CN102887945A - Bacteriocin and new bacterial isolates - Google Patents
Bacteriocin and new bacterial isolates Download PDFInfo
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Abstract
New bacteriocin and/or new lactic acid-producing strain are used for at least reducing the planting level of at least one target bacteria in animals, particularly in domestic fowl.
Description
The application Chinese patent application numbers 200580049168.0, denomination of invention that to be PCT International Application Serial No. PCT/US2005/008991 of submitting on March 18th, 2005 enter the China national stage on September 18th, 2007 are divided an application for the application for a patent for invention of " bacteriocin and new bacterial isolates ".
Technical field
The present invention relates to by the disease in new bacteriocin control animal, the particularly poultry of using new bacteriocinogeny lactic acid producing bacteria and/or being produced by these bacterial classifications.The invention still further relates to new bacteriocin, the aminoacid sequence of described new bacteriocin, and the lactic acid producing bacteria bacterial strain that produces described new bacteriocin.Further, the present invention relates to contain described new bacteriocin and/or produce the therapeutic composition of bacterial strain of their lactic acid producing bacteria, and the purposes of described therapeutic composition.
Background technology
The edible incorrect poultry prod of processing has caused people's intestinal disease.Early have recognized that Salmonellas (Salmonella spp.) is the pathogenic bacterium of this class disease, recently recognize that Campylobacter (Campylobacter spp.), particularly campylobacter jejuni (Campylobacter jejuni) also come into the picture wherein.These two kinds of microorganisms can be colonizated in the gi tract of poultry, and to these birds without any harmful effect, although some can be detected by the birds of field planting, but asymptomatic carrier can produce and treating processes in these microorganisms of Free propagation, cause further infecting birds and the corpse of work.Poultry in provand as the original storage master (Jones etc., Journal ofFood Protection (food protection magazine), the 54th volume, No.7,502-507, in July, 1991) of Salmonellas and Campylobacter.Can eliminate the problem that poultry pollutes forming the production period prevention field planting in live poultry.
Many factors facilitate bacterium in animal digestive tract 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 ofFood Production (foodstuff production magazine), the 42nd volume, 164-167,1979); (2) biliary 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 ofInternal Medicine (Archives of Internal Medicine), the 130th volume, 545-549,1972; Gilliland and Speck, Journal of Food Protection (food protection magazine), the 40th volume, 820-823,1977); Hugdahl etc., Infection and Immunity (infection and immunity), the 56th volume, 1560-1566,1988); (3) wriggling; (4) digestive ferment (Marmur, Journal ofMolecular Biology (molecular biology magazine), the 3rd volume, 208-218,1961); (5) immune response; (6) antimicrobial compounds of indigenous microorganism (indigenous microorganism) and their generations.Front four factors depends on host's phenotype, may not be in fact controlled variable.The immune response in stomach and intestine (GI) road is difficult for regulating.The factor that relates to indigenous microorganism and metabolite thereof depends on the normal microflora in GI road.
A kind of possible method of the field planting of control Campylobacter and/or Salmonellas is by using competitive exclusion (CE).Nurmi and Rantala (Nature (nature), the 241st volume, 210-211,1973) prove by resisting the Salmonellas field planting to the young chick (chick) that microflora does not also build up from the bacterium tube feed of healthy poultry enteral prods, effectively control the infection of Salmonellas.Use uncertain CE prepared product acceleration to chick and newly hatch the maturation of the archenteric flora of birds, and the replacer who transmits the natural process of microflora to its offspring by the hen that grows up is provided.The result of laboratory and field experiment provides by use the evidence that normal microflora falls to controlling the advantage of Campylobacter to chick; The flock of birds that reduces is the frequency of coli infections (Mulder and Bolder, IN:Colonization Control of human bacterial enteropathogens in poultry (field planting of the bacillary enteropathogen of people control in the poultry) by bending; L.C.Blankenship (volume), Academic Press, San Diego, California, 359-363,1991) and reduce had been reported (Stern, Poultry Science (poultry science), the 73rd volume by campylobacter jejuni (C.jejuni) level in the ight soil of the birds of field planting, 402-407,1994).
Schoeni and Wong (Appl.Environ.Microbiol., the 60th volume, 1191-1197,1994) reported the field planting that has significantly reduced campylobacter jejuni in the broiler chicken (broiler) by using carbohydrate additive and three kinds of certified antagonists (difference citric acid bacillus (Citrobacter diversus) 22, Cray Bai Shi pneumobacillus (Klebsiella pneumoniae) 23 and intestinal bacteria (Escherichia coli) 25).Also have after the poultry separation and Culture thing treatment with Lactobacillus acidophilus (Lactobacillus acidophilus) and streptococcus faecium (Streptococcus faecium), evidence (the Morishita etc. that campylobacter jejuni significantly reduces in the sample in the intestines of infection broiler chicken, Avian Diseases (poultry disease), the 41st volume, 850-855,1997).
Snoeyenbos etc. (U.S. Patent No. 4,335,107, June nineteen eighty-two) cultivate competitive exclusion (CE) the microflora technology that this prepared product has been developed the field planting of prevention Salmonellas by freeze-drying ight soil and anaerobism.Mikola etc. (U.S. Patent No. 4,657, in April, 762,1987) are used for the field planting of prevention Salmonellas with enteron stool and cecal content as CE microflora source.(the U.S. Patent No. 5 such as Stern; 451; 400; September nineteen ninety-five and United States Patent (USP) 6,241,335; April calendar year 2001) a kind of mucous membrane CE composition is disclosed with the field planting of protection poultry and livestock opposing Salmonellas and Campylobacter; wherein the Saliva Orthana 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.(the U.S. Patent No. 5 such as Nisbet, 478,557, in December, 1996) a kind of definite probiotic bacterium (probiotic) is disclosed, it can derive from various domestic animals, its be by cultured continuously directly from the ight soil of the target animal of growing up, the batch culture of caecum and/or large intestinal contents obtains.
The multiple proof of microorganisms has the compound of antibacterial property.The compound that one class is such, bacteriocin is comprised of sterilization protein, and its mechanism of action is similar to ion carrier antibiotic.Bacteriocin has the activity of opposing and the closely-related bacterial classification of its producer usually.They are by complicated microbial population, enteron aisle for example, and the extensive existence in oral cavity or the isolated bacteria culture of other epithelial surface shows that bacteriocin may have regulating effect aspect the population dynamics of bacteria ecological system.Bacteriocin is defined as having biological activity protein partly and the compound (Tagg etc., Bacteriological Reviews (Bacteriological Reviews), the 40th volume, 722-256,1976) of germicidal action by bacteriogenic.Other characteristics comprise: (1) narrow spectrum suppresses active, concentrates on closely-related bacterial classification; (2) be combined with specific cell receptor; And (3) plasmid carries that bacteriocin produces and the genetic determination of host cell bacteriocin immunity bunch.The Antagonism material of not exclusively determining is called as " bacteriocin sample material ".Some are the bacteriocin of antagonism gram positive bacterium, opposite non-confrontational gram negative bacterium effectively, has the activity than wide spectrum.Existing suggestion term bacteriocin, when being used for describing the inhibitor that is produced by gram positive bacterium, should satisfy (1) is the minimum standard (Tagg etc., the same) that a kind of peptide and (2) have fungicidal activity.
Lactic-acid-bacterium is one of most important probiotic microorganism.They are gram-positive, and are asporogenic, the biology of the shortage cytopigment of catalase feminine gender.They are anaerobism but oxytolerant, there have to be complicated nutritional requirement, acidproof and strictly fermentable, and with the main end product of lactic acid as sugar-fermenting.Lactic acid producing bacteria comprises lactobacillus (Lactobacillus) bacterial classification, bifidus bacillus (Bifidobacterium) bacterial classification, enterococcus faecalis (Enterococcus faecalis), faecium (Enterococcus faecium), Lactococcus lactis (Lactococcus lactis), Leuconostoc mesenteroides (Leuconostoc mesenteroides), pediococcus acidilactici (Pediococcus acidilactici), have a liking for sour lactic acid bacteria (SPorolactobacillus inulinus), thermophilus streptococcus (Streptococcus thermophilus) etc.These bacterial classifications extensively exist the bacteriocin aspect to be subject to special concern therein, also are widely used in fermented milk, food and the meat processing industry.They are in preservation and the existing detailed record of the effect in the flavor characteristic of food.Most of bacteriocin that is produced by these bacterial classifications only has the activity of other lactic-acid-bacteriums of opposing, but have several demonstrate to than macrospecies be difference gram positive bacterium and, under given conditions, to the anti-microbial activity of gram negative bacterium.
Lactobacillus has been widely studied the generation of its antagonist.Comprising bacteriocin (DeKlerk, 1967 fully qualitatively; Upreti and Hinsdill 1975; Barefoot and Klaenhammer 1983; Joerger and Klaenhammer 1986); Possible bacteriocin sample material (Vincent et al., 1959), with other and bacteriocin must be not relevant antagonist (Vakil and Shahni 1965; Hamdan and Mikolaj cik 1974; Mikolaj cik and Hamdan 1975; And Shahni etc., 1976).
Lactic-acid-bacterium bacteriocin known to Klaenhammer (1993) incites somebody to action at that time is divided into four primary categories:
I class---lantibiotics (Lantibiotics), it is＜the little peptide of 5kDa, contain uncommon amino acid L-lanthionine and Beta-methyl L-lanthionine.They are subject to special concern is because they have for other bacteriocins the very activity of wide spectrum.Example comprises Nisin, Nisin Z, carnocin U149, lacticin481, and lactocin5.
II class---the little peptide that does not contain L-lanthionine: a class is heterogeneous＜the little peptide of 10kDa.This class comprises have the opposing listeria spp peptide of activity of (Listeria spp.).
The III class---large heat-labile＞protein of 30kDa.Example is Helveticin.
