CN103865846A

CN103865846A - Enterococcus faecium and preparation method thereof

Info

Publication number: CN103865846A
Application number: CN201410067604.8A
Authority: CN
Inventors: 潘林福
Original assignee: YANGZHOU LYUBAO BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Current assignee: Jiangsu lvmu ecological environmental protection Co., Ltd
Priority date: 2014-02-27
Filing date: 2014-02-27
Publication date: 2014-06-18
Anticipated expiration: 2034-02-27
Also published as: CN103865846B

Abstract

The invention discloses enterococcus faecium and a preparation method thereof, relates to the field of microorganisms, and particularly relates to the enterococcus faecium and application thereof. A bacterium sample is acquired from the rumen of a slaughtered goat, subjected to directive breeding at the temperature of 32-42 DEG C under an anaerobic and photophobic condition, and separated and purified by use of a repeated divisional agar streak plate method so as to obtain enterococcus faecium L-01. The enterococcus faecium L-01 can be used as an animal microorganism fodder additive, has the effects of effectively inhibiting pathogenic bacterium in animal digestive tract, improving the micro-ecological environment of the digestive tract and enhancing the immunologic function of an animal body, can be used as a substitute product of antibiotics and can be used for effectively solving the existing serious situation.

Description

A kind of faecium and preparation method thereof

Technical field

The present invention relates to microorganism field, be specifically related to a kind of faecium and application thereof.

Background technology

The current appearance due to abuse of antibiotics and superbacteria etc. series of problems, make the unavoidable serious situation that caused thus that faces of the mankind, the task of top priority, must research and develop as early as possible Substitutes For Antibiotic, provide reliable technical guarantee for green, safety, efficiently animal-feed and human food prods produce.Probiotics is the nearly 20 years a kind of novel fodder additives that grow up, and it has the function of microbiotic and enzyme.Probiotics can be supplemented with bacteria group, improves archenteric flora balance, prevention and treatment dysbacteriosis; Can stimulate body immune system, improve animal body immunological competence; Can participate in flora struggle for existence, assist body to eliminate toxin and meta-bolites; Can improve organism metabolism, supplement body nutritive ingredient, promote growth of animals or poultry.

Faecium ( enterococcus faecium) be that the current Ministry of Agriculture makes (2013 No. 2045) animal microorganism fodder additives hurdle allow the probiotic bacterium kind using, research shows that this bacterium can be used as the application of animal microorganism fodder additives, can effectively suppress animal digestive tract pathogenic bacterium, improve digestive tube micro-ecological environment; Strengthen the immunologic function of animal body.

Summary of the invention

The present invention seeks to the appearance in order to solve current antibiotic abuse and superbacteria etc. series of problems, propose a kind of faecium L-01 that improves animal body metabolism, supplementary body nutritive ingredient, promotes growth of animals or poultry that is beneficial to.

The present invention propose faecium L-01 ( enterococcus faeciuml-01) be preserved in May 30 in 2012 the Chinese Typical Representative culture collection center that is positioned at Wuhan, China university, deposit number is CCTCC NO:M 2012194.

The present invention also proposes the preparation method of above faecium L-01, from the Rumen of butchering, gather bacterium sample, the condition of 37 ± 5 ℃ of outer employings, anaerobism, lucifuge is carried out directive breeding, is repeatedly rule after separation and purification and is obtained faecium L-01 by agar plate subregion.

Cud separation, the purifying of the present invention from healthy goat is butchered, cultivate, identify 1 Enterococcus faecalis conservation, can be used as the application of animal microorganism fodder additives, can effectively suppress animal digestive tract pathogenic bacterium, improve digestive tube micro-ecological environment; Strengthen the immunologic function of animal body, and can be used as Substitutes For Antibiotic, can effectively solve current faced serious situation.

The present invention screens the bacterial strain obtaining, different from the national conservation mechanism such as Chinese industrial microbial strains preservation administrative center existing bacterial classification source and biological characteristics.Through the preservation of Chinese industrial microbial strains, administrative center identifies, this bacterial strain is faecium, and has carried out genetic stability test in Chinese industrial microbial strains preservation administrative center, proves that this bacterial strain proterties can genetic stability.In the enzyme activity dynamic changing process of 8-12h, alpha-amylase activity and α-glucose transglucosidase vigor all reach peak value and maintain higher level.

Healthy Rumen after this strains separation is derived from and butchers, its tunning is applied to cultivated animals as animal microorganism fodder additives, especially to improving ruminating animal, its consistency and effect are ideal, in addition, also there is positive regulating and controlling effect for the foundation of piglet digestive tube micro-ecological environment stable state.

