Embodiment
One, the screening of bacterial strain
1. screening process
Bacterium sample is gathered from the cud that the goat raised is butchered, the specified conditions of external employing 37 ± 5 DEG C, anaerobism, lucifuge carry out directive breeding, tamed to single strain L-01 by the separation and purification of repeatedly ruling of agar plate subregion, study its morphology and biological characteristics.Colony characteristics, electron microscope observation single strain form and related keyword enzyme activity determination is observed by agar plate culture.
The configuration of 1.1 working stocks:
First be equipped with nutritional substrate with following table ratio:
| Composition | Weight percent (%) |
| Corn | 80 |
| Dregs of beans | 6 |
| Wheat bran | 10 |
| Salt | 1 |
| Stone flour | 1 |
| Secondary calcium phosphate | 2 |
Nutritional substrate 125g(2600r/min pulverizing 1.5min is taken again in the ratio of 5%) mix with water, digestion 1h under electromagnetic oven boiling state, after two-layer gauze extracting twice, after extract 2600r/min liquid making beating 1.5min, 500 order nylon-tissue bag are filtered, finally be settled to 2.5L, obtain working stocks.
1.2 artificial medium compositions: the formula with reference to Russell is improved.
Reductive agent (matching while using): 1NNaOH solution 4mL; Na2s7H2o570mg adding distil water is to 100mL.
Major element: Na2hPO45.70g; NaH2pO46.20g; MgSO47H2o0.60g adding distil water is to 1000mL.
Trace element: CaCl22H2o13.20g; MnCl24H2O10.00g; CoCl26H2o1.00g; FeCl36H2o0.8g adding distil water is to 100mL.
Damping fluid: NaHCO335.00g; NH4hCO34.00g; Adding distil water is to 1000mL.
Nutrient solution is prepared: major element 237mL; Trace element 0.12mL; Damping fluid 237mL; Reductive agent 49.5mL; Glucose 10g, adding distil water is to 1000mL.
1.3 culture medium prescription
Extractum carnis 0.9g, peptone 3g, NaCI1.5g, agar 10.5g, adds working stocks 300mL, supplements artificial medium to 600mL.
The cultural method of 1.4 bacteria distribution purifying
Take above-mentioned solid medium in Erlenmeyer flask according to formula, be placed on universal electric furnace and be heated to boiling, cover ventilative sieve and take out after autoclaving 20min at 121 DEG C, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after temperature is down to about 50 DEG C, about 10-20min solidifies.
Bacterium sample is gathered from the cud that the goat raised is butchered, and by this rumen bacteria liquid sterile saline doubling dilution to 10-8power, be then separated with transfering loop picking bacterium liquid sectional streak on solid medium, cultivate in 37 ± 5 DEG C of envrionment temperatures, anaerobism lucifuge, after colony growth goes out, the different bacterium colony of picking proceeds to rule and is separated, until purifying agaric.
2. the Senile Mouse of bacterial strain
Above-mentioned screening obtained strains photo on solid medium, as Fig. 5, MRS substratum, 37 DEG C, is cultivated 1 day, colonial morphology: oyster white, circular, tip-like, and smooth surface, projection are opaque, neat in edge.Oil Microscopic observation is spherical, and diameter is about 0.6 μm, paired or stack arrangement, Gram-positive (as Fig. 4).By 15000 times of S-4800 field emission scanning electron microscope photo this bacterium diameter known about 0.5 ~ 0.8um(as Fig. 2,3).
As seen from Figure 1: this bacterium is visible faint yellow circular small colonies on solid medium.
Two, the qualification of bacterial strain
The molecular biology identification of 1 sequence amplification
The extraction of 1.1 bacterial genomes DNA
Bacterial classification is growth 12 ~ 18h in enrichment liquid body is cultivated, and gets 1.5mL bacterium liquid and is placed in EP pipe, the centrifugal 5min of 5000r/min, extracts bacterial genomes DNA.
The amplimer of 1.2 bacterial strain 16SrDNA:
Use 16SrDNA universal primer to increase, by Dalian, precious biotechnology company limited synthesizes.
Reaction system: 0.25uLTaqDNA polysaccharase, 5uLl0 × PCR reaction buffer, 4uLdNTPMasterMix, luLPrimerF (upstream primer), luLPrimerR (downstream primer), 2uL template DNA, ddH2o supplies 50uL.
PCR reaction conditions: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30S, 55 DEG C of annealing 40S, 72 DEG C extend 90S, 30 circulations; 72 DEG C of ends extend 10min.
The mensuration of the detection of 1.3PCR product and l6SrDNA sequence
After 20g/L agarose gel electrophoresis, observe under ultraviolet lamp, if there is the single band of about 1.5kb, then prove Successful amplification target l6SrDNA sequence.Pcr amplification product is mailed to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's order-checking, then the 16SrDNA sequence obtained is submitted in NCBI nucleic acid database and carries out BLAST on-line analysis.
