BRPI0908664A2 - antimicrobial activity peptides, animal compositions for the treatment and prophylaxis of animals, compositions for the treatment and prophylaxis of plants, uses of said peptides and uses of the antimicrobial activity of paenibacillus elgii ourofinensis extract - Google Patents

Abstract

PEPTIDES WITH ANTIMICROBIAL ACTIVITY, MEDICINAL COMPOSITIONS FOR THE TREATMENT AND PROPHYLAXIS OF ANIMALS, COMPOSITIONS FOR THE TREATMENT AND PROPHYLAXIS OF PLANTS, USES OF PEPTIDE TITLES AND USES OF THE EXTRACT OF PAENIBACILLUS ELIGIBILITY AND ANIMAL FACILITY. The present invention aims to provide extract from a new strain of Paenibacillus sp., Here called Paenibacillus elgii ourofinensis. with antimicrobial activity and / or growth promoter of animals destined for slaughter for human consumption. More specifically, the invention provides new peptides obtained from fractions of the fermentation extract of Paenibacillus elgii ourofinensis, said new peptides having antimicrobial activity and promoting growth of organisms, for example, plants and animals. The invention also deals with the use of the extract of this new strain, Paenibacillus elgii ourofmensis, as a growth promoter and as an antimicrobial agent in the treatment and prophylaxis of animals intended for human consumption.

Description

ANTIMICROBIAN ACTIVITY PEPTIDES, MEDICAL COMPOSITIONS FOR TREATMENT AND PROPHILAXY OF ANIMALS, COMPOSITIONS FOR TREATMENT AND PROPHILAXY OF PLANTS, USES OF PEPTIDE TEXTS AND USES OF THE EXTRACT OF PAENIBACILLUS EINGIUSBIENSIROBIENS

Field of the Invention

The present invention relates to peptides with antimicrobial activity and growth promoting activity of organisms, especially plants and animals. The peptides of the invention were identified from extracts obtained from the culture of a new bacterial isolate of the genus Paenibacillus, more specifically from the isolate Paenibacillus elgii ourofinensis. The invention further relates to the use of Paenibacillus elgii ourofinensis isolate extract as a growth promoter and in the treatment and prophylaxis of organisms such as plants and animals. The invention also relates to the use of the peptides of the invention as growth promoter and in the treatment and prophylaxis of organisms such as plants and animals. Additionally, the present invention relates to animal compositions for the treatment and prophylaxis of animals, said composition comprising a therapeutically or prophylactically effective amount of the peptides of the invention in combination with a pharmaceutically acceptable carrier or excipient.

Background of the Invention

Antibiotics are the most exploited microbial products in the biotechnology industry. Today the technology used to treat veterinary infections and promote animal growth is based on conventional antibiotics. For example, it has been shown that Bacillus subtillis, Escheriehia coli and Salmonella enteritidis bacteria, which cause infections in chickens, have resistance to known antimicrobial agents such as tetracycline, nalidixic acid, ampicillin among others (see Ribeiro, AR, Kellermann, A., Santos, LR, Nascimento VP 2008. Antimicrobial resistance in Salmonella Enteritidis isolated from clinical and environmental samples of broiler chickens and broiler breeders (Arq. Bras. Med. Vet. Zootec. 60 (5): 1259-1262). Such conventional antibiotics are also used to treat human infections. This overlap leads to a large increase in resistant strains, which makes it difficult to treat infections in humans. Thus, the isolation of new chemical entities with antimicrobial activity for exclusive use in animals will bring significant advantages in the breeding of animals for slaughter, being said new entities used as growth promoters and treatment of veterinary infections, which vatangens will result not only in increase in producers' profits, but also in excluding the use of current antibiotics in man-eaten animals.

The emergence of resistant bacteria occurred soon after clinical use of penicillin-type antimicrobial agents. Since then it has become apparent that with each new antimicrobial agent that becomes available on the market rapidly bacteria resistant to these new agents appear and the speed with which this resistance is developed is commensurate with the extent of use of these new drugs. It has recently been found that the use of antimicrobials in human food has led to the emergence of antibiotic-resistant pathogenic bacteria used in the treatment of human infections (for more details, see Wegener HC et al. "Public Health Impacts of the Use of Antimicrobials in Animal Food, "Proceedings of the WBC Congress, 23rd World Bviiatrics Congress, Quebec City, Canada, 2004).

Antimicrobial agents may be natural or synthetic substances that inhibit or kill microorganisms, including bacteria. There are currently more than 15 different classes of antimicrobial substances, which differ in chemical structure and mechanism of action making them highly specific in the treatment of specific pathogens. The emergence of antimicrobial agents was the great victory of the 20th century in the fields of medicine, veterinary and agriculture, not only for their therapeutic value, but also for their ability to promote, when used in sub-therapeutic amounts, the growth of plants and animals ( see "Use of antimicrobials outside human medicine and antimicrobial resistance in humans", Fact sheet No. 268, 01/2002, available on the Web at http://www.who.int/mediacentre/factsheets/fs268/en/).

The problems of using antibiotics as antimicrobial agents for the treatment of humans and animals have become so great that government authorities in many countries have not only promoted research to minimize the impact of pathogenic bacteria resistance to current antibiotics, but also engaging in public awareness campaigns on the increasing risks of this increased resistance to reduce antibiotic use in animals, especially in sub-therapeutic amounts to promote animal growth and improve meat yields. Examples of such government initiatives are: (1) the UK Overarching AMR Report, published in 2004 (see "Overview of Antimicrobial Usage and Bacterial Resistance in Selected Human and Animal Pathogens in the UK: 2004", available on the web at h11p : //www.dardni.gov.uk/index/pubUcations/pubs-dard-animal-healtfa/pubs-vet-mcds.htm) and (2) the work done by San Martin, B. et al. Funded by the Chilean National Fund for Scientific and Technological Development (see San Martin, B. et al. "Evauation of Antimicrobial Resistance Using Indicator Bacteria Isolated from Pigs and Poultry in Chile", Intern J Appl Res Vet Med, Yol. 3, No 2, 171-178, 2005).

One of the most promising ways to achieve this goal is the use of biological material that is effective as an antimicrobial agent and as a growth promoter for animals, especially chickens and pigs. In 2004 it was identified by the Dal-Soo group (see Dal-Soo, K. et al. IiPaenibaciUus elgii sp. Nov., With broad antimicrobial activity ", International Journal of Systematic and Evolutionary Microbiology 54, 2031-2035, 2004) , two strains (SD17 and SD18) that have been classified as a new spore-forming bacterium with broad antimicrobial activity, said strains having been isolated from Perilla frutescens roots.Later, Dal-Soo, K. et al. (see Dal-Soo , K. et al. TiPaenibaciUus elgii SD17 as a Biocontrol Agent Against Soil-bome Turf Diseases ", Plant Pathol. J. 21 (4): 328-333, 2005) have identified broad-spectrum antimicrobial activity against diseases caused in the SD17 strain. by microorganisms in grass. Laboratory tests performed with granule formulation prepared with Paenibacillus elgii SD17 fermentation syrup showed similar efficacy to commercial fungicides, while field tests with the same formulation showed lower efficacy than commercial fungicides. Despite these field results, the authors considered the granule formulation made with Paenibacillus elgii SDl 7 an appropriate biocontrol agent.

