CN108179122A - A kind of probiotic enterococcus faecium of high adherency and its application - Google Patents
A kind of probiotic enterococcus faecium of high adherency and its application Download PDFInfo
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- CN108179122A CN108179122A CN201611125252.2A CN201611125252A CN108179122A CN 108179122 A CN108179122 A CN 108179122A CN 201611125252 A CN201611125252 A CN 201611125252A CN 108179122 A CN108179122 A CN 108179122A
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- enterococcus faecium
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- 241000194031 Enterococcus faecium Species 0.000 title claims abstract description 58
- 239000006041 probiotic Substances 0.000 title claims abstract description 16
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 16
- 230000000529 probiotic effect Effects 0.000 title abstract description 13
- 241001465754 Metazoa Species 0.000 claims abstract description 19
- 230000012010 growth Effects 0.000 claims abstract description 10
- 239000002068 microbial inoculum Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000000654 additive Substances 0.000 abstract description 5
- 230000000996 additive effect Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention discloses a kind of probiotic enterococcus faecium (Enterococcus faecium) of high adherency and its application, the deposit number of the enterococcus faecium is CGMCC NO.13229.The enterococcus faecium of the present invention has stronger resistance, can high temperature resistant, acidproof, Adhesion property is good, also with excellent probiotic, can improve efficiency of feed utilization, promote digesting and assimilating for nutriment in feed, promote growth of animal；Enhance the immune function of animal, improve daily gain, reduce feedstuff-meat ratio；With pollution-free, the features such as noresidue, biological environmental production.The enterococcus faecium of the present invention can be widely used in as a kind of novel probiotic additive in feed.
Description
Technical field
The invention belongs to technical field of microbe application, specifically, be related to a kind of probiotic enterococcus faecium of high adherency and
It is applied.
Background technology
Disease, which occurs, for animal in aquaculture can cause very big economic loss, such as enteritis, diarrhea, all be much by disease
Caused by opportunistic pathogen, and certain threat is also resulted in for the health of the mankind.As the discovery of antibiotic obtains this situation
To improve, antibiotic can kill pathogenic bacteria, can effectively treat bacteriosis, and antibiotic can also promote in a short time
Into the growth of animal.So antibiotic can be used for treating and preventing some diseases, it is also used as growth accelerator.But with
The drawbacks of progress of science, antibiotic also gradually by it is found that.
It is prebiotic as the substitute of antibiotic with continuous reinforcement of the people to livestock product safety and environmental protection consciousness
Bacterium is increasingly used in Animal nutrition and feed.Probiotics is that one kind can improve animal gastrointestinal tract microecological balance, is had
The microbe additive played beneficial to animal health and production performance.Its main function effect, which is embodied in, improves animal new old generation
It thanks, improves absorption of nutrient ingredients and utilization, improve immunity, reduce environmental pollution etc. plays a significant role.Probiotics quilt
Allow to be used in feed addition profit, it can be by preventing the harmful microbe in enteron aisle the effects that adherence mechanism and Reverse transcriptase
Growth and breeding, and its metabolite can kill pathogen, have the stable state for maintaining animal intestinal tract microorganism, the life for promoting animal
Long, prevention and treatment disease and other effects, and the death rate of animal can be reduced.It not only solves antibiotic to normal micro- in enteron aisle
The problem of harmful effect of biology, moreover it is possible to avoid generating side effect and drug resistance etc. using antibiotic.
Enterococcus faecium (Enterococcus faecium) is the normal lactic acid bacteria class of animal intestinal tract, and Gram-positive is facultative
The elliposoidal or ball bacteria of anaerobism, no gemma and flagellum.Growth temperature is 30~40 DEG C, and optimum temperature is 35~38 DEG C, suitable
PH for 5.0~7.5, resistance is poor, and metabolism generates a large amount of lactic acid.Research finds that enterococcus faecium can enhance the immune of piglet
System function, and improve intestinal microflora so as to promote the growth of weanling pig, broiler chicken caecum coliform count can be reduced
Amount increases the quantity of Bacillus acidi lactici, and intestinal flora is made to be in stable state, moreover it is possible to improve the laying rate in later stage of laying eggs, lay eggs
Quantity and egg size.Enterococcus faecium generates shadow to the Quantityanddiversity of immune organ, the development of enteron aisle villus, enteric microorganism
It rings, finds that enterococcus faecium is metabolized with 42 kinds of intestinal mucosa protein substances, exempts to the quantitative proteomics analysis of Intestine of Broiler mucous membrane
Epidemic disease is related with antioxidant system and eucaryotic cell structure.
