CN102304483A - Enterococcus faecium for feeding and applications thereof - Google Patents
Enterococcus faecium for feeding and applications thereof Download PDFInfo
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Abstract
The invention relates to enterococcus faecium for feeding as well as a freeze-drying fungicide and applications thereof, and discloses enterococcus faecium LAB12 CGMCC (China General Microbiological Culture Collection Center) No.4847 which is grampositive cocci, has no spores, grows well on an MRS agar plate, forms a round bacterial colony with the diameter of 0.5-1mm within 48 hours and is used for feeding, and the bacterial colony is round, smooth and upheaved, and is a shape of grey white dewdrop; the enterococcus faecium grows in a facultative anaerobic condition; the growth temperature range is 10 DEG C-45 DEG C; the optimum growth temperature is 30 DEG C-40 DEG C; and the growth pH value is 4-10, and the optimum pH value is 6.0. The freeze-drying fungicide formed by the bacterial strain is nontoxic and harmless, is gastric juice resistant, is cholate resistant, has a high inhibitory effect for multiple harmful bacteria, has a long quality guarantee period, can be widely applied to birds and livestock breeding to strengthen the animal disease-resistant capability, and is expected to be the substitution of antibiotics for feeding.
Description
Technical field
The invention belongs to microorganism strains, particularly relate to an Enterococcus faecalis bacterial strain and freeze-drying microbial inoculum thereof and its application in preparation feed and fodder additives.
Background technology
Faecium (Enterococcus faecium) belongs to enterococcus spp, is the part of normal microflora in people and the animal intestinal.Faecium is very strong to the adhesive power of animal intestine mucous membrane, can on the animal intestine mucous membrane, form layer of protecting property physiologic barrier, stops the field planting invasion of pathogenic bacterium.Faecium can also produce VITAMIN, amino acid, somatomedin, the required nutritive substance of growth of animal such as lactic acid acetate; Can produce simultaneously the material that lactolin, edeine, acidophilin and bacitracin etc. suppress the pathogenic bacterium growth and breeding.Faecium may be used in the nutritional supplementation of animal as a kind of feeding probiotic bacterium, and can be used as a kind of microbiotic substitute use.
The freeze-drying microbial inoculum is processed by vacuum freeze-drying method; It is freezing in advance soon to have added protectant bacterium liquid; Under vacuum condition, make ice crystal distillation wherein then, treat to remove part planar water wherein again after the ice crystal distillation, finally obtain the dry products that the residual water yield is 1%-4%.Microorganism cells after the lyophilize is not damaged basically, and the viable bacteria survival time is also longer, has the ideal instantly-soluble and replies immediately water-based.
Faecium is a kind of microorganism that can in feed, add that China Ministry of Agriculture announces.Patent documentation CN101629155A, CN 1831114A, CN 1314466A, CN 101492650A, CN 1314091A, CN1016683122A, CN 101701202A disclose an Enterococcus faecalis respectively, but all do not relate to the freeze-drying Research of Microorganisms Agent; Disclose solid-state cultivation of low moisture and the drying means of a kind of faecium among the patent documentation CN 101955900A, but this method is a solid-state fermentation process, the production operation mechanization degree is low, and fermenting process is wayward.
Summary of the invention
The purpose of this invention is to provide the faecium that a strain prepares feed and fodder additives to inhibited can be used for of pathogenic bacterium.
The name of manure enterococcin strain provided by the present invention (Enterococcus faecium) is called LAB12; This bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 05 16th, 2011, and deposit number is CGMCC No.4847.
Faecium (Enterococcus faecium) LAB12 CGMCC No.4847 is a gram-positive cocci, no gemma; Well-grown on the MRS agar plate, 48h forms the big small circular bacterium colony of diameter 0.5-1mm, and bacterium colony rounding, smooth, protuberance are canescence and reveal an appearance; The amphimicrobian growth, growth temperature range: 10 ℃-45 ℃, optimum growth temperature: 30 ℃-40 ℃; Growth potential of hydrogen scope: pH 4-10, optimum pH is 6.0; The part biochemical characteristic is as shown in table 1:
The part biochemical characteristic of table 1 faecium (Enterococcus faecium) LAB 12CGMCC No.4847
Annotate: "+" expression reacting positive; "-" expression reaction negative.
