CN102747003B - Screening and application of probiotic Enterococcus faecium - Google Patents
Screening and application of probiotic Enterococcus faecium Download PDFInfo
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Abstract
The invention belongs to the technical field of veterinary microbial additive preparation, and specifically relates to a strain of separated and screened Enterococcus faecium providing significant bacterial inhibition effects for common enteropathogenic bacteria such as staphylococcus aureus, escherichia coli and salmonella in breeding animals, and an application thereof. The probiotic Enterococcus faecium of the present invention is characterized in that: the strain is Enterococcus faecium HDRsEf1, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CCTCC NO:M2011031. The probiotic Enterococcus faecium of the present invention has characteristics of fast growth, high acid production capacity, strong stress resistance, safety, disease resistance and growth promotion, and can be used as the microbial feed additive for livestock and poultry feeds.
Description
Technical field
The invention belongs to microbe additive technical field for animals, relevant with the Antibiotic Additive field, the present invention relates to a kind of isolation identification, safety evaluation, anti-adversity and prebiotic Performance Testing of faecium (Enterococcus faecium) bacterial strain that common pathogen enterobacteria such as streptococcus aureus, intestinal bacteria, Salmonellas is had an obvious fungistatic effect and as the purposes of fodder additives.
Background technology
From nineteen forty-six Moore reported first is added microbiotic and can obviously be improved the day weight gain of broiler chicken in feed since, successively had more than 60 to plant microbiotic and be applied to livestock industry, at animal diseases control, improve the aspect such as livestock and poultry production performance and brought into play vital role.But along with antibiotic a large amount of uses, unscientific abuse particularly, cause the problem such as a large amount of generations, the imbalance of animal normal microflora, livestock product drug residue of Resistant strain day by day to highlight, the serious harm aquaculture develops in a healthy way, affect human food prods's safety, cause countries in the world government and insider's great attention.Under this background, probiotic bacterium is nontoxic with it, noresidue, without drug-fast characteristics, be considered to antibiotic best substitute in large-scale cultivation (Shanahan et al, 2000; Park et al., 2002).2008, the Ministry of Agriculture issued bulletin No. 1126, the kind of " fodder additives kind catalogue " clear microorganism fodder additive, and faecium is among them.
Although faecalis is widely used in food, medicine and feedstuff industry, but its use as probiotic bacterium is controversial topic always, the core of dispute is: the gene of antibiotics resistance gene or coding virulence factor may be transferred to GI other microorganism (Franz et al, 2003).Therefore, in screening during prebiotic faecalis, except will be to the first row filter of its probiotic properties, also must carry out virulence factor and antibiotics resistance detects to bacterial strain, to guarantee the security of bacterial strain.The present invention when bacterium, investigates detection to bacterial strain probiotic properties, virulence factor and antibiotics resistance at home first simultaneously, also considers in addition the anti-adversity of bacterium source animal varieties.Such Research Thinking is laid a good foundation for finally obtaining strain excellent.
Summary of the invention
The objective of the invention is the defect for prior art, separation screening one strain is safety but also the probiotic bacterium of obvious probiotic properties is arranged not only, this bacterial strain has obvious fungistatic effect to common pathogen enterobacteria such as streptococcus aureus, intestinal bacteria, Salmonellas, through identifying that separating this probiotic strain that obtains is faecium (Enterococcus faecium), 16SrDNA identifies to its microorganism mycology characteristic use, to its security, resistance and prebiotic property and as the purposes of fodder additives, carried out correlation test.
Technical scheme of the present invention is as follows:
Physio-biochemical characteristics and genetics characteristic according to screening target and probiotic bacterium, Physiology and biochemistry means and modern molecular biology method with classics, from the rectal contents of the Local Excellent kind " Tongcheng pig " of strong stress resistance, through a large amount of sortings, separation screening obtains an Enterococcus faecalis bacterial strain, the applicant is with its called after faecium (Enterococcus faecium) HDRsEf1, on January 24th, 2011, deliver Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province, its preserving number is CCTCC NO:M2011031.
The bacteria characteristic of faecium (Enterococcus faecium) HDRsEf1 is as described below:
This bacterial strain is Gram-positive, be singly, become oval coccus two or that short chain is arranged.Be red on KF suis nutrient agar, smooth bumps, periphery is neat, the bacterium colony of 1mm left and right size, periphery of bacterial colonies substratum color becomes yellow by purple.Peroxidase is negative.Bacterial strain can tolerate the growing environment of 6.5%NaCl, pH9.6; Can be positive in 45 ℃ of growths, 60 ℃ of tolerances 30min, bile-Vitamin C2 hydrolysis experiments, can be hydrolyzed pectinose, illustrate that this bacterial strain belongs to enterococcus spp faecium kind; Its 16S rRNA gene is carried out cloning and sequencing, and result is carried out Blastn relatively at NCBI, finds that the sequence homology of its 16S rRNA and faecium (AB362603.1) is the highest, and similarity is 99%; In addition, amplified faecium specific fragment ddl from this bacterium genome
E.faecium
The viability of this bacterial strain in acid and high cholate environment is strong, can tolerate 0.5% cholate, and after pH2.5 was hatched 2h, its viable count did not almost descend; In addition, this bacterial strain has obvious adhesivity (p＜0.05) to Saliva Orthana.Infer thus, this bacterial strain can be resisted the disadvantageous effect of hydrochloric acid in gastric juice and small intestine middle and high concentration cholate, adheres to the enteron aisle performance prebiotic effect of surviving, and this also is confirmed in animal experiment.
