Peptides having antibacterial activity

Abstract

The present invention is directed to a peptide. The peptide has an amino acid sequence that exhibits at least 95% sequence identity to SEQ ID NO.1 or SEQ ID NO.2. The present invention also discloses a composition comprising the peptide and specific uses.

Description

Technical Field

[0001] The present invention relates to antibacterial peptides that exhibit antibacterial activity against various bacterial strains and fungal strains, and more particularly to the technical field of peptides derived from natural plants. In particular, the present invention relates to natural peptides, recombinant peptides, or synthetic peptides derived from cacao plant vicilin-like proteins. The present invention further relates to the use of peptides for the treatment of bacterial and fungal infections in plants, animals, and humans.

Background Art

[0002] Antibiotic-resistant bacteria are a major problem faced by the food industry, agricultural industry, medical industry, veterinary industry, etc. In particular, there is concern about the potential transfer of potentially lethal antibiotic-resistant bacteria from food animals to human consumers.

[0003] Currently, methods for controlling the generation and spread of antibiotic-resistant bacteria include changing the amount and form of use of antibiotics, strengthening government monitoring and regulation, and continuously developing new or improved antibiotics. However, most bacteria are capable of adapting to the use of antibiotics and acquiring resistance to existing and new antibiotics, so in many cases, they exceed such conventional means. Therefore, it is necessary to continuously develop alternative means for controlling antibiotic resistance in bacteria.

[0004] Antibacterial peptides (AMPs) are a diverse group of natural compounds present in animals, plants, insects, and microorganisms. These peptides are responsible for defense against (other) microorganisms and can therefore be further utilized as alternatives to chemical preservatives. However, due to their poor availability in nature, high production costs, low stability, and complex formulation into suitable therapeutic products, their use in the treatment of microbial spread in plants, humans, and / or animals has been limited to date. Therefore, the use of antibacterial peptides in crop protection and for therapeutic purposes in human and veterinary medicine is also still limited.

[0005] Antimicrobial proteins exhibit various three-dimensional structures, and most of the activity and stability of antimicrobial proteins are determined by such three-dimensional structures. The stability of a specific protein is usually an important parameter in clinical applications and / or industrial applications.

[0006] The first interest in plant-derived molecules that are AMPs followed the isolation of the first plant-derived AMP, thionin. According to Caleya et al., Appl. Microbiol, 1972, 23(5) 998-100, thionin, a low-molecular-weight protein in wheat and barley flour, has been reported to be an AMP effective against many phytopathogenic bacteria such as Pseudomonas solanacearum, Xanthomonas phaseoli, X. campestris, Erwinia amylovora, and five Corynebacterium strains. Since then, several major groups of AMPs have been discovered, such as thionins (types I-V), defensins, cyclotides, 2S albumin-like proteins, lipid transfer proteins, etc.

[0007] Plant vicilins are generally present as a type of plant seed storage protein. It has been found that a certain vicilin can be processed to produce plant defense peptides. The most characteristic antimicrobial peptide produced from vicilin is from Macadamia integrifolia. The kernel of Macadamia nuts contains a 666aa vicilin protein, which includes a 212aa highly hydrophilic region close to the N-terminal signal sequence.

[0008] WO 1998 / 027805 discloses a family of penicillin-type peptides having antibacterial properties. The protein prototype is of natural origin, isolated from Macadamia integrifolia and also from other species including Theobroma cacao. In particular, two sequences designated as 47-amino acid TcAMP1 (Theobroma cacao antimicrobial protein 1) and 60-amino acid TcAMP2 (Theobroma cacao antimicrobial protein 2) are derived from the cocoa vicilin seed storage protein gene sequence encoding 556 amino acids (aa) and recombinantly expressed in E. coli. However, the isolated recombinant peptides and the compositions prepared with the recombinant peptides had effective antibacterial properties only at high doses (5 - 20 μg / ml).

[0009] Marcus et al. in Plant. Mol. Biol Rep (2008) 26, 75 - 87 investigated three Macadamia integrifolia peptide sequences and two Theobroma cacao peptide sequences that were identified as the N-proximal hydrophilic regions of vicilin seed proteins. The antibacterial activity of the peptides was predicted based on the presence of a C-X-X-X-C-(10 - 12)-C-X-X-X-C motif characteristic of the hydrophilic region proximal to the N-terminus. The His-tagged versions of the putative peptides were expressed in E. coli. The resulting recombinant peptides showed antibacterial activity against six plant pathogenic strains in vitro, but the effective amounts were very high.

[0010] Ecuador is the most important producer of fine flavor cocoa, accounting for approximately 50% of the world's production. The types of fine flavor cocoa produced in Ecuador mainly belong to the Nacional (often also called National or Arriba) variety. In Ecuador, in addition to the highly fragrant and traditional Nacional cocoa, the cocoa clone CCN-51 has been cultivated since the 1960s. Different from Nacional, CCN-51 has a weak aroma and is thus classified as a bulk cocoa type. However, CCN-51 is highly resistant to changes in climate conditions, has resistance to various pathogenic bacteria, and has a higher yield than other cocoa varieties. Therefore, CCN-51 hybrids are very popular among farmers in Ecuador. U.S. Patent No. 2004 / 0172683 describes polypeptides obtained from cocoa bean seeds that are factors contributing to the cocoa flavor.

[0011] A vicilin-derived peptide used in the production of cocoa and chocolate as a cocoa flavor enhancer is known from WO 2002 / 086125 A2.

[0012] The present invention thus describes an unknown peptide derived from cocoa that is highly effective and highly active against pathogenic microorganisms of a wide range of animals and plants. This peptide can be used safely and preferably does not contain corrosive chemicals that are toxic and potentially harmful to the environment. SUMMARY OF THE INVENTION

[0013] The present invention and its various embodiments contribute to providing solutions to one or more of the above-mentioned demerits. For this purpose, the present invention relates to a peptide having antibacterial activity as described in claim 1. More specifically, it provides a peptide showing 95% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2. This peptide is obtained from Theobroma cacao or produced recombinantly or synthetically, and surprisingly, it has been found to have antibacterial activity and / or antifungal activity against a wide range of pathogens. Preferred embodiments of this peptide are shown in any of claims 2 to 9.

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

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

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

[0017] In a fifth aspect, the present invention relates to a vector as described in claim 24 containing the coding sequence of the peptide enabling the expression of the peptide, a transgenic plant as described in claim 25, and a method for obtaining the transgenic plant as described in claim 26.

[0018] In the last aspect, the present invention relates to a seed coated with the peptide as described in claim 27. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

[0020] The present invention relates to a peptide or a composition containing the peptide as an active ingredient for medical or pharmaceutical use. In the present invention, the peptide has an amino acid sequence according to SEQ ID NO.1 or SEQ ID NO.2. Furthermore, the present invention relates to the use of the peptide, a method for obtaining the peptide, and a vector enabling the expression of the peptide.

[0021] Unless otherwise defined, all terms used in the description of the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the technical field to which the present invention belongs. As a further guide, definitions of terms are included to better understand the teachings of the present invention.

[0022] As used herein, the following terms have the following meanings.

[0023] As used herein, "a", "an", and "the" refer to the singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0024] As used herein, "about" is used when referring to measurable values such as parameters, amounts, durations, etc., and includes variations of no more than ±20%, preferably no more than ±10%, more preferably no more than ±5%, still more preferably no more than ±1%, and even more preferably no more than ±0.1% of the specified value, as appropriate for the practice of the present invention. It should be understood, however, that the value itself to which the modifier "about" refers is also specifically disclosed.

[0025] As used herein, "comprise", "comprising", and "comprises", as well as "comprised of", are synonymous with "include", "including", "includes", or "contain", "containing", "contains", and are inclusive or open-ended terms that identify the presence of what follows the component, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps known in the art or disclosed in the art.

[0026] Furthermore, in this specification and the claims, terms such as first, second, third, etc. are used to distinguish similar elements, unless otherwise specified, and are not necessarily used to describe a continuous or chronological order. When used in this way, the terms can be used interchangeably under appropriate circumstances, and it should be understood that the embodiments of the invention described herein can operate in an order other than that described or illustrated herein.

[0027] The description of a numerical range by endpoints includes all the numerical values and fractions included in that range, as well as the described endpoints.

[0028] The expressions "weight % (% by weight)", "weight percent", "% wt", or "wt%" refer to the relative weight of each component based on the total weight of the formulation throughout this specification, unless otherwise defined.

[0029] The term "one or more" or "at least one", for example, one or more elements in a group of elements, or at least one element, is clear in itself, and by way of further illustration, this term includes, inter alia, any one of the said elements, or any two or more references to the said elements, for example, any three or more, four or more, five or more, six or more, seven or more, etc. of the said elements, up to all of the said elements.

[0030] As used herein, the term "peptide" refers to a compound containing two or more amino acid residues linked by an amide bond formed between the carboxyl group derived from one amino acid residue and the amino group derived from an adjacent amino acid residue. The amino acid residues may be in the D-form or the L-form, may be naturally occurring or synthetic, and may be linear or cyclic.

[0031] The term "therapeutically effective amount" refers to an amount effective to improve any symptom of a disease. Since prevention can be considered a form of treatment, a therapeutically effective amount can also be referred to as a "prophylactically effective amount".

[0032] As used herein, the cocoa bean “slurry” is defined as the ground cocoa nib mass after separation of the cocoa butter.

[0033] Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. As a further guide, definitions of terms used herein are included to better understand the teachings of the present invention. The terms or definitions used herein are provided only to assist in understanding the present invention.

[0034] Throughout this specification, references to “one embodiment” or “an embodiment” mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, although they may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to one of ordinary skill in the art from this disclosure. Additionally, some embodiments described herein include some features of other embodiments and not others, but as will be understood by one of ordinary skill in the art, combinations of features of different embodiments are within the scope of the invention and are meant to form different embodiments. For example, in the following claims, any of the claimed embodiments may be used in any combination.

[0035] In a first aspect, the present invention relates to a peptide, which has an amino acid sequence showing 80% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2. Preferably, the peptide has an amino acid sequence showing 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or more preferably 100% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2. In a preferred embodiment, the peptide has an amino acid sequence showing 95% or more identity with SEQ ID NO.1 or SEQ ID NO.2.

[0036] As used herein, the term "sequence identity" refers to the identity of the amino acid sequences for each amino acid over a window of comparison. Thus, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the amino acids in both sequences are identical, dividing the number of identical positions by the total number of positions in the window of comparison (i.e., the size of the region), and multiplying the result by 100 to obtain the percentage of sequence identity. Gaps, i.e., positions in the alignment where a residue is present in one sequence but not in the other, are considered positions of non-identical residues. The determination of the percentage of sequence identity can be done manually or by using computer programs available in the art. An example of a useful algorithm is PILEUP. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0037] In another embodiment, the peptide has an amino acid sequence that differs from the sequence of SEQ ID NO.1 or SEQ ID NO.2 by at most 24, more preferably at most 23, even more preferably at most 22, 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, or even more preferably at most 1 amino acid residue.

[0038] In one embodiment, the peptide has an amino acid sequence according to SEQ ID NO.1, or an amino acid sequence that exhibits at least 98% sequence identity with SEQ ID NO.2 or an amino acid sequence that differs from SEQ ID NO.2 by at most 2 amino acids.

[0039] Alternatively, the peptide of the present invention has sequence identity with SEQ ID NO.1 or SEQ ID NO.2.

