Peptides having antimicrobial activity

Abstract

The present invention relates to a peptide wherein the peptide has an amino acid sequence exhibiting at least 95% sequence identity to SEQ ID N degree 1 or SEQ ID N degree 2. Compositions comprising the peptides, as well as specific uses, are also disclosed.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and more specifically to natural plant-derived peptides that exhibit antimicrobial activity against various bacterial and fungal strains. Specifically, the present invention relates to a natural, recombinant or synthetic peptide derived from vicilin of the cocoa plant. The present invention also relates to the use of the peptide in the treatment of bacterial and fungal infections in plants, animals and humans. Background Art

[0002] Bacterial antibiotic resistance is a major problem for the food and agricultural industries, as well as the medical and veterinary industries. Of particular concern is the potential for bacteria with lethal antibiotic resistance to be transferred from food animals to human consumers.

[0003] Current approaches to controlling the development and spread of antibiotic-resistant bacteria include changing antibiotic use and patterns of use of different antibiotics, increased government oversight and regulation, and the continued development of new and improved antibiotics. However, the ability of most bacteria to adapt to antibiotic use and to develop resistance to existing and new antibiotics often overcomes these conventional measures, and the continued development of alternative means of controlling bacterial antibiotic resistance is required.

[0004] Antimicrobial peptides (AMPs) are a diverse class of natural compounds found in animals, plants, insects and microorganisms. These peptides are responsible for the defense against (other) microorganisms and, therefore, they can further be used as an alternative to chemical preservatives. However, their use in the treatment of microbial infections in plants, humans and / or animals has been limited so far due to poor availability in nature, high production costs, low stability and complex formulation into suitable therapeutic products. Therefore, the use of antimicrobial peptides in crop protection as well as in human and veterinary treatments remains limited.

[0005] Antimicrobial proteins exhibit a variety of three-dimensional structures, which largely determine their activity and stability. The stability of a certain protein is often a critical parameter for clinical and / or industrial applications.

[0006] After the initial interest in plant-derived AMP molecules, the first plant-derived AMP, purinethionine, was isolated. Caleya et al. reported in Appl. Microbiol, 1972, 23 (5) 998-100 that low molecular weight protein purinethionine from wheat and barley flour is an AMP that is effective against a variety of plant pathogens, such as Pseudomonas solanacearum, Xanthomonas phaseoli, Xanthomonas campestris, Erwinia amylovora, and five strains of Corynebacterium. Since then, several major classes of AMPs have been discovered, including thioureas (type IV), defensins, cyclic peptides, 2S albumin-like proteins, and lipid transfer proteins.

[0007] Plant vicilin is a common class of plant seed storage proteins. It has been shown that some vicilins are processed to produce plant defense peptides. Among the antimicrobial peptides produced from vicilin, the most representative is the antimicrobial peptide from macadamia (Macadamia integrifolia). Macadamia kernels contain 666 amino acid vicilins, which include a highly hydrophilic region of 212 amino acids near the N-terminal signal sequence.

[0008] WO1998027805 discloses a class of vicilin-type peptides with antimicrobial properties. The prototype proteins are of natural origin and have been isolated from macadamia nuts and certain other species including cocoa (Theobroma cacao). In particular, two sequences labeled TcAMP1 (cocoa antimicrobial protein 1) of 47 amino acids and TcAMP2 (cocoa antimicrobial protein 2) of 60 amino acids were derived from the cocoa vicilin seed storage protein gene sequence encoding 566 amino acids (aa) and recombinantly expressed in Escherichia coli. However, the isolated recombinant peptides and compositions prepared therefrom have effective antimicrobial properties at high doses (5-20 μg / ml).

[0009] Marcus et al., Plant. Mol. Biol Rep (2008) 26, 75-87, studied three macadamia nut peptide sequences and two cocoa peptide sequences identified in the N-terminal hydrophilic region of vicilin seed proteins. The antimicrobial activity of the peptides was predicted based on the presence of a characteristic CXXXC-(10-12)-CXXXC motif in the N-terminal proximal hydrophilic region. A His-tagged version of the putative peptide was expressed in Escherichia coli. The recombinant peptides obtained showed in vitro antimicrobial activity against six plant pathogen strains, but the effective dose was very high.

[0010] Ecuador is the most important producer of fine cocoa, accounting for about 50% of the world's production. The fine cocoa produced in Ecuador mainly belongs to the Nacional variety (sometimes also called National or Arriba). In addition to the traditional Nacional cocoa with a strong aroma, Ecuador has been cultivating the cocoa clone CCN-51 since the 1960s. Unlike Nacional, CCN-51 is considered a bulk cocoa type because of its weaker aroma. However, it is more tolerant to changes in climatic conditions, is resistant to different pathogens and has a higher yield than other cocoa types. Therefore, the CCN-51 hybrid variety is very popular for Ecuadorian farmers to grow. US 2004 / 0172683 describes several polypeptides from cocoa beans seeds that are responsible for cocoa flavor.

[0011] WO2002086125A2 discloses a pea globulin-derived peptide as a cocoa flavor enhancer for use in the production of cocoa and chocolate.

[0012] The present invention describes an unknown peptide derived from cocoa that is highly effective and active against a large number of animal and plant pathogenic microorganisms. The peptide is safe to use and preferably does not contain corrosive chemicals that may be toxic and harmful to the environment. Summary of the invention

[0013] The present invention and its embodiments aim to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present invention relates to a peptide having antimicrobial activity as claimed in claim 1. More specifically, the present invention provides a peptide showing at least 95% sequence identity with SEQ ID N°1 or SEQ ID N°2. Surprisingly, it was found that such a peptide obtained from cocoa or produced by recombinant or synthetic methods has antibacterial and / or antifungal activity against a broad spectrum of pathogens. Preferred embodiments of the peptide are shown in any one of claims 2 to 9.

[0014] In a second aspect, the present invention relates to a composition comprising a peptide according to claim 10. Preferred embodiments of the composition are shown in claims 11 and 12.

[0015] In a third aspect, the present invention relates to the use of a peptide as claimed in claim 13 for therapeutic purposes. Preferred embodiments of the use are shown in any one of claims 14 to 21. More particularly, the peptide is used to treat infections with Gram-positive bacteria, Gram-negative bacteria and / or fungi, preferably hyphal fungi or yeasts, in humans, animals or plants.

[0016] In a fourth aspect, the present invention relates to a method for obtaining the peptide according to claims 22 and 23.

[0017] In a fifth aspect, the present invention relates to a vector comprising a gene sequence encoding a peptide, which allows the expression of the peptide described in claim 24, the present invention also relates to the transgenic plant described in claim 25, and the method for obtaining a transgenic plant described in claim 26.

[0018] In a final aspect, the invention relates to seeds according to claim 27 coated with said peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown are proteins isolated from cocoa beans after separation on a two-dimensional (2D) SDS polyacrylamide gel. Horizontal separation was based on their isoelectric point (pI, pH 4-7) and vertical separation was based on their molecular weight. Figure 2 As shown in A, the highlighted spots were analyzed by peptide mass fingerprinting. The numbers on the left indicate the molecular weight (kDa) of the reference marker.

[0020] Figure 2 A representative scheme of the detection and sequence localization of peptide mass fingerprint fragments determined by MALDI-ToF analysis is shown. Figure 2 The underlined amino acid sequence in A was identified as obtained from a 2D SDS polyacrylamide gel of cocoa protein according to SEQ ID NO: o Tryptic digestion product of peptide 1. Figure 2 The underlined amino acid sequence in B was identified as obtained from SDS polyacrylamide gel according to SEQ ID NO: o Tryptic digestion product of recombinant peptide 2.

[0021] Figure 3 shows the LC-MS results of a sample containing the peptide SEQ ID N°2. Figure 3A ) and restore ( Figure 3B ) samples, the mass of the detected peptides was 15.3 kDa.

[0022] Figure 4 The cytotoxicity of the peptide of SEQ ID N° 2 against Candida albicans and HaCaT cells according to the lactate dehydrogenase (LDH) release assay is shown. Each experiment was performed using 10,000 cells, which were incubated with different concentrations of the peptide for 1 hour. DETAILED DESCRIPTION

[0023] The present invention relates to a peptide for medical or pharmaceutical use or a composition comprising said peptide as an active ingredient, wherein said peptide has an amino acid sequence according to SEQ ID N° 1 or SEQ ID N° 2. Furthermore, the present invention relates to the use of said peptide, a method for obtaining said peptide, and a vector allowing the expression of said peptide.

[0024] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. For further guidance, term definitions are included to better understand the teachings of the present invention.

[0025] As used herein, the following terms have the following meanings:

[0026] As used herein, "a", "an" and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. For example, "a compartment" refers to one or more compartments.

[0027] As used herein, "about" refers to a measurable value, such as a parameter, amount, time interval, etc., and is intended to cover variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and more preferably + / -0.1% or less from the specified value, as long as such variations are suitable for implementation in the disclosed invention. However, it should be understood that the value referred to by the modifier "about" itself is also specifically disclosed.

[0028] As used herein, "includes," "comprising," "containing," and "consisting of" are synonymous with "including" and its variations or "containing" and its variations, and are inclusive or open-ended terms that specify the presence of what follows (e.g., components), and do not exclude or exclude the presence of additional, unlisted components, features, elements, members, steps known in the art or disclosed herein.

[0029] In addition, unless otherwise indicated, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and not necessarily to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operation in a sequence different from that described or illustrated herein.

[0030] The recitation of numerical ranges by endpoints includes all numbers and fractions within that range, as well as the recited endpoints.

[0031] Unless otherwise defined, the expressions "weight %", "weight percent", "% wt" or "wt %" here and throughout the specification refer to the relative weight of each component based on the total weight of the formulation.

[0032] Although the term "one or more" or "at least one", such as one or more or at least one member of a group of members, is clear in itself, by way of further example, the term specifically covers reference to any one of the members, or to any two or more of the members, for example, any ≥3, ≥4, ≥5, ≥6 or ≥7, etc., of the members, and up to all of the members.

[0033] The term "peptide" as used herein refers to any compound containing two or more amino acid residues, which are connected by an amide bond formed by the carboxyl group of one amino acid residue and the amino group of the adjacent amino acid residue. The amino acid residues can have L-form as well as D-form, and can be naturally occurring or synthetic, linear and cyclic.

[0034] The term "therapeutically effective amount" refers to an amount that is effective in ameliorating symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" because prevention can be considered treatment.

[0035] As used herein, cocoa bean "syrup" is defined as the mass of ground cocoa nibs after separation of the cocoa butter.

[0036] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical terms and scientific terms, have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. As a further guide, the terms used in the specification are defined to better understand the teachings of the present invention. The terms or definitions used herein are only used to help understand the present invention.

[0037] References to "one embodiment" or "an embodiment" throughout this specification mean that the specific features, structures, or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily all refer to the same embodiment, but may also be. In addition, it will be clear to those skilled in the art from this disclosure that specific features, structures, or characteristics may be combined in one or more embodiments in any suitable manner. In addition, although certain embodiments described herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is intended to be within the scope of the present invention and to form different embodiments, as understood by those skilled in the art. For example, in the following claims, any claimed embodiment may be used in any combination.

[0038] In a first aspect, the present invention relates to a peptide, wherein the peptide has an amino acid sequence that exhibits at least 80% sequence identity to SEQ ID N°1 or SEQ ID N°2. Preferably, the peptide has an amino acid sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more preferably 100% sequence identity to SEQ ID N°1 or SEQ ID N°2. In a preferred embodiment, the peptide has an amino acid sequence that exhibits at least 95% identity to SEQ ID N°1 or SEQ ID N°2.