The search for antimicrobial agents for the treatment of plant diseases caused by microorganisms has also resulted in the composition described in WO 09045023, which composition is based on the use of superabsorbent polymers as a support for (a) beneficial microorganisms that inhibit the growth of pathogens. in plants and (b) a nutrient medium for said beneficial microorganism. On page 13, line 2 of document W009045023 is cited as one of the beneficial microorganisms Paenibacillus elgii SDl7.

EP 1788074 describes new strains belonging to the genus Paenibacillus and their use, or the cutting of these new strains, to control plant diseases. Said new strains are Paenibacillus sp. BS-0048, Paenibacillus sp. BS-0074, Paenibacillus polymyxa BS-0105 and Paenibacillus sp. BS-0277. Formulations comprising crude fermentation extract using the novel strains and / or compounds produced in such fermentations are described herein, which compounds are called Fusaricidin A, Fusaricidin B, compound 3 and compound 4 having their chemical formulas as defined in claim 3.

As can be seen from the above state of the art there is no mention of the isolation of proteins or peptides with protective and plant growth promoting activity obtained from Paenibacillus sp. Moreover, there are no reports on obtaining protein or peptide obtained from Paenibacillus sp. with antimicrobial activity and / or growth promoter of animals intended for slaughter for human consumption. Such proteins or peptides are an important advance in the search for alternatives to reduce the increasing resistance of pathogenic bacteria to antibiotics in use to treat microbial infections in humans. Summary of the Invention

The present invention aims at providing extract of a new strain of Paenibacillus sp., Herein called Paenibacillus elgii ourofinensis, with antimicrobial and / or growth promoting activity of animals intended for slaughter for human consumption. More specifically, the invention provides novel peptides obtained from Paenibacillm elgii ourofinensis fermentation extract fractions, said novel peptides having antimicrobial and growth promoting activity of organisms, for example, plants and animals. The invention also concerns the use of the extract of this new strain, Paenibacillus elgii ourofinensis, as a growth promoter and antimicrobial agent in the treatment and prophylaxis of animals intended for human consumption.

A first embodiment of the invention relates to peptides having antimicrobial and / or growth promoting activity and having amino acid sequences as defined in SEQID No. 1 and SEQID No. 2, as follows:

SEQ ID No. 1: Trp Glu Tyr Gly Asn Cys Gly Pro Arg His Met Thr Gly Asn Ile

SEQ ID No. 2: Trp Glu Tyr Gly Asn Cys Gly Pro Axg His Met Arg Gly Asn Ile

A second embodiment of the invention relates to a medicament for treating and prophylaxing animals comprising: (i) a therapeutically effective amount of at least one peptide having amino acid sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2; (ii) optionally a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient.

A third embodiment of the invention relates to a medicated animal treatment and prophylaxis composition comprising: (i) a therapeutically effective amount of the culture extract of Paenibacillus elgii ourofinensis; (ii) optionally a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient. A fourth embodiment of the invention relates to a plant treatment and prophylaxis composition comprising: (i) an agriculturally effective amount of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2 ; (ii) optionally at least one additive; and (iii) an agriculturally acceptable carrier or substrate.

A fifth embodiment of the invention relates to a plant treatment and prophylaxis composition comprising: (i) an agriculturally effective amount of the culture extract of Paembacillus elgii ourofinensis; (ii) optionally at least one additive; and (iii) an agriculturally acceptable carrier or substrate.

A sixth embodiment of the invention relates to the uses of the peptides of the invention as growth promoters and antimicrobial agents in the treatment or prophylaxis of animals intended for human consumption.

A seventh embodiment of the invention concerns the uses of the peptides of the invention as growth promoters and in the treatment or prophylaxis of plant pathogen infections.

An eighth embodiment of the invention relates to the uses of Paenibacillus elgii ourofinensis culture extract as a growth promoter and in the treatment or prophylaxis of conditions involving animal or plant infections.

Brief Description of the Figures

Figure 1 illustrates the sequence cladogram of the 16S ribosomal RNA coding gene obtained by the MEGA4 program using Neighbor Joining - p-distance. Bootstrap = 1000.

Figure 2 shows agarose gel electrophoresis analysis of PCR amplified DNA with 1406F / 23SR oligonucleotides, as follows: (1) corresponds to Ladder Ikb plus marker; (2) and (3) correspond to P. elgii; (4) and (5) correspond to the novel isolate; (6) and (7) correspond to Paenibacillus sp .; and (8) and (9) correspond to Bacillus subtillis. Figure 3 shows the Intergenic Ribosomal Space Analysis on silver nitrate-stained 6% polyacrylamide gel, where: (1) corresponds to Ladder Ikb plus marker; (2) and (3) correspond to P. elgii; (4) and (5) correspond to the novel isolate; (6) and (7) correspond to Paenibacillus sp .; (8) and (9) correspond to Bacillus subtillis-, and (10) and (11) correspond to the Ladder Ikb plus marker.

Figure 4 shows the duplicate 1% agarose gel electrophoresis analysis of the RAPD amplification reaction product with primer 208. (M = Ladder Ikb plus marker; Pe = P. elgii-, No = new isolated (Psp = Paenibacillus sp. and Bs = B. subtillis).

Figure 5 illustrates the cladogram of rpoB gene sequences obtained through the MEGA 4 program using Neighbor Joining - p-distance. Bootstrap = 1000.

Figure 6 shows the SEPHADEX G-75 gel filtration chromatographic profile of the lyophilized fermented broth sample from the new isolate of Paenibacillus elgii from the Brazilian Cerrado.

Figure 7 shows the electrophoretic profile of the fractions of the new Paenibacillus elgii isolate after gel filtration (SEPHADEX G-75). Molecular Marker Peptide: Globin III (2,512); Globin II (6,214); Globin I (8,159); Globin H-III (10,700); Globin I + II (14,404); Globin (16,949).

Figure 8 illustrates the antimicrobial activity of post-SEPHADEX G-75 fractions of crude extract from the new Paenibacillus elgii isolate from the Brazilian Cerrado.

Figure 9 graphically shows the diameter measurement of the inhibition zone formed by the culture supernatant of the new cerrado isolate, Paenibacillus elgii ourofinensis, grown in LB culture medium, maintained at room temperature and at -20 ° C.

