AU659440B2 - Antimicrobial peptide and an antimicrobial agent - Google Patents

Description

P/0O/C)1 1' Regulation 3.2

AUSTRALIA

Patents Act 1 990 ^f IA U~fl4

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: ANTIMICROBIAL PEPTIDE AND AN ANTIMICROBIAL AGENT r c CC.

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The following statement is a full description of this invention, including the best method of performing it known to us: 4C S I a sa A S S a. a GH&CO REF: 22001-B:GJH:RK SC CC C S

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R8 S a C S C a ANTIMICROBIAL PEPTIDE AND ANTIMICROBIAL AGENT (FIELD OF THE INVENTION] The present invention concerns an antimicrobial peptide and an antimicrobial agent. More specifically, it concerns an antimicrobial agent and an antimicobial composition containing a novel antimicrobial peptide or a derivative of this peptide, as active components, in addition to a method for treating products which uses this antimicrobial agent.

In the specification of the present invention, the amino acids eaid peptides are represented by the abbreviations employed by IUPAC-IUB Committee on Biochemical Nomenclature, such as the following abbreviations.

Ala-: L-Alanine residue Arg-: L-Arginine residue Asn-: L-Asparagine residue Asp-: L-Aspartic acid residue Cys-: L-Cysteine residuc Gln-: L-Glutamine residue Glu-: L-Glutamic acid residue Gly-: Glycine residue His-: L-Histidine residue Ile-: L-Isoleucine residue Leu-: L-Leucine residue SLs-: L-Lvsine residue Mct-: L-Methionine residue t C Phe-: L-Phenylalanine residue Pro-: L-Proline residue Ser-: L-Serine residue Thr-: L-Threonine residue Trp-: L-Tryptophan residue Tyr-: L-Tyrosine residue Val-: L-Valine residue [PRIOR ART] Numerous inventions concerning peptides or their derivatives which possess antimicrobial properties against various microorganisms have so far been reported. Examples include a phosphonotripeptide (JapanesePatent Provisional Publication No.106689/82), a phosphonodipeptide derivative (Japanese Patent Provisional Publication No.13594/83) and a cyclic peptide derivative (Japanese Patent Provisional Publication No.213744/83) effective against gram-positive and gram-negative bacteria, a peptide demonstrating an antimicrobial cr and antiviral action (Japanese Patent Provisional Publication No.51247/84), a polypeptide effective against yeast (Japanese Patent Provisional Publication No.130599/85), a saccharopeptide derivative effective against gram-positive bacteria (Japanese Patent Provisional Publication No.172998/85, Japanese Patent Provisional Publication No.251699/86, Japanese Patent Provisional Publication No.44598/88), an oligopeptide effective I against gram-positive bacteria (Japanese Patent Provisional Publication No.22798/87), a peptidal antibiotic substance 2 Th[ r Trp-:~o L-rptpa resdu 11 (Japanese Patent Provisional Publication No.51697/87, Japanese Patent Provisional Publication No.17897/88) as well as an antimicrobial peptide extracted from blood cells of North American king crabs (Japanese Patent Provisional Publication No.53799/90) and an antimicrobial peptide isolated from blood lymph of honeybees (Japanese Patent Provisional Publication No.500084/90).

On the other hand, lactoferrin (hereinafter referred to as which is a natural iron-binding protein contained in tears, saliva, peripheral blood, milk etc. is known to demonstrate an antimicrobial activity against Escherichia coli, Candida, Clostridium and other harmful microorganisms (Journal of Pediatrics, Vol. 94, p. 1, 1979).

The inventors of the present invention, in planning to cheaply isolate from nature a substance which possesses strong antimicrobial properties, which has no undesirable side effects (such as antigenicity) and is heat-resistant, focused on whey, a by-product of cheese manufacturing, and conducted research regarding the antimicrobial properties of LF contained in it.

Surprisingly, they discovered that catabolites of LF produced by acid hydrolysis or by enzymatic cleavage of this protein have stronger antimicrobial properties and are more heat resistant than non-hydrolyzed LF, and have succeeded to isolate and synthesize potent antimicrobial peptides possessing specific amino acid sequences, and have filed Australian patent application No. 83704/91. Previously the amino acid sequences of these novel antimicrobial peptides have not been sufficiently understood, -r C Iit C I

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n _il 4 -4 however, and, therefore, the development of an effective antimicrobial agent had not yet been achieved.

SUMMARY OF THE INVENTION It is an advantage that the present invention embodies a novel antimicrobial peptide or a derivative thereof having a specific amino acid sequence which can be isolated from LF hydrolysate or chemically synthesized, and an antimicrobial agent, and an antimicrobial composition containing this peptide or its derivative as an active component, and a method for treating products which uses this antimicrobial agent.

In a first aspect of the present invention there is provided an antimicrobial peptide having an amino acid sequence selected from a group consisting of: Arg-Trp-Gln-Trp-Arg; Arg-Arg-Gln-Trp-Arg; Lys-Val-Ser-Trp-Arg; Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Lys; Arg-Arg-Trp-Gln-Trp-Arg; Arg-Arg-Arg-Gln-Trp-Arg; Lys-Thr-Val-Ser-Trp-Arg; t Lys-Arg-Asn-Met-Arg-Lys; r..p Arg-Trp-Gln-Glu-Met-Lys; 25 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 and Lys-Thr-Leu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Lys; or a derivative of the peptide, or a pharmaceutically or sitologically acceptable salt of the peptide.

In a second aspect of the present invention there is provided an antimicrobial agent containing an active component selected from a group consisting of an antimicrobial peptide, a derivative of the peptide, and a K pharmaceutically or sitologically acceptable salt of the S:22001-B 6/2/95 N*1t -4a peptide, wherein the antimicrobial peptide has an amino acid sequence selected from a group consisting of: Arg-Trp-Gln-Trp-Arg; Cb) Arg-Arg-Gln-Trp-Arg; Lys-Val-Ser-Trp-Arg; Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Lys; Arg-Arg-Trp,-Gln-Trp-Arg; Arg-Arg-Arg-Gln-Trp-Arg; Lys-Thr-Val-Ser-Trp-Arg; Lys-Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Met-Lys; 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 and Lys-Thr-Leu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Lyv; and wherein the active component is present at a concentration of at least 1 micromole/kg of the antimicrobial agent.