[0040] Amino acid sequence variants of the peptides contemplated herein can be substitution variants, insertion variants, or deletion variants. Deletion variants lack one or more residues in the peptide that may not be important for function. Substitution variants generally contain replacement amino acids at one or more sites within the peptide and can be designed to modulate one or more properties of the polypeptide, such as stability to proteolytic cleavage. The substitutions are preferably conservative substitutions, i.e., one amino acid is replaced with an amino acid having a similar size and a similar side chain or similar substituent. Conservative substitutions are known in the art and include, for example, the change from alanine to glycine, valine, or leucine; the change from arginine to lysine; the change from asparagine to glutamine; the change from cysteine to methionine; the change from glutamine to asparagine; the change from glutamic acid to aspartic acid; the change from glycine to proline; the change from histidine to glutamine, tyrosine, arginine, lysine, asparagine, or cysteine; the change from isoleucine to leucine or valine; the change from leucine to valine or isoleucine; the change from lysine to arginine; the change from phenylalanine to tyrosine, leucine, or methionine; the change from serine to threonine; the change from threonine to serine; the change from tryptophan to phenylalanine; the change from tyrosine to tryptophan or phenylalanine; the change from valine to isoleucine or leucine, etc.

[0041] Preferably, the peptide of the present invention is derived from Theobroma cacao or isolated from Theobroma cacao. More specifically, the peptide of the present invention is derived from the N-terminal region of the vicilin protein of Theobroma cacao. Vicilin is a storage protein present in several plant species and characterized by the plant species, such as Ananas comosus, Arachis hypogaea, Beta vulgaris, Capsicum annuum, Capsicum chinense, Carya illinoinensis, Chenopodium quinoa, Corchorus olitorius, Cucurbita maxima, Fragaria vesca, Glycine max, Gossipum arboreum, Herrania umbratica, Hordeum vulgare, Jatropha curcas, Juglans regia, Macadamia integrifolia, Macleaya cordata, Musa acuminata, Papaver somniferum, Ricinus communis, Solanum lycopersicum, Spinacia oleracea, Stenocarpus sinuatus, Theobroma cacao, Zea mays, etc., but not limited thereto. Such proteins typically contain highly hydrophilic N-proximal and C-terminal regions. Furthermore, they have 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) in an equidistant pattern.

[0042] In a further embodiment, the peptide described herein is derived from the Theobroma cacao variety CCN-51. Surprisingly, the cacao variety CCN-51 is known to have high resistance to microbial invasion, and it has been shown that the vicilin-derived N-terminal peptide is particularly abundant.

[0043] In another, or further, embodiment, the peptide comprises a signal peptide at its N-terminus. Preferably, the signal peptide has the sequence according to SEQ ID NO.3. In another embodiment, the signal peptide exhibits a sequence identity of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or more preferably 100% with respect to SEQ ID NO.3. Alternatively, the peptide has a signal peptide sequence that differs from SEQ ID NO.3 by a maximum of 3, more preferably a maximum of 2, and even more preferably 1 amino acid residue. The bisilinthan protein isolated from natural sources typically does not have the signal peptide. Surprisingly, it has been found that adding a signal peptide to the peptide enhances its antibacterial activity.

[0044] In another embodiment, the peptide is a recombinant peptide or a synthetic peptide. Inserting a vector containing the coding sequence of the peptide having SEQ ID NO.1 or SEQ ID NO.2 into a suitable expression system, such as Escherichia coli or another suitable expression system known in the art, yields the peptide. Any suitable protein / peptide synthesis method known in the art can be employed to synthesize the peptide of the present invention.

[0045] In a further, or alternative, embodiment, the peptides described herein are provided with affinity tags at their N-terminus and / or C-terminus. Affinity tags are used for the affinity purification of recombinant proteins and peptides expressed in Escherichia coli and other systems. The tag can be a polyhistidine tag, which is an amino acid motif consisting of at least six histidine (His) residues.

[0046] In one embodiment, the peptides containing a signal peptide disclosed herein have a sequence identity according to SEQ ID NO.4 or SEQ ID NO.5.

[0047] Alternatively, but not limited to, the peptide may be tagged with, for example, an HQ tag (HQHQHQ) in which histidine and glutamine residues alternate, an HN tag (HNHNHNHNHNHN) in which histidine and asparagine alternate, 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, an E tag (GAPVPYPDPLEPR), a FLAG tag (DYKDDDDK), an HA tag (YPYDVPDYA), a Myc tag (EQKLISEEDL), an NE tag (TKENPRSNQEESYDDNES), a Rho1D4 tag (TETSQVAPA), an S tag, an SBP tag (DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), a Strep tag (WSHPQFEK), etc.

[0048] As a result of analyzing the peptide of the present invention, it was found that this peptide has antibacterial activity and / or antifungal activity.

[0049] The antimicrobial peptide itself has a specific three-dimensional structure, which can be determined using X-ray crystallographic analysis technology or nuclear magnetic resonance spectroscopy technology. Although not wishing to be bound by theory, this structure is thought to play a role in the activity observed in the peptide. The α-helix type plant-derived antimicrobial peptides (AMPs) to which the peptides of the present invention belong often have amphipathic helices where one side of the helix is mainly hydrophilic and the other side is mainly hydrophobic. This structure is commonly seen in AMPs that disrupt cell membranes, causing leakage and lysis of cell contents, as well as AMPs that enter cells and attack other intracellular structures. α-Helix AMPs are usually structurally disordered in solution, which allows them to easily pass through the dense network of the cell wall. When binding to cell membranes that are hydrophobic and charged below the lipid heads, a secondary structure (α-helix) is formed, enabling the protein to penetrate the cell membrane. This order / disorder transition is mainly controlled by the length of the hydrophobic region within the α-helix. If the hydrophobic helix is too long, an ordered structure is formed in solution, which has an adverse effect on the activity of the AMP.

[0050] In certain embodiments, the peptide is active against Gram-positive and Gram-negative bacteria and fungi, preferably filamentous fungi and yeasts. Without limitation, the peptide is active against bacteria selected from the group including, but not limited to, 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., 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., Xylella spp., or Xylophilus spp., etc.

[0051] Although not limiting, the peptides of the present invention are active against fungi selected from the group comprising: 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., 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., 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., Torulaspora spp., Uromyces spp., Ustilago spp., Venturia spp., or Verticillium spp., etc.

[0052] In a second aspect, the present invention provides a composition comprising the peptide described in the above paragraph. This composition is particularly suitable for pharmaceutical and veterinary uses, crop protection uses, and / or cosmetic uses.

[0053] In certain embodiments, the composition comprises a peptide that exhibits at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or that differs from the sequence of SEQ ID NO.1 or SEQ ID NO.2 by at most five amino acids, and an excipient.

[0054] In some embodiments of the compositions described herein, the peptide comprises a signal peptide having at least 95% sequence identity with SEQ ID NO.3 or has the sequence of SEQ ID NO.3.

[0055] In certain embodiments, the composition is a liquid, semi-solid, solid, or gaseous composition, and / or the dosage form of the composition is a tablet, capsule, powder, granule, aerosol, paste, syrup, suspension, emulsion, or solution. Non-limiting examples of the above compositions are SP (soluble powders), SG (soluble granules), wettable granules, tablet formulations, dry flowables, aqueous flowables, granule wettables, OD (Oil dispersion), suspension concentrates, DC (Dispersible concentrate), emulsions, aqueous suspensions, fertilizer granules, sprayable compositions, etc. The composition can be formulated to be suitable for oral administration, injection administration, intravenous administration, intramuscular administration, cutaneous administration, inhalation administration, topical administration, or nasal administration. In other embodiments, the composition can be formulated such that coating, spraying, spray coating, evaporation, nebulizing, atomizing, suspension, dilution, etc. of one or more objects is possible.

[0056] In some embodiments, the composition includes a pharmaceutically acceptable carrier, excipient, or diluent. Optionally, the excipient is selected from fillers, binders, disintegrants, sweeteners, coating agents, lubricants, and / or glidants. The diluent or filler can increase the volume of the solid composition and make the dosage form containing the composition easier to handle for patients and caregivers. Examples of diluents suitable for the tablets according to the present invention include, for example, crystalline cellulose (e.g., Avicel®), microcrystalline cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dicalcium phosphate dihydrate, tricalcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylate (e.g., Eudragit®), potassium chloride, powdered cellulose, sodium chloride, sorbitol, talc, etc.

[0057] The solid composition compressed into a dosage form such as a tablet may contain an excipient. The excipient includes functions that assist in containing the active ingredient and other excipients together after compression. Suitable binders include acacia, alginic acid, carbomer (e.g., carbopol), sodium carboxymethyl cellulose, dextrin, ethyl cellulose, gelatin, guar gum, hydrogenated vegetable oil, hydroxyethyl cellulose (e.g., Klucel®), hydroxypropyl cellulose (e.g., Methocel®), liquid glucose, magnesium aluminum silicate, maltodextrin, methyl cellulose, polymethacrylate, povidone (e.g., Kollidon®, Plasdone®), pregelatinized starch, sodium alginate, starch, etc.

[0058] By adding a disintegrant to the composition, the dissolution rate of the compressed solid composition can be increased. Suitable disintegrants include alginic acid, calcium carboxymethylcellulose, sodium carboxymethylcellulose (e.g., Ac-Di-Sol®, Primellose®), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g., Kollidon®, Polyplasdone®), guar gum, magnesium aluminum silicate, methylcellulose, crystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g., Explotab®), starch, etc.

[0059] By adding a lubricant, the fluidity of the non-compressed solid composition can be improved and the dosing accuracy can be enhanced. Excipients that function as lubricants include colloidal silicon dioxide, magnesium trisilicate, magnesium stearate, powdered cellulose, starch, talc, tricalcium phosphate, etc.

[0060] When a dosage form such as a tablet is produced by compressing a powder composition, the composition is subjected to pressure by punches and dies. Some excipients and active ingredients are liable to adhere to the punches and dies, which may cause holes and surface irregularities in the product. Adding a lubricant to the composition can reduce adhesion and facilitate the release of the product from the die. Lubricants include magnesium stearate, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, zinc stearate, etc. Preferably, the lubricant is present at 0.25 - 1% by weight (w / w).

[0061] Flavors and flavor enhancers make the dosage form more palatable to the patient. Common flavors and flavor enhancers for pharmaceuticals that can be included in the compositions described herein include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, tartaric acid, and the like.

[0062] The solid composition can also be dyed using any pharmaceutically acceptable colorant to improve its appearance and / or to facilitate the identification of the product and unit dosage.

[0063] In certain embodiments, particularly when the composition is for agricultural use, the excipient is an agriculturally compatible excipient. The "agriculturally compatible carrier" or "agriculturally compatible excipient" can be regarded as a solvent and is generally inert but must be acceptable in agriculture. Thus, the term "agriculturally compatible" means a substance that, even when used routinely in the cultivated land, does not interfere with the producer's planting equipment and does not adversely affect the growth of the crop or the desired ecological balance in the cultivation area.

