ANTIMICROBIAL TUBULAR CONDUITS

Abstract

Tubular conduits are provided which have an outer surface and/or an inner surface functionalized with at least one antimicrobial peptide having the sequence X.sub.1X.sub.2WVX.sub.3IWVX.sub.4X.sub.5, wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are independently selected from K and R and wherein each amino acid is independently in the D or L configuration, or a salt or solvate thereof. The tubular conduits are preferably made of biodegradable polymeric material. The functionalization of the tubular conduits with the at least one antimicrobial peptide prevents contamination caused by Gram negative bacteria, Gram positive bacteria, fungi, yeasts and/or viruses.

Claims

1. A tubular conduit comprising an outer surface and an inner surface, wherein at least one portion of the outer surface and/or inner surface is functionalized with at least one antimicrobial peptide consisting of the amino acid sequence having the following general formula:
X.sub.1LX.sub.2WVX.sub.3IWVX.sub.4X.sub.5 wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are independently selected from the group consisting of K and R and wherein each amino acid is independently in the D or L configuration, or a salt or solvate thereof.

2. The tubular conduit of claim 1, wherein the at least one antimicrobial peptide is covalently linked to reactive groups that are present on the outer surface and/or inner surface of the tubular conduit or the at least one antimicrobial peptide is contained in a coating adhered to the least one portion of the outer surface and/or inner surface.

3. The tubular conduit of claim 1, wherein at least one of X.sub.1, X.sub.3, X.sub.4 and X.sub.5 has the meaning of R.

4. The tubular conduit of claim 1, wherein the at least one antimicrobial peptide consists of an amino acid sequence selected from the group consisting of RLKWVRIWRR (SEQ ID NO: 1), KLRWVRIWRR (SEQ ID NO: 2), RLRWVRIWRR (SEQ ID NO: 3), KLKWVRIWRR (SEQ ID NO: 4), RLKWVKIWRR (SEQ ID NO: 5), KLRWVKIWRR (SEQ ID NO: 6), RLRWVKIWRR (SEQ ID NO: 7), KLKWVKIWRR (SEQ ID NO: 8), RLKWVRIWKR (SEQ ID NO: 9), KLRWVRIWKR (SEQ ID NO: 10), RLRWVRIWKR (SEQ ID NO: 11), KLKWVRIWKR (SEQ ID NO: 12), RLKWVKIWKR (SEQ ID NO: 13), KLRWVKIWKR (SEQ ID NO: 14), RLRWVKIWKR (SEQ ID NO: 15), KLKWVKIWKR (SEQ ID NO: 16), RLKWVRIWRK (SEQ ID NO: 17), KLRWVRIWRK (SEQ ID NO: 18), RLRWVRIWRK (SEQ ID NO: 19), KLKWVRIWRK (SEQ ID NO: 20), RLKWVKIWRK (SEQ ID NO: 21), KLRWVKIWRK (SEQ ID NO: 22), RLRWVKIWRK (SEQ ID NO: 23), KLKWVKIWRK (SEQ ID NO: 24), RLKWVRIWKK (SEQ ID NO: 25), KLRWVRIWKK (SEQ ID NO: 26), RLRWVRIWKK (SEQ ID NO: 27), KLKWVRIWKK (SEQ ID NO: 28), RLKWVKIWKK (SEQ ID NO: 29), KLRWVKIWKK (SEQ ID NO: 30), RLRWVKIWKK (SEQ ID NO: 31) and KLKWVKIWKK (SEQ ID NO: 32), and wherein each amino acid is independently in the D or L configuration.

5. The tubular conduit of claim 1, wherein the tubular conduit is a drinking straw or a medical tube.

6. The tubular conduit of claim 1, wherein the number of moles of the at least one antimicrobial peptide present on the at least one portion of the outer surface and/or inner surface of the tubular conduit is between 1 and 10 nmol×cm.sup.2.

7. The tubular conduit of claim 1, wherein the tubular conduit is made of polymeric material, glass, or metal.

8. The tubular conduit of claim 7, wherein the tubular conduit is made of a polymeric material selected from the group consisting of styrene block copolymers, polyolefin mixtures, elastomeric mixtures, thermoplastic polyurethanes, thermoplastic copolyesters, thermoplastic polyamides, polypropylene, polyethylene, high density polyethylene, low density polyethylene, polyethylene terephthalate, poly-1,4 cyclohexanedimethylene terephthalate, polyethylene 2,6 naphthalate dibenzoate, polyolefin, polyvinylidene fluoride, polyethylene 2,6 naphthalate, acrylonitrile butadiene styrene, polyvinyl chloride, polyether block amide, biodegradable polymers, and mixtures thereof.

9. A method for preventing contamination of a tubular conduit by Gram negative bacteria, Gram positive bacteria, fungi, yeasts and/or viruses, said method comprising functionalizing at least one portion of an outer surface and/or of an inner surface of the tubular conduit with the antimicrobial peptide of claim 1.

10. The method of claim 9, wherein the tubular conduit is a drinking straw or a medical tube.

11. The tubular conduit of claim 1, wherein X.sub.2 has the meaning of K.

12. The tubular conduit of claim 1, wherein the tubular conduit is a drinking straw incorporated in and optionally removable from a container.

13. The tubular conduit of claim 7, wherein the tubular conduit is a biodegradable polymer selected from the group consisting of polylactic acid (PLA), polybutylene adipate-co-terephthalate (PBAT), polycaprolactone (PCL), modified starch (MaterBi®) or polyglycolic acid (PGA), polybutylene succinate (PBS), poly-hydroxy alkanoates (PHA), and mixtures thereof.

14. The tubular conduit of claim 7, wherein the tubular conduit is polylactic acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0038] FIG. 1 shows dose-response curves obtained with the RiLK1 peptide against S. aureus (A), L. monocytogenes (B), S. typhimurium (C) and E. coli (D); the abscissa axis shows the peptide concentration (μM), the ordinate axis shows the % of surviving cells, as described in Example 2a.

[0039] FIGS. 2a, 2b, 2c and 2d show for each bacterium tested, i.e., S. aureus, L. monocytogenes, S. typhimurium and E. coli, respectively, (In FIGS. 2c and 2d it is necessary to correct the caption relating to S. typhimurium and E. coli) photographic images of a control plate incubated with the bacterium without the addition of the RiLK1 peptide (1), a plate incubated with the bacterium and treated with a RiLK1 peptide concentration lower than its MBC (2), a plate incubated with the bacterium and treated with a RiLK1 peptide concentration corresponding to its MBC (3), as described in Example 2b.

[0040] FIG. 3 shows the reverse phase chromatographic plots, obtained using a C18 column through an HPLC system as described in Example 3, of the RiLK1 peptide present in the composition at time point=zero (Line A), and of the RiLK1 peptide remaining in the composition at time point=24 hours (Line B). The graph depicts the absorbance values of the RiLK1 peptide at 280 nm as a function of the elution time, measured in minutes (min).

