide derivatives (JP-A-58-13594), and cyclic peptide derivatives (JP-A-58-13594) effective against Gram-positive and Gram-negative bacteria 213744), a peptide exhibiting antibacterial and antiviral activities (Japanese Patent Application Laid-Open No. 59-51247), a polypeptide effective for yeast (Japanese Patent Application Laid-Open No. 60-130599)
Glycopeptide derivatives effective in Gram-positive bacteria (JP-A-60-172998, JP-A-61-251699, JP-A-63-44598), oligopeptides effective in Gram-positive bacteria (JP-A-62-22798), peptide antibiotics (JP-A-62-51697, JP-A-63-17897), and other antimicrobial peptides extracted from hemocytes of North American horseshoe crab (JP No. 2-53799) and antimicrobial peptides isolated from bee's haemolymph (Japanese Patent Laid-Open No. 2-500084) and the like are known.

[0004] On the other hand, lactoferrin (hereinafter referred to as LF) is a natural iron-binding protein contained in tears, saliva, peripheral blood, milk, etc., and against harmful microorganisms such as Escherichia coli, Candida and Clostridium. And is known to exhibit antibacterial activity [Journal of Pediatrics, Vol. 94, page 1,
1979]. However, the antibacterial effect of a peptide having a specific amino acid sequence isolated from a hydrolyzate of LF has not been described in the literature and has not been known. Further, in the peptide having the above amino acid sequence, a specific amino acid sequence having antibacterial activity is not known at all.

[0005]

SUMMARY OF THE INVENTION The inventors of the present invention have no undesirable side effects (eg, antigenicity),
Investigating the antimicrobial properties of LF contained in whey, a by-product of cheese production, with the aim of inexpensively isolating heat-resistant and strong antibacterial substances from nature. And found that a hydrolyzate obtained by hydrolyzing LF with an acid or an enzyme has higher heat resistance and antibacterial properties than undecomposed LF, and has already filed a patent application (Japanese Patent Application No. 2-13315. Request 1). After filing the prior application 1, the present inventors have further investigated the antibacterial substance present in the hydrolyzate of LF, but isolated a novel antibacterial peptide having a specific amino acid sequence, and A patent application was filed (Japanese Patent Application No. 2-238364; hereinafter referred to as the prior application 2), however, for such a novel antibacterial peptide, the amino acid sequence having the antibacterial property was not sufficiently elucidated.

The present invention has been made in view of the above circumstances, and can solve the problems of the prior art and the prior application, and can be isolated or chemically synthesized from a hydrolyzate of LF. A novel antimicrobial peptide having a specific antimicrobial amino acid sequence, an antimicrobial agent containing this peptide as an active ingredient, an antimicrobial compound containing this peptide as an active ingredient, and an antimicrobial agent containing this peptide as an active ingredient An object of the present invention is to provide a method for processing an article by using the method.

[0007] In order to solve the above-mentioned problems, the present invention provides a peptide having any one of the following amino acid sequences or a derivative in which the C-terminal is amidated. Further, the present invention comprises, as an active ingredient, a substance selected from the group consisting of a peptide having any one of the following amino acid sequences, a derivative in which the C-terminus is amidated, and a mixture of two or more of them. Antimicrobial agents and antimicrobial peptide formulations are provided. Further, the present invention provides a composition comprising, as an active ingredient, a substance selected from the group consisting of a peptide having any one of the following amino acid sequences, a derivative in which the C-terminus is amidated, and a mixture of two or more of them. Provided is a method of treating an article with a featured antimicrobial agent. Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg-Met-Lys-Lys Lys-Thr-Val-Ser- Trp-Gln-Thr-Tyr-Met-Lys-Lys Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys Lys-Thr-Leu-Arg-Trp-Gln-Asn-Glu- Met-Arg-Lys

The present invention also has a preferred embodiment in which the antibacterial agent and the antibacterial peptide compound contain an active ingredient in a concentration of at least 1 micromolar (μM). Hereinafter, the configuration, operation, and effect of the present invention will be described in detail. The antimicrobial peptide of the present invention can be produced by hydrolyzing an enzymatically isolated bovine LF or commercially available LF with an enzyme, or can be chemically synthesized by an ordinary method.

