CN105733977B - Probiotic feed additive - Google Patents
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Abstract
The invention discloses the probiotic feed additives that one plant of enterococcus faecium WEFA23 with external norcholesterol effect is made into, the bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place is Wuhan City's China typical culture collection center, deposit number is CCTCC M 2015785, and the deposit date is on December 28th, 2015.Enterococcus faecium of the present invention is located away from infant faeces, is gram-positive bacteria, and pole bile tolerance can keep higher survival rate in gastro-intestinal Fluid.And high yield bile salt hydrolase, there is extremely strong external Lowering cholesterol effect.Enterococcus faecium WEFA23 of the present invention can be used for developing the functional food of reducing blood lipid, prepare enteral microecological formulation, or treat or prevent enterogastric diseases with drug combination.
Description
Technical field
The invention belongs to microorganism field, the present invention is to disclose a kind of enterococcus faecium nterococcus faecium
WEFA23 and its application.
Background technique
Enterococcus faecium (Enterococcus faecium) is a kind of anaerobism, asporogenous gram-positive bacteria, thallus
It is rounded or oval, be in catenation, do not produce gemma.It can form that canescence, opaque, surface is smooth, straight on blood plate
Diameter is the circular colonies of 0.5~1mm.The life of enterococcus faecium and the mankind are closely related, in addition to being widely present in human intestine,
Also it is widely present in dairy products, meat, in vegetables.At present enterococcus faecium be widely used in food fermentation, Production of Livestock and Poultry and
Health care.
Summary of the invention
It is an object of the present invention to provide the probiotics enterococcus faecium and its application of one plant of norcholesterol function admirable.
The present invention isolates enterococcus faecium from infant faeces, has good prebiotic performance, especially pole bile tolerance, resistance to
By gastro-intestinal Fluid, high yield bile salt hydrolase and there is extremely strong external norcholesterol ability.And virulence gene detected is not carried,
There is no virulence phenotype detected, it is sensitive to common antibiotics, it is determined as safe bacterial strain.
Enterococcus faecium WEFA23 (Enterococcus faecium WEFA23) of the present invention was protected on December 28th, 2015
Ensconce the Wuhan Wuhan University, China China typical culture collection center, number: CCTCC NO:M 2015785.
Enterococcus faecium WEFA23 of the present invention can be grown in brain heart infusion (BHI) culture medium, belong to amphimicrobian
Growth, does not produce gemma.For the excellent performance for steadily saving enterococcus faecium of the present invention of holding time, we save it in containing
The mixing of 15% glycerol saves in liquid, freezes in -80 DEG C, or saves it in 10% skimmed milk and carry out freeze-drying preservation.
In order to be identified microorganism and be classified, 16S rRNA alkali has been carried out to enterococcus faecium WEFA23 of the present invention
Basic sequence analysis, result is to show with enterococcus faecium (Enterococcus faecium DO chromosome) highest
Homology (99%) is shown and the affiliation on the highest molecular systematics of enterococcus faecium.Also, by this microbial identification
For enterococcus faecium, and it is named as enterococcus faecium WEFA23.Wuhan City's Chinese Typical Representative culture is stored on December 28th, 2015
Collection, deposit number are CCTCC M2015785.The base sequence of the 16S rRNA gene of enterococcus faecium WEFA23 is as follows:
CTGCGGTGCTATAATGCAGTCGAACGCTTCTTTTTCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTG
GCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTAT
AACAATCAAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGC
TAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGA
GACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCG
CGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCT
TGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCC
GGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGT
CATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATG
GAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGA
TTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCT
AACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCCCAAGC
GGTGGAGCATGTGGTTTAATTGGAAGCAACGCGAAGACCCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGAT
AGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG
TCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGCCACTCTAGCAAGACTGCCGGTGACAAAC
CGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGCCCTGGGCTACACACGTGCTCCAATGGGAAGTAC
AACGAGTCGCGAAGTCGCGAGGTTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTCCAGGCTGCAACTCGCCT
GCATGAAGCCGGAATCGCTAGTAATCGCGGATCACCACGCCGCGGTGAATACGTTCCCGGCCCTTGTACACACCGCC
CGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCTAAGTGATGAAT
Enterococcus faecium WEFA23 of the present invention, which compares conventional enterococcus faecium, has significant higher bile tolerance ability, to stomach
The tolerance of intestinal juice is strong, can use as oral preparation.Enterococcus faecium high yield bile salt hydrolase of the present invention simultaneously, has
Stronger Lowering cholesterol effect has the potential of the probiotics of exploitation blood fat reducing function.From safe handling angle, the dung intestines
Coccus is free of virulence gene detected and phenotype, sensitive to common antibiotics, can use safely.
