US20140113817A1

US20140113817A1 - Peptides with antimicrobial activity, drug compositions for the treatment and prophylaxis of animals, compositions for the treatment and prophylaxis of plants, uses of said peptides, and uses of paenibacillus elgil ourofinensis extract

Info

Publication number: US20140113817A1
Application number: US14/111,707
Authority: US
Inventors: Carlos Henrique Henrique; Janaina Fernandez De Araújo; Alinne Pereira De Castro; Lucas Carvalho; Octávio Luis Franco; Adriane Kurokawa Silva; Ricardo Enrique Kruger
Original assignee: UNAIO BRASILIENSE DE EDUCACO E CULTURA-UBEC
Current assignee: UNAIO BRASILIENSE DE EDUCACO E CULTURA-UBEC; Ouro Fino Participacoes e Empreendimentos S/A; Uniao Brasiliense de Educacao e Cultura UBEC
Priority date: 2011-04-12
Filing date: 2011-04-12
Publication date: 2014-04-24
Also published as: AU2011365688B2; JP2014513080A; AU2011365688A1; MX2013012045A; NZ616686A; WO2012139179A1; US9534019B2; EP2708551A4; AU2011365688A8; KR20140058423A; RU2013150203A; US20150218226A1; EP2708551A1; CN103649107A; CO6940372A2; AU2011365688C1

Abstract

The present invention provides extracts of material fermented with Paenibacillus sp., preferably the one called here Paenibacillus elgii ourofinensis with antimicrobial activity and/or growth promoter in animals intended for slaughter for human consumption. The invention provides new peptides derived from the fractions of the fermentation of Paenibacillus elgii ourofinensis extract, said new peptides having antimicrobial activity and promoting the growth of organisms, for example, plants and animals. The invention is also related to the use of the extract of this new strain, Paenibacillus elgii ourofinensis, as a growth promoter and as an antimicrobial agent in the treatment and prophylaxis of animals intended for human consumption.

