MX2008013429A - Novel enterococcus and streptococcus strains and bacteriocins. - Google Patents

Abstract

Novel Enterococcus and Streptococcus bacteriocins produced by novel Enterococcus and Streptococcus strains are used for at least reducing the levels of colonization by at least one target bacteria in animals, especially poultry.

Description

NOVEDOUS CEPAS OF ENTEROCOCCUS AND STREPTOCOCCUS AND BACTERIOCINES BACKGROUND OF THE INVENTION Field of the Invention This invention relates to the control of diseases in animals, especially poultry, through the use of novel strains of Enterococcus and Streptococcus that produce bacteriocins and / or bacteriocins produced through these strains. It also refers to novel bacteriocins, amino acid sequences of novel bacteriocins, to strains of Enterococcus and Streptococcus that produce novel bacteriocins, and to Lactococcus-inducing strains. In addition, the invention relates to therapeutic compositions containing the novel bacteriocins and / or strains of Enterococcus or Streptococcus that produce them and to the uses of the therapeutic compositions.

Description of the Related Art The consumption of improperly prepared poultry products has resulted in intestinal diseases in humans. It has long been recognized that Salmonella spp. It is the causative agent of these diseases and more recently, Campylobacter spp., Especially Campylobacter jejuni, has also been implicated. Both of them ? microorganisms can colonize the gastrointestinal tracts of poultry without any harmful effect on birds, and although some colonized birds can be detected, asymptomatic carriers can freely expand during production and processing, giving as additional contamination to both live and corpses Poultry serve as the primary reservoir for Salmonella and Cam pylobacter in the food supply (Jones et al., Journal of Food Protection, Volume 54, No. 7, 502-507, July 1991). The prevention of colonization in live poultry during growth and production can reduce the problem of contamination of poultry. A number of factors contribute to the colonization and continuous presence of bacteria within the digestive tract of animals. These factors have been extensively reviewed by Savage (Progress in Food and Nutrition Science, Volume 7, 65-74, 1983). Among these factors are: (1) gastric acidity (Gilliland, Journal of Food Production, Volume 42, 164-167, 1979); (2) bile salts (Sharpe &Mattick, Milchwissenschaft, Volume 12, 348-349, 1967; Floch et al., American Journal of Clinical Nutrition, Volume 25, 1418-1426, 1972; Lewis &Gorbach, Archives of Internal Medicine, Volume 130, 545-549, 1972; Gilliland and Speck, Journal of Food Protection, Volume 40, 820-823, 1977); Hugdahl et al., Infection and Immunity, Volume 56, 1560-1566, 1988); (3) peristalsis; (4) digestive enzymes (Marmur, Journal of Molecular Biology, Volume 3, 208-218, 1961); (5) immune response; and (6) innate microorganisms and the antibacterial compounds that produce them. The first four factors depend on the host phenotype and may not be practically controllable variables. The immune response in the gastrointestinal tract (Gl) is not easily modulated. The factors that involve innate microorganisms and their metabolites depend on the normal flora of the gastrointestinal tract. A potential aspect to control the colonization of Campyiobacter and / or Salmonella is through the use of competitive exclusion (CE). Nurnii and Rantala (Nature, Volume 241, 210-211, 1973) demonstrated effective control of Salmonella infection by giving bacteria such as fattening of intestinal materials from healthy poultry to young chickens whose microflora has not yet been established, against colonization of Salmonella . The administration of CE preparations or defined to chickens accelerates the maturation of the intestinal flora in recently hatched birds and provides a substitute for the natural process of transmission of the microflora from the adult chicken to its progeny. Laboratory results and field investigations provide evidence of control benefits of Campyiobacter through the administration of normal microflora to chickens; reduced frequency of flocks infected by Campyiobacter (Mulder and Bolder, IN: Colonization Control of human bacterial enteropathogens in poultry; LC Blankenship (ed.), Academic Press, San Diego, Calif., 359-363, 1991) and levels have been reported Reduced from Campyiobacter jejuni (C. jejuni) in the feces of colonized birds (Stern, Poultry Science, Volume 73, 402-407, 1994). Schoeni and Wong (Appl. Environ Microbiol., Volume 60, 1191-1197, 1994) reported a significant reduction in colonization of C. jejuni in young chickens through the application of carbohydrate supplements along with three antagonists identified: Citrobacter diversus 22, Klebsiella pneumoniae 23, and Escherichia coli 25. There is also evidence of a significant reduction of C. jejuni in intestinal samples from infected young chickens after treatment with cultures, isolates from poultry, Lactobacillus acidophilus and Streptococcus faecium (Morishita et al. al., Avian Diseases, Volume 41, 850-855, 1997). Snoeynbos et al. (U.S. Patent No. 4,335,107, June 1982) developed a competitive exclusion (EC) microflora technique to prevent colonization of Salmonella by lyophilizing faecal droppings and cultivating this preparation anaerobically. Mikola et al. (U.S. Patent No. 4,657,762, April, 1987) used fecal and caecal contents as a source of EC microflora to prevent colonization of Salmonella. Stern et al. (U.S. Patent No. 5,451, 400, September, 1995, and U.S. Patent No. 6,241,335, April, 2001) describe a mucosal CE composition for the protection of poultry and livestock against colonization by Salmonella and Campylobacter. , where the pre-washed cecal mucin layer is discarded and the waste, maintained in an oxygen-free environment, is grown anaerobically. Nisbet et al. (U.S. Patent No. 5,478,557, December, 1996) describe a defined probiotic that can be obtained from a variety of domestic animals, which is obtained through continuous cultivation of a batch culture produced directly from droppings. Fecal, cecal contents and / or large intestine of the target adult animal. The microorganisms produce a variety of compounds, which demonstrate anti-bacterial properties. A group of these compounds, bacteriocins, consists of bactericidal proteins with a mechanism of action similar to ionophore antibiotics. Bacteriocins are usually active against species that are closely related to the producer. Its well-disseminated occurrence in bacterial species isolated from complex microbial communities such as the intestinal tract, oral surfaces and other epithelial ones, suggests that bacteriocins may have a regulatory role in terms of population dynamics within bacterial ecosystems. Bacteriocins are defined as compounds produced through bacteria that have a portion of biologically active protein and bactericidal action (Tagg et al., Bacteriological Reviews, Volume 40, 722-256, 1976). Other features may include: (1) a narrow spectrum inhibitor of activity centered around closely related species; (2) binding to cell-specific receptors; and (3) genetic determinants of plasmid origin of bacteriocin production and cell bacteriocin immunity Guest. The incompletely undefined antagonistic substances have been termed "bacteriocin-like substances". Some effective bacteriocins against Gram-positive bacteria, in contrast to the great-negative bacteria, have a broader spectrum of activity. It has been suggested that the term bacteriocin, when used to describe inhibitory agents produced against Gram-positive bacteria, must meet the minimum criteria of (1) being a peptide and (2) possessing bactericidal activity (Tagg et al, supra). Lactic acid bacteria are among the most important probiotic microorganisms. They are Gram-positive organisms, without spores, catalase negative lacking cytochromes. They are anaerobic but are aerotolerant, annoying, acid-tolerant, and strictly fermentative with lactic acid as the main final product of sugar fermentation. Bacteria that produce lactic acid include Lactobacillus species, Bifidobacterium species, Enterococcus species, Enterococcus faecium species, Lactococcus lactic, Streptococcus cricetus, Leuconostoc mesenteroides, Pediococcus acidilactici, Sporolactobacillus inulinus, Streptococcus thermophilus, etc. These species are of particular interest in terms of well-disseminated occurrence of bacteriocins within the group and are also of wide use throughout the fermented dairy, food and meat processing industries. Its role in the conservation and flavor characteristics of food has been well documented. Most of the Bacteriocins produced by this group are only active against other lactic acid bacteria, but several exhibit antibacterial activity towards more phylogenetically distant Gram-positive bacteria, and under certain circumstances, Gram-negative bacteria. Lactobacilli have been extensively studied for the production of antagonists. These include well-characterized bacteriocins (DeKIerk, Nature, Volume 214, 609, 1967; Upreti and Hinsdill, Anticicrob., Agents Chemother., Volume 7, 139-145, 1975; Barefoot and Klaenhammer, Antimicrob. Agents Chemother., Volume 45, 1808-1815, 1983; Joerger and Klaenhammer, Journal of Bacteriology, Volume 167, 439-446, 1986), bacteriocin-type potency substances (Vincent et al., Journal of Bacteriol., Volume 78, 479, 1959), and others. antagonists not necessarily related to bacteriocins (Vakil and Shahani, Bacteriology, Proc. 9, 1965; Hamdan and Mikolajcik, Journal of Antibiotics, Volume 8, 631-636, 1974; Mikolajcik and Hamdan ultured Dairy Products, Pag. 10, 1975; Shahani et al., Cultured Dairy Products Journal, Volume 11, 14-17, 1976). Klaenhammer (FEMS, Microbiol. Rev., Volume 12, 39-86, 1993) has classified the bacteriocins of lactic acid bacteria known to date in four major groups: Group I: Lantibiotics, which are small peptides of < 5 kDa containing the unusual amino acids, lanthionine and β-methyl lanthionine. They are of particular interest since they have a very broad spectrum of activity in relation to other bacteriocins. The examples include, Nisin, Nisin Z, carnocin U 149, lacticin 481 and lactocin 5. Group II: peptides that do not contain small lanthionine: a heterogeneous group of small peptides < 10 kDa. This group includes peptides against Listeria ssp. Group III: large heat-labile proteins of > 30 kDa. An example is Helveticin. Group IV: proteins of complex bacteriocins containing additional portions such as lipids and carbohydrates. Raczek (U.S. Patent No. US2002 / 0176, published on November 2002) describes the use of a composition containing live or dead microorganisms, which secrete bacteriocins, or the same bacteriocins or in combinations thereof, for use with forages for agricultural livestock. Several species of Enterococcus and Streptococcus that produce bacteriocins and their bacteriocins have been described in the related art. However, the present invention provides novel compositions containing at least one novel strain of an Enterococcus or Streptococcus and / or at least one novel bacteriocin produced by the novel strains; a method for using the strain and / or bacteriocin, the novel strains, amino acid sequences for novel bacteriocins, and methods of use, all of these are different from the strains of the related art, bacteriocins and methods of use.