Accompanying drawing explanation

Fig. 1 is the bacterial strain bacterium colony photo of faecium L-01 of the present invention.

Fig. 2, Fig. 3 are respectively the bacterial strain field emission scanning electron microscope photo of faecium L-01 of the present invention.

Fig. 4 is faecium L-01 bacterial strain MRS substratum thalli morphology photo of the present invention.

Fig. 5 is faecium L-01 bacterial strain MRS substratum colonial morphology photo of the present invention.

Fig. 6 is the time dependent graph of a relation of 39 ℃ of amylase (AMS) vigor.

Fig. 7 is the canonical plotting of p-NP.

Fig. 8 is 58 ℃ of time dependent graphs of a relation of α-glucose transglucosidase vigor.

Fig. 9 is that faecium L-01 grows tree to the 16S rDNA sequential system of relevant kind.

Figure 10 is that faecium L-01 plants to relevant pheSgene order phylogenetic tree.

Embodiment

One, the screening of bacterial strain

1. screening process

The cud of butchering from the goat of raising, gather bacterium sample, the specified conditions of 37 ± 5 ℃ of external employings, anaerobism, lucifuge carry out directive breeding, repeatedly rule separation and purification to single strain L-01 domestication by agar plate subregion, study its morphology and biological characteristics.Observe colony characteristics, electron microscope observation single strain form and relevant key enzyme vitality test by agar plate culture.

The configuration of 1.1 work mother liquors:

First following table ratio is equipped with nutrition substrate:

Composition	Weight percent (%)
		Corn	80
Dregs of beans	6
		Wheat bran	10
Salt	1
		Stone flour	1
Secondary calcium phosphate	2

Take nutrition substrate 125g(2600r/min in 5% ratio again and pulverize 1.5min) mix with water, digestion 1h under electromagnetic oven boiling state, after twice of two-layer gauze extracting, after extract 2600r/min liquid making beating 1.5min, 500 order nylon-tissue bag are filtered, finally be settled to 2.5L, obtain work mother liquor.

1.2 artificial medium compositions: the formula with reference to Russell is improved.

Reductive agent (matching while using): 1N NaOH solution 4mL; Na ₂s7H ₂o 570mg adding distil water is to 100mL.

Major element: Na ₂hPO ₄5.70g; NaH ₂pO ₄6.20g; MgSO ₄7H ₂o 0.60g adding distil water is to 1000mL.

Trace element: CaCl ₂2H ₂o 13.20g; MnCl ₂4H2O 10.00g; CoCl ₂6H ₂o 1.00g; FeCl ₃6H ₂o 0.8g adding distil water is to 100mL.

Damping fluid: NaHCO ₃35.00g; NH ₄hCO ₃4.00g; Adding distil water is to 1000mL.

Nutrient solution preparation: 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, NaCI 1.5g, agar 10.5g, adds work mother liquor 300mL, supplements artificial medium to 600mL.

The cultural method of 1.4 bacterium separation and purification

Above-mentioned solid medium is taken in Erlenmeyer flask according to formula, be placed on universal electric furnace and be heated to boiling, cover ventilative sieve and after autoclaving 20min, take out at 121 ℃, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after temperature is down to 50 ℃ of left and right, about 10-20min solidifies.

The cud of butchering from the goat of raising, gather bacterium sample, and by sterile saline doubling dilution to 10 for this rumen bacteria liquid ^-8power, then separates with transfering loop picking bacterium liquid sectional streak on solid medium, cultivates in 37 ± 5 ℃ of envrionment temperatures, anaerobism lucifuge, and after colony growth goes out, the different bacterium colonies of picking are proceeded to rule and separated, until purifying agaric.

2. the morphology of bacterial strain research

Above-mentioned screening obtained strains on solid medium photo as Fig. 5, MRS substratum, 37 ℃, cultivate colonial morphology 1 day: oyster white, circle, tip-like, smooth surface, projection, opaque, neat in edge.Oil Microscopic observation is spherical, and diameter is about 0.6 μ m, and paired or heap shape is arranged, Gram-positive (as Fig. 4).By 15000 times of known this bacterium diameter of S-4800 field emission scanning electron microscope photo 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 evaluation of bacterial strain

The molecular biology identification of 1 sequence amplification

The extraction of 1.1 bacterial genomes DNA

Bacterial classification is 12～18 h that grow in enrichment liquid body is cultivated, and get 1.5 mL bacterium liquid and are placed in EP pipe, and centrifugal 5 min of 5000 r/min, extract bacterial genomes DNA.

