CN101426515A - 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

New enterococcus and strains of streptococcus and bacteriocin

Background of invention

Invention field

[0001] the present invention relates to enterococcus by using new bacteriocinogeny and strains of streptococcus and/or the new bacteriocin control animal, the particularly poultry that produce by these bacterial strains in disease.The invention still further relates to new bacteriocin, the aminoacid sequence of described new bacteriocin, and relate to enterococcus or the streptococcus that produces described new bacteriocin, and relate to the inductor bacterial strain of Lactococcus.Further, the present invention relates to contain described new bacteriocin and/or produce their enterococcus or streptococcic therapeutic combination, and the application of described therapeutic combination.

Association area is described

[0002] the edible incorrect poultry prod of handling has caused people's intestinal diseases.Early have recognized that Salmonella (Salmonella spp.) is the pathogenic bacterium of this class disease, recognize that recently Campylobacter (Campylobacter spp.), particularly campylobacter jejuni (Campylobacter jejuni) also come into the picture wherein.These two kinds of microorganisms can be colonizated in the gastrointestinal tract of poultry, and to these birds without any harmful effect, though some can be detected by the birds of field planting, but asymptomatic bacillicarrier can freely propagate these microorganisms in production and processing procedure, causes further infecting the birds and the corpse of living.Poultry in provand as the original storage master of Salmonella and Campylobacter (Jones etc., Journalof Food Protection (food protection magazine), the 54th volume, No.7,502-507, in July, 1991).Can eliminate the poultry pollution problems forming the production period prevention field planting in live poultry.

[0003] many factors facilitate antibacterial in animal alimentary canal field planting and exist.These factors are at large commented by Savage (Progress in Food and Nutrition Science (food and nutrition science progress), the 7th volume, 65-74,1983).Includedly in these factors be: (1) gastric acidity (Gilliland, Journal of Food Production (food production magazine), the 42nd volume, 164-167,1979); (2) bile salts (Sharpe and Mattick, Milchwissenschaft, the 12nd volume, 348-349,1967; Floch etc., American Journal of Clinical Nutrition (U.S.'s clinical nutriology magazine), the 25th volume, 1418-1426,1972; Lewis and Gorbach, Archives of InternalMedicine (Archives of Internal Medicine), the 130th volume, 545-549,1972; Gilliland and Speck, Journal of FoodProtection (food protection magazine), the 40th volume, 820-823,1977); Hugdahl etc., Infection and Immunity (infecting and immunity), the 56th volume, 1560-1566,1988); (3) wriggling; (4) digestive enzyme (Marmur, Journalof Molecular Biology (molecular biology magazine), the 3rd volume, 208-218,1961); (5) immune response; (6) antimicrobial compound of indigenous microorganism (indigenous microorganism) and their production.Preceding four kinds of factors depend on host's Phenotype, may not be in fact controlled variablees.The immune response in gastrointestinal (GI) road is difficult for regulating.The factor that relates to indigenous microorganism and metabolite thereof depends on the normal flora in GI road.

[0004] a kind of possible method of the field planting of control Campylobacter and/or Salmonella is by using competitive exclusion (CE).Nurmi and Rantala (Nature (nature), the 241st volume, 210-211,1973) prove by resisting the Salmonella field planting to the young chickling (chick) that microbiologic population does not also build up from the antibacterial tube feed of healthy poultry enteral prods, effectively control the infection of Salmonella.Use uncertain CE prepared product acceleration to chickling and newly hatch the maturation of the archenteric flora of birds, and the replacer who transmits the natural process of microbiologic population to its offspring by the hen that grows up is provided.The result of laboratory and on-site inspection provides by use the evidence that normal microflora falls to controlling the advantage of Campylobacter to chickling; The flock of birds that reduces is the frequency of bacillus infection (Mulder and Bolder, IN:Colonization Control of human bacterialenteropathogens in poultry (field planting of the bacillary enteropathogen of people control in the poultry) by bending; L.C.Blankenship (volume), Academic Press, Santiago, California, 359-363,1991) and reduce by the existing report of campylobacter jejuni (C.jejuni) level in the feces of the birds of field planting (Stern, Poultry Science (poultry science), the 73rd volume, 402-407,1994).

[0005] Schoeni and Wong (Appl.Environ.Microbiol., the 60th volume, 1191-1197,1994) reported by using carbohydrate additive and three kinds of antagonisies that have been identified (difference citric acid bacillus (Citrobacterdiversus) 22, Cray Bai Shi pneumobacillus (Klebsiella pneumoniae) 23 and escherichia coli (Escherichia coli) 25) together and significantly reduced the field planting of campylobacter jejuni in the broiler chicken (broiler).Also have after poultry separation and Culture thing treatment with Lactobacillus acidophilus (Lactobacillus acidophilus) and streptococcus faecalis (Streptococcus faecium), evidence (the Morishita etc. that campylobacter jejuni significantly reduces in the enteral sample of infection broiler chicken, Avian Diseases (poultry disease), the 41st volume, 850-855,1997).

[0006] Snoeyenbos etc. (U.S. Patent number 4,335,107, June nineteen eighty-two) cultivates competitive exclusion (CE) the microbiologic population technology that this prepared product has been developed the field planting of prevention Salmonella by lyophilizing feces and anaerobism.Mikola etc. (U.S. Patent number 4,657, in April, 762,1987) are used to prevent the Salmonella field planting with enteron stool and cecal content as CE microbiologic population source.(U.S. Patent number 5 such as Stern; 451; 400; nineteen ninety-five JIUYUE and U.S. Patent number 6,241,335; April calendar year 2001) field planting of a kind of mucosa CE compositions with protection poultry and livestock opposing Salmonella and Campylobacter disclosed; wherein the mucin layer of the caecum of washing is scraped in advance, and the bits of scraping that scraped remain in the oxygen-free environment, carry out anaerobism and cultivate.(U.S. Patent number 5 such as Nisbet, in December, 478,557,1996) a kind of definite probiotic bacteria is disclosed, it can derive from various domestic animals, is directly to be obtained by the batch culture that feces, caecum and/or the big intestinal contents of the target animal of growing up produces by continuous culture.

[0007] the multiple proof of micro-organisms has the chemical compound of antibacterial property.The chemical compound that one class is such, bacteriocin is made up of sterilization albumen, and its mechanism of action is similar to ion carrier antibiotic.Bacteriocin has the activity of opposing and the closely-related strain of its Producer usually.They are by complicated microbial population, intestinal for example, and the extensive existence in oral cavity or the isolated bacteria culture of other epithelial surface shows that bacteriocin may have regulating action aspect the population dynamic of antibacterial ecosystem.Bacteriocin is defined as by the biologically active protein part of antibacterial production and the chemical compound of bactericidal action (Tagg etc., Bacteriological Reviews (Bacteriological Reviews), the 40th volume, 722-256,1976).Other characteristics can comprise; (1) narrow spectrum suppresses active, concentrates on closely-related strain; (2) combine with specific cell receptor; And (3) plasmid carries the hereditary determinant of bacteriocin production and host cell bacteriocin immunity.Not exclusively the antagonism material of determining is called as " bacteriocin sample material ".Some have than the broad-spectrum activity effectively to the bacteriocin of resisting gram-positive bacteria, opposite non-confrontational gram negative bacteria.Existing suggestion term bacteriocin, when being used to describe the inhibitor of being produced by gram positive bacteria, should satisfy (1) is the minimum standards (Tagg etc., the same) that a kind of peptide and (2) have bactericidal activity.

[0008] lactic acid bacteria is one of most important probiotic micro-organisms.They are gram-positive, and are asporogenic, the biology of the shortage cytochrome of catalase feminine gender.They be anaerobism but aerotolerant, complicated nutritional requirement, acid proof and strict fermentable is arranged, and with the main end-product of lactic acid as sugar fermentation.Lactic acid producing bacteria comprises lactobacillus (Lactobacillus) strain, bacillus bifidus (Bifidobacterium) strain, enterococcus faecalis (Enterococcus faecalis), enterococcus faecalis (Enterococcus faecium), lactococcus lactis (Lactococcus lactis), hamster streptococcus (Streptococcus cricetus), Leuconostoc mesenteroides (Leuconostoc mesenteroides), pediococcus acidilactici (Pediococcus acidilactici), have a liking for sour lactic acid bacteria (Sporolactobacillus inulinus), streptococcus thermophilus (Streptococcus thermophilus) etc.These strains extensively exist the bacteriocin aspect to be subjected to special concern therein, also are widely used in fermented milk, food and the meat processing industry.They are in the preservation and the existing detailed record of the effect in the flavor characteristic of food.Most of bacteriocin by these strain productions only has the activity of other lactic acid bacterias of opposing, but have several demonstrate to than macrospecies be difference gram positive bacteria and, under given conditions, to the antibacterial activity of gram negative bacteria.