Description

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Antibiotics are the most exploited microbial products in the biotechnology industry. Currently, the technology used for the treatment of veterinary infections and in the promotion of animal growth is based on conventional antibiotics. For example, it was shown that the bacteria Bacillus subtilis, Escherichia coli and Salmonella enteritidis that cause infections in chickens are resistant to known antimicrobial agents such as tetracycline, nalidixic acid, ampicillin, and others (see Ribeiro, A. R., Kellermann, A., Santos, L. R., Nascimento V. P. 2008. Resistência antimicrobiana em Salmonella enteritidis isoladas de amostras clínicas e ambientais de frangos de corte e matrizes pesadas. Arq. Bras. Med. Vet. Zootec. 60 (5): 1259-1262). Such conventional antibiotics are also used for the treatment of human infections. This overlay leads to the emergence of a large increase of resistant strains, which complicates the treatment of infections in humans. Thus, the isolation of new chemical entities with antimicrobial activity for exclusive use in animals will provide significant benefits in raising animals for slaughter, said entities being used as growth promoters and in the treatment of veterinary infections, and these benefits will result not only in increased earnings for producers, but also in the exclusion of the use of current antibiotics in animals consumed by men.
The emergence of resistant bacteria occurred soon after the clinical use of antimicrobial agents like penicillin. Since then it became clear that for each new antimicrobial agent that is available on the market a bacteria resistant to these new agents quickly appears, and the rapidity with which this resistance is developed is proportional to the size of the use of new drugs. Recently it was found that the use of antimicrobials in animals used to feed men has caused the emergence of pathogenic bacteria resistant to the antibiotics used to treat infections in humans (for more details, see Wegener H. C. et al. “Public Health Impacts of the Use of Antimicrobials in Food Animals”, Proceedings of the WBC Congress, 23rd World Buiatrics Congress, Quebec City, Canada, 2004).
The antimicrobial agents can be natural or synthetic substances which inhibit or kill microorganisms, including bacteria. Currently there are over 15 different classes of antimicrobial substances, which differ in chemical structure and mechanism of action, causing them to be 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 medicine and agriculture, not only for its therapeutic value, but also for its property to promote the growth of plants and animals when used in sub-therapeutic amounts (see “Use of antimicrobials outside human medicine and resultant antimicrobial resistance in humans”, Fact sheet No. 268, January 2002, available on the Web at http://www.who.int/mediacentre/factsheets/fs268/en/).
The problems of the use of antibiotics as antimicrobial agents for the treatment of humans and animals have assumed such a strong position that governmental authorities of different countries have not only promoted researches to minimize the impact of resistance of pathogenic bacteria to existing antibiotics, but also worked in campaigns to educate the public to the increasing risks of this increase in resistance, aiming at reducing the use of antibiotics in animals, especially in subtherapeutic quantities to promote animal growth and improve the yield of meat production. Examples of these government initiatives are: (1) the Report “Overarching AMR” in the UK, 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 http://www.dardni.gov.uk/index/publications/pubs-dard-animal-health/pubs-vet-meds.htm) and (2) the work performed by San Martín, B. et al. Funded by the Chilean National Fund for Scientific and Technological Development (see San Martín, B. et al. “Evaluation of Antimicrobial Resistance Using Indicator Bacteria Isolated from Pigs and Poultry in Chile”, Intern J Appl Res Vet Med, Vol. 3, No. 2, 171-178, 2005).
One of the most promising paths in the quest to achieve this goal is to use a biological material that is effective as an antimicrobial agent and as a growth promoter in animals, especially in chickens and pigs. In 2004 the group of Dal-Soo identified (see Dal-Soo, K. et al. “Paenibacillus elgii sp. nov., with broad antimicrobial activity”, International Journal of Systematic and Evolutionary Microbiology 54, 2031-2035, 2004), two strains (SD17 and SD18) which were classified as a new spore-forming bacteria with broad antimicrobial activity, said strains being isolated from roots of Perilla frutescens. Later, Dal-Soo, K. ela al. (see Dal-Soo, K. et al. “Paenibacillus elgii SD17 as a Biocontrol Agent Against Soil-borne Turf Diseases”, Plant Pathol. J. 21(4):328-333, 2005) identified in strain SD17 a broad-spectrum antimicrobial activity against diseases caused by microorganisms in grass. Laboratory tests conducted with a granule formulation prepared with the fermentation broth of Paenibacillus elgii SD17 showed efficacy similar to the commercial fungicides, while field tests with the same formulation showed an efficacy lower than the commercial fungicides. Despite these results on the field, the authors considered the granule formulation made with Paenibacillus elgii SD17 an appropriate biocontrol agent.
The search for antimicrobial agents for the treatment of plant diseases caused by microorganisms also resulted in the composition disclosed in WO 09045023, which composition is based on the use of superabsorbent polymers as a support for (a) beneficial microorganisms which inhibit the growth of pathogens in plants and (b) a nutrient medium for said beneficial microorganism. On page 13, line 2 of document WO09045023 Paenibacillus elgii SD17 is mentioned as one of the beneficial microorganisms.
EP 1788074 describes new strains belonging to the genus Paenibacillus and their use, or the culture of these new strains to control plant diseases. These new strains are Paenibacillus sp. BS-0048, Paenibacillus sp. BS-0074, Paenibacillus polymyxa BS-0105 and Paenibacillus sp. BS-0277. This document describes formulations comprising the crude fermentation extract using the new strains and/or compounds produced in these fermentations, such compounds called Fusaricidin A, Fusaricidin B, compound 3 and compound 4 with their chemical formulas defined in claim 3.
As can be seen in the prior art mentioned above there is no mention of the isolation of proteins or peptides with protective activity and promotes of plant growth obtained from Paenibacillus sp. And also, there is no report about getting a protein or a peptide derived from Paenibacillus sp. with antimicrobial activity and/or growth promoter in animals intended for slaughter for human consumption. Such proteins or peptides bring an important advance in the search for alternatives to reduce the increasing resistance of pathogenic bacteria to antibiotics in use for the treatment of microbial infections in humans.