The amplimer of 1.2 bacterial strain 16SrDNA:

Use 16SrDNA universal primer to increase, by Dalian, precious biotechnology company limited is synthetic.

Reaction system: 0.25uL Taq DNA polysaccharase, 5uLl0 × PCR reaction buffer, 4uL dNTP MasterMix, luLPrimer F (upstream primer), l uL Primer R (downstream primer), 2uL template DNA, ddH ₂o supplies 50uL.

PCR reaction conditions: 94 ℃ of denaturation 5 min; 94 ℃ of sex change 30 S, 55 ℃ of annealing 40 S, 72 ℃ are extended 90 S, 30 circulations; 72 ℃ are extended 10 min eventually.

The mensuration of 1.3 PCR products detections and l6S rDNA sequence

After 20g/L agarose gel electrophoresis, under ultraviolet lamp, observe, if there is the single band of 1.5 kb left and right, prove successfully to increase target l6S rDNA sequence.Mail pcr amplification product to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd order-checking, then the 16SrDNA sequence obtaining is submitted to and in NCBI nucleic acid database, carries out BLAST on-line analysis.

2 bacterial strain pheS gene primers and PCR reaction conditions

PheS: primer is synthetic by Dalian precious biotechnology company limited.

pheS-21-F CAYCCNGCHCGYGAYATGC；

pheS-22-R CCWARVCCRAARGCAAARCC。

PCR reaction conditions: 94 ℃ of denaturation 5 min; 94 ℃ of sex change 30 S, 50 ℃ of annealing 40 S, 72 ℃ are extended 40 S, 30 circulations; 72 ℃ are extended 10 min eventually.

The structure of 3 sequential analyses and genealogical tree

Adopt MEGA4.1 software, ortho position connection method shows bacterial classification and the relevant sequential system growth tree of planting, and carries out the Bootstraps statistical test repeating for 1000 times.

4 identification and analysis results

4.1 faecium L-01 grow tree to the 16S rDNA sequential system of relevant kind:

Adopt MEGA4.1 software, ortho position connection method shows L-01 and relevant 16S rDNA phylogenetic tree of planting, and carries out the similarity double counting of 1000 times, in figure, grow tree node and only show that Bootstrap value is greater than 50% numerical value, ( e., Enterococcus).

Shown by Fig. 9 Phylogenetic Analysis: bacterial strain L-01 and enterococcus spp ( enterococcussp.) in multiple kinds gather in a phylogeny branch, sequence homology is more than 98.3%, wherein with e.faeciumaTCC 19434 ^t(DQ411813) sequence homology is up to 99.9%.

Through identifying, the 16S rDNA sequence length of faecium L-01 is 1482bp, and qualification result is enterococcus spp.

4.2 faecium L-01 are to relevant kind pheSgene order phylogenetic tree:

Adopt MEGA4.1 software, ortho position connection method shows L-01 and relevant kind pheSphylogenetic Tree, carries out the similarity double counting of 1000 times, grows tree node and only show that Bootstrap value is greater than 50% numerical value in figure.

Shown by Figure 10 Phylogenetic Analysis: bacterial strain L-01 and faecium ( enterococcus faecium) gather in a phylogeny branch, sequence homology is 97.4%, with the sequence homology of other enterococcus spp bacterial classification type strain below 86.6%.

Through identifying, faecium L-01's pheSgene 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 definition: the amylase (AMS) in 100mL bacterium liquid, 39 ℃ with substrate-function 30 minutes, hydrolysis 10mg starch is 1 unit.Alpha-amylase activity temporal evolution is shown in that Fig. 6 is known, and in the enzyme activity dynamic changing process of 0-12 h, alpha-amylase activity continues to raise in 8-12 h dynamic changing process.

2. the mensuration of α-glucose transglucosidase vigor in bacterium liquid-chromophoric substrate is measured α-glucose transglucosidase vigor

Make reaction substrate with colourless p-NP-alpha-D-glucose glycosides (pNPG), through α-glucose transglucosidase hydrolyzing alpha-1, after 4-glucoside bond, discharge p-NP (pNP), under alkaline condition, the latter is yellow, the finally criterion as the active size of α-Isosorbide-5-Nitrae-glucose transglucosidase by the pNP generation under monitoring 405 or 410 nm.

1) preparation of reagent:

(1) 1.25mmol/L p-NP-alpha-D-glucose glycosides (pNPG) solution: 75.2mg pNPG is dissolved in 200mL

In 0.05mol/L acetate buffer (pH5.6).