This bacterial strain fecundity is very strong, and without lag phase, (0h～4h), enter stationary phase after 4h, be continued until 18h, and it is maximum that bacterial count reaches, and is 3.50 * 10 to enter very soon increased logarithmic phase
9CFU/mL, enter the paracme after 18h.
Although this bacterial strain does not produce the brood cell, relatively strong to the tolerance of high temperature, can tolerate 60 ℃, 30min; 65 ℃, the 10min survival rate is greater than 97%; This is applied to produce the actual good basis of having established for what this bacterial strain was made fodder additives.
This bacterial strain all has obvious bacteriostatic activity to common pathogen enterobacteria (golden yellow grape grape coccus, intestinal bacteria, Salmonellas).Use PCR method, estimated the security of this bacterial strain, find that this bacterial strain only contains efaA
fm1 virulence gene.To commonly using veterinary drug such as Ampicillin Trihydrate, vancomycin, tsiklomitsin, norfloxicin, furadantin, paraxin sensitivity, and to penicillin, erythromycin performance resistance; To the medium resistance of Ciprofloxacin performance.
Applicant's application is made faecium HDRsEf1 bacterial strain of the present invention by liquid fermenting and solid fermentation dual mode probiotics, as fodder additives, carried out the microbiotic alternate test, weanling pig is had and significantly controls in advance diarrhoea, promotion appetite, promote the effect of growth, produce a desired effect, thereby completed task of the present invention.
Faecium of the present invention has following advantage:
(1) bacterium source is in the good pig kind of the place of china pig of good stress resistance-be Hubei Province " Tongcheng pig "--the content of enteron aisle, Isolation and screening obtains from a large amount of candidate strain, this bacterial strain is named as faecium HDRsEf1, it has obvious fungistatic effect to common pathogen enterobacteria (streptococcus aureus, intestinal bacteria, Salmonellas), for the substitute antibiotics additive provides strong foundation.
(2) bacterial strain has been carried out the security elutriation with molecular biological method, so security is higher.
(3) this bacterial strain can tolerate hydrochloric acid in gastric juice and enteron aisle cholate high osmotic pressure environment fully, and has adhesive capacity, therefore can be at enteron aisle field planting performance prebiotic effect.
(4) although this bacterial strain does not produce the brood cell, relatively strong to the tolerance of high temperature, can tolerate 60 ℃, 30min; 65 ℃, the 10min survival rate is greater than 97%; 62 ℃, 55% survival in 30 seconds granulation feeds.90 ℃, 30% survival in 30 seconds granulation feeds; 80 ℃, 40% survival in 30 seconds granulation feeds.This is applied to produce the actual good basis of having laid for what this bacterial strain was made fodder additives.
(5) animal experiment proves, alternative microbiotic is made additive for farm animal feed and used, and production and use are all very simple, not only can improve livestock and poultry production performance, have more environmental friendliness, to person poultry safety's advantage.
Be prepared as probiotics preparation, substitute the Antibiotic Additive in pig feed, pig industry is played the prophyiaxis and promoting growth effect, greatly cut down the destruction on environment that the microbiotic in pig feed causes and the impact of human body health, with the generation of advancing to reduce resistance, with harmony, the Sustainable development that realizes livestock industry, the mankind and environment.
More detailed technical scheme is seen the content of " specific embodiments ".
Description of drawings
Sequence table SEQ ID NO:1 is the 16S rDNA Gene Partial sequence of strain isolated faecium strain HDRsEf1 of the present invention.
Fig. 1: the growth conditions of faecium strain on KF suis agar plate that is separation screening of the present invention.
Figure 1A is blank KF suis agar plate; Figure 1B. be that the faecium strain grows up to red bacterium colony on KF suis agar plate, the substratum of periphery of bacterial colonies becomes redness.
Fig. 2: the faecium strain Gram-positive reaction (* 1000) that is separation screening of the present invention
Fig. 3: 16S rRNA gene PCR augmentation detection result.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1: negative control; Swimming lane 2: bacterial strain of the present invention; Swimming lane 3:HB6 faecalis strain isolated; Swimming lane 4:TC12 faecalis strain isolated; Swimming lane 5:TC13 faecalis strain isolated
The species specificity gene ddl of Fig. 4 faecium
E.faeciumThe pcr amplification detected result.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1: faecium HDRsEf1 strain.
Fig. 5 is the In Vitro Bacteriostasis test-results of strain fermentation supernatant liquor of the present invention to common pathogen enterobacteria.
Fig. 5 A: to the In Vitro Bacteriostasis test of streptococcus aureus;
Fig. 5 B: to colibacillary In Vitro Bacteriostasis test;
Fig. 5 C: to the In Vitro Bacteriostasis test of Salmonellas.
Fig. 6: faecium HDRsEf1 virulence factor efaA
fmThe PCR detected result.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1: faecium HDRsEf1.
Fig. 7: the growth curve that is faecium TC3.