An example of chemically synthesizing the antimicrobial peptide of the present invention is as follows. Using an automatic peptide synthesizer (for example, Pharmacia LKB Biotechnology, Inc., LKB Biolynx 4170), shepard et al. [Journal of Chemical Society Parkin I
(Journal of Chemical Society Perkin I), 538
1981]. N, N'-dicyclohexylcarbodiimide is added to an amino acid whose amine function is protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group (hereinafter abbreviated as Fmoc-amino acid) to form an anhydride of the desired amino acid. This Fmoc-amino acid anhydride is used for the synthesis. To produce a peptide chain, an Fmoc-amino acid anhydride corresponding to a C-terminal amino acid residue is converted to an urtrosin A resin (manufactured by Pharmacia LKB Biotechnology) via its carboxyl group and dimethylaminopyridine as a catalyst.
Fixed to The resin is then washed with dimethylformamide containing piperidine to remove the protecting group for the amine function of the C-terminal amino acid. Corresponds to the second amino acid residue from the C-terminal of the amino acid sequence of the desired peptide
The Fmoc-amino acid anhydride is coupled via the C-terminal amino acid residue to the deprotected amine function of the first amino acid immobilized on the resin. Thereafter, desired amino acids are sequentially fixed in the same manner. After coupling of all amino acids is completed and a peptide chain having a desired amino acid sequence is formed, a solvent [for example, trifluoroacetic acid, 94% (weight; hereinafter the same unless otherwise noted), 5% phenol and 1% Consisting of ethanediol] to remove the protecting groups other than acetamidomethyl and to remove the peptide, and purify the peptide by high performance liquid chromatography.

An example of producing the antimicrobial peptide of the present invention by enzymatic hydrolysis is as follows. LF is dissolved in water, sterilized water, purified water or the like at a concentration of 0.5 to 20%, desirably 5 to 15%, an enzyme is added, and hydrolysis is performed. The enzyme to be used is not particularly limited, and a commercially available product, for example, Morcine F (trademark, manufactured by Seishin Pharmaceutical Co., Ltd., optimal pH 2.5-3.
0), pork pepsin (manufactured by Wako Pure Chemical Industries, Ltd., optimal pH 2-3), Sumiteam AP (trademark, manufactured by Shin Nippon Chemical Co., Ltd., optimal pH 3.
0), Amano A (trademark, manufactured by Amano Pharmaceutical Co., Ltd., optimal pH 7.
0) and endopeptidases such as trypsin (manufactured by Novo; optimum pH 8.0) can be used alone or in any combination. In addition to these enzymes, for example,
A commercially available soy sauce enzyme (manufactured by Tanabe Seiyaku Co., Ltd.) containing lactic acid bacteria-derived exopeptidase and peptidase obtained by the method described in Japanese Patent No. 43878 can also be used. The amount of the enzyme is preferably in the range of 0.1 to 5.0% based on the substrate.

The pH of the LF solution is adjusted to an optimum pH of the enzyme to be used, a predetermined amount of the enzyme is added, and the enzyme is added at 15 to 55 ° C., preferably 30 to 50 ° C. for 30 to 600 minutes, preferably 6 to 60 minutes.
Hold for 0-300 minutes and hydrolyze. Thereafter, the reaction solution can be used as it is or neutralized to inactivate the enzyme by a conventional method, and if necessary, neutralized and decolorized. It is possible to isolate the desired antimicrobial peptide by conventional chromatographic methods or the like from LF hydrolyzate obtained by <br/> more good sea urchin. For example, TSK gel ODS120T (manufactured by Tosoh Corporation)
In high-performance liquid chromatography using, it can be isolated with an acetonitrile gradient.