[0041] FIG. 4 shows the % decrease in bacterial load expressed as CFUs of a liquid culture of E. coli (A), S. typhimurium (B), and L. monocytogenes (C) in which a straw was inserted for 4, 6, 8 and 24 hours of contact.

[0042] FIG. 5 shows the % decrease in bacterial load expressed as CFUs of tap water contaminated by E. coli (A), S. typhimurium (B), and L. monocytogenes (C) with a bacterial concentration of 150 CFU/mL in which a straw was inserted for 4, 6, 8 and 24 hours of contact.

DETAILED DESCRIPTION OF THE INVENTION

[0043] A first object of the present invention is a tubular conduit comprising an inner surface and an outer surface, characterized in that at least one portion of the outer and/or inner surface(s) is functionalized with at least one antimicrobial peptide consisting of the formula (I).

[0044] In preferred embodiments, the at least one antimicrobial peptide is covalently linked to reactive groups which are present on the at least one portion of the outer and/or inner surface(s) of the tubular conduit; alternatively, the at least one antimicrobial peptide is contained in a coating attached to the at least one portion of the outer and/or inner surface(s) of the tubular conduit.

[0045] As indicated above, the at least one antimicrobial peptide consists of the amino acid sequence represented by the formula (I) shown below:

X.sub.1LX.sub.2WVX.sub.3IWX.sub.4X.sub.5  (I)

[0046] wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are independently selected from K and R and wherein each amino acid is independently in the D or L configuration, or a salt or a solvate thereof.

[0047] It should be noted that all amino acid sequences are represented in the present description by formulas whose orientation from left to right is in the conventional direction, i.e., from the amino terminus to the carboxyl terminus.

[0048] According to a preferred embodiment, the amino acids in the above formula (I) are all in the D configuration or in the L configuration.

[0049] According to another preferred embodiment, in the above formula (I), at least one of X.sub.1, X.sub.3, X.sub.4 and X.sub.5 has the meaning of R; more preferably, X.sub.4 and X.sub.5 have the meaning of R; still more preferably, X.sub.3, X.sub.4 and X.sub.5 have the meaning of R; even more preferably, X.sub.1, X.sub.3, X.sub.4 and X.sub.5 have the meaning of R.

[0050] In another preferred embodiment, X.sub.2 has the meaning of K.

[0051] In another preferred embodiment, at least one of X.sub.1, X.sub.3, X.sub.4 and X.sub.5 has the meaning of R and X.sub.2 has the meaning of K; more preferably, X.sub.4 and X.sub.5 have the meaning of R and X.sub.2 has the meaning of K; still more preferably, X.sub.3, X.sub.4 and X.sub.5 have the meaning of R and X.sub.2 has the meaning of K; even more preferably, X.sub.1, X.sub.3, X.sub.4 and X.sub.5 have the meaning of R and X.sub.2 has the meaning of K.

[0052] The following amino acid sequences are particularly preferred:

TABLE-US-00001 RLX.sub.2WVRIWX.sub.4X.sub.5, RLX.sub.2WVRIWRX.sub.5, RLX.sub.2WVRIWX.sub.4K, RLKX.sub.2WVRIWKK, X.sub.1LKWVX.sub.3IWRR, X.sub.1LKWVRIWRR, RLKWVX.sub.3IWRR, X.sub.1LRWVX.sub.3IWKK, X.sub.1LRWVKIWKK, KLRWVX.sub.1WKK,

[0053] wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 are independently selected from K and R and wherein each amino acid is independently in the D or L configuration.

[0054] The following specific amino acid sequences are further preferred:

TABLE-US-00002 (SEQ ID NO: 1) RLKWVRIWRR, (SEQ ID NO: 2) KLRWVRIWRR, (SEQ ID NO: 3) RLRWVRIWRR, (SEQ ID NO: 4) KLKWVRIWRR, (SEQ ID NO: 5) RLKWVKIWRR, (SEQ ID NO: 6) KLRWVKIWRR, (SEQ ID NO: 7) RLRWVKIWRR, (SEQ ID NO: 8) KLKWVKIWRR, (SEQ ID NO: 9) RLKWVRIWKR, (SEQ ID NO: 10) KLRWVRIWKR, (SEQ ID NO: 11) RLRWVRIWKR, (SEQ ID NO: 12) KLKWVRIWKR, (SEQ ID NO: 13) RLKWVKIWKR, (SEQ ID NO: 14) KLRWVKIWKR, (SEQ ID NO: 15) RLRWVKIWKR, (SEQ ID NO: 16) KLKWVKIWKR, (SEQ ID NO: 17) RLKWVRIWRK, (SEQ ID NO: 18) KLRWVRIWRK, (SEQ ID NO: 19) RLRWVRIWRK, (SEQ ID NO: 20) KLKWVRIWRK, (SEQ ID NO: 21) RLKWVKIWRK, (SEQ ID NO: 22) KLRWVKIWRK, (SEQ ID NO: 23) RLRWVKIWRK, (SEQ ID NO: 24) KLKWVKIWRK, (SEQ ID NO: 25) RLKWVRIWKK, (SEQ ID NO: 26) KLRWVRIWKK, (SEQ ID NO: 27) RLRWVRIWKK, (SEQ ID NO: 28) KLKWVRIWKK, (SEQ ID NO: 29) RLKWVKIWKK, (SEQ ID NO: 30) KLRWVKIWKK, (SEQ ID NO: 31) RLRWVKIWKK, (SEQ ID NO: 32) KLKWVKIWKK,

[0055] wherein each amino acid is independently in the D or L configuration.

[0056] The peptide with the sequence SEQ ID NO: 1 is the one indicated as “RiLK1” in the scope of the present description.

[0057] In the context of the present invention, the term “solvate” or “solvates” refers to complexes of the peptides of the invention with the solvents in which the synthesis reaction takes place or in which they are precipitated or crystallized. For example, a complex with water is known as a “hydrate”.

[0058] Salts and solvates suitable for the purposes of the invention are those that do not lead to a change in the conformation or stability of the peptides according to the invention, and therefore, do not interfere with their biological activity.

[0059] Preferred salts according to the invention are pharmaceutically acceptable salts that do not lead to a change in the conformation or stability of the peptides according to the invention. By way of illustrative and non-limiting examples, pharmaceutically acceptable acid addition salts include those formed with the hydrochloric, hydrobromic, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic and isethionic acids. Other acids, such as oxalic acid, although not pharmaceutically acceptable per se, can be useful as intermediates for obtaining the peptides of the invention and pharmaceutically acceptable salts thereof. Acceptable base salts include ammonium salts, alkali metal salts, for example potassium and sodium salts, alkaline earth metal salts, for example calcium and magnesium salts, and salts with organic bases, for example dicyclohexylamine and N-methyl-D-glucosamine.