[0064] An agricultural suitability carrier or an agricultural suitability excipient may be solid. Examples of solid carriers or excipients include, but are not limited to, clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, polymers, granular masses, perlite, perlite granules, peat, peat pellets, soil, vermiculite, charcoal, carbonation press mud from sugar factories, rice husks, carboxymethyl cellulose, fine sand, calcium carbonate, wheat flour, alum, starch, talc, polyvinyl pyrrolidone, or combinations thereof. An agricultural suitability carrier or an agricultural suitability excipient may be liquid. Examples of liquid carriers or excipients include, but are not limited to, water, alcohols, ketones, petroleum fractions, oils, aromatic hydrocarbons or paraffinic hydrocarbons, chlorinated hydrocarbons, liquefied gases, or combinations thereof. More specifically, an agricultural suitability carrier or an agricultural suitability excipient can include a dispersant, surfactant, additive, thickener, anti-caking agent, residue decomposer, composting agent, granular applicator, diatomaceous earth, colorant, stabilizer, preservative, polymer, coating, or combinations thereof. A person skilled in the art can easily determine the appropriate carrier or excipient to be used by considering factors such as specific compounds, the plants to which the inoculation material is applied, the type of soil, climate conditions, and whether the inoculation material is in liquid, solid, or powder form. Additives can include oils, gums, resins, clays, polyoxyethylene glycol, terpenes, viscous organic substances, fatty acid esters, sulfated alcohols, alkyl sulfonates, petroleum sulfonates, alcohol sulfates, sodium alkyl butanediamino acids, polyesters of sodium thiobutant dioate, benzene acetonitrile derivatives, proteinaceous substances, or combinations thereof. Examples of proteinaceous substances 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.The surfactant can include heavy petroleum oil, heavy petroleum distillate, polyol fatty acid ester, polyethoxylated fatty acid ester, arylalkyl polyoxyethylene glycol, alkylamine acetate, alkylaryl sulfonate, polyhydric alcohol, alkyl phosphate, or a combination thereof. Examples of the anti-caking agent include sodium salts such as sodium sulfite, sodium sulfate, and sodium salt of monomethylnaphthalenesulfonic acid, or a combination thereof; calcium salts such as calcium carbonate and diatomaceous earth, or a combination thereof, and the like.

[0065] In certain embodiments, the composition further comprises one or more selected from water, other nutrients, weak acids, vegetable oils, essential oils, metabolism promoters, emulsifiers, viscosity agents, colorants, suspending agents, dispersing agents, preservatives, complexing agents, stabilizers, carriers, solvents or wetting agents, or any combination thereof. In certain embodiments, the composition further comprises at least one oil, a surfactant, and a polymer.

[0066] The composition can be produced by conventional methods. In certain embodiments, the compositions provided herein include one or more preservatives that inhibit the activity of microorganisms. Suitable preservatives include mercury-containing substances such as merfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0067] The peptides described in this specification can be formulated in a composition in an amount of 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 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.

[0068] Alternatively, the peptides can be formulated in a composition in an amount of 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.

[0069] In another embodiment, the composition comprises at least 0.1% by weight of the peptide, more preferably at least 0.5% by weight, at least 1% by weight, at least 2% by weight, at least 3% by weight, at least 4% by weight, at least 5% by weight, at least 6% by weight, at least 7% by weight, at least 8% by weight, at least 9% by weight, at least 10% by weight, at least 11% by weight, at least 12% by weight, at least 13% by weight, at least 14% by weight, at least 15% by weight, at least 16% by weight, at least 17% by weight, at least 18% by weight, at least 19% by weight, at least 20% by weight, at least 21% by weight, at least 22% by weight, at least 23% by weight, at least 24% by weight, at least 25% by weight, at least 26% by weight, at least 27% by weight, at least 28% by weight, at least 29% by weight, at least 30% by weight, at least 31% by weight, at least 32% by weight, at least 33% by weight, at least 34% by weight, at least 35% by weight, at least 36% by weight, at least 37% by weight, at least 38% by weight, at least 39% by weight, at least 40% by weight, at least 41% by weight, at least 42% by weight, at least 43% by weight, at least 44% by weight, at least 45% by weight, at least 46% by weight, at least 47% by weight, at least 48% by weight, at least 49% by weight, at least 50% by weight, at least 51% by weight, at least 52% by weight, at least 53% by weight, at least 54% by weight, at least 55% by weight, at least 56% by weight, at least 57% by weight, at least 58% by weight, at least 59% by weight, at least 60% by weight, at least 61% by weight, at least 62% by weight, at least 63% by weight, at least 64% by weight, at least 65% by weight, at least 66% by weight, at least 67% by weight, at least 68% by weight, at least 69% by weight, at least 70% by weight, at least 71% by weight, at least 72% by weight, at least 73% by weight, at least 74% by weight, at least 75% by weight, at least 76% by weight, at least 77% by weight, at least 78% by weight, at least 79% by weight, at least 80% by weight, at least 81% by weight, at least 82% by weight, at least 83% by weight, at least 84% by weight, at least 85% by weight, at least 86% by weight, at least 87% by weight, at least 88% by weight, at least 89% by weight, at least 90% by weight, at least 91% by weight, at least 92% by weight, at least 93% by weight, at least 94% by weight, at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight or at least 99.5% by weight of the peptide.

[0070] In a third aspect, the peptides or compositions described herein are suitable for therapeutic use.

[0071] In one embodiment, the peptide or composition for therapeutic use comprises a peptide having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or having an amino acid sequence that differs from SEQ ID NO.1 or SEQ ID NO.2 by at most five amino acids.

[0072] In a further embodiment, the peptide comprises a signal peptide having at least 95% sequence identity with SEQ ID NO.3, or has the sequence according to SEQ ID NO.3.

[0073] As used herein, "therapeutic use" refers to the use of a peptide or a composition thereof for ameliorating the symptoms of a disease in a human or non-human animal. In practicing the therapeutic methods or methods of use provided herein, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject such as a mammal or non-mammal having a disease, disorder, or condition to be treated. In some embodiments, the subject is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents described herein, and optionally the compositions, can be used alone, or as components of a mixture, in combination with one or more therapeutic agents.

[0074] In one embodiment, the peptides described herein are used in a pharmaceutical composition for the treatment or prevention of infectious diseases caused by a wide range of microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and / or fungi, in a subject in need thereof. Here, the fungi are preferably filamentous fungi or yeasts, and the subject can be a human or an animal. The pharmaceutical composition can comprise a therapeutically effective amount of an antibacterial peptide and a suitable carrier.

[0075] In one embodiment, a peptide or composition for use in the treatment of bacterial and / or fungal infections in a subject in need thereof comprises a peptide having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or differing by a maximum of 5 amino acids from the sequence of SEQ ID NO.1 or SEQ ID NO.2.

[0076] In a further embodiment, the peptide comprises a signal peptide having at least 95% sequence identity with SEQ ID NO.3, or having the sequence of SEQ ID NO.3.

[0077] The peptides or compositions described herein can be administered to a subject by a suitable route of administration, including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, intraperitoneal routes, etc. Compositions described herein include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate release formulations, release controlled formulations, fast-dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and combinations of immediate release and release controlled formulations.

[0078] 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 can be formulated for oral, topical, or parenteral administration to treat microbial infections in veterinary medicine.

[0079] In another embodiment, the peptides or compositions described herein are suitable for use in crop protection as a feed or food additive, and for pharmaceutical use as a preservative and / or decontaminant.

[0080] In certain embodiments, the peptide or peptide composition comprises a peptide having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or a peptide having a sequence that differs from SEQ ID NO.1 or SEQ ID NO.2 by at most 5 amino acids.

[0081] In further embodiments, the peptide comprises a signal peptide having at least 95% sequence identity with SEQ ID NO.3, or has the sequence according to SEQ ID NO.3.

[0082] When used for crop protection, the peptides or compositions described herein are formulated with an agriculturally compatible carrier or excipient according to the above embodiments. Those skilled in the art will determine the appropriate dosage, dosage form, and method of application of the composition in the context of the crop to be treated and the function of the targeted pathogen.

[0083] In certain embodiments, the peptides or compositions described herein are incorporated into feed or food additives to prevent the spread of pathogens through feed and food. Feed is known to carry pathogens that can be harmful to the health and well-being of animals. Although it is widespread to use antiseptic products such as formaldehyde to disinfect feed, these products may instead have adverse effects. The antibacterial peptides described herein are a safe alternative.

[0084] In another embodiment, the peptides or compositions described herein are suitable for use as decontaminants or disinfectants for outer surfaces, tools, instruments, devices, objects, or body parts. In certain embodiments, such devices or objects include, but are not limited to, linens, fabrics, plastics, latex fabrics, natural rubbers, implanted devices, outer surfaces, or storage containers. In certain embodiments, the peptides or compositions disclosed herein are incorporated into detergents, soaps, or sprays.

[0085] In certain embodiments, the peptides described herein can also be incorporated into a variety of healthcare products, particularly cosmetics. For example, the peptides can be incorporated into toothpaste, mouthwash, shampoo, soap, cream, or antiperspirant to reduce or prevent microbial colonization or recolonization in the oral cavity or on the skin.

[0086] In another embodiment, the peptides and compositions described herein can be used as preservatives for foods, feeds, cosmetics or pharmaceuticals, or in the treatment of foods to control, reduce, or remove potential pathogens or contaminants.

[0087] In another embodiment, the peptides or compositions described herein are used to control pathogens in plants, preferably crops. Here, the pathogens are Gram-positive and Gram-negative bacteria and / or fungi, and the fungi are preferably filamentous fungi or yeasts. The plants can be monocotyledonous or dicotyledonous plants and include forage or forage legumes, ornamental plants, edible crops, trees, or shrubs. In certain embodiments, the plant is preferably a crop. Preferably, the plant is from the following groups, namely, Acer, Actinidia, Abelmoschus, Agave sisalana, Agropyron, Agrostis stolonifera, Allium, Amaranthus, Ammophila arenaria, Ananas comosus, Annona, Apium graveolens, Arachis, Artocarpus, Asparagus officinalis, Avena, Averrhoa carambola, Bambusa, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica, Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum, Carex elata, Carica papaya, Carissa macrocarpa, Carya, Carthamus tinctorius, Castanea, Ceiba pentandra, Cichorium endivia, Cinnamomum, Citrullus lanatus, Citrus, Cocos, Coffea, Colocasia esculenta, Cola, Corchorus, Coriandrum sativum, Corylus, Crataegus, Crocus sativus, Cucurbita, Cucumis, Cynara, Daucus carota, Desmodium, Dimocarpus longan, Dioscorea, Diospyros, Echinochloa, Elaeis, EleusinePearl millet, Eragrostis tef, genus Eriantus, Eriobotrya japonica, genus Eucalyptus, Eugenia uniflora, genus Fagopyrum, genus Fagus, Festuca arundinacea, Ficus carica, genus Fortunella, genus Fragaria, Ginkgo biloba, genus Glycine, Gossypium hirsutum, genus Helianthus, Hemerocallis fulva, genus Hibiscus, genus Hordeum, Ipomoea batatas, genus Juglans, Lactuca sativa, genus Lathyrus, Lens culinaris, Linum usitatissimum, Litchi chinensis, genus Lotus, Luffa acutangula, genus Lupinus, Luzula sylvatica, genus Lycopersicon, genus Macrotyloma, genus Malus, Malpighia emarginata, Mammea americana, Mangifera indica, genus Manihot, Manilkara zapota, Medicago sativa, genus Melilotus, genus Mentha, Miscanthus sinensis, genus Momordica, Morus nigra, genus Musa, genus Nicotiana, genus Olea, genus Opuntia, genus Ornithopus, genus Oryza, Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, genus Pennisetum, genus Persea, Petroselinum crispum, Phalaris arundinacea, genus Phaseolus, Phleum pratense, genus Phoenix, Phragmites australis, genus Physalis, genus Pinus, Pistacia vera, genus Pisum, genus Poa, genus Populus, genus Prosopis, genus Prunus, genus Psidium, Punica granatum, Pyrus communis, genus Quercus, Raphanus sativus, RheumIt belongs to Rheum rhabarbarum, Ribes, Ricinus communis, Rubus, Saccharum, Salix, Sambucus, Secale cereale, Sesamum, Sinapis, Solanum, Sorghum bicolor, Spinacia, Syzygium, Tagetes, Tamarindus indica, Theobroma cacao, Trifolium, Tripsacum dactyloides, Triticosecale rimpaui, Triticum, Tropaeolum minus, Tropaeolum majus, Vaccinium, Vicia, Vigna, Viola odorata, Vitis, Zea mays, Zizania palustris, Ziziphus, and includes their descendants and hybrids.