In, a third aspect of the present invention there is provided an antimicrobial composition comprising at least one active component selected from a group consisting of an antimicrobial peptide, a derivative of the peptide, and a pharmaceutically or sitologically acceptable salt of the peptide, weinthe atmcoilcomposition further comprises a suitable carrier medium and the peptide has an amino acid sequence selected from a group consisting of-, Ca) Arg-Trp-Gln-Trp-Arg; Cb) Arg-Arg-Gln-Trp-Arg; Lys-Val-Ser-Trp-Arg; d) Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln,-Glu-Lys; Arg-Arg-Trp-Gln-Trp-Arg; Arg-Arqj-Arg-Gln-Trp-Arg; 4, Lys-Thr-Val-Ser-Trp-Arg;4 C. S.22001-8 6/2195 -m 4b Arg-Trp-Gln-Glu-Met-Lys; Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys; L .,er-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 and Lys-Thr-Leu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Lys; and wherein the active component is present at a concentration of at least 1 microrn-ole/kg of said antimicrobial composition.

Derivatives may be derived by the addition of between 1 and X-1 amino aclids to any one of the per-tides embodied by the invention, or by the substitution of between 1 and X-1 amino acids of any one of the peptides of the invention, wherein X is the number of amino acids comprising the selected peptide.

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4 44 4 4 14 14 4 444 S,22OOI43 6/2/9S Tr,: Detailed Description of Embodiments of the Invention.

The antimicrobial peptide or the derivative thereof of the present invention may be prepared by enzymatic hydrolysis of bovine LF isolated by the conventional method from cow's milk or a commercially available LF. The antimicrobial peptide derivatives are those derivatives having minor amino acid partial substitutions or additions which do not abolish the antimicrobial properties of the peptide.

Alternatively, the antimicrobial peptide of the present invention can be chemically synthesized, and an example of chemical synthesis of the peptide is as follows. Using an automated peptide synthesizer (such as the one manufactured by Pharmacia LKB Biotechnology Co., LKB Biolynk 4170), the peptide is synthesized following the solid-phase peptide synthesis method of Sheppard et al. (Journal of Chemical Society Perkin I, S 538, 1981). N,N'-dicyclohexvlcarbodiimide is added to amino acids whose amine functional groups are protected by 9- S fluorenylmethoxycarbonyl (Fmoc) groups (hereinafter referred to as "Fmd-amino acid") and anhydrides of the desired amino acids 4 are prodi ed, and these Fmoc-amino acid anhydrides are used for synthesis., In order to produce a peptide chain, an Fmoc-amino acid anhydride corresponding to the C-terminal amino acid

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"i hamaiaLK BotcholgyCo, KBBilyk 17),th pptd t ;ji s y t ei e ol wigt esldp aepetd yt ei i i dimethylaminopyridine as a catalyst. Next, the resin is washed iwith dimethylformamide containing piperidine, and the protecting cgroup of the amine functional group of the C-terminal amino acid is removed. Next, an Fmoc-amino acid anhydride corresponding to the amino acid residue which is second from the C-terminal of the amino acid sequence of the desired peptide is coupled to the unprotected amine functional group of the first amino acid fixed to the resin through the above-mentioned C-terminal amino adid residue. Subsequently the successive desired amino acids are fixed in the same manner. After coupling of all the amino acids is completed and the npetide chaitalyst. of the desired amino acid sequence is formed, the protective groups other than acetoamidomethey are removed and the opetide is released with a solvent composed of, for exampe, 94%(weight. the same hereinafter unless otherwise indicated) trifluoroacetic acid, tr phenol and i ethandienc and the s peptide is purified using highperformance liquid chromotoa graphy. eir ano d ed ohAs aren examhe ouf the antimicrobia C-tide derminal amino athe p reseit invention, a peptide havin an amide at the carboxyl end is prepared as foleowsfnue sequentially amino acid residues e in the same manner as in the examle mentioned above excet for using Ultrosyn B resin (manufactured by Pharmacia LKB I iBiotechnology after the total completion of coupling of ino acited and the eultant formation of a pptirde chain having sequence is formed, the protective groups other than acetoamidomethyl are removed and the peptide is released with a solvent [composed of, for example, 94%(weight. the same a hereinafter unless otherwise indicated) trifluoroacetic acid, phenol and 1% ethandic\], and the peptide is purified using high-

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i" ,performance liquid chromotography.

i As an example of the antimicrobial pepxide derivative of the il i'^tt 4 oreselt invention, a peptide having an amide at the caroxyl end P is prepared as follows:fixcing sequntially amino acid residues in the same manner as in the example mentioned above except for 4 using Ultrosyn B resin (manufactured by Pharmacia LKB r i Biotechnology after the total completion of coupling of amino acid, and the resultant formation of a peptide chain having a desired amino acid sequence, eliminating the protecting groups d e 4. ni \i i, II GRIFFITH HACK CO

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other than acetoamidemethyl by means of a solvent which comprises 94% triruloroacetic acid, 5% phenol, and 1% ethandiol; then isolating peptides from the resin by means of a saturated ammonia/methanol solvent; and purifying peptides using highperformance liquid chromatography.

The antimicrobial peptide so obtained, the pharmaceutically or sitologically approved salts thereof, or a mixture of at least two of the above, is included as active components at a concentration of at least 1 micromole per kg and preferably 5 to micromoles per kg, in order to obtain the antimicrobial agent or the antimicrobial composition of the present invention.

Also, in the case of using the antimicrobial peptide derivative, the antimicrobial agent or composition:can be obtained in the same manner as described above.