2 bacterial strain pheS gene primers and PCR reaction conditions
PheS: primer is synthesized by the precious biotechnology company limited in Dalian.
pheS-21-FCAYCCNGCHCGYGAYATGC；
pheS-22-RCCWARVCCRAARGCAAARCC。
PCR reaction conditions: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30S, 50 DEG C of annealing 40S, 72 DEG C extend 40S, 30 circulations; 72 DEG C of ends extend 10min.
The structure of 3 sequential analyses and genealogical tree
Adopt MEGA4.1 software, ortho position connection method display bacterial classification is grown to relevant sequential system of planting and is set, and carries out the Bootstraps statistical test of 1000 repetitions.
4 identification and analysis results
4.1 faecium L-01 grow to relevant 16SrDNA sequential system of planting and set:
Adopt MEGA4.1 software, ortho position connection method display L-01 and the 16SrDNA phylogenetic tree of relevant kind, carry out the similarity double counting of 1000 times, grows tree node and only show Bootstrap value and be greater than 50% numerical value in figure, (e., Enterococcus).
Shown by Fig. 9 Phylogenetic Analysis: bacterial strain L-01 and enterococcus spp (enterococcussp.) in, multiple kinds are gathered in a phylogeny branch, and sequence homology is more than 98.3%, Qi Zhongyue.faeciumaTCC19434t(DQ411813) sequence homology is up to 99.9%.
Through qualification, the 16SrDNA sequence length of faecium L-01 is 1482bp, and qualification result is enterococcus spp.
4.2 faecium L-01 is to relevant kindpheSgene order phylogenetic tree:
Adopt MEGA4.1 software, ortho position connection method display L-01 and relevant kindpheSphylogenetic Tree, carries out the similarity double counting of 1000 times, grows tree node and only show Bootstrap value and be greater than 50% numerical value in figure.
Shown by Figure 10 Phylogenetic Analysis: bacterial strain L-01 and faecium (enterococcusfaecium) gather in a phylogeny branch, sequence homology is 97.4%, with the sequence homology of other Enterococcus species type strain below 86.6%.
Through qualification, faecium L-01'spheSgene order length is 509bp, and qualification result is faecium.
Three, faecium L-01 bacterial strain enzymic activity dynamic studies
1. the mensuration of alpha-amylase activity in bacterium liquid
Measuring method: iodine-starch colorimetry determination of amylase test kit (Bioengineering Research Institute is built up in Nanjing)
α-amylase (AMS) unit of activity defines: the amylase (AMS) in 100mL bacterium liquid, 39 DEG C with substrate-function 30 minutes, hydrolysis 10mg starch is 1 unit.Alpha-amylase activity changes in time sees that Fig. 6 is known, and in the enzyme activity dynamic changing process of 0-12h, alpha-amylase activity continues to raise in 8-12h dynamic changing process.
2. in bacterium liquid, the mensuration-chromophoric substrate of α-glucose transglucosidase vigor measures α-glucose transglucosidase vigor
Reaction substrate is made with colourless p-NP-alpha-D-glucose glycosides (pNPG), through α-glucose transglucosidase hydrolyzing alpha-1, p-NP (pNP) is discharged after 4-glucoside bond, the latter is yellow in the basic conditions, finally by the criterion of the pNP generation under monitoring 405 or 410nm as the active size of α-Isosorbide-5-Nitrae-glucose transglucosidase.
1) preparation of reagent:
(1) 1.25mmol/L p-NP-alpha-D-glucose glycosides (pNPG) solution: 75.2mgpNPG is dissolved in 200mL
In 0.05mol/L acetate buffer (pH5.6).
(2) 4mmol/L p-NP (pNP) solution: 111.3mgpNP is dissolved in 200mL0.05mol/L acetate buffer (pH5.6).
(3) 0.05mol/L acetate buffer (pH5.6): 16.4g sodium acetate adds 12mL acetic acid, is settled to 1000mL.
(4) 1mol/L sodium carbonate solution.
2) p-NP typical curve is made
Get the p-NP (4mmol/L) of different volumes by upper table respectively, be diluted to a series of concentration with 50mmol/L acetate buffer (pH5.6).Then on spectrophotometer, measure the optical density(OD) at 410nm place, take concentration as X-coordinate, and absorption photometric value is that ordinate zou makes curve.
The typical curve of p-NP is as Fig. 7.
3) mensuration of α-Isosorbide-5-Nitrae-glucose transglucosidase vigor
Get 0.8mL p-NP-alpha-D-glucose glycosides (pNPG) (1.25mmol/L), add 0.2mL bacterium liquid, in 58 DEG C of water-baths, 10min is incubated after rapid mixing, take out the sodium carbonate solution termination reaction also adding 2mL1mol/L immediately, by the optical density(OD) at spectrophotometric determination 410nm wavelength place, typical curve is found the amount being equivalent to p-NP, and calculates enzyme activity.