[0088] The peptides or compositions described herein are suitable for introduction into plants, parts of plants, or the substrates that constitute the plants or serve as hosts for the plants, thereby conferring pathogen resistance or disease control properties to the plants. The introduction may be artificial.

[0089] Examples of methods for introducing peptides or their compositions into plants and the like include treating plants and / or parts of plants and / or the growth medium in which the plants are grown, inoculating seeds, coating seeds, directly inoculating plants or parts of plants, spraying or moistening plants or parts of plants (e.g., ears). An appropriate method can be selected according to the type of plant into which the peptide or composition is introduced.

[0090] As another further non-limiting example, the peptides or compositions described herein can be applied in the form of a coating. The coating can be applied to seeds by spraying onto the seeds or by impregnating the seeds into a solution containing the peptide or composition. In another example, a binder, such as a binder consisting of a carbide (calcium carbonate), can be added to coat the seeds with the solution described in the previous example.

[0091] In some embodiments, the coating can be applied to exposed and untreated plant parts. In other embodiments, the coating can be applied as an overcoat to previously treated plant parts. Coating of seeds is particularly preferred in the treatment of soil-borne fungal diseases. In some embodiments, the coating of seeds can be applied to exposed and untreated seeds. In other embodiments, the coating of seeds can be applied as an overcoat to previously treated seeds.

[0092] In one embodiment, the peptides or compositions described herein can be applied to the soil or any other substrate on which the plant is grown, thereby removing pests and / or pathogens from the substrate.

[0093] Inoculation of the substrate that constitutes the plant or plant part, or the substrate that serves as the host for the plant or plant part, can be carried out, by way of example and not by way of limitation, by applying powders, granules, pellets, plugs, or soil drenches to the substrate. Inoculation can also be carried out by liquid application such as foliar spraying or liquid compositions. This application can be applied to growing plants or to plant substrates. Plants, particularly agricultural plants, can be grown in the substrate. In one embodiment, the substrate is soil, sand, gravel, polysaccharides, mulch, compost, peat moss, straw, logs, clay, or combinations thereof. In another embodiment, the substrate can also include a hydroponic system or an in vitro culture system. In some embodiments, combinations of different application methods as described herein are applied.

[0094] In certain embodiments, the peptides or compositions described herein are suitable for use in the treatment or prevention of pathogenic infections. The pathogen is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and / or fungi as described in any of the previous embodiments.

[0095] The present invention disclosed herein also relates to plant seeds coated with the peptides or compositions described in any of the previous embodiments.

[0096] In another aspect, the present invention relates to a method for obtaining the peptides disclosed herein, where the peptides are recombinant peptides, synthetic peptides, or those extracted or precipitated from Theobroma cacao.

[0097] In certain embodiments, the methods disclosed herein are used to obtain peptides having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or amino acid sequences that differ from SEQ ID NO.1 or SEQ ID NO.2 by at most five amino acids. Here, the peptides are recombinant peptides, synthetic peptides, or those extracted or precipitated from Theobroma cacao.

[0098] The variety Theobroma cacao CCN-51 has high levels of the peptides of the present invention and, thus, it has been found that the peptides of the present invention can be efficiently purified from CCN-51 cocoa beans using suitable extraction and precipitation techniques, followed by optimized chromatography techniques and other conventionally known methods. Accordingly, the present invention provides a solution for the direct valorization of CCN-51 cocoa beans, particularly non-fermented CCN-51 cocoa beans, for the isolation of peptides exhibiting antibacterial activity.

[0099] In a preferred embodiment, non-fermented and / or fermented dried cocoa beans are used to extract the peptides disclosed herein. In another embodiment, any form of Theobroma cacao tissue / organs are extracted to obtain the peptides of the present invention. It should be understood that, without departing from the scope of the present invention, cocoa pods and / or the husks, leaves, stems, and roots of cocoa beans can be used as plant material for the extraction of the peptides of the present invention. In another embodiment, the peptides disclosed herein can be extracted or precipitated from any processed Theobroma cacao material.

[0100] In one embodiment, the peptides described herein are isolated using cacao plant material collected from a selected cacao cultivation farm. Preferably, the samples are stored at 4 °C and further processed. Alternatively, the samples are stored at -80 °C prior to processing.

[0101] For peptide extraction, non-fermented, well-dried beans can be used. Usually, the mucilaginous pulp is preferably removed from the non-fermented beans by wiping. The seed coat is peeled off using a sterile scalpel, forceps, and / or other suitable instruments.

[0102] Beans are rich in lipids. In a preferred embodiment, cacao beans are defatted before peptide extraction. In one embodiment, the ground cacao beans can be defatted in acetone, the residual powdered beans can be dried, and then extracted in an aqueous buffer. By slowly adding very cold acetone during extraction, enzyme inactivation is reduced.

[0103] In certain embodiments, cacao beans are shelled before peptide extraction. The shelled bean material has the best peptide extractability compared to other parts of cacao such as shells and pulp, etc. (not limited thereto).

[0104] Cacao beans are finely ground using a suitable mill, grinder, or mortar and pestle. The device used is preferably pre-cooled by a cold atmosphere, a freezer, or cooled by liquid nitrogen. The ground powder is usually stored at a temperature not exceeding 4°C, preferably not exceeding 0°C, and most preferably -20°C for further processing.

[0105] In another embodiment, frozen plant material is used to extract the peptides described herein. All operations are carried out at 0°C to 4°C (i.e., in a refrigerator or on ice). The extraction procedure is carried out rapidly to minimize the exposure of the target protein to potentially harmful compounds and enzymes released during cell disruption.

[0106] In certain embodiments, cacao beans are ground without contacting the tissue with liquid nitrogen. To ensure the frozen state of the sample, 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). Materials that have never been frozen can be directly ground in an ice-cold buffer. In one embodiment, a tissue homogenizer is used to grind non-fermented cacao beans that have never been frozen. In another embodiment, acid-washed sand is added and the cacao beans are ground using a blender or mortar and pestle. Although foaming may be a problem during mixing, some control can be achieved by adding a few drops of n-octanol or other suitable organic solvents known in the art.

[0107] In certain embodiments, extraction is preferably performed using an extraction buffer at pH 8. The buffer maintains the stability of the isolated peptide from both the perspectives of pH and ionic strength. Non-limiting examples of buffers that can be used include N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid (HEPES), phosphate, 2-(N-morpholino)-ethanesulfonic acid (MES), trisaminomethane (TRIS), TAE buffer (tris base, acetic acid, and EDTA), TBE (tris base, boric acid, and EDTA), or mixtures thereof, including mixtures in a mixed solution with acetone, phenol, or other suitable solvents. Those skilled in the art should understand that the selection and type of buffer are optimized according to the variety of cacao and the use of fermented or unfermented beans in the extraction process. Without departing from the scope of the present invention, any suitable buffer commonly used in the art can be employed for the extraction of peptides. The amount of extraction buffer used per unit weight of plant material and / or cacao 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 increasing the ratio of the amount of buffer to the weight of the plant material and / or cacao beans (e.g., 10:1).

[0108] In certain embodiments, in the process of extracting the peptides described herein, first, the sample extract is concentrated. Concentration can be performed by precipitation, appropriate chromatography techniques, or any other method known in the art. In certain embodiments, 10% - 20% glycerol (v / v) is added to the extraction buffer to ensure the stability of the peptides.

[0109] In one embodiment, by adding an antioxidant to the extraction buffer, oxidation of the peptide is prevented, the reduced state of the free sulfhydryl groups is maintained, and oxidation of other components such as phenols is reduced. Non-limiting examples of such antioxidants include dithiothreitol (DTT), β-mercaptoethanol, ascorbic acid, and, depending on the situation, strong reducing agents such as sodium dithionite. In another embodiment, one or more reducing agents can be used, and without limitation, for example, DTT and β-mercaptoethanol can be used in combination.

[0110] In one embodiment, by adding a detergent to the buffer, disruption of the membrane and solubilization of the membrane protein are promoted. Some non-limiting examples include sodium dodecyl sulfate (e.g., 0.1 - 1.0%), Tween 80 (e.g., 0.1% - 1%), Triton X-100 (e.g., 0.1% - 1%), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS), octyl glucoside, or mixtures thereof. Those skilled in the art should understand that it is necessary to initially conduct empirical tests to determine the optimal combination of surfactants to maximize the concentration of the obtained bisilin (precursor) peptide in the extract.

[0111] Use of a protective agent such as polyvinylpolypyrrolidone (PVPP), soluble polyvinylpyrrolidone (PVP), and / or polyethylene glycol can prevent binding of polyphenols such as tannins to the antibacterial peptide of the present invention. It should be understood that the protective agent is not limited to these.

[0112] Furthermore, any protease inhibitor can be added to the buffer during the extraction of the antimicrobial peptide to control the degradation of the protein after extraction. Some non-limiting examples of protease inhibitors include phenylmethylsulfonyl fluoride (PMSF), bovine pancreatic trypsin inhibitor (also known as aprotinin), 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline-trans-epoxysuccinyl-L-leucyl-amide-(4-guanidino)butane, 2,2-bipyridyl-trans-epoxysuccinyl-L-leucyl-amide-(4-guanidino)butane, antipain and leupeptin, pepstatin, aspartic protease, commercially available protease inhibitors (e.g., those provided in tablet form by Roche, those provided as solutions by Sigma), those containing a cocktail of these inhibitors formulated for plants, and the like.

[0113] In a particularly preferred embodiment, extraction is carried out using a Tris-HCl buffer with dithiothreitol (DTT) as an antioxidant and sodium dodecyl sulfate (SDS) as an anionic surfactant, optionally in the presence of a protease inhibitor cocktail, under an alkaline environment.

[0114] The extraction / incubation time is optimized based on the extraction mixture and the concentration of antimicrobial peptides in the plant material and can be varied in the range of 15 minutes to 48 hours, preferably 30 minutes or more, most preferably 1 hour or more, more preferably 5 hours or less, and most preferably 3 hours or less. The extraction temperature preferably does not exceed 40°C, preferably does not exceed room temperature (23°C), and most preferably the extraction is carried out at a temperature not exceeding 15°C.

[0115] In certain embodiments, the peptides disclosed herein can be isolated from cocoa beans. The cocoa beans can be fermented or non-fermented. In another or further embodiment, these can be dried and / or non-dried cocoa beans. In a preferred embodiment, the peptides described herein can be isolated from non-fermented dried cocoa beans. In certain embodiments, the cocoa beans are ground, the husks are removed, and the nibs are pressed with an expeller. This yields a mixture of butter and slurry. The slurry is used for peptide extraction in some embodiments.