Figure 10 graphically shows the diameter measurement of the inhibition zone formed by the culture supernatant of the new Cerrado isolate, Paenibactllus elgii ourofinensis, grown in soybean peptone and sodium chloride culture medium maintained at room temperature and at room temperature. temperature of -20 ° C. Detailed Description of the Invention

To make the understanding of the invention clearer and more complete, the terms employed in this description are defined as follows:

- "antimicrobial" - a term meaning extract and peptides originating from Paenibacillus elgii ourofinensis that inhibit, prevent or destroy the growth or proliferation of microbes such as bacteria, fungi, protozoa or similar organisms.

- "growth promoter" means non-nutrient substances originating in Paenibacillus elgii ourofinensis which, whether administered to plants by foliar application or to soil, or to animals for human consumption orally (for example, incorporated in water or feed) or via parenteral, increase yield: (a) in plants through vertical root growth, leaf extension, early flowering, fruit augmentation; (b) in animals, by increasing body mass gain, increasing feed efficiency, decreasing the time required for the animal to reach the ideal slaughter weight, improving organoleptic qualities and decreasing fat, preventing infectious or parasitic pathologies; decreased mortality.

"therapeutically effective amount" means a quantity of the antimicrobial agent that is sufficient to effectively inhibit or prevent the establishment, growth, or spread of an infection caused by microorganisms sensitive to the peptides or extract of the Paenibacillus elgii ourofinensis of the invention. ; or an amount of the organism growth promoting agent that is sufficient to effectively promote the growth and development of such organisms, such as animals and plants.

- "plant pathogen" - a term which means any organism that may cause damage and / or disease to plants and includes fungi, prokaryotes (bacteria and mycoplasma), nematodes, protozoa and similar organisms.

- "therapeutic adjuvant" - a term which refers to agents that help or increase the action of the active ingredient or that affect the absorption, mechanism of action, metabolism or elimination of the active ingredient in order to increase its effects, whether in combat an infection caused by microbes, ie in the growth process of organisms such as plants and animals; furthermore, this term includes prophylactic action in enhancing the immune response developed by the peptides or Paenibacillus elgii ourofinensis extract of the invention.

- "additive" - a term meaning substances added to the plant treatment composition to increase the efficiency or modify certain properties of the formulation, such as absorption of the active ingredient, to facilitate application or minimize certain problems.

- "pharmaceutically acceptable carrier or excipient" - a term which means, in a therapeutically or prophylactically active drug composition, any substance which, at concentrations present in the pharmaceutical form, has no pharmacological activity, being employed in the composition for the purpose of providing the pharmaceutical form of characteristics that ensure its stability, bioavailability, acceptability and ease of administration or application, and which serves as the carrier of the active principle with antimicrobial action and / or growth promoter of the target anmalgam.

- "agriculturally acceptable carrier or substrate" - a term in a plant treatment composition means any substance which has no therapeutic or prophylactic action and which serves as the carrier of the active principle with antimicrobial action and / or growth promoter of Target plant.

The invention provides novel agents with antimicrobial activity and growth promoting organisms from the identification and isolation of these new agents from Paenibacillus elgii ourofinensis which is a bacterium now found in the Brazilian Cerrado region. The identification of these agents motivated the obtaining of the fermentation syrup extract and the preparation of peptides with antimicrobial activity and growth promoter of animals and plants. The importance of this preparation lies in the fact that it allows the use of said extract and peptides, among others, in the control of bacteria and fungi that cause loss in economically important livestock (for example, chickens and pigs). In addition to this antimicrobial activity, said extract and peptides also act as plant growth promoters and as inhibitors of plant infections by phytopathogens.

Initially, the invention is based on the isolation and identification of said novel bacterial isolate, herein called Paenibacillus elgii_ourofinensis. This new bacterial isolate is capable of producing antimicrobial activity (s) against numerous bacteria and fungi causing veterinary infections, strains that already have resistance to current antibiotics. Specifically, this invention proposes, in one of its embodiments, the use of the supernatant and / or culture extract of the novel bacterial isolate Paenibacillus elgii_ourofinensis in controlling veterinary infections and promoting animal growth.

As will be detailed below, after chromatographic and electrophoretic analyzes of the lyophilized sample of fermented broth from the new isolate, the separation process provided fractions that were analyzed, and at least two of the peptides responsible for antimicrobial activity were identified. This identification was performed by sequencing and the result of this analysis showed that there is no sequence similar to those of the present invention in the protein and peptide databases, indicating that new peptides with antimicrobial activity were isolated.

The present invention resulted from studies that began with the discovery and identification of a novel isolate that was later identified as a bacterium belonging to the genus Paenibacillus.

To identify the isolate, DNA extraction was performed. The isolate was grown under conventional culture conditions, for example in liquid LB medium at 37 ° C for 16 hours. Total DNA was extracted using a cell-based lysis protocol using dry ice and 65 ° C bath. For 16S gene analysis of ribosomal DNA an amplification reaction was performed using primer oligonucleotides 27F and 1492R. PCR was also performed with 1698F and 204IR oligonucleotides that are specific for amplifying the highly conserved rpoB gene encoding the bacterial RNA polymerase β subunit. From the amplification products of both rpoB and 16S gene, the ligation reaction with the pGEM-T Easy vector was performed by T4 Ligase (5μ1 of 10X buffer, 3μ1 of DNA; 1 μl of pGEM-T vector). T4 Ligase). These reactions remained overnight in a 16 ° C thermocycler and the enzyme was inactivated at 65 ° C for 20 min. The binding reaction was dialyzed for 30 min.

Transformations were made into Escherichia coli (DH5a) cells. In this procedure, 10 μl of the reaction and 50μ1 of competent E. coli (DH5a) cells were used, which were submitted to electroporation and, after this step, 1 ml of SOC culture medium was added. After incubation for 1 h at 37 ° C, the cell suspension was plated on Petri dishes containing LB medium plus ampicillin (150 μg / ml), since plasmid pGEM-T contains the gene that confers resistance to. this antibiotic. The plated cell suspension was incubated for 16 hours in an oven at 37 ° C. After incubation, plasmid DNA extraction was performed by Miniprep using the Qiagen kit. The plasmid DNA preparation was digested with restriction enzyme EcoR1 and analyzed by 1% agarose gel electrophoresis to confirm cloning. For 16S region sequence analysis, plasmid DNA preparation was subjected to sequencing reactions using the 27F oligonucleotide. The partial sequence obtained from the gene encoding 16S ribosomal RNA from the new isolate was deposited with GenBank and assigned accession number EU257517. For rpoB gene sequencing, two PCR reactions were performed using the plasmid DNA preparation, one with the T7 / SP6 primer set and the other with the 1698F / 2041R set of oligonucleotides. The partial sequence obtained from the new isolate rpoQ gene has also been deposited with GenBank and the accession number is EU257518.