The IV class---contain for example complicated bacteriocin-protein of lipid and carbohydrate of extra section.
The new composition of the new bacteriocin that the invention provides the new bacterial strain that contains at least a lactic acid producing bacteria bacterial strain and/or produced by at least a new bacterial strain; Use the method for described bacterial strain or bacteriocin, described new bacterial strain, the aminoacid sequence of described new bacteriocin, and using method, they are all different from bacterial strain, bacteriocin and using method in the correlation technique.
Summary of the invention
Therefore, an object of the present invention is to provide lactobacillus and the enterococcal new bacterial strain that produces new bacteriocin.
Another object of the present invention provides new saliva lactobacillus (Lactobacillus salivarius) the bacterial strain PVD32 that identification mark is NRRL B-30514.
Another purpose of the present invention provides the new Lactobacillus acidophilus that identification mark is NRRL B-30510 (Lactobacillus acidophilus) LWP320.
Another purpose of the present invention provides the new enterococcus faecalis that identification mark is NRRL B-30645 (Enterococcus faecalis) LWP21.
Another purpose of the present invention provides the new enterococcus durans that identification mark is NRRL B-30511 (Enterococcus durans) bacterial strain LWP26.
Another purpose of the present invention provides the lactobacillus that separates of the identification mark with the lactic bacilli strains that is selected from the group that is comprised of NRRL B-30514 and NRRL B-30510.
Another purpose of the present invention provides the lactobacillus that separates of the identification mark with the lactic bacilli strains that is selected from the group that is comprised of NRRL B-30645 and NRRL B-30511.
Another object of the present invention provides the new bacteriocin that is produced by lactobacillus and enterococcal new bacterial strain.
Another purpose of the present invention provides the new bacteriocin OR7 with aminoacid sequence shown in SEQ ID NO 1.
Another purpose of the present invention provides the new bacteriocin LWP320 with aminoacid sequence shown in SEQ ID NO 2.
Another purpose of the present invention provides the new bacteriocin LWP21 with aminoacid sequence shown in SEQ ID NO 3.
Another purpose of the present invention provides the new bacteriocin LWP26 with aminoacid sequence shown in SEQ ID NO 4.
Another object of the present invention provides therapeutic composition, comprise: (a) bacteriocin of at least a separation of significant quantity, being reduced at least less a kind of field planting level of target bacteria, the lactic acid producing bacteria bacterial strain of identification mark that described bacteriocin has the bacterial strain of the group that is selected from NRRL B-30514, NRRL B-30510, NRRL B-30645, NRRL B-30511 and its compositions of mixtures produces; (b) suitable treatment carrier.In one embodiment of the invention, described bacteriocin has the aminoacid sequence that is selected from by the group of SEQ ID NO.1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 or its compositions of mixtures.
Another object of the present invention provides the treatment feed for animal, comprise: (a) bacteriocin of at least a separation of significant quantity, to be reduced at least less a kind of field planting level of target bacteria, described bacteriocin has the lactic acid producing bacteria bacterial strain that is selected from by the identification mark of the bacterial strain of the group of NRRL B-30514, NRRL B-30510, NRRL B-30511, NRRL B-30645 and its compositions of mixtures and produces, (b) treatment carrier, and (c) animal-feed.In one embodiment of the invention, described at least a bacteriocin has the aminoacid sequence that is selected from by the group of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and its compositions of mixtures.
Another object of the present invention provides the treatment feed for animal, comprise: (a) bacteriocin of significant quantity, to be reduced at least less a kind of field planting level of target bacteria, described bacteriocin has the lactic bacilli strains that is selected from by the identification mark of the lactic bacilli strains of the group of NRRL B-30510, NRRLB-30514 and its compositions of mixtures and produces, (b) treatment carrier, and (c) animal-feed.In one embodiment of the invention, described bacteriocin has the aminoacid sequence that is selected from the group that is comprised of SEQ IDNO 1 and SEQ ID NO 2.
Another object of the present invention provides the therapeutic composition for animal, comprise: (a) bacteriocin of at least a separation of significant quantity, to be reduced at least less a kind of field planting level of target bacteria, described bacteriocin has the faecalis bacterial strain that is selected from by the identification mark of the faecalis bacterial strain of the group of NRRLB-30511, NRRL B-30645 and its compositions of mixtures and produces, (b) treatment carrier, and (c) animal-feed.
Another object of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, it is by using lactobacillus or the enterococcal new bacterial strain that comprises at least a product new bacteriocin to described animal, at least a new bacteriocin that is produced by lactobacillus or enterococcal new bacterial strain, the perhaps therapeutic composition of the combination of described new bacterial strain and/or new bacteriocin.
Another object of the present invention provides the method that lowers at least at least a target bacteria field planting level in animal, and it is to comprise the lactobacillus that is characterized as NRRL preserving number B-30514 and B-30510 by using to described animal; Diagnostic characteristics is the faecalis of NRRL B-30511 and NRRL B-30645; Therapeutic composition with the new bacterial strain of its mixture.
Another purpose of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, it is to be NRRL B-30510 by use for described animal comprising at least a diagnostic characteristics, NRRL B-30511, NRRL30514, NRRL B-30645, and the new bacterial strain of its mixture; At least a have aminoacid sequence shown in SEQ ID NO 1, SEQ ID NO2, SEQ ID NO 3, SEQ ID NO 4, and the bacteriocin of its mixture; Or the therapeutic composition of the combination of new bacterial strain and new bacteriocin.
Another purpose of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, and it is to comprise the therapeutic composition with new bacteriocin of aminoacid sequence shown in SEQ ID NO:1 by using to described animal.
Another purpose of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, and it is to comprise the therapeutic composition with new bacteriocin of aminoacid sequence shown in SEQ ID NO:2 by using to described animal.
Another purpose of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, and it is to comprise the therapeutic composition with new bacteriocin of aminoacid sequence shown in SEQ ID NO:3 by using to described animal.
Another purpose of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, and it is to comprise the therapeutic composition with new bacteriocin of aminoacid sequence shown in SEQ ID NO:4 by using to described animal.
Another object of the present invention provides the method that reduces at least at least a target bacteria field planting level in animal, and it is to comprise having the new bacterial strain of lactobacillus bacteria that diagnostic characteristics is NRRL B-30510 or NRRL B-30514 by using to described animal; Having diagnostic characteristics is the new bacterial strain of faecalis of NRRL B-30511 or NRRL B-30645, and the therapeutic composition of the bacteriocin of its mixture generation.
Other purposes of the present invention and advantage will become obvious according to following explanation.
The preservation of microorganism
The saliva lactobacillus is marked as NRRL B-30514 (bacterial strain PVD32), and the Lactobacillus acidophilus is marked as NRRL B-30510 (bacterial strain LWP320); Enterococcus durans is marked as NRRL B-30511 (bacterial strain LWP26) in calendar year 2001 preservation on August 3, and enterococcus faecalis is marked as NRRL B-30645 (bacterial strain LWP21) in preservation on April 1 in 2003.All above-mentioned bacterial strains are preserved in USDA farming research service centre patent culture collection center (national agricultural application research centre (National Center for Agricultural Utilization Research) according to the regulation of budapest treaty, 1815N.University Street, Peoria, Illinois 61604).
Description of drawings
Fig. 1 is to the photo of OR7 direct-detection behind the SDS-PAGE.Gel covers to determine which band has the molecular weight of anti-microbial activity and the activated band of tool with campylobacter jejuni.The 1st road---6,500-97,000 MMW molecular weight marker (AMERSHAM PHARMACIA BIOTECH): 97,000,66,000,43,000,30,000,21,000,14,400 and 6,500Da.The lactocin OR7 of the band in the 2nd road---purifying, the band in the 3rd road---the CAP lactocin OR7 after being absorbed by campylobacter jejuni, has anti-microbial activity, growth inhibiting zone (seeing arrow), quality is about 6,000Da, the band in the 4th road---CAP lactocin OR7.Other bands do not demonstrate anti-microbial activity.
Fig. 2 is to the photo of bacteriocin LWP320 and LWP26 direct-detection behind the SDS-PAGE.Gel covers to determine which band has the molecular weight of anti-microbial activity and the activated band of tool with campylobacter jejuni.The 3rd road---3,000-43,000 LMW molecular weight marker (AMERSHAM PHARMACIA BIOTECH): 3,000,11,400,18,000,21,000 and 43,000Da.The band in the 1st road---bacteriocin LWP320, the band in the 2nd road---bacteriocin LWP26 has anti-microbial activity, growth inhibiting zone, quality is respectively about 6,000 and about 3,000Da.Other bands do not demonstrate anti-microbial activity.
Fig. 3 is to the photo of enterococcin LWP21 direct-detection behind the SDS-PAGE.Gel covers to determine which band has the molecular weight of anti-microbial activity and active band with campylobacter jejuni.The 4th road---1060-43,000 LMW molecular weight marker (AMERSHAM PHARMACIA BIOTECH): 1,060,2,500,3,500,14,400,20,000,30,000 and 43,000Da.The band in the 1st road (the enterococcin LWP21 behind the SP-sepharose), and the band in the 2nd road (the 2nd peak behind phenyl-sepharose CL-4B) has anti-microbial activity, growth inhibiting zone (seeing arrow), quality is about 6,000Da.The band in the 3rd road (peak behind the phenyl-Sepharose CL-4B), have about 3, the quality of 000Da.Other bands do not demonstrate anti-microbial activity.
Embodiment
The importance that intestines infect in the people is recognized day by day fully that the relation between poultry pollution and people infect also has detailed record.The ability of eliminating this health risk by intervening poltry factory also is well-known.In broiler chicken production and the course of processing, the fecal matter that contains pathogenic agent is transferred on the meat, and persists in the kitchen of processed food.