[0116] In embodiments where other cocoa plant parts are used for peptide extraction, a number of 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 moderate minimum to avoid dilution of the protein extract. Since the cocoa fruit is acidic and rich in polyphenols, a high concentration of buffer is required to maintain the pH.

[0117] The protein extract obtained by the extraction process according to the present invention is preferably separated using sedimentation, centrifugation, filtration, or any other technique commonly used to obtain a protein extract free from residual plant material. Filtration through a cloth can be used, although it is not limited to microcrystalline cellulose. In another embodiment, a nylon mesh filter is preferred as it is very strong and can be washed and reused.

[0118] In certain embodiments, filtration is performed through a large flash chromatography column. In this way, various low molecular weight polyphenols that bind to the protein during mass production can be separated from the protein.

[0119] For the preparation of small tissue samples for crude assays, the extract can be desalted using a small gel filtration column, examples of which include, but are not limited to, PD-10 and NAP columns manufactured by Amersham Biosciences.

[0120] In another embodiment, the peptides described herein are isolated from cocoa beans by isoelectric precipitation. Typically, this precipitation is carried out by any suitable method known in the art after removing unwanted fractions such as carbohydrates, fats, and / or oils. This step concentrates the proteins in the remaining part of the plant material.

[0121] According to the present invention, the protein can be precipitated by dissolving the portion in a buffer of a specific pH and lowering the pH to the isoelectric point of the protein. In a preferred embodiment, the protein from the extract is precipitated using an ammonium sulfate solution, preferably an ammonium sulfate solution with a concentration of 10 - 20 wt%, most preferably an ammonium sulfate solution with a concentration of 12 - 14 wt%. The protein is recovered from the protein precipitate by centrifugation.

[0122] In a more preferred embodiment, the precipitated / extracted protein is further separated by electrophoresis. In a particularly preferred embodiment, the electrophoretic separation is two-dimensional electrophoresis. Electrophoresis is preferably carried out in a polyacrylamide gel. In one embodiment, electrophoresis is carried out in a 15% SDS-PAGE gel. Electrophoresis enables the high purification of peptides and overcomes the drawbacks of labor-intensive and costly non-electrophoretic techniques. The vertical field "separates" proteins mainly by their mobility in the field based on molecular weight.

[0123] In a further embodiment, electroelution or the like is carried out after separation, whereby the peptide is attracted to the surface of the gel, enabling the extraction and subsequent analysis of the peptide. In one embodiment, the band corresponding to the peptide is cut out from the electrophoretic gel, washed with Tris-EDTA, and extracted by sonication and centrifugation.

[0124] In a further preferred embodiment, the peptide of interest is dried by lyophilization or vacuum centrifugation. Alternatively, the peptide can be precipitated with a suitable agent. The lyophilized and / or precipitated peptide is optionally resuspended in distilled water and / or other suitable solvents and used for further analysis.

[0125] In another embodiment, the extracted or precipitated peptide is concentrated. Preferably, the peptide is concentrated by standard salt or organic solvent precipitation. Alternatively, the peptide can be concentrated by dialysis against a volatile buffer (e.g., ammonium carbonate), by using a filter-based concentrator, or by ion exchange chromatography. Concentration of the extract / precipitate is an important and time-consuming process, and thus it is known to those skilled in the art that special care needs to be taken to ensure that the peptide is not exposed to polyphenols or proteases during these processes.

[0126] Quantification of the peptide content is performed by spectrophotometric methods such as the Lowry method or Bradford assay, or any other method known in the art. In another embodiment, quantification of the peptide is performed by any suitable chromatographic method known in the art. In a preferred embodiment, LC-MS is used for quantification of the peptide content. In a preferred embodiment, MALDI-TOF analysis is performed on the fragments generated via tryptic digestion of the proteins obtained from the cocoa extract. In one embodiment, an immunochemical assay can be prepared and performed to further validate the data obtained from the MALDI-MS tryptic digest analysis for detecting specific differences between samples.

[0127] In a further preferred embodiment, the peptide is substantially free from other peptides. The term "substantially free" as used herein means being at least 95 wt% free from other peptides, preferably at least 99 wt% free from other peptides. In a preferred embodiment, the peptide is present in crystalline and / or solid form.

[0128] In another aspect, the present invention relates to a vector comprising a coding sequence of a peptide having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or having an amino acid sequence that differs from SEQ ID NO.1 or SEQ ID NO.2 by at most 5 amino acids, optionally comprising the sequence of an N-terminal signal peptide, wherein the signal peptide sequence has at least 95% sequence identity with SEQ ID NO.3 or is the sequence according to SEQ ID NO.3, wherein the vector is designed to enable the expression of the peptide in an expression system. In a preferred embodiment, the vector consists of a coding sequence of a peptide having 96%, more preferably 97%, more preferably 98%, more preferably 99%, or more preferably 100% identity with SEQ ID NO.1 or SEQ ID NO.2. In certain embodiments, the vector contains a purification tag as disclosed in the previous embodiments.

[0129] In certain embodiments, DNA extracted from cacao beans is used to amplify the coding sequence of the peptide and cloned into any suitable expression vector, such as a plasmid or virus, designed for gene expression in cells.

[0130] Accordingly, the peptides described herein can be expressed in any suitable system for the purpose of producing peptides for further use. Suitable hosts for protein expression include Escherichia coli, fungal cells, insect cells, mammalian cells, and plants. Standard methods for expressing proteins in such hosts are described in various texts, including Section 16 (Protein Expression) of Current Protocols in Molecular Biology (supra).

[0131] Generally, the DNA encoding the target amino acid sequence is contained in an expression vector, and optionally, is ligated in-frame at the 5' or 3' end to another coding sequence so as to encode a peptide having 95% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2, and optionally contains SEQ ID NO.3 at the N-terminus. The entire coding sequence is operably linked to a promoter such that the promoter drives the expression of the coding sequence. As used herein, the coding sequence is also referred to as the "target gene".

[0132] According to certain embodiments, the promoter is either a promoter native to the microorganism (e.g., the E. coli trpE promoter), a synthetic promoter such as the Tac promoter, or a promoter obtained from a bacteriophage such as phage λ or T7 that can function in a heterologous organism, e.g., a virus, bacterium, or microorganism. The promoter may be constitutive or, more preferably, inducible. The expression vector may also contain a selectable marker gene, which can be an antibiotic resistance gene such as the ampicillin, tetracycline, chloramphenicol, or kanamycin resistance gene.

[0133] Many promoter systems suitable for the expression of the peptides of the invention in E. coli are well commercially available. For example, the PBAD promoter from the araBAD (arabinose) operon has advantageous inducible properties and is inducible 1,200-fold over background (Guzman et al., 1995). Additionally, P LAC , P TAC , P TRC , PL, PR, etc. are available. The P TAC promoter is a hybrid derived from the E. coli trp and lac promoters and is one of the strongest known E. coli-based promoter systems. It is inducible by IPTG similar to the lac promoter.

[0134] A preferred host cell of the present invention is the E. coli M15 host strain. In another preferred embodiment, Pichia pastoris is used as the host cell for the expression of the peptides of the present invention.

[0135] In a further preferred embodiment, clones are selected and the overexpressed peptide is extracted via its affinity tag using Ni-NTA agarose. The recombinant peptide is separated and purified using the same method as applied to the peptides isolated from natural sources in the previous embodiments.

[0136] The expression of the novel peptide in the genetically engineered cells usually results in a product having the same three-dimensional structure as the naturally derived peptide isolated from cacao plant material. This three-dimensional structure contains correctly formed intramolecular disulfide bonds between cysteine residues. However, even when the protein is chemically synthesized, methods are known in the art to further process the protein in order to cleave unwanted disulfide bonds and form the desired bonds between the desired cysteine residues to give the desired three-dimensional structure.

[0137] 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 devices such as room temperature peptide synthesizers, microwave peptide synthesizers, parallel peptide synthesizers, etc.

[0138] In one embodiment of the present invention, it is possible to express the peptides described herein in transgenic plants. Accordingly, the present invention also relates to transgenic plants expressing one or more of the peptides described herein.

[0139] In certain embodiments, the transgenic plant expresses a peptide having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or having an amino acid sequence that differs from SEQ ID NO.1 or SEQ ID NO.2 by a maximum of five amino acids. In further embodiments, the peptide comprises the sequence of an N-terminal signal peptide, and the signal peptide sequence has 95% sequence identity with SEQ ID NO.3.

[0140] In further embodiments, the transgenic plant expresses a peptide having 96%, more preferably 97%, more preferably 98%, more preferably 99%, or more preferably 100% identity with SEQ ID NO.1 or SEQ ID NO.2.

[0141] In certain embodiments, the peptide comprises a purification tag as disclosed in the previous embodiments.

[0142] According to the present invention, plant cells can be transformed with a DNA construct by various known genetic engineering methods (Agrobacterium tumefaciens transformation, Ti plasmid, electroporation, microinjection, microprojectile gun, PEG-mediated transformation, etc.). The genetic engineering methods used herein are to be understood as any method used for introducing a foreign DNA sequence into a plant cell in order to regenerate a genetically modified plant that expresses a desired trait or characteristic.

[0143] In one embodiment of the present invention, a DNA sequence encoding a peptide having 95% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2 and optionally containing SEQ ID NO.3 at the N-terminus, and a DNA encoding a homolog derived from another plant species can be used in combination with a DNA sequence encoding a preprotein that produces a mature protein. This preprotein contains a native peptide sequence and targets the protein to a specific cell compartment (e.g., apoplast or vacuole). These coding sequences can be ligated to a plant promoter sequence that ensures strong expression in plant cells. This promoter sequence can guarantee strong constitutive expression of the protein in most or all plant cells, can be a promoter that guarantees expression in specific tissues or cells that are susceptible to the effects of microbial infection, or can be a promoter that guarantees strong induction of expression during the infection process. These types of gene cassettes also contain transcription termination and polyadenylation sequences 3' of the region encoding the antibacterial protein to ensure efficient production and stabilization of the mRNA encoding the antibacterial protein. Efficient expression of the antibacterial peptides disclosed herein can be facilitated by incorporating their individual DNA sequences into sequences encoding much larger peptides.

[0144] Using methods known in the art, a gene cassette encoding a peptide is expressed in plant cells. First, the gene cassette is ligated into a binary vector having the following: i) left and right border sequences flanking the T-DNA of the Agrobacterium tumefaciens Ti plasmid, ii) an appropriate selectable marker gene for the selection of antibiotic-resistant plant cells, iii) an origin of replication functional in both Agrobacterium tumefaciens and Escherichia coli, and iv) an antibiotic resistance gene enabling the selection of plasmid-containing cells of Agrobacterium tumefaciens and Escherichia coli. Without limitation, the DNA sequences of the peptides disclosed herein can be cloned into any binary vector known in the art, such as plasmids (pEXA128, pBR322, pUC19), bacteriophages (λ phage, M13 phage), cosmids, BACs or YACs. The binary vector having the DNA sequence of the peptide can be introduced into an Agrobacterium tumefaciens strain having a disarmed Ti plasmid such as strain LBA4404, GV3101, AGL1, or an Agrobacterium rhizogenes strain such as strain A4 or NCCP1885 by any of heat shock, electroporation, or triparental mating. These Agrobacterium strains are then co-cultured with appropriate plant extracts or intact plant tissues, and transformed plant cells and / or regenerated strains can be selected using antibiotic resistance. Alternatively, the binary vector having the DNA sequence of the peptide can also be overexpressed in bacterial or fungal cells.