By comparing the sequences obtained from the gene encoding the 16S rRNA region and also the rpoB gene sequences with data from the National Center for Biotechnology Information (NCBI) non-redundant sequence database using the "Nucleotide BLAST" program (see Altschul, SF, Gish3 W., Miller W., Myers, EW, Lipman, DJ 1990. Basic aligment search tool (J. Mol Biol. 215: 403-10)), the new isolate was identified as belonging to the genus Paenibacillus. This new isolate of the genus Paenibacillus now identified consists of variable gram-positive bacteria, optionally anaerobic or strictly aerobic, with formation of circular, white and opaque colonies. For phylogenetic studies the sequences of the gene encoding 16S ribosomal RNA from other Paenibacillus species were obtained from the NCBI website with which alignment was made using the BioEdit program, and the construction of the phylogenetic tree was performed using the MEGA4 program. using p-distance and neighbor joining with bootstrap 1000. Bacillus subtillis was used as external group. As can be seen from Figure 1, the obtained phylogenetic tree grouped the new isolate into the same copy as P. elgii, P. koreensis and P ehimensis.

To identify which species the new isolate belongs to, biochemical tests based on metabolic differences between species were performed. The catalase test was performed by adding 30% 2H2O2 in the presence of Paenibacillus and Staphylococcus epidermidis isolate as positive control. The isolate was unable to produce catalase as it did not convert H2O2 to oxygen, gas and water. To perform the oxidase test, filter paper containing a reducing agent that acts as the final acceptor of the electrons transferred by cytochrome oxidase C was used. This reagent has the characteristic of changing color from reduced to oxidized state causing a visible reaction. Thus, specific detection of the cytochrome enzyme in the analyzed bacteria is possible. Additionally, Pseudomonas aeruginosa was used as a positive control. The appearance of purple pink coloration in the isolate was not noticed, whereas in Pseudomonas aeuriginosa control this coloration is very visible. The following four detailed tests were performed using a positive strain of E. eoli as a positive control. The agar citrate test was used to verify if the isolate has citrate as the sole carbon source. The SIM Agar Test (Sulfur-indolemotility agar) was used for the possible identification of the ability to produce H2S for motility observation and indole production detected by the addition of Kovac reagent. The triple sugar iron (TSI) agar test was performed to measure the bacteria's ability to use the three sugars: glucose, lactose and sucrose at concentrations of 0.1%, 1.0% and 1.0%, respectively. The indole test was employed to verify the bacteria's ability to use pyruvic acid and ammonia to satisfy nutritional requirements while indole is accumulated in the medium. For the development of these four tests, three test tubes were prepared for each agar type. As for Kovac reagent, the E. coli positive control produced a bright red colored compound on the surface of the culture medium. However, the new Paenibacillus isolate did not show this coloration, meaning that tryptophan has not been hydrolyzed and therefore this new isolate is classified as indole negative. It has also been found that the isolate secretes substances capable of digesting starch forming the halo when iodine is added. Testing using medium to demonstrate fermentative (anaerobic) catabolism resulted in a yellow staining for E. coli (positive control) and for glycerol, D-xylose, glucose, arabinose and sugar-free isolates meaning that there was fermentative carbohydrate degradation.

Reduction from nitrate to nitrite was observed in E. coli and Paenibacillus isolate due to the occurrence of reaction in the presence of sulfanilic acid and N, N-Dimethyl-I-naphthylamine producing a red color (meaning NO2 is present ). In the casein test, the new isolate Paenibacillus was able to use the medium as a nutrient as well as Bacillus subtilis. However, E. coli did not show this ability. In a KOH rapid method (cell wall lysis) test, the E. coli strain showed the formation of viscous strand (Gram negative) because DNA was released when in saline solution. While the new isolate Paenibacillus did not increase viscosity (which means to be Gram positive).

Thus, it was found that: (i) the new isolate is facultative anaerobic; (ii) colonies grown on LB agar medium are circular or irregular, bright, convex and sticky; (iii) in Middlebrook3 agar culture medium colonies were circular to slightly irregular, slightly shiny, light yellow and sticky; (iv) the temperature for bacterial growth is 28-37 ° C and growth occurs at pH 7.0-8.0; (ν) the isolate also grows in the presence of 2% and 5% NaCl after 24 hours in LB culture medium and around 48 h in TSA culture medium; (vi) with growth in both liquid culture media (LB and TSA), colonies form whitish-colored clumps; (vii) the isolate has negative catalase, oxidase negative, and indole is not produced. It is important to note that all other species of the genus Paenibacillus listed in Table 1 have positive catalase results while the new isolate is negative in this test. Therefore, it can be concluded that the isolate inhibits the growth of E. coli, Salmonella enteritidis and Bacillus subtilis by producing an antimicrobial component.

References cited in Table 1 are: (a) Kim et al., 204 = Kim, Dal-Soo., Cheol-Yong Bae, Jae-Jin Jeon, Sam-Jae Chun, Hyun Woo Oh, Soon Gyu Hong, Keun- Sik Baek, Young Moon Eun & Kyung Sook Bae. 2004. Paenibacillus elgii sp. nov., with broadantimicrobial activity. Int J Syst Evol Microbiol. 54: 2031-2035; (b) Kuroshima et al., 1996 = Kuroshima, K., Sakane, T., Takata, R. & Yokota, A. 1996. Bacillus ehimensis sp. Nov and Bacillus ehitinolytieus sp. nov., new chitinolytic members of the genusBaeillus. IntJSyst Baeteriol. 46: 76-80; (c) Hendryckx et al., 1995 = Heyndrickx M., Vandemeulebroecke K., Scheldeman P., Hoste B., Kersters K., From Yos P., Logan NA, Aziz AM, Ali N., Berkeley RC 1995. Paenibacillus (formerly Bacillus) gordonae (Pichinoty et al. 1986) Ash et al. 1994 is a later subjective synonym of Paenibacillus (formerly Bacillus) validus (Nakamura 1984) Ash et al. 1994: amended description of P. validus. IntJSyst Baeteriol. 45 (4): 661-669; (d) Chung et al., 2000 = Chung, Y.R., Kim, C.H., Hwang, I. & Chun, J. 2000. Paenibacillus koreensis sp. nov., a new species that produces an iturin-like antifungal compound. Int J Syst Evol Microbiol. 50: 1495-1500; (e) Meehan et al, 2000 = Meehan, C., Bijourson, A.J. & Mcmullan, G. 2001. Paenibacillus azoredueens sp. nov., a synthetic azo dye decolorizing bacterium from industrial wastewater. Int J Syst Evol Microbiol. 51: 1681-1685. <table> table see original document page 16 </column> </row> <table> In order to obtain the profile of the new isolate, fatty acid methyl ester (FAME) analysis of the cell was performed. bacterial (total fatty acids). The extracted fatty acid preparation was analyzed using a microbial identification program (MIDI, Sherlock® TSBA 50 5.00 Library) and the obtained fatty acid profiles were compared with the data contained in this library. MIDI analyzes over 300 fatty acids and related compounds. The fatty acid profile of the new Paenibacillus isolate is presented in Table 2.