External antagonistic effect result shows: faecium (Enterococcus faecium) LAB12CGMCC No.4847 all has stronger restraining effect to streptococcus aureus (SA), salmonella typhi (ST 04Hi), pig source product enterotoxin type intestinal bacteria E.coli0139, pig source pathogenic escherichia coli (E.coli C83524, E.coli C83529).
Faecium (Enterococcus faecium) LAB12 CGMCC No.4847 can tolerate pH 2.5 synthetic gastric juice 180min; The highest tolerance gallbladder salinity is 3.0% (mass percent concentration).
LAB12CGMCC No.4847 is insensitive to fortum, cephalosporin, Prostaphlin, kantlex, cephalo oxime, gentamicin and Amikacin Sulphate for faecium (Enterococcus faecium), and is responsive to Pyocianil, cephazolin I, cephazolin, penbritin, Ceftriaxone, mould of happiness, penicillin, Cefoperazone, tsiklomitsin, erythromycin, Xin Meisu, doxycycline and Pipril.
Feed behind faecium (Enterococcus faecium) the LAB12CGMCC No.4847 intestinal bacteria quantity descend gradually (P＜0.05) in the animal intestinal.Feed continuously mouse faecium microbial inoculum 21 days is irritated stomach faecium (Enterococcus faecium) LAB12CGMCC No.4847 bacterium liquid 0.2mL/d, and bacterial concentration is 10
8CFU/mL, intestinal bacteria quantity reduces 99.9% in the stool in mice.
Second purpose of the present invention provides a kind of faecium (Enterococcus faecium) LAB12 CGMCCNo.4847 microbial inoculum.
Microbial inoculum provided by the present invention, its activeconstituents are faecium (Enterococcus faecium) LAB12CGMCC No.4847.
Another object of the present invention provides a kind of faecium (Enterococcus faecium) LAB12 CGMCCNo.4847 freeze-drying microbial inoculum.
The preparation method of faecium provided by the present invention (Enterococcus faecium) LAB12CGMCC No.4847 freeze-drying microbial inoculum can may further comprise the steps:
1) preparation MC liquid nutrient medium, it is subsequent use to sterilize;
2) preparation frozen solution: trehalose 10-100g/L, skimmed milk powder 10-200g/L, sucrose 10-200g/L, glycerine 10-100g/L, it is subsequent use to sterilize;
3) faecium (Enterococcus faecium) LAB12CGMCC No.4847 is inoculated in the MC liquid nutrient medium, leaves standstill under 37 ± 2 ℃ and cultivate 12-36 hour (being preferably 24 hours);
4) after cultivation finishes; Collect thalline; After cleaning thalline; Add frozen solution; The volume ratio 1 of thalline and frozen solution: 10-1: 100; Repeated oscillation; Frozen solution and thalline are mixed; Then; The bacterium liquid that will add cryoprotectant is packed in the freeze-drying bottle;-40 ± 2 ℃ of pre-freeze 3-5 hours (being preferably 4 hours), freeze fully after, make and freeze article temperature maintenance below 0 ℃ 36-48 hour; Open vacuum pump extraction freezing article moisture simultaneously; Wait to freeze article moisture content (freeze the article color and gradually become oyster white) when draining, elevated temperature to room temperature (10 ℃-30 ℃) gradually is to discharge remaining moisture; Avoid freeze-dried products to absorb airborne moisture; The goods moisture content is controlled in 0.5%-5% (quality percentage composition) scope after the freeze-drying, and freeze-drying seals after finishing, and obtains faecium (Enterococcus faecium) LAB 12CGMCC No.4847 freeze-drying microbial inoculum.