Competitive biological metabolite may help to control pathogenic agent for example campylobacter jejuni and Salmonellas.New antagonism bacterial classification of the present invention separates with the crop mucosal surface from the caecum of broiler chicken.The natural constituents of the antagonist that characterizes is low-molecular-weight peptide, bacteriocin, and it has the antagonistic activity of wide spectrum.
The invention provides new lactobacillus and faecalis bacterial strain, new bacteriocin, the aminoacid sequence of described bacteriocin contains described new bacteriocin and/or produces the therapeutic composition of their bacterial strain, and the method for using described new therapeutic composition.
Lactobacillus acidophilus LWP320 isolate is facultative anaerobe (microaerophile), the catalase feminine gender, gram-positive non-motile polymorphic bar shaped bacteria, its on MRS agar at about 37 ℃ of growth-delayings.When MRS agar is grown, it is that about 0.5mm is to the look that turns white, the translucent bacterium colony of about 0.8mm that this isolate produces diameter afterwards in about 24 hours little aerobic cultivation.In MRS meat soup, bacterial strain LWP320 is only in the growth of culture tube bottom.
Saliva lactobacillus PVD32 bacterial strain is facultative anaerobe (microaerophile), and is gram-positive, the catalase feminine gender, non-motile, polymorphic coryneform bacteria.Bacterium colony about 37 ℃ of little aerobic cultivations to produce diameter after about 18-24 hour be about 1mm to the circle of about 3mm to the bacterium colony with sharp edge regular shape, smooth, convex surface.This bacterial strain produces lactic acid and H
2O
2
Enterococcus faecalis LWP21 bacterial classification is facultative anaerobe (microaerophile), gram-positive cocci, and catalase is negative.On MRS agar at 37 ℃ of delayed growths.When MRS agar is cultivated, this bacterial strain produces the look that turns white that diameter is about 0.2mm, translucent bacterium colony at about 37 ℃ afterwards in about 24 hours little aerobic cultivation.About 37 ℃ after about 48 hours little aerobic cultivation, the diameter of bacterium colony is about 1mm to about 1.2mm.This bacterial strain only is grown in MRS meat soup in about 2/3 volume in culture tube bottom.
Enterococcus durans LWP26 bacterial classification is facultative anaerobe (microaerophile), gram-positive cocci, and the catalase feminine gender at about 37 ℃ of growth-delayings on MRS agar.When MRS agar is grown, this bacterial strain produces turn white look, translucent bacterium colony, and at about 37 ℃ of little aerobic cultivations bleach after about 24 hours.After about 48 hours, the about 0.8mm of bacterium colony size average out to is to about 1.0mm in about 37 ℃ of little aerobic cultivations.In MRS meat soup, the whole volumes of this strain growth in pipe.
Lactobacillus and the enterococcal species of separating produced the screening of bacteriocin activity by carrying out at the culture of nutrient agar medium with interested different target microbionation.Other detect bacterial strain and cultivated about 14-24 hour at about 37 ℃ under aerobic conditions.Caused by Yersinia enterocolitica (Yersinia enterocolitica) and pseudonodule yersinia enterocolitica (Y.pseudotuberculosis) were cultivated about 14-24 hour under aerobic conditions at about 28 ℃.The detection of the activity of opposing campylobacter jejuni is carried out at the Campylobacter agar that contains about 5% cracking blood with the campylobacter jejuni inoculation.The use of blood is that those of ordinary skills know, and comprises for example sheep, horse etc.The detection of the activity of opposing campylobacter jejuni is at about 5%O
2, about 10%CO
2, and about 85%N
2Micro-aerobic condition under carried out about 24-48 hour at about 42 ℃.The antagonistic bacterium plating that suspends in about 0.2ml physiological saline on lactobacillus agar, was cultivated about 24 hours.About 0.5cm
3Agar block cut and transfer to and be added with cracking blood, approximately 10mg/ml Rifampin, about 2.4U/ml polymyxin, and inoculated about 10
7On the brucella of individual campylobacter jejuni cell or the Campylobacter agar.Flat board was cultivated about 24-48 hour at about 42 ℃ under micro-aerobic condition.Activity is estimated by measuring growth inhibiting zone.
Discovery has the lactobacillus isolate of Antagonism to be evaluated the generation of its bacteriocin.Rough antibiotic prepared product (CAP) is by only preparing under aerobic conditions carrying out ammonium sulfate precipitation at about 37 ℃ of cultures at the about 18 hours antagonistic strain of hungry substratum (starvation medium) growth, and described hungry substratum is:
Adding distil water is to 1000ml, and the pH value is about 7.2.
Then with culture fluid with about 12,000 * g centrifugal about 10 minutes.Then the supernatant that obtains adds 1N NaOH and adjusts pH value extremely about 6.2 and add about 130U/ml catalase removing organic acid and hydrogen peroxide, and supressor.Carry out ammonium sulfate precipitation, desalination chromatogram and gel-filtration by combination and from supernatant, separate antagonistic peptide, obtain rough antibiotic prepared product (CAP).The CAP sample filters by 0.22 μ strainer (Millipore, Bedford, MA, USA).
Discovery has the faecalis isolate of Antagonism to be evaluated the generation of its bacteriocin.Rough antibiotic prepared product (CAP) is by only preparing carry out ammonium sulfate precipitation at about 37 ℃ about 18 hours antagonistic strain cultures of growing in MRS meat soup under aerobic conditions, and described MRS meat soup is:
MRS meat soup 15.0g
Lactose 0.05g
Adding distil water is to about 1000ml, and pH 7.2.
Then with culture fluid with about 12,000 * g centrifugal about 10 minutes.Then the supernatant that obtains adds 1N NaOH and adjusts pH value extremely about 6.2 and add about 130U/ml catalase removing organic acid and hydrogen peroxide, and supressor.Carry out ammonium sulfate precipitation, desalination chromatogram and gel-filtration by combination and from supernatant, separate antagonistic peptide, obtain rough antibiotic prepared product (CAP).The CAP sample filters by 0.22 μ strainer (Millipore, Bedford, MA, USA).
The molecular weight of all above-mentioned peptides is determined by the SDS-PAGE electrophoresis.The pI of described peptide determines by isoelectrofocusing.Aminoacid sequence uses by the Edman edman degradation Edman, and for example, 491cLC automatic sequencer (Applied Biosystems, Inc.) is determined.
Be purpose of the present invention, term " peptide " refers to the compound of two or more amino acid at least or amino acid analogue.Amino acid or amino acid analogue can link to each other by peptide bond.In another embodiment, 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, comprises D or L optical isomer, and amino acid analogue.
Thereby peptide derivant of the present invention and analogue include but not limited to that those comprise as the residue in its sequence that comprises of one-level aminoacid sequence is replaced all or part of aminoacid sequence of the peptide of the sequence cause the change that conservative amino acid replaces by the amino-acid residue of functional equivalent.
For example, one or more amino-acid residues in the sequence can be had similar polarity by another, as the amino acid substitution of function equivalent, cause reticent change.Amino acid whose replacement can be selected from other members of the affiliated classification of this amino acid in the sequence.For example, nonpolar (hydrophobic) amino acid comprises L-Ala, leucine, Isoleucine, α-amino-isovaleric acid, proline(Pro), phenylalanine, tryptophane, and methionine(Met).The amino acid that contains aromatic ring structure is phenylalanine, tryptophane and tyrosine.Polar neutral amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, asparagine, and glutamine.Positively charged (alkalescence) amino acid comprises arginine, Methionin, and Histidine.Electronegative (acidity) amino acid comprises aspartic acid and L-glutamic acid.This change can definite molecular weight or the iso-electric point of the poly-propionic acid amide gel electrophoresis of remarkably influenced.The non-conservation 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 two dimensional structure.
Peptide of the present invention can be by chemosynthesis.Synthetic peptide can be with the solid phase liquid technology of knowing, or concentrated (peptide condensation) technology of peptide, or its combination preparation, can comprise natural and/or synthetic amino acid.Being used for the synthetic amino acid of peptide can be the Boc (N of standard
αThe N of-amido protecting
α-t-butyl oxygen carbonyl) amino-acid resin, the standard of carrying out the original solid phase program of Merrifield goes to protect, neutralizes, is coupled and washing step (J.Am.Chem.Soc, the 85th volume, 2149-2154,1963), 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 synthesis), second edition, Pierce Chemical Co., Rockford, I11., 1984; Fields and Noble, Int.J.Pept.Protein Res., the 35th volume, 161-214,1990), or realize with automatic DNA synthesizer DNA.
According to the present invention, described peptide and/or described new bacteria bacterial strain can place part, parenteral in the treatment acceptable carrier, through mucous membrane, and be for example oral, intranasal, or per rectum, or through percutaneous drug delivery.Peptide of the present invention can modify to increase the ability that described peptide passes cytolemma if necessary, for example by increasing the hydrophobicity of described peptide, described peptide is engaged etc. with carrier (such as the part of specific receptors).
The present invention also provides target molecule is engaged with peptide of the present invention.For the target molecule of the object of the invention refers to when the vivo medicine-feeding, be positioned the molecule in desirable one or more zones.In various embodiments of the present invention, target molecule can be peptide or protein, antibody, lectin, carbohydrate or steroid.Target molecule can be peptide part or the antibody of the acceptor on target cell, such as monoclonal antibody.For the ease of crosslinked, antibody can reduce to the heterodimer of two heavy chains and light chain, perhaps F (ab ')
2Fragment can be reduced, and crosslinked by sulfydryl and the described peptide of reduction.