[0060] Preferably, according to the invention, the tubular conduit is a drinking straw or a medical tube; more preferably, it is a drinking straw incorporated in and optionally removable from a container such as, for example, a bottle, a canteen or a can.

[0061] In a preferred embodiment, the tubular conduit is a straw for food or beverages.

[0062] Preferably, according to the invention, the number of moles of the at least one antimicrobial peptide which are present on the at least one portion of the outer and/or inner surface(s) of the tubular conduit is between 1 and 10 nmol×cm.sup.2 of the surface, more preferably between 1.5 and 3 nmol×cm.sup.2 of the surface.

[0063] According to the invention, the material of the tubular conduit preferably is a polymer; suitable polymers are, by way of illustrative and non-limiting example, styrene block copolymers, polyolefin mixtures, elastomeric mixtures, thermoplastic polyurethanes, thermoplastic copolyesters, thermoplastic polyamides, polypropylene, polyethylene, high density polyethylene, low density polyethylene, polyethylene terephthalate, poly-1,4 cyclohexanedimethylene terephthalate, polyethylene 2,6 naphthalate dibenzoate, polyolefin, polyvinylidene fluoride, polyethylene 2,6 naphthalate, acrylonitrile butadiene styrene, polyvinyl chloride, polyether block amide, biodegradable polymers, and mixtures thereof. More preferably, the polymeric material is a biodegradable polymer such as, for example, polylactic acid (PLA), polybutylene adipate-co-terephthalate (PBAT), polycaprolactone (PCL), modified starch (MaterBi®) or polyglycolic acid (PGA), polybutylene succinate (PBS), poly-hydroxy alkanoates (PHA) (family of MaterBi® materials), and mixtures thereof. Even more preferably, the polymeric material is polylactic acid.

[0064] The present inventors investigated different types of polymers and found that PLA is particularly suitable for being functionalized for the purpose of the present invention. PLA is a thermoplastic polymer belonging to the aliphatic polyester family, with properties similar to those of polyethylene terephthalate (PET). It is derived from sugar, so it is a product derived from 100% natural resources and therefore has zero environmental impact. PLA can be easily processed with conventional machines for thermoforming and single or biaxial extrusion of polymeric films, by injection and by foaming. PLA is 100% biodegradable and compostable. PLA is stable in standard environmental conditions (20° C., 1 Atm), is degraded by hydrolysis at temperatures above 65° C. and humidity above 20%, so biodegradation times can vary considerably depending on the environmental conditions. At 65° C. and 95% humidity, which are the standard conditions of a normal composting station, PLA-based products are degraded in about 50 days. If left on the ground, PLA-based products degrade in 15 months, in 24 months if burned, in 48 months if placed in water.

[0065] Preferably, the tubular conduit according to the invention is a drinking straw or a medical tube made of PLA; more preferably, it is a drinking straw made of PLA and incorporated in and optionally removable from a container, for example, a bottle, a canteen or a can.

[0066] As indicated above, one embodiment of the invention is that in which the at least one antimicrobial peptide is covalently linked to reactive groups which are present on the at least one portion of the outer and/or inner surface(s) of the tubular conduit.

[0067] In this case, the N-terminal amino group of the peptide is covalently linked to at least one chemical group which is present on the at least one portion of the outer and/or inner surface(s) of the tubular conduit, said at least one chemical group being preferably selected from a carboxylic group, an excited hydroxyl radical, an activated alkoxy group, or an activated aldehyde or ketone group.

[0068] In fact, the present inventors found that when the N-terminal group of the peptide is covalently linked to chemical groups which are present on the surface of the tubular conduit, the bactericidal activity is maintained despite the locked conformation of the peptide, and that this bactericidal activity remains stable for long time periods.

[0069] Techniques for binding peptides to solid supports are known to those skilled in the art and vary depending on the material used. Mainly used surface functionalization methods are chemical or physical methods.

[0070] For example, in the case of materials having metal (gold, silver, platinum) or semiconductor (titanium, zinc, tin, zirconium, germanium) surfaces, a silanization process can be used. This involves, for example, reacting the material to be treated with a mixture of sulfuric acid (H.sub.2SO.sub.4) and oxygen peroxide (H.sub.2O.sub.2), which are able to activate the aforementioned surfaces by creating bonds of surface atoms and hydroxyl groups (—OH) easily replaceable by more stable bonds such as Si—C or Au—S. The activated surfaces can covalently bind the peptides of interest following treatment with a silanizing agent, such as aminopropyldimethylethoxysilane or aminopropyltriethoxysilane, and with a compound having two functional groups capable of forming the peptide covalent bond with the amino groups of the peptide, such as glutaraldehyde or bis-succinimide. These treatments are typical of the chemistry of aqueous solutions and for this reason they are referred to as wet processes, which are advantageous because they do not require particular technological equipment but only, preferably rigid, materials which can be wet and dried without difficulty.

[0071] In the case of plastic or polymeric surfaces, these can be activated to link the peptides of interest by applying both wet processes, such as those described above, and dry processes.

[0072] Wet activation, typical of the chemistry of aqueous solutions, is generally advantageous because it does not require particular technological equipment but only, preferably rigid, materials which can be wet and dried without difficulty. The activated surface is then reacted with a silanizing agent, such as aminopropyldimethylethoxysilane or aminopropyltriethoxysilane, and with a compound having two functional groups capable of forming the peptide covalent bond with the amino groups of the peptide, such as glutaraldehyde or bis-succinimide.

[0073] Dry activation is based on the interaction of the surface to be treated with electromagnetic radiation, for example laser, ultraviolet radiation, gamma rays, or with ionized gas (gas plasma). The interaction of the surface of a polymer with electromagnetic radiation causes surface activation, thereby allowing subsequent chemical modification of the surface itself. A similar operating principle also applies to the activation of polymeric surfaces by treatment with gas plasma. This method is particularly advantageous since, as the plasma is cold, the temperature of the treated material does not reach high values with respect to room temperature. This method requires low pressure (0.1-100 Pa) and the presence of a working gas (usually N.sub.2, O.sub.2 or Ar, CF.sub.4). [Hegemann, Dirk, Herwig Brunner, and Christian Oehr. “Plasma treatment of polymers for surface and adhesion improvement.” Nuclear instruments and methods in physics research section B: Beam interactions with materials and atoms 208 (2003): 281-286].

[0074] As indicated above, a further embodiment of the invention is that in which the at least one antimicrobial peptide is contained in a coating attached to at least one portion of the outer and/or inner surface(s) of the tubular conduit.