BRIEF DESCRIPTION OF THE INVENTION It is therefore an object of the present invention to provide at least one novel strain of Enterococcus or Streptococcus that produces novel bacteriocins. A further object of the present invention is to provide a novel strain of Streptococcus cricetus having the identification characteristics of NRRL B-30745. A further object of the present invention is to provide a novel strain of Enterococcus faecium having the identification characteristics of NRRL B-30746. Another object of the present invention is to provide novel bacteriocins produced by the novel strains of Enterococcus or Streptococcus. A still further object of the present invention is to provide a novel bacteriocin 50-52 having an amino acid sequence set forth in SEQ ID NO 1. Yet another object of the present invention is to provide a novel bacteriocin 760 having an amino acid sequence established in SEQ ID NO 2. An additional object of the present invention is to provide for at least reducing colonization levels through at least one target bacterium in animals, by administering to the animal a therapeutic composition that includes at least one novel strain of Enterococcus Streptococcus that produces a novel bacteriocin, at least one novel bacteriocin produced by a novel strain of Enterococcus or Streptococcus, or a combination of novel strains and / or novel bacteriocins. Yet another object of the present invention is to provide a method for at least reducing colonization levels through at least one target bacterium in animals by administering to the animal a therapeutic composition that includes a novel Enterococcus strain having the identification characteristics of NRRL No. Deposit B-30746, a novel strain of Streptococcus having the identification characteristics of NRRL No. B-30745, and mixtures thereof. A further object of the present invention is to provide a method for at least reducing levels of colonization through at least one target bacterium in animals, by administering to the animal a therapeutic composition that includes a novel bacteriocin 50-52 having an amino acid sequence as set forth in SEQ ID NO 1. A further object of the present invention is to provide a method for at least reducing colonization levels through at least one target bacterium in animals, by administering to the animal a therapeutic composition that includes a novel bacteriocin 760 having an amino acid sequence set forth in SEQ ID NO 2. Another object of the present invention is to provide a method to at least reduce colonization levels through at least one target bacterium in an animal, by administering to the animal a therapeutic composition comprising a bacteriocin produced through a novel strain of Enterococcus having the identification characteristics NRRL B-30746 , a novel strain of Streptococcus having the identification characteristics of NRRL B-30745, and mixtures thereof. Another object of the present invention is to provide a Lactococcus-inducing strain, which increases the production of bacteriocins through producing strains. A further object of the present invention is to provide an inducing strain of Lactococcus lactis having the identification characteristics of NRRL B-30744. A further object of the present invention is to provide a method for increasing the production of bacteriocins through producer strains, wherein the producer strains are co-cultured with a Lactococcus-inducing strain. Yet another object of the present invention is to provide a method for increasing the production of bacteriocins through producing strains, wherein said producing strains are co-cultured with a strain of Lactococcus having the identification characteristics of NRRL B-30744. A further object of the present invention is to provide a method for purifying bacteriocins that includes harvesting the culture fluid and the cells as separate samples, isolate the bacteriocin that has been adsorbed on the cell surface of both producing and inducing strains through the elution with pH buffer of sodium chloride-containing phosphate followed by an isolation step of ion exchange chromatography of the bacteriocin in the culture fluid through hydrophobic interaction chromatography. Other objects and advantages of the invention will be apparent from the following description.

Deposit of the Microorganisms Streptococcus cricetus, designated NRRL B-30745 (Strain 760); Enterococcus faecium designated NRRL B-30746 (Strain 50-52, and Lactococcus lactis designated NRRL B-30744 (Strain 320) have been deposited in accordance with the provisions of the Budapest Treaty on May 3, 2004 with USDA Agricultural Research Service Patent Culture Collection (National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, Illinois 61604).

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1B are photographs showing the direct detection of bacteriocin 760 after SDS-PAGE (1A) and isoelectric focusing (1B). Figures 2A and 2B are photographs showing the direct detection of bacteriocin 50-52 after SDS-PAGE (2A) and focusing isoelectric (2B).