The antimicrobial peptide or derivative thereof of the present invention can be administered to humans or to animals without further modifications, can be used in food products Si.' medicinal pharmaceutical products (such as eye medications, mastitis medications, diarrhea medications and athlete's foot medications), non-medicinal pharmaceutical products (such as Smouth washes, antiperspirants and hair tonics), various cosmetic products (such as hair conditioners), various tooth-brushing products (such as toothpastes and toothbrushes), various femini, ihygiene products, various bady products (such as S diaper), various geriatric products(such as denture cement and diapers), various cleaning agants (such as soaps, medicinal soaps, shampoos, rinses, lauidry detergents, kitchen detergents &ii t

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and house detergents), various sterilized products (such as sterilized kitchen paper and sterilized toilet'paper), feed and materials which serve as raw materials of the above, and they can also be added to, compounded with, sprayed onto, adhered to or used for coating or impregnation of any and all products wherein prevention or inhibition of microbial proliferation is generally desired.

The antimicrobial peptide or derivative thereof of the present invention can be used individually or concomitantly with other antimicrobial agents for treating any and all products whereicn arvention or inhibition of microbial proliferation is generally desired, for example, food products, medicinal pharmaceutical products (such as eye medications, mastitis medications, diarrhea medications and athlete's foot medications), non-medicinal pharmaceutical products (such as mouth washes, antiperspirants and hair tonics), various cosmetic products (such as hair conditioners), various tooth-brushing products (such as toothpastes and toothbrushes), various feminine hygiene products, various baby products (such as diapers), various geriatric products (such as denture cement and diapers), various cleaning agents (such as soaps, medicinal soaps, shampoos, rinses, laundry detergents, kitchen detergents and house detergents), various sterilized products (such as sterilized kitchen paper and sterilized toilet paper), feed and materials which serve as raw materials of the above.

Next, the present invention will be described in detail by means of Experiments.

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(Experiment 1) This experiment was performed in order to study the antimicrobial activity of an antimicrobial peptide.

Sample preparation Samples of Nos. 1 to 15 were chemically snythesized using the same methods as in Examples 1 to 15, respectively.

Experimental method 1. Preparation of a pre-incubation solution One platinum loop was collected from a stock slant of Escherichia coli, streaked on a standard agar medium (manufactured by Nissui Pharmaceutical Co.) and incubated under aerobic conditions for 16 hours at 37°C, the colonies which grew on the surface of the standard agar medium were collected using a platinum loop, suspended in sterilized physiological saline solution, the turbidity was measured using a spectrophotometer (manufactured by Hitachi Manufacturing Co.) and adjusted to 0.1 660 nm) and a pre-incubation solution was prepared.

2. Preparation of a basal medium C Bactocasitone (manufactured by Difco Laboratory Co.) was r c dissolved at a concentration of 1% in purified water, the pH was rtt adjusted to 7.0 with 1 M sodium hydroxide, the solution was S I

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sterilized at 1159C for 15 minutes and a basal medium (liquid medium) was prepared.

3. Preparation of the test media and of the control medium j Each sample was dissolved at a concentration of 0.01% in purified water, sterilization was performed by using a *i 2, 5, 10, 20, 50 and 100 micromol (pM) to the basal medium, as well as a control medium with no added samples, were prepared.

4. Antimicrobial activity test The above-mentioned pre-incubation solution was inoculated into the above-mentioned test media and the control medium at a concentration of cultured under aerobic conditions for 16 hours at 37°C, the turbidities of the culture media were measured using the above-mentioned method and the rate of inhibition of E. coli proliferation was calculated according to the following formula.

rate of inhibition of proliferation 100 (1 A/B) wherein A is the difference in turbidity of the test culture medium (the difference etween the turbidity of the test culture medium after 16 hours of culture and the turbidity of the test culture medium before the culturing) and B is the S turbidity of the control medium (the difference between the turbidity of the control culture medium after 16 hours of c culture and the turbidity of the control culture medium before the culturing). The percentages of the rate of inhibition of proliferation are not in weight (same hereinafter).

Results The results of this experiment are shown in Table 1.

As is clear from Table 1, all the samples Nos. 1 to 15 had an ,0 antimicrobial activity at concentration of 1 MM, and a high antimicrobial activity within a range of 5 to 20 MM. At 10 crcbrt t aI 'i ~1; Si 4 i\ l.j:

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residue, respectively.

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ii> [Table 1], Sample Added auantity (JIM) and inhibitory rate o. 0.5 1 2 5 10 20 60 i 0 6 0 '3 1 0 1 00 0 100 1 0 6 97 100 100 100 10071-00 0 5 ro95 9 0 1100 50 j73 88 95 100 10 0 21 r48 75f 89 }96 100 100I 6 0 [6 100 1001 1001 100 100 100 7 0 O 87 _li OO T00 oo6 100 ~100 84 72 83~ 91 95 100 9 I0 25 46 63~ 80 90 100 100 0 28 j55 811 90 ]97 {b00 100 11 0 65 100 100 100 100 j100 100__ 10 30 87[ 100 1!0 100 100 100 13 0 J42 72 83 1 91 95 100o 100 14 J0 j25 46 63j 80 90 100 100 [15 0 28 55 81 90 97 100 100 C, C 4C~

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a It4 I.e I 4 Q 0 a 41 ve 6 h s. v -11concentrations over 50 uM, such increase in concentration did not lead to increased antimicrobial activity. Antimicrobial activity of each peptide is therefore available at a concentration of at least 1 juM, and more preferably, within a range of 5 to 20 UM. Antimicrobial activity within this quantity range is almost equal to that of aminobenzylpenicillin.

Antimicrobial activity was tested also for peptides having amino acid sequences other than those of the above-mentioned samples Nos. 1 to 15, derivatives thereof and salts thereof, and similar results were obtained.

(Experiment 2) *oo* This experiment was performed in order to determine the amino acid sequence of the antimicrobial peptide used in Experiment 1.

The peptides of sample Nos. 1 to 15 used in Experiment 1 were hydrolyzed by 6 N hydrochloric acid and the amino acid .s compositions were analyzed by conventional methods, using an s" amino acid analyzer. Given cycles of Edman's degradation were performed on each sample, using a gas-phase sequencer (manufactured by Applied Biosystems and sequences of amino acid residues was determined.