[0009] lactobacillus has been widely studied the production of its antagonist.Comprising bacteriocin fully qualitatively (DeKlerk, Nature (nature), the 214th volume, 609,1967; Upreti and Hinsdill, Anticmicrob.Agents Chemother. the 7th volume, 139-145,1975; Barefoot and Klaenhammer, Anticmicrob.Agents Chemother. the 45th volume, 1808-1815,1983; Joerger and Klaenhammer, Journal of Bacteriology (bacteriology's magazine), the 167th volume, 439-446,1986); Possible bacteriocin sample material (Vincent etc., Journal of Bacterioll (antibacterial magazine), the 78th volume, 479,1959), with other and bacteriocin must be not relevant antagonist (Vakil and Shahani, Bacteriology (bacteriology), Proc.9,1965; Hamdan and Mikolajcik, Journal of Antibiotics (antibiotic magazine), the 8th volume, 631-636,1974; Mikolajcik and Hamdan, Cultured Dairy Products (fermented dairy product), 1975, the 10 pages; And Shahni etc., Cultured Dairy Products Journal (fermented dairy product magazine), the 11st volume, 14-17,1976).

[0010] Klaenhammer (FEMS, Microbiol.Rev. (microbiology summary), the 12nd volume, 39-86,1993) is divided into four primary categories with the lactic acid bacteria bacteriocin at that time:

I class---lantibiotics (Lantibiotics), it is＜the little peptide of 5kDa, contain uncommon aminoacid L-lanthionine and Beta-methyl L-lanthionine.They are subjected to special concern is because they have for other bacteriocins very broad-spectrum activity.Example comprises nisin (Nisin), nisin Z, beading rhzomorph (carnocin) U149, galactopoiesis bacillin (lacticin) 481 and galactopoiesis bacillin 5.

II class---the little peptide that does not contain L-lanthionine: a class is heterogeneous＜the little peptide of 10kDa.This class comprises the have the opposing listeria spp active peptide of (Listeria spp.).

The III class---big is heat-labile〉protein of 30kDa.Example is Helveticin.

The IV class---contain for example complicated bacteriocin-protein of lipid and carbohydrate of extra section.

[0011] Raczek (on November 28th, 2002 announced U.S. Patent application US2002/0176910) discloses to use to contain and has lived or the microorganism of dead secreting bacteria element or the compositions of bacteriocin self or its combining form are used with the feedstuff with agriculture domestic animal.

[0012] in correlative technology field, described the enterococcus of various bacteriocinogeny and Streptococcus with and bacteriocin.Yet, the invention provides new compositions, its contain at least a new enterococcus and streptococcus and or at least a new bacteriocin of producing by described new bacterial strain; A kind of aminoacid sequence that uses the method for described bacterial strain and/or bacteriocin, described new bacterial strain, described new bacteriocin with and using method, bacterial strain, bacteriocin and the using method of all these and correlative technology field are inequality.

The invention summary

Therefore [0013] the purpose of this invention is to provide the new enterococcus and the strains of streptococcus of at least a product new bacteriocin.

[0014] further aim of the present invention provides the new hamster streptococcus (Streptococcus cricetus) with NRRL B-30745 recognition feature.

[0015] the further again purpose of the present invention provides new hamster streptococcus (Streptococcus cricetus) bacterial strain with NRRL B-30746 recognition feature.

[0016] another object of the present invention provides the new bacteriocin of being produced by new enterococcus and streptococcic bacterial strain.

[0017] the further again purpose of the present invention provides the new bacteriocin 50-52 that has as the aminoacid sequence of listing in SEQ ID NO 1.

[0018] the further again purpose of the present invention provides the new bacteriocin 760 that has as the aminoacid sequence of listing in SEQ ID NO 2.

[0019] further aim of the present invention provides by reduce the method by the field planting level of at least a target bacteria in animal at least to animal administering therapeutic compositions, and described compositions comprises the new enterococcus and the streptococcic bacterial strain, at least a by new enterococcus and the new bacteriocin of streptococcic bacterial strain production or the combination of described new bacterial strain and/or described new bacteriocin of at least a product new bacteriocin.

[0020] further purpose of the present invention is by reducing the method by the field planting level of at least a target bacteria in animal at least to animal administering therapeutic compositions, and described compositions comprises the new enterococcal bacterial strain of the recognition feature with NRRL preserving number B-30746, the new streptococcic bacterial strain and composition thereof with recognition feature of NRRL preserving number B-30745.

[0021] further object of the present invention provides by reduce the method for the field planting level of at least a target bacteria in animal at least to animal administering therapeutic compositions, and described compositions comprises the new bacteriocin 50-52 that has as the aminoacid sequence of listing in SEQ ID NO 1.

[0022] further object of the present invention provides by reduce the method for the field planting level of at least a target bacteria in animal at least to animal administering therapeutic compositions, and described compositions comprises the new bacteriocin 760 that has as the aminoacid sequence of listing in SEQ ID NO 2.

[0023] another object of the present invention provides by reducing the method for the field planting level of at least a target bacteria in animal at least to animal administering therapeutic compositions, and described compositions comprises the bacteriocin of being produced by the new enterococcal bacterial strain with recognition feature NRRL B-30746, new streptococcic bacterial strain and composition thereof with recognition feature NRRL B-30745.

[0024] another object of the present invention provides Lactococcus (Lactococcus) inductor bacterial strain, and described inductor bacterial strain is by the output of the bacteriocin of raising Producer bacterial strain.

[0025] further purpose of the present invention provides the inductor bacterial strain of the lactococcus lactis (Lactococcus lactis) with recognition feature NRRL B-30744.

[0026] further purpose of the present invention provides the method that improves bacteriocin output by the Producer bacterial strain, and wherein said Producer bacterial strain and Lactococcus inductor bacterial strain are cultivated altogether.

[0027] further purpose of the present invention provides the method that improves bacteriocin output by the Producer bacterial strain, and wherein said Producer bacterial strain is cultivated altogether with the lactococcus strain with recognition feature of NRRL B-30744.

[0028] further object of the present invention provides the method that is used for the purification of bacterial element, described method comprises that results culture fluid and cell are as the sample that separates, by separate the bacteriocin on the cell surface that is adsorbed onto Producer and inductor bacterial strain with the phosphate buffer eluting that contains sodium chloride, then by go on foot the bacteriocin in the ion exchange chromatography separation and Culture liquid with hydrophobic interaction chromatography one.

[0029] further aim of the present invention and advantage will become obvious from following explanation.

The preservation of microorganism

[0030] hamster streptococcus (Streptococcus cricetus) is marked as NRRL B-30745 (bacterial strain 760); Enterococcus faecalis (Enterococcus faecium) is marked as NRRL B-30746 (bacterial strain 50-52) and lactococcus lactis (Lactococcus lactis) and is marked as NRRL B-30744 (bacterial strain 320) and was preserved in agricultural research service centre of United States Department of Agriculture (USDA) patent culture collection center (national agriculture applied research center (National Center for Agricultural Utilization Research on May 3rd, 2004 according to being specified in of budapest treaty, 1815 N.University Street, Peoria, Illinois 61604).

Brief description of drawings

[0031] Figure 1A and 1B are the photos that shows that SDS-PAGE (1A) and isoelectrofocusing (1B) back are directly detected bacteriocin 760.

[0032] Fig. 2 A and 2B are the photos that shows that SDS-PAGE (2A) and isoelectrofocusing (2B) back are directly detected bacteriocin 50-52.

Detailed Description Of The Invention

[0033] importance of intestines infection in the mankind is recognized day by day fully. Relation between poultry pollution and the human infection is existing detailed record also. The ability of eliminating this health risk by intervening poltry factory also is well-known. In the production of meat chick and process, the fecal matter that contains pathogen is transferred on the meat, and persists in the kitchen of processed food.

[0034] competitive biological metabolin may help the pathogen of control example such as campylobacter jejuni (Campylobacter jejuni) and salmonella. Isolated new antagonistic strain from caecum and the crop mucosal surface of meat chick. The natural constituents of the antagonist of identifying is the low molecular weight peptide with wide spectrum antagonistic activity, bacteriocin.

[0035] the invention provides new enterococcus bacterial strain, new strains of streptococcus, new lactococcus strain, new bacteriocin, the amino acid sequence of described bacteriocin, contain described new bacteriocin and/or produce the therapeutic combination of bacterial strain of described new bacteriocin and the method for using these new therapeutic combinations. The present invention also provides and produces and the method for the described new bacteriocin of purifying.

[0036] VREF, NRRL B-30746 is a kind of facultative aerobe and gram-positive cocci, and can be in about 37 ℃ of growths. Described bacterial strain produces erose edge at nutrient agar or plate count agar growth. In about 37 ℃ of little aerobic cultivations after about 24 hours, the about 2mm of described colony diameter.