The PCR product agarose gel electrophoresis figure of Fig. 8: cylA.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1:HB6 faecalis strain isolated; Swimming lane 2:TC12 faecalis strain isolated; Swimming lane 3:TC13 faecalis strain isolated.
The PCR product agarose gel electrophoresis figure of Fig. 9: esp.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1:HB6 faecalis strain isolated; Swimming lane 2:TC12 faecalis strain isolated; Swimming lane 3:TC13 faecalis strain isolated.
The PCR product agarose gel electrophoresis figure of Figure 10: agg.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1:TC12 faecalis strain isolated.
The PCR product agarose gel electrophoresis figure of Figure 11: efaAfs.
Each swimming lane in figure: M:DL2000DNA molecular weight standard; Swimming lane 1:HB6 faecalis strain isolated; Swimming lane 2:TC12 faecalis strain isolated; Swimming lane 3:TC13 faecalis strain isolated.
Specific embodiments
Embodiment 1: the separation of probiotic strain and evaluation
One, the separation of bacterial strain
Sample is taken from 100 ages in days, 21 age in days place of china pig variety Hubei " Tongcheng pig " (cultivar origin is in conservation field, Tongcheng County, Hubei Province), gathers the rectal contents of pig from anus with the disinfecting silk or cotton swab.After sample collecting, streak culture in KF suis nutrient agar (available from Hangzhou microorganism reagent company limited) with cotton swab, 37 ℃, 24h～48h chooses redness after cultivating, smooth bumps, and periphery is neat, and the bacterium colony of 1mm left and right size is done pure culture.Pure growth is carried out preliminary screening through observation, gramstaining, the peroxidase test of colonial morphology.The oval gram positive coccus that bacterium is is single, it is two to become or short chain is arranged and the bacterial strain of peroxidase test feminine gender, the preservation of all going down to posterity.The growth characteristics of the bacterial strain of this invention on KF suis nutrient agar are seen Fig. 1, and its gramstaining characteristic is seen Fig. 2.
Two, the evaluation that belongs to
With the bacterial strain of above-mentioned separation (hereinafter to be referred as strain isolated) carry out 6.5%NaCl growth test, pH9.6 meat soup growth test, 45 ℃ of growth tests, 60 ℃, 30min growth test, bile-Vitamin C2 hydrolysis experiment.The bacterial strain that can tolerate 6.5%NaCl, growth in pH9.6 meat soup, 45 ℃ can survives, 60 ℃ of effect 30min can be survived, bile-Vitamin C2 is hydrolyzed the positive namely is decided to be the enterococcus spp bacterium.Concrete steps are:
(1) anti-6.5%NaCl test is got children's strain isolated in age and is seeded in TSB substratum (available from the U.S. company BD) test tube that contains 6.5%NaCl of suitable growth, cultivates 24h for 37 ℃, with nonvaccinated control tube contrast, range estimation growing state.Muddiness is namely positive, otherwise negative.
(2) pH9.6 meat soup growth test is inoculated in strain isolated in the pH9.6TSB substratum, after 37 ℃ of cultivation 24h, and observations.It is muddy positive that substratum becomes, otherwise negative.
(3) 45 ℃ of growth tests, with the transfering loop liquid culture that a ring 24h cultivates of slinging, accesss in limpid BHI meat soup, and 45 ℃ of waters bath with thermostatic control are cultivated, and it is positive that substratum becomes muddiness, otherwise negative.Repeat this test 3 times, come to the same thing and just confirm.
(4) 60 ℃, 30min growth test picking children bacterium colony in age, be inoculated in the TSA inclined-plane, after 60 ℃ of cultivation 30min, changes 37 ℃ over to and cultivate 24h, has colony growth to be judged to the positive.
(5) bile Vitamin C2 hydrolysis experiment is inoculated in strain isolated in the bile esculin medium, 37 ℃ hatch 18-24h after, observations.The complete blackening of substratum is positive, and not blackening is negative.
Through above-mentioned enterococcus spp test, screen 4 strain faecalis bacterial strains, the applicant is with its difference called after HB6, TC12, TC13, HDRsEf1.
Three, strain isolated is carried out the preliminary evaluation of planting
To the 4 strain faecalis bacterial strain HB6 that filter out, TC12, TC13, HDRsEf1 carries out extracorporeal bacteria inhibitor test,, to the obvious bacterial strain of fungistatic effect,, by a series of biochemical test, further carries out the evaluation of planting.Comprise: glucose, wood sugar, sucrose, lactose, raffinose, sorbyl alcohol, N.F,USP MANNITOL, pectinose, arginine dihydrolase.With qualification result (in Table 1) with " common bacteria system identification handbook contrasts about the description of differentiating between the enterococcus spp kind in (eastern elegant pearl etc., 2001), determines that tentatively strain isolated HDRsEf1 is faecium.
Table 1 faecium strain HDRsEf1 biochemical identification result
Five, the conclusive evidence of strain isolated kind
On the basis of above-mentioned evaluation, further to above-mentioned faecalis strain isolated HB6, TC12, TC13, HDRsEf1 carry out the 16SrRNA gene order and detect and the species specificity gene test, and the kind under bacterial strain is proved conclusively evaluation.