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As a result it was determined that this peptide consisted of 5, 6 or 11 amino acid residues, and formed the following amino acid sequence.

SSample No., 1: Arg-Trp-Gln-Trp-Arg S ample No. 2: Arg-Arg-Gln-Trp-Arg -12- ljiz*.,~j a <'4 Sample No. 3: Samele No. 4: Sampel No. 5: Sample No. 6: Sample No. 7: Sample No. 8: Sample No. 9: Sample No.10: Sample No.11: Sample No.12: Sample No.13: Sample No.14: Lys-Val-ser-Trp.-Arg Arg-Asn-Met-Arg-Lvs Arg-Trp-Gln-G lu-Lys Arg-Arg-Trp-Gln-Trp-Arg Arg-Arg-Arg-G ln-Trp-Arg Lys-Thr-Val-Ser-TrD-Arg Lys-Arg-Asn-Met -Arg-Lys Arg-Trz-Gin-G lu-Met -Lvs Lys-Thr-Arg-Arg-Trp-G ln-Trp-Arg-Met -Lys-Lys Lys-Ser-Arg-Arg-Ar g-G ln-Trp-Arg-Met -Lys-Lys Ly" l-Thr-Val-Ser-Trp)-G ln-Thr-Tyr-Met -Lys-Lys Lys-.Thr-Phe-Gl n-Trp-Gl n-Arg-Asn-Met -Arg-Lvs 4, 4* 4 4 4 44. 4 4444 4, ('44 4, (2 44, 4 4,4, 4, 4, 4,4, I 4,4 4, 4, 4, t 4,4, 4, (4

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44 44 4 4 4 4 4 Sample No.15 :,Lvs-Thr-Leu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Lys (Experiment 3) This exneriment was performed to study the antimicrobial effect of an antimicrobial agent containing the peptide of the present invention.

Commercially available primary-processed vegetable (known as "cut vegetable") in an amount of 100 g was immezlsed in an aquaeous solution for 30 seconds, in which the antimicrobial peptide (sample No. 1 or 6) synthesized in the same manner as in Example 1 or 6 was added at a concentration of 10 p.M. After sufficiently eliminating water, the vegetable was held at and the viable count was measured by the conventional mC,,t-,o6 along with time lapse. Vegetable Immersed in city water with no antimicrobial peptide served as control.

The results of this experiment are' shown in Table 2. in L3 I-i [Table 2) Sample No. jViable count/g (control) in theII agent 0 hr. 12 hrs. 24 hrs. 36 hr's.

1.3xIO 3 '-.xO3 XO3 2. O3 (1.3x10 (2.9X10)4 (3.lxlO 5 (4.8xl10 6 1.3x10 3 1.4x10 1.7X10 3 2.1x10 3 (1.3X10 (3.2x10)4 (2.7xI0 t at Ct CCI I lets 1146 C C C

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-14this table, the figures in parentheses represent values for control. As in clear from Table 2, proliferation of bacteria was remarkably inhibited in the vegetable treated with the antimicrobial agent of the present invention. Almost the same results were obtained also for antimicrobial peptides synthesized in the same manner as in the other Examples and the salts thereof.

(Experiment 4) This experiment was performed to study the antimicrobial effect of an antimicrobial agent containing the antimicrobial peptide derivative of the present invention.

Commercially available primary-processed vegetable (known S as "cut vegetable") in an amount of 100' a was immersed in an 't I aqueous solution for 30 seconds, in which the antimicrobial tst, peptide derivative (sample No. 18) synthesized in the same manner as in Example 18 was added at a concentratin of 20 pM.

After sufficiently eliminating water, the vegetable was held at S 5°C, and dally changes in the viable count were measured by the conventional method. Vegetable immersed in city water with no St antimicrobial peptide derivative served as control.

The results of this experiment are shown in Table 3. As is clear from Table 3, proliferation of bacteria was remarkably i S inhibited in the vegetable treated with the antimicrobial agent containing the peptide derivative of the present invention.

Similar results were obtained also for antimicrobial peptide derivatives synthesized in the same manner as in the other Examples and the salts thereof.

4 8 1 u j i -i LLUII a group consisting of a antimicrobial peptide, a derivative of the peptide, and a pharmaceutical' or sitologically acceptable salt of the S:22001-B 6/2/95

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[Table 3] Viable count/g -1 Agent 7- 0 day 1 day 2 days 3 days 4 days lb I I cr1 I t £16 4~ I 1111

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-16- (2 (Experiment This experiment was performed to study the preservative effect in foods mixed with the antimicrobial peptide of the present invention.

Milk was pasteurized at 65°C for 30 minutes and poured separately into test tubes to an amount of 10 ml each. The antimicrobial peptide (Sample No. 2 or 7) synthesized in the same manner as in Example 2 or 7 was added to the milk at a concentration of 30 aM, and the mixture was uniformly mixed, and closely sealed. Milk in a sealed test tube with no S antimicrobial peptide served as control. All the test tubes were held at 25"C, and the number of days required for the milk 54*44 to solidify was measured.

SAs a result, while all the milk containing the antimicrobial peptide solidified in ten days, the control solidified in two days. This suggests that the antimicrobial peptides of the present invention largely retarded solidification of milk. An organoleptic test carried out on the C c c I tested milk and the control before preservation showed no difference in flavor or in exterior view between the two groups.

Similar results were obtained for antimicrobial peptides i. synthesized in the same manner as in the other Examples and the derivatives thereof.

(Experiment 6) This experiment was performed to study the antimicrobial spectrum of the antimicrobial peptides and the derivatives thereof of the present invention.

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Sample preparation The antimicrobial peptides (samples Nos. 1 to 15) were prepared using the same methods as in Examples 1 to 15, and antimicrobial peptide derivatives (samples Nos. 16 to 18) were prepared using the same methods as in Examples 16 to 18, and were sterilized by filtration using a 0.45 Am Millex filter prior to use.