Another aspect of the present invention provides therapeutic composition.Described therapeutic composition can be for oral, intranasal, pulmonary administration, injection etc.Described therapeutic composition comprises the of the present invention at least a bacteriocin of significant quantity and their derivative and/or at least a new bacterial strain to be reduced at least less a kind of level of target bacteria field planting, also comprises acceptable thinner, sanitas, solubilizing agent, emulsifying agent, adjuvant and/or carrier.Thinner can comprise damping fluid, as, for example, Tris-HCl, acetate, phosphoric acid salt; Additive can comprise washing agent and solubilising reagent, as, for example, Tween 80, polysorbate80 etc.; Antioxidant comprises, as, for example, xitix, Sodium Pyrosulfite etc.; Sanitas can comprise, for example, and Thimersol, phenylcarbinol etc.; And dilatant such as lactose, N.F,USP MANNITOL etc.
Therapeutic composition of the present invention can be merged enters polymkeric substance such as polyvinylpyrrolidone, and poly(lactic acid) in the particulate prepared product of polyglycolic acid etc., or mergedly enters in the liposome.Sealing of liposome comprises sealing of various polymkeric substance.Various polymer support all can be used for storage and contains and/or send one or more for the treatment of reagent discussed above, comprises the degradable and non-biodegradable composition of biological example.The representative example of Biodegradable compositions comprises albumin, collagen, gelatin, hyaluronic acid, starch, Mierocrystalline cellulose (methylcellulose gum, hydroxypropylcellulose, Vltra tears, carboxymethyl cellulose, cellulose acetate phthalic acid ester, the cellulose acetate succinate, hydroxypropyl methylcellulose phthalate), casein, dextran, polysaccharide, Fibrinogen, poly-(D, L rac-Lactide), poly-(D, the L-rac-Lactide is glycollide altogether), poly-(glycollide), poly-(butyric ester), poly-(alkyl carbonate) and poly-(ortho ester), polyester, poly-(hydroxypentanoic acid), Ju diethyleno dioxide ketone, poly-(ethylene glycol terephthalate), poly-(oxysuccinic acid), poly-(tartronic acid), polyanhydride, polyphosphonitrile, poly-(amino acid) and their multipolymer (usually referring to, Ilium, L., Davids, S.S. (volume) " Polymers in Controlled Drug Delivery (polymkeric substance in the controlled drug release) " Wright, Bristol, 1987; Arshady, J.Controlled Release 17:1-22,1991; Pitt, Int.J.Phar.59:173-196,1990; Holland etc., J.Controlled Release 4:155-0180,1986).
The representative example that can not degrade comprises poly-(ethylene-vinyl acetate) (" EVA ") multipolymer, silicon rubber, acrylate copolymer (polyacrylic acid, polymethyl acrylic acid, polymethylmethacrylate, Polyalkylcyanoacrylanano), polyethylene, polypropylene, polymeric amide (nylon 6,6), urethane, poly-(ester type polyurethane), poly-(ether-based polyurethane), poly-(ester-urea), polyethers (poly-(oxyethylene), poly-(propylene oxide), Pluronics and poly-(tetramethylene glycol), silicon rubber and vinyl polymer such as polyvinylpyrrolidone, poly-(vinyl alcohol), poly-(vinyl acetate between to for plastic phthalic acid ester).Polymkeric substance also can be developed, or (the alginate for example of negatively charged ion, carrageenin, carboxymethyl cellulose and polyacrylic acid), or cationic (chitosan for example, PLL, polyaziridine, and polyallylamine) (usually referring to Dunn etc., J.Applied Polymer Sci.50:353-365,1993; Cascone etc., J.Materials Sci.:Materials in Medicine (material in the medicine) 5:770-774,1994; Shiraishi etc., Biol.Pharm.Bull.16 (11): 1164-1168,1993; Thacharodi and Rao, Int ' 1J.Pharm.120:115-118,1995; Miyazaki etc., Int ' 1J.Pharm.118:257-263,1995).
Polymer support can be made various forms of, tool release characteristics likely and/or have specific desirable character.For example, polymer support can be made into when being exposed to specific trigger event (such as pH) to discharge treatment reagent (referring to such as Heller etc., " Chemically Self-Regulated Drug Delivery Systems; (with the self-adjusting drug delivery system of the mode of chemistry) ", from iPolymers in Medicine III (the polymkeric substance III in the medicine), Elsevier Science Publishers B.V., Amsterdam, 1988, pp.175-188; Kang etc., J.Applied Polymer Sci.48:343-354,1993; Dong etc., J.Controlled Release 19:171-178,1992; Dong and Hoffman, J.Controlled Release 15:141-152,1991; Kim etc., J.Controlled Release 28:143-152,1994; Cornejo-Bravo etc., J.Controlled Release 33:223-229,1995; Wu and Lee, Pharm.Res.10 (10): 1544-1547,1993; Serres etc., Pharm.Res.13 (2): 196-201,1996; Peppas, " Fundamentals of pH-and Temperature-Sensitive Delivery Systems; (pH and temperature sensitive delivery system basis) ", from (volumes) such as Gurny, Pulsatile Drug Delivery (drug delivery of pulsation), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 1993, pp.41-55; Doelker, " Cellulose Derivatives, (derivatived cellulose) " 1993, from Peppas and Langer (volume), Biopolymers I (biological polymer I), Springer-Verlag, Berlin).The representative example of the polymkeric substance of pH sensitivity comprises that polyacrylic acid and derivative thereof (comprise for example homopolymer such as polyaminocarboxylic acid; Polyacrylic acid; Polymethyl acrylic acid, the multipolymer of these homopolymer, and the multipolymer of polyacrylic acid and propylene monomer as discussed above.The polymkeric substance of other pH sensitivities comprises polysaccharide such as cellulose acetate phthalic acid ester; Hydroxypropyl methylcellulose phthalate; Hydroxypropyl methylcellulose acetate succinate; Trimellilate cellulose acetate trimellitate (trimellilate); And chitosan.The polymkeric substance of other pH sensitivities also comprises the polymkeric substance of any pH sensitivity and the mixture of water-soluble polymers.
Similarly, it is temperature sensitive (referring to for example that polymer support can be made into, Chen etc., " Novel Hydrogels of a Temperature-Sensitive Pluronic Grafted to a Bioadhesive Polyacrylic Acid Backbone for Vaginal Drug Delivery; (new type gel that is used for the responsive to temperature type Pluronic that is grafted to bioadhesion polyacrylic acid skeleton that intravaginal drug sends) ", from Proceed.Intern.Symp.Control.Rel.Bioact.Mater.22:167-168, Controlled Release Society, Inc., 1995; Okano, " Molecular Design of Stimuli-Responsive Hydrogels for Temporal Controlled Drug Delivery; (molecular designing that is used for the stimuli responsive type hydrogel that the sequential controlled drug sends) is " from Proceed.Intern.Symp.Control.Rel.Bioact.Mater.22:111-112, Controlled Release Society, Inc., 1995; Johnston etc., Pharm.Res.9 (3): 425-433,1992; Tung, Int ' 1J.Pharm.107:85-90,1994; Harsh and Gehrke, J.Controlled Release 17:175-186,1991; Bae etc., Pharm.Res.8 (4): 531-537,1991; Dinarvand and D ' Emanuele, J.Controlled Release36:221-227,1995; Yu and Grainger, " Novel Thermo-sensitive Amphiphilic Gels:Poly N-isopropylacrylamide-co-sodium acrylate-co-n-N-alkylacrylamide Network Synthesis and Physicochemical Characterization; (and the responsive type amphiphilic of Novel hot gel: poly N-isopropyl acrylamide-altogether-sodium acrylate-altogether-the synthetic and physicochemical characteristic of n-N-alkyl acrylamide network) ", Oregon science and technology graduate school chemistry and bio-science system, Beaverton, the Oregon, pp.820-821; Zhou and Smid, " Physical Hydrogels of Associative Star Polymers, (physical hydrogel of the star-type polymer of association) ", State University of New York's environmental science and polymkeric substance institute of forestry institute department of chemistry, Syracuse, New York, pp.822-823; Hoffman etc., " Characterizing Pore Sizes and Water ' Structure ' in Stimuli-Responsive Hydrogels; (aperture in the stimuli responsive type hydrogel and water ' structure ' feature) ", University of Washington biotechnology center, p.828; Yu and Grainger, " Thermo-sensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks:Cationic; Anionic and Ampholytic Hydrogels; (the thermo-responsive type swelling behavior in the crosslinked NIPA network) ", Oregon science and technology graduate school chemistry and bio-science system, Beaverton, Oregon, pp.829-830; Kim etc., Pharm.Res.9 (3): 283-290,1992; Bae etc., Pharm.Res.8 (5): 624-628,1991; Kono etc., J.Controlled Release 30:69-75,1994; Yoshida etc., J.Controlled Release 32:97-102,1994; Okano etc., J.Controlled Release 36:125-133,1995; Chun and Kim, J.Controlled Release 38:39-47,1996; D ' Emanuele and Dinarvand, Int ' 1J.Pharm.118:237-242,1995; Katono etc., J.Controlled Release16:215-228,1991; Hoffman, " Thermally Reversible Hydrogels Containing Biologically Active Species; (the hot reversible hydrogel that comprises biologically active substance) ", from (volumes) such as Migliaresi, Polymers in Medicine III (the polymkeric substance III in the medicine), Elsevier Science Publishers B.V., Amsterdam, 1988, pp.161-167; Hoffman, " Applications of Thermally-Reversible Polymers and Hydrogels in Therapeutics and Diagnostics; (hot reversible polymkeric substance and the application of gel in therapeutics and diagnostics) ", new development international symposium in the 3rd drug delivery system, the salt lake city, the Utah State, 24-27 day in February, 1987, pp.297-305; Gutowska etc., J.Controlled Release 22:95-104,1992; Palasis and Gehrke, J.Controlled Release 18:1-12,1992; Paavola etc., Pharm.Res.12 (12): 1997-2002,1995).