The premise that probiotics plays prebiotic effect is to stick field planting in gut epithelium, and it sticks field planting with host
Specificity, the feeding effect of homologous probiotics are better than heterologous, and bacterium can only be colonized in the gastrointestinal epithelial cells of isogenic animal
Surface.So the probiotics for going for good animal feeding needs to detach and screen out of animal body.
Invention content
The object of the present invention is to provide a kind of probiotic enterococcus faecium of high adherency and its applications.
In order to achieve the object of the present invention, enterococcus faecium of the invention is isolated from chicken intestinal Mixed Microbes, by bacterium
Fall morphologic observation, stick and bacteriostasis property experiment etc. obtain aimed strain, through 16S rDNA gene sequencings, enterococcus faecium
16SrDNA amplification electrophoretograms such as Fig. 1.Known array in the sequence of the 16S rDNA of bacterial strain enterococcus faecium and GenBank is carried out
Blast is compared, and the 16S rDNA of relevant kind is obtained from database, and special by the cellular morphology of strain, Physiology and biochemistry
The experimental datas such as 16S rDNA sequences of seeking peace synthesis determines that the bacterial strain belongs to enterococcus spp, is ultimately determined to Firmicutes, bacillus
Guiding principle, lactobacillus mesh, enterococcus section, the enterococcus faecium (Enterococcus faecium) of enterococcus spp.
The bacterial strain Adhesion property is good, and tolerance is strong, and external probiotic effects evaluation, the bacterial strain is can inhibit pathogen and tool
There is the effect of efficiency of feed utilization that is prebiotic and improving animal, 16S rDNA sequences are as shown in SEQ ID NO.1.The bacterial strain in
On November 2nd, 2016 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is dung
Enterococcus (Enterococcus faecium), preserving number are CGMCC No.13229.
The Microbiological Characteristics of enterococcus faecium (Enterococcus faecium) are：Gram-positive bacteria is cultivated in MRS
It is grown on base rapidly, 37 DEG C of cultures form milky, circle, the smooth moistening in surface, protrusion, neat in edge, diameter about 1mm for 24 hours
Left and right.
The present invention provides the microbial inoculum containing the enterococcus faecium.
The present invention provides the drug containing the enterococcus faecium.
The present invention provides the animal feed additives containing the enterococcus faecium.The feed addictive contains enterococcus faecium
Viable count is 5 × 108-1×109CFU/kg。
The present invention identifies the probiotic effects of enterococcus faecium by vitro method, the results showed that, enterococcus faecium can be acidproof, resistance to
High temperature, the interior environment that can resist gastrointestinal tract have the potentiality of probiotics.
The present invention also using bacteriostasis property, adhesion property and the security performance of in vitro method identification enterococcus faecium, is as a result shown：
The enterococcus faecium of the present invention, optimal pH 5.0-7.0, the bacterium can breed rapidly in MRS culture mediums, grow OD values
As shown in Fig. 2, the bacterium enters logarithmic phase after 2h, reach stationary phase after 8h, and after stationary phase bacteria concentration at longer one section
Between interior energy keep stablize.Bacterial concentration can reach 3.3 × 10 after stabilization8-5.0×108CFU/mL.This Enterococcus faecalis also has
Certain high temperature resistance, survival rate can reach more than 76% after heating 160s at 65 DEG C, and 75 DEG C are heated survival rate energy after 160s
Reach more than 30%, 85 DEG C of heating 160s survival rates can reach more than 23%.