[0039] The term "sequence identity" as used herein refers to the degree to which a sequence is identical on an amino acid-by-amino acid basis within a comparison window. Therefore, "sequence identity percentage" is calculated as follows: compare two optimally aligned sequences over a comparison window, determine the number of positions where identical amino acids occur in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 to obtain the sequence identity percentage. Gaps, i.e., positions where residues are present in one sequence but not in another sequence in the comparison, are considered to have positions with non-identical residues. Determining the percentage of sequence identity can be done manually, or using a computer program available in the art to complete. PILEUP is an example of a useful algorithm. Software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0040] In another embodiment, the peptide has a sequence which differs from one of the sequences SEQ ID N°1 or SEQ ID N°2 by at most 24, more preferably at most 23, even more preferably at most 22, even more preferably at most 21, even more preferably at most 20, even more preferably at most 19, even more preferably at most 18, even more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 11, even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, even more preferably at most 7, even more preferably at most 6, even more preferably at most 5, even more preferably at most 4, even more preferably at most 3, even more preferably at most 2, even more preferably at most 1 amino acid residue.

[0041] In one embodiment, the peptide has an amino acid sequence according to SEQ ID N°1, or exhibits at least 98% sequence identity with SEQ ID N°2 or differs from the sequence according to SEQ ID N°2 by at most 2 amino acids.

[0042] Alternatively, the peptides of the invention have a sequence identity according to SEQ ID N°1 or N°2.

[0043] The amino acid sequence variants of the peptides contemplated herein can be substitution, insertion or deletion variants. Deletion variants lack one or more peptide residues that may not be important for function. Substitution variants typically contain alternative amino acids at one or more sites within the peptide and can be designed to modify one or more properties of the polypeptide, such as stability against proteolytic cleavage. The substitution is preferably conservative, i.e. an amino acid is replaced by an amino acid with a similar size and side chain or functional group. Conservative substitutions are well known in the art and include, for example, alanine to glycine, valine or leucine; arginine to lysine; asparagine to glutamine; aspartic acid to glutamic acid; cysteine ​​to methionine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to glutamine, tyrosine, arginine, lysine, asparagine or cysteine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to phenylalanine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

[0044] Preferably, the peptides of the present invention are derived from or isolated from cocoa. More specifically, the peptides of the present invention are derived from the N-terminal region of vicilin from cocoa. Vicilins are storage proteins present and characterized in a variety of plant species, such as, but not limited to, pineapple (Ananas comosus), peanut (Arachis hypogaea), sugar beet (Betavulgaris), Mexican pepper (Capsicum annuum), Chinese pepper (Capsicum chinense), American pecan (Carya illinoinensis), quinoa (Chenopodium quinoa), long-capsule jute (Corchorus olitorius), winter squash (Cucurbita maxima), wild strawberry (Fragaria vesca), soybean (Glycine max), cottonwood (Gossipumarboreum), Colombian mallow (Herrania umbratica), barley (Hordeum vulgare), jatropha (Jatropha curcas), walnut (Juglans regia), macadamia (Macadamia integrifolia), Macleaya cordata, Musa acuminata, opium poppy (Papaver somniferum, castor (Ricinus communis), tomato (Solanum lycopersicum), spinach (Spinacia oleracea), fire wheel wood (Stenocarpus sinuatus), cocoa (Theobroma cacao) and corn (Zea mays). Such proteins usually contain extremely hydrophilic N-proximal and C-terminal regions. In addition, they carry a hydrophobic N-terminal signal peptide, which is usually removed during protein maturation. The N-proximal region of the precursor protein is particularly interesting because it contains at least two pairs, preferably four pairs of cysteine ​​motifs (CXXXC) with the same spacing pattern.

[0045] In other embodiments, the peptides disclosed herein are derived from the cocoa variety CCN-51. Surprisingly, the cocoa variety CCN-51, known to be highly resistant to microbial infection, was shown to be particularly rich in vicilin-derived N-terminal peptides.

[0046] In another or additional embodiment, the peptide comprises a signal peptide at its N-terminus. Preferably, the signal peptide has a sequence according to SEQ ID N°3. In another embodiment, the signal peptide exhibits at least 95%, 96%, 97%, 98%, 99% or more preferably 100% sequence identity with SEQ ID N°3. Alternatively, the peptide has a signal peptide sequence that differs from the sequence SEQ ID N°3 by at most 3 amino acid residues, more preferably at most 2 amino acid residues, even more preferably 1 amino acid residue. Vicilin isolated from natural sources generally do not have such a signal peptide. Surprisingly, when a signal peptide is added to the peptide, its antimicrobial activity is enhanced.

[0047] In another embodiment, the peptide is a recombinant peptide or a synthetic peptide. A suitable expression system, such as E. coli, or another suitable system known in the art, produces the peptide after inserting a vector containing the coding sequence of the peptide having SEQ ID N°1 or SEQ ID N°2. Any suitable protein / peptide synthesis method known in the art can be used to synthesize the peptides of the present invention.

[0048] In other or another embodiment, the peptides described herein are provided with affinity tags at their N and / or C termini. Affinity tags are used for affinity purification of recombinant proteins and peptides expressed in E. coli and other systems. The tag may be a polyhistidine tag, which is an amino acid motif consisting of at least six histidine (His) residues.

[0049] In one embodiment, the peptides disclosed herein containing a signal peptide have a sequence identity according to SEQ ID N°4 or N°5.

[0050] Alternatively and without limitation, the peptide can be tagged with an HQ tag (HQHQHQ) of alternating histidine and glutamine residues, an HN tag (HNHNHNHNHNHN) of alternating histidine and asparagine, a HAT tag (KDHLIHNVHKEEHAHAHNK), an ALFA tag (SRLEEELRRRLTE), an Avi tag (GLNDIFEAQKIEWHE), a C tag (EPEA), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), a polyglutamic acid tag, a polyarginine tag , E tag (GAPVPYPDPLEPR), FLAG tag (DYKDDDDK), HA tag (YPYDVPDYA), Myc tag (EQKLISEEDL), NE tag (TKENPRSNQEESYDDNES), Rho1D4 tag (TETSQVAPA), S tag (KETAAAKFERQHMDS), SBP tag (DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) or Strep tag (WSHPQFEK).

[0051] Analysis of the peptides of the present invention showed that the peptides have antibacterial and / or antifungal activities.

[0052] Antimicrobial peptides themselves have a specific three-dimensional structure that can be determined using X-ray crystallography or nuclear magnetic resonance techniques. Although not wishing to be bound by theory, it is believed that this structure plays a role in the observed activity of the peptides. The alpha helical plant-derived antimicrobial peptides (AMPs) to which the peptides of the present invention belong generally have an amphipathic helix, wherein one side of the helix is ​​primarily hydrophilic, and the other side is primarily hydrophobic. This structure is generally found in AMPs that damage the cell membrane, causing leakage and cleavage of cell contents, as well as in AMPs that enter cells to attack other structures within the cell. Alpha-helical AMPs are generally disordered in structure in solution, which helps to pass through the dense network of the cell wall. After binding to the hydrophobic and charged cell membrane below the lipid head, a secondary structure (alpha-helix) is formed, which enables the protein to penetrate the cell membrane. This order / disorder transition is mainly regulated by the length of the hydrophobic domain within the alpha helix. Excessively long hydrophobic helices can lead to ordered structures in solution, which have a negative impact on AMP activity.

[0053] In one embodiment, the peptides are active against Gram-positive and Gram-negative bacteria and fungi, preferably hyphal fungi or yeasts. Without limitation, the peptides are active against bacteria selected from the group consisting of Acinetobacter spp., Bartonella spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter spp., Chlamydia spp., Clostridium spp., Corynebacterium spp., Enterococcus spp., Enterobacter spp., Erwinia spp., Escherichia spp., Francisella spp., Haemophilus spp., Helicobacter spp. spp.), Klebsiella spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Treponema spp., Ureaplasma spp., Vibrio spp., Yersinia spp., Acidovorax spp.), Agrobacterium spp., Arthrobacter spp., Bacillus spp., Burkholderia spp., Clavibacter spp., Cronobacter spp., Curtobacterium spp.), Lefisonia spp., Pantoea spp., Paenibacillus spp., Pectobacterium spp., Phytoplasma spp., Proteus spp., Pseudomonas spp., Ralstonia spp., Rhizobacter spp., Rhizomonas spp., Rhodococcus spp., Serratia spp., Sphingomonas spp., Spiroplasma spp., Streptomyces spp., Xanthomonas spp. spp.), Xylella spp. or Xylophilus spp.

[0054] Without limitation, the peptides of the present invention are active against fungi selected from the group consisting of Ajellomyces spp., Aspergillus spp., Basidiobolus spp., Blastomyces spp., Candida spp., Coccidioides spp., Conidiobolus spp., Cryptococcus spp., Emmonsia spp., Histoplasma spp., Hanseniaspora spp., Lacazia spp., Paracoccidioides spp., Pneumocystis spp., Sporothrix spp. spp.), Stachybotrys spp., Talaromyces spp., Acrocalymma spp., Aecidium spp., Albonectria spp., Allodus spp., Alternaria spp., Amphobotrys spp., Apiosporina spp., Armillaria spp., Blumeria spp., Botryotinia spp., Botrytis spp., Ceratosystis spp., Colletotrichum spp., Cryptosporiopsis spp. spp.), Exobasidium spp., Fusarium spp., Hypocrea spp., Leptosphaeria spp., Magnaporthe spp., Melampsora spp., Meyerozyma spp., Monilinia spp., Mycospharella spp., Microsphaera spp.), Mucor spp., Penicillium spp., Pichia spp., Phytophtora spp., Saccharomyces spp., Sporobolomyces spp., Plasmodiophora spp., Podosphaera spp., Puccinia spp., Pythium spp., Rhizoctonia spp., Sclerotinia spp., Septoria spp., Taphrina spp., Thanatephorus spp., Torulasporas spp., Uromyces spp.), Ustilago spp., Venturia spp., or Verticillium spp.

[0055] In a second aspect, the present invention provides a composition comprising a peptide as described in the above paragraph. The composition is particularly suitable for pharmaceutical and veterinary applications, for crop protection and / or for cosmetic applications.

[0056] In one embodiment, the composition comprises a peptide exhibiting at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or differing from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids and an excipient.

[0057] In certain embodiments of the compositions disclosed herein, the peptide comprises a signal peptide having at least 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

[0058] In one embodiment, the composition is liquid, semisolid, solid or gas, and / or is the dosage form of tablet, capsule, powder, granule, aerosol, paste, syrup, suspension, emulsion or solution. The non-limiting examples of the composition are soluble powder, soluble granule, wettable granule, tablet formulation, dry flow agent, water flow agent, wettable dispersible granule, oil dispersion, suspension concentrate, dispersible concentrate, emulsifiable concentrate, aqueous suspension, fertilizer granule or sprayable composition. The composition can be formulated to be suitable for oral, injection, intravenous, intramuscular, skin, suction, local or intranasal administration. In other embodiments, the composition is formulated to allow coating one or more objects, spraying, spray coating, evaporation, atomization, atomization, suspension, dilution, etc.

[0059] In certain embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or diluent. Preferably, the excipient is selected from a filler, a binder, a disintegrant, a sweetener, a coating agent, a lubricant and / or a glidant. A diluent or filler can increase the volume of a solid composition and can make a dosage form containing the composition easier for patients and caregivers to handle. Diluents suitable for tablets of the present invention include, for example, microcrystalline cellulose (e.g. ), microcellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, glucose, calcium hydrogen phosphate dihydrate, calcium phosphate trihydrate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates (e.g. ), potassium chloride, powdered cellulose, sodium chloride, sorbitol, and talc.

[0060] Solid compositions compressed into dosage forms (e.g., tablets) may contain excipients, the functions of which include helping to contain the active ingredient and other excipients after compression. Suitable binders include acacia, alginic acid, carbomers (e.g., carbopol), sodium carboxymethylcellulose, dextrin, ethylcellulose, gelatin, guar gum, hydrogenated vegetable oils, hydroxyethylcellulose, hydroxypropylcellulose (e.g., ), hydroxypropyl methylcellulose (e.g. ), liquid glucose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylate, povidone (e.g. ), pregelatinized starch, sodium alginate and starch.