(1) faecalis strain isolated genome extraction step is as follows:
(1) get respectively 1mL strain isolated pure growth, add in 1.5mL EP pipe, the centrifugal 5min of room temperature 8000rpm, abandon supernatant, and the precipitation Eddy diffusion is in 1mL TE (pH8.0).
(2) add the N,O-Diacetylmuramidase of 6 μ L 50mg/mL, 37 ℃ of effect 2h.
(3) add again 2MNaCl 50 μ L, 10% sodium lauryl sulphate (being SDS), 110 μ L, the Proteinase K 3 μ L of 20mg/mL, 50 ℃ of effects 3h or 37 ℃ spend the night.
(4) bacterium liquid is all assigned to two 1.5mLEP pipes, added isopyknic phenol: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1), mix, room temperature is placed 5-10min; The centrifugal 10min of 12000rpm; So repeat twice of extracting.
(5) add the Virahol of 0.6 times of volume, mix, room temperature is placed 10min.The centrifugal 10min of 12000rpm.
(6) precipitate with 75% washing with alcohol.
(7) after air-dry, be dissolved in 50 μ L ddH
2In O, add 1 μ L 10mg/mL RNase A, 37 ℃ of digestion 2-3h.
(8) get 2-5 μ L electrophoresis detection.Be stored in-20 ℃ standby, be designated hereinafter simply as " faecalis genome ".
(2) design of primers of amplification 16S rRNA gene:
With reference to the faecalis 16S rRNA gene order (gene accession number AB362603.1) of having delivered, application Primer 5.0 analysis software design primers, primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, and the DNA sequence dna of primer is as described below:
Forward primer F:5 '-CGT GCC TAA TAC ATG CAA GTC GAA C-3 ',
Reverse primer R:5 '-ACG ACT TCA CCC CAA TCA TCT ATC C-3 '.
(3) pcr amplification 16S rRNA gene
With the above-mentioned primer pair genomic 16S rRNA of the above-mentioned faecalis strain isolated gene that increases respectively.Reaction system is in Table 2.The PCR program is: 94 ℃ of 5min, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 30 rear 72 ℃ of extension 10min of circulation.Get the PCR product and carry out electrophoresis detection on 0.8% sepharose (containing ethidium bromide), the clip size of amplification conforms to expection, totally 1475 base pairs (seeing Fig. 3).
Table 2 strain HD RsEf16S rRNA PCR system
(4) clone and the order-checking of 16S rRNA gene PCR product
Reclaim DNA with DNA purification kit (available from TIANGEN company), concrete steps are with reference to the specification sheets of this test kit.Be connected to pDM18-T carrier (Pu Luomaige (Beijing) Bioisystech Co., Ltd after the PCR product purification, be U.S. Promega company), transform DH5 α competent cell, be applied to the enterprising row filter of agar plate that contains ampicillin (Ampicillin, Amp) 50 μ g/mL.The picking white colony is in the LB liquid nutrient medium that contains Amp (50 μ g/mL), and 37 ℃ of constant-temperature tables spend the night, and directly gets bacterium liquid and does template and carry out PCR and identify., to the bacterium liquid of positive colony, deliver to the order-checking of Jin Site Science and Technology Ltd..Sequencing result is carried out Blast in ncbi database relatively.Retrieval is found, sequence (AB362603.1) homology of the 16SrRNA of the HDRsEf1 bacterial strain that the present invention separates and the faecium of report is the highest, similarity is 99%, and specifically referring to SEQ ID NO:1: the present invention separates the 16S rRNA Gene Partial sequence of manure enterococcin strain HDRsEf1.
(5) species specificity gene identification
The species specificity fragment of faecium is ddl
E.faecium(Dutka-Malen et al., 1995), fragment designs primer accordingly, take enterococcal genome as template, pcr amplification faecium species specificity fragment.
Amplification ddl
E.faeciumThe DNA sequence dna of fragment primer pair is as follows:
Forward primer F1:5 '-GCA AGG CTT CTT AGA-GA-3 '
Reverse primer F2:5 '-CAT CGT GTA AGC TAA CTT C-3 '
The PCR program is: 94 ℃ of 5min, 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 1min, 30 rear 72 ℃ of extension 10min of circulation.
From the 4 strain faecalis strain isolated HB6 that detect, TC12, TC13, in HDRsEf1, only amplified the specific fragment ddlE.faecium (seeing Fig. 4) of faecium from strain HD RsEf1 of the present invention, proved that further strain isolated HDRsEf1 is faecium (Enterococcus faecium).
Embodiment 2: the bacteriostasis property test of manure enterococcin strain HDRsEf1
With streptococcus aureus (ATCC25923, available from Ministry of Health's visiting center), Salmonella choleraesuls C78-1 (available from China Veterinery Drug Inspection Office) and pig pathogenic colon bacillus O138:K88 (C83902, available from China Veterinery Drug Inspection Office) be indicator, take HDRsEf1 strain fermentation supernatant of the present invention as fungistat, detect the In Vitro Bacteriostasis performance of manure enterococcin strain HDRsEf1, concrete operations are as follows:
1) preparation of ferment product and processing: fresh faecium HDRsEf1 bacterium liquid is inoculated in MRS broth culture (U.S. company BD product) by 2% (v/v), standing cultivation 18～24h, the centrifuging and taking supernatant liquor, 10000rpm, 30min are centrifugal, draw supernatant; Once centrifugal again; And measure each fermented liquid pH; 4 ℃ standby, and the viable count of this liquid microbe starter is 3.5 * 10
9CFU/mL.