This plant of bacterium has the ability of tolerance cholate and pepsin.0.1%, 0.2%, 0.3% is added in the medium
And 0.5% cholate culture for 24 hours after, bacterial concentration can reach 3.5 × 107-5.8×107CFU/mL.In the medium
The pepsin of addition 0.5% is as simulate the gastric juice, and after 37 DEG C are cultivated 2h, the survival rate of bacterium can reach more than 82%.It can be resistant to
By vivo environment, there is higher adhesion strength, there are good probiotic effects.Pass through application effect in being added in broiler fodder
Research, shows that enterococcus faecium can be widely used in as a kind of novel probiotic additive in feed.
The enterococcus faecium that present invention screening obtains, which has, improves efficiency of feed utilization, and the digestion of nutriment in feed is promoted to inhale
It receives；Enhance the immune function of animal, improve daily gain, reduce feedstuff-meat ratio；It is pollution-free, the features such as noresidue, biological environmental production, have
Better probiotic properties and the potentiality as feed addictive.
Description of the drawings
Fig. 1 expands electrophoretogram for enterococcus faecium 16SrDNA.Left side is maker, and right side is purpose band.
Fig. 2, which is that bacterium is extended at any time in MRS culture mediums, grows OD value figures.
6 plants of bacterium that Fig. 3 is obtained for primary dcreening operation, number A-F, the growing state figure under different pH condition.
Fig. 4 is 6 plants of bacterium that primary dcreening operation obtains, number A-F, the growing state figure under condition of different temperatures.
Fig. 5 is the adhesion result of the test of A bacterial strains and F bacterial strains.
Fig. 6 is influence of the F bacterial strains (PNC01) to Caco-2 cell supernatant LDH contents.Control group is does not add F bacterium.
Fig. 7 is bile tolerance result of the test figure of the F bacterial strains (PNC01) under different gallbladder salinities.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The separation and identification of 1 enterococcus faecium of embodiment
1st, enterococcus faecium isolating and purifying and preserves
From the crop of chicken, jejunum, ileum, caecum acquisition mucous membrane and a little chyme as in sterilized centrifuge tube, sample
Middle addition sterile saline (PBS buffer solution) mixing, it is dilute with sterile saline (PBS buffer solution) multiple proportions to draw suspension
It releases, diluted concentration gradient is 10-1To 10-10, 10 are chosen after dilution-4To 10-10Sample, each diluted concentration takes 100uL uniform
Being coated in the selective agar medium of lactic acid bacteria, (MRS agar mediums, MRS agar mediums used composition are：Powdered beef
5.0g, peptone 10.0g, dusty yeast 4.0g, glucose 20.0g, sodium acetate 5.0g, potassium dihydrogen phosphate 2.0g, Triammonium citrate
2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, distilled water 1000mL, pH6.2 ± 0.1.) on, culture dish is fallen
It puts and 48h is cultivated in 37 DEG C of incubators, obtain single bacterium colony.
Choosing colony form differs single bacterium colony and is seeded in the MRS fluid nutrient mediums of sterilizing (MRS used from culture dish
Fluid nutrient medium forms：Powdered beef 5.0g, peptone 10.0g, dusty yeast 4.0g, glucose 20.0g, sodium acetate 5.0g, phosphorus
Acid dihydride potassium 2.0g, Triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, distilled water 1000mL,
PH6.2 ± 0.1), it is incubated overnight in 37 DEG C of incubators (about 12h).Bacterium solution becomes cloudy after 12h, with the inoculation of sterilizing after shaking up
Ring is crossed to after dipping bacterium solution on MRS agar mediums, and culture dish is inverted into 37 DEG C of incubators and cultivates 48h, thus then
Complete primary purifying.
Above-mentioned purification step is repeated, until the last colonial morphology of tablet is in the same size, general purifying 3-4 times.It draws
The bacterium solution and the sterile glycerol of 500 μ L 60% that 500 μ L are obtained after purification are with 1:1 volume ratio mixes in 2ml sterile centrifugation tubes
Conjunction shakes up, and -80 DEG C of refrigerator cryopreservations are put into after being sealed with sealed membrane, each general bacterium at least preserves 3 pipes.