DETAILED DESCRIPTION OF THE INVENTION The importance of enteric infections in humans has been greatly recognized. The relationship of contamination of poultry and infection in humans is well documented. The ability to reduce this health hazard through interventions in poultry processing plants is also well known. During the production and processing of tender chicken, faecal materials containing pathogens are transferred into the meat and persist in the kitchens that process food. The metabolites of competing organisms may contribute to the control of taled pathogens such as Campyiobacter jejuni and Salmonella. The novel antagonistic strains were isolated from caecal surfaces and from the mucous membranes of young chickens. The native components of the antagonists characterized with low molecular weight peptides, bacteriocins, which have a broad spectrum of antagonistic activity. The present invention provides a novel strain of Enterococcus, a novel strain of Streptococcus, a novel strain of Lactococcus, novel bacteriocins, amino acid sequences of said bacteriocins, therapeutic compositions containing the novel bacteriocins and / or strains that produce them, and methods for using the novel therapeutic compositions. The present invention also provides a method for the production and purification of novel bacteriocins. Enterococcus faecium, NRRL B-30746, in an facultative aerobic with highly positive cocci, and is capable of growing at approximately 37 ° C. The strain grows on irregularly shaped edges producing nutrient or plaque account agar. The colonies have a diameter of about 2 mm after the microaerophilic culture for about 24 hours at about 37 ° C. Streptococcus cricetus, NRRL B-30745, is an facultative aerobic with gram-positive cocci, and is capable of growing at approximately 37 ° C. The strain grows on edges with regular form nutrient or plate count agar producers. The colonies have a diameter of about 1 mm after microaerophilic culture for about 24 hours at about 37 ° C. The classification of strains of Enterococcus and Streptococcus isolated for the production of bacteriocin activity was performed on nutrient agar seeded with different target bacteria of interest. Other test strains were cultured under aerobic conditions at approximately 37 ° C for approximately 18-24 hours. Yersinia enterocolitica and Y. pseudotuberculosis were cultured at approximately 28 ° C under aerobic conditions for approximately 18-24 hours. The tests for activity against Campylobacter jejuni were carried out in C. jejuni seeded on agar from Campylobacter containing approximately 5% of blood used. The use of blood is within the ordinary experience in the art and includes for example, of sheep, horse, etc. The tests for activity against Campylobacter jejuni are performed under microaerobic conditions of about 5% 02, about 10% C02, and about 85% N2 for about 24-48 hours at about 42 ° C. Approximately 0.1 ml of the antagonistic bacteria suspended in normal saline were plated on MRS agar and incubated for approximately 24-48 hours. MRS agar cubes of approximately 0.5 cm3 were cut and transferred to Brucella or Campylobacter agar supplemented with lysed blood, approximately 10 micrograms / ml rifamycin, approximately 2.4 U / ml polymyxin, and seeded with approximately 107 Campylobacter cells. jejuni Plates were incubated at approximately 42 ° C for approximately 24-48 hours under microaerobic conditions. The activity was evaluated by measuring the areas of growth inhibition. The isolates found as antagonists were evaluated for the production of bacteriocin. Crude antimicrobial preparations (CAPs) were prepared by precipitation of ammonium sulfate from cultures of antagonistic strains developed in approximately 10% Brucella broth together with an enhancing amount of Lactobacillus lactis bacteriocin (Strain 320; NRRL B-30744 ) used as an inductor, to approximately 37 ° C for approximately 14 hours under aerobic conditions. An enhancer amount of the inducer bacteriocin is defined as the amount of inducing bacteria required to at least increase the production of bacteriocin from a producer strain as compared to the producer strain grown without the inducing strain. An example of concentration of inducer to producer strain in co-culture is approximately 10: 1 (inducer: producer). The cultures were then centrifuged at approximately 2,500 X g for approximately 10 minutes. Antagonistic peptides were isolated from the supernatant through a combination of ammonium sulfate precipitation, gel filtration using Superóse 12 HR, cation ion exchange chromatography using Sepharose SP FF. With co-cultures, secreted bacteriocins can be adsorbed on the cell surface of both producing and inducing cells. In order to harvest these bacteriocins, the cell pellet of the centrifugation step is mixed with an elution pH regulator which is made of phosphate pH regulator with about 0.7% NaCl, pH of about 8.0. The suspension was mixed and incubated for approximately 20 minutes followed by centrifugation at approximately 10,000 g for approximately 15 minutes. The bacteriocin was isolated from the supernatant through ion exchange chromatography on SuperOse SP FF. The molecular weights of the peptides are determined by SDS-PAGE electrophoresis. The values of pls of the peptides were determined through isoelectric focusing. The amino acid sequences were determined through Edman degradation using, for example, an automatic 491 cLC sequencer (Applied Biosystems, Inc.). For the purposes of the present invention, the term "peptide" means a compound of at least two or more amino acids or amino acid analogues. The amino acids or amino acid analogs can be linked through peptide bonds. In another embodiment, the amino acids can be linked through other bonds, for example, ester, ether, etc. The peptides can be in any structural configuration including linear, branched or cyclic configurations. As used herein, the term "amino acids" refers to either natural or synthetic amino acids, including the optical isomers of both D and L, and amino acid analogs. The peptide derivatives and analogs of the present invention include, but are not limited to, those which contain, as a primary amino acid sequence, all or part of the amino acid sequence of the peptide including altered sequences wherein the amino acid residues are functionally equivalents are substituted by residues within the sequence that result in conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be replaced by another amino acid of a similar polarity, which acts as a functional equivalent, giving as a result a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The amino acids that contain aromatic ring structures are phenylalanine, tryptophan and tyrosine. Polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include Arginine, Usin, and Histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. It is not expected that such alterations significantly affect the apparent molecular weight, as determined by polyacrylamide gel electrophoresis or isoelectric point. Non-conservative amino acid substitutions can also be introduced to substitute an amino acid with a particularly preferred property. For example, Cys can be introduced at a potential site for disulfide bridges with another Cys. You can enter Pro due to its particularly flat structure. The peptides of the present invention can be chemically synthesized. Synthetic peptides can be prepared using the well known techniques of peptide condensation, solid phase, or liquid phase techniques, or any combination thereof, and can include natural and / or synthetic amino acids. The amino acids used for peptide synthesis can be resin from Boc amino acid (Na-t-butyloxycarbonyl N-protected amino) with the standard deprotection, neutralization, coupling and washing protocols of the original Merrifield solid phase process (J. Am. Chem. Soc, Volume 85, 2149-2154 , 1963), or the protected labile-based Na-amino acid 9-fluorenylmethoxycarbonyl (Fmoc) (Carpino and Han, J. Org. Chem., Volume 37, 3403-3409, 1972). In addition, the method of the present invention can be used with other Na-protected groups that are familiar to those skilled in the art. Solid phase peptide synthesis can be achieved through techniques within ordinary skill in the art (See, for example, Stewart and Young, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, III, 1984; and Noble, Int. J. Pept. Protein Res., Volume 35, 161-214, 1990), or using automatic synthesizers. In accordance with the present invention, peptides and / or novel bacterial strains can be administered in a therapeutically acceptable, topical, parenteral, transmucosally, such as orally, nasally, rectally or transdermally vehicle. The peptides of the present invention can be modified if necessary to increase the ability of the peptide to cross cell membranes such as by increasing the hydrophobic nature of the peptide, introducing the peptide as a conjugate to a carrier, such as a ligand to a specific receptor, etc. The present invention also provides conjugating a target molecule to a peptide of the invention. The molecules of activation for the purposes of the present invention represent a molecule that when administered in vivo, is located at a desired location or locations. In various embodiments of the present invention, the activation molecule may be a peptide or protein, antibody, lectin, carbohydrate or steroid. The activation molecule can be a peptide ligand of a receptor in the target cell or an antibody such as a monoclonal antibody. To facilitate entanglement, the antibody can be reduced to two heavy and light chain hetero-dimers, or the F (ab ') 2 fragment can be reduced, and crosslinked to the peptide via the reduced sulfhydryl. Another aspect of the present invention is to provide therapeutic compositions. The compositions may be for oral, nasal, pulmonary, injection, etc. administration. The therapeutic compositions include effective amounts of at least one bacteriocin of the present invention and its derivatives and / or at least one novel strain to at least reduce colonization levels through at least one objective bacteria together with diluents, preservatives , solubilizers, emulsifiers, auxiliaries and / or acceptable carriers. The diluents may include pH regulators such as Tris-HCl, acetate, phosphate, for example; the additives may include detergents and solubilizing agents such as Tween 80, Polysorbate 80, etc., for example; antioxidants include, for example, ascorbic acid, sodium metabisulfite, etc .; Conservatives may include, for example, Thimersol, alcohol benzyl, etc .; and substances that provide volume such as lactose, mannitol, etc. The therapeutic composition of the present invention can be incorporated into the preparation into particles of polymeric compounds such as polyvinylpyrrolidone, polylactic acid, polyglycolic acid, etc., or in liposomes. The liposomal encapsuiation includes encapsuination through several polymers. A wide variety of polymeric carriers can be used to contain and / or deliver one or more of the therapeutic agents presented above, including, for example, both biodegradable and non-biodegradable compositions. Representative examples of biodegradable compositions include albumin, collagen, gelatin, hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropylcellulose), hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate), casein, dextrans, polysaccharides, fibrinogen, po Ii (D, L lactide), poly (D, L-lactide-co- glycolide), poly (glycolide), poly (hydroxybutyrate), poly (alkylcarbonate) and poly (orthoesters), polyesters, po I i (hydroxyvaleric acid), polydioxanone, po Ii (ref st ethylene lat), pol i (malic acid), poly (tartronic acid), polyanhydrides, polyphosphazenes, poM (amino acids) and their copolymers (see generally, llium, L., Davids, SS (eds.) "Polymers in Controlled Drug Delivery" Wright, Bristol , 1987; Arshady, J. Controlled Reeléase 17: 1 -22, 1991; Pitt, M. J. Phar. 59: 173-196, 1990; Holland et al., J. Controlled Relay 4: 155-0180, 1986). Representative examples of non-degradable polymers include copolymers of poly (ethylene-vinyl acetate) ("EVA"), silicone rubber, acrylic polymers (polyacrylic acid, polymethacrylic acid, polymethylmethacrylate, polyalkylene cyanoacrylate), polyethylene, polypropylene, polyamides (nylon 6). , 6), polyurethane, poly (urethane urethanes), poly (ether urethanes), poly (ester urea), polyethers (poly (ethylene oxide), pluronics and poly (tetramethylene glycol)), silicone rubbers and vinyl polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate phthalate. Polymers that are both anionic (e.g., alginate, carrageenan, carboxymethylcellulose and polyl (acrylic acid), and cationic (e.g., chitosan, poly-L-lysine, polyethyleneimine, and poly (allylamine)) can also be developed ( see generally, Dunn et al., J. Applied Polymer Sci. 50: 353-365, 1993; Cascone et al., J. Materials Sci .: Materials in Medicine 5: 770-774, 1994; Shiraishi et al., Biol. Pharm. Bull 16 (11): 1164-1168, 1993, Thacharodi and Rao, Infl J. Pharm. 120: 115-118, 1995, Miyazaki et al., Infl J. Pharm. 118: 257-263, 1995) Polymeric vehicles can be developed in a variety of ways, with desired release characteristics and / or with specific desired properties For example, polymeric carriers can be designed to release a therapeutic agent after exposure to an activation event. specific such as pH (see, for example, Heller et al., "Chemcally Self-Regulated Drug Delivery Systems," in Polyme rs in Medicine III, Elsevier Science Publishers B. V., Amsterdam, 1988, pp. 175-188; Kang et al., J. Applied Polymer Sci. 48: 343-354, 1993; Dong et al., J. Controlled Release 19: 171-178, 1992; Dong and Hoffman, J. Controlled Release 15: 141-152, 1991; Kim et al., J. Controlled Reléase 28: 143-152, 1994; Cornejo-Bravo et al., J. Controlled Reléase 33: 223-229, 1995; Wu and Lee, Pharm. Res. 10 (10): 1544-1547, 1993; Serres et al., Pharm. Res. 13 (2): 196-201, 1996; Peppas, "Fundamentals of pH- and Temperature-Sensitive Delivery Systems," in Gurny et al. (eds.), Pulsatile Drug Delivery, Wissenschaftliche Verlagsgesellschaft GmbH, Stuttgart, 1993, pp. 41-55; Doelker, "Cellulose Derivatives," 1993, in Peppas and Langer (eds), Biopolymers I, Springer-Verlag, Berlin). Representative examples of pH-sensitive polymers include poly (acrylic acid) and its derivatives (including, for example, homopolymers such as poly (aminocarboxylic acid), poly (acrylic acid), poly (methylacrylic acid), copolymers. of said homopolymers, and poly (acrylic acid) copolymers and acrylonomers such as those discussed above Other pH sensitive polymers include polysaccharides such as cellulose acetate phthalate; hydroxypropylmethylcellulose phthalate; hydroxypropylmethyl cellulose acetate succinate; cellulose and chitosan Other more pH-sensitive polymers include any mixture of a pH-sensitive polymer and a water-soluble polymer, and polymeric vehicles that are sensitive to temperature can be designed (see, for example, Chen et al. , "Novel Hydrogels of a Temperature-Sensitive Pluronic Grafted to a Bioadhesive Polyacrylic Acid Backbone for Vaginal Drug Delivery, "in Proceed, Intern Symp.Control, Bioactive Relativity, Mater. 22: 167-168, Controlled Relay Society, Inc., 1995; Okano, "Molecular Design of Stimuli-Responsive Hydrogels for Temporary Controlled Drug Delivery," in Proceed, Intern Symp.Control, Rei. Bioact .Mater.22: 111-112, Controlled Relay Society, Inc., 1995; Johnston et al. ., Pharm. Res. 9 (3): 425-433, 1992; Tung, Int'l J. Pharm. 107: 85-90, 1994; Harsh and Gehrke, J. Controlled Release 17: 175-186, 1991; Bae et al., Pharm. Res. 8 (4): 531-537, 1991; Dinarvand and D'Emanuele, J. Controlled Release 36: 221-227, 1995; Yu and Grainger, "Novel Thermo-sensitive Amphiphilic Gels: PolyN-isopropylacrylamide-co-sodium acrylate-co-nN-alkylacrylamide Network Synthesis and Physicochemical Characterization, "Dept. of Chemical &; Biological S c i. , Oregon Graduate Institute of Science & Technology, Beaverton, OR, pp. 820-821; Zhou and Smid, "Physical Hydro gels of Associative Star Polymers," Polymer Research Institute, Dept. of Chemistry, College of Environmental Science and Forestry, State Univ. Of New York, Syracuse, N. Y., pp. 822-823; Hoffman et al., "Characterizing Pore Sizes and Water" Structure 'in Stimuli-Responsive Hydrogels, "Center for Bioengineering, Univ. Of Washington, Seattle, Wash., P.828; Yu and Grainger," Thermo-sensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks: Cationic, Anionic and Ampholytic Hydrogels, "Dept. of Chemical &Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton, OR, pp. 829-830; Kim et al., Pharm. Res. 9 (3): 283-290, 1992; Bae et al., Pharm. Res. 8 (5): 624-628, 1991; Kono et al., J. Controlled Reléase 30: 69-75, 1994; Yoshida et al., J. Controlled Reléase 32: 97-102, 1994; Okano et al., J. Controlled Reléase 36: 125-133, 1995; Chun and Kim, J. Controlled Reeléase 38: 39-47, 1996; D'Emanuele and Dinarvand, Int'l J. Pharm. 118: 237-242, 1995; Katono et al., J. Controlled Release 16: 215-228, 1991; Hoffman, "Thermally Reversible Hydrogels Containing Biologically Active Species," in Migliaresi et al. (eds.), Polymers in Medicine MI, Elsevier Science Publishers B.V., Amsterdam, 1988, pp. 161-167; Hoffman, "Applications of Thermally Reversible Polymers and Hydrogels in Therapeutics and Diagnostics," in Third International Symposium on Recent Advances in Drug Delivery Systems, Salt Lake City, Utah, Feb. 24-27, 1987, p. 297-305; Gutowska et al., J. Controlled Reléase 22: 95-104, 1992; Palasis and Gehrke, J. Controlled Relay 18: 1-12, 1992; Paavola et al., Pharm. Res. 12 (12): 1997-2002, 1995). Representative examples of thermogelling polymers, and their gelatin temperature (LCST (degrees centigrade)) include homopolymers such as poly (N-methyl-N-n-propylacrylamide), 19.8; poly (N-n-propylacrylamide), 21.5; poly (N-methyl-N-isopropylacrylamide), 22.3; poly (N-n-propylmethacrylamide), 28.0; poly (N-isopropylacrylamide), 30.9; poly (N, n-diethylacrylamide), 32.0; p or I i (N -isopropylmethacrylamide), 44.0; poly (N-cyclopropylacrylamide), 45.5; poly (N-ethylmethylacrylamide), 50.0; poly (N-methyl-N-ethylacrylamide), 56.0; poly (N-cyclopropylmetacnlamide), 59.0; poly (N-ethylacrylamide), 72.0. In addition, thermogelling polymers can be made by preparing copolymers between monomers of the above, or by combining said homopolymers with other water-soluble polymers such as acryl monomers (e.g., acrylic acid and its derivatives such as methacrylic acid, acrylate and its derivatives such as butyl methacrylate, acrylamide, and Nn-butylacrylamide). Other representative examples of thermogelling polymers include cellulose ether derivatives, such as hydroxypropylcellulose, 41 ° C; methylcellulose, 55 ° C; hydroxypropylmethylcellulose, 66 ° C; and ethylhydroxyethylcellulose, and Pluronics such as F-127, 10-15 ° C; L-122, 19 ° C; L-92, 26 ° C; L-81, 20 ° C; and L-61, 24 ° C. A variety of shapes can be designed through the polymeric carriers of the present invention, including, for example, bar-shaped devices, pellets, slices or capsules (see, for example, Goodell et al, Am. J. Hosp. Pharm. 43: 1454-1461, 1986; Langer et al., "Controlled release of macromolecules from polymers", in Biomedical Polymers, Polymeric Materials and Pharmaceuticals for Biomedical Use, Goldberg, EP, Nakagim, A. (eds.) Academic Press , pp. 113-137, 1980; Rhine et al., J. Pharm. Sci. 69: 265-270, 1980; Brown et al., J. Pharm. Sci. 72: 1181-1185, 1983; and Bawa et al. al., J. Controlled Reeléase 1: 259-267, 1985). The therapeutic agents can be linked through occlusion in the polymer matrices, linked through bonds covalent, or encapsulated in microcapsules. Within certain preferred embodiments of the invention, the therapeutic compositions are provided in non-capsular formulations such as microspheres (varying the size from nanometers to micrometers), pastes and strips of various sizes, films and sprays. Another aspect of the present invention is to provide a therapeutic composition and animal feed. The therapeutic composition of the present invention can be encapsulated using a polymeric carrier as described above and then added to a food through known means of applying it to the food, such as, for example, through mechanical mixing, spraying, etc. The therapeutic composition includes, for example, an amount of at least one bacteriocin effective to at least reduce colonization levels through at least one target bacterium in an animal, such as, for example, about 0.5 grams of bacteriocin ( s) / 100 grams, about 1.25 grams of a polymer vehicle such as polyvinylpyrrolidone / 100 grams, and about 8.6% of a diluent such as water / 100 grams mixed with any granular component that is digestible, such as, for example, grain of ground corn; ground grains such as, for example, oats, wheat, alforjon; ground fruits such as, for example, pears, etc. The therapeutic composition is then added to any type of animal feed in effective amounts for at least reducing the colonization levels of at least one target bacterium such as, for example, at ratios of bacteriocin to feed of about 1:10 to about 1: 100. For the purposes of the present invention, examples of animal feed include green fodder, silages, dry green fodder, roots, tubers, fresh fruits, grains, seeds, beer grains, apple pulp, brewer's yeast, distillation residues, byproducts of milling, byproducts of sugar production, starch or oil production, and various food wastes. The product can be added to animal feed for raising livestock, poultry, rabbits, pigs, or sheep. It can be mixed with other food additives for these cattle. The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