SUMMARY OF THE INVENTION

The present invention aims at providing Paenibacillus sp extract with antimicrobial activity and/or growth promoter in animals intended for slaughter for human consumption comprising new lipopeptides not described in the prior art with antimicrobial activity. More specifically, the invention provides new peptides derived from the fractions of the fermentation of Paenibacillus extract, preferably Paenibacillus elgii, said novel peptides having antimicrobial activity and promoting the growth of organisms, such as plants and animals. The invention is also related to the use of the extract as a growth promoter and as an antimicrobial agent in the treatment and prophylaxis of animals intended for human consumption.
In an initial embodiment, the present invention comprises the process for obtaining the extract comprising at least one of the following compounds shown:
wherein the process comprises the step of performing the technique of extracting Paenibacillus from the fermentation broth.
In a second embodiment, the present invention comprises extracts of Paenibacillus comprising at least one of the following compounds:
A third embodiment of the invention relates to lipopeptides having antimicrobial activity and/or growth promoter, represented below:
A fourth embodiment of the invention relates to a drug composition for the prophylaxis and treatment of animals comprising: (i) a therapeutically effective amount of at least one of the following compounds:

- (ii) optionally, a therapeutic adjuvant; and (iii) a pharmaceutically acceptable carrier or excipient.

A fifth embodiment of the invention relates to a drug composition for the prophylaxis and treatment of animals comprising: (i) a therapeutically effective amount of the culture extract of Paenibacillus comprising at least one of the following compounds:

- and (ii) a pharmaceutically acceptable carrier or excipient.

A sixth embodiment of the invention relates to a composition for the prophylaxis and treatment of plants comprising: (i) an agriculturally effective amount of at least one of the following compounds:

- and (ii) an agriculturally acceptable carrier or substrate.

A seventh embodiment of the invention relates to the use of the lipopeptides of the present invention as growth promoters and as antimicrobial agents in the treatment or prophylaxis of animals intended for human consumption.
An eighth embodiment of the invention relates to uses of the lipopeptides of the present invention as growth promoters and in the treatment or prophylaxis of the infections caused by phytopathogens in plants.
A ninth embodiment of the invention relates to the use of the extract of the culture of Paenibacillus comprising at least one of the following compounds:

- as a growth promoter and in the treatment or prophylaxis of conditions involving infections in animals or plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the FT-MS spectrum of the crude extract produced by P. elgii.

FIG. 2 depicts the formula of Permetina A.

FIG. 3 depicts the spectrum of CID of the ion m/z of 1101.7 of Permetina A.

FIG. 4 depicts the proposed cleavage of Permetina A.

FIG. 5 depicts the structural formula of the lipopeptides a) BMY-28160 and b) Permetina A.

FIG. 6 depicts the structural formula of a) Permetina A and b) novel Compound 1.

FIG. 7 depicts the spectrum of CID of the ion of m/z 1067.7 of the novel compound 1.

FIG. 8 depicts a comparative study of fragmentation patterns for a) compound 1 and b) Permetina A.

FIG. 9 depicts the molecular formula of the lipopeptides a) BMY-28160 and b) Compound 2.

FIG. 10 depicts the spectrum of CID of the ion of m/z 1053.7 corresponding to compound 2.

FIG. 11 depicts a comparative study of fragmentation patterns for the lipopeptides a) BMY-28160 and b) compound 2.