2) the about 25mL (thickness is about 4mm) of the every flat-plate inverted LB of the preparation of LB flat board nutrient agar (U.S. company BD product), make plate dry before test, take indicator evenly after coating without visible water droplet as standard.
3) the indicator bacterium solution preparation is streak culture respectively with indicator, obtains single bacterium colony; Picking list bacterium colony is in LB liquid nutrient medium (U.S. company BD product), and 37 ℃ of shaking table shaking culture 12h are used for this test.
4) dilution of indicator bacterium liquid is 10 with Maxwell opacity tube adjustment indicator bacterial concentration
8CFU/mL; Again with bacterium liquid dilution 10
7CFU/mL.Bacterium liquid after dilution is evenly coated on the LB flat board with sterilizing cotton swab.
5) after the agar surface moisture drying, with tweezers, aseptic Oxford cup is put into culture dish gently, draw fermented supernatant fluid 0.25mL and (note the bacterium hydrorrhea not being gone out outside cup) to the cup of Oxford.
6) plate is placed 4 ℃ of refrigerator diffusion numbers hour, changes in 37 ℃ of incubators.Viewing test result and measure the size of inhibition zone after 10-12h.
Test-results shows that fungistatic effect effect in the bacterial strain that is compared of strain HD RsEf1 of the present invention is best, the results are shown in Figure 5 and table 3.
Table 3 faecium strain HDRsEf1 fermented supernatant fluid extracorporeal bacteria inhibitor test
Embodiment 3: the safety evaluation of faecium strain
One, the amplification of virulence factor
The amplification of virulence factor mainly comprises cylA (accession number: AAA62652.1; Gilmore et al., 1994), gelE (accession number: D85392; Mannu et al., 2003), esp (accession number: AF034779; Shankar et al., 1999), agg (without accession number: Galli et al., 1990), ace (accession number: AF26083; Mannu et al., 2003), efaAfs (accession number: EFU03756) and efaA
fm(accession number: FJ609170.1; Mannu et al., 2003).
Take the faecalis genome of aforementioned preparation as template, special primer pair with each virulence factor carries out pcr amplification, each primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, and its sequence, PCR product size, amplification condition and reference are in Table 4, and virulence gene pcr amplification system is in Table 5.
Table 4 test primer sequence, PCR product size and amplification condition
Table 5 virulence gene pcr amplification system
Get the PCR product and carry out electrophoresis on 0.8% sepharose (containing ethidium bromide), observe in gel imaging system and take pictures.Electrophoresis result shows: at 4 strain faecalis HB6, and TC12, TC13, in HDRsEf1, coamplification has gone out 5 kinds of virulence gene cylA, esp, agg, efaAfs and efaA
fm, amplification clip size out is all consistent with expection, and gelE, ace all do not increase out.HB6 has 3 virulence genes, is cylA
+esp
+EfaA
fs +TC12 has 4 virulence genes, is agg
+CylA
+esp
+EfaA
fs +TC13 has 3 virulence genes, is cylA
+esp
+EfaA
fs +Faecium HDRsEf1 of the present invention only contains 1 virulence factor efaA
fm +, faecium HDRsEf1 virulence factor efaA
fmThe PCR detected result is seen Fig. 6, other 3 strain faecalis HB6, and TC12, the TC13PCR detected result is seen Fig. 8～Figure 11.Need to prove that at present all there is efaA in the business faecium of selling on the market of detecting with PCR method
fmVirulence factor (Kieun et al., 2008), and the actual result of using shows that the existence of this virulence factor does not show toxic side effect to people and animals, also is proven in this embodiment at this specification sheets 5.
Two, hemolytic test and gelatin hydrolysis test
Faecium strain HDRsEf1 is seeded on 5% defiber rabbit blood LB flat board, cultivates 18～24h for 37 ℃, observe the haemolysis situation, occur that the transparent beta zone of hemolysis is that the haemolysis phenotype is positive.Streptococcus aureus (ATCC25923) is cooked positive control.
Bacterium is inoculated in the BHI agar plate that contains 10g/L peptone and 30g/L gelatin, after 37 ℃ of cultivation 18～24h, then flat board is put in 4 ℃, 5h, the dizzy namely positive of muddiness appears in periphery of bacterial colonies.(do positive control with Riemerlla anatipestifer Yunmeng strain isolated.
Faecium strain HDRsEf1 does not show hemolytic reaction on 5% defiber rabbit blood agar, the gelatin hydrolysis test is also negative.