The antimicrobial peptide, pharmacologically acceptable salts thereof, or a mixture of two or more thereof obtained as described above is used as an active ingredient in at least 1 macromol, preferably 5 to 20 micromol. The antimicrobial agent of the present invention can be obtained by adding the antimicrobial agent of the present invention at a desired concentration. The antimicrobial peptide or a derivative thereof according to the present invention can be directly administered to humans or animals, or can be used as foods, pharmaceuticals (eg, eye drops, mastitis therapeutics, diarrhea preventives, athlete's foot drugs, etc.), quasi-drugs ( For example, mouthwashes, antiperspirants, hair nourishes, etc.), various cosmetics (eg, hairdressing agents, etc.), various toothpastes (eg, toothpastes, toothbrushes, etc.), various sanitary products, various baby products (eg, diapers, etc.), Various elderly products (eg, denture fixatives, diapers, etc.), various detergents (eg,
Soaps, medicated soaps, shampoos, rinses, laundry detergents, kitchen detergents, home detergents, etc., various sanitizing products (eg, sanitizing paper for kitchen, sanitizing paper for toilet, etc.), feed, and their raw materials It may be added, blended, sprayed, adhered, coated, impregnated, or the like to any material, or any other article in which prevention or suppression of microbial growth is generally desired.

The antimicrobial peptides or derivatives thereof according to the present invention may be used alone or in combination with other antimicrobial agents for foods, pharmaceuticals (eg, eye drops, mastitis therapeutics, diarrhea preventives, athlete's foot drugs, etc.), pharmaceuticals Quasi-drugs (e.g. mouthwash, antiperspirant, hair nourisher, etc.), various cosmetics (e.g. hair styling, etc.),
Various toothpaste products (eg, toothpaste, toothbrush, etc.), various sanitary products, various baby products (eg, diapers, etc.), various elderly products (eg, denture fixatives, diapers, etc.), various detergents (eg, soaps, medicated soaps) , Shampoos, rinses, laundry detergents, kitchen detergents, household detergents, etc., various sanitizing products (eg, sanitizing paper for kitchen, sanitizing paper for toilets, etc.), feed, raw materials for them, etc. In general, it can be used for treating any article for which prevention or suppression of the growth of microorganisms is desired.

Next, the present invention will be described in more detail with reference to test examples. (Test Example 1) This test was performed to examine the antibacterial activity of an antibacterial peptide. (1) Preparation of Samples Samples 1 to 5 chemically synthesized by the same method as in Examples 1 to 5 were used.

(2) Test method 1) Preparation of pre-culture solution One platinum loop was collected from a stock slant of Escherichia coli, smeared on a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and treated at 35 ° C. for 16 hours. The colonies aerobically cultured and grown on the surface of a standard agar medium were scraped with a platinum loop, suspended in sterile physiological saline, and the turbidity (OD; 660 nm) measured with a spectrophotometer (manufactured by Hitachi, Ltd.) was adjusted to 1.0. It was adjusted to prepare a preculture.

2) Preparation of basal medium Bactocaditon (manufactured by Difco) is dissolved in purified water at a concentration of 1%, and the pH is adjusted to 7.0 with 1M sodium hydroxide.
The solution was sterilized at 115 ° C. for 15 minutes to prepare a basal medium (liquid medium). 3) Preparation of test medium and control medium Each sample was dissolved in purified water at a concentration of 0.01%, and sterilized with a sterile filter (manufactured by Advantech).
Test media added to the basal media at concentrations of 5, 10, 20, and 100 micromolar (μM) and control media without additives were prepared.

4) Antibacterial test The above culture medium and control medium were inoculated with the above-mentioned preculture solution at a concentration of 1%, aerobically cultured at 35 ° C for 16 hours, and the turbidity of the culture solution was measured in the same manner as described above. The E. coli growth inhibition rate was calculated from the following equation. Growth inhibition rate (%) = 100 (1−A / B) [where A is the turbidity difference of the test culture (turbidity of the test culture after 16 hours of culture and turbidity of the test culture before culture) Difference with
And B, the turbidity difference of the control medium (the difference between the turbidity of the control culture solution after 16 hours of culturing and the turbidity of the control culture solution before culturing), respectively. The percentage of the growth inhibition rate is not based on the weight (the same applies hereinafter). (3) Test Results The results of this test are as shown in Table 1.