The representative example of hot glue cohesion compound, and their gelling temp (LCST (℃)) comprise that homopolymer is such as poly-(N-methyl-N-n-propyl group acrylamide), 19.8; Poly-(N-n-propyl group acrylamide), 21.5; Poly-(N-methyl-N-isopropyl propyl group acrylamide), 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.And, hot glue cohesion compound can be by the multipolymer between the above-mentioned two or more monomers of preparation, perhaps with this homopolymer and other water-soluble polymerss such as acryl monomer (acrylmonomer) (for example vinylformic acid and derivative thereof such as methacrylic acid, acrylate and derivative thereof such as butyl methacrylate, acrylamide, and N-n-butyl acrylamide) make up and make.Other representative example of hot glue cohesion compound comprise cellulose ether derivative such as hydroxypropylcellulose, 41 ℃; Methylcellulose gum, 55 ℃; Vltra tears, 66 ℃; And Type 3U, and Pluronics such as F-127,10-15 ℃; L-122,19 ℃; L-92,26 ℃; L-81,20 ℃; And L-61,24 ℃.
Utilize and to make multiple different form with polymer support of the present invention, comprise that for example bar-shaped design (rod-shaped device), ball, sheet (slab) or capsule are (referring to for example, Goodell et al., Am.J.Hosp.Pharm.43:1454-1461,1986; Langer etc., " Controlled release of macromolecules from polymers (macromole is from the controlled release of polymkeric substance) ", from Biomedical Polymers, Polymeric Materials and Pharmaceuticals for Biomedical Use (biomedicine is for polymer materials and the medicine of biomedical applications), Goldberg, E.P., Nakagim, A. (volume) Academic Press, pp.113-137,1980; Rhine etc., J.Pharm.Sci.69:265-270,1980; Brown etc., J.Pharm.Sci.72:1181-1185,1983; And Bawa etc., J.Controlled Release 1:259-267,1985).
Treatment reagent can be connected with polymkeric substance is covalently bound by being enclosed in the polymeric matrix, perhaps can be encapsulated in the microcapsule.In certain preferred embodiment of the present invention, therapeutic composition is provided with thread dose of (thread), membrane agent and sprays of the preparation of non-capsule such as microballoon (size range in nanometer to micron), paste, various size.
Another aspect of the present invention provides therapeutic composition and animal-feed.Therapeutic composition of the present invention can be sealed with above-mentioned polymer support, then adds in the feed by any known method that is applied in the feed, for example by mechanical stirring, sprays etc.Described therapeutic composition comprises, for example, can effectively reduce at least at least a bacteriocin and/or the antagonistic bacterium of the amount of at least a target bacteria level of field planting in animal, for example approximately lactocin and/or the enterococcin/1000g of every kind of 0.5g, about 1.25g polymer support is polyvinylpyrrolidone/1000g for example, with about 8.6% thinner water/1000g for example, with digestible any grain fraction, the corn cereal that for example grinds; Cereal is oat for example, wheat, buckwheat; The fruit that grinds is such as mixing such as pears.Then the amount of described therapeutic composition with the field planting level that effectively reduces at least at least a target bacteria added in the animal-feed of any type, for example bacteriocin is about 1: 10 to about 1: 100 to the ratio of feed.Be purpose of the present invention, the example of animal-feed comprises green fodder, silage, dry green fodder, root, stem tuber, fleshy fruit, cereal, seed, brewer's grains, pomace, cereuisiae fermentum, distillation residue, milling by product, the byproduct that sugar, starch or oil are produced, and various castoff.This product can add in the animal-feed of rear cattle, poultry, rabbit, pig or sheep etc.It can mix use with other fodder additivess that are used for these domestic animals.
Following embodiment only is in order further to illustrate the present invention, not really want to limit the scope of the present invention of being determined by claim.
Produce two kinds of new antagonistic strains of bacteriocin, the saliva lactobacillus is marked as NRRL B-30514 (bacterial strain PVD32) and Lactobacillus acidophilus, is marked as NRRL B-30510 (bacterial strain LWP320) and separates from the caecum of broiler chicken and crop.Described caecum and crop are emptying and with aseptic 0.85%w/v salt brine solution (physiological saline) washed twice of about 100ml in advance.It is in about 7.0 the sterile saline solution that caecum and crop thing are suspended in pH.The suspension direct flat plate of 10 times of dilutions of about 0.1ml is inoculated on the selectivity MRS agar.Flat board was cultivated about 16-18 hour in about 37 ℃ of anaerobism.Select at least 10 bacterium colonies that lactobacillus is special from each sample.Lactobacillus is differentiated with the little detection system of APO 50CHL (bioMerieux, France).
Bacterial strain saliva lactobacillus PVD32 and Lactobacillus acidophilus LWP320 cultivated about 24 hours at MRS agar at about 37 ℃.These two bacterial strains are facultative anaerobes, and gram-positive non-motile bar shaped bacteria can be at about 30 ℃, 37 ℃, and 42 ℃ of growths.Bacterial strain PVD32 grows at MRS agar, in about 37 ℃ of little aerobic cultivations after about 18-24 hour, produces diameter and be the circle of about 1-3mm to the bacterium colony with sharp edge regular shape, smooth, convex surface.Bacterial strain LWP320 grows at MRS agar, 37 ℃ of little aerobic cultivations after about 24 hours, produce diameter be approximately 0.5 to 0.8mm turn white translucent bacterium colony.
Enterococcus faecalis LWP21 and enterococcus durans LWP26 about 37 ℃ about 24 hours of MRS agar growth.They are gram-positive facultative anaerobes, and non-motile coccus can be at about 30 ℃, 37 ℃, and 42 ℃ of growths.Bacterial strain LWP21 grows at MRS agar, produces the translucent colony of the look that turns white, and little aerobic cultivation becomes transparent after about 24 hours.In about 37 ℃ of little aerobic cultivations after about 48 hours, colony diameter is about 0.8 to about 1.0mm.
Use API 50CHL suspension medium, the result in API 50CHL and EN-COCCUS galleries as described in Table 1.The result that following table 2 shows shows that bacterial strain PVD32 is the saliva lactobacillus, and bacterial strain LWP320 is the Lactobacillus acidophilus, and bacterial strain LWP21 is enterococcus faecalis, and bacterial strain LWP26 is enterococcus durans.
The target bacteria of estimating the antagonistic activity of antagonism isolate comprises bacterial strain L4 and the B1 of the campylobacter jejuni that separates from Muscovite broiler chicken, campylobacter jejuni ATCC 11168, several kinds of enterobacteriaceae (Enterobacteriaceae), Pseudomonas aeruginosa (Pseudomonas aeruginosa) strains A TCC 9027, streptococcus aureus (Staphylococcus aureus) and Listeria monocytogenes (Listeria monocytongenes) are as detecting culture to estimate the antagonistic activity of isolate.Campylobacter jejuni about 37 ℃ at about 5%O
2, about 10%CO
2, and about 85%N
2Little aerobic condition under, be added with about 0.02% sodium bisulfite, about 0.05% ferrous sulfate, and on the brucella agar of about 5% part cracking sheep blood or the Campylobacter agar growth about 18-24 hour.Other bacterial strain Caused by Yersinia enterocoliticas (Y.enterocolitica) and pseudonodule yersinia enterocolitica (Y.pseudotuberculosis) were being cultivated about 18-24 hour under aerobic conditions at about 37 ℃ or about 28 ℃ on the nutrient agar medium.Isolate is evaluated the antagonistic activity of Campylobacter.Approximately 0.2ml physiological saline suspension, was cultivated about 24 hours at about 37 ℃ on MRS agar by plating.About 0.5cm
3Agar block be cut and transfer to and be added with about 5%-10% part cracking sheep blood, about 10mg/ml Rifampin, and about 2.5u/ml polymyxin, inoculum size be each flat board about 10
7On the brucella agar or Campylobacter agar of individual campylobacter jejuni cell.Flat board was cultivated about 24 hours under aforesaid micro-aerobic condition at about 42 ℃.Antagonistic activity is estimated by the diameter of measuring the campylobacter jejuni inhibition zone.
Antagonistic activity to campylobacter jejuni is estimated with 11,790 kinds of isolates, and wherein 279 kinds of isolates demonstrate the antagonistic action to campylobacter jejuni.