[0061] The dissolution rate of a compacted solid composition can be increased by adding a disintegrant to the composition. Suitable disintegrants include alginic acid, calcium carboxymethylcellulose, sodium carboxymethylcellulose (e.g. ), colloidal silicon dioxide, cross-linked sodium carboxymethylcellulose, cross-linked polyvinylpyrrolidone (e.g. ), guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g. ) and starch.

[0062] Glidants may be added to improve the flowability of non-compacted solid compositions and to increase dosage accuracy. Excipients that may be used as glidants include colloidal silicon dioxide, magnesium trisilicate, magnesium stearate, powdered cellulose, starch, talc, and tricalcium phosphate.

[0063] When making dosage forms such as tablets by compressing powdered compositions, the compositions are subject to pressure from the punch and die. Certain excipients and active ingredients tend to adhere to the surface of the punch and dye, which can cause the product to have pits and other surface irregularities. Lubricants can be added to the compositions to reduce adhesion and simplify the release of the product from the die. Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, palmitic acid stearyl glyceride, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc and zinc stearate. Preferably, the lubricant is present in 0.25 % by weight to 1 % by weight (w / w).

[0064] Flavoring agents and flavor enhancers make the dosage form more palatable to the patient. Common flavoring agents and flavor enhancers for pharmaceutical products that can be included in the compositions described herein include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, and tartaric acid.

[0065] The solid composition may also be dyed using any pharmaceutically acceptable colorant to improve its appearance and / or facilitate patient identification of the product and unit dosage level.

[0066] In one embodiment, particularly when the composition is intended for agricultural use, the excipient is an agriculturally compatible excipient. The "agriculturally compatible carrier" or "agriculturally compatible adjuvant" can be considered a carrier, which is generally inert and must be agriculturally acceptable. Therefore, the term "agriculturally compatible" means a substance that can be routinely used under field conditions without interfering with the grower's planting equipment and does not adversely affect the growth of crops or the desired ecological balance in the planting area.

[0067] Agriculturally compatible carriers or excipients can be solid.Solid carriers or excipients can include but are not limited to clay, natural or synthetic silicates, silicon dioxide, resins, waxes, solid fertilizers, polymers, granular materials, perlite, perlite particles, peat, peat particles, soil, vermiculite, charcoal, sugar mill carbonization squeezing mud, rice husks, carboxymethyl cellulose, fine sand, calcium carbonate, flour, alum, starch, talcum, polyvinyl pyrrolidone or its combination.Agriculturally compatible carriers or excipients can be liquid.Liquid carriers or excipients can include but are not limited to water, alcohol, ketones, petroleum fractions, oils, aromatic hydrocarbons or paraffin hydrocarbons, chlorinated hydrocarbons, liquefied gas or its combination.More specifically, agriculturally compatible carriers or excipients can include dispersants, surfactants, additives, thickeners, anti-caking agents, residue decomposition agents, composting formulas, particle applications, diatomaceous earth, coloring agents, stabilizers, preservatives, polymers, coatings or or its combination. Those skilled in the art can consider various factors, such as specific compounds, plants to be applied inoculants, soil types, climatic conditions, whether the inoculants are liquid, solid or powdered forms, etc., and easily determine the appropriate carrier or excipient to be used. Additives can include oils, gums, resins, clays, polyoxyethylene glycols, terpenes, viscous organic matter, fatty acid esters, sulfated alcohols, alkyl sulfonates, petroleum sulfonates, alcohol sulfates, alkyl butane dioic acid sodium, polyesters of thiobutane dioic acid sodium, benzyl cyanide derivatives, protein materials or combinations thereof. Protein materials can include dairy products, wheat flour, soybean meal, blood, albumin, gelatin or combinations thereof. Thickeners can include long-chain alkyl sulfonates of polyethylene glycol, polyoxyethylene oleate or combinations thereof. Surfactants can contain heavy petroleum, heavy petroleum fractions, polyol fatty acid esters, polyethoxylated fatty acid esters, aryl alkyl polyoxyethylene glycols, alkylamine acetates, alkyl aryl sulfonates, polyols, alkyl phosphates or combinations thereof. The anti-caking agent may include a sodium salt, such as sodium sulfite, sodium sulfate, sodium monomethylnaphthalene sulfonate, or a combination thereof; or a calcium salt, such as calcium carbonate, diatomaceous earth, or a combination thereof.

[0068] In one embodiment, the composition further comprises one or more of the following: water, other nutrients, weak acid, vegetable oil, essential oil, metabolic promoter, emulsifier, viscosity agent, coloring material, suspending agent, dispersant, preservative, complexing agent, stabilizer, carrier, excipient or wetting agent or any combination thereof. In one embodiment, the composition further comprises at least one oil, surfactant and polymer.

[0069] The composition can be manufactured in a conventional manner. In certain embodiments, the composition provided herein comprises one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as metfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0070] The peptides described herein can be used at 0.1 to 50 μg / ml, preferably 0.1 to 45 μg / ml, preferably 0.1 to 40 μg / ml, preferably 0.1 to 35 μg / ml, preferably 0.1 to 30 μg / ml, preferably 0.1 to 25 μg / ml, preferably 0.1 to 20 μg / ml, preferably 0.1 to 15 μg / ml, preferably 0.1 to 10 μg / ml, preferably 0.1 to 9 μg / ml, preferably 0.1 to 8 μg / ml, preferably 0.1 to 7 μg / ml, preferably 0.1 to 8 μg / ml, preferably 0.1 to 9 μg / ml, preferably 0.1 to 10 μg / ml, preferably 0.1 to 15 μg / ml, preferably 0.1 to 10 ... The amount of 0.1 to 6 μg / ml, preferably 0.1 to 5 μg / ml, preferably 0.1 to 4 μg / ml, preferably 0.1 to 3 μg / ml, preferably 0.1 to 2 μg / ml, preferably 0.1 to 1 μg / ml, preferably 0.1 to 0.9 μg / ml, preferably 0.1 to 0.8 μg / ml, preferably 0.1 to 0.7 μg / ml, preferably 0.1 to 0.6 μg / ml, preferably 0.1 to 0.5 μg / ml or preferably 0.1 to 0.4 μg / ml is formulated in the composition.

[0071] Alternatively, the peptide can be used at 0.2 to 50 μg / ml, preferably 0.3 to 50 μg / ml, preferably 0.4 to 50 μg / ml, preferably 0.5 to 50 μg / ml, preferably 0.6 to 50 μg / ml, preferably 0.7 to 50 μg / ml, preferably 0.8 to 50 μg / ml, preferably 0.9 to 50 μg / ml, preferably 1 to 50 μg / ml, preferably 2 to 50 μg / ml, preferably 3 to 50 μg / ml, preferably 4 to 50 μg / ml, preferably 5 to 50 μg / ml, preferably 6 to 50 μg / ml, preferably 7 to 50 μg / ml, preferably 8 to 50 μg / ml, preferably 9 to 50 μg / ml, preferably 10 to 50 μg / ml, preferably 15 to 50 μg / ml, preferably 0.5 to 20 μg / ml, preferably 25 to 50 μg / ml, preferably 30 to 50 μg / ml, preferably 35 to 50 μg / ml, preferably 40 to 50 μg / ml, preferably 45 to 50 μg / ml is formulated in the composition.

[0072] In another embodiment, the composition contains at least 0.1% by weight of the peptide, more preferably at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, 70 wt%, 71 wt%, 72 wt%, 73 wt%, 74 wt%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% peptide by weight.

[0073] In a third aspect, the peptides or compositions described herein are suitable for therapeutic use.

[0074] In one embodiment, the peptide or composition for therapeutic use comprises a peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2, or differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acid differences.

[0075] In other embodiments, the peptide comprises a signal peptide having at least 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

[0076] "Therapeutic use" as described herein refers to the use of a peptide or a composition thereof to improve disease symptoms in a human or non-human animal. In practicing the treatment or use methods provided herein, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject, such as a mammal or non-mammal suffering from a disease, disorder or condition to be treated. In certain embodiments, the subject is a human. The therapeutically effective amount may vary greatly depending on the severity of the disease, the age and relative health of the subject, the efficacy of the therapeutic agent used, and other factors. The therapeutic agents described herein, and in some cases the compositions, can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0077] In one embodiment, peptides as described herein are used in pharmaceutical compositions to treat or prevent infections caused by various microorganisms (including gram-positive bacteria, gram-negative bacteria and / or fungi, preferably hyphae fungi or yeast) in a subject in need. The subject can be a human or an animal. The pharmaceutical composition can comprise a therapeutically effective amount of the antimicrobial peptide and a suitable carrier.

[0078] In one embodiment, the peptide or composition for treating bacterial and / or fungal infections in a subject in need thereof comprises a peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or that differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids.

[0079] In other embodiments, the peptide comprises a signal peptide having at least 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

[0080] The peptides or compositions described herein can be administered to a subject by an appropriate route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation or intraperitoneal administration. The compositions described herein can include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsed release formulations, multi-particulate formulations, and mixed immediate and controlled release formulations.

[0081] In one embodiment, the peptides or compositions described herein can be added to animal feed to reduce potential microbial infections in livestock. In another embodiment, the peptides or compositions described herein are formulated for oral, topical or parenteral use to treat microbial infections in veterinary medicine.

[0082] In another embodiment, the peptide or composition described herein is suitable for use in crop protection, as a feed or food additive, for pharmaceutical use, as a preservative and / or as a detergent.

[0083] In one embodiment, the peptide or peptide composition comprises a peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids.

[0084] In other embodiments, the peptide comprises a signal peptide having at least 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

[0085] When used for crop protection, the peptides or compositions described herein are formulated with an agriculturally compatible carrier or agriculturally compatible excipient according to the above embodiments. Those skilled in the art will determine the correct dosage, form and method of application of the composition according to the crop being treated and the effect of the target pathogen.

[0086] In one embodiment, the peptides or compositions described herein are incorporated into feed or food additives to prevent pathogens from spreading through feed and food. It is well known that feed may carry pathogens that are harmful to animal health and welfare. Preservative products such as formaldehyde are commonly used to disinfect feed, but these products can have a negative impact in turn. The antimicrobial peptides described herein are a safe alternative.

[0087] In another embodiment, the peptides or compositions described herein are suitable for use as a detergent or disinfectant for surfaces, tools, instruments, devices, objects, or body parts. In one embodiment, such devices or objects include, but are not limited to, linens, cloths, plastics, latex fabrics, natural rubber, implantable devices, surfaces, or storage containers. In a specific embodiment, the peptides or compositions disclosed herein are incorporated into a cleaning agent, detergent, soap, or spray.

[0088] In certain embodiments, the peptides described herein can also be added to various health products, particularly cosmetics. For example, the peptides can be added to toothpaste, mouthwash, shampoo, soap, cream or antiperspirant to reduce or prevent the colonization or establishment of microorganisms in the oral cavity or skin.

[0089] In another embodiment, the peptides and compositions described herein can be used as food, feed, cosmetic or pharmaceutical preservatives or for treating food to control, reduce or eliminate potential pathogens or contaminants.