EXAMPLE 1 Two novel antagonistic strains, Enterococcus faecium strain 50-52 (NRRL B-30746) and Streptococcus cricetus strain 760 (NRRL B-30745) were isolated from mucosal surfaces of approximately 1.0 gram of tender chicken cecal which was suspended in approximately 10 minutes. mi of a sterile saline solution at 0.85% w / v (normal saline) and heated to approximately 80 ° C for approximately 15 minutes. Approximately 0.10 mi of about 1:50, and 1: 2,500 of suspensions were sprayed on plates either from plate count agar or MRS Agar. Plates were incubated at approximately 37 ° C for approximately 24 hours under microaerobic conditions. Colonies with different morphology were plotted on MRS agar. These cultures were incubated under microaerobic conditions for approximately 24 hours at about 37 ° C. Strain 760 was grown at approximately 37 ° C for approximately 24 hours on MRS agar. The strain is an facultative aerobic with great-positive cocci, it is capable of growing between approximately 37 ° C and 45 ° C. The strain grew on nutrient agar or plate-counting agar producing gray colonies, with regular circular shape, little convex, with wavy margins with a diameter of approximately 2 mm after aerobic incubation at approximately 37 ° C for approximately 24 hours. The biochemical properties of strain 760 were determined with an APJ 50 CLH system (Bio-Merieux, France). The strain degrades lactose, mannitol, ribose, salicin, sorbitol, trehalose, arabinose, and melibiose. Slightly hydrolyzes raffinose and inulin, and does not hydrolyze against arginine and esculin. It is not capable of a and ß hemolysis. It does not grow in the presence of approximately 6.5% sodium chloride. The strain is negative for catalase. Strain 50-52 was grown on MRS agar at approximately 37 ° C for approximately 24 hours. The strain is an facultative aerobic with large-positive cocci, it is capable of growing on nutrient agar or agar plate count producing grayish colonies, with an irregular circular shape, slightly convex, with wavy margins with a diameter of approximately 1 mm after aerobic incubation at approximately 37 ° C for approximately 24 hours. The biochemical properties of strain 50-52 were determined with the EN-Coccus test system. The strain degrades arginine, arabinose, and mannitol; and does not hydrolyze sorbose, sorbitol, melibiose, raffinose, and methycitose. It grows in the presence of approximately 65% sodium chloride. Bacterial strains, to assess the antagonistic activity of strains 50-52 and 760, included isolates of Cam pylobacter jejuni (C. jejuni) NCTC 11168, S. enteritidis, and E. coli 0157: H7. Cultures of C. jejuni were grown on Brucella agar or Campylobacter agar containing about 5% partially used blood at about 42 ° C for about 24-48 hours under microaerobic conditions of about 5% 02, about 10% C02, and approximately 85% N2. The antagonistic activity of the isolates against Campylobacter was evaluated. About 2.0 ml of the normal saline suspensions were placed on MRS agar plates and incubated at about 37 ° C for approximately 24 hours. MRS agar cubes of approximately 0.5 cm3 were cut and transferred onto Brucella agar or Campylobacter agar supplemented with approximately 5% -10% partially lysed blood, about 10 micrograms / ml of rifampicin, and about 2.5 u / ml of polymyxin and inoculated with approximately 107 Campylobacter jejuni cells per plate. Plates were incubated at approximately 42 ° C for approximately 24 to 48 hours under microaerobic conditions as described above. Antagonistic activity was evaluated by measuring the size of the diameter of the zones of inhibition of C. jejuni.