[0075] In this case, the functionalization process involves the deposition of a liquid antimicrobial composition containing the antimicrobial peptide onto the surface portion of the tubular conduit, after which the liquid antimicrobial composition is allowed to dry. The liquid antimicrobial composition can optionally comprise film-forming agents which form a film on the surface of the antimicrobial conduit, which film favours the permanence of the peptide on the surface. Therefore, the aforementioned coating may comprise a film-forming polymer.

[0076] Preferably, the concentration of the at least one antimicrobial peptide in the liquid antimicrobial composition is between 10 and 100 μM, more preferably between 20 and 80 μM, 30 and 60 μM, 40 and 60 μM.

[0077] Preferably, the incubation time of the tubular conduit in the liquid antimicrobial composition containing the at least one antimicrobial peptide is between 18 and 36 hours, more preferably between 20 and 30 hours.

[0078] As indicated above, the characteristics of the tubular conduit according to the invention are suitable for preventing contamination by Gram negative bacteria, Gram positive bacteria, fungi, yeasts and/or viruses.

[0079] Therefore, a second object of the present invention is the use of an antimicrobial peptide as defined above, for the prevention of contamination of a tubular conduit by Gram negative bacteria, Gram positive bacteria, fungi, yeasts and/or viruses.

[0080] In this context, the Gram negative bacteria are preferably selected from the group consisting of Campylobacter such as, for example, Campylobacter coli, Campylobacter concisus, Campylobacter jejuni, Campylobacter C. rectus; Arcobacter such as, for example, Arcobacter butzleri, Arcobacter cryaerophilus; Citrobacter such as, for example, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Citrobacter gillenii, Citrobacter koseri; Enterobacter such as, for example, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, Enterobacter cowanii, Enterobacter gergoviae; Escherichia such as, for example, Escherichia coli; Klebsiella; Morganella such as, for example, Morganella Morganii; Proteus such as, for example, Proteus vulgaris, Proteus mirabilis; Shigella such as, for example, Shigella dysenteriae; Salmonella such as, for example, Salmonella typhi, Salmonella typhimurium; Yersinia such as, for example, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica; Serratia marcescens; Aerobacter aerogenes; Enterobacter sakazakii; Acinetobacter, such as, for example, Acinetobacter baumannii, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter boissieri; Moraxella such as, for example, Moraxella catarrhalis (synonym Branhamella catarrhalis); Neisseria such as, for example, Neisseria meningitidis; Haemophilus such as, for example, Haemophilus influenzae; Pasteurella such as, for example, Pasteurella multocida; Pseudomonas such as, for example, Pseudomonas aeruginosa; Vibrio such as, for example, Vibrio cholerae, Vibrio fischeri, Stenotrophomonas maltophilia, and combinations thereof. More preferably, the Gram negative bacteria are selected from Salmonella typhimurium and Escherichia coli.

[0081] The Gram positive bacteria are preferably selected from the group consisting of Actinobacteria such as, for example, Tropheryma whipplei; Bacillus; Carnobacterium; Clostridium; Corynebacterium diphtheria; Enterococcus such as, for example, Enterococcus faecalis; Gardnerella vaginalis; Lactobacillus; Lactococcus; Listeria such as, for example, Listeria monocytogenes; Micrococcus; Staphylococcus such as, for example, Staphylococcus aureus; Streptococcus such as, for example, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, and combinations thereof. More preferably, the Gram positive bacteria are selected from Listeria monocytogenes and Staphylococcus aureus.

[0082] The fungi are preferably Aspergillus brasiliensis and the yeasts are preferably Candida albicans.

[0083] The viruses are preferably viruses equipped with a capsid and an additional coating called pericapsid; they can be either DNA or RNA viruses. Preferably, the virus is an adenovirus, papilloma virus (HPV), herpes virus, coronavirus, influenza virus, cytomegalovirus (CMV), HIV or Ebola virus.

[0084] The examples that follow are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims.

EXAMPLES

Example 1—Synthesis of the RiLK1 Peptide

[0085] The RiLK1 peptide having the sequence RLKWVRIWRR (SEQ ID NO: 1) was synthesized by solid phase peptide synthesis using the protective group Fluoromethoxycarbonyl (Fmoc).

[0086] Rink-Amide MBHA resin with a degree of substitution of 0.5 mmol/g was used as a solid support. The resin has a linker that provides an amide bond and releases the peptide amidated at the C-terminus.

[0087] At the end of the synthesis, the protective group was removed by treatment with a 40% (v/v) solution of piperidine in DMF, while the detachment from the resin and the removal of the protective groups from the amino acid side chains were obtained by treatment with an acidic solution consisting of 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% H.sub.2O (v/v/v).

[0088] After the detachment from the solid support, the peptide was precipitated in cold ethyl ether, at −20° C. The sample was centrifuged at 3500 rpm for 5 minutes in order to collect the precipitate. The precipitate was dissolved in a mixture of CH.sub.3CN/H.sub.2O (95:5), frozen and lyophilized.

[0089] This procedure can be used for the synthesis of all AMP peptides employed in the invention.

Example 2—Analysis of the Bactericidal Activity of RiLK1

Example 2a—Assessment of the Concentration Capable of 50% Bacterial Growth Inhibition (IC.SUB.50.)

[0090] The bactericidal activity of RiLK1 peptides was assessed against both Gram positive (Listeria monocytogenes LM2 and Staphylococcus aureus) and Gram negative (Salmonella typhimurium and Escherichia coli) pathogenic bacteria.

[0091] For the four bacterial species selected, certified and characterized strains, as indicated in the following Tables 1 and 2, were used.

[0092] The assessment of the concentration of RiLK1 peptides capable of inhibiting 50% of bacterial growth (IC.sub.50) was performed by the broth microdilution assay as described in Wang HX and Ng TB (2003, Peptides 24:969-972).

[0093] Standard deviations were obtained from triplicate experiments for each peptide dilution, and the IC50s were determined using GraphPad Prism version 6.00 (Graph-Pad Software, La Jolla Calif. USA).

[0094] All bactericidal activity assays were performed using 2-3 log CFUs (Colony Forming Units), which represents a realistic approximation of the contamination levels that may be contained in the tubular conduits for food or medical use.

[0095] Staphylococcus aureus

[0096] A control stock suspension was prepared in which 10′ CFUs of S. aureus were inoculated in 10 ml of BPW. 5 mM stock solutions of the RiLK1 peptides were then prepared and serial dilutions (100 to 1 μM) were carried out in BPW and inoculated with 10.sup.3 CFUs of S. aureus, and then incubated for 6 hours at 37° C. At the same time, control samples were prepared and treated in the same way but without the addition of the peptides.

[0097] 50 μl of each bacterial suspension were poured onto blood agar or rabbit plasma fibrinogen agar Petri dishes and incubated for 20 hours at 37° C.