In the preparation method of above-mentioned faecium (Enterococcus faecium) LAB12CGMCC No.4847 freeze-drying microbial inoculum, the MC liquid culture based formulas in the said step 1) is: glucose 5-10g/L, peptone 5-10g/L, pawpaw peptone 5-10g/L, yeast extract 5-10g/L, dibasic ammonium citrate 1-5g/L, extractum carnis 5-10g/L, tween 80 1-5g/L, K
2HPO
41-5g/L, pH 6.0 ± 0.1.
The invention provides an Enterococcus faecalis (Enterococcus faecium) LAB 12CGMCC No.4847 and microbial inoculum thereof; Wherein the frozen solution through screening has been adopted in the preparation of freeze-drying microbial inoculum; This frozen solution is good to the preservation effect of faecium viable bacteria body, and the shelf time is long.Microbial inoculum by this bacterial strain is processed is nontoxic, resistant to gastric juice, bile tolerance; Multiple harmful bacterium there is very strong restraining effect, can be widely applied to fowl, poultry aquaculture (directly add in the animal daily ration and get final product), strengthen the animal disease resistant ability; Be expected to become the surrogate of feeding antibiotic, have a extensive future.
The biomaterial explanation:
The name of faecium of the present invention (Enterococcus faecium) is called LAB12; This bacterial strain has been preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center that the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City (being called for short CGMCC) on 05 16th, 2011, deposit number is CGMCC No.4847.
Below in conjunction with specific embodiment the present invention is explained further details.
Embodiment
Embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Method therefor is ordinary method if no special instructions among the following embodiment.
Separation, evaluation and the preservation of embodiment 1, faecium (Enterococcus faecium) LAB12 CGMCC No.4847
One, the separation of faecium (Enterococcus faecium) LAB12 CGMCC No.4847
MSC substratum: peptone 5g, extractum carnis 5g, yeast extract paste 5g, Trisodium Citrate 2g, sodium acetate 5g, glucose 5g, potassium primary phosphate 2g, soil temperature 801.0mL, lime carbonate 20g, sal epsom 0.58g, manganous sulfate 0.25g, ammonium sulfate 1g; Water 1000mL; Transfer 6.5,115 ℃ of sterilizations of pH 20min.
Enrichment medium: the liquid MSC substratum that contains 40 μ g/mL cycloheximides.
CaCO
3MSC substratum: the MSC solid medium that contains 0.5% (mass percent concentration) light calcium carbonate.
Acidproof MSC substratum: the pH value of MSC substratum is transferred to 3.0.
Bile tolerance screening MSC substratum: the pig cholate that in the MSC substratum, adds 0.3% (mass percent concentration).
YPD solid medium (live bacterial count is used): glucose 20g, Tryptones 20g, yeast powder 10g, agar powder 18g, water 1000mL transfers 7.0,115 ℃ of sterilizations of pH 20min.
Gather the piglet fresh excreta from Hua Zhong Agriculture University boar mensuration center; After the enrichment medium enrichment culture (cultivating 36 hours down for 37 ℃); Get in the acidproof MSC liquid nutrient medium that 1mL is inoculated in 10mL pH3.0; Behind 37 ℃ of cultivation 8h; Get 1mL and be inoculated in 10mL and contain in the bile tolerance screening MSC liquid nutrient medium of 0.3% (mass percent concentration) pig cholate and cultivate 12h, get the 1mL dilution again and coat CaCO
3On the MSC flat board, pick out the strongest bacterial strain of acid producing ability, called after LAB12.
Two, the Physiology and biochemistry of LAB12 bacterial strain is identified
The LAB12 bacterial strain is carried out Physiology and biochemistry identify result: gram-positive cocci, no gemma; Well-grown on the MRS agar plate, 48h forms the big small circular bacterium colony of diameter 0.5-1mm, and bacterium colony rounding, smooth, protuberance are canescence and reveal an appearance; The amphimicrobian growth, growth temperature range: 10 ℃-45 ℃, optimum growth temperature: 30 ℃-40 ℃; Growth potential of hydrogen scope: pH 4-10, optimum pH is 6.0; The part biochemical characteristic is as shown in table 2:
The part biochemical characteristic of table 2LAB12 bacterial strain
Annotate: "+" expression reacting positive; "-" expression reaction negative.