EXAMPLE 2 Crude antimicrobial preparations were extracted from cultures of the antagonistic strains: Enterococcus 50-52 and Stre ptococcus 760. Antagonists were developed in approximately 250 ml of 10% Brucella broth together with the Lactococcus lactis strain 320 induction strain (NRRL B- 30744, supra) at about 37 ° C for about 14 hours under conditions 14 hours under aerobic conditions. The concentration of the producing strain was about 10 6 CFU / ml and for the inducing strain about 10 7 CFU / ml. The resulting cultures were centrifuged at approximately 2,500 X g for approximately 10 minutes, removing most of the viable cells. The decanted supernatant was mixed with about 60% saturated ammonium sulfate and incubated at about 4 ° C for about 24 hours to precipitate the bacteriocin compounds. After centrifugation at approximately 10,000 X g for approximately 20 minutes, the pellet was resuspended in about 1.5 ml of about 10 mM sodium phosphate buffer, pH about 7.0, and dialyzed overnight against about 2.5 liters of the same pH buffer. The solution was designated as a crude antimicrobial preparation (CAP). Each sample of the preparation was sterilized by passing it through a filter with a pore of 0.22 microns (Millipore, Bedford, MA, USDA). Table 1 shows a production of bacteriocin with and without the use of the inducing strain.