[0090] In another embodiment, the peptides or compositions described herein are used to control pathogens in plants, preferably pathogens in crops, wherein the pathogens are Gram-positive and Gram-negative bacteria and / or fungi, preferably hyphal fungi or yeast. The plants may be monocots or dicots, including forage or forage legumes, ornamental plants, food crops, trees or shrubs. In one embodiment, the plants are preferably crops. Preferably, the plant belongs to the group consisting of Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananascomosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., Asparagus officinalis, Avena spp., Averrhoa carambola, Bambusa spp., Benincasa hispida), Brazil chestnut (Bertholletia excelsea), beet (Beta vulgaris), mustard (Brassica spp.), pink leaf leech (Cadaba farinosa), tea tree (Camellia sinensis), canna (Canna indica), hemp (Cannabis sativa), pepper (Capsicum spp.), lip grass (Carex elata), papaya (Carica papaya), large flowered tiger thorn (Carissa macrocarpa), pecan (Carya spp.), safflower (Carthamus tinctorius,), chestnut (Castanea spp.), gibbey (Ceiba pentandra), chicory (Cichorium endivia), camphor (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus (Citrus spp.), coconut (Cocosspp.), coffee (Coffea spp.), Colocasia esculenta, Cola spp., Jute (Corchorus spp.), Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp., Eleusine coracana, Eragrostistef, Erianthus spp.), loquat (Eriobotrya japonica), eucalyptus (Eucalyptus spp.), red fruit (Eugenia uniflora), buckwheat (Fagopyrum spp.), beech (Fagus spp.), tall fescue (Festuca arundinacea), fig (Ficus carica), kumquat (Fortunella spp.), strawberry (Fragaria spp.), ginkgo (Ginkgo biloba), soybean (Glycine spp.), upland cotton (Gossypium hirsutum), sunflower (Helianthus spp.), daylily (Hemerocallis fulva), hibiscus (Hibiscus spp.), barley (Hordeum spp.), sweet potato (Ipomoea batatas), walnut (Juglans spp.), lettuce (Lactuca sativa), Lathyrus spp.), Lens culinaris, Linumusitatissimum, Litchi chinensis, Lotus spp., Luffaacutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp., Macrotyloma spp., Malus spp.), Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordicaspp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp., Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa), Pennisetum spp., Avocado spp., Parsley (Petroselinum crispum), Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp.), radish (Raphanus sativus), rhubarb (Rheum rhabarbarum), Ribes spp., castor (Ricinus communis), Rubus spp., Saccharum spp., willow (Salix spp.), elderberry (Sambucus spp.), Secale cereale, Sesamum spp.), Sinapis spp., Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecalerimpaui, Triticum spp., Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, mays), Zizania palustris, Ziziphus spp.; including their descendants and hybrids.

[0091] The peptides or compositions described herein are suitable for introduction into a plant, a plant part or a matrix comprising or carrying the plant, thereby conferring pathogen resistance or pathogen control to the plant. The introduction may be artificial.

[0092] The method of introducing the peptide or its composition into the plant may include: treating the plant and / or plant part and / or the growth medium in which the plant grows, inoculating seeds, coating seeds, directly inoculating the plant or plant part, spraying or wetting the plant or plant part (e.g., ear of corn). The appropriate method can be selected according to the type of plant into which the peptide or composition is to be introduced.

[0093] As another non-limiting example, peptide as herein described or composition can be applied in the form of coating.Coating can be applied to seed by spraying on seed or soaking the seed in a solution containing peptide or composition.In another example, in order to coat seed with the solution as described in the previous example, an adhesive can be added, for example an adhesive comprising carbide (calcium carbonate).

[0094] In embodiments, the coating can be applied to exposed and untreated plant parts. In other embodiments, the coating can be applied as an outer coating to previously treated plant parts. Seed coatings are particularly preferred in the treatment of soil fungal diseases. In embodiments, the seed coating can be applied to exposed and untreated seeds. In other embodiments, the seed coating can be applied as a seed coat to previously treated seeds.

[0095] In one embodiment, the peptides or compositions described herein may be applied to the soil or any other substrate in which the plant grows to remove pests and / or pathogens from the substrate.

[0096] Inoculation of the matrix containing or carrying the plant or plant part can be carried out by, for example, but not limited to, using a powder, granule, pellet, plug or soil soak applied to the matrix. Inoculation can also be carried out by liquid application, such as foliar spray or liquid composition. Application can be applied to growing plants or to the matrix. Plants, particularly crops, can grow in the matrix. In one embodiment, the matrix is ​​soil, sand, gravel, polysaccharide, mulch, compost, peat moss, straw, log, clay or a combination thereof. In another embodiment, the matrix can also include a hydroponic system or an in vitro culture system. In some embodiments, a combination of different application methods as described herein has been employed.

[0097] In one embodiment, the peptide or composition as described herein is suitable for treating or preventing infection by a pathogen, wherein the pathogen is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and / or fungi as described in any of the preceding embodiments.

[0098] The invention disclosed herein also relates to plant seeds coated with the peptide or composition described in any of the preceding embodiments.

[0099] In another aspect, the present invention relates to a method for obtaining a peptide disclosed herein, wherein the peptide is a recombinant peptide, a synthetic peptide, or is extracted or precipitated from cocoa.

[0100] In one embodiment, the method disclosed herein is used to obtain a peptide that exhibits at least 95% sequence identity with SEQ ID N°1 or SEQ ID N°2 or differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by at most 5 amino acids, and wherein the peptide is a recombinant peptide, a synthetic peptide, or extracted or precipitated from cocoa. It was found that the peptide of the present invention is present in a high content in the CCN-51 cocoa variety, so the peptide of the present invention can be economically purified from CCN-51 cocoa beans using suitable extraction and precipitation techniques, followed by optimized chromatography techniques and other methods known in the prior art. Therefore, the present invention provides a solution for isolating peptides exhibiting antimicrobial activity directly using CCN-51 cocoa beans, especially unfermented CCN-51 cocoa beans.

[0101] In a preferred embodiment, unfermented and / or fermented and dried cocoa beans are used to extract peptides as disclosed herein. In another embodiment, any type of cocoa tissue / organ is extracted to obtain the peptides of the present invention. It should be understood that cocoa pods and / or cocoa shells, leaves, stems and roots can be used as plant materials for extracting the peptides of the present invention without departing from the scope of the present invention. In another embodiment, the peptides disclosed herein can be extracted or precipitated from any processed cocoa material.

[0102] In one embodiment, cocoa plant material is collected from selected cocoa growing farms and used to isolate peptides as described herein. Preferably, the samples are stored at 4°C for further processing. Alternatively, the samples are stored at -80°C prior to processing.

[0103] For the extraction of peptides, unfermented, dried beans can be used. The sticky pulp of unfermented beans is usually removed, preferably completely. The seed coat is peeled off with a sterile scalpel, forceps and / or any other suitable instrument.

[0104] Beans are rich in lipids and in a preferred embodiment, the cocoa beans are defatted prior to peptide extraction. In one embodiment, ground cocoa beans can be defatted in acetone, the remaining powdered beans dried, and then extracted in an aqueous buffer. Slow addition of very cold acetone during extraction can reduce enzyme inactivation.

[0105] In one embodiment, the cocoa beans are shelled prior to peptide extraction. Compared to other parts of cocoa (such as but not limited to shell and pulp), shelled bean material shows the best peptide extraction rate.

[0106] The cocoa beans are ground into a fine powder using a suitable mill, grinder or mortar and pestle, whereby the apparatus is preferably pre-cooled in cold air, a freezer, or cooled by liquid nitrogen. The ground powder is typically stored in an environment at a temperature not exceeding 4°C, preferably not exceeding 0°C, most preferably at -20°C, for further processing.

[0107] In another embodiment, frozen plant material is used to extract the peptides described herein. All operations are performed at 0°C to 4°C (ie, in a cold room or on ice). The extraction process is performed rapidly to minimize exposure of the target protein to potentially harmful compounds and enzymes released when cells are disrupted.

[0108] In one embodiment, the cocoa beans are ground without contacting the tissue with liquid nitrogen. To ensure that the sample remains frozen, the mortar / mill and / or grinder can be placed in a shallow pool of liquid nitrogen (e.g., in the lid of a standard polystyrene box). Fresh material can be ground directly in ice-cold buffer. In one embodiment, a tissue homogenizer is used to grind unfermented fresh cocoa beans. In another embodiment, the cocoa beans are ground in a blender or mortar and pestle, and acid-washed sand is added. Foaming during mixing may be a problem, which can be controlled to a certain extent by adding a few drops of n-octanol or any other suitable organic solvent known in the art.

[0109] In one embodiment, an extraction buffer having a pH value of preferably 8 is used for extraction. The buffer maintains the stability of the isolated peptide from the perspective of pH and ionic strength. Some non-limiting examples of the buffer used include N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid (HEPES), phosphate, 2-(N-morpholine)-ethanesulfonic acid (MES), trisaminomethane (TRIS), TAE buffer (Tris base, acetic acid and EDTA), TBE (Tris base, boric acid and EDTA) or any mixture thereof in acetone, phenol or any other suitable solvent or mixture thereof. It should be understood by those skilled in the art that the selection and type of buffer is optimized according to the cocoa variety, fermented or non-fermented beans used in the extraction process. Any suitable buffer commonly used in the art can be used to extract peptides without departing from the scope of the present invention. The volume of extraction buffer used per unit weight of plant material and / or cocoa beans depends on the type of plant material and the peptide concentration. According to one embodiment of the present invention, better extraction can be achieved by using a higher ratio of buffer volume to plant material and / or cocoa bean weight (e.g., 10:1).

[0110] In one embodiment, in the extraction process of peptides described herein, the sample extract is first concentrated. This can be achieved by precipitation, suitable chromatographic techniques or any other method known in the art. In one embodiment, 10% to 20% glycerol (v / v) is added to the extraction buffer to ensure the stability of the peptide.

[0111] In one embodiment, an antioxidant is added to the extraction buffer to prevent oxidation of the peptides, maintain the reduced state of free sulfhydryls, and reduce oxidation of other components such as phenols. Certain non-limiting examples of such antioxidants include dithiothreitol (DTT), β-mercaptoethanol or ascorbic acid, and in some cases, stronger reducing agents such as sodium dithionite are also included if necessary. In another embodiment, more than one reducing agent can be used, such as, but not limited to, a combination of DTT and β-mercaptoethanol.

[0112] In one embodiment, a detergent is added to the buffer to enhance membrane disruption and solubilization of membrane proteins. Certain non-limiting examples include sodium dodecyl sulfate (e.g., 0.1 to 1.0%), Tween 80 (e.g., 0.1% to 1%), Triton X-100 (e.g., 0.1% to 1%), 3-[(3-cholamidopropyl)dimethylammonium]-2-hydroxy-1-propanesulfonate (CHAPS) or octyl glucoside, or any mixture thereof. It will be appreciated by those skilled in the art that preliminary empirical testing is required to determine the optimal combination of surfactants to maximize the concentration of vicilin (precursor) peptides obtained in the extract.

[0113] It should be understood that protective agents such as, but not limited to, polyvinylpolypyrrolidone (PVPP), soluble polyvinylpyrrolidone (PVP) and / or polyethylene glycol may be used to prevent polyphenols such as tannins from binding to the antimicrobial peptides of the present invention.

[0114] In addition, any protease inhibitor can be added to the buffer during the extraction of antimicrobial peptides to control the degradation of protein after extraction. Some non-limiting examples of the protease inhibitor are phenylmethylsulfonyl fluoride (PMSF), bovine trypsin inhibitor (also referred to as aprotinin), 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or 2,2-bipyridine, trans-epoxysuccinyl-L-leucyl-amide-(4-guanidino) butane, antipain and leupeptin, pepstatin, aspartic protease, commercially available protease inhibitors containing these inhibitor mixtures customized for plants (for example, the inhibitor provided by Roche in tablet form and the inhibitor provided by Sigma in solution form) etc.

[0115] In a particularly preferred embodiment, the extraction is performed in an alkaline environment and optionally in the presence of a protease inhibitor cocktail using Tris-HCl buffer supplemented with dithiothreitol (DTT) as an antioxidant and sodium dodecyl sulfate (SDS) as an anionic surfactant.

[0116] Extraction / incubation time is optimized according to the antimicrobial peptide concentration in the extraction mixture and the plant material, can be 15 minutes to 48 hours, preferably be not less than 30 minutes, most preferably be not less than 1 hour, and preferably be no more than 5 hours, most preferably be no more than 3 hours.The extraction temperature is preferably no more than 40 ℃, preferably no more than room temperature (23 ℃), most preferably completes at a temperature no more than 15 ℃.