[0018]

[Table 1]

As is clear from Table 1, Samples 1 to
5 showed antimicrobial activity at 1 μM and 5 to 20 μM
M showed strong antibacterial activity in the range, and at 50 μM or more, no increase in antibacterial activity due to an increase in concentration was observed. Therefore,
The concentration having antibacterial properties is at least 1 μM, preferably 5 to 20 μM. This amount is about the same antimicrobial activity as aminobenzylpenicillin.

The antibacterial properties of peptides having an amino acid sequence other than those of Samples 1 to 5, derivatives thereof, and salts thereof were tested, and almost the same results were obtained. (Test Example 2) This test was performed to confirm the amino acid sequence of the antimicrobial peptide used in Test Example 1.

The peptides of Samples 1 to 5 obtained in Test Example 1
The mixture was hydrolyzed with N 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 using a gas-phase sequencer (manufactured by Applied Biosystems), and the sequence of 11 amino acid residues was determined. As a result, this peptide was composed of 11 amino acid residues and had the following amino acid sequence.

Sample 1: Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Ly
s Sample 2: Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg-Met-Lys-Ly
s Sample 3: Lys-Thr-Val-Ser-Trp-Gln-Thr-Tyr-Met-Lys-Ly
s Sample 4: Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Ly
s Sample 5: Lys-Thr-Leu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Ly
s (Test Example 3) This test was performed to examine the antibacterial spectrum of the antibacterial peptide of the present invention.

(1) Preparation of Sample An antimicrobial peptide was prepared by the same method as in Examples 1 to 5, and was sterilized by filtration with a filter (0.45 μm Myrex filter) before use. Note that these samples were referred to as Sample 1 to Sample 5, respectively. (2) Test method The logarithmic phase strains of the various microorganisms shown in Table 2 were used for 0, 1.5,
3,6,12,25,50, and peptone medium composed of 1% Bacto-peptone (manufactured by Difco Laboratory Co.) containing each sample at a rate of 100 [mu] M were inoculated at a rate of 10 ⁶ / ml, the 160Myu l The cells were cultured at 37 ° C. or 30 ° C. for 17 hours using a 96-well microtiter plate (Falcon). The growth status of various microorganisms in various samples and concentrations was measured by absorbance at 660 nm, and the minimum growth inhibition concentration (MI
C; μM) was determined.

The microorganisms used in this test were all of the Institute of Medical Science, University of Tokyo (IID), the Institute of Physical and Chemical Research (JCM),
It is readily available from the Japan International Dairy Federation (IDF), and MMI is an applicant's laboratory stock. (3) Test results The results of this test are as shown in Table 2. As is clear from Table 2, the antimicrobial peptide was obtained from Pseudomonas aeruginosa MMI 603, a gram-negative bacterium (PA in Table 2).
And Klebsiella pneumoniae JCM 1662T
(Indicated as KP in Table 2) at 50 μM or less, showing antibacterial activity and being a gram-positive bacterium, Listeria monoc.
ytogenes IDF 1b (indicated as LM in Table 2), and St
It exhibited antibacterial activity against aphylococcus aureus JCM 2151 (denoted as SA in Table 2) at a low concentration of 12 μM or less.

It should be noted that substantially the same results were obtained with other antimicrobial peptides of the present invention, their derivatives and their salts.

[0026]

[Table 2]

Test Example 4 This test was conducted to examine the effect of treating the article with the antibacterial agent of the present invention. The commercially available primary processed vegetables (so-called cut vegetables) are divided into two groups of 100 g each, immersed in a 20 micromolar aqueous solution of the antimicrobial peptide derivative produced by the same method as in Example 30 for 30 seconds, and drained sufficiently. ,
After storage at 5 ° C., the change in the number of viable cells was measured over time by a conventional method. Even with the sample was immersed in tap water without added antimicrobial peptides as controls, were identical test as above.