Table 1. Bacteriocin production with and without the use of the Lactococcus lactis strain 320 induction strain EXAMPLE 3 The spectrum of the antimicrobial activity of the CAPs was determined using a spot or spot test. Approximately 1 ml of sterile crude antimicrobial preparations (CAP), obtained as in Example 2 above, were diluted with about 1 ml of sodium phosphate pH buffer (pH about 7.0) and sterilized as previously done in Example 2 Approximately 10 microliters of each sample is plated on Campyiobacter agar supplemented with blood or Nutrient agar (MPA or Meta peptone agar) previously seeded with target bacteria cells. Plates containing C. jejuni cultures were grown at approximately 42 ° C under microaerobic conditions, Y. enterocolitica and V. pseudotuberculosis were aerobically cultivated at about 28 ° C, and other bacterial strains were incubated aerobically at about 37 ° C during approximately 24 or 48 hours. The identification was based on areas of inhibition produced by the target bacteria. The activity of CAP was expressed in arbitrary units (AU) per one milliliter of the preparation where a visible zone of growth inhibition of the crop appeared (Henderson et al., Archives of Biochemistry and Biophysics, Volume 295, 5-12, 1992 incorporated herein by reference). All the experiments were conducted in duplicate. See Table 2 in Example 4 below.

EXAMPLE 4 The CPAs and bacteriocins were electrophoresed in approximately 15% by weight of agarose gel, about 1% SDS (9 X 12 cm) in Tri-glycine pH regulator. After electrophoresis at approximately 100 mA for about 4 hours, the gels were fixed with a solution containing approximately 15% ethanol and 1% acetic acid. The gels were then washed with distilled water for about 4 hours. To determine the molecular weights of fractions of protein, the gel was stained with a solution containing about 0.21% Coomassi Blue G-250, about 40% ethanol, and about 7% acetic acid. Washed gels were tested against three target bacteria, C. jejuni NCTC 11168, E. coli 0157: H7 904, and S. enteritidis 204 through the method of Bhunia et al. (Journal of Industrial Microbiology, Volume 2, 319-322, 1987; incorporated herein by reference). The gels were placed in Peri boxes, covered with 5% Campyiobacter agar from semi-solid blood (about 0.75%) or semi-solid MPA, and seeded with cells from the test strains. Plates containing C. jejuni were incubated at about 42 ° C for about 48 hours under microaerobic conditions, E. coli 0157: H7 and S. enteriditidis at about 37 ° C for about 24 hours. The assessment was based on the visualization of zones of inhibited growth of the test strains in the presence of bacteriocins. The isoelectric signal identified two different fractions, which differed in isoelectric points (pl: CAP 70, contained fractions with pl = approximately 9.2 and approximately 9.5) CAP 50-52 contained fractions with pl = approximately 7.7 and 8.4. observed the antagonistic activity of C. jejuni in the fraction with pl = approximately 9.5 in the preparation 760, while in the preparation 50-52 this inhibition was observed in the fraction with p = approximately 8.4 (Figures 1A-B and 2a-B , Table 1 below.) The gels covered with Campyiobacter jejuni were used to determine which band (s) corresponds to the antimicrobial activity, the molecular weight and the isoelectric point. Figure 1A and 1B for Bacteriocin 760 show the molecular weight of the active fraction (1A) and the isoelectric point for the active fraction (1B). The gels were covered with Campylobacter jejuni to determine the antimicrobial activity, molecular weight and isoelectric point. In Figure 1A, lane 1 shows LMW molecular weight markers ranging from 14,400-94,000 (Amersham Pharmacia Biotech): 14,000; 20,100; 30,000; 43,000; 67,000; and 94,000 Da. Lane 2 contains the molecular weight markers for insulin, β chain (Sigma, USA): 3,500 Da. Lane 3 shows pure 760 bacteriocin corresponding to the antimicrobial activity, the zone of growth inhibition (arrow) has a mass of about 5,500 Da. Figure 1B, lane 1 shows pl standards (Protein Test Mixture, pl Marker Proteins, Serva). Lane 2 shows pure 760 bacteriocin corresponding to the antimicrobial activity, the zone of growth inhibition (arrow) had a pl of about 9.5. The other bands did not show antimicrobial activity. Figures 2A and 2B show the direct detection of bacteriocin 50-52 using SDS-PAGE (2A) and isoelectric focusing (2B). The gel was coated with Campylobacter jejuni to determine which band (s) corresponds to the antimicrobial activity and molecular weight as shown in Figure 2A. Lane 1 shows LMW Molecular Weight Markers of scale 1,600-26,000 (Amersham Pharmacia Biotech): 1,600; 3,500; 6,500; 14,200; 17,000; and 26,000 Da. The band in lane 2 containing pure 50-52 bacteriocin corresponds to the antimicrobial activity, the zone of growth inhibition and had a mass of approximately 3,900 Da. Figure 2B, lane 1 contained pl standards (Protein Test Mixture, pl Marker Proteins, Serva). The band in lane 2 (pure 50-52 bacteriocin), corresponding to the antimicrobial activity, the zone of growth inhibition (arrow) had a pl of about 8.4. The other bands did not show antimicrobial activity. Specimens of bacteriocins were placed on IEF gels (pH about 4.4-10-0) (Novex, San Diego, CA). The gels were run at approximately 100V for approximately 1 hour, 200V for approximately 2 hours, and 500V for approximately 30 minutes in XCM II ™ Mini-Cell (Novex). The gels were washed with distilled water for approximately 4 hours without fixation followed by staining with Coomassie Blue G-250 to determine isoelectric points (pl) of the bacteriocins and their ability to inhibit test growth as presented in Figures 1 and 2 and Table 2.