[0117] In one embodiment, the peptides disclosed herein can be isolated from cocoa beans. The cocoa beans can be fermented or non-fermented. In another or other embodiment, they can be dried and / or non-dried cocoa beans. In a preferred embodiment, the peptides described herein can be isolated from unfermented dry cocoa beans. In one embodiment, the cocoa beans are crushed, the shells are removed, and the broken pieces are then placed in a press and pressed. Thereby a mixture of butter and slurry is obtained. In certain embodiments, the slurry is used for peptide extraction.

[0118] In embodiments where other cocoa plant parts are used to extract peptides, various protective agents (such as buffers, antioxidants, protease inhibitors, etc.) are used to stabilize the proteins in the extract. The amount of extraction solvent is kept to a reasonable minimum to avoid diluting the protein extract. Cocoa fruit is acidic and high in polyphenols, requiring high buffer concentrations to maintain pH.

[0119] Preferably, the protein extract obtained according to the extraction step of the present invention is separated by sedimentation, centrifugation, filtration or any other commonly used technique to obtain a protein extract free of residual plant material. Cloth (such as but not limited to Miracloth, etc.) can be used for filtration. In another embodiment, a nylon mesh filter is preferably used because it is very strong and can be cleaned and reused.

[0120] In one embodiment, the filtrate is run through a large flash chromatography column. In this way, various low molecular weight polyphenols that bind to proteins can be separated from the proteins during large-scale production.

[0121] For preparation of small tissue samples for crude analysis, extracts can be desalted using small gel filtration columns, such as, but not limited to, PD-10 or NAP columns, Amersham Biosciences, etc.

[0122] In another embodiment, the peptides described herein are isolated from cocoa beans by isoelectric precipitation. Typically, such precipitation is performed once unwanted portions (e.g., carbohydrates, fats, and / or oils) have been removed by any suitable method known in the art. This step concentrates the proteins in the remaining portion of the plant material.

[0123] According to the invention, the fraction is dissolved in a buffer at a specific pH value and the pH is lowered to the isoelectric point of the proteins so that they can precipitate. In a preferred embodiment, the proteins from the extract are precipitated using an ammonium sulfate solution, preferably an ammonium sulfate solution with a concentration of at least 10% by weight and at most 20% by weight, most preferably an ammonium sulfate solution with a concentration of 12% to 14% by weight. The proteins are collected from the protein precipitate by centrifugation.

[0124] In other preferred embodiments, the precipitated / extracted proteins are further separated by electrophoresis. In particularly preferred embodiments, the electrophoretic separation is a two-dimensional electrophoresis. The electrophoresis is preferably performed in a polyacrylamide gel. In one embodiment, the electrophoresis is performed in a 15% SDS-PAGE gel. Electrophoretic techniques allow for a high degree of purification of peptides, eliminating the labor-intensive and costly disadvantages of non-electrophoretic techniques. The vertical field "separates" the proteins according to their mobility in the field, which is primarily based on the molecular weight of the protein.

[0125] In other embodiments, separation is followed by electroelution or the like, whereby the peptides are attracted to the surface of the gel, thereby enabling extraction and subsequent analysis of the peptides. In one embodiment, bands corresponding to the peptides are excised from the electrophoresis gel, washed in Tris-EDTA, extracted by sonication and centrifugation.

[0126] In other preferred embodiments, the peptide of interest is dried by freeze drying or vacuum centrifugation. Alternatively, the peptide can be precipitated with a suitable reagent. The freeze dried and / or precipitated peptide can be selectively resuspended in distilled water and / or any other suitable solvent and used for further analysis.

[0127] In another embodiment, the extracted or precipitated peptides are concentrated. Preferably, the peptides are concentrated using standard salt or organic solvent precipitation methods. Alternatively, the peptides are concentrated by dialysis against a volatile buffer (e.g., ammonium carbonate), by using a filter-based concentrator, or by ion exchange chromatography. It will be appreciated by those skilled in the art that concentration of the extract / precipitate is a slow and critical process, so special care should be taken not to expose the peptides to polyphenols and proteases during these processes.

[0128] The quantification of peptide content is performed by spectrophotometry (such as the Lowry method or Bradford assay) or any other method known in the art. In another embodiment, the quantification of peptides is performed by any suitable chromatographic method known in the art. In a preferred embodiment, the LC-MS method is used to quantify the peptide content. In a preferred embodiment, the fragments produced by the protein obtained from the cocoa extract by trypsin digestion are subjected to MALDI-TOF analysis. In one embodiment, the data obtained from the MALDI-MS trypsin digestion analysis are further confirmed by preparing and performing immunochemical analysis to detect specific differences between samples.

[0129] In other preferred embodiments, the peptide is substantially free of other peptides. As used herein, the term "substantially free" means at least 95% by weight free of other peptides; preferably at least 99% by weight free of other peptides. In a preferred embodiment, the peptide is present in a crystalline and / or solid form.

[0130] In another aspect, the present invention relates to a vector comprising a coding sequence for a peptide having at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or differing from the sequence according to SEQ ID N°1 or SEQ ID N°2 by at most 5 amino acids, and optionally comprising a sequence encoding an N-terminal signal peptide having 95% sequence identity to SEQ ID N°3 or being a sequence according to SEQ ID N°3, wherein the vector is designed to allow the expression of the peptide in an expression system. In a preferred embodiment, the vector comprises a coding sequence for a peptide having 96%, more preferably 97%, more preferably 98%, more preferably 99% or more preferably 100% identity to SEQ ID N°1 or SEQ ID N°2. In one embodiment, the vector comprises a purification tag as disclosed in the previous embodiment.

[0131] In one embodiment, DNA extracted from cocoa beans is used to amplify the coding sequence of the peptide and cloned into a suitable expression vector, such as any plasmid or virus designed for gene expression in cells.

[0132] Therefore, the peptides described herein can be expressed in any suitable system for the purpose of producing peptides for further use. Suitable hosts for expressing the protein include Escherichia coli, fungal cells, insect cells, mammalian cells and plants. Standard methods for expressing proteins in these hosts are described in a variety of texts, including Section 16 (Protein Expression) of Current Protocols in Molecular Biology (above).

[0133] Typically, the DNA encoding the amino acid sequence of interest is contained in an expression vector, and in some cases, is linked to another coding sequence in frame at the 5' or 3' end, thereby encoding a peptide having at least 95% sequence identity with SEQ ID N°1 or SEQ ID N°2, and optionally comprising SEQ ID N°3 at the N-terminus. The total coding sequence is operably linked to a promoter so that the promoter drives the expression of the coding sequence. The coding sequence is also referred to herein as a "target gene".

[0134] According to one embodiment, the promoter is a promoter natural to the microorganism (e.g., E. coli trpE promoter), a synthetic promoter such as a Tac promoter, or a promoter obtainable from a heterologous organism, such as a virus, a bacterium, or a bacteriophage, such as a bacteriophage lambda or T7 that can function in a microorganism. The promoter can be constitutive, or more preferably inducible. The expression vector can also contain a selective marker gene, which can be an antibiotic resistance gene, such as an ampicillin, tetracycline, chloramphenicol, or kanamycin resistance gene.

[0135] There are many promoter systems available, usually from commercial sources, that are suitable for expressing the peptides of the invention in E. coli. For example, P of the araBAD (arabinose) operon BAD The promoter has good induction properties, with an induction rate of 1,200-fold above background (Guzman et al., 1995). Other promoters include P LAC , P TAC , P TRC , PL and PR. TAC The promoter is a hybrid of the E. coli trp and lac promoters and is one of the most efficient E. coli-based promoter systems known. It is inducible by IPTG, like the lac promoter.

[0136] The preferred host cell of the present invention is the E. coli M15 host strain. In another preferred embodiment, Pichia pastoris is used as a host cell for expressing the peptides of the present invention.

[0137] In other preferred embodiments, selection of clones is done and the overexpressed peptide is extracted via its affinity tag using Ni-NTA agarose.The recombinant peptide is isolated and purified using the same methods as the peptide isolated from natural sources in the previous embodiments.

[0138] Expression of the novel peptide in genetically engineered cells will generally produce a product having the same three-dimensional structure as the native peptide isolated from the cocoa plant material. The three-dimensional structure includes correctly formed intramolecular disulfide bonds between cysteine ​​residues. However, even if the protein is chemically synthesized, methods are known in the art for further processing of the protein to break undesirable disulfide bonds and form bridges between desired cysteine ​​residues to obtain the desired three-dimensional structure, if this is necessary.

[0139] In another embodiment of the present invention, the peptide can be obtained by chemical synthesis using known peptide synthesis techniques, such as solid phase synthesis on a room temperature peptide synthesizer, a microwave peptide synthesizer, a parallel peptide synthesizer, or the like.

[0140] In one embodiment of the present invention, the peptides as described herein may be expressed in transgenic plants. Therefore, the present invention also relates to transgenic plants expressing one or more peptides as described herein.

[0141] In one embodiment, the transgenic plant expresses a peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or that differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids. In other embodiments, the peptide comprises the sequence of an N-terminal signal peptide, the signal peptide sequence having 95% sequence identity to SEQ ID N°3.

[0142] In other embodiments, the transgenic plant expresses a peptide that is 96%, more preferably 97%, more preferably 98%, more preferably 99% or more preferably 100% identical to SEQ ID N°1 or SEQ ID N°2.

[0143] In one embodiment, the peptide comprises a purification tag as disclosed in the previous embodiment.

[0144] According to the present invention, plant cells can be transformed with DNA constructs according to a variety of known genetic engineering methods (Agrobacterium tumefaciens transformation, Ti plasmid, electroporation, microinjection, microgun, PEG-mediated transformation, etc.). As used herein, genetic engineering methods can be understood as any method for introducing foreign DNA sequences into plant cells to regenerate transgenic plants expressing desired traits or characteristics.

[0145] In one embodiment of the invention, a DNA sequence encoding a peptide having at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 and optionally comprising SEQ ID N°3 at the N-terminus (), as well as DNA encoding homologs from other plant species, can be used in combination with a DNA sequence encoding a precursor protein that produces a mature protein. The precursor protein contains a natural peptide sequence that targets the protein to a specific cell compartment (e.g., apoplast or vacuole). These coding sequences can be linked to a plant promoter sequence to ensure strong expression in plant cells. The promoter sequence can ensure strong constitutive expression of the protein in most or all plant cells, it can be a promoter that ensures expression in specific tissues or cells susceptible to microbial infection, and it can also be a promoter that ensures strong induction of expression during infection. These types of gene cassettes will also include transcription termination and polyadenylation sequences 3' of the antimicrobial protein coding region to ensure efficient production and stability of the mRNA encoding the antimicrobial protein. By incorporating their respective DNA sequences into a sequence encoding a larger peptide, the effective expression of the antimicrobial peptides disclosed herein may be promoted.

[0146] The gene cassette encoding the peptide is expressed in plant cells using methods known in the art. First, the gene cassette is connected to a binary vector carrying the following: i) the left border sequence and the right border sequence of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens; ii) a suitable selection marker gene for selecting antibiotic-resistant plant cells; iii) a replication origin that is functional in both Agrobacterium tumefaciens and Escherichia coli; and iv) an antibiotic resistance gene that allows selection of Agrobacterium tumefaciens and Escherichia coli cells carrying plasmids. Without wishing to be limited, the DNA sequence of the peptide disclosed herein can be cloned into any binary vector known in the art, such as plasmids (pEXA128, pBR322, pUC19), phages (λ phage, M13 phage), cosmids, BACs or YACs. The binary vector carrying the DNA sequence of the peptide can be introduced into the Agrobacterium tumefaciens strain (such as strain LBA4404, GV3101 and AGL1) or Agrobacterium rhizogenes strain (such as A4 or NCCP1885) carrying the disarmed Ti plasmid by heat shock, electroporation or triparental mating. These Agrobacterium strains can then be co-cultured with suitable plant explants or intact plant tissues, and the transformed plant cells and / or regenerated bodies can be selected by antibiotic resistance. As an alternative, the binary vector carrying the DNA sequence of the peptide can be overexpressed in bacteria or fungal cells.