DETAILED DESCRIPTION OF THE INVENTION

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

- “antimicrobial”—a term which means extract and peptides originating from Paenibacillus, preferably Paenibacillus elgii, more preferably Paenibacillus elgii ourofinensis that inhibit, prevent or destroy the growth or proliferation of microbes such as bacteria, fungi, protozoa or similar bodies.
- “growth promoter”—non-nutrient substances originating from Paenibacillus that, when administered to plants by foliar or soil application, or to animals for human consumption orally (for example, incorporated in the feed or water) or by parenteral route, increase the productivity: (a) in the plants through the root vertical growth, extension of leaves, earlier flowering, increased fruit; (b) in the animals, through increased body weight gain, increased feed efficiency, decreased time required for the animal to reach the ideal weight for slaughter, improvement in organoleptic qualities and decreased fat, prevention of infectious or parasitic diseases and decreased mortality.
- “therapeutically effective amount”—the term means an amount of 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 the Paenibacillus extract of the invention; or an amount of the growth promoter agent of bodies which is sufficient to effectively promote the growth and development of these organisms, such as animals and plants.
- “plant pathogen”—a term that means any and every organism that can cause damage and/or illness to plants and includes fungi, prokaryotes (bacteria and mycoplasma), nematodes, protozoa and similar bodies.
- “adjuvant therapy”—a term that corresponds to the agents that aid or increase the action of the active ingredient, or that affect the absorption, the mechanism of action, the metabolism or elimination of the active ingredient in order to increase its effects, in order to fight an infection caused by microbes, that is, the process of growth of organisms such as plants and animals; additionally, this term includes prophylactic activity in the elevation of the immune response developed by the peptides or the Paenibacillus extract of the invention.
- “additive”—a term that means substances added to the composition for the treatment of plants in order to increase efficiency or modify certain properties of the formulation, such as the absorption of the active ingredient, aiming at facilitating the application or minimizing certain problems.
- “pharmaceutically acceptable vehicle or excipient”—a term that means, in a drug composition for therapeutic or prophylactic activity, any and every substance that, at the concentrations present in the dosage form, does not have pharmacological activity, being employed in the composition in order to provide the dosage form with characteristics which ensure its stability, bioavailability, acceptability and ease of administration or application, and which works as carrier of the active ingredient with antimicrobial and/or growth promoter activity of the target animal.
- “agriculturally acceptable carrier or substrate”—a term that means, in a composition for the treatment of plants, any and every substance that has no therapeutic or prophylactic action and serves as a carrier of the active ingredient with antimicrobial and/or growth promoter action of the target plant.