Experimental methods Various microbial strains shown in Table 4 in the logarithmic phase of growth were inoculated in a peptone medium which consisted of 1% Bactopeptone (manufactured by OAfco Laboratory Co.) at a cell concentration of 10 /ml, and 160 1 S thereof were incubated for 17 hours at 37C (or 30"C) using a 96hole microtiter plate (manufactured by ?alcon Each sample was added to each-medium at a ratio of 0, 1.5, 3, 6, 12, 25, 100, 125 or 250 9M. The growth of the various microorganisms in the various samples at various concentrations was studied by measuring the light absorption at 660 nm. The minimum concentration of the antimicrobial peptide which completely S inhibited the growth of the various microorganisms was considered the minimum inhibitory concentration (MIC: uM).

The microorganisms used in this experment are available from The Medical School Laboratories of Toklyo University (lID), Japanese Physicochemical Laboratories (JCM), Japanese international Dairy Federation (IDF), and the storae at the laboratoryof the applicant (MMI).

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[Table 41 4 4 941# 4 4 444$ 4 4 4~4 4 44 9 44 4 44 4 44444 4 4 Sample Minimum inhibitory concentration (jaM) No. L M S A I P A K P 1 50 2 6 25 3 100 250 4 50 250 50 25 6 25 50 7 12 25- 8 25 f 100 25 2 50 100 o 11 I 1.5 f 3 625 12 1.5 I 3 6 1 13 6 1 12 50 14 I 6 12 25 3 I 6 25 00 16 12 100 17 36 18 1.5 I 3 I 12 4 note) *~Cultured at 30'0 Not tested SI The results of this experiment are shown in Table 4. As is clear from Table 4, the antimicrobial peptideo of samples Nos. 1 to 5 showed an antimicrobial activity at concentrations "of up to 250 UM against the tested Gram-positive bacteria, Listeria monocytogenes IDF lb (represented by LM in the table) and Staphylococcus aureus JCM 2151 (represented by SA in the table), whereas samples Nos. 6 to 10 showed an antimicrobial activity at concentrations of up to 100 lM against these bacteria.

SSample No. 16 which is the derivative of sample No. 1 showed a strong antimicrobial activity about 2.5 to 4 times as high as that of sample No. 1, and sample No. 17 which is the derivative of sample No. 6 showed a strong antimicrobial activity about eight times as high as that of sample No. 6.

Vurthermore, samples Nos. 11 to 15 showed an antimicrobial activity at concentrations of up to 12 MM against Gram-positive bacteria LM and SA, and showed an antimicrobial activity at S concentrations of up to 50 M. against the tested Gram-negative bacteria, Pseudomonas aeruginosa MMI 603 (represented by PA in the table) and Kiebsiella pneumbniae JCM 1662 T (represented by KP in the table).

Sample No. 18 which 'is the derivative of sample No. 11 si a strong antimicrobial activity about twice as high-?s i that of sample io. 11 against Gram-negative strains. This S sample No. 18 showed also an antimicrobial activity at low concientrations of 6 MM and 3 JM, respectively, against In addition, virtually idntical results were obtained with the other antimicrobial peptides of the present invention, other antimicrobial peptides derivatives and salts thereof.

[EFFECTS OF THE INVENTION] Since the antimicrobial peptide or derivative thereof of the present invention possesses an antimicrobial activity which is considerably better than that of natural LF or LF hydrolysate and is effective against a wide range of microorganisms, it is suitable for a wide range of applications, and since it I r demonstrates an antimicrobial effect even in small amounts, it 4 can be applied to food products etc. with hardly any effect on S4, their flavor.

The present invention will now be explained in further detail by means of examples. Of course, the present inventiont S is not limited to or by these examples.

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1 IP I Example 1 A peptide was synthesized using an automated peptide synthesizer (manufactured by Pharmacia LKB Biotechnology Co., Trademark: LKB Biolynx 4710) in accordance with the solid-phase peptide synthesis method of Sheppard et al. (the Journal of Chemical Society Perkin I, p. 536, 1981).

N,N'-dicyclohexylcarbodiimide was added to amino acids whose the amine functional groups were protected by Fmoc groups and anhydrides of the desired amino acids were duced, and these Fmoc-amino acid anhydrides were used for synthesis. In c, order to produce a peptide chain, about 0.1 mmol of Fmoct$n arginine anhydride corresponding to the C-terminal arginine ct residue was fixed, to 1 a of Ultrosyn A resin (manufactured by 'Pharmacia LKB Biotechnology Co.) throuah the carboxyl arouo c r 'thereof, using dimethylaminopyridine as a catalyst. Next, the resin in an amount of 1 g was washed with dimethylformamide containing piperidine, and the protecting group of the amine .[functional group of the C-terminal amino acid was removed. The i Fmoc-tryptophan anhydride corresponding to the second amino acid c residue from the C-terminal was then coupled to the unprotected amine functional group of the above-mentioned arginine residue.

,,..ubsequentlv, alutamine, tryptophan and arginine were .sequentially fixed in the same manner.

After the completion of coupling of all amino acids and formation of a peptide chain of the desired amino acid ;equence, protecting groups other than acetoamidomethyl were removed and Sthe peptide was released with a solvent comprising 94% -22- I trifluoroacetic acid, 5% phenol and 1% ethandiol, the peptide was purified by using high-performance liquid chromatography.

This solution was conceitrated and dried, and about 3 mg of peptide was obtained.

Example 2 About 4 mg of a peptide having the amino acid sequence Arg- Arg-Gln-Trp-Arg was obtained in the same manner as in Example 1 except that the fourth amino acid residue from the C-terminal was replaced with arginine residue.

Example 3 About 5 mg of a peptide having the amino acid sequence Lys- W Val-Ser-Trp-Arg was obtained in the same manner as in Example 1 c except that the third, the fourth and the fifth amino acid .residues from the C-terminal were replaced with serine residue, valine residue and lysine residue, respectively.

Example 4 About 4 mg of peptide having the amino acid sequence Arg- ,Asn-Met-Arg-Lys was obtained in the same manner as in Example 1 except that the first, the second, the third and the fourth 4 'amino acid residues from the C-terminal were replaced with lysine residue, arginine, methionine, and asparagine residues, r espectively.