2nd, colony morphological observation
Lactic acid bacteria is gram-positive bacteria, dark blue or purple can be dyed by Gram's staining, and can be in electricity after dyeing
Microscopic observation distinguishes rod bacterium and ball dress bacterium.The slant strains that step 1 is preserved, through 3 activation.Activation step will freeze
Strain is placed to thaw in the ice chest in super-clean bench, is crossed to after dipping bacterium solution with the oese of sterilizing on MRS agar mediums, will
Culture dish is inverted into 37 DEG C of incubators and cultivates 48h.Then the good microbionation of picking colony form is in MRS Liquid Cultures
In base, 12h or so is cultivated in 37 DEG C of incubators, thus activation is completed.If the bacterial activity of gained is not high, can will obtain
Bacterium solution is inoculated into the inoculum concentration of 2%-5% in MRS fluid nutrient mediums and cultivates 12h again.Clean glass slide is taken after activation, is dripped
One drop zymotic fluid is smoothened on glass slide, fixed after dry, carries out Gram's staining.Microscopy chooses Gram's staining as the positive
Pellet form Strain as spare.
3rd, the screening of Lactic Acid Bacteria
(1) it is acidproof
Selection pH value is 2,3,3.5 and 4 four gradients.After MRS fluid nutrient mediums have been configured, culture medium is adjusted with HCl
Into required target ph, high pressure sterilization after isometric each gradient media is fitted into triangular flask.By the lactic acid after activation
Bacterium bacterium solution is inoculated in identical inoculum concentration in each processing, is compareed and is inoculated in original pH with identical inoculum concentration and obtains MRS liquid
In culture medium.8h is cultivated in 37 DEG C of incubator.Time shakes up after, by bacterium solution doubling dilution into taking after different concentration
100 μ L bacterium solutions are uniformly coated onto on MRS agar mediums, and culture dish meter of the clump count in 30-300 is chosen after cultivating 48h in 37 DEG C
Number, is compared with a control, and calculates the percentage that each processing quantity accounts for control quantity.The preferable 6 plants of bacterium of result of the test are picked, are compiled
Number for A-F, result of the test is as shown in Figure 3.As a result show that the acid resistance of 6 bacterial strains is all preferable, when pH value is 3,6 plants of bacterium growths
Rate reaches more than 40%, and when pH value is 3.5,6 plants of bacterium growth rates reach more than 70%, when pH value is 4,6 plants of bacterium growth rates
Reach more than 80%.
(2) high temperature resistant
65,75,85 DEG C of three gradients are chosen, 6 plants of bacterium bacterium solutions of A-F after activation are put respectively in equal volume and are packed into three temperature
In the triangular flask of degree, control be put into 37 DEG C of water-baths in equal volume, with steady temperature in water-bath heating water bath 2min 40s.
Time after by bacterium solution doubling dilution into 100 μ L bacterium solutions being taken uniformly to be coated onto on MRS agar mediums after different concentration, in 37 DEG C
Culture dish counting of the clump count in 30-300 is chosen after cultivating 48h, is compared with a control, each processing quantity is calculated and accounts for control quantity
Percentage.Result of the test is as shown in figure 4, display number is A and two plants of bacterium of F survival rate after 65 DEG C of heating 160s can reach
Survival rate can reach more than 30% after more than 76%, 75 DEG C of heating 160s, and 85 DEG C of heating 160s survival rates can reach more than 23%
The bacterial strain that acidproof and high-temperature resistant result preferably number is A, F is selected according to acidproof and high temperature resistant result of the test to carry out
Follow-up test.