[0147] The present disclosure also relates to a method for obtaining a transgenic plant, which expresses a peptide that exhibits at least 95% sequence identity with SEQ ID N°1 or SEQ ID N°2 or that differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids, and optionally comprises a sequence encoding an N-terminal signal peptide, said signal peptide sequence having 95% sequence identity with SEQ ID N°3.

[0148] The method comprises the following steps:

[0149] (a) optionally isolating or synthesizing a nucleic acid sequence encoding a peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or that differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids;

[0150] and incorporating the nucleic acid sequence into a suitable expression vector;

[0151] (b) introducing the nucleic acid into at least one plant cell by genetic engineering methods to produce a transformed plant cell expressing the peptide;

[0152] (c) regenerating the transformed plant cells into intact transgenic plants; and

[0153] (d) selecting and identifying transgenic plants expressing the peptide using a suitable screening method.

[0154] In one embodiment of the methods disclosed herein, the nucleic acid sequence encoding the peptide is isolated using any method known in the art (such as a gene cloning method). Non-limiting examples of gene cloning include PCR-based cloning and restriction enzyme-based cloning.

[0155] In another embodiment of the method disclosed herein, the DNA sequence of the peptide is introduced into the plant cell by gene gun bombardment. Gene gun bombardment involves the use of a gene gun to introduce foreign DNA sequences into plant cells. The DNA sequence encoding the antimicrobial peptide disclosed herein is attached to metal particles such as gold particles and then bombarded into the plant cell.

[0156] In another embodiment, the DNA sequence of the peptide is introduced into the plant cell by electroporation, which involves the use of electric pulses to produce pores on the cell membrane, thereby making the DNA sequence encoding the antimicrobial peptide enter the cell. As an alternative, the DNA sequence encoding the antimicrobial peptide can be directly injected into the plant cell (microinjection).

[0157] In another alternative embodiment, the DNA sequence encoding the peptide is introduced into the plant cell by PEG-mediated transformation, which involves the use of polyethylene glycol (PEG) to create temporary pores in the cell membrane, thereby allowing the DNA sequence to enter the cell.

[0158] Any plant organ, tissue or explant can be genetically engineered to obtain the transgenic plants disclosed herein. Non-limiting examples include: single cells, callus, seeds, leaf explants, stem explants, root explants, embryonic tissue, meristem or pollen.

[0159] In general, the specific genetic engineering methods and explants used to obtain transgenic plants expressing the peptides disclosed herein depend on factors such as the plant species, the efficiency of the transformation method, and the desired level of antimicrobial peptide expression.

[0160] In other embodiments of the methods disclosed herein, the transformed cells are regenerated into whole plants expressing the peptide.

[0161] Identify and select transgenic plants expressing the peptide. This step is critical to eliminate non-transformed plants. Antibiotic or herbicide resistance selection is one of the most commonly used selection methods for transgenic plants. Transgenic plants are transformed with genes that confer resistance to antibiotics or herbicides and then grown in a medium containing the antibiotics or herbicides, which would kill or inhibit the growth of non-transformed plants. In certain embodiments, transgenic plants are transformed with a gene encoding a reporter protein, which can then be easily identified and selected based on the expression of the reporter gene.

[0162] In other embodiments, transgenic plants are selected by nutritional selection. This method is based on the ability of transgenic plants to grow in a medium lacking specific nutrients necessary for the growth of non-transformed plants. Transgenic plants are transformed with genes that give the plant the ability to synthesize the missing nutrients.

[0163] Marker-free selection involves the use of selectable markers that can be removed from transgenic plants after selection. This can be achieved by using site-specific recombination systems, such as the Cre-lox system, which allows the removal of selectable marker genes without leaving any traces of foreign DNA.

[0164] The present invention is further described by the following non-limiting examples, which further illustrate the present invention but are not intended to, nor should they be construed to, limit the scope of the present invention.

[0165] Example

[0166] Example 1: Peptide extraction, purification and identification

[0167] Cocoa bean samples derived from the cocoa hybrid CCN-51 were collected from a small, 30-year-old traditional cocoa plantation in Ecuador. Samples were provided by Barry Callebaut AG in non-fermented and fermented / dried form. Non-fermented samples were shipped on dry ice and stored at -80°C upon arrival, while fermented dry samples were shipped and stored at 4°C for further processing. The sticky pulp was wiped off the unfermented beans and the seed coat was peeled off with a sterile scalpel. The seed coat of the fermented beans was removed with tweezers. The beans were ground using a Retsch Grindomix GM200 knife grinder at 10,000 rpm. The ground powder was used directly (or stored at -20°C for further processing).

[0168] 250 mg of finely ground powder was transferred to a 2 ml polypropylene microcentrifuge tube and suspended in 1 ml of protein extraction buffer (100 mM Tris HCl, 1% DTT and 1% SDS; pH adjusted to 8.1; protease inhibitor cocktail according to the manufacturer's instructions). The mixture was thoroughly vortexed for 60 seconds and incubated at 4°C with gentle shaking for 60 minutes, then centrifuged at 13,200 rpm for 20 minutes at 4°C in a benchtop 5810R centrifuge. The supernatant containing the protein fraction was gently transferred to a new cup using a pipette and stored at -20°C for further analysis.

[0169] The extracted cocoa protein content was estimated using the method disclosed by Bradford MM in "Analytical Biochemistry" 1976;72:248-54.

[0170] According to the Bradford method, 300 μl of Bradford reagent was added to 10 μl of diluted protein solution in a microtiter plate and vortexed briefly. After incubation for 10 min at room temperature, the absorbance was measured at 595 nm in triplicate. The extracted protein concentration was quantified by a standard curve using bovine serum albumin in a concentration range of 1–1500 μg / ml.

[0171] The extracted cocoa proteins and / or peptides were separated according to molecular weight using SDS-PAGE. Briefly, 25 μl of protein sample was mixed with 5 μl of 6X sample buffer containing bromophenol blue as a tracking dye. The mixture was heated at 95°C for 5 minutes and then loaded onto an SDS-PAGE gel (83 mm × 65 mm × 1 mm) containing 12.5% ​​or 15% (w / v) acrylamide. Electrophoresis was performed at 130 V for 90 minutes. After electrophoresis, the gel was washed with Blue (45% (v / v) methanol, 10% acetic acid, 2.93 x 10 -3 M Brilliant Blue G-250) staining for 20 minutes, or blotted onto a PVDF membrane by electrophoresis, as previously described by Towbin in Proc. Natl. Acad. Sci. USA, 1979, 76, 4350-4354. The blot was treated with 5% (w / v) milk in PBST at 4°C overnight. Subsequently, the protein blot was probed with a polyclonal antibody diluted 1:2,000 in PBST containing 5% (w / v) milk powder and incubated for 2 hours (h) at room temperature. In addition, the blot was washed 6 times, each time gently shaking at room temperature for 5 minutes. Subsequently, the blot was incubated for 1 hour at room temperature with an HRP-conjugated goat anti-rabbit IgG antibody, which was diluted 1:10,000 in PBST containing 5% (w / v) milk powder. After incubation, the blot was washed 6 times with PBST, each time gently shaking at room temperature for 5 minutes. Finally, indirectly bound HRP was detected by enzymatic activity using luminol as substrate in the presence of hydrogen peroxide as described by Mruk and Cheng in Spermatogenesis, 2011, 1, 121-122.

[0172] Before two-dimensional electrophoresis, total protein extracts were purified using the Ready repTM 2D Clean-up Kit. In order to remove interfering substances such as ionic detergents, lipids or phenolic compounds, the total protein extracts were purified using this kit according to the manufacturer's recommendations. After precipitation and washing in the wash buffer provided by the kit, the protein samples were resuspended in 130 μL of rehydration buffer (2 M thiourea, 6 M urea, 16.2 x 10 -3 M CHAPS, 25.9x 10 -3M DTT) and supplemented with ampholytes.

[0173] Cocoa proteins were subjected to 2D gel electrophoresis using isoelectric focusing and subsequent SDS-PAGE. For this purpose, 80 μg of protein were applied to immobilized pH gradient (IPG) strips (7 cm, pH 3-10) and soaked for 14 h at room temperature. Isoelectric focusing was performed in an i12TM IEF electrophoresis tank (50 V, 70 min; 150 V, 20 min; 300 V, 15 min; gradient to 600 V, 10 min; 600 V, 15 min; gradient to 1,500 V, 10 min; 1,500 V, 30 min; gradient to 3,000 V, 20 min; 3,000 V, 210 min; pause at 50 V). Next, the IPG strips were centrifuged at room temperature at 6.48 x 10 -2 The equilibration was carried out for 15 min in a 0.216 M iodoacetamide solution dissolved in an equilibration buffer (6 M urea, 30% (w / v) glycerol, 69.2 x 10 -3 MSDS was dissolved in 0.05 M Tris-HCl buffer, pH 8.8. The samples were separated by 12.5% ​​or 15% polyacrylamide gels on a Bio-Rad Molecular weight separation was performed on a Tetra system (110 V, 10 min; continued at 130 V). The molecular weight of the proteins was estimated by visual mobility in the polyacrylamide gel and the predicted amino acid sequence weight. After staining and destaining, the resulting 2D gel was scanned with an Epson scanner.

[0174] Scanned 2D gels were processed by ImageMaster 2D Platinum software 6.0 (GE Healthcare). During this process, the parameters for automatic spot detection were set for all samples as follows: smoothness = 6, significance = 100, and minimum area = 5. During manual spot selection, detected non-protein spots (i.e., contamination artifacts including streaks and dust particles) were removed. By default, spot volumes are background-subtracted from the spot by excluding the lowest tenth percentile of pixel values ​​on the spot border from all other pixel values ​​within the spot border. To match sets of 2D gels, a reference sample (called master gel) was defined, which allowed identification of the same protein spots in different gels. The resulting matching report lists the matching spots in the individual 2D gels with the corresponding spots in the master gel and the respective normalized relative spot volumes.

[0175] Protein spots of interest were cut from the 2D gel, cut into small pieces, and washed twice for 15 minutes in 100 μL 0.05M ammonium bicarbonate buffer (containing 50% ACN (v / v)). The gel fragments were dehydrated by adding 500 μL acetonitrile and incubated at room temperature for 10 minutes. After decantation and short air drying, the samples were supplemented with trypsin digestion buffer and incubated at 55°C for 30 minutes according to the previously determined method. The samples were directly used for matrix-assisted laser desorption ionization-TOF mass spectrometry (MALDI-TOF-MS) analysis.

[0176] For spectral identification of peptide patterns, peptides were reduced with 15 mM TCEP or used in non-reduced form. Peptides were loaded onto a Proshell 300SB-C8 column (1 mm x 75 mm, 5 μl particles) equipped with a narrow-pore guard column Zorbax 300SB-C8 (2.1 mm x 12.5 mm, 5 μm particles). Proteins were eluted from 95% solvent A (0.1% formic acid (FA), 0.05% trifluoroacetic acid (TFA) in water) to 90% solvent B (0.1% FA, 0.05% TFA in acetonitrile) by a 5-minute linear gradient, followed by a 3-minute wash with 90% solvent B, and after 2 minutes, returned to 5% solvent B and equilibrated for 2.5 minutes. The mass spectrometer was operated in MS mode with a resolution of 60,000 (m / z 400) and a mass range of 600 to 4000 m / z. The AGC setting used was 1E7, allowing the trap to be filled in 1 second. The source settings were SID of 15 V, capillary temperature of 325 °C, and voltage of 4.2 kV.

[0177] The recorded spectra were deconvoluted using the Xtract TM algorithm in Freestyle software (Thermo fisher scientific, USA).