Three, the 16S rDNA of LAB12 bacterial strain order-checking comparison
Total DNA with bacterial strain LAB12 is a template; The 16S rDNA sequence of this bacterial strain of pcr amplification under the guiding of primer P0 (5 '-GAGTTTGATCMTGGCTCAG-3 ') and PC3 (5 '-TACHAGGGTATCTAATCCT-3 '); The PCR reaction system is 10 * Buffer, 5.0 μ L; DNTP (10mmol/L) 1.0 μ L, Primer 1 (20 μ mol/L) 1.0 μ L, Primer 2 (20 μ mol/L) 1.0 μ L; Template DNA 2.5 μ L; Taq enzyme (5.0U/ μ L) 2.5 μ L, ultrapure water 34 μ L, totally 50 μ L.The PCR reaction conditions is: 95 ℃ of 5min of elder generation; 94 ℃ of 1min then, 50 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations; Last 72 ℃ are extended 8min.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result has obtained the specific band of big or small about 800bp through amplification.Use sepharose to reclaim test kit recovery and this purpose band of purifying available from Axygen company; Send precious biotechnology (Dalian) company limited to carry out determined dna sequence, sequencing result shows the nucleotide sequence that the 16S rDNA sequence of bacterial strain LAB12 has sequence 1 in the sequence table.Measured sequence and the 16S rDNA sequence among the Genbank are carried out the Blastn similarity analysis relatively, and the homology of the 16SrDNA sequence of results strain LAB12 and Enterococcus faecium has reached 99.8%.
Comprehensive Physiology and biochemistry is identified and 16S rDNA sequencing result; Can confirm that bacterial strain LAB12 is faecium (Enterococcus faecium); This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 16th, 2011, and deposit number is CGMCC No.4847.
The resistant to gastric juice property analysis of embodiment 2, faecium (Enterococcus faecium) LAB12 CGMCC No.4847
The synthetic gastric juice of preparation: Tryptones 8.3g, glucose 3.5g, sodium-chlor 2.05g, calcium chloride 0.11g, Repone K 0.37g, potassium primary phosphate 0.6g, pig cholate 0.05g, N,O-Diacetylmuramidase 0.1g, stomach en-13.3mg, adding distil water 1000mL; Be respectively 1.5 and 2.5,115 ℃ of sterilization 20min with 1mol/L hydrochloric acid adjust pH.Wherein N,O-Diacetylmuramidase and stomach en-filtration sterilization.
Actication of culture; By 1% (mass/volume percentage concentration; The g/100mL of unit) inoculum size is transferred to faecium (Enterococcus faecium) LAB12 CGMCC No.4847 in the 100mL liquid MSC substratum; 37 ℃, 200r/min overnight incubation; Get 1mL bacterium liquid and add in the aseptic synthetic gastric juice of 9mL, detect number of viable with the viable bacteria plate count respectively at 0min, 30min, 180min.
The pH of hydrochloric acid in gastric juice is about 3.0, and liquid food retention time under one's belt is 1-2h, and in very short time, stomach inner pH value can be raised to about 6.0 after on the feed.Faecium (Enterococcus faecium) LAB12 CGMCC No.4847 does not have behind the 30min in the simulated gastric fluid of pH 1.5 and does not have survival; But tolerance 30min survival rate is 95% in the simulated gastric fluid of pH2.5, and tolerance 180min survival rate is 16.2%.