[0145] The present disclosure also relates to a method for obtaining a transgenic plant that expresses a peptide having an amino acid sequence that exhibits 95% or more sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or that differs from SEQ ID NO.1 or SEQ ID NO.2 by a maximum of five amino acids, optionally including the sequence of an N-terminal signal peptide, and wherein the signal peptide sequence has 95% sequence identity with SEQ ID NO.3.

[0146] This method includes the following steps: (a) Optionally, isolating or synthesizing a nucleic acid sequence encoding a peptide having at least 95% sequence identity with SEQ ID NO.1 or SEQ ID NO.2, or having a sequence that differs from SEQ ID NO.1 or SEQ ID NO.2 by a maximum of five amino acids, and incorporating the nucleic acid sequence into an appropriate expression vector; (b) Introducing the nucleic acid into at least plant cells by genetic engineering methods to produce transformed plant cells that express the peptide; (c) Regenerating the transformed plant cells into whole transgenic plants, and (d) Selecting and identifying transgenic plants that express the peptide using appropriate screening methods.

[0147] In certain embodiments of the methods disclosed herein, a nucleic acid sequence encoding a peptide is isolated using any method known in the art, such as gene cloning methods. Non-limiting examples of gene cloning include PCR-based cloning and restriction enzyme-based cloning.

[0148] In another embodiment of the methods disclosed herein, the DNA sequence of the peptide is introduced into plant cells by biolistic bombardment. Biolistic bombardment involves using a gene gun to introduce a foreign DNA sequence 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 plant cells.

[0149] In yet another embodiment, the DNA sequence of the peptide is introduced into plant cells by electroporation. Electroporation uses an electrical pulse to create pores in the cell membrane, allowing the DNA sequence encoding the antimicrobial peptide to enter the cell. Alternatively, the DNA sequence encoding the antimicrobial peptide may be directly injected into plant cells (microinjection). In yet another embodiment, a DNA sequence encoding a peptide is introduced into a plant cell by PEG-mediated transformation, which uses polyethylene glycol (PEG) to form temporary pores in the cell membrane to allow the DNA sequence to enter the cell.

[0150] To obtain the transgenic plants disclosed herein, any plant organ, tissue or extract can be used in combination with genetic engineering methods. Non-limiting examples include somatic cells, callus, seeds, leaf extracts, stem extracts, root extracts, embryonic tissue, meristematic tissue, pollen.

[0151] Generally, the specific genetic engineering methods and extracts 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 expression level of the antimicrobial peptide.

[0152] In a further embodiment of the methods disclosed herein, the transformed cells are regenerated into whole plants expressing the peptide.

[0153] The transformed plants expressing the peptide are identified and selected. This step is essential to eliminate non-transformation programs. Antibiotic resistance selection or herbicide resistance selection is one of the most commonly used selection methods for transgenic plants. Transgenic plants are transformed with a gene conferring resistance to an antibiotic or herbicide and then grown on a medium containing the antibiotic or herbicide to kill or inhibit the growth of non-transformed plants. In some embodiments, transgenic plants can be transformed with a gene encoding a reporter protein and easily identified and selected based on the expression of the reporter gene.

[0154] In other embodiments, transgenic plants are selected by nutrient selection. This method is based on the ability of transgenic plants to grow on a medium lacking specific nutrients that are essential for the growth of non-transformed plants. The transgenic plants are transformed with a gene that confers the ability to synthesize the missing nutrient.

[0155] Marker-free selection uses a selection marker that can be removed from the transgenic plant after selection. This can be achieved using a site-specific recombination system such as the Cre-lox system, which can remove the selection marker gene without leaving a trace of foreign DNA.

[0156] The present invention is further illustrated by the following non-limiting examples, which are not intended to limit the scope of the invention and should not be construed as limiting.

Example

[0157] (Example 1: Extraction, purification, and identification of peptides) Cocoa bean samples from the cocoa hybrid CCN-51 were collected from a small-scale, traditional cocoa farm in Ecuador that has been in business for over 30 years. The samples were provided by Barry Callebaut AG in non-fermented and fermented / dried forms. The non-fermented samples were transported on dry ice and stored at -80 °C upon arrival. On the other hand, the fermented-dried samples were transported at 4 °C and stored for further processing. The mucilaginous pulp of the non-fermented beans was wiped off, and the seed coats were peeled off with a sterile scalpel. The seed coats of the fermented beans were removed with forceps. The beans were finely ground at 10,000 rpm using a Retsch Grindomix GM200 knife mill. The ground powder was used as is (or stored at -20 °C for further processing).

[0158] Transfer 250 mg from the micronized powder into a 2 mL polypropylene microcentrifuge tube, and suspend it in 1 mL of protein extraction buffer (100 mM Tris HCl, 1% DTT, 1% SDS, pH adjusted to 8.1, protease inhibitor cocktail was used according to the manufacturer's instructions). Vortex the mixture thoroughly for 60 seconds, incubate for 60 minutes with gentle shaking at 4 °C, and then centrifuge at 13,200 rpm for 20 minutes at 4 °C using a tabletop 5810R centrifuge. Gently transfer the supernatant containing the protein fraction to a new cup by pipetting and store it at -20 °C for further analysis.

[0159] The content of the extracted cocoa protein was evaluated using the method by Bradford MM disclosed in "Analytical Biochemistry" 1976; 72: 248 - 54.

[0160] According to the Bradford method, 300 μl of Bradford reagent was added to 10 μl of the diluted protein solution in a microtiter plate and vortexed briefly. After incubating at room temperature for 10 minutes, the absorbance was measured three times at 595 nm. The extracted protein concentration was quantified in the concentration range of 1 - 1500 μg / ml from the standard curve using bovine serum albumin.

[0161] The extracted cocoa protein and / or peptide was separated according to the molecular weight using SDS-PAGE. That is, 25 μl of the 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 loaded onto an SDS-PAGE gel (83 mm × 65 mm × 1 mm) containing 12.5% or 15% (w / v) acrylamide. Electrophoresis was carried out at 130 V for 90 minutes. After electrophoresis, the gel was stained with Coomassie® Blue (45% (v / v) methanol, 10% acetic acid, 2.93 x 10-3 M Coomassie® Brilliant Blue G-250) for 20 minutes, or electrophoresed onto a PVDF membrane by the method previously described by Towbin et al. 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. Then, the protein blot was probed with a polyclonal antibody diluted 1:2,000 with PBST containing 5% (w / v) powdered milk and incubated at room temperature for 2 hours (h). Furthermore, the blot was washed 6 times for 5 minutes each at room temperature with gentle shaking. Then, the blot was diluted 1:10,000 with HRP-labeled goat anti-rabbit IgG antibody in PBST containing 5% (w / v) powdered milk and incubated at room temperature for 1 hour. After incubation, the blot was washed 6 times with PBST for 5 minutes each at room temperature with gentle shaking. Finally, as described by Mruk and Cheng in Spermatogenesis, 2011, 1, 121-122, indirectly bound HRP was detected by its enzyme activity using luminol as a substrate in the presence of hydrogen peroxide.

[0162] Prior to proceeding with two-dimensional electrophoresis, the total protein extract was purified using the Ready PrepTM 2D Cleanup Kit. To remove interfering substances such as ionic detergents, lipids, and phenolic compounds, the total protein extract was purified using the kit recommended by the manufacturer. After precipitation and washing with the wash buffer included in the kit, the protein sample was suspended in 130 μL of rehydration buffer (2 M thiourea, 6 M urea, 16.2 x 10-3 M CHAPS, 25.9 x 10-3 M DTT), and an amphoteric solvent was added.

[0163] Two-dimensional gel electrophoresis of cacao proteins was performed by isoelectric focusing followed by SDS-PAGE. For this purpose, 80 μg of protein was applied to an immobilized pH gradient (IPG) strip (7 cm, pH 3-10) and immersed at room temperature for 14 hours. Isoelectric electrophoresis was carried out using a Bio-Rad Protean® i12TM IEF Cell (50 V, 70 minutes; 150 V, 20 minutes; 300 V, 15 minutes; gradient to 600 V, 10 minutes; 600 V, 15 minutes; gradient to 1,500 V, 10 minutes; 1,500 V, 30 minutes; gradient to 3,000 V, 20 minutes; 3,000 V, 210 minutes; stopped temporarily at 50 V). Next, 6.48 x 10-2 M DTT and 0.216 M iodoacetamide solution were dissolved in the equilibration buffer (6 M urea, 30% (w / v) glycerol, 69.2 x 10-3 M SDS, 0.05 M Tris-HCl buffer, pH 8.8), and the IPG strip was equilibrated at room temperature for 15 minutes. Molecular weight separation was performed using a Bio-Rad Mini-Protean® Tetra System (110 V, 10 minutes, then 130 V) on a 12.5% or 15% polyacrylamide gel. The molecular weight of the protein was evaluated by visual movement in the polyacrylamide gel and the predicted weight of the amino acid sequence. After staining and destaining, the obtained 2D gel was scanned with an Epson Scanner.

[0164] The scanned 2D gels were processed using ImageMaster 2D Platinum software 6.0 software (GE Healthcare). At that time, for all samples, the automatic spot detection parameter settings were Smooth = 6, Saliency = 100, and Min Area = 5. Detected non-protein spots, i.e., contamination artifacts including streaks and dust, were removed during manual spot selection. By default, the spot volume was background-subtracted on a spot-by-spot basis, and the lowest 10 percentile pixel values on the spot boundary were excluded from all other pixel values within the spot boundary. For the matching of 2D gel sets, a reference sample called the master gel was defined so that the same protein spots could be identified between different gels. The resulting match report lists the matched spots in each individual 2D gel and the corresponding spots in the master gel, along with their normalized relative spot volumes.

[0165] The target protein spots were excised from the 2D gels, minced, and washed twice with 100 μL of 0.05 M ammonium bicarbonate buffer containing 50% ACN (v / v) for 15 minutes. 500 μL of acetonitrile was added to dehydrate the gel pieces, and they were incubated at room temperature for 10 minutes. After decantation and brief air drying, trypsin digestion buffer was added to the samples, and they were incubated at 55 °C for 30 minutes as established previously. The samples were used directly for matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis.

[0166] For spectrometric identification of peptide patterns, peptides were used either reduced with 15 mM TCEP or without reduction. Peptides were loaded onto a Proshell 300SB-C8 column (inner diameter 1 mm x 75 mm, particle size 5 μl) equipped with a narrow bore guard column Zorbax 300SB-C8 (inner diameter 2.1 mm x 12.5 mm, particle size 5 μm). Proteins were eluted from the column with a linear 5-minute gradient from 95% solvent A (0.1% formic acid (FA), 0.05% trifluoroacetic acid (TFA), water) to 90% solvent B (0.1% FA, 0.05% TFA, acetonitrile), then washed with 90% solvent B for 3 minutes, returned to 5% solvent B in 2 minutes, and equilibrated for 2.5 minutes. The mass spectrometer was operated in MS mode with a resolution of 60,000 (m / z 400) in the mass range of 600 - 4000 m / z. AGC was set to 1E7 to fill the trap in 1 second. The source settings were set to SID15V, capillary temperature 325 °C, and voltage 4.2 kV.

[0167] The recorded spectra were deconvoluted with the Xtract TM algorithm of Freestyle software (Thermo fisher scientific, USA).

