CN108641979A - A kind of enterococcus faecium, its high density fermentation cultural method and probiotics prepared therefrom - Google Patents
A kind of enterococcus faecium, its high density fermentation cultural method and probiotics prepared therefrom Download PDFInfo
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Abstract
A kind of enterococcus faecium of present invention proposition, its fermentation process in high density and probiotics prepared therefrom, belong to lactic acid bacteria culture technique field, the significant manure enterococcin strain of probiotic and resistance is filtered out as feed addictive, can effectively replace part antibiotic.The enterococcus faecium Enterococcus faecium that the technical solution provides are E13 5, and deposit number is CCTCC M2017191.Enterococcus faecium provided by the invention can be used as safe and efficient novel fodder additive and carry out partial alternative antibiotic usage, so as to meet the feed industry of modernization and the demand of livestock breeding industry.
Description
Technical field
The invention belongs to lactic acid bacteria culture technique field more particularly to a kind of enterococcus faecium, its high density fermentation culture sides
Method and probiotics prepared therefrom.
Background technology
Enterococcus faecium belongs to Streptococcaceae, Enterococcus, Gram-positive, amphimicrobian.It is with excellent life
Object characteristic is commensal gut bacterium, can form dominant microflora in enteron aisle, since growth speed is fast, has preferable adhesion strength,
Lactic acid and some antibacterial materials are generated, take advantage status in new born 2-3 days of many animals, beneficial to increase
The quantity of bacterium inhibits harmful bacteria, promotes intestinal health, adjusts the microecological balance of enteron aisle and can prevent diarrhea, is grown improving
Aspect of performance has notable effect, effect suitable with antibiotic.
Enterococcus faecium not only has good biological characteristics, additionally it is possible to improve the immunocompetence of animal body.By dung intestines ball
The probiotics preparation of bacterium composition can improve the conversion ratio of feed, improve livestock products quality, reduce diarrhea rate and the death rate.
Therefore, enterococcus faecium has a wide range of applications in livestock and poultry breeding industry, and probiotics are because having environmental-friendly, nothing
The advantages that residual, is increasingly being applied to Animal husbandry production, the protection and aquaculture to breeding ecological environment it is sustainable
Development has great importance.
However, the heat resistance of existing enterococcus faecium, stomach juice-resistant, bile tolerance and fermenting property etc. do not adapt to it is growing
Industrialization demand, the feed industry of modernization and the demand of livestock breeding industry cannot be met, therefore, filtered out probiotic
And the significant manure enterococcin strain of resistance becomes a big bottleneck problem of animal husbandry development.In addition, the lasting feeding of antibiotic
The medicament residue etc. resulted in the drug resistance of bacterium, livestock products increasingly aggravates.Probiotics is as a kind of nontoxic, noresidue, nothing
The feed addictive of drug resistance, it has also become one of effective antibiotic substitute products.Therefore, safe and efficient novel feeding is found
Feed additives, which carry out partial alternative antibiotic, becomes aquaculture urgent problem to be solved.
Invention content
A kind of enterococcus faecium of present invention proposition, its high density fermentation cultural method and probiotics prepared therefrom, sieve
The significant manure enterococcin strain of probiotic and resistance is selected as feed addictive, can effectively replace part antibiotic.
In order to achieve the above object, the present invention provides an Enterococcus faecalis, the enterococcus faecium Enterococcus
Faecium is E13-5, and deposit number is CCTCC M2017191.
The enterococcus faecium Enterococcus faecium that the present invention also provides a kind of as described in above-mentioned technical proposal
The high density fermentation cultural method of E13-5, includes the following steps：
Level-one produces seed culture：Take the inclined-plane that MRS culture mediums are inoculated under the basic bacteria aseptic condition of enterococcus faecium
Carry out pure culture, test tube is after 37 DEG C of -40 DEG C of stationary cultures 24-48 hour, microscopy qualification, as level-one production seed；
Two level produces seed culture：Level-one production seed inclined-plane is taken, sterile saline is added into strain inclined plane will be oblique
Bacterium colony on face is washed down, is inoculated into secondary seed culture solution according to 1% inoculum concentration, and every bottle of liquid amount is 300-
400mL, 37 DEG C of -40 DEG C of stationary cultures 14-16 hours, microscopy is without miscellaneous bacteria, and thalline is in division stage, after the assay was approved, as
Two level produces seed；
Three-level produces seed culture：Secondary seed culture solution is taken to be inoculated into the training of three-level seed according to the inoculum concentration of 5-10%
In nutrient solution, fermentation temperature is 37 DEG C -40 DEG C, and pH controls are cultivated 6-8 hours, fermented in 7.0, speed of agitator 50r/min
Tank pressure is set as 0.05mpa in journey, and when the content of sugar is less than 0.6%, and microscopy is without miscellaneous bacteria, transferred species carries out tank fermentation；
Upper tank fermentation：Three-level seed culture fluid is taken to be inoculated into fermented and cultured tank according to the inoculum concentration of 5-10%, fermentation temperature
Degree is 37 DEG C -40 DEG C, and pH controls are 6.5, speed of agitator 150r/min, in fermentation process dissolved oxygen amount control 10% or more,
Tank pressure, tank pressure not more than 0.1mpa are increased according to dissolved oxygen；It is mended using fed-batch mode when sugared content is less than 1.0%
Material, when sugared content no longer reduces, bacterium turbidity is not further added by, and microscopy is without miscellaneous bacteria, fermented and cultured terminates.