(3) adhesion is tested
The centrifugation of enterococcus faecium bacterium solution is abandoned after supernatant after cleaning centrifugation with PBS, be resuspended with the cell culture fluid of antibiotic-free
After be added in tissue culture plate, co-culture 3h after cleaned 3 times with PBS, wash away the bacterium do not sticked.It then will with trypsase
Cell dissociation is counted into single shape, coated plate, calculates the percentage for being attached on bacterium on cell.Result of the test is as shown in figure 5, F bacterium
Adherence rate for 74.33%, far above A bacterium, in summary experimental result selection F bacterium.
(4) safety testing
This plant is screened obtained F bacterium safety testing using CaCo-2 cells, verifies the poison of F bacterium and its products upon cell
Property.By the passage of Caco-2 cells in tissue culture plate, cultivated in 37 DEG C of incubators, until covering with cell monolayer.Take the bacterium of F bacterium
Supernatant is sucked out after liquid centrifugation and is retained for use, bacterium is resuspended with isometric fresh cell medium, will contain germy
Culture solution and bacterial supernatant are added separately in the culture plate for covering with cell monolayer, are put into 37 DEG C of incubator cultures, in 1h and
4.5h draws cell supernatant and measures LDH values.It can find that this plant of bacterium F can significantly reduce the LDH contents in cell supernatant, subtract
Slow Apoptosis.Result of the test adds LDH after F bacterium as shown in fig. 6, LDH contents in control group supernatant are 182.805U/L
It is reduced to 65.11U/L.
(5) bile tolerance is tested
It is separately added into cholate 0.1%, 0.2%, 0.3%, 0.5% in liquid medium, is inoculated with and lives by 10% amount
The F actication of culture liquid of change, spread plate counts after culture for 24 hours.It is compared simultaneously with being free of the fluid nutrient medium of cholate.Experiment
The results are shown in Figure 7, and the control group bacterial concentration that cholate additive amount is 0 is 3.8 × 108CFU/mL, the bacterium solution of each processing group are dense
Spend is 3.5 × 107-5.8×107CFU/mL。
(6) resistance to pepsin experiment
50ml fluid nutrient mediums is taken to add in pepsin as simulate the gastric juice according to 0.5% amount, are then connect according to 10%
The F bacterium cultures of kind amount access activation, 37 DEG C are cultivated 2h sampling and measuring viable counts, and the Liquid Culture of pepsin is not added with equivalent
Base is as control.The viable count of processing group accounts for the 82.33% of control group.
(7) purpose strain is determining
High temperature resistant, acidproof, bacteriostatic activity, the adhesion for considering each lactic acid bacteria finally obtain an Enterococcus faecalis F bacterium,
FNC01 is named as, high temperature resistant, acid resistance are strong, and Adhesion property is good and safe and non-toxic.
MRS solid mediums composition used is (being dissolved in 1000mL distilled water)：Powdered beef 5.0g, peptone 10.0g, yeast
Powder 4.0g, glucose 20.0g, sodium acetate 5.0g, potassium dihydrogen phosphate 2.0g, Triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate
0.05g, Tween 80 1mL, pH6.2 ± 0.1；Shake flask culture conditions are：37 DEG C, rotating speed 80-120r/m cultures 12h.For fermenting
The medium component of liquid is (being dissolved in 1000mL distilled water)：Powdered beef 5.0g, peptone 10.0g, dusty yeast 4.0g, glucose
20.0g, sodium acetate 5.0g, potassium dihydrogen phosphate 2.0g, Triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween-80
1mL, agar 17-18g, pH6.2 ± 0.1.
MRS fluid nutrient mediums composition used is (being dissolved in 1000mL distilled water)：Powdered beef 5.0g, peptone 10.0g, yeast
Powder 4.0g, glucose 20.0g, sodium acetate 5.0g, potassium dihydrogen phosphate 2.0g, Triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate
0.05g, Tween 80 1mL, pH6.2 ± 0.1；Shake flask culture conditions are：37 DEG C, rotating speed 80-120r/m cultures 12h.For fermenting
The medium component of liquid is (being dissolved in 1000mL distilled water)：Powdered beef 5.0g, peptone 10.0g, dusty yeast 4.0g, glucose
20.0g, sodium acetate 5.0g, potassium dihydrogen phosphate 2.0g, Triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween-80
1mL, pH6.2 ± 0.1.