Example About 5 mg of a peptide having the amino acid sequence Arg- Trp-Gln-Glu-Lys was obtained in the same manner as in Example 1 except that the first and the second amino residues from the Cterminal were replaced with lysine residue and glutamic acid S- -23residue, respectively.

residue, res e, te r e ad l e r e, i residue, respectively.

Example 6 About 6 mg of a peptide having the amino acid sequence Arg- Arg-Trp-Gln-Trp-Arg was obtained in the same manner as in Example 1 except that an arginine e e e was additionall fixed fat the sixth position from the C-terminal.

Example 1 About 4 mg of a peptide having the amino acid sequence Arg- -24- Arg-Arg-Gln-Trp-Arg was obtained in the same manner as in Example 6 except that the fourth amino acid residue from the C- 'terminal was replaced with arginine residue.

Example 8 0 About 6 mg of a pept'-e having the amino acid sequence Lyst Thr-Val-Ser-Trp-Ara was obtained in the same manner as in Example 6 excepot that the third, the fourth, the fifth and the sixth residues from the C-terminal were replaced with serine residue, valine residue, threonine residue and iysine residue, r respectively.

S Example 9 About 5 ma of a peptide having the amino acid sequence Lvs- ArgASn-Met-rg-Lys was obtained in the same manner as in Example 6 except that the first, the second, the third, the fourth and the sixth amino acid residues from the C-terminal i were replaced with lysine residue, arginine residue, methionine residue, aoparagine residue and lysine residue, respectively.

Example jr About 7 mg of a peptide having the amino acid sequence Arg- 1 -24- Trp-Gln-Glu-Met-Lys was obtained in the same manner as in Example 6 except that the first, the second, the third, the fourth and the fifth amino acid residues from the C-terminal were replaced with lysine residue, methionine residue, glutamic acid residue, glutamine residue and tryptophan residue, respectively.

Example 11 About 16 mg of a peptide having the amino acid sequence Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lvs was obtained by fixina 11 amino acid residues sequentially from the C-terminal in the same manner as in Example 1.

S

Example 12 About 20 mg of a peptide having the amino acid sequence SLys-Ser-Arg-Arg-Arg-GIn-Trp-Arg-Met-Lys-Lys was obtained in the same manner as in Example 11 except that the seventh and the tenth a.I"no acid residues from the C-terminal were replaced with arginine residue and serine residue.

Example 13 About 17 mg of a peptide having the amino acid sequence Lys-Thr-Val-Set-Trp-Gln-Thr-Tyr-Met-Ls-Lys was obtained in the same manner as in Example 11 except that the fourth, the fifth, the eighth and the ninth amino acid residues from the C-terminal were replaced With tvrosine residue, threonine residue, serine residue, and valine residue, respectively.

Example 14 About 13 mg of a peptide having the amino acl' sequence Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys was obtained in the 0i 0 N, i same manner as in Example 11 except that the second, the fourth, the fifth, the eighth and the ninth amino acid residues were replaced with arginine residue, asparagine residue, arginine residue, glutamine residue, and phenylalanine residue, respectively, Example About 16 mg of a peptide having the amino acid sequence Lys-Thr-Lcu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Lys was obta.ned in the same manner as in Example 11 except that the second, the fourth, the fifth and the ninth amino acid residues were replaced with arginine residue, glutamic acid residue, asparagine residue, and .leucine residue, respectively.

Example 16 Amino acid residues were seauentiallv fixed in the same t. manner as in Example 1 except for the use of Ultrosyn B resin (manufactured by Pharmacia LKB After the completion of coupling of all amino acids, the protecting group other than Z, acetoamidomethyl were removed by means of a solvent comprising S94% trifluoroacetic acid, 5% phenol, and 1% ethandiol, and a peptide was released by the use of saturated ammonia/methanol solution. Then, the peptide was purified by high-performance liquid chromatography, concentrated and dried to obtain about 4 mg of a peptide drivative having the amino acid sequence Arg-Trp- Gln-Trp-Arg-NH Example 17 About mg of a peptide derivative having the amino acid -26- I? i sequence Arg-Arg-Trp-Gln-Trp-Arg-NH 2 was obtained in the same manner as in Example 16 except that amino acid residues were fixed sequentially in the same manner as in Example 6.

Example 18 About 15 mg of a peptide derivative having the amino acid sequence Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-NH was obtained in the same manner as in Example 16 except that amino acid residues were fixed in the same manner as in Example 11.

Example 19 About 9.6 kg of pet feed were produced by adding 120 mg of S the antimicrobial peptide obtained by the same method as in Example 3 to 10 kg of pet feed of the following composition and n uniformly mixed.

e Powdery meat 8.0 Scc s Fish meal 5.0 Corn 5.0 Soybean oil meal :12.0 Wheat germ 8.0 Wheat :51.23% IC Skim milk powder 4.0 Beef tallow 2.0 Brewer's veast powder 2.0 Bone meal 2.0 Common salt 0.5 Mixed vitamins and minerals 0.27%.

Example A skin cream of the following composition was produced by I I L LI-

I

the conventional method: Stearic acid :14.25% Ethanol 1.90% Squalene 2.85 Octyldodecyl myristate 4.75 Glycerin 9.50% 1,3-butylenoglycol monostearate 3.80% Polyoxyethylene (20 mol) sorbitane 2.85 Antimicrobial peptide of Example 4 0.0005 Purified water :60.10% Example 21 A mixture of 7.4 kg of a commercially available honey (19% sugar, 20% water and 0.2% protein), 2.6 kg of a commercially "e available butter (15% water) and 240 mg of an antimicrobial Sce peptide obtained in the same manner as in Example 5 was uniformly mixed in a container while heating to 40°C, then rapidly cooled to 10 0 C, and whipped by stirring while blowing Snitrogen gas to obtain a honey food product having plasticity in :an amount of about 9.7 kg.