Preferably, in terms of mass fraction, the formula of the secondary seed culture solution includes 1% glucose, 1% albumen
Peptone, 1% yeast extract and 4% trace salt, pH7.0；The formula of the three-level seed culture fluid includes 2% glucose, 1.5%
Peptone, 1.5% yeast extract and 4% trace salt, pH7.0.
Preferably, in terms of mass fraction, fermented and cultured formula of liquid that when upper tank fermentation uses include 2% glucose,
2% peptone, 2% yeast extract and 4% trace salt, pH7.0.
Preferably, feed supplement state modulator when upper tank fermentation is：Sugared content control range is 0.5-1.0%, peristaltic pump
Rotating speed be 120-150rpm, the feed supplement period be 100s, aperture 50-60s.
The present invention provides a kind of enterococcus faecium Enterococcus faecium with described in technical solution as above again
E13-5 is the probiotics that primary raw material is prepared.
The preparation method for the probiotics that invention further provides a kind of as described in above-mentioned technical proposal, including it is following
Step：
Take the enterococcus faecium Enterococcus faecium E13-5 of predetermined amount under the conditions of rotating speed 10000r/min into
Row centrifugation, obtains bacterium mud, by bacterium mud and protective agent with mass ratio 1:3 ratio is sufficiently mixed, and is then lyophilized, and
Bacterium mud after freeze-drying is thoroughly mixed with auxiliary material, obtains probiotics.
Preferably, in terms of mass fraction, the protective agent include 5% sucrose, 4% sorbierite, 3% skimmed milk power,
1% gelatin and 0.5%Vc, the auxiliary material includes 49% starch, 20% lactose, 15% dextrin, 10% PVP K30,5% micro-
Crystalline cellulose and 1% chlorate.
Preferably, will be successively lyophilized in the following conditions with the well-mixed bacterium mud of protective agent：In -40 DEG C of pre-freezes
3-4 hours, it is evacuated to 20Pa or so at -40 DEG C to -45 DEG C or less, in 5 DEG C of lyophilizations 20-24 hours, is distilled at 0 DEG C
Dry 8-10 hour, in 25 DEG C of parsing-desiccations 4-6 hours, last closing vacuum 2-4 hours.
Compared with prior art, the advantages and positive effects of the present invention are：
1, enterococcus faecium provided by the invention has significant probiotic and resistance, has preferable heat-resisting, resistance to stomach
The biological characteristics of acid, bile tolerance, adapt to growing industrialization demand, meet feed industry and the poultry of modernization
Herd the demand of aquaculture；
2, present invention optimizes the trainings of the seed of the high density fermentation of enterococcus faecium Enterococcus faecium E13-5
Base and fermentation medium are supported, by the pH of control zymotic fluid, speed of agitator, dissolved oxygen amount in fermentation process and passes through control sugar
Content using stream plus by the way of carry out the strategy of batch feeding, realize the enterococcus faecium for being used to prepare probiotics
Inexpensive, efficient production；
3, the present invention is to the optimal screening of freeze drying protectant and lyophilized technique so that enterococcus faecium bacterium after freeze drying
The survival rate of strain may be up to 70% or more, and 12 months viable bacteria rates of -20 DEG C of storages solve known newborn bar still 60% or more
Bacterium survival rate in freeze-drying process is greatly reduced and loses serious problem during preserving, meanwhile, micro- life of the enterococcus faecium
State preparation has abandoned the glucose that previous preparation uses, and has selected lactose, both ensure that the energy needed for Strain survival, together
When lactose than glucose more stablize, it is not hygroscopic so that preparation drying be not easy the moisture absorption；
4, the probiotics of enterococcus faecium provided by the invention can be both added as solid can also use liquid
The oral mode of body, while also assuring that the survival of bacterial strain is unaffected.