The bile tolerance property analysis of embodiment 3, faecium (Enterococcus faecium) LAB 12CGMCC No.4847
Prepare the MSC liquid nutrient medium, wherein be added with 0.1%, 0.3%, 0.5%, 1.0%, 2.0%, 3.0%, 5.0% (mass percent concentration) cholate (available from medicine company limited-liability company of traditional Chinese medicines group) respectively.Get the thalline fermentation culture (10 of faecium (Enterococcus faecium) LAB 12CGMCC No.4847 after 37 ℃ of overnight incubation of 1mL
9CFU/mL), add 9mL and be added with in the substratum of above concentration cholate, 37 ℃ leave standstill cultivation 12h, dilution 10
4Get 0.1mL doubly and be coated with MSC solid agar plate, observe the growing state of thalline, to detect its bile tolerance character.(the bacterium number is 3 * 10 if growing number surpasses 30
6CFU/mL) then show its well-grown, the cholate of this concentration of ability; If growing number shows that then its growth is relatively poor, anti-this concentration cholate ability at 5-30; Then show it as if growing number less than 5 and do not grow, not this concentration cholate of ability.Gallbladder salinity fluctuates in 0.03%-0.3% (mass percent concentration) scope in the piglet small intestine.
Detected result shows that the highest tolerance gallbladder salinity of bacterial strain of the present invention is 3.0% (mass percent concentration), far above the scope of 0.03%-0.3% (mass percent concentration).
The antibiotics resistant property analysis of embodiment 4, faecium (Enterococcus faecium) LAB12CGMCC No.4847
The MSC solid medium is melted postcooling to 50 ℃, add good faecium (Enterococcus faecium) the LAB12CGMCC No.4847 bacterium liquid 0.1mL (10 of activation in every 30mL substratum
9CFU/mL) pour plate; After treating that planar surface is dried slightly; Put into common antibiotics (penicillin; Prostaphlin; Penbritin; Pyocianil; Pipril; Cephalosporin; Cephalexin; Cephazolin; Cephalo skin oxime; Reach new again; Ceftriaxone; Cefoperazone; Amikacin Sulphate; Gentamicin; Kantlex; Xin Meisu; Tsiklomitsin; Doxycycline; Minomycin; Erythromycin; Paraxin) 37 ℃ of cultivations of drug sensitive test paper (available from Hangzhou microorganism reagent company limited of antibacterials bacterial drug resistance inspection center of the Ministry of Health); Observe its growing state, measure antibacterial circle diameter.Antibacterials drug sensitive test paper and experiment judging criterion with reference to Hangzhou microorganism reagent company limited of antibacterials bacterial drug resistance inspection center of the Ministry of Health formulates obtain faecium (Enterococcus faecium) LAB12 CGMCC No.4847 to 24 kinds of antibiotic resistances.
Detected result shows; Faecium (Enterococcus faecium) LAB12 CGMCC No.4847 is insensitive to fortum, cephalosporin, Prostaphlin, kantlex, cephalo oxime, gentamicin and Amikacin Sulphate, and is responsive to Pyocianil, cephazolin I, cephazolin, penbritin, Ceftriaxone, mould of happiness, penicillin, Cefoperazone, tsiklomitsin, erythromycin, Xin Meisu, doxycycline and Pipril.
The bacteriostatic test plate of embodiment 5, faecium (Enterococcus faecium) LAB12 CGMCC No.4847
Preparation LB solid medium: Tryptones 10g/L, yeast soak powder 5g/L, sodium-chlor 10g/L, agar 18g/L, 121 ℃, sterilization 30min.
Indicator streptococcus aureus (SA), salmonella typhi (ST 04Hi), pig source product enterotoxin type intestinal bacteria (E.coli 0139), pig source pathogenic escherichia coli (E.coliC83524, E.coliC83529) are inoculated in respectively in the nutrient broth medium (extractum carnis 5g, peptone 10g, NaCl 5g, water 1000mL, pH 7.2-7.4), and 37 ℃ of cultivation 20h are subsequent use.LAB12 CGMCCNo.4847 is inoculated in the MSC liquid nutrient medium with faecium (Enterococcus faecium), and 37 ℃ of cultivation 20h are subsequent use.Transfer the pH value of MSC liquid to make it identical with lactic acid with faecium (Enterococcus faecium) LAB12CGMCC No.4847 fermented liquid pH value, with it as contrast.With aseptic cotton carrier 0.1mL indicator nutrient solution is evenly spread upon on the solid LB substratum, treat to punch equably with metal tube after dull and stereotyped the doing, the aperture is 10mm ± 0.1mm, with aseptic nipper agar block in the hole is chosen again, uses hot glass rod back cover then.In each hole, add streptococcus acidi lactici fermented solution respectively and contrast liquid, cultivate 24h for 37 ℃, observation has or not antibacterial ring, and measures the bacteriostatic diameter of contrast and streptococcus acidi lactici fermented solution.