Table 2. Antimicrobial activity of crude antimicrobial preparations (CAP) of bacteriocins evaluated by spot or spot test methods, SDS-PAGE. and Isoelectric focusing (IEF) EXAMPLE 5 The production of Enterococcus strain 50-52 and Streptococcus strain 760 were cultured simultaneously with a bacterial-inducing strain, Lactococcus actis (Strand 320; supra) in Brucella Broth (Difco). About 1.32 X 107 cells of Enterococcus faecium or Streptococcus cricetus were placed together with approximately 3.9 X 10 6 Lactococcus lactis cells in 250 ml flasks and cultured for approximately 24 hours. The concentrations of producing and inducing strains were determined every two hours together with the specific activity of the bacteriocin to determine the optimal time to obtain usable amounts of the bacteriocin. The bacteriocins were isolated and purified by two methods. The first was sedimentation of the bacteriocin using ammonium sulfate followed by three-step chromatography: gel filtration in 12 HR Superóse, ion exchange chromatography in Superoff SPFF, and hydrophobic interaction chromatography in Octil Sepharose 4 FF. This is Method A. For Method B, after the simultaneous cultivation of producing and inducing strains, most of the bacteriocin produced is secreted into the culture fluid. A portion of the bacteriocin will adsorb on the cells of the producer and inducer strains. In order to avoid the loss of the adsorbed bacteriocin, the bacteriocins are eluted from the cells. Method B involves two steps: (1) the isolation of bacteriocins from the culture fluid supernatant; and (2) the isolation of bacteriocin from the precipitate of cells from both inducing and producing strains. In step 1, the cultures are harvested and separated by centrifugation at approximately 10,000 g for approximately 15 minutes to precipitate the cells. The supernatant is applied to an Octil Sepharose 4 FF column to recover the bacteriocins. The cell pellet is used in step 2. The pellet was suspended in pH phosphate buffer with approximately 0.7% NaCl, pH (elution pH regulator) and the suspension was mixed and incubated for about 20 minutes. After incubation, the suspension was centrifuged at approximately 10,000 g for approximately 15 minutes. The bacteriocins were isolated from the supernatant using ion exchange chromatography on Superóse SP FF. The purified products of both methods were analyzed for their antagonistic activity against Campylobacter jejuni. SDS-PAGE and isoelectric focusing (IEF) were performed. The results are summarized below in Tables 3 and 4. It was found that the specific activity of the purified preparations through Method B is 4 times higher than the activity of the same product obtained through Method A. It took approximately 12.5 hours produce the preparation through Method B against 52 hours using Method A. Method B reduced the number of steps required for the purification of bacteriocins from CAP eliminating gel filtration and hydrophobic interaction chromatography and only applying exchange chromatography of ion. 3. Comparative data on the specific activity of bacteriocins 760 and 50-52 isolated using Methods A and B Table 4. Antimicrobial activity of 760 and 50-52 purified bacteriocins determined through the Stain Test, SDS-PAGE, and Isoelectroenf oq ue (IEF) The amino acid sequences of purified bacteriocins were determined through Edman degradation using an automatic 491 cLC sequencer (Applied Biosystems, USA). The bacteriocins were hydrolysed in approximately 6M HCl under a vacuum at approximately 110 ° C for about 72 hours. The molecular weights of bacteriocins 50-52 and 760 were determined by mass spectrometry using a Voyager-DERP (Perkin-Elmer, USA). The MALDI-TOF system, a matrix-assisted laser desorption ionization time, was used together with a matrix, 2-cyano-hydroxycinnamic acid. The amino acid sequences are: 50-52: TTKNYG G VC NSVNWCQCGNVWASCNLATGCAAWL CKLA SEQ ID NO 1 760: N RWYCNSAAGGVGGAAVCGLAG YVGEAKEN I AG EVRKGWGMAGG FTHNKACKS SEQ ID 2 The calculated molecular weights of the peptides were approximately 3.9 kDa for bacteriocins 50-52 and approximately 5.5 kDa for bacteriocin 760. Analysis through MALDI-TOF revealed the following molecular weights: approximately 3,932 Da for bacteriocin 50-52 and approximately 5,362 Da for bacteriocin 760.

EXAMPLE 6 The influence of enzymes, temperature, and pH on bacteriocin activity was determined. About 10 mi from one of the The following enzymes were transferred to tubes containing approximately 20 ml of bacteriocins: beta-chymotrypsin-approximately 100 mg / ml, proteinase K-approximately 200 mg / ml, papain-approximately 60 mg / ml, lysozyme-approximately 750 mg / ml, and lipase -about 100 mg / l (all from Sigma-Aldrich Corp., St. Louis MO). After an incubation of about three hours at about 37 ° C, the bacteriocin-enzyme mixture was analyzed for antimicrobial activity using the spot test as in Example 3. The untreated bacteriocins served as positive controls. To study the thermostability of bacteriocins, a sample of approximately 2 mg / ml was boiled in a water bath for approximately 15 minutes, cooled and evaluated in terms of its antimicrobial activity. Approximately 2 mg / ml of bacteriocin were used to evaluate the effect of pH. About 2 milliliters of sterile solutions, about 10 mM NaOH or about 10 mM HCl were added to samples to test the pH from about 3 to about 10. The samples were incubated at about 37 ° C for about 2 hours and 24 hours , and at approximately 90 ° C for about 20 minutes. The samples were adjusted to a pH of about 7.2 through the addition of about 4 mM of sterile phosphate buffer and analyzed for their antimicrobial activity using the spot test as described above in Example 3.

The bacteriocins 50-52 and 760 lost their antimicrobial activity after being treated with beta-chymotrypsin, proteinase K, and papain, but were retained when treated with lysozyme, lipase, or by heating at approximately 90 ° C (Table 5). They were stable at different pH values, varying from about 3.0 to about 9.0, but were inactive at about a pH of 10 (Tables 5 and 6).