[0098] All experimental conditions investigated used the plate count method to estimate the bactericidal activity of the peptides. Specifically, the numbers of colonies grown on agar plates seeded with the bacterial suspensions in the absence or presence of the individual peptide dilutions were counted and compared. Standard deviations were determined using statistical software.

[0099] Listeria monocytogenes

[0100] A control stock suspension was prepared in which 10.sup.3 CFUs of L. monocytogenes were inoculated in 10 ml of Half Fraser Broth and serial dilutions (100 to 0.01 μM) of the suspension were performed. 5 mM stock solutions of the peptides in DMSO were then prepared and serial dilutions (100 to 0.01 μM) were carried out in Fraser Broth and inoculated with 10.sup.3 CFUs of L. monocytogenes, and then incubated for 6 hours at 37° C. At the same time, control samples were prepared and treated in the same way but without the addition of the peptides. 50 μl of each bacterial suspension were seeded on different culture plates: blood agar and ALOA (Oxoid, Basingstoke, UK), which were then incubated for 24-48 hours at 37° C. Each dilution series included control plates inoculated with DMSO without the peptide and control plates with bacteria only.

[0101] Salmonella typhimurium

[0102] A control stock suspension was prepared in which 10.sup.3 CFUs of S. typhimurium were inoculated in 10 ml of BPW (Oxoid, Basingstoke, UK). 5 mM stock solutions of the peptides were then prepared and serial dilutions (100 to 1 μM) were carried out in BPW and inoculated with 10.sup.3 CFUs of S. typhimurium, and then incubated for 6 hours at 37° C. 50 μl of each bacterial suspension were seeded on Petri dishes with blood agar or chromogenic agar (Oxoid, Basingstoke, UK) and incubated for 20 hours at 37° C. Each dilution series included control plates inoculated with DMSO without the peptide and control plates with bacteria only.

[0103] Escherichia coli

[0104] A control stock suspension was prepared in which 10.sup.3 CFUs of E. coli were inoculated in 10 ml of BPW (Oxoid, Basingstoke, UK). 5 mM stock solutions of the peptides were then prepared and serial dilutions (100 to 1 μM) were carried out in BPW and inoculated with 10.sup.3 CFUs of E. coli, and then incubated for 6 hours at 37° C. 50 μl of each bacterial suspension were seeded on Petri dishes with blood agar or chromogenic agar (Oxoid, Basingstoke, UK) and incubated for 20 hours at 37° C. Each dilution series included control plates inoculated with DMSO without the peptide and control plates with bacteria only.

[0105] FIG. 1 depicts the dose-response curve obtained with the RiLK1 peptide against S. aureus (A), L. monocytogenes (B), S. typhimurium (C), and Escherichia coli (D).

[0106] On the basis of the dose-response curves obtained, the IC.sub.50 values (peptide concentration capable of inhibiting 50% of bacterial growth) against the bacterial strains mentioned above were determined.

[0107] The data reported in Table 1 show that the RiLK1 peptide exhibits strong bactericidal activity against all the tested bacteria (IC.sub.50<2 μM); in particular, a stronger bactericidal activity is observed against S. typhimurium, E. coli and L. monocytogenes (IC.sub.50<1.5 μM) and an even stronger bactericidal activity is observed against L. monocytogenes (IC.sub.50 0.46 μM).

TABLE-US-00003 TABLE 1 RiLK1 Bacterium IC.sub.50 [μM] S. aureus (ATCC) 1.98 L. monocytogenes (LM2) 0.46 (field strain) S. Typhimurium (ATCC) 1.30 E. coli (ATCC) 1.20

Example 2b—Evaluation of the Minimum Bactericidal Concentration (MBC)

[0108] The evaluation of the Minimum Bactericidal Concentration (MBC), i.e., the lowest concentration of antimicrobial agent capable of 99.9% inhibition of bacterial growth on plates, was carried out as described in Bilikova et al, (2015, Peptides 68:190-196).

[0109] The data reported in Table 2 below show that the RiLK1 peptide exhibits strong bactericidal activity against all the tested bacteria (MBC <20 μM); in particular, a stronger bactericidal activity is observed against L. monocytogenes, S. typhimurium and E. coli (MBC <5 μM) and an even stronger bactericidal activity is observed against L. monocytogenes and E. coli (MBC 2 μM).

TABLE-US-00004 TABLE 2 RiLK1 Bacterium MBC [μM] S. aureus (ATCC) 16 L. monocytogenes (LM2) 2 (field strain) S. typhimurium (ATCC) 4 E. coli (ATCC) 2

[0110] The images in FIG. 2a represent photographic images of (1) the control plate incubated with S. aureus without the addition of the RiLK1 peptide, (2) the plate incubated with S. aureus and treated with 2 μM RiLK1 peptide, (3) the plate incubated with S. aureus and treated with 16 μM RiLK1 peptide, which shows that at a concentration of 16 μM this peptide is able to inhibit almost 100% of the bacterial growth.

[0111] The images in FIG. 2b represent photographic images of (1) the control plate incubated with L. monocytogenes without the addition of the RiLK1 peptide, (2) the plate incubated with L. monocytogenes and treated with 0.8 μM RiLK1 peptide, (3) the plate incubated with L. monocytogenes and treated with 2 μM RiLK1 peptide, which shows that at a concentration of 2 μM this peptide is able to inhibit almost 100% of the bacterial growth.

[0112] The images in FIG. 2c represent photographic images of (1) the control plate incubated with S. typhimurium without the addition of the RiLK1 peptide, (2) the plate incubated with S. typhimurium and treated with 0.8 μM RiLK1 peptide, (3) the plate incubated with S. typhimurium and treated with 2 μM RiLK1 peptide, which shows that at a concentration of 2 μM this peptide is able to inhibit almost 100% of the bacterial growth.

[0113] The images in FIG. 2d represent photographic images of (1) the control plate incubated with E. coli without the addition of the RiLK1 peptide, (2) the plate incubated with E. coli and treated with 0.8 μM RiLK1 peptide, (3) the plate incubated with E. coli and treated with 2 μM RiLK1 peptide, which shows that at a concentration of 2 μM this peptide is able to inhibit almost 100% of the bacterial growth.

Example 2c—Analysis of the Activity Against Fungi and Yeasts

[0114] Evaluation of the RiLK1 peptide concentration capable of inhibiting the growth of the fungus Aspergillus brasiliensis

[0115] The antifungal activity of the RiLK1 peptide was determined against Aspergillus brasiliensis. For this purpose, a stock culture was prepared in which 10.sup.5 CFUs of A. brasiliensis were inoculated in 10 ml of buffered peptone water. A reference strain (ATCC 9341) was used for this fungal species. The culture was incubated for 6 h at 37° C. with the RiLK1 peptide at 25 μM concentrations.

[0116] At the same time, control samples were incubated without adding the peptide. 100 μl of the samples thus prepared were seeded on DG18 plates (Dichloran 18% Glycerol Agar—ISO 21527-2) and incubated at 25° C. for 7 days.