The comparison of the results obtained from the fatty acid analysis of the new isolate Paenibacillus with that of other species of the same genus is presented in Table 3.

Preliminary identification using the MIDI library demonstrated that the new strain belongs to the genus Paenibacillus, but cannot be clearly identified as a known species due to low relative similarity (0.633) (see Table 4). Quantitative (iso-C15: 0) and qualitative (C16: lW7c alcohol) differences were observed between the new isolate and its nearest neighbor P. elgii. This fact shows that this new strain belongs to a new species. <table> table see original document page 18 </column> </row> <table> <table> table see original document page 19 </column> </row> <table> Table 4. similarity index using MIDI

<table> table see original document page 20 </column> </row> <table>

Additionally, to undoubtedly confirm the novelty of the novel isolate, it was grown in a Petri dish containing solid LB medium. A Bacillus subtillis inhibition test and a Gram stain test were performed to confirm that there was no contamination before sending the strain to a reference center for identification of new bacterial isolates located in Belgium. Confirmed the absence of contamination, the isolate was grown in 1.5 ml LB soft-agar in cryotubes and sent to the center for the following analyzes: DNA / DNA hybridization with other species belonging to the genus Paenibacillus and percentage of GC content. In addition to the strain of the new isolate, cultures of Paenibacillus elgii LMG 24465, Paenibacillus ehimensis LMG 18048 and Paenibacillus validus LMG 11161 were analyzed. After the extraction of total DNA, the G + C content of the isolate was determined using HPLC. The percentage obtained in mol was 53.4%, which represents the average of three independent analyzes of the same DNA sample. DNArDNA hybridizations were made at 44 ° C and the homologies reported in Table 5 represent the average of at least two hybridizations. The average standard deviation was 14 units, but deviations of 20-25 units were still accepted. The submitted strain presented a homology above 70%, generally accepted limit for species delineation, with the P. elgii strain LMG 24465. From this result it could be concluded that the strain of the new isolate belongs to the species Paenibacillus elgii. However, depending on the results obtained, it is likely that the new isolate may be a subspecies of P. elgii not yet identified. Table 5. DNA hybridization result: DNA between strains in percentage of homology

<table> table see original document page 21 </column> </row> <table>

The strain of the novel Brazilian Cerrado isolate of the present invention was compared with the strain of Paenibacillus elgii LGM 24465T, KCTCl 0016BP, NBRC100335T obtained from the Belgium Collection Laboratorium Voor Microbiologie Bacterium. P. elgii DNA was extracted from the novel isolate of the present invention, Paenibacillus sp. and Bacillus subtillis using the MOBIO UltraClean ™ Mierobial DNA Isolation extraction kit. A PCR was performed with 1406F / L1R oligonucleotides that amplify the ITS region (intergenic space region between the 16S and 23S ribosomal RNA coding regions). After checking the PCR results, the product was electrophoresed on 6% polyacrylamide gel for 1 hour and 30 minutes using constant amperage of 45W. These analyzes were performed with repetitions to confirm the veracity of the results. The result of the total DNA amplification of the bacterial strains with the 1406F / 23SR oligonucleotides is shown in Figure 2. It can be seen in the duplicate assays that the product fragments for P. elgii for the novel isolate of the invention and for Paenibacillus sp are the same size, and the fragment for the B. subtillis amplified product is a different size. In the polyacrylamide gel (see Figure 3), the samples are also in duplicate to confirm the veracity of the results, in addition to the fact that amplification and RISA were performed with three repetitions. As can be seen from Figure 3, the P. elgii sample has a very different band pattern from the novel isolate of the invention, although there are some equivalent bands. On the other hand, the novel isolate of the invention is Paenibacillus sp. presented a very similar band pattern, with a few different bands. It could be observed that the band pattern of the genus Paenibacillus is totally different from the band pattern of Bacillus. This result showed that P. elgii and the new isolate showed differences among themselves through the analysis of intergenic regions. Each band presented in the gel may be representing a ribosomal operon that served as a template for the amplification of the intergenic region, thus, these two strains have different operons, confirming that there are differences between P. elgii strains from the Collection Laboratorium Voor Mierobiologie Bacterium and the strain of the new Brazilian Cerrado isolate that corresponds to the bacteria from which the extract and peptides of the invention are obtained.

Differences were also observed in RAPD analyzes comparing P. elgii, the new isolate of the invention, Paenibacillus sp. and Bacillus subtillis (see Figure 4). Once again, it was observed that the B. subtillis DNA band profile is totally different from the others. In the case of Paenibacillus sp., The profile is different, but some bands are observed in common with the other Paenibacillus, and there is a common band for P. elgii and Paenibacillus sp., Which is absent in the profile of the new isolate of the invention. . When comparing the band profile of P. elgii and the new isolate of the invention, we observed the absence of bands in one sample and presence in another, as well as differences in the intensity of some bands, but the band profiles of these two strains are quite different. similar to each other, with several bands in common.

The RAPD technique allows the use of low stringency primer oligonucleotides with few nucleotides, which arbitrarily select sequences from specific sites of genomic DNA, where complete or semi-complete pairing from amplified DNA occurs, allowing for the distinction of DNA samples. same species by comparing the genomic pattern obtained. This is one of the simplest methods because the DNA sample does not need to have large fragments or be highly purified, requiring only a few nanograms of the sample to perform RAPD (see Welsh, J. & McClelland, M. 1990. Fingerprinting). genomes using PCR with arbitrary primers Nucleic Acids Research 18 (24): 7213-7213; Kersulyte, D., Struelens, MJ, Deplano, A.; Berg, DE 1995. Comparison of arbitrarily primed PCR and macrorestriction (pulsed-fiild) gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients (Clinical Journal Microbiology. 33: 2216-9). The use of this technique in the comparison of P. elgii with the new isolate of the invention was important to show some differences between them, although they belong to the same species compared to the other analyzes.