Three, antibiotic susceptibility test
Choose 9 kinds of drug sensitive test papers such as penicillin, Ampicillin Trihydrate, vancomycin, erythromycin, tsiklomitsin, Ciprofloxacin, norfloxicin, furadantin, paraxin and carry out drug sensitive test from (Hangzhou microorganism reagent company limited).With M-H substratum (available from Britain BD company), standard sensitive strain streptococcus aureus ATCC25923 is as the Quality Control bacterium, and the test judging criterion is carried out (2009 editions) with reference to the standard of the NCCLS latest edition that WHO provides.Testing sequence is as follows:
(1), with transfering loop picking faecalis bacterium colony on the MRS agar plate, be inoculated in 3～5mLMRS broth culture, be put in 37 ℃ of standing cultivations of incubator;
(2) cultivate 2～8h,, take blank sheet of paper that surplus is arranged as background, adjust turbidity to 0.5 Maxwell standard opacity tube turbidity.When if bacterial concentration is too dense, available meat soup or normal saline dilution (the bacterium liquid of correction will use in 15min);
(3) dip bacterium liquid with aseptic cotton carrier, and coat on the M-H agar plate squeezing on tube wall after removing unnecessary bacterium liquid, at every turn with flat board rotation 60 degree, last along periphery around Liang Quan, repeatedly several times, guarantee to be coated with evenly;
(4) after the moisture on flat board is absorbed fully by agar, be attached to planar surface with the aseptic nipper quick scraps of paper of getting it filled, the scraps of paper can not be picked up once pasting again.5 scraps of paper of each dull and stereotyped subsides, every scraps of paper spacing is no less than 24mm, and scraps of paper width between centers plate edge is no less than 15mm.
(5) stick in scraps of paper 15min, flat-plate inverted is placed in 37 ℃ of incubators; Cultivate 16～18h observations, survey vancomycin and need 24h.
Test-results is in Table 6, and faecium HDRsEf1 is to Ampicillin Trihydrate, vancomycin, tsiklomitsin, norfloxicin, furadantin, paraxin sensitivity; And to penicillin, erythromycin performance resistance; To the medium resistance of Ciprofloxacin performance.
The antibiotic susceptibility test result of table 6 faecium strain HDRsEf1
Above-mentioned virulence factor detection, hemolytic test and the results such as gelatin hydrolysis test and antibiotic susceptibility test, show that faecium strain HDRsEf1 is safe.
Embodiment 4: the adverse-resistant characteristic of faecium HDRsEf1 and growth characteristics
One, resistance test
(1) cholate tolerance test
With after 2 generations of HDRsEf1 activation, get 1mL and be inoculated in 9mL and contain 0%, 0.15%, in 0.3%, 0.5% cholate MRS meat soup, cultivate 12h for 37 ℃, nutrient solution is done respectively continuous 10 times of dilutions, getting 0.1mL from each extent of dilution, to be applied to MRS flat
The tolerance of table 7 faecium HDRsEf1 to cholate
Plate, observe growing state and carry out enumeration.Result shows that HDRsEf1 can tolerate 0.15%, 0.3%, 0.5% gallbladder salinity (in Table 7), and its viable count all reaches 10
7More than CFU/mL.In Small Intestine of Piglets, the content of cholate fluctuates in 0.03%～0.3% scope, and therefore, the HDRsEf1 bacterial strain can tolerate the environment of enteron aisle cholate.
(2) acid resistance test
The faecium HDRsEf1 of activation is inoculated respectively pH2.0 with 2% inoculum size, the hydrochloric acid soln of pH2.5 and pH6.8 distilled water, 37 ℃ of standing cultivations.Get inoculation 0h, 0.5h, 1h, 1.5h, the nutrient solution of 2h, do respectively continuous 10 times of dilutions, gets 0.1mL from each extent of dilution and be applied to the MRS flat board, observes growing state and carry out enumeration.
The tolerance of table 8 faecium strain HDRsEf1 to different pH values
The results are shown in Table 8, HDRsEf1 after pH2.5 is hatched 2h, viable count is 4.10 * 10
7CFU/mL, compare with control group, descends very little; After pH2.0 was hatched 2h, the bacterial count order of magnitude that only descended, be 2.20 * 10
6CFU/mL.
During the piglet birth, in stomach, the pH value is generally 5～6, and rear field planting because of milk-acid bacteria makes the pH value drop to gradually 4 left and right, and before 2 monthly ages, the pH value remains on 3 left and right.Usually hydrochloric acid in gastric juice pH value is in 3.0 left and right, and the time that slop stops under one's belt is 1～2h, and after feed, in very short time, stomach inner pH value can rise to 6.0 left and right.When probiotic bacterium passed through gi tract, chyme reduced as a kind of protective material the degree that probiotic bacterium comes to harm, in case under surviving in passing through stomach and duodenum, the bacterium that enters ileum, caecum in company with chyme can sharply increase.Hence one can see that, and strain HD RsEf1 of the present invention is under low pH value gastric acid environment, and survival odds is very large, has abundant bacteria living to get off, and then arrives enteron aisle smoothly.
(3) high temperature tolerance test.
In MRS meat soup, the inoculation 2% HDRsEf1 bacterial strain that has activated, cultivate 3～4h under 37 ℃ of conditions.Bacterium liquid is through 60 ℃, 65 ℃ and 70 ℃ of thermal treatment 0min, 5min, and 10min, 15min, 20min, 25min, 37 ℃ are made as contrast, then bacterium liquid are done continuous 10 times of dilutions, with dull and stereotyped survey of MRS, contain the bacterium number.Contain the bacterium number with 1g
CFU/ mL represents, test-results shows faecium HDRsEf1 10min survival rate more than 97% (in Table 9) under 65 ℃ of conditions.