Further, the invention discloses:
A kind of probiotic feed additive, including enterococcus faecium WEFA23 are stored in Wuhan City on December 28th, 2015
China typical culture collection center, deposit number are CCTCC M2015785.
The probiotic feed additive further includes lactobacillus plantarum and Lactobacillus rhamnosus；Compare dung by freeze-dried powder quality
Enterococcus WEFA23: lactobacillus bulgaricus: streptococcus thermophilus=1:1:1；To guarantee better active effect, three kinds of freeze-dried vaccines
The viable count of powder is all not less than 109cfu/g。
Preparation method is as follows:
1) preparation of enterococcus faecium WEFA23 freeze-dried powder: brain heart infusion will be inoculated into from enterococcus faecium WEFA23 glycerol tube
(BHI) in culture medium, 37 DEG C of constant temperature stationary culture 12h, the strain activated.Seed liquor is inoculated by 5% inoculum concentration
The fermentation liquid of acquisition for 24 hours, is finally centrifuged by 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture in 4 DEG C of 10000rpm in BHI culture medium
10min collects thallus and skimmed milk power progress frozen drying is added.
2) brain heart infusion (BHI) culture the preparation of lactobacillus plantarum freeze-dried powder: will be inoculated into from lactobacillus plantarum glycerol tube
In base, 37 DEG C of constant temperature stationary culture 12h, the strain activated.Seed liquor is inoculated into BHI culture medium by 5% inoculum concentration
In 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture for 24 hours, the fermentation liquid of acquisition is finally centrifuged 10min in 4 DEG C of 10000rpm, is collected
Thallus is added skimmed milk power and carries out frozen drying.
3) brain heart infusion (BHI) training the preparation of Lactobacillus rhamnosus freeze-dried powder: will be inoculated into from lactobacillus plantarum glycerol tube
It supports in base, 37 DEG C of constant temperature stationary culture 12h, the strain activated.Seed liquor is inoculated into BHI culture by 5% inoculum concentration
The fermentation liquid of acquisition for 24 hours, is finally centrifuged 10min in 4 DEG C of 10000rpm, received by 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture in base
Collect thallus and skimmed milk power progress frozen drying is added.
4) 3 kinds of dry powder formulations are mixed by 1:1:1 to get this product.
Using probiotic feed additive of the present invention, diarrhea caused by domestic animal bacterial infection can be prevented and treated, and
And reach stimulation animal body immunity, improve animal gastrointestinal tract digestion power, increase the conversion ratio of feed, promotes growth of animal
Effect.
Detailed description of the invention
The bile tolerance of Fig. 1 enterococcus faecium WEFA23
Fig. 2 enterococcus faecium WEFA23 is to the survival condition after in-vitro simulated gastrointestinal transit
The external degrading rate of cholesterol of Fig. 3 enterococcus faecium WEFA23
Each bacterium survival rate of Fig. 4 freeze-dried powder.
Specific embodiment
The present invention is explained further below in conjunction with example, but embodiment is merely to illustrate the present invention, and of the invention answers
It cannot be limited in any way by these embodiments with range.
Embodiment 1: the separation and identification of enterococcus faecium WEFA23
The dilution of infant faeces (sampling from healthcare hospital for women & children, Jiangxi Province) is coated on brain heart infusion (BHI) agar plate
On, for 24 hours, the bacterium colony for using oese picking single carries out bacterium colony PCR as template to Anaerobic culturel.Positive PCR product is sent to public affairs
Department's sequencing, 16s rRNA base sequence is compared with database, the base sequence and dung intestines of enterococcus faecium WEFA23
Coccus (Enterococcus faecium strain pq117) has highest homology (99.9%), therefore is confirmed to be
Enterococcus faecium is named as enterococcus faecium WEFA23, is stored in Wuhan City's China typical culture collection on December 28th, 2015
Center, deposit number are CCTCC M 2015785.