Total P. elgii DNA was PCR amplified using 1698F / 2041R oligonucleotides that are specific for amplifying the highly conserved rpoB gene encoding the β subunit of bacterial RNA polymerase. From the PCR product, the binding reaction with the pGEM-T Easy vector was performed by T4 Ligase (5μ1 10X buffer, 3μ1 DNA; Ιμΐ of the pGEM-T Easy vector; Ιμΐ of T4 Ligase). This reaction remained overnight in a 16 ° C thermocycler and the enzyme was inactivated at 65 ° C for 20 min. The binding reaction was dialyzed for 30 minutes. Transformation into Escherichia coli cells (Epi 300) was performed. In this procedure, ΙμΙΟ of the reaction and 50 μΐ of competent E. coli cells (Epi 300) were used and the mixture was submitted to electroporation and after this procedure, 1 ml of SOC culture medium was added. After incubation for 1 h at 37 ° C, the cell suspension was plated in Petri dishes containing LB medium plus ampicillin (150 μg / ml), as plasmid pGEM-T contains the gene that confers resistance to this antibiotic. , and the plates incubated for 16 h in an oven at 37 ° C. Next, plasmid DNA extraction was performed by Miniprep using the Qiagen kit. Plasmid DNA preparation was digested with restriction enzyme EcoR1 and analyzed by 1% agarose gel electrophoresis to confirm cloning. Two PCR reactions were performed using the plasmid DNA preparation, one with the T7 / SP6 primer set and the other with the 1698F / 2041R set of oligonucleotides. The products of these reactions were sequenced. Sequences of the rpoB gene from other Paenibacillus species were obtained from the NCBI website with which, through the BioEdit program, the alignment and construction of a phylogenetic tree using the MEGA4 program using p-distance and neighbor joining with bootstrap were made. 1000. Bacillus subtillis was used as an external group.

The rpoB gene sequencing result showed 98% sequence identity between P. elgii and the novel isolate of the invention by analyzing the sequence obtained from P. elgii by the nucleotide BLAST program (http: //blast.ncbi.nlm. nih.gov/Blast.cgi')· The partial sequence of the P. elgii rpoB gene has been deposited with GenBank and the accession number is FJ407093. The phylogenetic tree shows extreme proximity between P. elgii and the novel isolate of the invention (see Figure 5). From confirmation of this result, the novel isolate of the invention was named Paenibacillus and / gi / isolate ourofinensis or Paenibacillus elgii ourofinensis. Phylogenetic analyzes based on the 16S rDNA gene sequence were of great importance in defining the Paenibacillus genus (see Ash, C., Priest, FG & Collins, MD 1993. Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins ) using a PCR probe test Antonie van Leeuwenhoek 64: 253—260). However, limitations in 16S rDNA-based phylogenetic analyzes due to the presence of different copies of this gene restrict its use in Paenibacillus species (see Berge, O., Guinebretière, MH, Achouak, W., Normand, P. & Heulin, T. 2002 Paenibacillus graminis Nov., and Paenibacillus odorifer Nov., isolated from plant roots, soil and food (JSyst Evol Microbiol 52: 607-616). The rpoB gene has been successfully used to construct phylogenetic relationships in various groups of bacteria, and usually has greater discriminatory power than 16S rDNA sequences (see Dahllof, I., Baillie, H. & Kjelleberg, S. 2000). rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity (Appl Environ Microbiol 66: 3376-3380). The high identity between the P. elgii rpoB gene sequences and the novel isolate of the invention confirms the DNA-DNA hybridization result, indicating that these strains can be separated as different species.

The present invention relates, in a first embodiment, to a novel bacterial extract having at least one biological activity, among antimicrobial activity with respect to microorganisms causing disease or damage to animals and plants and growth promoting activity of said animals and plants. Said new bacterial extract being characterized by originating from the new isolate Paenibacillus elgii ourofinensis. The term "bacterial extract" is intended in this description to mean the fraction that is soluble in water or an aqueous solution, said fraction being obtained by separation of the bacteria by centrifugation, the bacterial constituents having antimicrobial and growth promoting activity. in the liquid phase, also known as the supernatant.

According to the invention, Paenibacillus and / gn_ourofinensis bacteria are grown in an appropriate medium so that they can multiply, the pH of the medium being adjusted to the conditions of its natural environment (pH 7.0-8.0) and temperature for the growth range, corresponding to 28-37 ° C. Many bacterial culture media are described in the literature and those skilled in the art will be able to select and adjust fermentation conditions to obtain the extract of the invention. After fermentation, the bacteria obtained are separated from the supernatant. Paenibacillus and / gz / _ourofmensis bacteria can be separated by the application of various methods, in particular by centrifugation or filtration. After the separation step, the liquid phase (supernatant) is subjected to a drying process, for example lyophilization, to separate the mixture of antimicrobial and growth promoting constituents, this mixture corresponds to the bacterial extract of the invention.

The present invention also relates to specific peptides having the sequences SEQ ID No. 1 and SEQ ID No. 2, both peptides characterized by having antimicrobial and / or growth promoting activity of plants and animals, especially animals such as chickens and pigs. . The peptides of the invention were identified and characterized by fractionation of the liquid fermentation phase of PaenibaciUus and / gzi_ourofínensis bacteria by an appropriate method such as gel filtration (SEPHADEX G-75) of lyophilized sample of fermented Paenibacillus broth and / gzzi solado ourofinensis, obtaining the chromatographic profile as shown in Figure 6. In the analyzes made from the electrophoretic profile of Paenibacillus and / gz'z'_isolado ourofmensis filtration post-gel fractions (SEPHADEX G-75) (see Figure 7), some fractions were chosen for the antimicrobial activity test of the crude extract of the invention using Bacillus subtilis and activity was observed in fraction 24 (see Figure 8). This fraction was used to obtain the peptide sequences responsible for this activity. The sequences obtained were: WEYGNCGPRHMTGNI and WEYGNCGPRHMRGNI, corresponding to SEQ ID NO. 1 (Trp Glu Tyr Gly As. Met Arg Gly Asn Ile), respectively. Such sequences were analyzed by the blastx program and the result of this analysis demonstrated that there is no sequence similar to SEDQ ID No. 1 and SEQ ID No. 2 sequences of the present invention in the database, indicating that new peptides with antimicrobial activity were isolated.

The peptides of the present invention may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized manually or in an automated peptide synthesizer or may be isolated from the Paenibacillus elgiiourofmensis bacterial fermentation syrup. It is also possible to produce the peptides by genetic engineering techniques. Codons coding for specific amino acids are known to those skilled in the art, and therefore DNA molecules encoding the peptides of the invention may be constructed by appropriate techniques, and such a DNA molecule may be cloned into an appropriate expression vector (such as such as plasmid or phage), which is transfected into a system suitable for expression of the peptides of the invention.

The present invention also relates to medicaments for the treatment and prophylaxis of animal animals for human consumption, said compositions comprising: (i) a therapeutically effective amount of at least one peptide having amino acid sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2; (ii) optionally a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient. As mentioned above, the peptides of the invention having amino acid sequences as represented in SEQ ID No. 1 and SEQ DD No. 2 possess antimicorbial activity and / or growth promoting activity of said animals.

The invention also encompasses a medicament composition for the treatment and prophylaxis of animals for human consumption, said compositions comprising: (i) a therapeutically effective amount of the culture extract of Paenibacillus elgii ourofinensis; (ii) optionally a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient. Said extract may be in liquid form, corresponding to the liquid phase (supernatant) separated from the fermentation syrup, or may be in the dry state after drying, for example by lyophilization, of said liquid phase.