Specific implementation mode
Below in conjunction with specific embodiment to enterococcus faecium provided by the present invention, its fermentation process in high density and by it
The probiotics of preparation are clearly and completely described, and described embodiments are only a part of the embodiments of the present invention,
Instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative labor
The every other embodiment obtained under the premise of dynamic, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of new enterococcus faecium is present embodiments provided, separation screening and identification are as follows：
The intestinal contents 1g for acquiring healthy animal rectum with sterile small spoon under aseptic condition as possible, is placed in and fills
30mL MRS culture mediums (formula peptone 10.0g/L, powdered beef 8.0g/L, yeast powder 4.0g/L, glucose 20.0g/L,
K2HPO42.0g/L, citric acid hydrogen diamine 2.0g/L, sodium acetate 5.0g/L, MgSO4 0.2g/L、MnSO40.04g/L, it spits
- 80 1.0g/L of temperature.) 100mL conical flasks in, enrichment culture 8h, then draw 1mL and 9mL sterile salines be configured to
1:10 dilutions (are configured to 10-1Dilution), 3-5min is vibrated, dilution 1mL is accurately drawn with micropipettor to Sheng
Have in the physiological saline test tube that 9 mL sterilize, vibrates 1-2min with turbula shaker, be configured to 10-2Dilution, carry out successively
10-3-10-6Dilution selects 10-3-10-6Three dilutions draw 200 μ L and are coated on MRS culture mediums respectively, and training is inverted in 37 DEG C
After supporting 48h, after repeating passage pure culture 3 times on MRS slant mediums again with oese picking single bacterium colony, picking list
It cultivates in a bacterium colony to MRS culture mediums, is saved backup at -72 DEG C with final concentration of 30% glycerine.
The single bacterium colony for taking bacterial strain after purifying 3 times simultaneously, is transferred on MRS solid mediums, in 37 DEG C of constant incubators
Culture, the features such as observing the edge of its bacterium colony, smoothness, surface gloss, viscosity, transparency, color, shape.As a result it shows
Show, bacterial strain is in well-grown on the MRS agar mediums of pH6.2-6.4, and neat in edge is smooth, and lustrous surface is sticky, impermeable
It is bright, form linen dew drop-wise circular colonies.
Picking cultivates fresh cultured object for 24 hours on MRS solid mediums, carries out Gram's staining.The results show that should
Bacterial strain is gram-positive cocci.
Physiology and biochemistry identification, catalase test, nitrate reduction examination are carried out to the gram-positive cocci bacterial strain
Test, indole test, gelatin liquefaction test and hydrogen sulfide production test are feminine gender, show that the bacterial strain belongs to genus lactubacillus.Pass through smart ammonia
Sour hydrolysis experiment, hippurate hydrolysis experiment, all kinds of sugar fermentating tests and 4 DEG C and 65 DEG C of growth tests, and with《It is common thin
Fungus strain system identification handbook》With《The taxonomic identification of lactic acid bacteria》Control, the qualification result bacterial strain are enterococcus faecium.
To specifications the step of, requires, and is extracted using the bacterial genomes of TIANGEN Biotech (Beijing) Co., Ltd.
Kit (TIANAMP Bacteria DNA Kit) extracts the genomic DNA of the Enterococcus faecalis.
The genome DNA of the Enterococcus faecalis bacterial strain is template, utilizes bacterial universal primers 27F： 5'-
AGAGTTTGATCCTGGCTCAG-3', 1492R：5'-TACCTTGTTACGACTT-3'PCR expands the 16S rRNA of the bacterial strain
Segment, PCR reaction systems are 10 × Buffer, 5.0 1.0 μ L, Primer1 (20 μm of ol/L) 1.0 of μ L, dNTP (10m mol/L)
μ L, Primer2 (20 μm of ol/L) 1.0 μ L, 2.5 μ L of template DNA, Taq enzyme (5.0U/ μ L) 2.5 μ L, 34 μ L of ultra-pure water, totally 50 μ
L。
PCR reaction conditions are：First 95 DEG C of 5min；Then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min30 s, totally 35 recycle；
Last 72 DEG C of extensions 5min.After reaction, 1% agarose gel electrophoresis detection is carried out to pcr amplification product, as a result through expanding
Increase the specific band for obtaining size about 1500bp.With the Ago-Gel purchased from TIANGEN Biotech (Beijing) Co., Ltd.
QIAquick Gel Extraction Kit recycles and purifies the purpose band, and Sangon Biotech (Shanghai) Co., Ltd. is sent to carry out sequencing.