The result shows that faecium (Enterococcus faecium) LAB12 CGMCC No.4847 bacterium liquid all has restraining effect to indicator strain, especially to the pig source produce enterotoxin type intestinal bacteria (E.coli 0139), pig source pathogenic escherichia coli (E.coli C83524, E.coli C83529) restraining effect is obvious.
The experiment of feeding of the mouse of embodiment 6, faecium (Enterococcus faecium) LAB12 CGMCC No.4847
Preparation maconkey agar substratum: peptone 20g/L, lactose 10g/L, bovine bile 5g/L, sodium-chlor 5g/L, toluylene red 0.03g/L, agar 14g/L, pH value 7.0-7.2,121 ℃ of sterilization 30min.
Laboratory animal is selected regular grade Kunming system 40 of (KM) mouse (available from Hubei Province Preventive Medicine Academy's Center for Disease Control Experimental Animal Center) for use, and male, body weight 18-20g is divided into 4 groups at random, 10 every group.The faecium group is irritated stomach faecium (Enterococcus faecium) LAB12 CGMCC No.4847 bacterium liquid 0.2mL/d, and bacterial concentration is 10
8CFU/mL, control group irritate stomach MSC culture medium solution, continuous irrigation stomach 21d.Surveyed in per 5 days and organize stool in mice intestinal bacteria quantity, adopt to force method get the about 0.1g of stool in mice, add the 5mL diluent; And in the aseptic technique platform, add 3 granulated glass spherees; At vibrator vibration 3-5min, the vibration rotating speed is 250r/min, dilutes and is inoculated on the maconkey agar substratum.Calculate the wet coliform count in just of every gram.
The result shows that intestinal bacteria quantity is all the time 10 in the control group mice ight soil
7About CFU/g, change not significantly (P＞0.05), intestinal bacteria quantity drops to 10 gradually from the 6th day by the 21st day in the faecium group stool in mice
4About CFU/g (intestinal bacteria quantity reduces 99.9% in the stool in mice), (P＜0.05) changes noticeably.
The preparation of embodiment 7, faecium (Enterococcus faecium) LAB 12CGMCC No.4847 freeze-drying microbial inoculum and quality guaranteed period are detected
1) preparation MC liquid nutrient medium: glucose 5-10g/L, peptone 5-10g/L, pawpaw peptone 5-10g/L, yeast extract 5-10g/L, dibasic ammonium citrate 1-5g/L, extractum carnis 5-10g/L, tween 80 1-5g/L, K
2HP0
41-5g/L, pH6.0 ± 0.1,115 ℃, sterilization 20min.
2) preparation frozen solution: trehalose 10-100g/L, skimmed milk powder 10-200g/L, sucrose 10-200g/L, glycerine 10-100g/L, 115 ℃, sterilization 20min.
3) faecium (Enterococcus faecium) LAB12CGMCC No.4847 is inoculated in the MC liquid nutrient medium, leaves standstill under 37 ± 2 ℃ and cultivate 24 hours (12-36 hour all can).