Brain heart infusion (BHI) fluid nutrient medium is formulated as follows: egg the preparation method comprises the following steps: finished commercial prod (the rich biology in sea, Qingdao)
White peptone 10g is dehydrated small bovine brain leaching powder 12.5g, is dehydrated beef heart infusion 5.0g, sodium chloride 5.0g, glucose 2.0g, disodium hydrogen phosphate
2.5g, pH value 7.4 ± 0.2；38.5g finished powder is weighed, stirring and dissolving is in 1000mL distilled water, 121 DEG C of high pressure sterilizations
15min is saved backup.
Brain heart infusion (BHI) agar medium the preparation method comprises the following steps: BHI fluid nutrient medium be added 1.5% (w/v) agar powder,
121 DEG C of high pressure sterilization 15min, pour plate when culture medium temperature is down to about 50 DEG C.
Embodiment 2: bile salt hydrolase vitality test
Qualitative analysis: preparing containing 5mM Glycocine deoxycholic acid GDCA (or 0.5mM Taurodeoxycholic acid TDCA) and
0.37g/L CaCl2BHI solid medium tablets, take 2 μ L enterococcus faecium WEFA23 bacteria suspensions (about 108Cfu/mL it) puts to upper
It states in solid medium tablets, is compareed so that the MRS solid plate of cholate is not added, every plant of bacterium is cooked three in parallel.37 DEG C of anaerobism trainings
After supporting 72h, whether observation culture basal part or periphery of bacterial colonies have the precipitating circle of white to generate.Wherein "+" representative has precipitating to enclose
It generates；"-" is represented to be generated without precipitating circle.
Quantitative analysis: the bacterium solution sufficiently activated is inoculated by 1% inoculum concentration containing 30mL BHI fluid nutrient medium
In conical flask, 37 DEG C of stationary culture 12h.Last thalline were collected by centrifugation (10,000 × g；4℃；10min), then with 0.1M phosphate
Twice, adjustment cell concentration makes its absorbance 10 or so at 600nm for buffer (pH 7.0) washing centrifugation.In order to prevent
The oxidation of BSH is usually added into the dithiothreitol (DTT) (DTT) of 10mM.Take the above-mentioned cell suspension of 1mL, ultrasonication 20min (work
Make the time: the intermittent time=1:3), it is centrifuged (10,000 × g immediately；10min；4 DEG C) removal cell fragment, obtain cell-free mention
Liquid (cell free extract) is taken, -20 DEG C of preservations can be placed on.Above-mentioned cell extract is taken, detects cholate using ninhydrin method
Hydrolase BSH enzyme activity.
1 enterococcus faecium WEFA23 bile salt hydrolase activity of table
Experimental result: as shown in table 1, enterococcus faecium WEFA23 in the BHI solid medium tablets containing 5mM GDCA and
There is apparent white precipitate circle in the BHI solid medium tablets of 5mM GDCA, shows enterococcus faecium WEFA23 to TDCA
There is hydrolysis with GDCA.It is determined by BSH activity of the ninhydrin method to bacterial strain, finds WEFA23 pairs of enterococcus faecium
The activity of GDCA is higher than the activity of TDCA, reaches 1.86 ± 0.005U/mg. compared to existing conventional enterococcus faecium, enzyme activity
It is considerably higher.Bile salt hydrolase not only assists in self tolerance cholate, and can reduce human serum cholesterol.The bacterium high yield
Bile salt hydrolase implies that the bacterium has the potential of exploitation cholesterol-lowering products.
Embodiment 3: external norcholesterol effect
Cholesterol assimilation: the bacterium solution sufficiently activated is inoculated by 1% inoculum concentration containing 100 μ g/mL water solubilitys
In the BHI fluid nutrient medium of cholesterol, the nonvaccinated BHI fluid nutrient medium containing 100 μ g/mL water-soluble cholesterols is pair
According to.37 DEG C of Anaerobic culturels for 24 hours after, 10000rpm/min be centrifuged 5min, take supernatant, with O-phthalic acid system measurement supernatant in gallbladder
Sterol content.