Compared with measured sequence is carried out Blastn similarity analysis with the 16S rRNA sequences in Genbank, as a result
The 16S ribosomal RNA gene sequences such as the bacterial strain and Enterococcus faecium strain PIS01/021 it is same
Source property has reached 99%, and sequence refers to sequence table, which is enterococcus faecium Enterococcus faecium E13-
5, deposit number is CCTCC M:2017191, it is preserved in the China typical culture collection for being located at China, Wuhan, Wuhan University
Center, the deposit date is on April 13rd, 2017.
Embodiment 2
Present embodiments provide a kind of high density fermentation training of the enterococcus faecium obtained such as the separated identification of above-described embodiment
The method of supporting, it is specific as follows：
Level-one produces the fermentation culture method of seed：
The basic bacteria of enterococcus faecium Enterococcus faecium E13-5 is taken aseptically to be inoculated with MRS fine jades
The inclined-plane of fat culture medium carries out pure culture, and test tube was in 37 DEG C of -40 DEG C of stationary cultures 24-48 hours.Pass through bacterium colony observation, microscopy
Deng after the assay was approved, seed is produced as level-one, in 2-8 DEG C of preservation, the holding time is usually no more than 1 month；
Two level produces the fermentation culture method of seed：
The level-one of enterococcus faecium Enterococcus faecium E13-5 is taken to produce seed inclined-plane, into strain inclined plane
The sterile physiological saline of 4mL is added to wash down the bacterium colony on inclined-plane, secondary seed culture solution is inoculated into according to 1% inoculum concentration
In, every bottle of liquid amount is 300-400mL, 37 DEG C of -40 DEG C of stationary cultures 14-16 hours.Microscopy should be without miscellaneous bacteria, and at thalline
In division stage, after the assay was approved, seed is produced as two level, in 2-8 DEG C of preservation, the holding time is usually no more than 3 days；
Wherein, in terms of mass fraction, the formula of secondary seed culture solution includes 1% glucose, 1% peptone, 1% ferment
Mother's leaching powder and 4% trace salt, pH7.0；
Three-level produces the fermentation culture method of seed：
Take the secondary seed culture solution of enterococcus faecium Enterococcus faecium E13-5 according to the inoculation of 5-10%
Amount is inoculated into three-level seed culture fluid, and fermentation temperature is 37 DEG C -40 DEG C, and pH is controlled in 7.0, speed of agitator 50r/min,
Culture 6-8 hours, tank pressure is set as 0.05mpa in fermentation process；When the content of sugar is less than 0.6%, and microscopy should be without miscellaneous bacteria
When, can transferred species carry out tank fermentation；
Wherein, in terms of mass fraction, the formula of three-level seed culture fluid include 2% glucose, 1.5% peptone,
1.5% yeast extract and 4% trace salt, pH7.0.
Upper tank fermented and cultured is specific as follows：
The seed culture fluid of enterococcus faecium Enterococcus faecium E13-5 is taken to be connect according to the inoculum concentration of 5-10%
Kind in fermented and cultured tank, fermentation temperature is 37 DEG C -40 DEG C, and pH controls are 6.5, speed of agitator 150r/min, fermentation process
Middle dissolved oxygen amount control increases tank pressure, tank pressure not more than 0.1mpa 10% or more according to dissolved oxygen；When the content of sugar is less than
Feed supplement is carried out using fed-batch mode when 1.0%, when sugared content no longer reduces, bacterium turbidity is not further added by, and microscopy is without miscellaneous bacteria,
Fermented and cultured terminates；
Wherein, feed supplement state modulator when upper tank fermentation is：Sugared content control range is 0.5-1.0%, and peristaltic pump turns
Speed is 120-150rpm, and the feed supplement period is 100s, aperture 50-60s；In terms of mass fraction, fermentation that when upper tank fermentation uses
It includes 2% glucose, 2% peptone, 2% yeast extract and 4% trace salt, pH7.0 to cultivate formula of liquid.
The seed of the high density fermentation of enterococcus faecium Enterococcus faecium E13-5 is optimized in the present embodiment
Culture medium and fermentation medium by the pH of control zymotic fluid, speed of agitator, dissolved oxygen amount in fermentation process and pass through control
The content of sugar carries out the strategy of batch feeding in such a way that stream adds, and realizes enterococcus faecium Enterococcus faecium
The high density fermentation of E13-5,50L fermentation tanks viable count under the fermentation condition optimized reach 2.3 × 1010CFU/mL, 1 ton
Viable count may be up to 2.7 × 10 under fermentation tank optimal conditions10CFU/mL is tradition MRS trainings under the conditions of identical fermentation-scale respectively
Support the fermented viable count of base 31 times and 34 times, viable count are higher, training in the enterococcus faecium high density fermentation reported
Support the cost of base also reduces nearly 20% than MRS culture medium, realizes the low of the enterococcus faecium for being used to prepare probiotics
Cost, efficient production.