[0117] All experimental conditions investigated used the plate count method to estimate the fungicidal activity of the peptide. Specifically, the numbers of colonies grown on agar plates seeded with the fungal suspensions in the absence or presence of the individual peptide dilutions were counted and compared. The standard deviations of the triplicate incubations of each plate were determined using statistical software. The evaluation of the Minimum Fungicidal Concentration (MFC), i.e., the lowest concentration of antifungal agent capable of 99.9% inhibition of fungal growth on plates, was carried out as described in Bilikova et al, (2015, Peptides 68:190-196).

[0118] The RiLK1 peptide has strong fungicidal activity against the tested fungus, with an MFC value less than or equal to 25 μM. It should be noted that the peptide IDR-1018-K6 (object of patent WO2019012158) against the same fungus and under the same experimental conditions is not active, with a post-treatment decrease in the fungal load of 0 Log compared to the significant decrease induced by RiLK1 (5 Log reduction).

[0119] Evaluation of the RiLK1 Peptide Concentration Capable of Inhibiting the Growth of the Yeast Candida albicans

[0120] The inhibitory activity of the RiLK1 peptide was determined against the yeast Candida albicans. A stock culture was prepared in which 10.sup.5 CFUs of C. albicans were inoculated in 10 ml of buffered peptone water. A reference strain (ATCC 14053) was used for this species. The culture was incubated for 6 h at 37° C. with the RiLK1 peptide at 25 μM concentrations. At the same time, control samples were incubated without adding the peptide. 100 μl of the samples thus prepared were seeded on DG18 plates (Dichloran 18% Glycerol Agar—ISO 21527-2) and incubated at 25° C. for 7 days.

[0121] All experimental conditions investigated used the plate count method to estimate the antimycotic activity of the peptide. Specifically, the numbers of colonies grown on agar plates seeded with the cultures in the absence or presence of the individual peptide dilutions were counted and compared. The standard deviations of the triplicate incubations of each plate were determined using statistical software. The evaluation of the Minimum Fungicidal Concentration (MFC), i.e., the lowest concentration of antifungal agent capable of 99.9% inhibition of fungal growth on plates, was carried out as described in Bilikova et al, (2015, Peptides 68:190-196).

[0122] The RiLK1 peptide has strong antimycotic activity against C. albicans, with an MFC value less than or equal to 25 μM. It should be noted that, as shown in Table 3, the peptide IDR-1018-K6 (described in patent application WO2019012158) against the same fungus and under the same experimental conditions exhibits low efficiency, with a post-treatment decrease in the fungal load of 1.0 Log compared to the significant decrease induced by RiLK1 (5 Log reduction).

TABLE-US-00005 TABLE 3 RiLK1 IDR-1018-K6 Drop Drop Fungus Log CFU/ml Log CFU/ml A. brasiliensis 5 0 C. albicans 5 1.0

Example 3—Functionalization of Straws with the Antimicrobial Peptide

[0123] Polylactic acid (PLA) straws prepared by extrusion with equipment from Hangzhou Depth Machinery Co., Ltd. were functionalized with the RiLK1 peptide prepared in Example 1. Specifically, the straws were first dry activated by plasma treatment (50-100 W for 5 min) using an oxygen plasma (Atmospheric Plasma Surface Treatment Machine with Three Rotary Nozzles—Shenzhen Fangrui Technology Co., Ltd.) and subsequently incubated for 24 hours in a 50 μM RiLK1 solution. At the end of the process, the peptide was covalently linked to reactive groups present on the surfaces of the polylactic acid straw.

[0124] FIG. 3 shows the reverse phase chromatographic plots obtained using a C18 column and an HPLC system. Line A shows the absorption peak of the RiLK1 peptide present in the composition at time point=zero, that is, before the incubation of the activated polylactic acid straw. Line B shows the absorption peak of the RiLK1 peptide remaining in the composition at time point=24 hours, that is, after 24 hours of incubation of the straw. After 24 hours of incubation, the yield of the bond between the RiLK1 peptide and the straw could be estimated and was found to be 17%. Knowing the surface area of the functionalized straw, which is 11.304 cm.sup.2, the number of surface-binding peptide moles was calculated and found to be 2.25 nmol×cm.sup.2 of the straw.

[0125] The reverse phase chromatography performed on a C18 column through an HPLC system, after placing the straws functionalized with the RiLK1 peptide in water for 24 hours, showed the total lack of release of the peptide in the water.

Example 4—Analysis of the Bactericidal Activity of the Straws Functionalized with the Antimicrobial Peptide

[0126] The straws functionalized as described in Example 3 were tested against E. coli (A), S. typhimurium (B) and L. monocytogenes (C), starting from a value of 10.sup.1-10.sup.2 CFUs (Colony Forming Units); the effectiveness was assessed at 4, 6, 8 and 24 hours.

[0127] The graph in FIG. 5 shows the case in which the straw was inserted into a culture broth, with an average of 10-100 CFUs/ml. For up to 4 hours, no reduction in the CFUs was observed, as the concentration of pathogens is still too low. At 6 hours, the functionalized straw carries out its antibacterial action against E. coli (100% reduction), S. typhimurium (99% reduction) and L. monocytogenes (73% reduction). For up to 6 hours, the functionalized straw still carries out its antibacterial action against E. coli (25% reduction). After 6 hours, at 8 and 24 hours, the bactericidal activity of the straw is zero against S. typhimurium and L. monocytogenes.

[0128] The graph in FIG. 6 shows the real case in which the straw is inserted in drinking water contaminated with 15-150 CFUs pathogens/ml.

[0129] In this case, since the liquid is not a culture broth, the growth of the pathogen is much less, and the functionalized straw carries out a bactericidal activity for up to 24 hours against S. typhimurium (95% reduction) and L. monocytogenes (99% reduction), and for up to 8 hours against E. coli (81.5% reduction). In this regard, it should be considered that, if the straw is integrated into a container, bottle or canteen used to drink water, this container is generally emptied and/or refilled within 8/12 hours.

[0130] Furthermore, from the above data, it is clear that the functionalized straw is a device capable of sanitizing water by causing a drop in Listeria and Salmonella, against which it is still active even after 24 hours.

[0131] As shown by the data reported in Examples 2a and 2b, respectively, the RiLK1 peptide, which is a representative of the peptides used in the present invention, has strong bactericidal activity against all the tested bacteria (IC.sub.50<2 μM; MBC <20 μM).

[0132] As shown in Example 3, the RiLK1 peptide representative of the peptides used in the present invention is able to bind to the polymeric surface of a PLA straw with a 17% bond yield, resulting in a polymeric straw functionalized with the antimicrobial peptide; the functionalized straw placed in water for 24 hours remains stable without release of the antimicrobial peptide.