4) after cultivation finishes; The centrifugal 5min of 5000r/min collects thalline; Behind twice of sterile distilled water cleaning thalline, add frozen solution, the volume ratio 1 of thalline and frozen solution: 10-1: 100; Repeated oscillation; Frozen solution and thalline are mixed, and then, the bacterium liquid that will the add cryoprotectant capacity of packing into is in the freeze-drying bottle of 10mL; Every bottled 2-5mL is in-40 ± 2 ℃ of pre-freezes 4 hours (3-5 hour all can).After freezing fully, make and freeze the article temperature, open vacuum pump extraction freezing article moisture simultaneously maintenance below 0 ℃ 36-48 hour.Wait to freeze article moisture content (freeze the article color and gradually become oyster white) when draining; Elevated temperature to room temperature (10 ℃-30 ℃) gradually; To discharge remaining moisture; Avoid freeze-dried products to absorb airborne moisture; The goods moisture content is controlled in 0.5%-5% (quality percentage composition) scope after the freeze-drying; Freeze-drying seals after finishing, and obtains faecium (Enterococcus faecium) LAB12 CGMCC No.4847 freeze-drying microbial inoculum.
5) freeze-drying microbial inoculum preservation effect and quality guaranteed period are detected:
Method: faecium (Enterococcus faecium) the LAB12 CGMCC No.4847 freeze-drying microbial inoculum of processing is preserved under the normal temperature and pressure state, after five months, measured viable count.The live bacterial count substratum is the YPD solid medium: glucose 20g, Tryptones 20g, yeast powder 10g, agar powder 18g, water 1000mL transfers 7.0,115 ℃ of sterilizations of pH 20min.
The result: after preserving 5 months under the normal temperature and pressure state, faecium (Enterococcus faecium) LAB12CGMCC No.4847 freeze-drying microbial inoculum viable bacteria survival rate is respectively 68.5%.The freeze-drying microbial inoculum preservation effect that the inventive method preparation is described is better, and the shelf time is longer.
Embodiment 8, clostridium butylicum (Clostridium butyricum) the B1 CGMCC No.4845 weanling pig effect analysis of feeding
With the weanling pig effect analysis of feeding of faecium (Enterococcus faecium) the LAB12 CGMCC No.4847 freeze-drying microbial inoculum of embodiment 7 preparation; Method is: select 40 of the close weanling pigs of 21 age in days body weight, respectively handle different not remarkable (± 0.20kg) (P＞0.05) of the mesosome method of double differences through check.The piglet random number is divided into 4 groups, 10 every group (public mother at random).4 groups are respectively:
Blank group: basal diet;
Microbiotic group (positive control): basal diet+feeding antibiotic (Xin Meisu 120mg/kg, Quinocetone 75mg/kg, mass percent concentration);
Low dose group: (dosage is 2 * 10 to basal diet+low dosage faecium preparation
8CFU/kg);
Middle dose groups: basal diet+(dosage is 10 to middle dosage faecium preparation
9CFU/kg);
High dose group: (dosage is 5 * 10 to basal diet+high dosage faecium preparation
9CFU/kg).
Closed pig house is raised, and temperature is controlled at 20 ℃.Test pig free choice feeding, drinking-water.Carry out immunization by the pig farm conventional procedure.Trial period is 30d.
The result is as shown in table 3; Compare with the microbiotic positive controls, the microbial inoculum of processing with bacterial strain of the present invention can improve efficiency of feed utilization, promotes the growth (feedstuff-meat ratio reduces by 17.2%) of weanling pig; Reduce diarrhea of weaned piglets rate (diarrhea rate reduces by 83.9%), can play the antibiotic effect that replaces.
The feed effect of faecium (Enterococcus faecium) LAB12 CGMCC No.4847 of table 3 weanling pig
Claims (10)
1. faecium (Enterococcus ffaecium) LAB12 CGMCC No.4847.
2. faecium according to claim 1 (Enterococcus faecium) LAB12 CGMCCNo.4847 is characterized in that: faecium (Enterococcus faecium) LAB12 CGMCC No.4847 is a gram-positive cocci, no gemma; Well-grown on the MRS agar plate, 48h forms the big small circular bacterium colony of diameter 0.5-1mm, and bacterium colony rounding, smooth, protuberance are canescence and reveal an appearance; The amphimicrobian growth, growth temperature range: 10 ℃-45 ℃, optimum growth temperature: 30 ℃-40 ℃; Growth potential of hydrogen scope: pH 4-10, optimum pH is 6.0.