[0168] (Results) The peptides disclosed herein were identified from the total protein fraction isolated from the beans of the CCN-51 cacao variety. After 2D separation of the proteins isolated from cacao beans, the spots corresponding to 15 kDa were excised and digested with trypsin (Figure 1). The digested peptides were identified by MALDI-TOF-MS) analysis (Figure 2A). The recombinant peptide of SEQ ID NO.2 was separated by SDS polyacrylamide gel electrophoresis, excised from the gel, and subjected to trypsin digestion, resulting in the MALDI-TOF-MS peptide fingerprint shown in Figure 2B. The mass of 15.3 kDa was detected using the intact mass fingerprint in both the reduced and non-reduced samples containing the peptide according to SEQ ID NO.2. The LC-MS results of the sample according to SEQ ID NO.2 are shown in Figure 3.

[0169] (Example 2: Production of Recombinant Peptide) Total DNA was extracted from the embryos of CCN-51 cacao beans, and the nucleotide sequence encoding the peptide was amplified using the primers Forward_BamH1-5’CGCGGATCCTATGGCAGAAAACAATAT3’ and Reverse_Kpn1-5’CGCGTACCTTTGTGATTATGGTAATT3’. As a result, 300 bp of DNA (the target DNA) was amplified.

[0170] The amplified DNA fragment was ligated into the BamH1 / Kpn1 site of the cloning vector pEXA128. Both the plasmid retaining the peptide DNA and the pQE30 overexpression plasmid were transformed into Escherichia coli DH5α cells for amplification. The plasmid was extracted from the overnight culture, digested with BamHI and KpnI at 37°C for 2 hours, and separated on a 1% agarose gel. The inserts from the linearized pQE30 plasmid and pEXA128 plasmid were extracted from the gel and ligated into the pQE30 plasmid. The pQE30 overexpression plasmid encodes a HIS tag, and overexpression of the peptide of the present invention results in a fusion peptide consisting of the peptide and an N-terminal HIS tag. The resulting plasmid was transformed into Escherichia coli M15 overexpression cells. The nucleotide sequence of the plasmid extracted after overnight culture was determined, and only the clones that matched the expected sequence were used for overexpression. Using 16 ml of the overnight culture, the cells were inoculated into 800 ml of liquid LB medium at 37°C. After the OD600 reached 0.6 - 0.8, IPTG was added to a final concentration of 1 mM to induce overexpression. Cells overexpressing the peptide containing the signal peptide were incubated at 20°C for 24 hours, and cells overexpressing the peptide without the signal peptide were incubated at 37°C for 5 hours.

[0171] The recombinant peptide was purified and identified as described in Example 1.

[0172] (Example 3: Peptide Synthesis) Peptides with the same sequences as SEQ ID NO.1 and SEQ ID NO.2 were chemically synthesized by Seramun Diagnostica GmbH (Heidesee, Germany).

[0173] (Example 4: Minimum Inhibitory Concentration (MIC) Assay) The MIC assay determines the lowest concentration at which a compound inhibits the growth of a microbial population. The antibacterial activity of the peptides described herein was compared to the activities of two other AMPs, TcAMP1 and TcAMP2, discussed in WO 1998 / 027805, as well as antibiotics or bactericides. Comparative MIC assays were performed against several bacterial and fungal species (Table 1).

[0174] Peptides according to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.7 were serially diluted to create a concentration series ranging from 0.39 μg / ml to 303 μg / ml.

[0175] Pantoea, Staphylococcus, and Listeria were cultured in Trypticase 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.

[0176] In the MIC assay, bacteria and fungi were cultured on agar plates using their respective media. The preculture was prepared by inoculating a single bacterial colony or a small piece of fungus into 5 ml of each liquid medium. The optical density (OD) at 600 nm was measured after 1 to 5 days and adjusted so that the OD600 became 1. Using this cell suspension, a 1:500 dilution was prepared, corresponding to approximately 2×106 microbial cells per milliliter.

[0177] To evaluate the MIC, 96-well plates were prepared with the test antimicrobial peptides. Each well was filled with 100 μl of the microbial cell culture, 90 μl of the culture medium, and 10 μl of the peptide or the corresponding dilution of the control. The plates were cultured overnight (ON) at the optimal growth temperature for each test bacterium (Table 1). The results were visually evaluated, and the MIC was determined based on the presence or absence of growth.

[0178] Table 1 shows the organisms tested in the MIC assay of various antimicrobial peptides. The species name, classification, and growth conditions are indicated. DSM is the order number of the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). SF refers to the organisms obtained from the Jena Microbial Resource Collection (JMRC, Jena, Germany).

[0179]

Table 1

[0180] (Results) All of the peptides according to the present invention showed strong antibacterial activity against all of the organisms tested.

[0181] The MIC values obtained for the peptides according to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7 against fungal species are shown in Table 2. All of the peptides of the present invention inhibited the growth of fungi more efficiently at lower concentrations than the peptides of the prior art.

[0182] Table 2 shows the results of MIC assays using the peptides of the present invention (SEQ ID NO.1, NO.2, NO.4) and the peptides of the prior art (SEQ ID NO.6, NO.7) with fungi as the test organisms. Clotrimazole was used as a reference.

[0183]

Table 2

[0184] The MIC values of the peptides according to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7 against bacterial species are shown in Table 3. All of the peptides of the present invention inhibited the growth of bacteria, and the peptides according to SEQ ID NO.1, NO.2 and NO.4 were more efficient than the peptides of the prior art in all of the bacterial species tested.

[0185] Table 3 shows the results of MIC assays using the peptides of the present invention (SEQ ID NO.1, NO.2, NO.4) and the peptides of the prior art (SEQ ID NO.6, NO.7) with bacterial species as the test organisms. Gentamicin and streptomycin were used as references.

[0186]

Table 3

[0187] (Example 5: Stability of the peptide in body fluids) Sheep blood assay In vitro experiments were conducted to test the behavior of the peptides disclosed herein in blood against C. albicans. C. albicans isolated from human blood was used (DSMZ 6569). The peptide according to SEQ ID NO.1 was adjusted to a concentration of 1 mg / ml and mixed with an equal volume of sheep blood. This mixture was incubated overnight at 30 °C. To remove the solid part of the blood, the mixture was centrifuged, and the resulting plasma supernatant was used for the MIC.

[0188] Urine assay In vitro experiments were conducted to test the behavior of the peptides disclosed herein in urine against C. albicans. The peptide according to SEQ ID NO.1 was adjusted to a concentration of 1 mg / ml and mixed with an equal volume 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 the MIC.

[0189] Milk assay In vitro experiments were conducted to test the behavior of the peptides disclosed herein in 3.5% fat milk against Listeria monocytogenes. The peptide according to SEQ ID NO.1 was adjusted to a concentration of 1 mg / ml and mixed with an equal volume of milk. This mixture was cultured overnight at 37 °C. To remove the solid part of the milk, the mixture was centrifuged, and the resulting supernatant was used for the MIC.

[0190] (Results) When the peptide was mixed with sheep blood, a decrease in the ability to inhibit C. albicans cell growth was observed. However, the peptides described herein still showed activity at 25 μg / ml in sheep blood (Table 4).

[0191] The ability to inhibit the cell growth of C. albicans was not much affected by mixing the peptide with urine. Efficient antibacterial activity was obtained with 2 μg / ml of the peptide in synthetic urine and 5 μg / ml of the peptide in human urine, compared to 0.4 μg / ml under control conditions (Table 4).

[0192] Similarly, when the peptide was mixed with milk, the concentration inhibiting the growth of L. monocytogenes increased from 3.2 μg / ml in the absence of milk to 80 μg / ml (Table 4).

[0193] This peptide was suggested to be suitable for therapeutic use as it did not completely lose its ability to inhibit the growth of bacteria and yeast in body fluids.

[0194] Table 4 shows the results of the MIC assay when the peptide of the present invention (SEQ ID NO.1) was mixed with sheep blood, urine or milk. The control was a peptide without body fluid, and caspofungin and tetracycline were used as references.

[0195]

Table 4

[0196] (Example 6: MIC assay in soil slurry) To test the behavior of the peptides disclosed herein in soil slurry against Erwinia amylovora, in vitro experiments were conducted. The soil slurry was generated by mixing 5 g of soil with 20 ml of water. This mixture was incubated on an overhead shaker for 1 hour. Solids were removed by centrifugation, and the resulting supernatant (referred to as soil slurry) was used for MIC measurement. An equal volume of the peptide according to SEQ ID NO.1 (8 mg / ml) was mixed with the soil slurry and incubated overnight at 28°C. This mixture was used for the MIC against Erwinia amylovora.

[0197] (Results) The peptides described in this specification inhibited the growth of bacteria at 25 μg / ml in soil slurry, compared to 15 μg / ml under control conditions. Since the ability to inhibit bacterial growth in soil is not completely lost, the peptides described in this specification are suitable for agricultural use.

[0198] (Example 7: Applicability of Peptides to Technical Surfaces) Experiments were conducted to test the behavior of the peptides disclosed in this specification on technical surfaces against Candida albicans.

[0199] For the surfaces of ceramic (pore size 1100 μm), stainless steel, and silicon, the biofilm formation of this organism was tested in a biofilm reactor. Four discs with discs of the three types of surfaces were placed in each reactor and tightened with screws. With all the discs attached, the system was autoclaved. Each reactor was filled with 297 mL of SU medium, and 3 mL of the SU preculture solution (OD600 = 0.1) of C. albicans (vaginal isolate) was added. The biofilm reactor was cultured at 30 °C with stirring at 200 rpm. A total of three bioreactor systems were operated: control, treatment with peptide-enriched cocoa extract from day 0, and treatment with the same extract from day 3. The final total protein concentration of the cocoa extract was 100 μg / mL.

[0200] On days 1, 3, 5, and 7, the stems of the discs were removed from each reaction. The screws of the discs were removed and immersed in 2 mL of SU. These liquids were diluted with SU medium to prepare a dilution series of 10 -1 ~10 -5 . This diluted solution was plated on YPD solid medium and cultured at 30 °C for 48 hours. The colonies formed on the plate were counted, and the colony-forming units (CFU) / cm 2 of the disc were calculated.

[0201] (Results) Growth of C. albicans was not observed on any of the test surfaces. These results suggest that the peptides disclosed herein are very stable and effective as surface disinfectants or decontaminants.

[0202] (Experiment 8: In vitro assays in yeast and mammalian cells) To examine whether the growth inhibitory effect of the peptides is bacteriostatic or bactericidal, a lactate dehydrogenase (LDH) release assay was performed using C. albicans (vaginal isolate) and human keratinocyte (HaCaT) cells as selected model organisms. LDH is an enzyme present in many cell types. Damage to the cell membrane causes LDH to be released into the cell culture medium and can then be detected and quantified by a subsequent coupled enzyme color reaction.

[0203] HaCaT cells were cultured in DMEM medium supplemented with 4.5 g / L glucose, 2 mM L-glutamine, and 10% fetal bovine serum (FCS) at 37 °C and 8% CO2. The cells were cultured in 75 cm2 cell culture flasks containing approximately 20 ml of DMEM. The medium was changed twice a week. When confluence reached 90% or more, the cells were passaged at a ratio of 1:10. The passage solution was a 1:1 mixture of EDTA (0.05% stock) and trypsin (0.1% stock) in PBS solution without Ca 2+ and Mg 2+ containing PBS solution.