Enzyme activity unit is defined as: the α-glucose transglucosidase in 1L bacterium liquid, 58 DEG C with p-NP-α-D glucoside effect 10 minutes, the enzyme amount that per minute catalysis is formed required for 1umoL p-NP is 1 unit of activity.
Enzyme activity=(A × n)/(V × t);
V in formula-enzyme liquid measure (mL); N-enzyme liquid extension rate; T-reaction times (min).
α-glucose transglucosidase vigor changes in time sees Fig. 8, and as shown in Figure 8, in the enzyme activity dynamic changing process of 0-12h, α-glucose transglucosidase vigor continues to raise in the dynamic changing process of 8-12h.
Sum up: by above test, prove that faecium L-01 of the present invention has genetic stability good, bacterial strain is energetic, the amylase of secretion and the high characteristic of the enzyme activity of transglucosidase.
Due to above feature, the present invention is expected the fodder additives being applied to preparation animal, cultivated animals, and pig, bird, ruminating animal, aquatic animal, effectively can replace microbiotic, plays diseases prevention, resistant effect.
Lv Bao bio tech ltd, <110> Yangzhou
<120> faecium L-01 and preparation method thereof
<160>2
<210>1
<211>1482
<212>DNA
<213> artificial sequence
<220>
The 16SrDNA sequence of <223> faecium L-01 bacterial strain
<400>1
gacgaacgctggcggcgtgcctaatacatgcaagtcgaacgcttctttttccaccggagc60
ttgctccaccggaaaaagaggagtggcgaacgggtgagtaacacgtgggtaacctgccca120
tcagaaggggataacacttggaaacaggtgctaataccgtataacaatcnaaaccgcatg180
gttttgatttgaaaggcgctttcgggtgtcgctgatggatggacccgcggtgcattagct240
agttggtgaggtaacggctcaccaaggccacgatgcatagccgacctgagagggtgatcg300
gccacattgggactgagacacggcccaaactcctacgggaggcagcagtagggaatcttc360
ggcaatggacgaaagtctgaccgagcaacgccgcgtgagtgaagaaggttttcggatcgt420
aaaactctgttgttagagaagaacaaggatgagagtaactgttcatcccttgacggtatc480
taaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagc540
gttgtccggatttattgggcgtaaagcgagcgcaggcggtttcttaagtctgatgtgaaa600
gcccccggctcaaccggggagggtcattggaaactgggagacttgagtgcagaagaggag660
agtggaattccatgtgtagcggtgaaatgcgtagatatatggaggaacaccagtggcgaa720
ggcggctctctggtctgtaactgacgctgaggctcgaaagcgtggggagcaaacaggatt780
agataccctggtagtccacgccgtaaacgatgagtgctaagtgttggagggtttccgccc840
ttcagtgctgcagctaacgcattaagcactccgcctggggagtacgaccgcaaggttgaa900
actcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagca960
acgcgaagaaccttaccaggtcttgacatcctttgaccactctagagatagagcttcccc1020
ttcgggggcaaagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgg1080
gttaagtcccgcaacgagcgcaacccttattgttagttgccatcattcagttgggcactc1140
tagcaagactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccc1200
cttatgacctgggctacacacgtgctacaatgggaagtacaacgagttgcgaagtcgcga1260
ggttaagctaatctcttaaagcttttctcagttcggattgcaggctgcaactcgcctgca1320
tgaagccggaatcgctagtaatcgcggatcagcacgccgcggtgaatacgttcccgggcc1380
ttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaacc1440
ttttgagccagccgcctaaggtgggatagatgattggggtgg
<210>2
<211>509
<212>DNA
<213> artificial sequence
<220>
The pheS gene order of <223> faecium L-01 bacterial strain
<400>2
catattatatatgacccgagtattatcatccgggcccgatatgcaagatactttctatat60
ttcagacgagatattgattcggacacatacttcaccagtccaagcacggacaatggaaaa120
gcatgatttctctaagggtgctctacgaatgatctcgcctggaaaagttttccgcagaga180
tacagatgatgcgacccacagccatcagttccatcaaattgaagggctagttgttgataa240
aaacatcacgatgggcgatcttaaaggaacattagaagtcgtaatgaaaaaaatgtttgg300
ggaagatcgtgaaatccgtttgcgtccaagttatttcccatttacggagccatcagtaga360
agtagatgtcagttgtttcaaatgtggtggtgccggttgtaacgtatgtaaatacactgg420
atggatcgagattttaggagctggcatggtgcacccgaatgtgttgaagatgtcaggaat480
cgatccagaagaatattcaggttttgctt