[0037] hamster streptococcus, NRRL B-30745 is a kind of facultative aerobe and gram-positive cocci, and can be in about 37 ℃ of growths. Described bacterial strain is at the edge of nutrient agar or plate count agar growth generation rule shape. In about 37 ℃ of little aerobic cultivations after about 24 hours, the about 1mm of described colony diameter.

[0038] enterococcus that separates and streptococcus are to carry out at the nutrient agar of inoculation by the different target bacteria of paying close attention to for the production of the screening of eomycin activity. Other test strains were cultivated about 18-24 hour under about 37 ℃ of aerobic conditions. Yersinia enterocolitica (Yersinia enterocolitica) and yersinia pseudotuberculosis (Y.pseudotuberculosis) were cultivated about 18-24 hour under about 28 ℃ of aerobic conditions. The activity test of opposing campylobacter jejuni is to carry out at the campylobacter agar that contains about 5% cytolysis blood (lysed blood) by the campylobacter jejuni inoculation. The use of blood is fully in ordinary skill and comprises such as sheep, horse etc. The activity test of opposing campylobacter jejuni is at about 5% O₂, about 10% CO₂And about 85% N₂Little aerobic condition under at about 42 ℃ of about 24-48 hours. About 0.1ml is suspended in that described antagonistic bacterium in the physiological saline is applied on the MRS agar and the about 24-48 of incubation hour. About 0.5cm³MRS agar cube cut and transfer to replenished by the polymyxins of cytolysis blood, the rifampin of about 10 ug/ml, about 2.4U/ml and by about 10⁷On the brucella agar or campylobacter agar of individual campylobacter jejuni cell inoculation. Dull and stereotyped under about 42 ℃ of little aerobic conditions the about 24-48 of incubation hour. Active by measuring growth inhibiting regional evaluation.

[0039] estimates the separator that is found to be Antagonism that is used for bacteriocin production. By ammonium sulfate thick antimicrobial preparations of precipitation (CAPs) from the culture of antagonistic strain, described antagonistic strain under about 37 ℃ of aerobic conditions at the Lactococcus lactis (bacterial strain 320 as the inducer of about 10% brucella broth and bacteriocin enhancing amount; NRRL-30744) growth is about 14 hours in. The inducer of bacteriocin enhancing amount is defined as producer's bacterial strain of cultivating when not having inducer's bacterial strain and compares the amount that the bacteriocin that has improved at least producer's bacterial strain is produced required inducer bacterium. The inducer is about 10:1 (inducer: the producer) to the embodiment of the concentration ratio of producer's bacterial strain in the coculture. Described culture then under about 2,500 * g centrifugal about 10 minutes. Antagonistic peptide is by ammonium sulfate precipitation, with Superose 12 HR gel filtrations, separate from supernatant with the combination of Sepharose SP FF cation-exchange chromatography. The bacteriocin of secretion may be adsorbed onto on the cell surface of the producer and inducer's cell with coculture. In order to obtain these bacteriocins, by the little group of cell of centrifugation step gained with have about 0.7% NaCl, the elution buffer that the phosphate buffer of pH about 8.0 consists of mixes. Mixed this suspension and incubation about 20 minutes, then about 10, centrifugal about 15 minutes of 000g. Plain from the supernatant separation of bacterial through the ion-exchange chromatography on Superose SP FF. Determined the molecular weight of described peptide by the SDS-PAGE electrophoresis. Determined the pIs of described peptide by isoelectric focusing. Employing for example 491 cLC automatic sequencers (Applied Biosystems, Inc.) is determined amino acid sequence by the Edman degraded.

[0040] for the purposes of the present invention, term " peptide " means the compound of two or more amino acid at least or amino acid analogue. Described amino acid or amino acid analogue can link to each other by peptide bond. In another embodiment, described amino acid can pass through other keys, ester for example, and ether etc. link to each other. Peptide can be any node configuration, comprises linearity, branch, or cyclic configuration. Term used herein " amino acid " refers to natural or synthetic amino acid, comprise D or L optical isomer both, and amino acid analogue.

[0041] peptide derivant of the present invention and analog include but not limited to comprise as those of all or part of amino acid sequence of the peptide of one-level amino acid sequence, replace the sequence that residue in the sequence causes the change that conserved amino acid replaces thereby all or part of amino acid sequence of described peptide comprises wherein with the amino acid residue of functional equivalent.

[0042] for example, one or more amino acid residue in the described sequence can be by the other 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor with similar polarity, and described amino acid plays function equivalent, causes reticent the change. Amino acid whose substitute can be selected from other members of the affiliated classification of this amino acid in the sequence. For example, nonpolar (hydrophobic) amino acid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The amino acid that contains aromatic ring structure is phenylalanine, tryptophan and tyrosine. Polar neutral amino acid comprises glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (alkalescence) amino acid comprises arginine, lysine, and histidine. Electronegative (acidity) amino acid comprises aspartic acid and glutamic acid. Expect that this change can definite apparent molecular weight or the isoelectric point of the poly-propionamide gel electrophoresis of appreciable impact. Non-conservative amino acid substitution also can be introduced into to replace amino acid, makes it to have the character of special preference. For example, Cys be directed into possible position to form disulphide bridges with another Cys. Pro can be introduced into because of its special planar structure.

[0043] peptide of the present invention can be by chemical synthesis. Synthetic peptide can be with the solid phase of knowing, liquid phase, or peptide condensation (peptide condensation) technology, or any its combination preparation, and can comprise natural and/or synthetic amino acid. Be used for the synthetic amino acid of peptide and can be using the standard of the original solid phase program of Merrifield to go to protect, neutralize, be coupled and the standard Boc (N of washing methods (J.Am.Chem.Soc, the 85th volume, 2149-2154,1963)^αThe N of-amido protecting^α-t-butyl oxygen carbonyl) amino-acid resin, or the N of alkali sensitivity^α9-fluorenylmethyloxycarbonyl (Fmoc) amino acid (Carpino and Han, J.Org.Chem., the 37th volume, 3403-3409,1972) of-amido protecting. In addition, method of the present invention can be used other N well known to those skilled in the art^αThe group of-protection. Solid-phase peptide synthetic can with the ordinary skill of this area (referring to for example Stewart and Young, Solid Phase Synthesis (solid phase is synthetic), second edition, Pierce Chemical Co., Rockford, I11., 1984; Fields and Noble, Int.J.Pept.Protein Res., the 35th volume, 161-214,1990) or finish with automatic synthesizer.

[0044] according to the present invention, described polypeptide and new bacterial isolates can with treatment acceptable carrier form partly, enteron aisle other places, (for example per os, intranasal, per rectum or through skin) administration through mucous membrane ground. The ability that peptide of the present invention can be modified to increase described peptide if necessary and passes cell membrane, described modification for example the hydrophobic property by increasing described peptide, described peptide is introduced as the conjugate of the carrier part of certain special receptor (for example for), etc.

[0045] the present invention also provides and target molecule is puted together (conjugate) operation to the peptide of the present invention. The target molecule of the object of the invention means to navigate to the molecule of one or more positions of expectation when vivo medicine-feeding. In various embodiments of the present invention, described target molecule can be peptide or albumen, antibody, agglutinin, carbohydrate, or steroids. Target molecule can be peptide part or the antibody of acceptor on the target cell, for example monoclonal antibody. Crosslinked for promoting, described antibody can be simplified attaches most importance to and light chain heterozygosis dimer, perhaps F (ab ')₂Fragment can be simplified, and is linked on the described peptide by the sulfydryl of reduction.

[0046] another aspect of the present invention relates to provides therapeutic combination. Described composition can be for mouth, nose, lung administration, injection etc. The bacteriocin at least a of the present invention that described therapeutic combination comprises effective dose with and derivative and/or at least a new bacterial strain being reduced at least less a kind of field planting level of target bacteria, and acceptable diluent, anticorrisive agent, solubilizer, emulsifying agent, adjuvant and/or carrier. Diluent can comprise for example Tris-HCl, acetate, phosphatic buffer solution; Additive for example can comprise detergent and solubilising reagent, Tween 80 for example, polyoxyethylene sorbitan monoleate etc.; Antioxidant comprises for example ascorbic acid, sodium pyrosulfite etc.; Anticorrisive agent can comprise, for example Thimersol, phenmethylol etc.; And increment material for example lactose, mannitol etc.