Example 22 Commercially available sodium casein (manufactured by S. \\Nisse! Kyoeki Co.) in an amount of 1 kg and commercially .available maltodextrin (manufactured by Matsutani Chemical Co.) I in an amount of 3 kg were dissolved in about 20 kg of hot water, added with minerals in a given amount, and heated to Then, 0.5 kg of commercially available corn oil (manufactured by Talyo Fats and Oils Co.) and 0.3 kg of commercially availabie -28- 4 s I tf L 1 coconut oil (manufactured by Taiyo Fats and oils Co.) both containing dissolved fat-soluble vitamins in a given amount were added to this solution, and the mixture was emulsified by means of a high-pressure equalizer and added with water-soluble vitamins in a given amount. An antimicrobial peptide synthesized in the same manner as in Example 6 was added in an amount of 36 mg to this emulsified solution, and the mixture was sterilized at 135"C for two deconds, and aseptically charged into a 500 ml sterile container, to obtain 49 units of an enteral nutrient.

I Example 23 t tt L An ointment of the following composition was produced by the conventional method: t t Vaseline :26.3% Paraffin 5.3% Ce4ostearylalcohol 2.1% Prdpylene glycol :10.5% Polyoxyethylene polyoxypropylene 3.2% Glycol ether Antimicrobial peptide of Example 7 0.0005% Purified water :52.6%.

r Example 24 S An antimicrobial peptide synthesized in the same manner as in Example 8 in an amount of 140 mg was added to 10 kg of a commercially available assorted feed for adult hog of the following'composition, and the mixture was uniformly mixed to obtain a hog-raising feed: 9i i! -29- i iu Corn 34.7% Milo 30.0% Soybean oil meal Fish meal Wheat bran :10.0% Alfalfa meal Refinery molasses Tricalcium phosphate 1.1% Calcium carbonate 0.4% Common salt 0.4% c Vitamin mixture 0.2% 1 Minetal mixture 0.2% ExamPle c A commercially available cellulase agent "Cellulose AMANO" £4.

(manufactured by Amano Pharmaceutical Co.; cellulose saccharifying ability: 15,000 units/g; the unit is hereafter expressed as in an amount of 105 g was added to 120 tons of phosphoric acid buffer solution (pH: Then, 6.0 kg of S konjak powder (manufactured by Shimizu Chemical Co.) was j dissolved by stirring into the mixture while heating the same to After holding at 40"C for 16 hours, the mixture was heated to 95"C for ten minutes and the reaction was stopped.

The resultant solution was continuously separated centrifugally i at 6,000 x g, and the supernatant liquid was passed sequentially through 1,500 ml of cation exchance resin DOWEX SOW x 8 (H+ type; manufactured by Dow Chemical Co.) and 6,000 ml of anion exchange resin DOWEX 1 x 8 (OH- type; manufactured by Dow i

I

Chemical for desalting, then concentrated and spray-dried, to obtain about 4,200g of a decomposed neutral polysaccharide.

Subsequently, 3:000 g of this a decomposed neutral polysaccharide, 57.91 g of sodium chloride (manufactured by Ishizu 2.57 g of calcium chloride (manufactured by Ishizu 1.53 g of magnesium chloride hexahydrate (manufactured by Kokusan Chemical 39.2 g of sodium lactate (manufactured by Wako Junyaku Kogyo 50 g of amino acid mixture (manufactured by Ajinomoto and 0.4 g of an antimicrobial peptide prepared in the same manner as in Example 13 were t dissolved into 10 tons of water, and the resultant solution was t* subjected to ultrafiltration through an ultrafiltration membrane SACL-1050 (manufactured by Asahi Chemical Co.) having a differential molecular weight of 13,000, and the resultant filtrate was sterilized at 121C for ten minutes, thus obtaining a peritoneum dialysate which was aermfree and did not contain any pyrogenic substances.

Example 26 Sixty units of blueberry jam (sugar concentration: S were manufactured by mixing 4.5 kg of blueberry fruit, 4.5 kg of sugar, 0.07 kg of pectin, 0.01 kg of citric acid, 0.01 kg of sucrose ester of fatty acid (HLB: 10), and 400 mg of an 7 i antimicrobial peptide prepared in the same manner as in Example with 1 kg of water, dissolving the same, sterilizing the same at 102 0 C for five minutes by means of a scrubber type heat i i exchanger,,cooling the mixture to 85 4 C, chargi g 150 g of the same into glass bottles, sealing the bottles, and cooling the -31same.

Example 27 A tooth-paste of the following composition was produced: Sorb itol 1:47.0% Glycerin .15.0%Q Carboxymethylcellulose sodium Sorbitan ester of fatty acid Saccharin sodium Antimicrobial peptide derivative of Example 16 .0.002% *',,Examp~le 28 A skin cream rinsing agent of the following composition produced: 'aSodium monolauryl phosphate :35.5% Sodium monomethkriphosphate 10.0% Sodium chloride r. lyethylene glycol (molecular Weight: 8,000) Sorbltol Aromatic .0.7% Antimicrobial peptide derivative of 4 ;44Examp~le 18 .0.002%.

3 2a

Claims (15)

1. An antimicrobial peptide having an amino acid sequence selected from a group consisting of: Arg-Trp-Gln-Trp-Arg; Arg-Arg-Gln-Trp-Arg; Lys-Val-Ser-Trp-Arg; Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Lys; Arg-Arg-Trp-Gln-Trp-Arg; Arg-Arg-Arg-Gln-Trp-Arg,, Lys-Thr-Val-Ser-Trp-Arg; Lys-Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Met-Lys; Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys; is Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg-Met-Lys-Lys; (in) Lys-Thr-Val-Ser-Trp-Gln-Thr-Tlyr-Met-Lys-Lys; Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg- Lys and Lys-Thr-Leu-Arg-Trp-Gln,-Asn-Glu-Met-Arg-Lys; or a derivative of the peplitide, or a pharmaceutically or sitologically, acceptable salt of the peptide.

2. An antimicrobial peptide according to claim or a derivative thereof derived by an addition of between 1 and X-1 amino acids to any one of amino acid sequences to wherein X is the number of amino acids a8 comprising the amino Aquence.