(2) 4mmol/L p-NP (pNP) solution: 111.3mg pNP is dissolved in 200mL 0.05mol/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) make p-NP typical curve

Get respectively the p-NP (4mmol/L) of different volumes by upper table, 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, absorption photometric value is that ordinate zou is made 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, after mixing rapidly, in 58 ℃ of water-baths, be incubated 10min, take out and add immediately the sodium carbonate solution termination reaction of 2mL 1mol/L, by the optical density(OD) at spectrophotometric determination 410nm wavelength place, on typical curve, find the amount that is equivalent to p-NP, and calculate enzyme activity.

Enzyme activity unit is defined as: the α-glucose transglucosidase in 1L bacterium liquid, 58 ℃ with p-NP-α-D glucoside effect 10 minutes, it is 1 unit of activity that per minute catalysis forms the needed enzyme amount of 1umoL p-NP.

Enzyme activity=(A × n)/(V × t);

V-enzyme liquid measure (mL) in formula; N-enzyme liquid extension rate; T-reaction times (min).

α-glucose transglucosidase vigor temporal evolution is shown in Fig. 8, and as shown in Figure 8, in the enzyme activity dynamic changing process of 0-12 h, α-glucose transglucosidase vigor continues to raise in the dynamic changing process of 8-12 h.

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 to be applied to the fodder additives of preparation animal, cultivated animals, and pig, bird, ruminating animal, aquatic animal, can effectively replace microbiotic, performance 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 16S rDNA sequence of <223> faecium L-01 bacterial strain

<400>1

gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gcttcttttt ccaccggagc 60

ttgctccacc ggaaaaagag gagtggcgaa cgggtgagta acacgtgggt aacctgccca 120

tcagaagggg ataacacttg gaaacaggtg ctaataccgt ataacaatcn aaaccgcatg 180

gttttgattt gaaaggcgct ttcgggtgtc gctgatggat ggacccgcgg tgcattagct 240

agttggtgag gtaacggctc accaaggcca cgatgcatag ccgacctgag agggtgatcg 300

gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360

ggcaatggac gaaagtctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt 420

aaaactctgt tgttagagaa gaacaaggat gagagtaact gttcatccct tgacggtatc 480

taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540

gttgtccgga tttattgggc gtaaagcgag cgcaggcggt ttcttaagtc tgatgtgaaa 600

gcccccggct caaccgggga gggtcattgg aaactgggag acttgagtgc agaagaggag 660

agtggaattc catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cagtggcgaa 720

ggcggctctc tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt 780

agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttggagg gtttccgccc 840

ttcagtgctg cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa 900

actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 960

acgcgaagaa ccttaccagg tcttgacatc ctttgaccac tctagagata gagcttcccc 1020

ttcgggggca aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080

gttaagtccc gcaacgagcg caacccttat tgttagttgc catcattcag ttgggcactc 1140

tagcaagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200

cttatgacct gggctacaca cgtgctacaa tgggaagtac aacgagttgc gaagtcgcga 1260

ggttaagcta atctcttaaa gcttttctca gttcggattg caggctgcaa ctcgcctgca 1320

tgaagccgga atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggcc 1380

ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc 1440

ttttgagcca gccgcctaag gtgggataga tgattggggt gg

<210>2

<211>509

<212>DNA

<213> artificial sequence

<220>

The pheS gene order of <223> faecium L-01 bacterial strain

<400>2

catattatat atgacccgag tattatcatc cgggcccgat atgcaagata ctttctatat 60

ttcagacgag atattgattc ggacacatac ttcaccagtc caagcacgga caatggaaaa 120

gcatgatttc tctaagggtg ctctacgaat gatctcgcct ggaaaagttt tccgcagaga 180

tacagatgat gcgacccaca gccatcagtt ccatcaaatt gaagggctag ttgttgataa 240

aaacatcacg atgggcgatc ttaaaggaac attagaagtc gtaatgaaaa aaatgtttgg 300

ggaagatcgt gaaatccgtt tgcgtccaag ttatttccca tttacggagc catcagtaga 360

agtagatgtc agttgtttca aatgtggtgg tgccggttgt aacgtatgta aatacactgg 420

atggatcgag attttaggag ctggcatggt gcacccgaat gtgttgaaga tgtcaggaat 480

cgatccagaa gaatattcag gttttgctt

Claims

Faecium L-01 ( enterococcus faeciuml-01), its deposit number is CCTCC M 2012194.
2. the preparation method of faecium L-01 as claimed in claim 1, it is characterized in that gathering the Rumen from butchering bacterium sample, the condition of 37 ± 5 ℃ of external employings, anaerobism, lucifuge is carried out directive breeding, is repeatedly rule after separation and purification and is obtained faecium L-01 by agar plate subregion.