[0178] Results: The peptides disclosed herein were identified in the total protein fraction isolated from beans of the CCN-51 cocoa variety. After 2D separation of proteins isolated from cocoa beans, the spot corresponding to 15 kDa was excised and digested with trypsin ( Figure 1 The digested peptides were identified using MALDI-TOF-MS analysis ( Figure 2 A) The recombinant peptide of SEQ ID No 2 was separated by SDS polyacrylamide gel electrophoresis, excised from the gel, and digested with trypsin to obtain Figure 2 MALDI-TOF-MS peptide fingerprint shown in B. The mass detected using intact mass fingerprinting was 15.3 kDa in both the reduced and non-reduced samples containing the peptide according to SEQ ID N° 2. The LC-MS results for the sample according to SEQ ID N° 2 are shown in FIG3 .

[0179] Example 2: Recombinant peptide production

[0180] Total DNA was extracted from CCN-51 cocoa bean embryos and the nucleotide sequence encoding the peptide was amplified using primers forward_BamH1-5'CGCGGATCCTATGGCAGAAAACAATAT3' and reverse_Kpn1-5'CGCGGTACCTTTGTGATTATGGTAATT3' This produced an amplified fragment of 300 bp DNA (target DNA).

[0181] The amplified DNA fragment was ligated to the BamH1 / Kpn1 site of the cloning vector pEXA128. Both the plasmid carrying the peptide DNA and the pQE30 overexpression plasmid were transformed into E. coli DH5α cells for amplification. The plasmids were extracted from the overnight culture, digested with BamHI and KpnI for 2 hours at 37°C, and then separated on a 1% agarose gel. The linearized pQE30 plasmid and the insert fragment from the pEXA128 plasmid were extracted from the gel and the insert fragment was ligated into the pQE30 plasmid. The pQE30 overexpression plasmid encodes a HIS tag, and overexpression of the peptide of the present invention will produce a fusion peptide consisting of the peptide and an N-terminal HIS tag. The resulting plasmid was transformed into E. coli M15 overexpression cells. The plasmid extracted from the overnight culture was sequenced, and only clones matching the expected sequence were used for overexpression. 16 ml of the overnight culture was inoculated into 800 ml of liquid LB medium at 37°C. Once the OD 600 When it reached between 0.6 and 0.8, IPTG was added to a final concentration of 1 mM to induce overexpression. Cells overexpressing peptides containing signal peptides were incubated at 20°C for 24 hours, and cells overexpressing peptides without signal peptides were incubated at 37°C for 5 hours.

[0182] The recombinant peptides were purified and identified as described in Example 1.

[0183] Example 3: Synthesis of peptides

[0184] Peptides with the same sequence as SEQ ID N°1 and SEQ ID N°2 were chemically synthesized by Seramun Diagnostica GmbH (Heidesee, Germany).

[0185] Example 4: Minimum Inhibitory Concentration (MIC) Determination

[0186] MIC assays determine the lowest concentration at which a compound inhibits the growth of a microbial population. The antimicrobial potential of the peptides described herein was compared with the activity of two other AMPs (TcAMP1 and TcAMP2) discussed in WO1998027805, as well as antibiotics or fungicides. Comparative MIC assays were performed against several bacteria and fungi (Table 1).

[0187] The peptides according to SEQ ID N°1, SEQ ID N°2, SEQ ID N°4, SEQ ID N°6 and SEQ ID N°7 were serially diluted to create a concentration sequence ranging from 0.39 μg / ml to 303 μg / ml.

[0188] Pantoea, Staphylococcus, and Listeria were cultured in Tryptic Soy Broth (TSY) medium consisting of 1.7% casein peptone, 0.3% soy peptone, 0.25% glucose, 0.5% NaCl, 0.25% K2HPO4, and optionally 1.5% agar. Candida cells were cultured in Yeast Extract Peptone Dextrose (YPD) broth medium consisting of 1% yeast extract, 2% peptone, 2% glucose, and optionally 2% agar. Ceratocystis, Botrytis, Leptosphaeria, Mycosphaerella, Sclerotinia, and Verticillium were cultured in Potato Dextrose Agar (PDA) medium consisting of 2.4% Difco premix broth and optionally 1.5% agar.

[0189] For MIC determination, bacteria and fungi were grown on agar plates with the respective culture medium. Precultures were prepared by inoculating a single bacterial colony or a small piece of fungus in 5 ml of the respective liquid medium. The optical density (OD) at 600 nm was measured after one to five days and the OD 600 Adjust to 1. This cell suspension is used to prepare a 1:500 dilution, equivalent to approximately 2x10 6 microbial cells / ml.

[0190] In order to assess MIC, 96 well plates were prepared with the test antimicrobial peptides. Each well contained 100 μl microbial cell cultures, 90 μl culture medium and 10 μl corresponding dilutions of peptide or control. The plates were incubated overnight (ON) (Table 1) at the optimal growth temperature of every kind of tested organism. Visual assessment results were determined according to growth or no growth of MIC.

[0191] Table 1. Organisms for which a range of antimicrobial peptides have been tested in the MIC assay. Species name, classification and growth conditions are given. DSM refers to the ordering number of the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). SF refers to the organism obtained from the Jena Microbial Resources Collection (JMRC, Jena, Germany).

[0192]

[0193] MIC determinations were performed in at least two independent replicates using different peptide starting concentrations. Preliminary tests showed that any steps to purify the tested proteins did not affect the MICs obtained.

[0194] Results: All peptides of the present invention showed strong antimicrobial activity against all tested organisms.

[0195] Table 2 shows the MIC values ​​obtained against fungal species for the peptides according to SEQ ID N°1, SEQ ID N°2, SEQ ID N°4, SEQ ID N°6 and SEQ ID N°7. All peptides of the present invention inhibited fungal growth more effectively than the peptides of the prior art at lower concentrations.

[0196] Table 2. Results of MIC assays using fungal species as test organisms and peptides of the invention (SEQ ID N°1, N°2, N°4) and prior art peptides (SEQ ID N°6 and N°7). Clotrimazole was used as reference.

[0197]

[0198] Table 3 shows the MIC values ​​obtained against bacterial species for the peptides according to SEQ ID N°1, SEQ ID N°2, SEQ ID N°4, SEQ ID N°6 and SEQ ID N°7. All peptides of the invention inhibited bacterial growth, and in all tested species, the peptides according to SEQ N°1, N°2 and N°4 were more effective than the peptides of the prior art.

[0199] Table 3. Results of MIC assays using bacterial species as test organisms and peptides of the invention (SEQ ID N°1, N°2 and N°4) and prior art peptides (SEQ ID N°6 and N°7). Gentamycin and streptomycin were used as references.

[0200]

[0201] Example 5: Stability of peptides in body fluids

[0202] Sheep blood assay

[0203] In vitro experiments were performed to test the behavior of the peptides disclosed herein against Candida albicans in blood. Candida albicans isolates from human blood (DSMZ 6569) were used. The peptide according to SEQ ID N°1 was adjusted to a concentration of 1 mg / ml and mixed with an equal amount of sheep blood. This mixture was incubated overnight at 30°C. In order to remove the solid part of the blood, the mixture was centrifuged and the plasma supernatant obtained was used for MIC.

[0204] Urine test

[0205] In vitro experiments were performed to test the behavior of the peptides disclosed herein against Candida albicans in urine. The peptide according to SEQ ID N°1 was adjusted to a concentration of 1 mg / ml and mixed with an equal amount of synthetic urine or human urine. These mixtures were incubated overnight at 30°C. To remove the solid part of the urine, the mixture was centrifuged and the resulting supernatant was used for MIC.

[0206] Milk assay

[0207] In vitro experiments were performed to test the behavior of the peptides disclosed herein against Listeria monocytogenes in 3.5% fat bovine milk. The peptide according to SEQ ID N°1 was adjusted to a concentration of 1 mg / ml and mixed with an equal amount of milk. This mixture was incubated overnight at 37°C. To remove the solid part of the emulsion, the mixture was centrifuged and the resulting supernatant was used for MIC.

[0208] Results: When the peptides were mixed with sheep blood, the ability to inhibit the growth of Candida albicans cells decreased. However, the peptides described herein were still active at 25 μg / ml in sheep blood (Table 4).

[0209] Mixing the peptide with urine had little effect on the ability to inhibit the growth of Candida albicans cells. Effective antimicrobial activity was achieved at 2 μg / ml of the peptide in synthetic urine and at 5 μg / ml of the peptide in human urine, compared to 0.4 μg / ml under control conditions (Table 4).

[0210] Similarly, when the peptides were mixed with milk, the concentration that inhibited the growth of L. monocytogenes increased from 3.2 μg / ml in the absence of milk to 80 μg / ml (Table 4).

[0211] The peptides did not completely lose their ability to inhibit bacterial and yeast growth in body fluids, indicating that they are suitable for therapeutic use.

[0212] Table 4. MIC determination results of the peptide of the present invention (SEQ ID N° 1) when mixed with sheep blood, urine or cow milk. The control was peptide without body fluid, and caspofungin and tetracycline were used as references.

[0213]

[0214] Example 6: MIC determination in soil slurry

[0215] In vitro experiments were performed to test the behavior of the peptides disclosed herein against Erwinia amylovora in soil slurries. Soil slurries were generated by mixing 5 g of soil with 20 ml of water. The mixture was incubated on an overhead shaker for 1 hour. The solid portion was removed by centrifugation, and the supernatant obtained (referred to as soil slurry) was used for MIC determination. An equal volume of SEQ ID N°1 peptide (8 mg / ml) and soil slurry were mixed and incubated overnight at 28°C. The mixture was used for MIC testing of Erwinia amylovora.

[0216] Results: The peptides described herein inhibited bacterial growth at a concentration of 25 μg / ml in soil slurries compared to 15 μg / ml in control conditions. Since they do not completely lose their ability to inhibit bacterial growth in soil, the peptides described herein are suitable for agricultural use.

[0217] Example 7: Suitability of peptides on technical surfaces.

[0218] Experiments were performed to test the behavior of the peptides disclosed herein against Candida albicans on technical surfaces.

[0219] Biofilm formation of this organism was tested on ceramic (pore size 1100 μm), stainless steel and silicon surfaces in biofilm reactors. Four discs were placed into each reactor and tightened, each containing three discs from each surface. The system was autoclaved after all discs were installed. Each reactor was filled with 297 ml of SU medium and 3 ml of a preculture of Candida albicans (vaginal isolate) in SU (OD 1.04) was added. 600 =0.1). The biofilm reactor was incubated at 30°C and 200 rpm stirring. A total of three bioreactor systems were run: a control system, a system treated with peptide-rich cocoa extract from day 0, and a system treated with the extract from day 3. The final total protein concentration of the cocoa extract was 100 μg / mL.

[0220] On days 1, 3, 5, and 7, one disk was removed from each reactor. The disk was unscrewed and immersed in 2 mL of SU. These liquids were diluted in SU medium and 10 -1 -10 -5 The dilutions were plated onto YPD solid medium and cultured at 30°C for 48 hours. The colonies formed on the plates were counted and the colony forming units (CFU) / cm 2 plate.

[0221] Results: No growth of Candida albicans was observed on any of the surfaces tested. These results demonstrate that the peptides disclosed herein are highly stable and can be effectively used as surface disinfectants or detergents.

[0222] Experiment 8: In vitro experiments in yeast and mammalian cells

[0223] To investigate whether the growth inhibitory effect of the peptides was bacteriostatic or microbicidal, lactate dehydrogenase (LDH) release assays were performed on Candida albicans (vaginal isolate) as the model organism of choice and on human keratinocytes (HaCaT). LDH is an enzyme present in a variety of cell types. Cell membrane damage results in the release of LDH into the cell culture medium, which can subsequently be detected and quantified by a coupled enzyme colorimetric reaction.