The results of this test are as shown in Table 3. As is clear from Table 3, in the vegetables treated with the antibacterial peptide derivative of the present invention, the growth of bacteria was remarkably suppressed. In addition, about the same result was obtained also about the antimicrobial peptide synthesize | combined by the same method as another Example, its derivative (s), and its salt.

[0029]

[Table 3]

(Test Example 5) This test was performed to examine the antibacterial effect of the antibacterial peptide derivative of the present invention. (1) Preparation of Sample An antimicrobial peptide derivative was prepared in the same manner as in Example 6. (2) Test method The same method as in Test example 3 was used. However, Escherichia
coli MMI O 111 and Escherichia coli IID 861 were added. The source of the strain is the same as in Test Example 3. (3) Test results The minimum growth inhibitory concentration (MIC; μM) for each microorganism is Es
cherichia coli MMI O 111 is 6, Escherichia coli IID
861 for 3, Pseudomonas aeruginosa MMI 6 O 3 for 3, Kleb
siella pneumoniae JCM 1662T 12, Listeria monocyt
ogenes IDF 1b 1.5 and Staphylococcus aureus J
CM 2151 was 3. In addition, about the same result was obtained also about the antimicrobial peptide synthesize | combined by the same method as another Example, its derivative (s), and its salt.

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

[0032]

【Example】

Example 1 Using an automatic peptide synthesizer (Pharmacia LKB Biotechnology Inc., LKB Biolynx 4170), shepard et al. [Journal of Chemical Society Perkin I
I), p. 538, 1981] based on the solid phase peptide synthesis method 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. It was used for. To produce a peptide chain, Fmoc-lysine anhydride corresponding to the C-terminal lysine residue is immobilized via its carboxyl group on Ultrosin A resin (Pharmacia LKB Biotechnology) using dimethylaminopyridine as a catalyst. . The resin is then washed with dimethylformamide containing piperidine to remove the protecting group for the amine function of the C-terminal amino acid. The Fmoc-lysine anhydride corresponding to the second from the C-terminus of the amino acid sequence was then coupled via the C-terminal amino acid residue to the lysine-deprotected amine functional group immobilized on the resin. Hereinafter, methionine, arginine, tryptophan, glutamine, tryptophan, arginine, arginine, threonine, and lysine were immobilized in the same manner.

When the coupling of all amino acids is completed,
After a peptide chain having the desired amino acid sequence is formed, 94
The protective group other than acetamidomethyl was removed and the peptide was eliminated with a solvent consisting of 5% trifluoroacetic acid, 5% phenol, and 1% ethanediol, and the peptide was purified by high performance liquid chromatography. This solution was concentrated and dried to obtain about 16 mg of the peptide. Example 2 Example 1 was repeated except that the 7th and 10th amino acid residues from the C-terminus were changed to an arginine residue and a serine residue.
Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg-
About 20 mg of peptide having the amino acid sequence of Met-Lys-Lys
Obtained. Example 3 Same as Example 1 except that the 4, 5, 8, and 9th amino acid residues from the C-terminus were changed to tyrosine residue, threonine residue, serine residue and valine residue, respectively. Lys-Thr-Val-Ser-Trp-Gln-Thr-Tyr-Met-
About 17 mg of a peptide having the amino acid sequence of Lys-Lys was obtained. Example 4 Amino acid residues at positions 2, 4, 5, 8, and 9 from the C-terminus were replaced with an arginine residue, an asparagine residue,
Amino acid sequence of Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys by the same method as in Example 1 except that the amino acid sequence was changed to arginine residue, glutamine residue and phenylalanine residue. About 13 mg of a peptide having Example 5 Example 1 except that the types of the second, fourth, fifth and ninth amino acid residues from the C-terminal were changed to arginine residue, glutamic acid residue, asparagine residue and leucine residue, respectively. Lys-Thr-Leu-Arg-
About 16 mg of a peptide having the amino acid sequence of Trp-Gln-Asn-Glu-Met-Arg-Lys was obtained. Example 6 Amino acid residues were sequentially fixed in the same manner as in Example 1 except that Ultrathin B resin (Pharmacia LKB) was used. After binding of all amino acids is complete, remove the protecting groups other than acetamidomethyl with a solvent consisting of 94% trifluoroacetic acid, 5% phenol, and 1% ethanedithiol. Purify the peptide by chromatography, concentrate, dry and Lys-Thr-Arg-Arg-Trp-Gln-Trp-Ar
About a peptide having the amino acid sequence of g-Met-Lys-Lys-NH2
15 mg were obtained. Example 7 A skin cream having the following composition was produced.