The qualification result that table 1.API 50CHL and EN-COCCUS detect
| Bacterial strain | Detect (score) | Bacterial classification |
| PVD32 | API?50CHL(99.9％) | The saliva lactobacillus |
| LWP320 | API?50CHL(96.6％) | The Lactobacillus acidophilus |
| LWP21 | EN-COCCUS-detects (96%) | Enterococcus faecalis |
| LWP26 | EN-COCCUS-detects (98%) | Enterococcus durans |
The feature (result of API 50CHL) of table 2a. lactobacillus bacterial classification
| Isolate and character thereof | |
| Carbohydrate | PVD32LWP320 |
| The 0-contrast | |
| Glycerine | |
| Erythritol (Erythriol) | |
| D-R | |
| L-arabinose | |
| Ribose | |
| The D-wood sugar | |
| The L-wood sugar | |
| Adonit | |
| Beta-methyl-xyloside | |
| Semi-lactosi | ++ |
| D-Glucose | ++ |
| D-Fructose | ++ |
| D-MANNOSE | +- |
| The L-sorbose | |
| Rhamnosyl | +- |
| Melampyrum | |
| Inositol | |
| N.F,USP MANNITOL | +- |
| Sorbitol Powder | +- |
| Alpha-Methyl D-MANNOSE glycosides | -？ |
| Alpha-Methyl D-glucoside | -？ |
| The N-acetyl glucosamine | ++ |
| Vitamin B17 (Amigdaline) | -+ |
| Arbutin | -？ |
| Aesculin | |
| Saligenin | -+ |
| Cellobiose | -+ |
| Maltose | ++ |
| Lactose | ++ |
| Melibiose | +- |
| Sucrose | ++ |
| Trehalose | +- |
| O14-Methyldelectine (Inuline) | |
| Melizitose | |
| The D-raffinose | +？ |
| Amidone (Amidon) | |
| Glycogen | |
| Xylitol | |
| Gentiobiose | |
| The D-turanose | |
| The D-lyxose | |
| D-Tag | |
| The D-trehalose | |
| The L-trehalose | ？- |
| D-R alcohol | ？- |
| L-arabinose alcohol | |
| Gluconate | |
| ?2ceto?gluconane | |
| ?5ceto?gluconane | |
| Be accredited as | Saliva lactobacillus Lactobacillus acidophilus |
Table 2b. detects the characteristic of the enterococcal species that obtains with EN-COCCUS
| Isolate and character thereof | |
| Detect | LWP21LWP26 |
| L-arabinose | |
| Ribose | ++ |
| The L-sorbose | |
| N.F,USP MANNITOL | +- |
| Sorbitol Powder | +- |
| Lactose | ++ |
| Melibiose | -d |
| Sucrose | +- |
| Melizitose | (+)- |
| The D-raffinose | |
| Arginine | ++ |
| Be accredited as | The enterococcus faecalis enterococcus durans |
Table 3. isolate is active to the inhibition that campylobacter jejuni detects bacterial strain
| Antagonism is differentiated | The bacterial classification source | The growth-inhibiting diameter (mm) of campylobacter jejuni |
| 11168B1L4F2KI UW3 | ||
| LWP21 | The broiler chicken caecum | 754524 |
| LWP26 | The broiler chicken caecum | 254637 |
| LWP320 | The broiler chicken caecum | 454536 |
| PVD32 | The broiler chicken crop | 554546 |
The new antagonism bacterial classification of two kinds of bacteriocinogeny, enterococcus faecalis 21 (NRRL B-30645), and enterococcus durans 26 (NRRL B-30511) are separated from caecum and the crop smear of healthy broiler chicken.Caecum and crop are drained and with 0.085%w/v salt brine solution (physiological saline) washed twice of aseptic about pH7.0.Caecum and crop thing are suspended in the stroke-physiological saline solution of about pH7.0.Approximately the suspension of 10 times of dilutions of 0.1ml is inoculated on the MRS selective medium by direct flat plate, and is dull and stereotyped about 37 ℃ of cultivations about 24-48 hour.Faecalis is differentiated with little detection kit EN-COCCUS.
Campylobacter jejuni ATCC 11168 is used as detecting culture to estimate the antagonistic activity of isolate, described in above-mentioned embodiment 1.33 kinds of other bacterial classifications are used as detecting culture to estimate the antagonistic activity of isolate, described in above-mentioned embodiment 1.Isolate is also estimated as described in above-mentioned embodiment 1 antagonistic activity of Campylobacter.
Antagonistic activity to campylobacter jejuni is estimated with 1, the 200 kind of isolate that is derived from about 125 broiler chicken.Wherein 63 kinds of isolates demonstrate the Antagonism to campylobacter jejuni ATCC 11168.12 kinds of isolates the most activated (seeing above-mentioned table 1-3) are studied it in MRS meat soup, produced the ability of bacteriocin between incubation period.
Rough antibiotic prepared product by only on the hungry substratum 37 ℃ under aerobic conditions the about 18 hours lactobacillus antagonistic strain cultures of growth carry out ammonium sulfate precipitation and prepare, described hungry substratum is:
Adding distil water is to 1000ml, and the pH value is about 7.2
With culture fluid with about 12,000 * g centrifugal about 10 minutes.The supernatant that obtains adds 1NNaOH and adjusts pH to about 6.2, and adds about 130U/ml catalase to remove organic acid, hydrogen peroxide, and supressor.Carry out ammonium sulfate precipitation by combination, the desalination chromatogram is separated antagonistic peptide with gel-filtration from supernatant, obtains rough prepared product (CAP).The CAP sample filters by 0.22 μ strainer (Millipore, Bedford, MA).
The faecalis isolate that demonstrates anti-microbial activity in above-described embodiment 2 in cultivated about 18 hours under aerobic conditions at about 37 ℃, described MRS meat soup (HiMedia) is:
MRS meat soup 15.0g
Lactose 0.05g
Adding distil water is to about 1000ml, and pH about 7.2
Results culture fluid and with about 12,000 * g centrifugal about 10 minutes.The supernatant that obtains adds 1N NaOH and adjusts pH to about 6.2, and adds about 130U/ml catalase removing organic acid and hydrogen peroxide, and supressor.Carry out ammonium sulfate precipitation by combination, the desalination chromatogram is separated antagonistic peptide with gel-filtration from supernatant, obtains rough antibiotic prepared product (CAP).The CAP sample filters by 0.22 μ strainer (Millipore).
Embodiment 5
The anti-microbial activity spectrum of CAP is determined by Spot Jest.Approximately the aseptic rough antibiotic prepared product (CAP) as obtaining in above-mentioned embodiment 3 and 4 of 1ml dilutes with about 1ml sodium phosphate buffer (pH about 7.0), and sterilization, described in above-mentioned embodiment 3 and 4.Approximately every kind of sample plate of 10ml is inoculated in the Campylobacter agar that adds blood or has inoculated in advance on the nutrient agar medium of target bacteria cell.The flat board that contains the campylobacter jejuni culture is grown under little aerobic condition at about 42 ℃, and Caused by Yersinia enterocolitica and pseudonodule yersinia enterocolitica are cultivated at about 28 ℃ of aerobics, and other bacterial isolateses are cultivated at about 37 ℃ of aerobics, cultivate about 24 or 48 hours.Discriminating is based on the inhibition zone that target bacteria produces.The activity expression of CAP is arbitrary unit (the AU) (Henderson etc. of culture growth each milliliter prepared product when demonstrating visible inhibition zone, Archives of Biochemistry and Biophysics (biological chemistry and biophysics archives), the 295th volume, 5-12,1992; Incorporate by reference this paper at this).All experiments all repeat twice.
Following table 4 and 5 has provided the antagonistic activity for the rough antibiotic prepared product of the preparation of the faecalis among the lactobacillus among the embodiment 3 and the embodiment 4.
Two kinds of isolates of lactobacillus bacterial classification suppress the growth (table 3) of campylobacter jejuni.Isolate detects (bioMerieux, France) with API 50CHL and differentiates to be saliva lactobacillus-PVD32 (NRRL B-30514) and Lactobacillus acidophilus LWP320 (NRRL B-30510).As shown in table 4, all these bacterial strains produce the lactocin with broad spectrum antibiotic activity.They suppress the growth of Gram-positive and gram-negative micro-organism.The activity of Lactocin OR-7 opposing campylobacter jejuni is the strongest.
Two kinds of isolates of enterococcal species suppress the growth (table 3) of campylobacter jejuni.Isolate is enterococcus faecalis (isolate LWP21) (NRRL B-30645) and enterococcus durans (isolate LWP26) (NRRL B-30511) with the ENCOCCUS detection and identification.Two kinds of bacterial strains produce the enterococcin (enterocins) of broad spectrum activity, suppress Gram-positive and gram-negative micro-organism.The enterococcin 21 that enterococcus faecalis produces is that all 33 kinds of unique inhibition detect bacterial strain, comprise especially the bacteriocin that the bacteriocin of different sources is had the growth of the proteus vulgaris (Proteus vulgaris) of resistance and morganella morganii (Morganella morganii).
The lactocin that is produced by lactobacillus and lactic acid coccus (Lactococcus spp.) in table 4. Spot Jest and the activity (AU/ml) of lactococcin
| Detect bacterial strain | OR-7Lactcin | LWP320Latocin |
| Campylobacter jejuni ATTC 11168 | 3200 | 1600 |
| Campylobacter jejuni L4 | 3200 | 1600 |
| Campylobacter jejuni UV3 | 3200 | 800 |
| Campylobacter jejuni F2 | 3200 | 800 |
| Salmonella enteritidis (S.enteritidis) 1 | 1600 | 800 |
| |
1600 | 800 |
| Salmonella enteritidis 204 | 800 | 800 |
| Salmonella enteritidis 237 | 1600 | 800 |
| Chicken Salmonella pullorum (S.gallinarumpullorum) | 800 | 400 |
| Salmonella typhimurium (S.typhimurium) 320 | 1600 | 400 |
| Salmonella typhimurium 383/60 | 1600 | 800 |
| Salmonella choleraesuls (S.choleraesuis) 370 | 1600 | 800 |
| Salmonella choleraesuls 434/4 | 800 | 400 |
| Intestinal bacteria (E.coli) 0157:7EDL 933 | 1600 | 1600 |
| Colon bacillus 0157: 7904 | 3200 | 3200 |
| Colon bacillus 0157: 7J61 | 3200 | 400 |
| Colon bacillus 0157: 7131 | 3200 | 800 |
| Formula citric acid bacillus (Citrobacter freundii) not | 1600 | 400 |
| Klebsiella pneumoniae | 1600 | 200 |
| Dysentery will is congratulated formula bacterium (Sh.Dysenteriae) | 1600 | 200 |
| Caused by Yersinia enterocolitica 03 | 1600 | 400 |
| Caused by Yersinia enterocolitica 09 | 1600 | 200 |
| Caused by Yersinia enterocolitica 11 | 800 | 400 |
| Pseudonodule yersinia enterocolitica ser4 | 1600 | 200 |
| Pseudonodule yersinia enterocolitica ser 14 | 1600 | 200 |
The activity of the enterococcin that table 5. enterococcus faecalis 21 and enterococcus durans 26 produce
Embodiment 6
CAP and bacteriocin with the sepharose of about 15% weight and approximately 1%SDS (9 * 12cm) carry out the SDS-PAGE electrophoresis in the triglycine damping fluid.After approximately 100mA carried out about 4 hours electrophoresis, gel was fixed with the solution that contains about 15% ethanol and about 1% acetic acid.Then this gel uses distilled water wash 4 hours.In order to determine the molecular weight of protein component, gel is with containing about 0.15% coomassie brilliant blue R_250 (Sigma, USA), about 40% ethanol, and the solution-dyed of about 7% acetic acid.Gel was used phosphate buffered saline buffer (PBS) washing about 1.5 hours then, and with deionized water wash about 3 hours.Gel after the washing with the method for Bhunia etc. (Journal ofIndustrial Microbiology, Volume 2,319-322,1987; Incorporate by reference text into) detect it to three kinds of target bacteria, campylobacter jejuni ATCC11168, colon bacillus 0157: H7904, and the resistance of Salmonella enteritidis 237.Gel places petri diss (Petri dishes), covers with about 5% cracking sheep blood-semi-solid Campylobacter agar (about 0.75%) or semi-solid agar (0.7%), with the cell inoculation that detects bacterial strain.The flat board that contains campylobacter jejuni was cultivated about 48 hours at about 42 ℃ under little aerobic condition, and Escherichia coli O 157: H7 and Salmonella enteritidis were cultivated about 24 hours at about 37 ℃.Detection is based on to observe under the condition that bacteriocin exists and detects the strain growth inhibition zone.The activity of purification of bacterial element is evaluated (table 6 is lactobacillus bacterial classifications, and table 7 is enterococcal species).