The tolerance of table 9 faecium HDRsEf1 to high temperature
Two, the mensuration of growth curve
In the inoculum size access MRS meat soup of faecium strain HDRsEf1 by 1% (v/v) that has activated, 37 ℃ of standing cultivations, carry out plate count every the 2h sampling; Take the time as X-coordinate, the logarithm of bacterium is that ordinate zou is drawn growth curve.
Table 10 faecium strain HDRsEf1 is at the growing state of different time
Result shows, the faecium strain HDRsEf1 growth that the present invention separates is without lag phase, and (0h～4h), enter stationary phase after 4h, be continued until 18h, and it is maximum that bacterial count reaches, and is 3.50 * 10 to enter very soon increased logarithmic phase
9CFU/mL.This bacterial strain fecundity is very strong, is conducive to industrialized large scale fermentation production, and its growth curve is seen Fig. 7 and table 10.
Three, adhesiveness test
Saliva Orthana Mucin (Mucin-HRP with horseradish peroxidase-labeled, Sigma company) make adhesiveness test, the bovine serum albumin of horseradish peroxidase-labeled (BSA-HRP) compares, with ELISA method (Repentigny L, 2000) measure faecium strain HDRsEf1 to mucinous adhesivity, result shows, its OD
450Can reach 0.062, compared significant difference (p＜0.05) (in Table 11) with control group, faecium and Saliva Orthana that the present invention separates have obvious adhesivity, and this is the field planting survival of faecium strain HDRsEf1 at enteron aisle, brings into play its prebiotic effect and lays a good foundation.
Table 11 faecium HDRsEf1 adheres to mucinous experimental result
Annotate: compared with the control
*: p＜0.05
The lower gastric acid environment of opposing pH and to the tolerance of bile, be the primary index of screening probiotic bacterium; when probiotic bacterium passes through gi tract; chyme reduces as the degree that a kind of protective material makes probiotic bacterium be subject to the acid injury; in case under survival, the bacterium that enters ileum, caecum in company with chyme can sharply increase after passing through stomach and duodenum.Faecium HDRsEf1 of the present invention is under very low pH value gastric acid environment, survival odds is very large, have abundant bacteria living to get off, and then arrive enteron aisle smoothly, and this bacterium makes it can surely grow smoothly finally and bring into play prebiotic effect in host's enteron aisle to the tolerance of enteron aisle cholate.
In addition, probiotic bacterium in concentrated or pelletization, all will carry out high-temperature heating treatment at bacterium liquid, therefore, the tolerance of high temperature is become another key index of selecting probiotic strain.Although faecium strain HDRsEf1 of the present invention can not produce the brood cell, can be under 65 ℃ of conditions the 10min survival rate more than 97%; This provides another favourable condition for faecium strain HDRsEf1 performance prebiotic effect.
Embodiment 5: Application Example of the present invention
One, sucking piglets, weanling pig feeding experiment
With the Antibiotic Additive in the alternative sucking pig material of faecium strain HDRsEf1, the host animal of this bacterial strain of feeding (pig), purpose is the interior probiotic properties of body of part checking bacterial strain of the present invention.
1. test materials and grouping
The two-way cross piglet (, from Hua Zhong Agriculture University's test pig farm, being conventional commodity Hybrid) of first getting landrace and Large White is experimental animal, raises 10 ± 2, every nest take nest as unit.On the same group be birth on the same day, on the same group piglet age in days does not differ 1-3 days.Used with feed and grouping situation in Table 12, wherein organize the 3 every grams of feed and contain faecium HDRsEf15 * 10
6CFU.Ai Li feed (creep feed and child care material) is produced and is provided by Wuhan Ai Li Animal nutrition company limited, contains in antibiotic feed and has added colistin 40ppm, kitasamycin 20ppm.
Table 12 test grouping
2. process of the test
Feeding experiment the time span Piglet Development lactation and two stages of wean, specifically since 7 ages in days, to 37 ages in days, finishes, can be divided into religion groove early stage: 7-22 age in days and teaching the groove later stage: two periods of 22-37 age in days.The duration of test animal is all fed in the free choice feeding mode, and it is as follows to carry out following vaccine inoculation:
10 ages in days: haemophilus parasuis deactivation vaccine (available from Wuhan Keqian Animal Biological Products Co., Ltd.'s product), mycoplasma deactivation vaccine (available from the product of Schering Plough company production) is exempted from;
15 ages in days: mycoplasma deactivation vaccine two is exempted from, circovurus type 2 deactivation vaccine (available from U.S. Pu Taike national defence company limited product);
20 ages in days: swine fever cell vaccine (available from Guangdong Yongsheng biological products company limited product) is exempted from.
3. testing index
Record feed actual consumption every day, calculate average feed consumption rate; On-test (7 age in days), mid-term (22 age in days) and all weigh on an empty stomach while finishing (37 age in days), calculate average daily gain; Calculate feed conversion rate (being feedstuff-meat ratio) according to feed intake and weight gain of piglets; The dead number of record also calculates mortality ratio; After off-test (i.e. 35 ages in days), in 20% ratio blood sampling, with the antibody horizontal after corresponding ELISA antibody assay kit detection swine fever, circovirus-II vaccine immunity, two kinds of all test kits are produced by Wuhan Keqian Animal Biological Products Co., Ltd..