3. An antimicrobial peptide according to claim 1. or a derivative thereof derived by a substitution of between 1 t It and X-1 amino acids of any one of amino acid sequences to wherein X is the number of amino acids comprising the amino acid sequence. f

4. An antimicrobial peptide according to any one of claims 1 to 3 or a derivative thereof wherein the derivative is an amide. St An ntimicrobial peptide according to claim 1. 0~ S:22001-13 612195 34

6. An antimicrobial agent containing an active component slected from a group consisting of an antimicrobia, peptide, a derivative of the peptide, and a pharmaceutically or sitologically acceptable salt of the peptide, wherein the antimicrobial peptide has an amino acid sequence selected from a group consisting of: Arg-Trp-Gln-Trp-Arg; Arg-Arg-Gln-Trp-Arg; Lys-Val-Ser-Trp-Arg; Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Lys; Arg-Arg-Trp-Gln-Trp-Arg; Arg-Arg-Arg-Gln-Trp-Arg; Lys-Thr-Val-Ser-Trp-Arg; Lys-Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Met-Lys; 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 and Lys-Thr-Leu-Arg-Trp-Gln-Asn-Glu-Met-Arg-Lys; and wherein the active component is present at a concentration of at least 1 micromole/kg of the antimicrobial agent.

7. An antimicrobial agent according to claim 6 wherein a the derivative of the peptide is derived by an addition of between 1 and X- 1 amino acids to any one of amino sequences to wherein X is the number of amino acids comprising the amino acid sequence. Ntt t

8. An antimicrobial agent according to claim 6 wherein the derivative of the peptide is derived by a substitution of between 1 and X-1 amino acids of any one of amino acid sequences to wherein X is the number of amino acids comprising the aminoaicid sequence. VAL

9. An antimicrobial agent according to any one of I t/ i claims 6 to 8 wherein the derivative is an amide. S:22001-B 6/2 35 An antimicrobial agent according to claim 6 wherein the active component is the antimicrobial peptide or a pharmaceutically or sitologically acceptable salt thereof.

11. An antimicrobial agent according to any one of claims 6 to 10 wherein the active component is present at a concentration of from 5 to 20 micromoles/kg of the antimicrobial agent.

12. An antimicrobial composition comprising at least one active component selected from a group consisting of an antimicrobial peptide, a derivative of the peptide, and a pharmaceutically or sitologically acceptable salt of the peptide, wherein the antimicrobial composition further comprises a suitable carrier medium and the peptide has an amino acid sequence selected from a group consisting of: Arg-Trp-Gln-Trp-Arg; Arg-Arg-Gln-Trp-Arg; Lys-Val-Ser-Trp-Arg; Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Lys; Arg-Arg-Trp-Gln-Trp-Arg; Arg-Arg-Arg-Gln-Trp-Arg; Vtc Ii h) Lys-Thr-Val-Ser-Trp-Arg; Lyg-Arg-Asn-Met-Arg-Lys; Arg-Trp-Gln-Glu-Met-Lys; Lys-Thr-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys; Lys-Ser-Arg-Arg-Arg-Gln-Trp-Arg-Met-Lys-Lys; 0 Lys-Thr-Val-Ser-,Trp-Gln-Thr-Tyr-Met-Lys-Lys; Lys-Thr-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys and Lys-Thr-LeuArg-Trp-Gln-Asn-Glu-Met-Arg-Lys; and wherein the active component is present at a concentration of at least 1 micromole/kg of said antimicrobial composition. 4ili~"" S:22001-B 6/2/95 36 1,3. An antimicrobial composition according to claim 12 'wherein the derivative of the peptide is derived by an addition of between 1 and X-l amino acids to any one of amino acid sequences to wherein X is the number of amino acids comprising the amino sequence.

14. An antimicrobial composition according to claim 12 wherein the derivative of the peptide is derived by a substitution of between 1 and X-l amino acids of any one of amino acid sequences to wherein X is the number of amino acids comprising the amino acid sequence, An antimicrobial composition according to any one of claims 12 to 14 wherein the derivative is an amide.

16. An antimicrobial composition according to claim 12 wherein the active component is the antimicrobial peptide or a pharmaceutically or sitologically acceptable salt t'lereof.

17. An antimicrobial composition according to any one of claims 12 to 16 wherein the active component is present at a concentration of from 5 to 20 micromoles/kg of said t 20 antimicrobial composition.

18. A method for preventing or inhibiting microbial il proliferation in or on a product comprising the step of treating the product with a antimicrobial peptide as defined in any one of claims 1 to I' 25 19. A method for preventing or inhibiting microbial t proliferation in or on a product comprising the step of treating the product with an antimicrobial agent as defined in any one of claims 6 to 11. C i 4 S:22001-B 6/2/95 TZT~7~ 37 A method for preventing or inhibiting microbial proliferation in or on a product comprising the step of treating the product with an antimicrobial composition as defined in any one of claims 12 to 17. Dated this 6th day of February 1995 MORINAGA MILK INDUSTRY CO.. LTD By their Patent Attorney GRIFFITH HACK CO. #4 4 4 ~tt I C CCC I C CCI. C #44 C I 4 C 49 I. II 4 4. C *4 4 I CCI;44 C I I II 4 4 I II. 4

44.. S II I 4* 4. I. I 44 4~ I I C. I' .44 S:22001-B 6/2/95 4 ABSTRACT ANTIMICROBIAL PEPTIDE AND ANTIMICROBIAL AGENT An antimicrobial peptide containing at least the following amino acid sequence, or a derivative thereof, an antimicrobial agent containing said antimicrobial peptide or a derivative thereof as active components at a concentration of at least 1 M, an antimicrobial composition containing said antimicrobial peptide or a derivative thereof, and a method for processing Sa\ products which uses the antimicrobial agent containing at least said antimicrobial peptide or a derivative thereof: A X A a, (where, A is an arginine residue or a lysine residue; and c P"S X is an amino acid sequence comprising at least from three to nine arbitrary amino acid residues other than cysteine residues.) i' JS