[0133] As shown in Example 4, the PLA straw functionalized with the antimicrobial peptide RiLK1 and inserted in drinking water contaminated with pathogens exhibits bactericidal activity both against Gram negative bacteria, such as S. typhimurium and E. coli, and against Gram positive bacteria, such as L. monocytogenes. In particular, the functionalized straw carries out a bactericidal activity for up to 24 hours against S. typhimurium (95% reduction) and L. monocytogenes (99% reduction), and for up to 8 hours against E. coli (81.5% reduction).

[0134] Overall, the experimental data reported above show that tubular conduits made of polymeric material whose outer and/or inner surface(s) is/are functionalized with the specific antimicrobial peptides used in the invention are particularly suitable for use in both the food and medical fields for the prevention of contamination by Gram negative and/or Gram positive bacteria.

[0135] Therefore, functionalization with the specific antimicrobial peptides used in the invention finds useful application, for example, in the prevention of contamination on the surface of straws, preferably on the surface of straws incorporated in and optionally removable from bottles, in both the food and medical fields.

[0136] Functionalization with the specific antimicrobial peptides used in the invention also finds useful application in the sanitization of liquid foods or beverages, for example water, which pass through straws, preferably through straws incorporated in and optionally removable from bottles.

[0137] The antimicrobial peptides used in the invention have the additional advantages of selectively interacting with the lipid bilayer of the bacterial membrane, causing the death of the microorganisms, and of not easily selecting mutants and not inducing antibiotic-resistance phenomena.

[0138] The polymer used in the invention has the additional advantages of being derived from 100% natural resources, therefore having zero environmental impact and being 100% biodegradable and compostable.

Example 5: Analysis of the Bactericidal Activity of Further AMP Peptides Used in the Invention

[0139] The bactericidal activity of further AMP peptides used in the invention, in particular of the RiLK31 peptide (RLRWVKIWKK, SEQ ID NO:31) and the RiLK3 peptide (RLRWVRIWRR, SEQ ID NO:3), was tested. The bactericidal activity was expressed as a percentage of viable cells in terms of CFU (Colony Forming Units) in a bacterial sample exposed to each peptide compared to those in a control bacterial sample in the absence of the peptide. All tests were carried out with the same peptide concentration, corresponding to 15 μM. The results obtained are shown in Table 4.

TABLE-US-00006 TABLE 4 Peptide E. coli Staphylococcus Salmonella Listeria RiLK1 100% 96.9% 100% 99.5% RiLK31 100%   83% 100% 98.8% RiLK3 100%   96% 100%   99%

[0140] Table 4 shows that the further tested AMP peptides have a bactericidal activity comparable to that of the RiLK1 peptide used as the representative peptide in experiments 1˜4 of the present description.

Example 6: Characterization of Further AMP Peptides Used in the Invention by Computational Analysis and Comparison with the HE10 Peptide of the Prior Art

[0141] In bioinformatics, sequence alignment is a way to organize DNA, RNA or protein sequences in order to identify similarity regions which may result from functional, structural or evolutionary relationships between the sequences. Aligned sequences of amino acid residues are typically represented as rows in a matrix [Analysis Tool Web Services from the EMBL-EBI. (2013) McWilliam H, Li W, Uludag M, Squizzato S, Park Y M, Buso N, Cowley A P, Lopez R. Nucleic acids research 2013 July; 41 (Web Server issue):W597-600 doi:10.1093/nar/gkt376; Principles and Methods of Sequence Analysis—Sequence—Evolution—Function—NCBI Bookshelf (Chapter 4) Koonin E V, Galperin M Y. Sequence—Evolution—Function: Computational Approaches in Comparative Genomics. Boston: Kluwer Academic; 2003; Hans G. Boman (1995). Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 1995. 13:61-92]. Multiple sequence alignment highlights similarity areas, which may be associated with specific features, i.e., structural/functional biases that can be more conserved than other regions.

[0142] In protein sequence alignments, the degree of similarity between amino acids occupying a particular position in the sequence can be interpreted as an approximate measure of a particular conserved region or motif in the sequence. The absence of substitutions, or the presence of highly conservative substitutions alone (i.e., the substitution of amino acids whose side chains have similar biochemical properties) in a particular region of the sequence suggests that this region may have structural or functional importance [Hans G. Boman (1995). Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 1995. 13:61-92; Markéta Pazderková, Petr Malo, Vlastimil Zima, Katerina Hofbauerová, Vladimir Kopecký Jr., Eva Kocišová, Tomáš Pazderka, Václav Cerovský and Lucie Bednárová (2019). Interaction of Halictine-Related Antimicrobial Peptides with Membrane Models. Int. J. Mol. Sci., 20, 631; doi:10.3390/ijms20030631; Igor Zelezetsky, Alessandro Tossi (2006). Alpha-helical antimicrobial peptides—Using a sequence template to guide structure—activity relationship studies. Biochimica et Biophysica Acta 1758: 1436-1449; Yang Wang, Jianbo Chen, Xin Zheng, Xiaoli Yang, Panpan M, Ying Cai, Bangzhi Zhang and Yuan Chena (2014). Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity. J. Pept. Sci.; 20: 945-951. DOI 10.1002/psc.2705].

[0143] The presence of the same amino acid residues but in different positions in two amino acid sequences is not a gain but on the contrary leads to a decrease in the similarity score between the two peptides, as it can cause a profound structural and functional alteration.

[0144] When comparing two peptides, the correct way to identify variations and define similarity is to align the two sequences. This means placing one sequence on top of the other, so that it is easier to highlight which positions are the same and which are different. After performing the alignment, two quantitative parameters can be extracted from each pairwise comparison, namely identity and similarity. Identity defines the percentage of directly matching amino acids in the alignment. Similarity occurs when an amino acid is replaced with a similar residue, so that the physical-chemical properties are retained. For example, a change from arginine to lysine maintains the +1 positive charge. This change is much more likely to be acceptable, as the two residues have similar properties and do not impair the function of the peptide. Therefore, the percentage similarity of two sequences is the sum of the identical matches plus the similar ones. The similarity degree depends on the criteria that are used to compare two amino acid residues.

[0145] For example, the RiLK1 peptide (RLKWVRIWRR, SEQ ID NO:1) and the prior art HE10 peptide (VRLIVRIWRR, SEQ ID NO: 33) differ from one another in four positions out of a total of 10 positions. Accordingly, they are 60% identical. However, considering the four substitution pairs, R>V, L>R, K>L, W>I, it appears that the biochemical properties and the size of the residues in each pair are significantly different. Therefore, as illustrated below, the substitution of the first four residues in the RiLK1 peptide compared to HE10 significantly modifies the physical-chemical and structural properties of the peptide.