4th, 16S rDNA sequencings
The centrifugation column type bacterial genomes DNA extraction kit produced using TIANGEN Biotech (Beijing) Co., Ltd.,
Catalog number (Cat.No.)：DP302.After the bacterium solution culture 12-14h of FNC01 bacterium, bacterium solution 1ml is drawn for carrying DNA.
The amplification of 16SrDNA, using lactic acid bacteria universal primer 27f and 1525R,
27f:5'-AGAGTTTGATCCTGGCTCAG-3'；1525R：
5'-AGAAAGGAGGTGATCCAGCCC-3', primer are synthesized in Sangon Biotech (Shanghai) Co., Ltd..
PCR reaction systems：10×Buffer：5μl；Primer：Each 1 μ l of up/down；dNTP：4μl；enzyme：0.25μl；template：
1μl；ddH2O：37.75 μ l, Total volume：50μl.
During PCR, first by Buffer, dNTP, Primer, ddH2O adds in mixing in centrifuge tube, then is separately added into masterplate, most
After be separately added into enzyme, prevent from acutely shaking with centrifuge gentle centrifugation after adding in enzyme.Whole process in super-clean bench on ice
Operation.60 DEG C of annealing temperature, 95 DEG C of denaturation temperature 3-5 minutes, 72 DEG C of elongating temperature, 35 cycles.Archaeal dna polymerase is Thermo
The DreamTaq of scientific-fermentas productionsTMDNA Polymerase.Electrophoresis is carried out after PCR amplification, with 1 × TAE
1.5% agarose, micro-wave oven heating and melting are prepared, 100ml glue adds 5ul dyeing liquor redgel, treats that temperature drops to not after mixing
Glue is spread after scalding one's hand.Electrophoresis liquid is 1 × TAE, and voltage first is transferred to 120v, voltage is transferred to 90v after sample runs out of hole.5ul PCR
Reaction solution adds in 1ul Loading Buffer.Giving PCR product to raw work bioengineering (Shanghai) share with part primer has
Limit company is sequenced, and Microbes is selected to carry out BLAST comparisons in NCBI sequencing result.Finally determining FNC01 bacterium (F
Bacterium) it is enterococcus faecium.It is commonly micro- that the bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms on November 2nd, 2016
Bio-Centers (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
Postcode 100101), Classification And Nomenclature is enterococcus faecium (Enterococcus faecium), and preserving number is CGMCC No.13229.
2 broiler feeding experiment of embodiment
Isolated enterococcus faecium FNC01 bacterium to be crossed and are detached, picking single bacterium colony is in MRS culture mediums, in 37 DEG C,
Culture is recovered in the shaking table of rotating speed 100rpm, expands culture with 5% inoculum concentration inoculation after recovery.Obtain using after bacterium solution Ah
Primary glue and skimmed milk powder are drawn with 1:The additive amount addition of 1 ratio 20% is spray-dried to obtain bacterium powder.The bacterium finally obtained
Powder number of viable is 5 × 109CFU/g.1 400, age in days section Baoji is selected, 5 processing groups of random arrangement each handle 8 weights
It is multiple, 10 chickens are each repeated, respectively：Blank control group and enterococcus faecium group (1 × 109CFU/kg), the test period is 42 days,
Record the feed consumption weight and body weight gains of broiler chicken.
As shown in table 1, enterococcus faecium does not make significant difference to Broiler chicks average daily gain and average daily gain (P>0.05),
Significantly affect 1-21 and 1-42d Broiler chicks feedstuff-meat ratios (P<0.05).Compared with blank control group, adding enterococcus faecium in daily ration can
The reduction broiler chicken feedstuff-meat ratio (P of 1-21 days and 1-42 days<0.05).Enterococcus faecium falls below the feedstuff-meat ratio of 1-21 days by 1.57
1.45, the feedstuff-meat ratio of 1-42 days is fallen below 1.69 by 1.75.It can be seen that the obtained enterococcus faecium FNC01 bacterium of present invention screening can be with
The feedstuff-meat ratio of broiler chicken is significantly reduced, reduces the feeding cost of broiler chicken, has and improves efficiency of feed utilization, promotes nutriment in feed
Digest and assimilate, enhance the immune function of animal, improve the effect of daily gain.