3. faecium according to claim 1 (Enterococcus faecium) LAB12 CGMCCNo.4847 is characterized in that: enterotoxin type intestinal bacteria E.coli0139 is produced in streptococcus aureus (SA), salmonella typhi (ST 04Hi), pig source to faecium (Enterococcus faecium) LAB12CGMCC No.4847 and pig source pathogenic escherichia coli (E.coli C83524, E.coli C83529) all has stronger restraining effect.
4. faecium according to claim 1 (Enterococcus faecium) LAB12 CGMCCNo.4847 is characterized in that: faecium (Enterococcus faecium) LAB12CGMCC No.4847 can tolerate pH 2.5 synthetic gastric juice 180min; The highest tolerance gallbladder salinity is 3.0% (mass percent concentration).
5. faecium according to claim 1 (Enterococcus faecium) LAB12 CGMCCNo.4847; It is characterized in that: faecium (Enterococcus faecium) LAB12 CGMCC No.4847 is insensitive to fortum, cephalosporin, Prostaphlin, kantlex, cephalo oxime, gentamicin and Amikacin Sulphate, and is responsive to Pyocianil, cephazolin I, cephazolin, penbritin, Ceftriaxone, mould of happiness, penicillin, Cefoperazone, tsiklomitsin, erythromycin, Xin Meisu, doxycycline and Pipril.
6. faecium according to claim 1 (Enterococcus faecium) LAB12 CGMCCNo.4847; It is characterized in that: behind the faecium of feeding (Enterococcus faecium) the LAB12 CGMCC No.4847, intestinal bacteria quantity descend gradually (P＜0.05) in the animal intestinal.
7. a faecium (Enterococcus faecium) LAB12 CGMCC No.4847 freeze-drying microbial inoculum, its activeconstituents is faecium (Enterococcus faecium) LAB12 CGMCC No.4847.
8. the preparation method of the said faecium of claim 7 (Enterococcus faecium) LAB12CGMCC No.4847 freeze-drying microbial inoculum may further comprise the steps:
1) preparation MC liquid nutrient medium, it is subsequent use to sterilize;
2) preparation frozen solution: trehalose 10-100g/L, skimmed milk powder 10-200g/L, sucrose 10-200g/L, glycerine 10-100g/L, it is subsequent use to sterilize;
3) faecium (Enterococcus faecium) LAB12 CGMCC No.4847 is inoculated in the MC liquid nutrient medium, leaves standstill under 37 ± 2 ℃ and cultivated 12-36 hour;
4) after cultivation finishes, collect thalline, behind the cleaning thalline; Add frozen solution, the volume ratio 1 of thalline and frozen solution: 10-1: 100, repeated oscillation; Frozen solution and thalline are mixed, and the bacterium liquid that will add cryoprotectant was packed in the freeze-drying bottle, at-40 ± 2 ℃ of pre-freeze 3-5 hours; After freezing fully, make and freeze the article temperature, open vacuum pump extraction freezing moisture in the article simultaneously maintenance below 0 ℃ 36-48 hour; Wait to freeze article moisture content when draining; Elevated temperature to room temperature is to discharge remaining moisture gradually; Avoid freeze-dried products to absorb airborne moisture; The goods moisture content is controlled in 0.5%-5% (quality percentage composition) scope after the freeze-drying; Freeze-drying seals after finishing, and obtains faecium (Enterococcus faecium) LAB 12CGMCC No.4847 freeze-drying microbial inoculum.
9. preparation method according to claim 8 is characterized in that: the MC liquid culture based formulas in the said step 1) is: glucose 5-10g/L, peptone 5-10g/L, pawpaw peptone 5-10g/L, yeast extract 5-10g/L, dibasic ammonium citrate 1-5g/L, extractum carnis 5-10g/L, tween 80 1-5g/L, K
2HPO
41-5g/L, pH6.0 ± 0.1.
10. claim 8 or 9 described faeciums (Enterococcus faecium) the LAB12 CGMCC No.4847 freeze-drying microbial inoculum application in preparation fodder additives or veterinary drug, the instead microbiotic.
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