Cholesterol co-precipitation: the bacterium solution sufficiently activated is inoculated by 1% inoculum concentration water-soluble containing 100ug/ml
In the BHI fluid nutrient medium of property cholesterol and 0.3% bovine bile.It is nonvaccinated containing 100ug/ml water-soluble cholesterol and
The BHI fluid nutrient medium of 0.3% bovine bile is control.Remaining operation is same as above.
Degrading rate of cholesterol (%)=(A-B)/A × 100%
Cholesterol level in culture solution after wherein A- is inoculated with；Cholesterol level in culture solution after B- culture 48h.
Experimental result: as shown in figure 3, enterococcus faecium WEFA23 presents high degrading rate of cholesterol.Be free of 0.3%
Cholate in the case where the degradation rate of cholesterol reach (51.5 ± 1.18) %, the cholesterol in the case where containing 0.3% cholate
Degradation rate is up to (86.1 ± 1.36) %.Compared to conventional enterococcus faecium, degradation rate is higher.Illustrate that the bacterium can effectively drop
Low cholesterol can be studied further, and norcholesterol functional food and probiotics are developed.
Embodiment 4: bile tolerance evaluation
The single colonie of enterococcus faecium is inoculated in 5mL BHI fluid nutrient medium, for 24 hours, passage is twice for 37 DEG C of anaerobic fermentations.With
1% inoculum concentration is inoculated in respectively in the BHI fluid nutrient medium that gallbladder salinity is 0,0.3%, 0.5% and 1.0%, in 37 DEG C of anaerobism
Culture for 24 hours, measures OD value at 600nm, using the OD value of 0% bovine bile culture as positive control, calculates relative survival rate.
Relative survival rate (%)=(A-A0)/(A1-A0)
In formula: A0The OD value of 0h under-difference gallbladder salinity；OD value under A-difference gallbladder salinity for 24 hours；A1- 0% cholate
OD value under concentration for 24 hours.
Experimental result:
As shown in Figure 1, enterococcus faecium WEFA23 cholate solubility be 0.3%, 0.5%, 1.0% culture medium culture for 24 hours
Afterwards, relative survival rate is respectively 99.01%, 80.75%, 75.06%, illustrates that enterococcus faecium WEFA23 has extremely strong cholate
Tolerance.
Embodiment 5: in-vitro simulated gastro-intestinal Fluid transhipment
It takes the enterococcus faecium WEFA23 activated entirely to be incubated overnight with 1% inoculum concentration, collects thallus, be resuspended in 10%
In skimmed milk.And it is inoculated in simulate the gastric juice and intestinal juice respectively with 10% inoculum concentration, it is incubated for 120min, counts viable count.
Experimental result:
As shown in Figure 2 A and 2 B, enterococcus faecium WEFA23 is only having dropped 0.11lg through gastric juice 120min viable count
Cfu/mL is only having dropped 0.42lg cfu/mL through gastric juice 120min viable count.As a result it shows that enterococcus faecium WEFA23 has to open
Send out the potential of oral probiotics.
Embodiment 6: the screening of enterococcus faecium WEFA23 virulence gene and phenotype
The screening of virulence gene: mesh is extracted using gram-positive bacterium genome DNA extracting reagent kit (Beijing Zhuan Meng)
After the genomic DNA for marking bacterial strain enterococcus faecium WEFA23, virulence and antibiotic resistance genes in PCR screening aimed strain are utilized.
Amplified production is observed in gel imaging system after 1.0% agarose gel electrophoresis.Selected cls gene and primer relevant information
See Table 2 for details.
2 virulence gene of table and primer relevant information
Specific steps are as follows:
The reaction system of PCR amplification is as follows:
PCR amplification program:
Haemolysis Phenotypic examination: it completely after activation strain to be tested, is drawn with oese and is being added to 5% sterile de- fiber Sheep Blood
BHI plate on, 37 DEG C of constant temperature stationary cultures are for 24 hours；It is grown to its single colonie, its haemolysis circle situation is observed, if periphery of bacterial colonies goes out
Now clearly transparent ring is judged as positive.Every group is repeated 3 times.