Table 5. Effect of enzymes and temperature on the antimicrobial activity of bacteriocins * activity determined through the spot test, with C.jejuni 11168 as the indicator strain. + presence of activity absence of activity after treatment with enzymes or exposure to temperature Table 6. Effect of pH on the activity of bacteriocins PH Activity determined through the spot test with C. jejuni NCTC 11168 20 min (c § 90 ° C 2 h (c 37 ° C 24h @ 37 ° C 3.0 + + + 5.0 + + + 6.2 + + + 7.0 + + + 8.4 + + + 9.1 + + + 10.0 - + presence of activity absence of activity EXAMPLE 7 The susceptibility of Campylobacter spp. For purified preparations of bacteriocins 760 and E50-52 using strains isolated from young chickens as described above in Example 1. the antagonistic activity of the bacteriocins was determined based on minimum inhibitory concentrations (MICs), which were determined through of agar diffusion. Table 7 below shows the MICs of bacteriocin for the strains tested. All bacteriocins tested are highly antagonistic to campylobacter spp strains. The 760 strain is much more active than the rest, with MICs between approximately 0.05 to approximately 0.1 μ / ml Table 7. MICs of bacteriocin for strains of Cam pylobacter spp EXAMPLE 8 The susceptibility of Gram-positive and Gram-negative bacteria to bacteriocins 760 and 50-52 was determined using a spot test as described above in Example 3. The results are presented in Tables 8 and 9. As shown in FIGS. see in Table 7, bacteriocin 760 has a broad spectrum and a high level of antagonistic activity. Bacteriocin inhibits the growth of both Gram-positive and Gram-negative bacteria. Its MIC for the test strains ranges from about 0.1 μ / ml to about 3.2 μ / ml. As seen in Table 9, bacteriocin 50-52 has a broad spectrum and high level of antagonistic activity. Bacteriocin inhibits the growth of both Gram-positive and Gram-negative bacteria. Its MIC for test strains ranges from approximately 0.1 μ / ml to approximately 3.2 μ / ml.

TABLE 8. Bacteriocin 760 antibacterial activity determined through the Stain Test MIC test strains, mg / ml S. enteritidis 1 0.2 S. enteritidis 4 0.4 S. enteritidis 204 0.2 S. enteritidis 237 0.2 S. choleraesuis 434/4 0.4 S. choleraesuis 370 0.4 S. typhimurium 383/60 0.4 S. typhimurium 320 0.2 S. gallinarum pullorum 0.4 E. colt HB101 0.1 E. colt C600 0.1 E. colt 0157: 117 Y-63 1.6 E. colt0157: H7 G-3 1.6 E. colt0157: H7 OD-3 1.6 E. colt 0157: 1-17 lab.39 1.6 Y. enterocolitica 03 0.1 Y. enterolithic 09 0.1 Y. enterolithic 04 0.1 Citrobacter fi-eundi 1.6 Klebsiella pneumoniae 3.2 Sh. dysenteriae 0.1 Staphylococcus aureus 1.6 Staphylococcus epidermis 1.6 And pseudotuberculosis 3.2 Y. pseudotuberculosis 3.2 Pseudomonas aeruginosa 25583 0.4 Proteus mirabilis 3.2 Morganella morganii 3.2 L. monocytogenes 9-72 0.1 C. jejuni L4 0.1 TABLE 9. Bacteriocin 50-52 antibacterial activity determined through the Stain Test Staphylococcus epidermis 3.2 Y. pseudotuberculosis 0.4 Pseudornonas aeruginosa 25583 3.2 Proteus mirabilis 1.6 Morganella morganii 3.2 L. monocytogenes 9-72 0.2 C. jejuni L-4 0.2 EXAMPLE 9 The minimum inhibitory concentrations (MICs) of bacteriocins 760 and 50-52 and Methicillin were determined through the Stain Test. Several concentrations of purified bacteriocins (μ / ml), in a volume of 10 μ ?, were added to cultures of Staphylococcus aureus, Staphylococcus epidermidis, Pseudornonas aeruginosa, and Helicobacter pylori. The cultures were incubated for approximately 24 hours at about 37 ° C under aerobic conditions for all, except Helicobacter pylori which was incubated under microaerobic conditions. The results are shown below in Table 10.

Table 10. MIC of bacteriocins for Staphylococcus aureus, Staphylococcus epidermidis, Pseudornonas aeruginosa, and Helicobacter pylori Bacteriocin or MIC of purified Bacteriocins and Antibiotic (ng / ml) antibiotic S. aureus S. epidermidis P. aeruginosa H. pylori 760 1.6 1.6 0.4 0.2 50-52 0.1 0.4 0.8 0.8 Methicillin 52.5 64.8 78.4 93.8 The detailed description above is for the purpose of illustration. Said detail is only for those purposes and those skilled in the art can make variations without departing from the spirit and scope of the invention.

Claims (15)

1. An isolated bacteriocin produced through a strain of Enterococcus or a strain of Streptococcus having the identification characteristics of a strain selected from the group consisting of NRRL B-30746 and NRRL B-30745.

2. The bacteriocin according to claim 1, having an amino acid sequence of SEQ ID NO 1.

3. The bacteriocin according to claim 1, having an amino acid sequence of SEQ ID NO 2.

4. A strain of isolated Enterococcus faecium that has the identification characteristics of NRRL B-30746.

5. A strain of Streptococcus cricetus that has the identification characteristics of NRRL B-30745.

6. A therapeutic composition comprising: (a) at least one isolated bacteriocin produced through a strain of Enterococcus or a strain of Streptococcus having the identification characteristics of a strain selected from the group consisting of NRRL B-30746, NRRL B-3745, and mixtures thereof in an amount effective to at least reduce colonization levels through at least one target bacterium; and (b) a suitable therapeutic vehicle. The therapeutic composition according to claim 6, wherein said bacteriocin has a sequence of amino acid selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, or mixtures thereof. 8. A therapeutic food comprising: (a) at least one isolated bacteriocin produced through a strain of Enterococcus or a strain of Streptococcus having the identification characteristics of a strain selected from the group consisting of NRRL B-30746 and NRRL B-30745, and mixtures thereof, in effective amounts to at least reduce levels of colonization through at least one target bacterium, (b) a therapeutic vehicle, and (c) an animal feed. The therapeutic food according to claim 8, wherein at least said bacteriocin has an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, or mixtures thereof. 10. A method for at least reducing levels of colonization through at least one target bacterium in an animal, comprising: administering to an animal, a therapeutic composition comprising at least one isolated bacteriocin produced through an animal strain of Enterococcus or a strain of Streptococcus having the identification characteristics of a strain selected from the group consisting of NRRL B-30746 and NRRL B-30745, and mixtures thereof, in effective amounts to at least reduce the levels of colonization through at least one target bacterium and a therapeutic vehicle. The method according to claim 10, wherein said bacteriocin has an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, or mixtures thereof. 12. An isolated strain of Lactococcus lactis that has the identification characteristics of NRRL B-30744. 13. A method for increasing the production of bacteriocins through bacteria, comprising: (a) adding an enhancing amount of Lactococcus lactis bacteriocin having the identification characteristics of NRRL B-30744 to a culture of a bacteriocin-producing bacterium for form a co-culture, and (b) cultivate said co-culture at approximately 37 ° C. 14. A method for isolating a bacteriocin, comprising: (a) co-culturing a bacteriocin-producing bacterium with an inducing bacterium to produce bacteriocin in a culture medium, (b) harvesting said culture medium and cells through centrifugation to obtain a supernatant and a cell pellet, (c) apply said supernatant containing the bacteriocin to a hydrophobic interaction chromatography column to obtain an isolated bacteriocin preparation, (d) incubate said cell pellet from step (b) in a regulator of elution pH to form a first mixture, (e) separating the cells from said cell pellet and said pH regulator by centrifugation, (f) isolating the bacteriocin from said pH regulator through ion exchange chromatography. 15. The method according to claim 15, wherein said ion exchange chromatography uses a Superóse SP FF column.