The invention provides new agents with antimicrobial and growth promoter activity of organisms from the identification and isolation of these new agents of Paenibacillus. The identification of these agents led to obtaining the extract of the fermentation broth 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 permits the use of said extract and peptides, among others, to control bacteria and fungi that cause loss in raising of economically important animals (e.g., chickens and pigs). In addition to this antimicrobial activity, said extract and peptides also act as growth promoters of plants and as inhibitors of plant infection by phytopathogens.
At first, the invention is based on the process of obtaining, isolating and identifying new lipopeptide found in bacterial isolate extracts. These new bacterial compounds are capable of producing antimicrobial activity against numerous bacteria and fungi which cause veterinary infections, these strains already being resistant to the current antibiotics. Specifically, this invention provides, in one of its embodiments, the use of supernatant and/or extract of the culture comprising new lipopeptide found in bacterial isolate extracts to control veterinary infections and promote the growth of animals.
As it will be detailed later, after electrophoretic and chromatographic analyzes of the lyophilized sample of the fermentation broth of the new isolate, the separation process provided moieties which were analyzed, and at least two of the lipopeptides responsible for the antimicrobial activity were identified. This identification was performed by sequencing, and the results of this analysis demonstrated that there is no other sequence similar to the ones of the present invention in the databases of proteins and peptides, indicating that new lipopeptides with antimicrobial activity were isolated.
The present invention relates in a first embodiment to the process of obtaining a new bacterial extract having at least one biological activity among antimicrobial activity with respect to microorganisms that cause disease or damage in animals and plants and activity growth promoter of said animals and plants, said new bacterial extract being characterized in that it comprises at least one of the new compounds described below:
The terms “compound” and “lipoprotein” are used interchangeably in the present invention to refer to the new chemical elements found in the extract from Paenibacillus, preferably in the extract from Paenibacillus elgii, more preferably in the extract from Paenibacillus elgii ourofinensis.
The term “bacterial extract” is intended to mean, in this description, the extract obtained from processing Paenibacillus, preferably from fermented Paenibacillus. In a preferred embodiment, the processing of fermented Paenibacillus for the extraction is carried out by methods known in the art such as, for example, liquid-liquid extraction using an organic solvent such as, but not limited to, n-butanol, or by extraction with ion-exchange cartridge.
The present invention also relates to specific peptides having compounds 1 and/or 2 described below:
both having antimicrobial activity and/or promoting the growth of plants and animals, particularly animals such as chickens and pigs.
The lipopeptides of the present invention may be produced by known techniques and obtained in a substantially pure form. For example, lipopeptides can be synthesized manually or in an automated synthesizer, or they may be isolated from the fermentation broth of bacteria Paenibacillus.
The present invention also relates to drug compositions for the treatment and prophylaxis of animals from animals intended for human consumption, said compositions comprising: (i) a therapeutically effective amount of at least one of the compounds of the present invention; and (ii) a pharmaceutically acceptable carrier or excipient; and (iii) optionally, a therapeutic adjuvant. As mentioned before, the lipopeptides of the invention have antimicrobial activity and/or activity growth promoter of said animals.
The invention also relates to a drug composition for the treatment and prophylaxis of animals intended for human consumption, said compositions comprising: (i) a therapeutically effective amount of the culture extract of Paenibacillus having at least one of the compounds of the present invention; (ii) a pharmaceutically acceptable carrier or excipient; (iii) optionally, a therapeutic adjuvant. Said extract can be in the liquid form, corresponding to the liquid phase (supernatant) separated from the fermentation broth, or it may be in the dry state after drying, for example, by lyophilization of said liquid phase.
The drug compositions of the invention contain as active ingredient at least one of the lipopeptides of the present invention, or mixtures thereof, in combination with a pharmaceutically acceptable carrier or diluent, and optionally one or more therapeutic adjuvants. Therefore, the active ingredients according to the invention can be administered individually or together in any conventional dosage/veterinary form of oral or parenteral administration. For oral administration, the composition of the invention may be in the form of a solution, suspension, tablet, pill, capsule, powder, and the solid forms can be of quick release, controlled release or delayed release, or any combination of these types, and said forms may be added to the water or feed of animals.
The tablet form contains various excipients such as sodium citrate, calcium carbonate, calcium phosphate and the like, along with disintegrants such as starch or complex silicates. It is also common the use of binding agents in this dosage form such as polyvinylpyrrolidone, sucrose, gelatin and acacia, and additionally lubricating agents such as magnesium stearate, sodium lauryl sulfate and acacia.
Other appropriate solid dosage/veterinary forms include powders which can be either water dispersible or they may be enclosed in gelatin capsules as hard-type capsules or soft-type capsules. In this case, the excipient includes lactose or polyethylene glycols of varying molecular weights.
In the case of liquid dosage forms for oral administration, sweeteners, flavoring agents, dyes, emulsifying agents or suspending enhancers and diluents may be used in the excipient, such as water, ethanol, propylene glycol, glycerin and combinations thereof.
The pharmaceutical compositions containing the active ingredients of the invention are usually prepared following conventional methods, and are administered in the appropriate dosage/veterinary form.
As an example, the oral solid dosage/veterinary forms may contain, along with the active ingredient, diluents, lubricants, binders, disintegrating agents and others. Examples of diluents that may be used in the pharmaceutical/veterinary compositions of the invention are: lactose, dextrose, saccharose, cellulose, corn starch 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 that may be used in the pharmaceutical/veterinary compositions of the invention are: starches, arabic gum, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone. Examples of disaggregating agents that may be used in the pharmaceutical/veterinary compositions of the invention are: starch, alginic acid, starch or sodium alginates or glycolates; effervescent mixtures; dyes; sweeteners. Additionally, the pharmaceutical/veterinary compositions of the invention can use wetting agents such as lectin, polysorbates, laurylsulphates, and in general, pharmacologically inactive and non-toxic substances commonly used in pharmaceutical/veterinary formulations. The preparation of said pharmaceutical/veterinary compositions of the invention can be carried out in a known manner, for example by means of mixing, granulating, pressing into tablets, sugar-coating, or film-coating processes. The liquid dispersions for oral administration may be e.g. syrups, emulsions and suspensions. In addition to the active ingredient of the present invention, the syrups may contain one or more carrier agents, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol. The suspensions and the emulsions may contain as carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. For parenteral administration, it is conventional to use sterile, pharmaceutically acceptable oils, and propylene glycol may also be employed, as well as sterile aqueous solutions.
The drug compositions of the present invention may also contain one or more therapeutic adjuvants. Examples of such adjuvants are: phosphatide-polysaccharide conjugate as described in U.S. Pat. No. 5,785,975; Veterinary emulsion-based adjuvants, e.g. Emulsigen®, Carbigen®, Polygen®, available from the company MPV Technologies; adjuvants such as the acrylic polymer and the dimethyl dioctadecyl ammonium bromide, disclosed in US2009/0017067; sub-micron oil-in-water squalene emulsion, polyoxyethylene, sorbitan monooleate and sorbitan trioleate, known by the brand name MF59™, available from the company Novartis, and other examples may also be mentioned.
Additionally, the invention also relates to a composition for the prophylaxis and treatment of plants comprising: (i) a therapeutically effective amount of at least one of the lipopeptides of the present invention; (ii) an agriculturally acceptable carrier or substrate; and (iii) optionally, at least one additive.
In another embodiment of the invention, said composition for the treatment and prophylaxis of plants comprises: (i) an agriculturally effective amount of the culture extract of Paenibacillus comprising the lipopeptide of the present invention; (ii) an agriculturally acceptable carrier or substrate; and (iii) optionally, at least one additive.
The formulation of the invention may be in liquid or solid form, for example in the form of a wettable powder. Both formulations, dry or liquid, shall be packed in containers and in conditions that preserve the integrity of the active ingredients. Liquid formulations have lower shelf life, but the dry formulations (e.g., wettable powder) require less stringent conditions, such as storage in a cool place in the absence of light and moisture, and the product may be stored from one crop to another, since packed in freezer or refrigerator.
The formulations of the invention generally contain an amount of active ingredient effective to control microorganisms harmful to plants and/or promote plant growth, said active ingredient comprising the lipopeptides of the present invention described below:

- or mixtures thereof, in combination with an agriculturally acceptable carrier in the form of a solid or liquid diluent, preferably solid. Formulations that may be used include powders, granules, pellets, suspensions, emulsions, wettable powders, flowable solids, and the like, provided that such forms are consistent with the physical chemical properties of the active ingredient and compatible with the lipopeptides of the present invention, with the mode of application and environmental factors such as soil type, humidity and temperature. Formulations in spray may also be used, especially when the application areas are large. The more concentrated compositions are also within the scope of protection of the present invention, and said compositions are suitable for use as intermediates to the diluted final 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 appropriate additives to obtain the desired product characteristics. Furthermore, it should be clear that the active ingredient used in the present invention can also be used alone in special applications where a high concentration of the composition of the invention is required.
Other formulating agents present in minor amounts can be added to the compositions of the invention such as additives to reduce foaming, clumps, corrosion, unwanted microbial growth, and other similar agents. Another essential feature of the formulation of the present invention is the need for ensuring that all ingredients of the composition are mutually compatible and do not contribute to loss of antimicrobial and/or growth promoter activity characteristic of the active ingredient of the invention.
The compositions of the present invention can also be mixed with other agricultural chemicals, fertilizers, soil conditioners, stabilizers, or with one or more insecticides, fungicides, acaricides, or other biologically active compounds to form a multicomponent formulation. Examples of other protection products in agriculture as the active ingredient of the formulation of the present invention that may be mixed are insecticide-type sodium channel agonist (e.g., pyrethroid), sodium channel blocking agents (for example, pyrazolines), acetylcholinesterase inhibitors (e.g., organophosphates and carbamates), nicotinic acetylcholine binding agents, gabaergic binders, agonists or antagonists of octapine (e.g., formamidine) insecticides from the pyrrole group and others.
Below is presented by way of example the isolation of the compounds comprised in the Paenibacillus extract. Among these are described and characterized the new and inventive compounds of the present invention. However, it should be understood that this example is provided for illustrative purposes only and that various modifications or changes in light of the embodiments disclosed herein are suggestive to those skilled in the art and are to be included within the spirit and scope of this disclosure and the scope of the accompanying claims.