The detection of Gelatin phenotype: taking and activate strain to be tested completely, is drawn with oese and is being added to 1.5% skimmed milk
BHI plate on, 37 DEG C of constant temperature stationary cultures are for 24 hours.It grows, visually observes to its single colonie, if periphery of bacterial colonies occurs clearly
Halation is judged as positive.Every group is repeated 3 times.
The Forming ability of biomembrane detects: taking and activates strain to be tested completely, bacterial concentration is adjusted to about 108cfu/ml。
It is diluted with fresh BHI culture medium by 1:100,200uL is taken to be seeded in 96 hole steril cell culture plates, separately take that 200uL's is new
Fresh BHI culture medium is as control.37 DEG C of stationary cultures remove the dung intestines ball that swims for 24 hours, with PBS (pH=7.2) buffer board-washing 3 times
Bacterium is inverted drying, is washed twice with 1% violet staining 15min, PBS to colourless, with acetone and alcohol mixeding liquid (20:80vol/
Vol) dissolving crystallized purple reads each hole optical density (OD) value at 595nm wavelength.If the surveyed OD value of control wells is ODc, bacterial strain is raw
Object film Forming ability is divided into four classes: OD≤ODc is negative (-), i.e. inanimate object film Forming ability；ODc < OD≤DD is weakly positive
(+)；2ODc < OD≤4ODc is moderate positive (++)；OD >=4ODc is strong positive (+++)
Experimental result: enterococcus faecium WEFA23 is both without any virulence gene detected, also without any virulence detected
Phenotype, preliminary judgement are safe.
Embodiment 6: antibiotics sensitivity experiment
Its sensibility to antibiotic is detected using microorganism medicine susceptability test paper (safe biology).It is 10 by solubility7cfu/mL
Lactobacillus plantarum be coated on MRS agar plate, drug sensitive test paper is placed on it, in 37 DEG C Anaerobic culturel 18 hours, with card
Ruler measures its transparent loop diameter, compares its sensibility to corresponding antibiotic referring to susceptibility judgment criteria table (safe biology).
Experimental result:
Sensibility of the 3 enterococcus faecium WEFA23 of table to 7 kinds of antibiotic
S: sensitive, I: neutral, R: tolerance
Experimental result: enterococcus faecium WEFA23 is resistant to 3 kinds of antibiotic, and respectively rifampin, Ciprofloxacin, celebrating is big mould
Element.To 3 kinds of antibiotic sensitives, respectively ampicillin, vancomycin, chloramphenicol.To erythromycin intermediary.(such as table 3).
Embodiment 7: composite probiotics feed additive preparation process
Composite probiotics feed additive, including enterococcus faecium WEFA23, lactobacillus plantarum and Lactobacillus rhamnosus；By jelly
Dry powder quality is than enterococcus faecium WEFA23: lactobacillus bulgaricus: streptococcus thermophilus=1:1:1.Wherein three kinds of bacterium are all not less than
109cfu/g。
Preparation method is as follows:
1) preparation of enterococcus faecium WEFA23 freeze-dried powder: brain heart infusion will be inoculated into from enterococcus faecium WEFA23 glycerol tube
(BHI) in culture medium, 37 DEG C of constant temperature stationary culture 12h, the strain activated.Seed liquor is inoculated by 5% inoculum concentration
The fermentation liquid of acquisition, is finally centrifuged by 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture 18h in BHI culture medium in 4 DEG C of 10000rpm
10min collects thallus, and skimmed milk power is added and concentrates it to 1010Cfu/ml carries out frozen drying.
2) brain heart infusion (BHI) culture the preparation of lactobacillus plantarum freeze-dried powder: will be inoculated into from lactobacillus plantarum glycerol tube
In base, 37 DEG C of constant temperature stationary culture 12h, the strain activated.Seed liquor is inoculated into BHI culture medium by 5% inoculum concentration
In 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture 18h, the fermentation liquid of acquisition is finally centrifuged 10min in 4 DEG C of 10000rpm, is added
Skimmed milk power concentrates it to 1010Cfu/ml carries out frozen drying.