The pharmaceutical compositions of the invention contain the active ingredient chosen from the bacterial fermentation extract Paenibacillus elgii ourofinensis, and the petid having amino acid sequence chosen from SEQ ID No. 1 and SEQ ID No. 2, or mixtures thereof, in combination with a carrier. or pharmaceutically acceptable diluent and optionally one or more therapeutic adjuvants. Therefore, the active ingredients according to the invention may be administered individually or together in any conventional oral / parenteral pharmaceutical / veterinary form. For oral administration, the composition of the invention may be in the form of solution, suspension, tablet, pill, capsule, powder, the solid forms being of the rapid release, controlled release or extended release type or any combination of these types, such forms being added to the water or animal feed.

The tablet form contains various excipients such as sodium citrate, calcium carbonate, calcium phosphate and the like together with disintegrants such as starch or complex silicates. It is also common in this pharmaceutical form for the use of binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia and additionally lubricating agents such as magnesium stearate, sodium lauryl sulfate and acacia. Other suitable solid pharmaceutical / veterinary forms include powders which may be of the water dispersible type or may be packaged in hard capsule or soft capsule gelatin capsules, in which case the excipient may include lactose or polyethylene glycols of molecular weight. varied.

In the case of liquid oral administration dosage forms, sweeteners, flavoring agents, colorants, emulsifying or suspending enhancers and diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof may be used in the excipient.

Pharmaceutical compositions containing the active ingredients of the invention are usually prepared following conventional methods and are administered in appropriate pharmaceutical / veterinary form.

For example, solid oral pharmaceutical / veterinary forms may contain, along with the active ingredient, diluents, lubricants, binding agents, disintegrating agents and the like. Examples of diluents that may be used in the pharmaceutical / veterinary compositions of the invention are: lactose, dextrose, sucrose, cellulose, cornstarch or potato starch. Examples of lubricants that may be used in the pharmaceutical / veterinary compositions of the invention are: silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycols. Examples of binding agents which may be used in the pharmaceutical / veterinary compositions of the invention are: starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone. Examples of disintegrating agents which may be used in the pharmaceutical / veterinary compositions of the invention are: starch, alginic acid, starch or sodium starch glycolate; effervescent mixtures; dyes; sugary. Additionally, wetting agents such as lectin, polysorbates, lauryl sulfates may be used in the pharmaceutical / veterinary compositions of the invention; and, in general, non-toxic pharmacologically inactive substances commonly used in pharmaceutical / veterinary formulations. The preparation of said pharmaceutical / veterinary compositions of the invention may be carried out in known manner, for example, by mixing, granulating, tableting, sugar coating, or film coating processes. Liquid dispersions for oral administration may be, for example, syrups, emulsions and suspensions. In addition to the active principle of the present invention, syrups may contain one or more carrier agents, for example sucrose or sucrose with glycerin and / or manita and / or sorbitol. Suspensions and emulsions may contain as a carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. For parenteral administration, the use of sterile and pharmaceutically acceptable oils is conventional and propylene glycol as well as sterile aqueous solutions may be employed.

The pharmaceutical compositions of the present invention may further contain one or more therapeutic adjuvants. Examples of such adjuvants are: polysaccharide-phosphidium conjugate as described in US Patent 5785975; emulsion-based veterinary adjuvants, for example Emulsigen®, Carbigen®, Polygen®, available from MPV Technologies; adjuvants such as acrylic polymer and dimethyl dioctadecyl ammonium bromide, described in US20090017067; sub-micron oil-in-water emulsion of squalene, polyoxyethylene, sorbitan monooleate and sorbitan trioleate, known under the brand name MF59 ™, available from Novartis, and other examples may be mentioned.

Additionally, the invention also relates to a plant treatment and prophylaxis composition comprising: (i) a therapeutically effective amount of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2 ; (ii) optionally at least one additive; and (iii) an agriculturally acceptable carrier or substrate.

In another embodiment of the invention, said plant treatment and prophylaxis composition comprises: (i) an agriculturally effective amount of the crop extract of PaenibaciUus elgii ourofinensis; (ii) optionally at least one additive; and (iii) an agriculturally acceptable carrier or substrate. The plant treatment and prophylaxis compositions of the invention contain the active ingredient chosen from the bacterial fermentation extract Paenibacillus elgii ourofínensis, and petid having amino acid sequence chosen from SEQ ID No. 1 and SEQ ID No. 2, or mixtures thereof, in combination with an agriculturally acceptable carrier or substrate and optionally one or more additives.

The formulation of the invention may be in liquid form or solid form, for example in wettable powder form. Both formulations, dry or liquid, must be packaged in packaging and conditions that preserve the integrity of the active ingredients. Liquid formulations have shorter shelf life, but dry formulations (eg wettable powder) require less rigid conditions such as storage in a cool place with no light and moisture, and the product can be stored from crop to crop, provided it is stored in a freezer or refrigerator.

The formulations of the invention generally contain an amount of active ingredient effective in controlling plant-damaging microorganisms and / or promoting plant growth, said active ingredient being selected from bacterial fermentation extract Paenibacillus elgii ourofínensis, and petid having an amino acid sequence chosen from SEQ ID No. 1 and SEQ ID No. 2, or mixtures thereof, in combination with an agriculturally acceptable carrier, in the form of a liquid or solid, preferably solid, diluent. Formulations that may be used include powders, granules, pellets, suspensions, emulsions, wettable powders, flowable solids and the like, provided such forms are consistent with the physicochemical properties of the active ingredient and compatible with the bacterial fermentation extract Paenibacillus elgii. ourofínensis, or with the petid having amino acid sequence chosen from SEQ ID No. 1 and SEQ ID No. 2, the mode of application and environmental factors such as soil type, air humidity and temperature. Spray formulations can also be used, especially when application areas are large. More concentrated compositions are also within the protection scope of the present invention, and said compositions are suitable for use as intermediates for the diluted end product to be applied. The formulations of the invention will typically contain effective amounts of the active ingredient, one or more diluents, one or more surfactants and other suitable additives to achieve desirable product characteristics. Furthermore, it should be clear that the active ingredient used in the present invention may also be used alone in special applications where high concentration of the composition of the invention is required.

Other formulating agents, present in smaller amounts, may be added to the compositions of the invention, such as additives to reduce foaming, blunting, corrosion, unwanted microbial growth, and the like. Another essential feature of the formulation of the present invention is the need to ensure that all ingredients of the composition are mutually compatible and do not contribute to the loss of antimicrobial and / or growth promoting activity characteristic of the active principle of the invention.