TABLE-US-00007 (SEQ ID NO: 1) RLKWVRIWRR Peptide-RiLKI (SEQ ID NO: 33) VRLIVRIWRR Peptide HE10

[0146] The underlined residues correspond to the positions where the two peptides differ.

[0147] As clearly shown in Table 5, the hydrophobicity, hydropathicity, amphipathicity, hydrophilicity, net charge and propensity for a disordered conformation of the claimed RiLK1 peptide are significantly different from those of HE10. These properties are known to strongly affect the characteristics of the peptides and modulate their physical-chemical characteristics, with a corresponding influence on the antimicrobial activity [Yang Wang, Jianbo Chen, Xin Zheng, Xiaoli Yang, Panpan M, Ying Cai, Bangzhi Zhang and Yuan Chena (2014). Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity. J. Pept. Sci.; 20: 945-951. DOI 10.1002/psc.2705; Hyung-Sik Won, Min-Duk Seo, Seo-Jeong Jung, Sang-Jae Lee, Su-Jin Kang, Woo-Sung Son, Hyun-Jung Kim, Tae-Kyu Park, Sung-Jean Park, and Bong-Jin Lee, Structural Determinants for the Membrane Interaction of Novel Bioactive Undecapeptides Derived from Gaegurin 5. J. Med. Chem. 2006, 49, 4886-4895]. Hydrophobicity is known to affect both mammalian cell toxicity and antimicrobial activity. Hydrophobic residues facilitate interactions with fatty acyl chains. Relatively low hydrophobicity prevents binding to zwitterionic membranes found in mammalian cells, resulting in low toxicity. However, hydrophobicity is required for permeabilization of the bacterial membrane, but some studies have shown that beyond an optimal level of hydrophobicity, a further increase leads to a loss of antimicrobial activity and an increase in toxicity. Amphipathicity reflects the possibility of an amino acid sequence to form well-structured hydrophobic and hydrophilic domains on opposite faces. The Boman index was originally designated as the protein binding potential and was later renamed as the Boman index. This function calculates the sum of the solubility values for all residues in a sequence and, for normalization, is divided by the number of residues. The Boman index provides an overall estimate of the membrane binding potential of a peptide. The propensity of proteins or peptides for an (intrinsically) disordered conformation plays a key role in cellular regulation and signalling processes. Proteins and peptides under different biological conditions show marginal structural stability, and repeatedly unfold and fold in vivo. Indeed, numerous studies have shown that the effects of the denatured state, such as residual structure, excluded volume and intrinsic conformational propensities, play a key role in molecular recognition, allosteric signalling, folding, and stability [Peter Tompa, Eva Schad, Agnes Tantos and Lajos Kalmar (2015). Intrinsically disordered proteins: emerging interaction specialists. Current Opinion in Structural Biology, 35:49-59. doi.org/10.1016/j.sbi.2015.08.009].

TABLE-US-00008 TABLE 5 RiLK1 HE10 Half-life 855.71 835.91 (sec) Hydrophobicity −0.56 −0.36 Hydropathicity −1.12 0.23 Amphipaticity 1.35 0.98 Hydrophilicity 0.31 0.02 Net charge 5.0 4.0 Boman index 4.67 3.45 Propensity for a −0.61 −0.09 disordered conformation

[0148] Table 5 therefore clearly shows that the RiLK1 peptide used in the invention and the HE10 peptide of the prior art exhibit considerable differences in terms of chemical-physical properties. It therefore appears that a 60% identity percentage (which should be considered low in any case) cannot in any way be considered predictive of a chemical-physical and therefore functional similarity between the two peptides.

[0149] This is further confirmed by the data in Table 6, which shows the same prediction, carried out with the same algorithms, on 4 other AMP peptides of the invention. These 4 peptides also exhibit values of hydrophobicity, hydropathicity, amphiphilicity, hydrophilicity, net charge, Boman index and propensity for a disordered conformation similar to each other and to RiLK1 but completely different compared to HE10.

TABLE-US-00009 TABLE 6 RiLK1 RiLK2 RiLK4 RiLK7 RiLK23 HE10 Half-life 855.71 855.71 920.11 855.71 920.11 835.91 (sec) Hydrophobicity −0.56 −0.56 −0.49 −0.56 −0.49 −0.36 Hydropathicity −1.12 −1.12 −1.06 −1.12 −1.06 0.23 Amphipaticity 1.35 0.98 Hydrophilicity 0.31 0.31 0.31 0.31 0.31 0.02 Net charge 5.0 5.0 5.0 5.0 5.0 4.0 Boman index 4.66 4.66 5.6 4.66 3.73 3.45 Propensity for a −0.61 −0.61 −0.62 −0.61 −0.59 −0.09 disordered conformation

[0150] The inventors also found that the presence of more than one tryptophan (W) residue—a feature common to all the AMP peptides of the invention—plays an important role in determining the antimicrobial properties, as it represents an advantageous and distinctive feature of the interface region between the lipid bilayers. It is also known that the side chains of tryptophan residues are involved in peptide folding in aqueous solution [David I. Chan, Elmar J. Prenner, Hans J. Vogel (2006). Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action. Biochimica et Biophysica Acta 1758; 1184-1202 doi:10.1016/ibbamem.2006.04.006]. Considering not only the effects of the primary structure but also those of the secondary structure on the antimicrobial activity, the ability to assume an amphipathic structure is a functionally important feature for AMP incorporation into bacterial membranes [Marlon H. Cardoso, Karen G. N. Oshiro, Samilla B. Rezende, Elizabete S. Candido, Octávio L. Franco. The Structure/Function Relationship in Antimicrobial Peptides: What Can we Obtain From Structural Data? Advances in Protein Chemistry and Structural Biology—February 2018, DOI: 10.1016/bs.apcsb.2018.01.008]. All the AMP peptides of the invention have at least a second tryptophan residue inserted in the central part of the sequence, which has the purpose of enhancing the amphipathicity of the helix.

[0151] NMR analysis of the RiLK1 peptide in a micellar environment (SDS) has clearly demonstrated the existence of a well-defined structure in the helical arrangement, which places the positively charged side chains and the hydrophobic side chains on opposite sides. In this arrangement, all charged side chains are iso-oriented and prone to interact with the negatively charged surface of the SDS micelles.

[0152] Furthermore, the two side chains of the tryptophan residues, which are present in the W-X-X-X-W motif that characterizes all the AMP peptides used in the present invention, are located at a suitable distance for mutual stacking, thus contributing to the stabilization of the helix.

[0153] The HE10 decapeptide described in WO2015038339 and all the 10 amino acid-long analogues thereof, on the other hand, do not contain the W-X-X-X-W motif which characterizes the AMP peptides used in the invention and which, for the reasons illustrated above, represents a functionally important feature for the bactericidal activity.

[0154] Therefore, the AMP peptides used in the invention are not similar to the HE10 peptide of the prior art neither in structural nor in functional terms.