[0047] therapeutic combination of the present invention can be merged in polymerizable compound (such as PVP, PLA, polyglycolic acid etc.) the special preparation thing, or incorporates in the liposome. Liposomal encapsulatedly comprise sealing of various polymer. Multiple macromolecule carrier can be used for comprising and/or send one or more of therapeutic agents discussed above, comprises for example biodegradable and not biodegradable both compositions. The representative example of biodegradable composition comprises albumin, collagen, gelatin, hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, CAP, cellulose acetate succinate, Hydroxypropyl Methylcellulose Phathalate), casein, dextran, polysaccharide, fibrinogen, poly-(D, L lactic acid), poly-(D, Pfansteihl-copolymerization-Glycolic acid), poly-(glycolide), poly-(butyric ester), poly-(alkyl carbonate) and poly-(ortho esters), polyester, poly-(hydroxypentanoic acid), poly-dioxy cyclohexanone, poly-(ethylene glycol terephthalate), poly-(malic acid), poly-(tartronic acid), poly-acid anhydrides, polyphosphazene, poly-(amino acid) and copolymer thereof are (usually, see Ilium, L., Davids, S.S. " Polymers in Controlled Drug Delivery (controlled drug send in polymer) " of (editor), Wright, Bristol, 1987; Arshady, J.Controlled Release (controlled release magazine) 17:1-22,1991; Pitt, Int.J.Phar. (International Journal of Pharmaceutical Medicine) 59:173-196,1990; The people such as Holland, J.Controlled Release (controlled release magazine) 4:155-0180,1986).

[0048] representative embodiment of nondegradable polymer comprises poly-(ethylene-vinyl acetate) (" EVA ") copolymer, silicon rubber, acrylate copolymer (polyacrylic acid, polymethylacrylic acid, polymethyl methacrylate, Polyalkylcyanoacrylanano (polyalkylcynoacrylate)), polyethylene, polypropylene, polyamide (nylon 6,6), polyurethane, poly-(ester polyurethane), poly-(ether polyurethane), poly-(ester-urea), polyethers (poly-(oxirane), poly-(expoxy propane), Pluronics and poly-(tetramethylene glycol)), silicon rubber and as PVP, polyvinyl alcohol, gathers the polyvinyl of acetic acid O-phthalic vinyl acetate etc.). Also can develop anion (such as alginate, carrageenan, carboxymethyl cellulose and poly-(acrylic acid)) or cation (for example, shitosan, poly-L-Lysine, polymine and polyallylamine) (usually, see Dunn etc., J.Applied Polymer Sci. (using polymer magazine) 50:353-365,1993; The people such as Cascone, J.Materials Sci.:Materials in Medicine (material science: medical material) 5:770-774,1994; Shiraishi etc., Biol.Pharm.Bull. (bio-pharmaceutical wall bulletin) 16 (11): 1164-1168,1993; Thacharodi and Rao, Int ' l J. Pharm. (International Journal of Pharmaceutical Medicine) 120:115-118,1995; Miyazaki etc., Int ' l J.Pharm. (International Journal of Pharmaceutical Medicine) 118:257-263,1995).

[0049] polymer support can fashion into the various forms of release characteristic with expectation and/or specific desirable properties. For example, in case polymer support can make be exposed to specific trigger event for example the pH form that just discharges therapeutic agent (see, for example, polymer III in medicine (Polymers in Medicine III), Heller etc. " Chemically Self-Regulated Drug Delivery Systems (with the self-regulating drug delivery system of the mode of chemistry) ", Elsevier Science Publisher B.V., Amsterdam, 1988, the 175-188 pages or leaves; The people such as Kang, J.Applied Polymer Sci. (journal of applied) 48:343-354,1993; The people such as Dong, J.Controlled Release (controlled release magazine) 19:171-178,1992; Dong and Hoffman, J.Controlled Release (controlled release magazine) 15:141-152,1991; The people such as Kim, J.Controlled Release (controlled release magazine) 28:143-152,1994; The people such as Cornejo-Bravo, J. Controlled Release (controlled release magazine) 33:223-229,1995; Wu and Lee, Pharm.Res. (drug research) 10 (10): 1544-1547,1993; The people such as Serres, Pharm.Res. (drug research) 13 (2): 196-201,1996; In the Pulsatile Drug Delivery (pulse drug delivery) that the people such as Gurny edit Peppas " Fundamentals of pH-and Temperature-Sensitive Delivery Systems (the responsive and temperature sensitive delivery system of pH basis) ", Wissenschaftliche Verlagsgesellschaft GmbH, the Stuttgart, 1993, the 41-55 pages or leaves; " Cellulose Derivatives (cellulose derivative) " 1993 of Doelker in the Biopolymers I (biopolymer I) that Peppas and Langer edit, Springer-Verlag, Berlin). The embodiment of representational pH sensitive polymer comprise polyacrylic acid with and derivative (comprise, for instance, homopolymers, for example polyaminocarboxylic acid, polyacrylic acid, polymethylacrylic acid, the copolymer of these homopolymers, and polyacrylic acid and for example copolymer of propylene Monomer of acyls discussed above (acrylmonomer)). The polymer of other pH sensitivities comprises polysaccharide for example CAP, Hydroxypropyl Methylcellulose Phathalate, acetic acid butanedioic acid hydroxypropyl methylcellulose, acetic acid trimellitic acid (trimellilate) cellulose; And shitosan. Also have the polymer of other pH sensitivity to comprise the polymer of pH sensitivity and any mixture of water-soluble polymer.

[0050] similarly, polymer support can be made into temperature sensitive form (to be seen, for example, " Novel Hydrogels of a Temperature-Sensitive Pluronic Grafted to a Bioadhesive Polyacrylic Acid Backbone for Vaginal Drug Delivery (be used for Novel temperature-sensitive pluronic hydrogel the polyacrylic acid skeleton that be grafted to bio-adhesive that intravaginal drug send on) " of the people such as Chen in controlled release bioactive materials international conference collection of thesis 22:167-168, Controlled Release Society, Inc., 1995; Okano is in controlled release bioactive materials international conference collection of thesis 22:111-112 " Molecular Design of Stimuli-Responsive Hydrogels for Temporal Controlled Drug Delivery (MOLECULE DESIGN that is used for the corresponding hydrogel of stimulation that time control medicine discharges) " Controlled Release Society, Inc., 1995; People Pharm.Res. (drug research) 9 (3): the 425-433 such as Johnston, 1992; Tung, Int ' l J.Pharm. (International Journal of Pharmaceutical Medicine) 107:85-90,1994; Harsh and Gehrke, J.Controlled Release (controlled release magazine) 17:175-186,1991; The people such as Bae, Pharm.Res. (drug research) 8 (4): 531-537,1991; Dinarvand and D ' Emanuele, J.Controlled Release (controlled release magazine) 36:221-227,1995; Yu and Grainger, ＂ Novel Thermo-sensitive Amphiphilic Gels:Poly N-isopropylacrylamide-co-sodium acrylate-co-n-N-alkylacrylamide Network Synthesis and Physicochemical Characterization (novel thermosensitive type amphipathic gel: the synthetic and Physico-Chemical Characterization of poly-N-isopropyl acrylamide-copolymerization-PAA-copolymerization-n-N-alkyl acrylamide network) "; Oregon Graduate Institute of Science ﹠ Technology chemistry and bioscience system;; Oregon; pause than not, 820-821 page or leaf; Zhou and Smid, " Physical Hydrogels of Associative Star Polymers (physical hydrogel of association type star-shape polymer) ", the New York State University's environmental science and polymer research institute of department of chemistry of forestry institute, Syracuse, New York (Syracuse), the 822-823 page or leaf; The people such as Hoffman, " Characterizing Pore Sizes and Water ' Structure ' in Stimuli-Responsive Hydrogels (sign of stimulating responsive hydrogel mesoporous and water ' structure ') ", University of Washington bioengineering center, Seattle, the State of Washington, the 828th page; Yu and Grainger, " Thermo-sensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks:Cationic; Anionic and Ampholytic Hydrogels (the temperature-sensitive expansion behavior in the crosslinked NIPA network: cation, anion and ampholytic hydrogel) ", Oregon Graduate Institute of Science ﹠ Technology chemistry and bioscience system, pause than not in the Oregon, the 829-830 page or leaf; The people such as Kim, Pharm.Res. (drug research) 9 (3): 283-290,1992; The people such as Bae, Pharm. Res. (drug research), 8 (5): 624-628,1991; The people such as Kono, J.Controlled Release (controlled release magazine) 30:69-75,1994; The people such as Yoshida, J.Controlled Release (controlled release magazine) 32:97-102,1994; The people such as Okano, J.Controlled Release (controlled release magazine) 36:125-133,1995; Chun and Kim, J.Controlled Release (controlled release magazine) 38:39-47,1996; D ' Emanuele and Dinarvand, Int ' l J.Pharm. (International Journal of Pharmaceutical Medicine) 118:237-242,1995; The people such as Katono, J.Controlled Release (controlled release magazine) 16:215-228,1991; In " Polymers in Medicine (polymer in the medicine) III " that the people such as Migliaresi edit Hoffman " Thermally Reversible Hydrogels Containing Biologically Active Species (the hot reversible hydrogel that contains bioactive species) ", Elsevier Science Publisher B.V., Amsterdam, 1988, the 161-167 pages or leaves; The 3rd drug delivery system new development international symposium, salt lake city, the Utah State, 24-27 day in February, 1987, the 297-305 page or leaf, " the Applications of Thermally Reversible Polymers and Hydrogels in Therapeutics and Diagnostics (application of thermally reversible polymer and hydrogel in acology and diagnostics) " of Hoffman; The people such as Gutowska, J. Controlled Release (controlled release magazine) 22:95-104,1992; Palasis and Gehrke, J.Controlled Release (controlled release magazine) 18:1-12,1992; The people such as Paavola, Pharm.Res. (drug research) 12 (12): 1997-2002,1995).