The compositions of the present invention may also be mixed with other agricultural chemicals, fertilizers, soil conditioners, stabilizers, or with one or more biologically active insecticides, fungicides, acaricides or other compounds to form a multicomponent formulation. Examples of other agricultural protection products with the active principle of the formulation of the present invention may be mixed are sodium channel agonist type insecticides (e.g. pyrethroids), sodium channel blocking agents (e.g. pyrazolines), acetylcholine esterase inhibitors (e.g. organophosphates and carbamates), nicotinic acetylcholine binding agents, gabaergic binding agents, octapine agonists or antagonists (e.g. formamidines), pyrrol group insecticides and others.

The following is an example of the isolation, cultivation and antimicrobial activity assays of the isolate of the invention, Paenibacillus elgii ourofinensis. However, it should be understood that such an example is provided for illustrative purposes only and that various modifications or changes, in light of the embodiments disclosed herein, will be suggestive to those skilled in the art and should be included within the spirit and scope of this description and scope of the claims. that accompany it. EXAMPLE

Isolation, cultivation and antimicrobial activity

A new bacterial strain was isolated from Cerrado soil. The isolate showed antimicrobial activity in relation to the bacterial strains Bacillus subtillis, Escherichia eoli and Salmonella enteritidis. For inhibition tests the isolate was cultured for 48 h at 28 ° C in liquid LB (Luria Bertani) culture medium. The cell suspension was centrifuged and the supernatant recovered. Both the isolate and its supernatant were plated in the center of Petri dishes containing solid LB culture medium. The bacterial strains mentioned above were plated and after 24 h incubation zones of inhibition were observed in Petri dishes with approximately 3cm in diameter. Physical and chemical tests were also performed with the new isolate culture supernatant, where one sample of the supernatant was kept at 95 ° C for 20 minutes and another sample of the supernatant was treated with proteinase K for 1 h at 37 ° C. Aliquots of each supernatant sample subjected to the described treatments were applied to the center of Petri dishes containing the solid LB culture medium and a S. enteritidis culture was spread on each of these dishes with the aid of the Drigalsky loop. After 24h of incubation, inhibition halo was observed on the temperature treatment plates, but no inhibition halo was observed on the plate containing the proteinase K-treated supernatant. This result indicates that this antimicrobial activity may be associated with a peptide because The fact that the plate containing the material of the invention did not have this activity after proteinase K treatment may have been due to the degradation of peptides by this enzyme (data not shown). The culture supernatant of the new isolate was also tested for its temporal stability for antimicrobial activity. Culture supernatants recovered from the isolate grown in LB culture medium and in soybean peptone and sodium chloride culture medium were kept at room temperature for a period of 100 days. Every two days, and thereafter each week, 500 μΐ of this supernatant was removed and placed in a hole made in solid LB medium until completely absorbed, with three repetitions. The control was kept frozen at -20 ° C and aliquots were thawed and applied to the petri dish also in triplicate. After distribution of these supernatants, a 200 μl aliquot of Bacillus subtillis inoculum was applied to each plate and spread throughout the plate with the aid of the Drigalsky loop. The plates were kept in an oven at 37 ° C for a period of 16 hours. The inhibition halo was measured on each plate and the average halos value was obtained for each sample. Antimicrobial activity testing using the supernatant stored at room temperature for 70 days resulted in a halo diameter decrease of approximately 0.5 cm, and the control remained stable (see Figure 9). Activity testing using the supernatant obtained in soybean peptone and sodium chloride medium resulted in a B. subtillis inhibition halo of about 2 to 2.5 cm. After 20 days of storage of the supernatant kept at room temperature, a drop of approximately 0.5 cm in diameter of the inhibition zone was observed. It was found that after 84 days, the isolate supernatant grown in medium containing soybean peptone and sodium chloride no longer inhibited the presence of B. subtillis (see Figure 10). For the supernatant stored at -20 ° C, which corresponds to the control, the inhibition zone was kept constant during all antimicrobial activity tests performed (see Figure 10).

All publications and patent applications mentioned in the description are indicative of the level of those skilled in the art to which the invention relates. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be made within the scope of the claims accompanying this description.

Claims (19)

1. Peptide having antimicrobial and / or growth promoting activity of animals or plants characterized by comprising an amino acid sequence? selected from group SEQID No. 1 and SEQID No. 2. Peptide according to claim 1, characterized in that said? animals are intended for human consumption. Peptide according to claim 2, characterized in that said animals are chickens or pigs. A pharmaceutical composition for the treatment and / or prophylaxis of animals, comprising: (i) a therapeutically effective amount of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 2; (ii) optionally a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient. Composition according to Claim 4, characterized in that said treatment or prophylaxis is performed under veterinary conditions requiring antimicrobial activity. Composition according to Claim 4, characterized in that said treatment or prophylaxis is performed under veterinary conditions requiring growth promoting activity. A pharmaceutical composition for the treatment and / or prophylaxis of animals, comprising: (i) the highest therapeutically effective amount of Paenibacillus elgii ourofinensis culture extract; (ii) optionally a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient. Composition according to Claim 7, characterized in that said treatment or prophylaxis is performed under veterinary conditions requiring antimicrobial activity. Composition according to Claim 7, characterized in that said treatment or prophylaxis is performed under veterinary conditions requiring growth promoting activity. A plant treatment and / or prophylaxis composition comprising: (i) an agriculturally effective amount of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ED No. 1 and SEQ ID No. 2; (ii) optionally at least one additive; and (iii) an agriculturally acceptable carrier or substrate. Composition according to Claim 10, characterized in that said treatment or prophylaxis is performed under agronomic conditions requiring antimicrobial activity. Composition according to Claim 10, characterized in that said treatment or prophylaxis is performed under agronomic conditions which require growth promoting activity. Composition for plant treatment and prophylaxis, comprising: (i) an agriculturally effective amount of the crop extract of Paenibacillus elgii ourofinensis; (ii) optionally at least one additive; and (iii) an agriculturally acceptable carrier or substrate. Composition according to Claim 13, characterized in that said treatment or prophylaxis is performed under agronomic conditions requiring antimicrobial activity. Composition according to Claim 13, characterized in that said treatment or prophylaxis is performed under agronomic conditions requiring growth promoting activity. Use of the peptides defined in claim 1, characterized in that they are used as growth promoters and / or as antimicrobial agents in the treatment or prophylaxis of animals intended for human consumption. Use of the peptides defined in claim 1, characterized in that said use is as growth promoter and in the treatment or prophylaxis of plant pathogen infections. 18. Use of extract from the culture extract of Paenibacillus elgii ourofinensis, characterized in that its use is as a growth promoter and in the treatment or prophylaxis of conditions involving infections in animals. 19. Use of extract from culture extract of Paenibacillus elgii ourofmensis, characterized in that its use is as a growth promoter and in the treatment or prophylaxis of conditions involving plant infections.