Stearic acid 150 g Cetanol 20 g Squalane 30 g Octyldodecyl myristate 50 g Glycerin 100 g 1,3-butylene glycol monostearate boroxyethylene ethylene (20 mol) Sorbitan 30 g Example 1 40 mg Purified water 530 g Example 8 Blueberry fruit 4.5 kg of sugar, 4.5 kg of sugar, 0.07 kg of pectin, 0.01 kg of citric acid, 0.01 kg of sucrose fatty acid ester (HLB: 10), and 400 mg of the antibacterial peptide produced by the same method as in Example 5 were added to water. Mix with 1 kg, dissolve, sterilize at 102 ° C. for 5 minutes using a scraping type heat exchanger, cool to 85 ° C., fill 150 g crow bottles, seal, cool, and add blueberry jam (sugar content: (50 ° Bx) 60 pieces were produced. Example 9 400 mg of the antimicrobial peptide produced by the same method as in Example 4 was added to 10 kg of a compound feed for adult pig having the following composition, and the mixture was uniformly mixed to obtain a feed for adult pig.

Maize 34.7 (%) Mylo 30.0 Soybean oil cake 9.0 Fish meal 5.0 Bran 10.0 Alfalfa meal 6.0 Molasses 3.0 Tricalcium phosphate 1.1 Calcium carbonate 0.4 Salt 0.0 4 Vitamin Mixture 0.2 Mineral Mixture 0.2 Example 10 An external ointment for skin having the following formulation was produced.

Vaseline 250 g Paraffin 50 g Cetostearyl alcohol 20 g Propylene glycol 100 g Polyoxyethylene / polyoxypropylene glycol ether 30 g Antimicrobial peptide of Example 2 40 mg Purified water 500 g Example 11 The following formulation of a creamy cleansing agent for skin was produced. did.

Sodium monolauryl phosphate 35.5 (%) Sodium monocetyl phosphate 10.0 Sodium chloride 7.0 Polyethylene glycol (molecular weight 8000) 5.0 Sorbitol 5.0 Flavor 0.7 Antimicrobial peptide of Example 6 0.002 Example 12 Phosphate buffer (pH 6.5) 120 l is a commercially available cellulase preparation cellulase "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.
00 units / g. Hereinafter, the unit is described as u) 105 g,
Konjac powder (Shimizu Chemical Co., Ltd.) 6.0k while heating to 40 ℃
g was dissolved by stirring and kept at 40 ° C for 16 hours.
The reaction was stopped by heating for minutes. This reaction solution is 6,000 ×
g), and the supernatant is cation exchange resin Dowex 50W × 8 (H + type; Dow Chemical) 1,500ml
And anion exchange resin Dowex 1 × 8 (OH-
Type. The solution was sequentially passed through 6,000 ml of desalting, desalted, concentrated, and spray-dried to obtain about 4,200 g of a neutral polysaccharide hydrolyzate.

3000 g of the neutralized polysaccharide decomposed product, 57.91 g of sodium chloride (manufactured by Ishizu), 2.57 g of calcium chloride dihydrate (manufactured by Ishizu), 1.53 g of magnesium chloride hexahydrate (manufactured by Kokusan Chemical), lactic acid Sodium (Wako Pure Chemical Industries, Ltd.) 39.2
g, 50 g of amino acid mixture (manufactured by Ajinomoto Co.) and Example 3
0.4 g of the antimicrobial peptide produced by the same method as above was dissolved in 10 tons of water, and the ultrafiltration membrane ACL-1050 having a molecular weight cut off of 13,000 was obtained.
Ultrafiltered using (manufactured by Asahi Kasei Corporation), the resulting permeate was sterilized 10 minutes at 121 ° C., sterile, and to obtain a peritoneal dialysis solution about 9 tons of pyrogen.