[0051] representative embodiment of hot glue cohesion compound with and gelling temp (LCST (degree centigrade)) comprise homopolymers, for example poly-(N-methyl-N-n-propyl group acrylamide), 19.8; Poly-(N-n-propyl group acrylamide), 21.5; Poly-(N-methyl-N-N-isopropylacrylamide), 22.3; Poly-(N-n-propyl methyl acid amides), 28.0; NIPA, 30.9; Poly-(N, n-diethyl acrylamide), 32.0; Poly-(N-isopropyl methyl acrylamide), 44.0; Poly-(N-cyclopropyl acrylamide), 45.5; Poly-(N-ethyl-methyl acrylamide), 50.0; Poly-(N-methyl-N-ethyl acrylamide), 56.0; Poly-(N-cyclopropyl Methacrylamide), 59.0; Poly-(N-ethyl acrylamide), 72.0. In addition, hot glue cohesion compound can be made by the copolymer between above two kinds of (multiple) monomers of preparation, perhaps prepare by the water-soluble polymer that makes up these homopolymers and other, for example the propylene Monomer of acyls is (for example for described water-soluble polymer, acrylic acid and derivative thereof, for example methacrylic acid, acrylate and its derivative, for example butyl methacrylate, acrylamide, and N-n-butyl acrylamide). The representative embodiment of other hot glues cohesion compounds comprises cellulose ether derivative, hydroxypropyl cellulose for example, 41 degrees centigrade; Methylcellulose, 55 degrees centigrade; Hydroxypropyl methylcellulose, 66 degrees centigrade; And ethylhydroxyethylcellulose and pluronics, such as F-127,10-15 degree centigrade; L-122,19 degrees centigrade; L-92,26 degrees centigrade; L-81,2O degree centigrade; And L-61,24 degrees centigrade.

[0052] can make various forms by polymer support of the present invention, for example comprise equipment, piller, sheet or the capsule of clavate (see, for example, people such as Goodell, Am.J.Hosp.Pharm. (United States Hospital pharmaceutical journal) 43:1454-1461,1986; At Biomedical Polymer, Polymeric Materials and Pharmaceuticals for Biomedical Use (biomedical polymer, the polymeric material of biomedical applications and medicine) in people such as Langer, " Controlledrelease of macromolecules from polymers (from polymer controlled release macromole) ", Goldberg, E.P., Nakagim, A. edits, Academic Press, the 113-137 page or leaf, 1980; People such as Rhine, J.Pharm.Sci. (pharmaceutical science magazine) 69:265-270,1980; People such as Brown, J.Pharm.Sci. (pharmaceutical science magazine) 72:1181-1185,1983; And people such as Bawa, J.Controlled Release (controlled release magazine) 1:259-267,1985).

[0053] therapeutic agent can fetter by covalently bound by containing (occlusion) in polymeric matrix, perhaps is encapsulated in the microcapsule and is connected.In some embodiment preferred of the present invention, therapeutic combination provides with non-capsule formula form, and described non-capsule formula form is microsphere (size is from the nanometer to the micron) for example, the paste of various sizes and filament (thread), film and spraying.

[0054] another aspect of the present invention provides therapeutic combination and animal feed.Therapeutic combination of the present invention can coat and known its method that is applied to feedstuff is added into feedstuff by any subsequently as mentioned above with polymer support, and described known method is for example by mechanical mixture, spraying or the like.Described therapeutic combination comprises, at least a bacteriocin of one consumption, described consumption is effective to reducing the field planting level of at least a target bacteria in animal at least, one or more bacteriocins of for example about 0.5 gram/100 grams, polymer support (for example polyvinyl pyrrolidone)/100 gram of about 1.25 grams, and about 8.6% diluent (for example water/100 grams), mix with digestible any particulate component, described digestible granular substance comprises, the corn kernel of milling, grind grain (Herba bromi japonici for example, Semen Tritici aestivi, Semen Fagopyri Esculenti), grind fruit (for example pears) etc.Described then therapeutic combination with to the field planting level that reduces at least a target bacteria at least effectively amount be added in the animal feed of any kind, described effective amount for example, bacteriocin arrives about 1:100 to the about 1:10 of the ratio of feedstuff for instance.For the purposes of the present invention, the example of animal feed comprises greenfeed, ensilage, dried greenfeed, root, tuber, succulent fruit, grain, seed, distiller's grains of beer (brewer ' s grain), marc, beer yeast, bottoms, the side-product of milling, produces the side-product of sugar, starch or oil, and various food waste.This product can add the animal foodstuff that is used for cattle, poultry, rabbit, pig or sheep raising etc. to.Can be mixed for these domestic animals with other feed additives.

[0055] following embodiment only is in order further to illustrate the present invention, not really want to limit the scope of being determined by claim of the present invention.

Embodiment 1

[0056] from the isolated two kinds of new antagonistic strain of mucomembranous surface of caecum of about 1.0 gram broiler chicken, manure enterococcin strain 50-52 (NRRL B-30746) and hamster strains of streptococcus 760 (NRRL B-30745), be suspended in the saline solution (normal saline) of 0.85% (w/v) of about 10ml sterilization and be heated to about 80 ℃ about 15 minutes.The about 1:50 of about 0.10ml and the suspension of 1:2500 are sprawled to be applied on plate count agar or the MRS agar.Dull and stereotyped under little aerobic condition about 24 hours of about 37 ℃ of incubations.Bacterium colony with different shape is rule on MRS agar.These cultures under little aerobic condition about 24 hours of about 37 ℃ of incubations.

[0057] bacterial strain 760 was grown about 24 hours on MRS agar at about 37 ℃.Described bacterial strain is facultative aerobe and gram-positive cocci, can grow between about 37 ℃ and 45 ℃.Described bacterial strain is grown on Nutrient agar or plate count agar, at about 37 ℃ of aerobic incubations generation rule circle after about 24 hours, low projection, slightly gray bacterium colony, has waviness, and diameter is about 2mm.The biochemical property of bacterial strain 760 is determined (Bio-Merieux, France) by APJ 50 CLH systems.Described bacterium colony reduces lactose, mannitol, ribose, salicin, Sorbitol, trehalose, arabinose and 6-(.alpha.-D-galactosido)-D-glucose..It is hydrolysis Raffinose and inulin (inulin) slightly, not hydrolysis arginine and Esculin.It can not α and β haemolysis.It is not grown in the presence of about 6.5% sodium chloride.This bacterial strain is the catalase feminine gender.

[0058] bacterial strain 50-52 cultivated about 24 hours on MRS agar at about 37 ℃.Described bacterial strain is facultative aerobe and gram-positive cocci, can on Nutrient agar or plate count agar, grow, produce irregular cycle, low projection, slightly gray bacterium colony after about 24 hours at about 37 ℃ of aerobic incubations, have waviness, diameter is about 1mm.The biochemical property of bacterial strain 50-52 is determined by the EN-Coccus test.Described bacterial strain decomposes arginine, arabinose and mannitol; Not hydrolysis sorbose, Sorbitol, 6-(.alpha.-D-galactosido)-D-glucose., Raffinose and meticitose.It is grown in the presence of about 6.5% sodium chloride.

[0059] target bacteria that is used to estimate bacterial strain 50-52 and 760 antagonistic activities comprises campylobacter jejuni (C.jejuni) NCTC 11168, the separator of Salmonella enteritidis (S.enteritidis) and escherichia coli (E.coli) 0157:H7.At about 5% O ₂, about 10% CO ₂And about 85% N ₂Little aerobic condition under, with the culture of campylobacter jejuni on brucella agar that contains about 5% parts of fine cellular lysis blood or Campylobacter agar in about 42 ℃ of growths about 24-48 hour down.Other bacterial strain was cultivated about 24 hours down at about 37 ℃ on Nutrient agar.Estimate the antagonistic activity of separator to Campylobacter.The suspension of about 0.2ml normal saline is applied on the MRS agar and about 24 hours of about 37 ℃ of incubations.Downcut about 0.5cm ³MRS agar cubic block and transfer to every block of plate with the polymyxin of the rifampicin of the part cell solution blood of about 5%-10%, about 10mg/ml and about 2.5u/ml additional, with about 10 ⁷On the brucella agar or Campylobacter agar of the campylobacter jejuni inoculation of individual cell.Dull and stereotyped under little aerobic condition as described above at about 42 ℃ of about 24-48 of following incubation hours.The size of the diameter by measuring the campylobacter jejuni inhibition zone is estimated antagonistic activity.