CAP sample and bacteriocin place on the IEF gel (the about 3.1-10.0 of pH) (Novex, San Diego, CA).Gel electrophoresis is at XCMII
TMCarried out about 1 hour with about 100V among the Mini-Cell (Novex), carried out about 2 hours with 200V, carried out about 30 minutes with 500V.Gel about 30 seconds with distilled water wash, unfixing, then use coomassie brilliant blue R_250 (Sigma, USA) dyeing is with the iso-electric point (pI) of definite bacteriocin and the ability of their inhibition detection strain growths, shown in following table 6 and table 7, table 6 is lactocins, and table 7 is enterococcin.
As shown in Figure 1, the CAP OR7 of saliva lactobacillus PVD 32 contains about 6kDa, approximately 16kDa, and three protein ingredients of about 40kDa.In order to detect their activity, the dyeing electrophoretic band that contains described peptide is placed in to cultivate campylobacter jejuni ATCC11168, on the semi-solid Campylobacter agar of the cell of Salmonella enteritidis, and Escherichia coli O 157: H7.Approximately the component of 6kDa is found to have all three kinds activity that detect bacterial strain of opposing.Isoelectrofocusing has differentiated that a kind of pI equals about 9.0 component and shows all three kinds activity that detect bacterial strains of opposing.
As shown in Figure 2, the enterococcin LWP26 of the lactocin LWP320 of pure Lactobacillus acidophilus LWP320 and pure enterococcus durans LWP26 contains respectively about 6kDa and the about protein component of 3kDa.In order to detect their activity, the dyeing electrophoretic band that contains described peptide is placed in to cultivate campylobacter jejuni ATCC 11168, on the semi-solid Campylobacter agar of the cell of Salmonella enteritidis, and Escherichia coli O 157: H7.Approximately the component of 6kDa and about 3kDa is found to have all three kinds activity that detect bacterial strains of opposing.Isoelectrofocusing has differentiated that a kind of pI equals respectively about 8.5 and about 7.8 component and shows all three kinds activity that detect bacterial strains of opposing.
As shown in Figure 3, the enterococcin LWP21 of the enterococcus faecalis LWP21 of separation contains two protein ingredients of about 3.0kDa (the 1st peak) and about 6.0kDa (the 2nd peak).In order to determine anti-microbial activity, electrophoretic band is placed in to cultivate campylobacter jejuni ATCC11168, on the semi-solid Campylobacter agar of the cell of Salmonella enteritidis, and Escherichia coli O 157: H7.Approximately the component of 6.0kDa has all three kinds activity that detect bacterial strain of opposing.The component of not observing about 3.0kDa has anti-microbial activity.For the enterococcin LWP26 that separates, isoelectrofocusing has differentiated that a kind of pI is that about 7.8 single protein ingredient detects bacterial strains to all three kinds Antagonism (table 7) is arranged.High reactivity and enterococcin LWP21 in Spot Jest the 1st peak+the 2nd peak component contacts, and lowest activity is presented among SDS-PAGE and the IEF.
Table 6. is by Spot Jest, SDS-PAGE, and the anti-microbial activity of the lactocins CAP that determines of IEF
Table 7. is based on the characteristic of the active ingredient of SDS-PAGE and isoelectrofocusing result's enterococcin LWP21 and LWP26
Embodiment 7
Such as the rough antibiotic prepared product of the bacteriocin of acquisition as described in the above-mentioned embodiment 2 with gel-filtration and ion-exchange chromatogram purification.The rough antibiotic prepared product (CAP) of lactocin OR7 and lactocin LWP320 is injected into about 75ml sodium phosphate buffer, in Superose 12HR 16/50 post of about 5.9 balances of pH (Pharmacia, 1.6X 50cm).Described prepared product is with the flow velocity wash-out of identical damping fluid with about 8ml/ minute.Detect the activity of elution fraction opposing campylobacter jejuni ATCC 11168.The concentration of protein detects with the method (Journal ofBiological Chemistry, Volume 193,1951) of Lowry etc.The component that suppresses the campylobacter jejuni growth is further purified by cation-exchange chromatography with CM-Sepharose post (Pharmacia, 2.5X 20cm).The CM-Sepharose post uses damping fluid C (50mM Citrate trianion-phosphate buffered saline buffer, pH about 5.0) with about 8ml/ minute flow velocity balance.Bacteriocin is that the damping fluid C of about 8mM of about 0.1% to about 0.8% was with about 8ml/ minute flow velocity wash-out with NaCl concentration wherein.Determine anti-microbial activity and the protein concn of each component.The component of purifying is called lactocins OR7 and LWP320.
The OR7 of purifying and LWP320 estimate with SDS-PAGE and isoelectrofocusing, described in above-mentioned embodiment 4.The highest active component (OR7) is that molecular weight is about 6kDa, and pI is about 9.0 peptide (Fig. 1 and table 6), and active ingredient LWP320 is that molecular weight is about 6kDa, and pI is about 8.5 peptide (Fig. 3 and table 6).
The faecalis bacteriocin of CAP passes through two step purifying: ion-exchange chromatography and hydrophobic chromatography.The active CAP component that obtains among the embodiment 4 is injected into in the SP-Sepharose Fast Flow post (V-30ml) of damping fluid C (20mM sodium phosphate, pH about 7.8) with about 18ml/ minute flow velocity balance.This post washs with the damping fluid C of 4 times of volumes, and component contains the damping fluid C wash-out of 0.5M NaCl with about 100ml.Composition activity passes through with campylobacter jejuni ATCC 11168 as above-mentioned embodiment 5 described Spot Jests are determined.The component that can suppress the growth of campylobacter jejuni is injected in the Phenyl-Sepharose CL-4B post of using damping fluid D (0.06M borate buffer solution, the pH about 8.5) balance that contains 0.2M NaCl.This post washs with the damping fluid D of 4 times of volumes.Component is with the damping fluid D wash-out of about 70% ethanol of containing of about 4ml (v/v).The antagonistic activity of every kind of component is measured with Spot Jest.Again carry out the prepared product that cation-exchange chromatography obtains purifying with SP-Sepharose or Phenyl-Sepharose CL-4B.Protein concn is determined with the method (as mentioned above) of Lowry.The component of separating is called enterococcin LWP21 and LWP26.
LWP21 and the LWP26 component of separating are estimated with SDS-PAGE and isoelectrofocusing, described in above-mentioned embodiment 4.The component 2 that the activity of LWP21 is the highest is that molecular weight is about 6kDa, and pI is about 8.4 peptide (Fig. 2 and table 7), and the activated component 1 that do not have of LWP21 is that molecular weight is about 3kDa, and pI is about 5.6 peptide (Fig. 2 and table 7).The highest component of activity of LWP26 is that molecular weight is about 3kDa, and pI is about 7.8 peptide (Fig. 3 and table 7).
The aminoacid sequence of purification of bacterial element is determined by the Edman edman degradation Edman with 491cLC automatic sequencer (Applied Biosystems, USA).Bacteriocin approximately is being hydrolyzed about 72 hours among the 6M HCl at about 110 ℃ in a vacuum.Determine molecular weight with Voyager-DERP (Perkin-Elmer, USA) by mass spectrum.The MALDI-TOF system, a kind of matrix assisted laser desorption ionization ionizes the flight time system, and with matrix, 2-cyano group-hydroxycinnamic acid uses together.The aminoacid sequence of four kinds of bacteriocins is:
OR7：
KTYYGTNGVHCTKNSLWGKVRLKNMKYDQNTTYMGRLQDILLGWATGAFGKTFH
SEQ?ID?NO?1
LWP320：
ARKYGNGVCGSKWINNGGFQVIGNNAAANLTNWGEAFASATKSGCSATTCIINAMA
SEQ?ID?NO?2
LWP21 (component 1): FNIRGGYNFGKSVRHWDAIGNMAGIIKL SEQ ID NO 3
LWP21 (component 2):
VFHAYSARGVRNNYKCAGVPDAIGCAVRGIFIHGYSLQWMQVKWGWLFK?SEQ?ID?NO4
LWP26：TKTTRGNGVNKECWETYKAGTVDILWASWSK SEQ?ID?NO?5
The calculating molecular weight of OR7 peptide is about 6,000Da.MALDI-TOF the analysis showed that the molecular weight of OR7 is 5,123Da.
The calculating molecular weight of LWP320 peptide is about 6,000Da.MALDI-TOF the analysis showed that the molecular weight of LWP320 is 5,858Da.
The calculating molecular weight of LWP21 peptide (component 1) is about 3,000Da.MALDI-TOF the analysis showed that the molecular weight of LWP21 (component 1) is 2,786Da.