[0224] HaCaT cells were grown in DMEM medium supplemented with 4.5 g / L glucose, 2 mM L-glutamine, and 10% fetal calf serum (FCS) at 37°C and 8% CO2. The cells were placed in a 75 cm 2 Cells are grown in culture flasks. The medium is changed twice a week. Once more than 90% confluence is reached, cells are passaged at a ratio of 1:10. The passage solution consists of a 1:1 mixture of EDTA (0.05% stock) and trypsin (0.1% stock) in PBS without Ca 2+ and Mg 2+ .

[0225] LDH Cytotoxicity Assay CyQUANT TM (Invitrogen, No. C20300 and C20301) was carried out according to the protocol provided by the company. According to the standard protocol described in the kit, the optimal inoculation density of HaCaT or Candida albicans cells was determined to be 10,000 cells / 100 μL. The cells were exposed to the peptide of SEQ ID N°2 for 1 h or overnight, with a concentration range of 1.95 μg / ml to 250 μg / ml, and LDH release was detected. Cytotoxicity was calculated using the following formula provided by the company: Cytotoxicity % = [(peptide-treated LDH activity)-(spontaneous LDH activity) (maximum LDH activity)-(spontaneous LDH activity)] * 100, spontaneous LDH activity was measured in untreated (water added) samples, and maximum LDH activity was measured using the lysing agent provided by the kit supplier.

[0226] Results: The cell death rate of the target organism Candida albicans detected was linear from 0 to 250 μg / ml, with cytotoxicity reaching 92% at the highest tested protein concentration ( Figure 4In contrast, when HaCaT cells were treated with the peptide, LDH release was hardly induced. The cytotoxicity of the peptide to HaCaT cells was 0% to 1% at a peptide concentration of 125 μg / ml and reached 6% at a peptide concentration of 250 μg / ml ( Figure 4 ).

[0227] It is proposed that the present invention is not limited to any of the previously described implementations, and that certain modifications may be added to the manufacturing examples presented without re-evaluating the appended claims. For example, the preferred embodiment of the present invention is directed to the CCN-51 variety of the cocoa tree, but any cocoa variety that produces sufficient amounts of vicilin for further processing may be used without departing from the scope of the present invention.

[0228] Example 9: Extraction of peptides from unfermented cocoa beans

[0229] The peptides disclosed herein are isolated from unfermented but fully dried cocoa beans. The cocoa beans are mechanically crushed using a bean crusher to obtain a mixture of crushed grains and shells. The shells are removed and the shelled crushed grains are transferred to a press. A mixture of butter and slurry is thus obtained, which is centrifuged at 40°C at a speed of 16,600xg for 10 minutes. The butter fraction collected at the top is discarded, and the solid slurry particles are crushed and stored at 4°C until further use.

[0230] The peptides were then extracted in 3 ways.

[0231] Method 1: PEB (Protein Extraction Buffer) Extraction

[0232] The crushed slurry particles were preheated in a 50°C water bath for 10 minutes and then dissolved in protein extraction buffer (100 mM Tris, 1% SDS and 1% DTT, pH 8.1) and added at a particle to buffer weight ratio of 1:4. The dissolved slurry particles were incubated with stirring at room temperature for 1 hour and then centrifuged at 16,600 x g for 20 minutes. The peptide-containing liquid phase was collected and purified by dialysis or ultrafiltration (Centricon Plus-70, Merck Chemicals GmbH, a subsidiary of Merck KGaA, Darmstadt, Germany) to remove PEB components.

[0233] Method 2: PEB extraction followed by acid precipitation

[0234] The crushed slurry particles were treated as described in method 1, that is, the crushed slurry particles were preheated in a 50°C water bath for 10 minutes and then dissolved in PEB. The dissolved slurry precipitate was incubated with stirring at room temperature for 1 hour and then centrifuged at 16,600xg for 20 minutes. The liquid phase containing the peptides was collected and acid precipitated to enrich the peptides of the present invention in the protein mixture according to their isoelectric points. Therefore, the pH value of the protein extract was reduced to 6.0 with 1M citric acid, resulting in a color change from dark gray to red. The mixture was then centrifuged at 16,600xg for 10 minutes. The supernatant was discarded and the protein particles were resuspended in PEB using ultrasound to fully dissolve the particles. Subsequently, harmful substances were removed by 5kDa MWCO ultrafiltration (CentriconPlus-70, Merck Chemicals GmbH, a subsidiary of Merck KGaA, Darmstadt, Germany).

[0235] Method 3: Hot Extraction

[0236] The crushed slurry particles were heated to 60°C in tap water with a weight volume ratio of particles to water of 1:4 and stirred for 10 minutes. The obtained mixture was centrifuged at 16,600 x g for 20 minutes at 10°C.

[0237] The peptide-containing supernatant obtained after extraction by any of methods 1 to 3 is collected and quantified or analyzed by SDS-PAGE and MIC. Before use, the protein extract is lyophilized or vacuum centrifuged to increase the concentration.

[0238] Sequence Listing

[0239] SEQ ID N°1 (peptide according to one embodiment of the present invention)

[0240] YGRKQYERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKN

[0241] SEQ ID N°2 (peptide according to one embodiment of the present invention)

[0242] YGRKQYERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHK

[0243] SEQ ID N°3 (signal peptide according to one embodiment of the present invention)

[0244] MVISKSPFIVLIFSLLLSFALLCSGVSA

[0245] SEQ ID N°4 (peptide according to one embodiment of the present invention)

[0246] MVISKSPFIVLIFSLLLSFALLCSGVSAYGRKQYERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKN

[0247] SEQ ID N°5 (peptide according to one embodiment of the present invention)

[0248] MVISKSPFIVLIFSLLLSFALLCSGVSAYGRKQYERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHK

[0249] SEQ ID N°6 (prior art peptide)

[0250] YERDPRQQYEQCQRRCSEATEEREQEQCEQRCEREYKEQQRQQEEE

[0251] SEQ ID N°7 (prior art peptide)

[0252] LQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKNRSEEEEGQQR

[0253] SEQ ID N°8 (Forward_BamH1 forward primer)

[0254] CGCGGATCCTATGGCAGAAAACAATAT

[0255] SEQ ID N°9 (Reverse_Kpn1 forward primer)

[0256] CGCGGTACCTTTGTGATTATGGTAATT

[0257] SEQ ID N°10 (HQ tag)

[0258] HQHQHQ

[0259] SEQ ID N°11 (HN tag)

[0260] HNHNHNHNHNHNH

[0261] SEQ ID N°12 (HAT tag)

[0262] KDHLIHNVHKEEHAHAHNK

[0263] SEQ ID N°13 (ALFA tag)

[0264] SRLEEELRRRLTE

[0265] SEQ ID N°14 (Avi tag)

[0266] GLNDIFEAQKIEWHE

[0267] SEQ ID N°15 (C-tag)

[0268] EPEA

[0269] SEQ ID N°16 (Calmodulin-Tag)

[0270] KRRWKKNFIAVSAANRFKKISSSGAL

[0271] SEQ ID N°17 (E-tag)

[0272] GAPVPYPDPLEPR

[0273] SEQ ID N°18 (FLAG-tag)

[0274] DYKDDDDK

[0275] SEQ ID N°19 (HA-tag)

[0276] YPYDVPDYA

[0277] SEQ ID N°20 (Myc-tag)

[0278] EQKLISEEDL

[0279] SEQ ID N°21 (NE-tag)

[0280] TKENPRSNQEESYDDNES

[0281] SEQ ID N°22 (Rho1D4 tag)

[0282] TETSQVAPA

[0283] SEQ ID N°23 (S-tag)

[0284] KETAAAKFERQHMDS

[0285] SEQ ID N°24 (SBP tag)

[0286] DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP

[0287] SEQ ID N°25 (Streptomycin-tag)

[0288] WSHQ

Claims

1. A peptide, wherein Said peptide has an amino acid sequence according to SEQ ID N°1, or exhibits at least 98% sequence identity with SEQ ID N°2, or differs from the sequence according to SEQ ID N°2 by at most 2 amino acids.

2. The peptide according to claim 1, wherein The peptide is the peptide according to SEQ ID N°1 or SEQ ID N°2.

3. The peptide according to claim 1 or 2, wherein The peptide comprises a signal peptide at its N-terminus.

4. A peptide as claimed in any one of claims 1 to 3, wherein The signal peptide has at least 95% sequence identity to SEQ ID N°3 or has a sequence according to SEQ ID N°3.

5. A peptide as claimed in any one of claims 1 to 4, wherein The peptides have antibacterial and / or antifungal activity.

6. The peptide according to claim 5, wherein The activity is against Gram-positive and Gram-negative bacteria and / or fungi, preferably hyphal fungi and yeasts.

7. A peptide as claimed in any one of the preceding claims, wherein The peptide is derived from or isolated from Theobromacacao, preferably the cocoa variety CCN-51.

8. A peptide as claimed in any one of the preceding claims, wherein The peptide is a recombinant peptide or a synthetic peptide.

9. A peptide as claimed in any one of the preceding claims, wherein The peptide is provided with an affinity tag.

10. A composition comprising a peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or that differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids, and an excipient.

11. The composition according to claim 10, wherein The peptide comprises a signal peptide having at least 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

12. The composition according to any one of claims 10 or 11, wherein The composition is in liquid, semi-solid, solid or gaseous form, and / or wherein The composition is in the form of tablets, capsules, powders, granules, aerosols, pastes, syrups, suspensions, emulsions or solutions.

13. A peptide exhibiting at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or differing from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids or a composition as claimed in any one of claims 10 to 12 for therapeutic use.

14. The peptide or composition for therapeutic use according to claim 13, wherein The peptide comprises a signal peptide having at least 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

15. A peptide that exhibits at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or that differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids or a composition as claimed in claims 10 to 12 for use in treating bacterial and / or fungal infections in a subject in need thereof.

16. The peptide or composition for use according to claim 15, wherein: The peptide comprises a signal peptide having 95% sequence identity to SEQ ID N°3 or has a sequence according to SEQ ID N°3.

17. The peptide or composition for use according to claims 15 to 16, wherein: The subject is a human or an animal.

18. Use of a peptide showing at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or differing from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids or a composition according to claims 10 to 11 for crop protection, as a feed or food additive, preservative, as a detergent or in cosmetic applications.

19. The use according to claim 18, wherein The peptide comprises a signal peptide having 95% sequence identity to SEQ ID N°3 or having a sequence according to SEQ ID N°3.

20. The use according to any one of claims 18 to 19, wherein The peptide or composition is used for controlling pathogens in plants, preferably crops.

21. The use according to claim 20, wherein The pathogen is selected from the group of Gram-positive bacteria, Gram-negative bacteria and / or fungi.

22. A method for obtaining a peptide which exhibits at least 95% sequence identity with SEQ ID N°1 or SEQ ID N°2 or which differs from the sequence according to SEQ ID N°1 or SEQ ID N°2 by a maximum of 5 amino acids, wherein: The peptide is a recombinant peptide, a synthetic peptide, or is extracted or precipitated from cocoa.

23. The method of claim 22, wherein: The peptides are extracted from cocoa bean slurry.

24. A vector comprising a coding sequence for a peptide having at least 95% sequence identity to SEQ ID N°1 or SEQ ID N°2 or differing from the sequence according to SEQ ID N°1 or SEQ ID N°2 by at most 5 amino acids, and optionally comprising an N-terminal signal peptide sequence having 95% sequence identity to SEQ ID N°3, wherein The vector is designed to allow expression of the peptide in an expression system.

25. A transgenic plant expressing the peptide of any one of claims 1 to 9.

26. A method for producing the transgenic plant of claim 25, wherein: The method comprises the steps of introducing a nucleic acid encoding the peptide into a plant cell, thereby producing a transformed plant cell expressing the peptide, and regenerating the transformed plant cell into a transgenic plant.

27. A plant seed coated with the peptide or composition of any one of claims 1 to 12.

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