Effect of 1 enterococcus faecium of table to meat chicken production performance
Although above having used general explanation, specific embodiment and experiment, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
SEQUENCE LISTING
<110>China Agricultural University
<120>A kind of probiotic enterococcus faecium of high adherency and its application
<130> KHP161117095.3
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1455
<212> DNA
<213> Enterococcus faecium
<400> 1
gtgctataca tgcaagtcga acgcttcttt ttccaccgga gcttgctcca ccggaaaaag 60
aggagtggcg aacgggtgag taacacgtgg gtaacctgcc catcagaagg ggataacact 120
tggaaacagg tgctaatacc gtataacaat caaaaccgca tggttttgat ttgaaaggcg 180
ctttcgggtg tcgctgatgg atggacccgc ggtgcattag ctagttggtg aggtaacggc 240
tcaccaaggc cacgatgcat agccgacctg agagggtgat cggccacatt gggactgaga 300
cacggcccaa actcctacgg gaggcagcag tagggaatct tcggcaatgg acgaaagtct 360
gaccgagcaa cgccgcgtga gtgaagaagg ttttcggatc gtaaaactct gttgttagag 420
aagaacaagg atgagagtaa ctgttcatcc cttgacggta tctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gatttattgg 540
gcgtaaagcg agcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600
gagggtcatt ggaaactggg agacttgagt gcagaagagg agagtggaat tccatgtgta 660
gcggtgaaat gcgtagatat atggaggaac accagtggcg aaggcggctc tctggtctgt 720
aactgacgct gaggctcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttgga gggtttccgc ccttcagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacgac cgcaaggttg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctttgacc actctagaga tagagcttcc ccttcggggg caaagtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt attgttagtt gccatcattc agttgggcac tctagcaaga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatgggaagt acaacgagtt gcgaagtcgc gaggctaagc taatctctta 1260
aagcttctct cagttcggat tgcaggctgc aactcgcctg catgaagccg gaatcgctag 1320
taatcgcgga tcagcacgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttggag ccagccgcct 1440
aaggtgggat agatg 1455
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
agaaaggagg tgatccagcc c 21
Claims (8)
1. a kind of enterococcus faecium (Enterococcus faecium), entitled PNC01, deposit number CGMCC
NO.13229。
2. the microbial inoculum containing enterococcus faecium described in claim 1.
3. the application of enterococcus faecium described in claim 1 or the microbial inoculum described in claim 2 in probiotics is prepared.
4. the drug containing enterococcus faecium described in claim 1 or the microbial inoculum described in claim 2.
5. application of the microbial inoculum described in enterococcus faecium described in claim 1 or claim 2 in feed addictive is prepared.
6. the feed addictive containing enterococcus faecium described in claim 1 or the microbial inoculum described in claim 2.
7. the application of enterococcus faecium described in claim 1 or the microbial inoculum described in claim 2 in growth of animal is promoted.
8. the use as claimed in claim 7, which is characterized in that enterococcus faecium described in claim 1 is added in animal feed
Or the microbial inoculum described in claim 2, the viable count for making enterococcus faecium in feed are 5 × 108-1×109CFU/kg。
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| CN112063566A (en) * | 2020-09-23 | 2020-12-11 | 江苏澳华生物科技研究院有限公司 | Enterococcus faecium and application thereof |
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| CN107828706A (en) * | 2017-10-18 | 2018-03-23 | 东北农业大学 | A kind of eGFP is marked, anti-swine infectious enterogastritis and the restructuring VREF vaccine strain of pig epidemic diarrhea and its application |
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