Embodiment 2:

[0060] by two kinds of antagonistic strains: the culture of enterococcus 50-52 and streptococcus 760 extracts thick antimicrobial preparations.In that (NRRL B-30744 as above) under aerobic conditions cultivates about 14 hours together with inductor bacterial strain lactococcal strain 320 in 10% the brucella broth of antagonist at about 250ml under 37 ℃.The concentration of Producer bacterial strain is about 106CFU/mL and the inductor bacterial strain is about 10 ⁷CFU/mL.The culture of gained centrifugal about 10 minutes at about 2,500 * g removes most of living cells.Supernatant that decant falls and about 60% saturated ammonium sulfate mix and are incorporated in about 24 hours of about 4 ℃ of incubations with the plain chemical compound of precipitum.After centrifugal about 20 minutes with about 10,000 * g, precipitum is suspended in the sodium phosphate buffer of the about 10mM pH about 7.0 of about 1.5ml again, and identical buffer dialysis is spent the night to about 2.5L.Gained solution is called as thick antimicrobial preparations (CAP).The sample of described prepared product is by filter membrane (Millipore, Massachusetts Bedford, USDA) sterilization through 0.22 micron pore size.Table 1 shows the bacteriocin production of using and not using the inductor bacterial strain.

Table 1: use and do not use the bacteriocin production of inductor bacterial strain lactococcal strain 320

Embodiment 3

[0061] adopt spot test to determine the microbial resistance spectrum of described CAPs.The thick antimicrobial preparations of sterilization (CAP) that about 1ml is obtained among the embodiment 2 in the above is with about 1ml phosphate-sodium salt buffer (pH about 7.0) dilution and as sterilizing among the embodiment 2.Every kind of about 10 microlitres of sample are applied on the Campylobacter agar or Nutrient agar (MPA or Meta peptone agar) that the blood of the cell inoculation of usefulness target bacteria replenishes in advance.Under little aerobic condition, grow under about 42 ℃ and contain the flat board of campylobacter jejuni, about 28 ℃ of following aerobic cultivation yersinia enterocoliticas (Y.enterocolitica) and yersinia pseudotuberculosis (Y.pseudotuberculosis), other bacterial isolateses descended aerobic incubations about 24 or 48 hours at about 37 ℃.The inhibition zone that identification based target antibacterial is produced.The activity of CAP is represented (people such as Henderson with the every milliliter of prepared product of arbitrary unit (AU) when the growth inhibiting visibility region of culture occurs, Archives of Biochemistryand Biophysics (biochemistry and biophysics archives), the 295th volume, 5-12,1992, incorporate this paper into by reference).All experiments are all carried out twice.Table 2 among the face embodiment 4 as follows.

Embodiment 4

[0062] CAPs and bacteriocin in the triglycine buffer in about 15% agarose gel weight, about 1%SDS (electrophoresis in 9 * 12cm).After about 4 hours, gel is fixed with the solution that contains have an appointment 15% ethanol and about 1% acetic acid at about 100mA electrophoresis.Used the described gel of distilled water wash then about 4 hours.In order to determine the molecular weight of albumen level part, described gel is with containing 0.21% the blue G-250 of coomassie of having an appointment, about 40% ethanol and about 7% acetic acid dyeing.The method of washed gel by Bhunia etc. is to three kinds of target bacteria: campylobacter jejuni NCTC 11168, colon bacillus 0157: H7904 and Salmonella enteritidis 204 are tested (Journal of Industrial Microbiology (industrial microorganism magazine), the 2nd volume, 319-322,1987; Incorporate this paper into by reference).Described gel is placed in the Micro-Organism Culture Dish (Petri Dishes), covers with 5% blood-semisolid Campylobacter agar (about 0.75%) or semisolid MPA, and with the cell inoculation of test strain.About 48 hours of about 42 ℃ of incubations, colon bacillus 0157: H7 and Salmonella enteritidis were about 24 hours of about 37 ℃ of incubations under little aerobic condition for the flat board that contains campylobacter jejuni.Evaluation is based on the visibility region of the time test bacteriostatic growth that has bacteriocin.

[0063] (pI:CAP760 contains the level part with pI=about 9,2 and about 9,5 for different totally different level part on two kinds of isoelectric point, IPs of isoelectrofocusing identification.CAP 50-52 contains the level part with pI=about 7.7 and 8.4.Have in prepared product 760 in the level part of pI=about 9.5 and observe antagonistic activity to campylobacter jejuni, and in prepared product 50-52, this is suppressed in the level part of pI=about 8.4 and is observed (Figure 1A-B and 2A-B, following table 1).

[0064] with the gel of campylobacter jejuni blanketing determine which or which be with corresponding microbial resistance, its molecular weight and isoelectric point, IP.Figure 1A of directed toward bacteria element 760 and 1B show the molecular weight (1A) of active component and the isoelectric point, IP (1B) of active component.With campylobacter jejuni blanketing gel to determine antimicrobial acivity, molecular weight and isoelectric point, IP.In Figure 1A, road 1 shows that the LMW scope is 14,400-94, and 000 molecular weight marker thing (Amersham Pharmacia Biotech): 14,000,20,100,30,000,43,000,67,000 and 94,000Da.Be insulin in the road 2, and the molecular weight marker thing of β chain (Sigma, USA): 3,500Da.Road 3 shows the pure bacteriocin 760 of corresponding antimicrobial acivity, and growth inhibited zone (arrow) has about 5, the quality of 500Da.Figure 1B, and road 1 demonstration pI reference material (the albumen test mixture, pI label albumen, Serva).What road 2 showed is the pure bacteriocin 760 of corresponding antimicrobial acivity, and growth inhibiting zone (arrow) has about 9.5 pI.Other bands do not show antimicrobial acivity.

[0065] Fig. 2 A and 2B demonstration utilizes the directly plain 50-52 of bacterial detection of SDS-PAGE (2A) and isoelectrofocusing (2B).With campylobacter jejuni blanketing gel with determine which or which be with corresponding antimicrobial acivity and molecular weight (shown in Fig. 2 A).Road 1 shows that the LMW scope is 1,600-26, and 000 molecular weight marker thing (Amersham Pharmacia Biotech): 1,600,3,500,6,500,14,200,17,000 and 26,000Da.Contain the band in the road 2 of pure bacteriocin 50-52 of corresponding antimicrobial acivity, it is about 3 that the growth inhibited zone has, the molecular weight of 900Da.Fig. 2 B, road 1 contain the pI reference material (the albumen test mixture, pI label albumen, Serva).Band (pure bacteriocin 50-52) in the road 2 of corresponding antimicrobial acivity, growth inhibiting zone (arrow) has about 8.4 pI.Other bands do not show antimicrobial acivity.

[0066] the bacteriocin specimen is placed on (the about 4.4-10.0 of pH) (Novex, California San Diego) on the IEF gel.At XCM II ^TMDescribed gel was run about 1 hour at about 100V, and 200V ran about 2 hours, ran about 30 minutes at 500V.Fixing with about 4 hours of distilled water wash gel, then dye with the isoelectric point, IP (Pi) of definite described bacteriocin and the ability of their inhibition test strain growths, as in Fig. 1 and 2 and as shown in the table 2 with Coomassie blue G-250.

Table 2: the antibacterial activity of the thick antimicrobial preparations of bacteriocin that the method by spot test, SDS-PAGE and isoelectrofocusing (IEF) is estimated

Bacteriocin	Test strain	Inhibition activity (AU/ml) in the spot test	The inhibition activity that SDS-PAGE measures	The inhibition activity that IEF measures
					760	Campylobacter jejuni NCTC 11168 Salmonella enteritidis 204 Escherichia coli O 157s: H7 904	51,200 12,80012,800	M.W.5.5kDa M.W.5.5kDaM.W.5.5kDa	Be with 1 pI=9.5 to be with 1 pI=9.5 to be with 1 pI=9.5
50-52	Campylobacter jejuni ATCC 11168 Salmonella enteritidis 204 Escherichia coli O 157s: H7904	12,800 12,8003,200	M.W.3.9kDa M.W.3.9kDaM.W.3.9kDa	Be with 1 pI=8.4 to be with 1 pI=8.4 to be with 1 pI=8.4

Embodiment 5

[0067] (bacterial strain 320 as above) is cultivated simultaneously in brucella broth (Difco) lining with inducing the bacterial isolates lactococcus lactis will to produce enterococcus bacterial strain 50-52 and strains of streptococcus 760.About 1.32 * 10 ⁷Enterococcus faecalis or the streptococcic cell of hamster and about 3.9 * 10 ⁶The lactococcus lactis cell be placed in the flask of 250ml together and cultivated about 24 hours.The concentration of production and inducible strain and the ratio of bacteriocin are lived and are every two hours determined once with definite optimum time that obtains the amount of available bacteriocin.Separate and the purification of bacterial element by two kinds of methods.The firstth, then be triphasic chromatograph with the ammonium sulfate precipitation bacteriocin: the hydrophobic interaction chromatography on ion exchange chromatography on the gel filtration on the Superose 12HR, the Superose SPFF and octyl group Sepharose 4FF.This is method A.