EXAMPLE

The organic extract obtained by liquid-liquid extraction of Paenibacillus elgii fermented in n-butanol shows a mixture of 5 lipopeptides belonging to the class of Permetinas. Among these, compounds of m/z 1067.7 and 1053.7 are unpublished. The signal of m/z 1101.7 was assigned to the presence of permetina A, the signal of m/z 1087.7 was assigned to the presence of compound BMY-28160, and the signal of m/z 1073.7 was assigned to the compound Pelgipeptin A. These compounds were characterized by ultra resolution mass spectrometry using a spectrometer FT-ICR-MS and collision-induced dissociation experiments (CID). These two experiments confirm the proposed structure for all lipopeptides present in the organic extract produced by Paenibacillus elgii, as detailed below.
2.1. Structural Characterization of Lipopeptides Produced by Paenibacillus elgii.
The organic extract obtained by liquid-liquid extraction of fermented Paenibacillus elgii in n-butanol was analyzed by ultrasonic resolution mass spectrometry. The spectrum obtained is shown in FIG. 1.
The signals of m/z observed in the mass spectrum of FIG. 1 were characterized as belonging to the class of Permetinas. Permetinas are lipopeptide containing 9 amino acids linked to a lipid chain of a 6-carbon β-hydroxy acid.
The signal of m/z 1101.7024 was assigned to the presence of Permetina A (FIG. 2). The signal was obtained with a variation of ΔMM=0.5 ppm, which is in accordance with the structural formula proposed for Permetina A.
The signal of m/z 1101.7 was subjected to sequence mass spectrometry experiments (MS/MS) for fragmentation and structural characterization studies. The spectrum of collision-induced fragmentation (ICD) is shown in FIG. 3.
The spectrum of MS/MS obtained for the ion of m/z 1101.7 shown in FIG. 3 is identical to the spectrum obtained by Wu and colleagues in the characterization studies of permetina A. The major ion fragments which show the presence of Permetina A are shown in FIG. 4.
The signal of m/z 1087.6868, which was attributed to the presence of the lipopeptide BMY-28160, is a lipopeptide analogue of Permetina A. In compound BMY-28160 the amino acid at position 2 is Valine (Val), different from Permetina A, where the amino acid in this second position is a Leucine/Isoleucine (Leu/Ile) as shown in FIG. 5.
Signals of m/z 1073.6734, 1067.7193 and 1053.7042 were also observed in the mass spectrum shown in FIG. 1. Wu and colleagues²have identified the signal of m/z 1073.6734, which was assigned to a lipopeptide analog of BMY-28160 with a difference of 14 Da, which was assigned as a methylene group (—CH₂—).
However, the signs of m/z 1067.7193 and 1053.7042 were first identified in this crude extract, and are considered novel lipopeptides.
The signal of m/z 1067.7193 was attributed to the presence of the lipopeptide analogue of Permetina A (FIG. 6). The signal was obtained with a variation of ΔMM=0.5 ppm, which is in accordance with the molecular formula proposed for Compound 1.
Experiments of MS/MS were performed for the structural characterization and sequencing of the amino acids present in the compound 1. The spectrum obtained in this CID experiment is shown in FIG. 7.
Based on the fragmentation patterns, the amino acid sequence of compound 1 was characterized as an analog of Permetina A in which the amino acid phenylalanine (Phe) in position 4 of Permetina A was replaced by the amino acid leucine/isoleucine (Leu/Ile) in FIG. 6
A comparative study of fragmentation patterns for Permetina A and compound 1 is presented in FIG. 8.
The formation of fragment ions of m/ z 327 and 440 in the CID spectrum of compound 1 (FIG. 7) confirm the presence of the amino acid leucine/isoleucine at positions 2 and 4.
The signal of m/z 1053.7 observed in the mass spectrum of the crude extract (FIG. 1) was assigned to a second novel compound. This compound was characterized as being a lipopeptide analogue to lipopeptide BMY-28160. The signal was obtained with a variation of ΔMM=1.1 ppm, which is in accordance with the molecular formula proposed for compound 2.
The structural difference between the two compounds is that for the lipopeptide BMY-28160 the amino acid at position 4 is a Phenylalanine (Phe) and for the novel compound the amino acid at position 4 is a leucine/isoleucine (Leu/Ile) as shown in FIG. 9.
The signal of m/z 1053.7 was subjected to CID experiments with the aim of obtaining information about the amino acid sequencing of this new compound. The fragmentation spectrum obtained is shown in FIG. 10.
The fragments of ion of m/ z 227 and 426 observed in the spectrum of FIG. 10 as well as the comparative study of the fragmentation patterns between lipopeptide BMY-28160 and compound 2 shown in FIG. 11 confirm the structural assignment presented for compound 2 in FIG. 9.
All publications and patent applications mentioned in the specification 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 way of reference to the same extent as if each individual publication or each patent application were specifically and individually indicated to be incorporated by way of reference.
Although the foregoing invention has been described in some detail by means of illustration and examples for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the claims accompanying this description.