4 results
The data presentation of table 13, the difference of each group of test initial weight is not remarkable; After lactation and teaching groove to raise early stage, mean body weight from high to low: group 3＞group 1＞group 2, the result that later stage religion groove is raised: group 3＞group 1＞group 2, the mean body weight of group 3 and group 1 is significantly higher than group 2.
In two stages, religion groove early stage, a head all day weight gain is arranged from high to low: group 3＞group 1＞group 2, and group 3 is significantly higher than group 2, and group 1 and group 2 differences are not remarkable.The religion groove later stage, group 3＞group 1＞group 2, group 3 and group 1 all are significantly higher than group 2.
From the beginning all daily ingestion amount is seen, in earlier stage, group 3＞group 1＞group 2, wherein organize 3 and exceed an order of magnitude than other two groups the religion groove.The religion groove later stage, group 3＞group 1＞group 2, group 3 utmost points are significantly higher than other two groups.Material/meat is than aspect, and due to lactation period, the weightening finish of piglet is relevant with what with breast milk nutrition, and the feed of searching for food seldom, is not therefore relatively taught the groove feedstuff-meat ratio in early stage.After wean, weight gain of piglets, fully by the feed of searching for food, organize 3＞group 2＞group 1, but it is not remarkable respectively to organize difference; The feedstuff-meat ratio of group 1 is minimum, and it is the highest to organize 3 feedstuff-meat ratios.
Duration of test, have piglet to occur dead, and the statistics mortality ratio is followed successively by group 1＞group 3＞group 2.
Table 13 piglet growth performance relatively
Antibody horizontal after table 14 swine fever and circovirus-II vaccine immunity
The result demonstration of table 14, the hog cholera antibody level is followed successively by group 3＞group 1＞group 2 from high to low, but it is not remarkable respectively to organize difference; The circovirus 2 type antibody level is followed successively by group 3＞group 1＞group 2 from high to low.The level that can find out 3 two kinds of antibody of group is all the highest.
The hog cholera antibody positive rate is group 3＞group 2＞group 1; The circovirus 2 type antibody positive rate is group 3＞group 1=group 2.Result shows: the hog cholera antibody positive rate 100% of group 3, the positive rate of group 3 is higher than group 1 and group 2., although the circovirus 2 type antibody positive rate is low, organize 3 positive rate still higher than other each groups.
Test is since 7 age in days religion grooves, and 22 age in days wean, last till 37 ages in days.From the statistics of growth performance, group 3 food consumptions all are significantly higher than other two groups, and its head all day weight gain also higher than other groups, but on feedstuff-meat ratio not as good as other groups.But for being in lactation and the child care piglet in early stage, food consumption and day weight gain seem most important, because the physical state of piglet is very crucial to fattening in the future, feedstuff-meat ratio is not but the emphasis of considering the period of growing seedlings.
The antibody test data show, use the group 3 of probiotics of the present invention, and antibody positive rate is all higher than two control groups.Especially organize 3 and all belong to the highest at two kinds of antibody horizontals and positive rate, as seen, faecium strain HDRsEf1 of the present invention can strengthen immune effect of vaccine, improves body protective power; And use antibiotic group 1, although can growth promotion, raising that can not the effective stimulus human body immune function.
In sum, add faecium strain HDRsEf1 of the present invention and can not only significantly improve the growth performance of piglet, can also improve the immunologic function of body.Be expected to apply in pig industry as a kind of microbiotic substitute technology, the addition that is recommended in the preparation of faecium strain HDRsEf1 of the present invention in piglet diet is 5 * 10
6CFU/g.
Two, broiler feeding test
1 104 of the yellow chickens of age in days three, be divided into two groups at random, and feed is Wuhan honest feed Group Co.,Ltd 803,813 types, and 40d feeds.The grouping situation is in Table 15, and wherein the every gram of test group feed contains faecium HDRsEf15 * 10
6CFU
Empty stomach when on-test and off-test is weighed, and calculates average daily gain; Calculate feedstuff-meat ratio according to feed intake and broiler growth.
Table 15 test grouping
The impact of table 16 faecium HDRsEf1 on the growth of meat chicken performance
Faecium HDRsEf1 on the impact of growth of meat chicken performance in Table 16, the test group feedstuff-meat ratio is 1.972, control group is 2.145, both significant differences (P＜0.05), show that in daily ration, interpolation faecium HDRsEf1 is remarkable for improving the effect of growth of meat chicken performance, the addition that is recommended in faecium strain HDRsEf1 preparation of the present invention in daily ration of broiler is 5 * 10
6CFU/g.
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Claims (1)
1. a separation screening has the manure enterococcin strain of obvious fungistatic effect to the streptococcus aureus of causing a disease, intestinal bacteria, Salmonellas, it is characterized in that, described manure enterococcin strain (Enterococcus faecium) HDRsEf1, be deposited in Chinese Typical Representative culture collection center (CCTCC), its preserving number is CCTCC NO:M2011031, this bacterial strain is high temperature tolerance type manure enterococcin strain, its 16S rDNA gene order is as shown in sequence table SEQ ID NO:1, and it also contains 1 virulence factor efaA
fm +Gene.
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