3) brain heart infusion (BHI) training the preparation of Lactobacillus rhamnosus freeze-dried powder: will be inoculated into from lactobacillus plantarum glycerol tube
It supports in base, 37 DEG C of constant temperature stationary culture 12h, the strain activated.Seed liquor is inoculated into BHI culture by 5% inoculum concentration
The fermentation liquid of acquisition is finally centrifuged 10min in 4 DEG C of 10000rpm, added by 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture 18h in base
Enter skimmed milk power and concentrates it to 1010Cfu/ml carries out frozen drying.
4) count plate
0.1g freeze-dried powder is accurately weighed, mortar grinder crushes, until 9.9 sterile salines, stirring 20min is mixed.Take lml
Bacterium solution after mixing is added in the test tube equipped with 1mL physiological saline, and concussion mixes, by sample to be tested through a series of 10 times of gradients
Dilution, takes 0.2mL to be added on the plate prepared diluted bacterium solution, bacterium solution is then coated with entire plate table with spreading rod
Face is cultivated at 37 DEG C, calculates clump count, then calculate total viable count contained by every gram of freeze-dried powder by upper formula.Every gram of original bacteria liquid is living
Bacterium number=same dilution three or more repeats flat-plate bacterial colony average × extension rate × 5.
4) 3 kinds of dry powder formulations are mixed by 1:1:1 to get this product.
Experimental result is shown in Fig. 4: the survival rate of enterococcus faecium WEFA23 freeze-dried powder is 12%, the survival of lactobacillus plantarum freeze-dried powder
Rate is 8%, and Lactobacillus rhamnosus freeze-dried powder survival rate reaches 15%.Finally, the final viable count of three kinds of bacterium is all up to 109cfu/
g。
Using probiotic feed additive of the present invention due to the participation of enterococcus faecium WEFA23, compared to conventional enterococcus faecium
With stronger reduction cholesterol and acid-fast ability；Diarrhea caused by domestic animal bacterial infection can be prevented and treated, and is reached
To stimulation animal body immunity, animal gastrointestinal tract digestion power is improved, promotes growth of animal.
SEQUENCE LISTING
<110>University Of Nanchang
<120>probiotic feed additive
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1452
<212> DNA
<213> Enterococcus faecium
<400> 1
ctgcggtgct ataatgcagt cgaacgcttc tttttcaccg gagcttgctc caccggaaaa 60
agaggagtgg cgaacgggtg agtaacacgt gggtaacctg cccatcagaa ggggataaca 120
cttggaaaca ggtgctaata ccgtataaca atcaaaaccg catggttttg atttgaaagg 180
cgctttcggg tgtcgctgat ggatggaccc gcggtgcatt agctagttgg tgaggtaacg 240
gctcaccaag gccacgatgc atagccgacc tgagagggtg atcggccaca ttgggactga 300
gacacggccc aaactcctac gggaggcagc agtagggaat cttcggcaat ggacgaaagt 360
ctgaccgagc aacgccgcgt gagtgaagaa ggttttcgga tcgtaaaact ctgttgttag 420
agaagaacaa ggatgagagt aactgttcat cccttgacgg tatctaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggatttatt 540
gggcgtaaag cgagcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600
gggagggtca ttggaaactg ggagacttga gtgcagaaga ggagagtgga attccatgtg 660
tagcggtgaa atgcgtagat atatggagga acaccagtgg cgaaggcggc tctctggtct 720
gtaactgacg ctgaggctcg aaagcgtggg gagcaaacag gattagatac cctggtagtc 780
cacgccgtaa acgatgagtg ctaagtgttg gagggtttcc gcccttcagt gctgcagcta 840
acgcattaag cattccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac 900
gggggcccgc ccaagcggtg gagcatgtgg tttaattgga agcaacgcga agacccttac 960
caggtcttga catcctttga ccactctaga gatagagctt ccccttcggg ggcaaagtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttattgttag ttgccatcat tcagttggcc actctagcaa gactgccggt 1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg ccctgggcta 1200
cacacgtgct ccaatgggaa gtacaacgag tcgcgaagtc gcgaggttaa gctaatctct 1260
taaagcttct ctcagttcgg attccaggct gcaactcgcc tgcatgaagc cggaatcgct 1320
agtaatcgcg gatcaccacg ccgcggtgaa tacgttcccg gcccttgtac acaccgcccg 1380
tcacaccacg agagtttgta acacccgaag tcggtgaggt aacctttgga gccagccgcc 1440
taagtgatga at 1452
Claims (3)
1. probiotic feed additive, it is characterised in that including enterococcus faecium (Enterococcus faecium) WEFA23；
Enterococcus faecium (Enterococcus faecium) WEFA23 was stored in China, Wuhan City on December 28th, 2015
Type Tissue Collection, deposit number are CCTCC NO:M 2015785.