[0040]

As described in detail above, the present invention has the following effects. (1) The antimicrobial peptide of the present invention has significantly better antimicrobial activity than natural LF and LF hydrolyzate. (2) Since the antimicrobial peptide of the present invention exhibits an antimicrobial effect in a small amount, it has almost no effect on flavor when used in foods and the like.

──────────────────────────────────────────────────の Continuation of front page (51) Int.Cl. ⁶ Identification symbol FI A61K 38/00 ADZ A61K 37/02 ADZ (72) Inventor Tsuneharu Yamauchi 4-2 Tamano, Kamakura City, Kanagawa Prefecture Garden Heights 405 Tamano Kamakura (72) Inventor Hiroyuki Wakabayashi 118 Minamikibogaoka, Asahi-ku, Yokohama-shi, Kanagawa Prefecture Morinaga Nobogaoka dormitory (72) Inventor Yukiko Tokita 3-6 Asahimachi, Sagamihara-shi, Kanagawa 201 201 Flat Stone Sagami 201 , VOL. 18, NO. 23 (1990) p. 7167 NUCLEIC ACIDS RES EARCH, VOL. 18, NO. 13 (1990) p. 4013 (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 7/06 A01N 37/44 A01N 63/00 A23K 1/16 A23L 3/3526 A61K 38/00 CA (STN) REGISTRY (STN)

Claims (5)

(57) [Claims] 1. A amino acid sequence of any of the following, Lys-Thr-Arg-Arg -Trp-Gln-Trp-Arg-Met-Lys-Lys, Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg -Met-Lys-Lys , Lys-Thr-Val-Ser-Trp-Gln-Thr-Tyr-Met-Lys-Lys , Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys , C-terminal, the peptide or its those consisting of Lys-Thr-Leu-Arg- Trp-Gln-Asn-Glu-Met-Arg-Lys is amidated
Derivative. Wherein any of the amino acid sequence of the following, Lys-Thr-Arg-Arg -Trp-Gln-Trp-Arg-Met-Lys-Lys, Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg -Met-Lys-Lys , Lys-Thr-Val-Ser-Trp-Gln-Thr-Tyr-Met-Lys-Lys , Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys , C-terminal, the peptide or its those consisting of Lys-Thr-Leu-Arg- Trp-Gln-Asn-Glu-Met-Arg-Lys is amidated
An antibacterial agent comprising, as an active ingredient, a substance selected from the group consisting of a derivative thereof, and a mixture of two or more thereof. 3. The antibacterial agent according to claim 2, wherein the active ingredient is contained at a concentration of at least 1 micromolar (μM). Wherein any of the amino acid sequence of the following, Lys-Thr-Arg-Arg -Trp-Gln-Trp-Arg-Met-Lys-Lys, Lys-Ser-Arg-Arg-Arg-Gln-Trp -Arg-Met-Lys-Lys , Lys-Thr-Val-Ser-Trp-Gln-Thr-Tyr-Met-Lys-Lys , Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg -Lys, C-terminus of the peptide or its those consisting of Lys-Thr-Leu-Arg- Trp-Gln-Asn-Glu-Met-Arg-Lys is amidated
An antimicrobial peptide formulation comprising, as an active ingredient, a substance selected from the group consisting of a derivative thereof, and a mixture of two or more thereof. 5. The amino acid sequence of any of the following, Lys-Thr-Arg-Arg -Trp-Gln-Trp-Arg-Met-Lys-Lys, Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg -Met-Lys-Lys , Lys-Thr-Val-Ser-Trp-Gln-Thr-Tyr-Met-Lys-Lys , Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys , C-terminal, the peptide or its those consisting of Lys-Thr-Leu-Arg- Trp-Gln-Asn-Glu-Met-Arg-Lys is amidated
A method for treating an article with an antibacterial agent, characterized by containing, as an active ingredient, a substance selected from the group consisting of a derivative obtained from the above, and a mixture of two or more thereof.