[0068] for method B, when cultivating Producer and inductor bacterial strain at the same time, most of bacteriocin of being produced all is secreted in the culture fluid.The bacteriocin of part will be adsorbed onto on inductor and the Producer bacterial strain.For fear of the loss of bacteriocin of absorption, the described bacteriocin of eluting from the cell.Method B comprised for two steps: (1) is the separation of bacterial element from the supernatant of culture fluid; And (2) are from inducing and produce separation of bacterial element the cell precipitation of bacterial strain.In step 1, obtain described culture and by about 10, centrifugal about 15 minutes sedimentation cells of 000g and separating.Supernatant adds on the octyl group Sepharose4FF post to reclaim bacteriocin.In step 2, use cell precipitation.Described precipitation is suspended in the phosphate buffer with about 0.7%NaCl, pH (eluent), about 20 minutes of mixing suspension and incubation.Behind the incubation, described suspension is about 10, centrifugal about 15 minutes of 000g.Employing is ion exchange chromatography separation of bacterial element from supernatant on Superose SP FF.Product by two kinds of method purification is analyzed its antagonistic activity to campylobacter jejuni.Carry out SDS-PAGE and etc. point focusing (IEF).The results are summarized in the following table 3 and table 4.

[0069] finds by live active high 4 times than the like products that obtains by method A of the ratio of the described prepared product of method B purification.Need about 52 hours than usefulness method A, the prepared product of production method B needs about 12.5 hours.Method B is by eliminating gel filtration and hydrophobic interaction chromatography, and only uses ion exchange chromatography and reduced and need be used for from the step of CAP purification of bacterial element.

Table 3: usefulness method A and B isolated bacterial plain 760 and 50-52 are than the comparative data of living

Table 4: purification of bacterial of determining by spot test, SDS-PAGE and isoelectrofocusing plain 760 and the antimicrobial acivity of 50-52

Bacteriocin	Test strain	Suppress active, spot test (AU/ml)	Suppress active, SDS-PAGE (kDa)	Suppress active, IEF
					760	Campylobacter jejuni	51,200	+ be with 1 m.w.=5.5	+ be with 1 pI=9.5
	Salmonella enteritidis 204	12,800	+ be with 1 m.w.=5.5	+ be with 1 pI=9.5
						Escherichia coli O 157: H7	12,800	+ be with 1 m.w.=5.5	+ be with 1 pI=9.5
50-52	Campylobacter jejuni	12,800	+ be with 1 m.w.=3.9	+ be with 1 pI=8.4
						Salmonella enteritidis 204	12,800	+ be with 1 m.w.=3.9	+ be with 1 pI=8.4
	Escherichia coli O 157: H7	6,400	+ be with 1 m.w.=3.9	+ be with 1 pI=8.4

[0070] (Applied Biosystems (applying biological system) USA) determines the aminoacid sequence of the bacteriocin of purification with the Edman degraded to adopt 491 cLC automatic sequencers.Bacteriocin is about 72 hours of about 110 ℃ of hydrolysis under vacuum in the HCl of about 6M.Bacteriocin 50-52 and 760 molecular weight adopt Voyager-DERP, and (Perkin-Elmer USA) is determined by mass spectrum.The MALDI-TOF system, substance assistant laser desorpted ionized flight time system, use with substrate 2-cyano group-hydroxycinnamic acid.Aminoacid sequence is:

50-52：TTKNYGNGVCNSVNWCQCGNVWASCNLATGCAAWLCKLA SEQ ID NO 1

760：NRWYCNSAAGGVGGAAVCGLAGYVGEAKENIAGEVRKGWGMAGGFTHNKA

CKS SEQ ID 2

The calculating molecular weight of described peptide is about 3.9kDa for bacteriocin 50-52, and is about 5.5kDa for bacteriocin 760.The analysis showed that following molecular weight with MALDI-TOF: for bacteriocin 50-52 is about 3, and 932Da is about 5 for bacteriocin 760,362Da.

Embodiment 6

[0071] determines that enzyme, temperature and pH are for the active influence of bacteriocin.A kind of in the following enzyme of about 10ml is transferred in the test tube that contains the 20ml bacteriocin of having an appointment: β-chymase-Yue 100mg/ml, E.C. 3.4.21.64-Yue 200mg/ml, papain-Yue 60mg/ml, lysozyme-Yue 750mg/ml and lipase-Yue 100mg/ml (all from Sigma-Aldrich Corp., St. Louis, the Missouri State).After about 3 hours, the mixture of bacteriocin and enzyme adopts as the spot test among the embodiment 3 and analyzes antimicrobial acivity at about 37 ℃ of incubations.Undressed bacteriocin plays positive control.

[0072] in order to study the heat stability of bacteriocin, the sample of about 2mg/ml boiled in water-bath about 15 minutes, and cooling is also pressed the evaluation of its antimicrobial acivity.The bacteriocin of about 2mg/ml is used to estimate the pH effect.With the sterile solution of about 2ml, the NaOH of about 10mM or the HCl of about 10mM add to the pH of sample with test from about 3 to about 10.Sample is about 37 ℃ of incubations about 2 hours and 24 hours, and about 20 minutes of about 90 ℃ of incubations.Phosphate buffer by adding about 4mM sterilization with sample be adjusted to about 7.2 pH and with as top embodiment 3 in spot test analyze its antimicrobial acivity.

[0073] after handling with β-chymase, E.C. 3.4.21.64 and papain, bacteriocin 50-52 and 760 has lost its antimicrobial acivity, but when its with lysozyme, lipase or maintenance (table 5) when being heated to about 90 ℃ of processing.When from about 3.0 to about 9.0 different pH, they are stable, do not have activity in (table 5 and 6) but become when about pH10.

Table 5: enzyme and temperature are to the influence of bacteriocin antibacterial activity

Handle	Active *
		β-chymase	-
E.C. 3.4.21.64	-
		Papain	-
Lysozyme	+
		Lipase	+
100 ℃, 15 minutes	+

^*Active definite by spot test, use campylobacter jejuni NCTC 11168 as indicator strain

With enzyme or after being exposed to Temperature Treatment:

+ exist active

-there is not an activity

Table 6:pH is to the active effect of bacteriocin

pH	With the definite activity of campylobacter jejuni NCTC 11168 spot tests
			20min @ 90℃ 2h @ 37℃ 24h @ 37℃
3.0	+ + +
		5.0	+ + +
6.2	+ + +
		7.0	+ + +
8.4	+ + +
		9.1	+ + +
10.0	- - -

+ exist active

-there is not an activity

Embodiment 7

[0074] campylobacter is used as top being determined by strain separated in the broiler chicken poult of describing in embodiment 1 bacteriocin 760 of purification and the susceptibility of E50-52 prepared product.The antagonistic activity of bacteriocin is based on minimal inhibitory concentration (MICs) evaluation, and described minimal inhibitory concentration is determined by agar diffusion.Table 7 shows the MICs of bacteriocin at the bacterial strain of being tested.To some extent the bacteriocin of test all be the height antagonism for the campylobacter bacterial strain.Bacterial strain 760 will more have much active than the bacterial strain that remaining has at about 0.05 to about 0.1 μ g/ml MICs.

The bacteriocin MIC of table 7. pair campylobacter bacterial strain

Embodiment 8

[0075] Gram-positive and gram negative bacteria adopt for the susceptibility of bacteriocin 760 and 50-52 as top spot test described in embodiment 3 is determined.The results are shown in table 8 and 9.As from table 8 as seen, bacteriocin 760 has wide spectrum and high-caliber antagonistic activity.Described bacteriocin suppresses the growth of Gram-positive and gram negative bacteria.Its MIC for test strain changes in about 3.2 μ g/ml scopes at about 0.1 μ g/ml.As from table 9 as seen, bacteriocin 50-52 has wide spectrum and high-caliber antagonistic activity.Described bacteriocin suppresses the growth of Gram-positive and gram negative bacteria.Its MIC for test strain changes in about 3.2 μ g/ml scopes at about 0.1 μ g/ml.