[0204] The LDH cytotoxicity assay CyQUANT® (Invitrogen, numbers C20300 and C20301) was performed according to the protocol provided by the company. The optimal seeding density was determined to be 10,000 cells per 100 μL for HaCaT cells or C. albicans cells according to the standard protocol described in the kit. Cells were exposed to the peptide according to SEQ ID NO.2 at concentrations from 1.95 μg / ml to 250 μg / ml for 1 hour or overnight, and LDH release was detected. Cytotoxicity was calculated using the following formula provided by the company. % Cytotoxicity = [(LDH activity treated with peptide) / (maximum LDH activity) - (spontaneous LDH activity)] * 100. Spontaneous LDH activity was measured in untreated (water added) samples, and maximum LDH activity was measured with the lysis agent provided by the kit supplier.

[0205] (Results) Cell death of the detected target organism C. albicans was linear in the range of 0 - 250 μg / ml and showed 92% cytotoxicity at the highest concentration of the tested protein (Figure 4). In contrast, when HaCaT cells were treated with this peptide, almost no induction of LDH release was observed. The cytotoxicity of the peptide against HaCaT cells was 0% - 1% up to a peptide concentration of 125 μg / ml and reached 6% at a peptide concentration of 250 μg / ml (Figure 4).

[0206] The present invention is not limited to any of the embodiments described above, and it is considered that several modifications can be made to the presented production examples without re-evaluating the appended claims. For example, although the preferred embodiment of the present invention is directed to the CCN-51 variety of Theobroma cacao, any cacao variety that produces a sufficient amount of visinin for further processing can be used without departing from the scope of the present invention.

[0207] (Example 9: Peptide Extraction from Non-Fermented Cocoa Beans) The peptides disclosed in this specification were isolated from non-fermented but sufficiently dried cocoa beans. The cocoa beans were mechanically crushed with a bean breaker to obtain a mixture of nibs and husks. The husks were removed, and the dehusked nibs were transferred to an expeller. This yielded a mixture of butter and slurry, which was separated by centrifugation at 16,600×g for 10 minutes at 40°C. The butter fraction that collected on top was discarded, the solid slurry pellet was ground, and stored at 4°C until the next use.

[0208] Subsequently, the peptides were extracted in three ways.

[0209] (Method 1: PEB (Protein Extraction Buffer) extraction) The ground slurry pellet was preheated in a water bath at 50°C for 10 minutes and dissolved in protein extraction buffer (100 mM Tris, 1% SDS, 1% DTT, pH 8.1). The dissolved slurry pellet was incubated at room temperature for 1 hour with stirring and then centrifuged at 16,600 x g for 20 minutes. The liquid phase containing the peptides was collected and purified by dialysis or ultrafiltration (Centricon Plus-70, Merck Chemicals GmbH, an affiliate of Merck KGaA, Darmstadt, Germany) to remove the components of PEB.

[0210] (Method 2: PEB extraction and acid precipitation) The crushed slurry pellets were treated in the same manner as in Method 1, i.e., the crushed slurry pellets were preheated in a water bath at 50 °C for 10 minutes and then dissolved in PEB. Subsequently, the dissolved slurry pellets were incubated at room temperature for 1 hour with stirring and then centrifuged at 16,600 × g for 20 minutes. The peptide-containing liquid phase was recovered and subjected to acid precipitation to concentrate the peptides of the present invention in the protein mix based on the isoelectric point. Thereby, the pH of the protein extract was lowered to 6.0 with 1 M citric acid, changing the color from dark gray to red. This mixture was centrifuged at 16,600 x g for 10 minutes. The supernatant was discarded, and the protein pellet was resuspended in PEB by sonication. Thereafter, harmful substances were removed by ultrafiltration with a 5 kDa MWCO (Centricon Plus-70, Merck Chemicals GmbH, an affiliate of Merck KGaA, Darmstadt, Germany).

[0211] (Method 3: Thermal extraction) The crushed slurry pellets were heated to 60 °C for 10 minutes with stirring in tap water at a pellet-to-water ratio of 1:4 w / v. The resulting mixture was centrifuged at 16,600 x g at 10 °C for 20 minutes. The peptide-containing supernatant obtained after extraction by any of the methods 1 to 3 was recovered and quantified or analyzed by SDS-PAGE and MIC. The protein extract was lyophilized or vacuum centrifuged before application and further concentrated (up-concentration). Sequence Listing SEQ ID NO.1 (Peptide of an embodiment of the present invention) YGRKQYERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKN SEQ ID NO.2 (Peptide of an embodiment of the present invention) YGRKQYERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHK SEQ ID NO.3 (Signal peptide of an embodiment of the present invention) MVISKSPFIVLIFSLLLSFALLCSGVSA SEQ ID NO.4 (Peptide of an embodiment of the present invention) MVISKSPFIVLIFSLLLSFALLCSGVSAYGRKQYERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKN SEQ ID NO.5 (Peptide of an embodiment of the present invention) MVISKSPFIVLIFSLLLSFALLCSGVSAYGRKQYERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEELQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHK SEQ ID NO.6 (Peptide of the prior art) YERDPRQQYEQCQRRCESEATEEREQEQCEQRCEREYKEQQRQQEEE SEQ ID NO.7 (Peptide of the prior art) LQRQYQQCQGRCQEQQQGQREQQQCQRKCWEQYKEQERGEHENYHNHKKNRSEEEEGQQR SEQ ID NO.8 (Forward_BamH1 forward primer) CGCGGATCCTATGGCAGAAAACAATAT SEQ ID NO.9 (Reverse_Kpn1 forward primer) CGCGGTACCTTTGTGATTATGGTAATT SEQ ID NO.10 (HQ tag) HQHQHQ SEQ ID NO.11 (HN tag) HNHNHNHNHNHN SEQ ID NO.12 (HAT tag) KDHLIHNVHKEEHAHAHNK SEQ ID NO.13 (ALFA tag) SRLEEELRRRLTE SEQ ID NO.14 (Avi tag) GLNDIFEAQKIEWHE SEQ ID NO.15 (C-tag) EPEA SEQ ID NO.16 (Calmodulin-tag) KRRWKKNFIAVSAANRFKKISSSGAL SEQ ID NO.17 (E-tag) GAPVPYPDPLEPR SEQ ID NO.18 (FLAG-tag) DYKDDDDK SEQ ID NO.19 (HA-tag) YPYDVPDYA SEQ ID NO.20 (Myc-tag) EQKLISEEDL SEQ ID NO.21 (NE-tag) TKENPRSNQEESYDDNES SEQ ID NO.22 (Rho1D4 tag) TETSQVAPA SEQ ID NO.23 (S-tag) KETAAAKFERQHMDS SEQ ID NO.24 (SBP-tag) DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP SEQ ID NO.25 (Strep-tag) WSHPQFEK

Claims

1. A peptide having an amino acid sequence according to SEQ ID NO. 1, an amino acid sequence showing 98% or more sequence identity with SEQ ID NO. 2, or an amino acid sequence having up to two amino acids different from SEQ ID NO.

2.

2. The peptide according to claim 1, wherein the peptide is a peptide according to SEQ ID NO. 1 or SEQ ID NO.

2.

3. The peptide according to claim 1, wherein the peptide is fused with a signal peptide at its N-terminus.

4. The peptide according to claim 3, wherein the signal peptide has a sequence that shows 95% or more sequence identity with SEQ ID NO. 3 or a sequence according to SEQ ID NO.

3.

5. The peptide according to claim 1, wherein the peptide has antibacterial activity and / or antifungal activity.

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

7. The peptide according to claim 1, wherein the peptide is derived from or isolated from theobroma cacao, preferably derived from or isolated from theobroma cacao species CCN-51.

8. The peptide according to claim 1, wherein the peptide is a recombinant peptide or a synthetic peptide.

9. A composition comprising a peptide exhibiting 95% or more sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2, or a peptide differing from SEQ ID NO. 1 or SEQ ID NO. 2 by up to five amino acids, and an excipient.

10. The composition according to claim 9, wherein the peptide comprises a signal peptide exhibiting 95% or more sequence identity with SEQ ID NO. 3, or a signal peptide having the sequence according to SEQ ID NO.

3.

11. The composition according to claim 9, wherein the composition is a liquid, semi-solid, solid, or gaseous composition, and / or the composition is in the form of a tablet, capsule, powder, granules, aerosol, paste, syrup, suspension, emulsion, or solution.

12. A therapeutic peptide or composition, wherein the peptide exhibits 95% or more sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2, or the peptide differs from SEQ ID NO. 1 or SEQ ID NO. 2 by up to 5 amino acids, or the composition according to claim 9.

13. The therapeutic peptide or composition according to claim 12, wherein the peptide is fused with a signal peptide at its N-terminus, and the signal peptide preferably exhibits 95% or more sequence identity with SEQ ID NO. 3, or has the sequence according to SEQ ID NO.

3.

14. A peptide or composition for use in subjects requiring treatment of bacterial and / or fungal infections, wherein the peptide exhibits 95% or more sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2, or differs from SEQ ID NO. 1 or SEQ ID NO. 2 by up to five amino acids, or the composition according to claim 9.

15. The peptide or composition for the application according to claim 14, wherein the peptide is fused with a signal peptide at its N-terminus, and the signal peptide preferably exhibits 95% or more sequence identity with SEQ ID NO. 3, or has the sequence according to SEQ ID NO.

3.

16. The peptide or composition for the use described in claim 14, wherein the subject is a human or an animal.

17. The use of a peptide exhibiting 95% or more sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2, or the use of a peptide that differs from SEQ ID NO. 1 or SEQ ID NO. 2 by up to 5 amino acids, or the use of the composition according to claim 9, for use as feed or food additive in crop protection, as a decontamination agent, as a preservative, or for cosmetic purposes.

18. The use according to claim 17, wherein the peptide is fused with a signal peptide at its N-terminus, and the signal peptide preferably exhibits 95% or more sequence identity with SEQ ID NO. 3, or has the sequence according to SEQ ID NO.

3.

19. The use according to claim 17, wherein the peptide or composition is used for controlling pathogens in plants, and the plant is preferably a crop.

20. The use according to claim 19, wherein the pathogen is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, and / or fungi.

21. A method for producing a peptide that exhibits 95% or more sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2, or a peptide that differs from SEQ ID NO. 1 or SEQ ID NO. 2 by up to 5 amino acids, wherein the peptide is produced by recombinant technology, synthesis technology, or extraction or precipitation from theobroma cacao.

22. The method according to claim 21, wherein the peptide is extracted from a cocoa bean slurry.

23. A vector comprising a coding sequence of a peptide exhibiting 95% or more sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2, or a coding sequence of a peptide differing by up to five amino acids from SEQ ID NO. 1 or SEQ ID NO. 2, wherein the vector is optionally fused with a signal peptide sequence at its N-terminus, the signal peptide sequence preferably exhibiting 95% or more sequence identity with SEQ ID NO. 3, and the vector is designed to enable the expression of the peptide in an expression system.

24. A transgenic plant expressing the peptide described in any one of claims 1 to 8.

25. A method for producing a transgenic plant according to claim 24, the method comprising the steps of introducing a nucleic acid encoding the peptide into plant cells, thereby producing transformed plant cells expressing the peptide, and regenerating the transformed plant cells into a transgenic plant.

26. Plant seeds coated with the peptide according to any one of claims 1 to 8 or the composition according to any one of claims 9 to 11.

Images