The calculating molecular weight of LWP21 peptide (component 2) is about 6,000Da.MALDI-TOF the analysis showed that the molecular weight of LWP21 (component 2) is 4,986Da.
The calculating molecular weight of LWP26 peptide is about 3,000Da.MALDI-TOF the analysis showed that the molecular weight of LWP26 is 3,231Da.
The biological chemistry purifying of table 8.lactocin OR7
The biological chemistry purifying of table 9. enterococcin LWP21
Embodiment 8
Measured enzyme, temperature and the pH impact on the bacteriocin activity.One of following enzyme of about 10 μ l is transferred in the pipe that contains about 20ml bacteriocin: β-Quimotrase-about 100mg/ml, Proteinase K-about 200mg/ml, papoid-about 60mg/ml, N,O-Diacetylmuramidase-about 75mg/ml, and lipase-about 100mg/ml (all are from Sigma-Aldrich Corp., St.Louis MO).Cultivate after about 3 hours for about 37 ℃, the mixture of bacteriocin and enzyme is analyzed its anti-microbial activity with Spot Jest as described in Example 5.Untreated bacteriocin is as positive control.
In order to study the thermostability of bacteriocin, approximately the sample of 2mg/ml boiled in water-bath about 15 minutes, and cooling is estimated its anti-microbial activity by Spot Jest.Approximately the 2mg/ml bacteriocin is used for estimating the effect of pH.The about sterile solution of 2ml, approximately 10mMNaOH or approximately 10mMHCl be added in the sample pH to detect about 3 to about 10.Sample was cultivated about 2 hours and 24 hours at about 37 ℃, cultivated about 20 minutes at about 90 ℃.To adjust sample about 7.2 to pH by adding about 4mM sterile phosphate damping fluid, with analyzing its anti-microbial activity such as above-mentioned embodiment 5 described Spot Jests.
Described bacteriocin has lost activity after processing with β-Quimotrase, Proteinase K and papoid, but at retentive activity (table 10 and 11) with N,O-Diacetylmuramidase, lipase or when being heated to about 90 ℃.They are stable in from about 3.0 to about 9.0 different pH values, but at about pH 10 non-activity (table 12 and 13) that becomes.Enterococcin loses its activity at pH about 9.1 and about 10.
Table 10. enzyme and temperature are to the effect of the anti-microbial activity of OR7
| Process | Active * |
| β-Quimotrase | |
| Proteinase K | |
| Papoid | |
| Quimotrase | |
| Lipase | |
| 90 ℃, 15 minutes |
* resist the activity of campylobacter jejuni ATCC11168 in the Spot Jest
(+) exists active
(-) loses activity after processing
Table 11. enzyme and temperature are to the effect of the anti-microbial activity of enterococcin LWP21 and enterococcin LWP26
| Process | Active * |
| β-Quimotrase | |
| Proteinase K | |
| Papoid | |
| N,O-Diacetylmuramidase | + |
| Lipase | + |
| 90 ℃, 15 minutes | + |
* resist the activity of campylobacter jejuni ATCC11168 in the Spot Jest
Exist active after (+) processes
(-) loses activity after processing
Table 12.pH is to the effect of the activity of lactocin OR7
| pH | 37 ℃ of active * after 2 hours | 37 ℃ of active * after 24 hours | 90 ℃ of activity after 20 minutes |
| 3.0 | + | + | |
| 5.0 | + | + | + |
| 6.2 | + | + | + |
| 7.0 | + | + | + |
| 8.4 | + | + | + |
| 9.1 | + | ||
| 10.0 |
* resist the activity of campylobacter jejuni ATCC11168 in the Spot Jest
Exist active after (+) processes
(-) loses activity after processing
Table 13.pH is to the effect of the activity of enterococcin LWP21 and enterococcin LWP26
* resist the activity of campylobacter jejuni ATCC11168 in the Spot Jest
Exist active after (+) processes
(-) loses activity after processing
Embodiment 9
Bacteriocin OR7 is purifying as described in example 5 above.By known in the art, the bacteriocin OR7 of purifying places polyvinylpyrrolidone to add commodity poultry feed, the about 250mg/kg commodity of concentration poultry feed.The therapeutic composition that about 100g is made by the corn cereal that grinds contains about 0.5g bacteriocin OR7, approximately 1.25g polyvinylpyrrolidone, and about 8.6% water.Approximately this composition of 100g is added in about 2kg poultry feed.The chick in 1 day age, 6 day age and 18 day age is grouped the unit of being furnished with feed appliance, water and filtrated air supply that places separately isolation.Food and water are arbitrarily supplied.Chick (table 14) for 1 day age:
First groupChick is organized in contrast, freely obtains and does not add putting somebody on a diet of bacteriocin.These chick just are subject to stimulation (challenge) such as above-mentioned embodiment 1 described campylobacter jejuni L4 and B1 at the first day of life.These bacterial strains by the per os tube feed with in the 0.2ml volume about 2 * 10
6Concentration feed.Five chick were put to death (sacrificed) in about 7 days afterwards in stimulation, and all the other five in afterwards execution in about 10 days of stimulation.
Second groupChick is in the stimulation that was subject to campylobacter jejuni L4 and B1 on the 1st day of its life, with above-mentioned first group identical.These chick freely obtain in rose in the 4th day three days of life and contain putting somebody on a diet of about 250mg bacteriocin OR7/kg food.Second group of chick execution in about 7 days after campylobacter jejuni stimulates.
The 3rd groupThe stimulation that chick is accepted is identical with second group of chick with feeding, puts to death in rear 10 days in stimulation, and the result is as shown in Table 15.
The result for the treatment of that table 14. bacteriocin OR-7 induces campylobacter jejuni to infect for experiment in the broiler chicken
* put somebody on a diet for the chick in 1-10 days ages prepares therapeutic on the basis of commodity food.
For the chick in 6 day age, control group freely obtains and does not add putting somebody on a diet of bacteriocin.These chick accepted in about 0.2ml volume about 10 on the 2nd day experiment
6The campylobacter jejuni bacterial strain L-4 as described in example 1 above of CFU, and the stimulation of B-1.Campylobacter jejuni is by per os tube feed feeding.Chick was put to death in stimulation in rear 9 days, 11 days and 14 days.Two groups of experiment chick freely obtain from beginning in the 6th day of life has putting somebody on a diet of the bacteriocin OR7 that is encapsulated in the polyvinylpyrrolidone.Coexist mutually the 2nd day of experiment of these chick and above-mentioned contrast chick is upset.Chick is in the 9th day, 11 days and execution in 14 days of life.The result is shown in following table 15.
Add in table 15. feed bacteriocin OR7 the infection relevant with campylobacter jejuni 6 day age chick experiment process
* the pure dosage of giving the chick feeding be 3 days time=about 26.4mg; 5 days=about 50.6mg; 8 days=about 80.5mg
For the chick in 18 day age, the contrast chick freely obtains does not add putting somebody on a diet of bacteriocin.These chick are as mentioned above at 10 of the 15th day about 0.2ml volume of per os tube feed feeding of testing
7The dosage of CFU campylobacter jejuni bacterial strain L-4 and B-1.Chick is in execution in the 24th day of experiment.The chick of accepting the aforesaid regular diet that contains bacteriocin OR7 freely obtains the feed that contains about 0.25g bacteriocin OR7/kg feed from about five days of beginning in about the 19th day of life, pure therapeutic dose is every about 109mg of chick.Chick is in execution in the 24th day of life.The result is shown in following table 16.
Add in table 16. feed bacteriocin OR7 the infection relevant with campylobacter jejuni 18 day age chick experiment process
Chick is upset the 1st day of life, and the contrast chick freely obtains food and the water that does not add bacteriocin.Chick is subject to about 10 at the 1st day per os tube feed of life
6The campylobacter jejuni bacterial strain L4 of CFU and the stimulation of B1.The contrast chick is in execution in the 17th day of life.In experimental group I, the chick in 1 day age is subject to the stimulation identical with contrasting chick, freely obtains the feed that contains about 0.250g bacteriocin OR7/kg feed from beginning in the 14th day of life, in execution in about the 17th day of life; The chick of II group was subject to the stimulation identical with contrasting chick the 1st day of life, freely obtain the feed that contains about 0.500g bacteriocin OR7/kg feed the 14th day of life; Execution in the 17th day at life.The result is shown in table 17.
The experiment of the chick of the infection campylobacter jejuni of interpolation bacteriocin OR7 is processed in table 17. feed
Aforementioned detailed description is for illustrational purpose, and 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 (6)
1. bacteriocin, the aminoacid sequence of described bacteriocin is SEQ ID NO 2.
2. bacteriocin, the aminoacid sequence of described bacteriocin is SEQ ID NO 3.
3. bacteriocin, the aminoacid sequence of described bacteriocin is SEQ ID NO 4.
4. therapeutic composition comprises:
(a) bacteriocin of at least a separation of significant quantity, to be reduced at least less a kind of field planting level of target bacteria, the aminoacid sequence of described bacteriocin is the aminoacid sequence that is selected from the group that is comprised of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4; With
(b) suitable treatment carrier.
5. treatment feed that is used for animal comprises:
(a) bacteriocin of at least a separation of significant quantity, to be reduced at least less a kind of field planting level of target bacteria, the aminoacid sequence of described bacteriocin is the aminoacid sequence that is selected from the group that is comprised of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4
(b) treatment carrier; With
(c) animal-feed.
6. the application of therapeutic composition in making the medicine that reduces at least the field planting level of at least a target bacteria in animal, wherein said therapeutic composition comprises:
At least a bacteriocin of significant quantity, to be reduced at least less a kind of field planting level of target bacteria, the aminoacid sequence of described bacteriocin is the aminoacid sequence that is selected from the group that is comprised of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4; With
The treatment carrier.
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