Table 8. is by the antibacterial activity of the definite bacteriocin 760 of spot test

Test strain	MIC，mg/ml
		Salmonella enteritidis 1 (S.enteritidis 1)	0.2
Salmonella enteritidis 4 (S.enteritidis 4)	0.4
		Salmonella enteritidis 204 (S.enteritidis 204)	0.2
Salmonella enteritidis 237 (S.enteritidis 237)	0.2
		Salmonella choleraesuls 434/4 (S.choleraesuis 434/4)	0.4
Salmonella choleraesuls 370 (S.choleraesuis 370)	0.4
		Salmonella typhimurium 383/60 (S.typhimurium 383/60)	0.4
Salmonella typhimurium 320 (S.typhimurium 320)	0.2
		Fowl typhoid Hakuri Salmonella (S.gallinarum pullorum)	0.4
Escherichia coli HB101 (E.coli HB101)	0.1
		Escherichia coli C600 (E.coli C600)	0.1
Escherichia coli O 157: H7Y-63 (E.coli O157:H7 Y-63)	1.6
		Escherichia coli O 157: H7G-3 (E.coli O157:H7 G-3)	1.6
Escherichia coli O 157: H7OD-3 (E.coli O157:H7 OD-3)	1.6
		Escherichia coli O 157: H7lab.39 (E.coli O157:H7 lab.39)	1.6
Yersinia enterocolitica 03 (Y.enterocolitica 03)	0.1
		Ye Ersenshi enterobacteria 09 (Y.enterolitica 09)	0.1
Ye Ersenshi enterobacteria 04 (Y.enterolitica 04)	0.1
		Citrobacter freundii (Citrobacter freundi)	1.6
Klebsiella pneumoniae (Klebsiella pneumoniae)	3.2
		Shigella dysenteriae (Sh.dysenteriae)	0.1
Staphylococcus aureus (Staphylococcus aureus)	1.6
		Staphylococcus epidermidis (Staphylococcus epidermidis)	1.6
Yersinia pseudotuberculosis (Y.pseudotuberculosis)	3.2
		Yersinia pseudotuberculosis (Y.pseudotuberculosis)	3.2
Pseudomonas aeruginosa 25583 (Pseudomonas aeruginosa 25583)	0.4
		Proteus mirabilis (Proteus mirabilis)	3.2
Morganella morganii strain (Morganella morganii)	3.2
		Listeria monocytogenes 9-72 (L.monocytogenes 9-72)	0.1
Campylobacter jejuni L4 (C.jejuni L4)	0.1

Table 9. is by the antibacterial activity of the definite bacteriocin 50-52 of spot test

Bacterial strain	MICs(μg/ml)
		Salmonella enteritidis 1 (S.enteritidis 1)	0.1
Salmonella enteritidis 4 (S.enteritidis 4)	0.1
		Salmonella enteritidis 204 (S.enteritidis 204)	0.2
Salmonella enteritidis 237 (S.enteritidis 237)	0.1
		Salmonella choleraesuls 434/4 (S.choleraesuis 434/4)	0.1
Salmonella choleraesuls 370 (S.choleraesuis 370)	0.1
		Salmonella typhimurium 383/60 (S.typhimurium 383/60)	0.1
Salmonella typhimurium 320 (S.typhimurium 320)	0.1
		Fowl typhoid Hakuri Salmonella (S.gallinarum pullorum)	0.1
Escherichia coli HB101 (E.coli HB101)	0.1
		Escherichia coli C600 (E.coli C600)	0.1
Escherichia coli O 157: H7Y-63 (E.coli O157:H7 Y-63)	0.1
		Escherichia coli O 157: H7G-3 (E.coli O157:H7 G-3)	0.1
Escherichia coli O 157: H7OD-3 (E.coli O157:H7 OD-3)	0.1
		Escherichia coli O 157: H7lab.39 (E.coli O157:H7 lab.39)	0.1
Yersinia enterocolitica 03 (Y.enterocolitica 03)	0.1
		Ye Ersenshi enterobacteria 09 (Y.enterolitica 09)	0.4
Ye Ersenshi enterobacteria 04 (Y.enterolitica 04)	0.1
		Citrobacter freundii (Citrobacter freundi)	0.1
Klebsiella pneumoniae (Klebsiella pneumoniae)	0.2
		Shigella dysenteriae (Sh.dysenteriae)	0.4
Staphylococcus aureus (Staphylococcus aureus)	0.4
		Staphylococcus epidermidis (Staphylococcus epidermidis)	3.2
Yersinia pseudotuberculosis (Y.pseudotuberculosis)	0.4
		Pseudomonas aeruginosa 25583 (Pseudomonas aeruginosa 25583)	3.2
Proteus mirabilis (Proteus mirabilis)	1.6
		Morganella morganii strain (Morganella morganii)	3.2
Listeria monocytogenes 9-72 (L.monocytogenes 9-72)	0.2
		Campylobacter jejuni L4 (C.jejuni L4)	0.2

Embodiment 9

[0076] minimal inhibitory concentration of bacteriocin 760 and 50-52 and methicillin (MICs) is determined by spot test.Each concentration of the bacteriocin of the purification in 10 μ l volumes is added in the culture of staphylococcus aureus, staphylococcus epidermidis, Pseudomonas aeruginosa and helicobacter pylori.For except all culture of helicobacter pylori (under little aerobic condition) all about 24 hours of the aerobic incubation down of about 37C.Show in result's table 10 below.

Table 10: bacteriocin is to the MIC of staphylococcus aureus, staphylococcus epidermidis, Pseudomonas aeruginosa, helicobacter pylori

[0077] aforementioned detailed description is an illustrative purposes for example.These details only are for this purpose, and those skilled in the art can make variation and can not deviate from the spirit and scope of the present invention.

Claims (15)

1. isolating bacteriocin of being produced by enterococcus bacterial strain or strains of streptococcus, described enterococcus or strains of streptococcus have the recognition feature of the bacterial strain that is selected from the group of being made up of NRRL B-30746 and NRRL B-30745.

2. bacteriocin as claimed in claim 1 has the aminoacid sequence of SEQ ID NO 1.

3. bacteriocin as claimed in claim 1 has the aminoacid sequence of SEQ ID NO 2.

5. the isolating manure enterococcin strain that has the recognition feature of NRRL B-30746.

6. the isolating hamster strains of streptococcus that has the recognition feature of NRRL B-30745.

7. therapeutic combination comprises:

(a) reduce at least a isolating bacteriocin of being produced by enterococcus bacterial strain or strains of streptococcus of the effective dose of at least a target bacteria field planting level at least, described enterococcus bacterial strain or strains of streptococcus have the recognition feature of the bacterial strain that is selected from the group of being made up of NRRL B-30746, NRRL B-30745 and composition thereof; And

(b) suitable treatment carrier.

8. therapeutic combination as claimed in claim 7, wherein said bacteriocin have the aminoacid sequence that is selected from the group of being made up of SEQ ID NO.1, SEQID NO 2 or its mixture.

9. therapeutic feedstuff that is used for animal comprises:

(a) reduce at least a isolated bacterial element of producing by enterococcus bacterial strain or strains of streptococcus of effective dose of the field planting level of at least a target bacteria at least, described enterococcus bacterial strain or strains of streptococcus have the recognition feature of the bacterial strain that is selected from the group of being made up of NRRL B-30746, NRRL B-30745 and composition thereof

(b) treatment carrier, and

(c) animal feed.

10. therapeutic feedstuff as claimed in claim 11, wherein said at least a bacteriocin have the aminoacid sequence that is selected from the group of being made up of SEQ IDNO1, SEQ ID NO 2 and composition thereof.

11. one kind is used for reducing at least the method for at least a target bacteria in the field planting level of animal, comprises:

Be reduced at least less a kind of target bacteria the field planting level effective dose comprise at least a isolating by enterococcus bacterial strain or strains of streptococcus production bacteriocin and the therapeutic combination of treatment carrier be administered to animal, described enterococcus bacterial strain or strains of streptococcus have the recognition feature of the bacterial strain that is selected from the group of being made up of NRRL B-30746, NRRL B-30745 and composition thereof.

12. method as claimed in claim 11, wherein said bacteriocin have the aminoacid sequence that is selected from the group of being made up of SEQ ID NO 1, SEQ IDNO 2 and composition thereof.

13. have the isolated strains of lactococcus lactis of the recognition feature of NRRL B-30744.

14. a method that is used for being improved by antibacterial bacteriocin output comprises:

(a) lactococcus lactis that adds the bacteriocin lifting capacity in the culture of bacteriocinogeny antibacterial is to form the co-cultivation thing, and described lactococcus lactis has the recognition feature of NRRL B-30744, and

(b) cultivate described co-cultivation thing at about 37 ℃.

15. a method that is used for the separation of bacterial element, it comprises:

(a) co-cultivation bacteriocinogeny antibacterial and inductor antibacterial to be producing bacteriocin in culture medium,

(b) by the described culture medium of centrifugal results and cell obtaining supernatant and the little group of cell,

(c) the described supernatant that contains bacteriocin is applied to the hydrophobic interaction chromatography post with the plain prepared product of acquisition isolated bacterial,

(d) in elution buffer the little group of the described described cell of incubation from step b forming first mixture,

(e) by centrifugal from little group of described cell and described buffer isolated cell,

(f) by ion exchange chromatography from described buffer separation of bacterial element.

16. method as claimed in claim 15, wherein said ion exchange chromatography are used Superose SP FF post.

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