Claims

1. A process of obtaining an extract with lipopeptides comprising fermenting Paenibacillus and extracting at least one of the following compounds:

2. A process according to claim 1, wherein said Paenibacillus is Paenibacillus elgii and/or Paenibacillus elgii ourofinensis.

3. A bacterial extract of Paenibacillus comprising at least one of the following compounds:

4. A lipopeptide isolated with antimicrobial activity and/or growth promoter comprising at least one of the following compounds:

5. A drug composition for treatment and/or prophylaxis of animals comprising the lipopeptide of claim 4:

a pharmaceutically acceptable carrier or excipient.

6. A composition according to claim 5, comprising extracts of material fermented with Paenibacillus.

7. A composition according to claim 6, wherein said Paenibacillus is Paenibacillus elgii and/or Paenibacillus elgii ourofinensis.

8. A drug composition for treatment and/or prophylaxis of animals comprising the bacterial extract of Paenibacillus of claim 3 and:

a pharmaceutically acceptable carrier or excipient.

9. A composition according to claim 8, wherein said Paenibacillus is Paenibacillus elgii and/or Paenibacillus elgii ourofinensis.

10. A composition for the prophylaxis and treatment of plants comprising the lipopeptide of claim 4 and

an agriculturally acceptable carrier or substrate.

11. A composition according to claim 10, comprising extracts of material fermented with Paenibacillus.

12. A composition according to claim 11, wherein said Paenibacillus is Paenibacillus elgii and/or Paenibacillus elgii ourofinensis.

13. A method of treatment or prophylaxis of animals intended for human consumption, the method comprising the use of at least one of the lipopeptides of claim 4

as a growth promoter or antimicrobial agent.

14. A method for the treatment or prophylaxis of infections caused by phytopathogens in plants, the method comprising the use of at least one of the lipopeptides of claim 4

as a growth promoter or antimicrobial agent.

15. A method for the treatment or prophylaxis of conditions involving infections in animals or plants, the method comprising the use of at least one of the lipopeptides of claim 4

as a growth promoter or antimicrobial agent.