2. probiotic feed additive according to claim 1, it is characterised in that further include lactobacillus plantarum and rhamnose cream
Bacillus；By freeze-dried powder quality than enterococcus faecium WEFA23: lactobacillus plantarum: Lactobacillus rhamnosus=1:1:1.
3. probiotic feed additive according to claim 2, preparation method is as follows:
1) enterococcus faecium WEFA23 the preparation of enterococcus faecium WEFA23 freeze-dried powder: is inoculated into brain heart infusion (BHI) from glycerol tube
In culture medium, 37 DEG C of constant temperature stationary culture 12h, the strain activated；Seed liquor is inoculated into BHI training by 5% inoculum concentration
It supports 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture in base and for 24 hours, the fermentation liquid of acquisition is finally centrifuged 10min in 4 DEG C of 10000rpm,
It collects thallus and skimmed milk power progress frozen drying is added；
2) preparation of lactobacillus plantarum freeze-dried powder: lactobacillus plantarum is inoculated into brain heart infusion (BHI) culture medium from glycerol tube,
Seed liquor is inoculated into BHI culture medium 37 DEG C by 5% inoculum concentration by 37 DEG C of constant temperature stationary culture 12h, the strain activated
The fermentation liquid of acquisition for 24 hours, is finally centrifuged 10min in 4 DEG C of 10000rpm by anaerobic fermentation, that is, deep layer stationary culture, is collected thallus and is added
Enter skimmed milk power and carries out frozen drying；
3) Lactobacillus rhamnosus the preparation of Lactobacillus rhamnosus freeze-dried powder: is inoculated into brain heart infusion (BHI) culture from glycerol tube
In base, seed liquor is inoculated into BHI culture medium by 5% inoculum concentration by 37 DEG C of constant temperature stationary culture 12h, the strain activated
In 37 DEG C of anaerobic fermentations, that is, deep layer stationary culture for 24 hours, the fermentation liquid of acquisition is finally centrifuged 10min in 4 DEG C of 10000rpm, is collected
Thallus is added skimmed milk power and carries out frozen drying；
4) by enterococcus faecium WEFA23: lactobacillus plantarum: Lactobacillus rhamnosus freeze-dried powder preparation 1:1:1 in mass ratio mixing, i.e.,
?.
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| CN102093967A (en) * | 2010-12-02 | 2011-06-15 | 中国农业科学院特产研究所 | Mink source enterococcus faecium and application thereof |
| CN102559546A (en) * | 2011-12-15 | 2012-07-11 | 李绩 | Composite microbial leaven |
| CN103843972A (en) * | 2014-02-28 | 2014-06-11 | 广州优锐生物科技有限公司 | Feed additive, preparation method and application of feed additive |
| CN104560820A (en) * | 2014-12-30 | 2015-04-29 | 杭州师范大学 | Enterococcus faecium KQ2.6 and application thereof |
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| CN102093967A (en) * | 2010-12-02 | 2011-06-15 | 中国农业科学院特产研究所 | Mink source enterococcus faecium and application thereof |
| CN102559546A (en) * | 2011-12-15 | 2012-07-11 | 李绩 | Composite microbial leaven |
| CN103843972A (en) * | 2014-02-28 | 2014-06-11 | 广州优锐生物科技有限公司 | Feed additive, preparation method and application of feed additive |
| CN104560820A (en) * | 2014-12-30 | 2015-04-29 | 杭州师范大学 | Enterococcus faecium KQ2.6 and application thereof |
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