Embodiment 3
Present embodiments provide a kind of enterococcus faecium Enterococcus with the separated identification of embodiment as above
Faecium E13-5 are the preparation process for the probiotics that primary raw material is prepared, specific as follows：
Take the enterococcus faecium Enterococcus faecium E13-5 of predetermined amount under the conditions of rotating speed 10000r/min into
Row centrifugation, obtains bacterium mud, and the deslagging period is 50-60min, then by bacterium mud and protective agent (in terms of mass fraction, the protection
Agent includes 5% sucrose, 4% sorbierite, 3% skimmed milk power, 1% gelatin and 0.5%Vc) with mass ratio 1:3 ratio carries out
It is sufficiently mixed, is then lyophilized successively in the following conditions：In -40 DEG C of pre-freezes 3-4 hours, taken out at -40 DEG C to -45 DEG C or less
Vacuum is to 20Pa or so, in 5 DEG C of lyophilizations 20-25 hours, in 0 DEG C of lyophilization 8-10 hours, in 25 DEG C of parsing-desiccation 4-
6 hours, finally close vacuum 2-4 hours, and (in terms of mass fraction, the auxiliary material includes with auxiliary material by the bacterium mud after freeze-drying
49% starch, 20% lactose, 15% dextrin, 10% PVP K30,5% microcrystalline cellulose and 1% chlorate) carry out it is abundant
It is stirred, obtains probiotics.
Pass through the optimal screening to freeze drying protectant and lyophilized technique in the present embodiment so that the enterococcus faecium is being frozen
The survival rate of bacterial strain may be up to 70% or more after dry, and 12 months viable bacteria rates of -20 DEG C of storages solve known still 60% or more
Lactobacillus in freeze-drying process survival rate be greatly reduced and lose serious problem during preserving.Meanwhile the enterococcus faecium
Probiotics abandoned the glucose that previous preparation uses, and selected lactose, both ensure that the energy needed for Strain survival
Amount, while lactose is more stablized than glucose, it is not hygroscopic so that preparation drying is not easy the moisture absorption, efficiently avoids miscellaneous bacteria
Growth and breeding, the also adjustable crystallization behavior of lactose, form micro crystal so that preparation is more stablized, and bacterial strain is wrapped in fine
Its time-to-live is also just more permanent in crystal.Lactose can be transformed into lactic acid, acetic acid by the lactic acid bacterias such as enterococcus faecium, make enteron aisle PH
Value declines, these organic acids can stimulate intestines peristalsis, has whole intestines effect, further increases the function and effect of probiotics；
PVP K30, with pharmaceutically there is relatively broad application, for international one of the three big medicinal new accessories advocated, can be used in people
Make adhesive, cosolvent, dispersant, the stabilizer of enzyme and heat-sensitive drug also acts as cryopreservative, PVP K30 makes
With so that probiotics of the enterococcus faecium can be both added as solid can also by the way of liquid oral,
Also assure that the survival of bacterial strain is unaffected simultaneously.
Embodiment 4
Present embodiments provide a kind of enterococcus faecium Enterococcus obtained such as above-described embodiment separation identification
The probiotic and resistance performance detection of faecium E13-5, wherein, stomach juice-resistant heat-resisting to its, bile tolerance biological characteristics
Testing result is as shown in table 1：
1 enterococcus faecium of table is heat-resisting, stomach juice-resistant, bile tolerance testing result
| Viable count | Survival rate (based on 100%) | |
| Physiological saline | 6.9×109cfu | -- |
| 60 DEG C of effect 10min | 6.7×109cfu | 97.10% |
| 60 DEG C of effect 30min | 4.7×109cfu | 68.12% |
| Simulated gastric fluid (pH2.0) acts on 2.5h | 2.4×109cfu | 34.78% |
| Simulated intestinal fluid (0.3%) acts on 2.5h | 3.3×109cfu | 47.83% |
By the testing result of table 1, as it can be seen that the Enterococcus faecalis is with existing other enterococcus faecium, (NFER-5 is in 60 DEG C of warm
The survival rate for handling 10min is only 1.68 × 109cfu/9.40×109Cfu=1.24%, Zhang Honggang and Li Li separated dung
Enterococcus acts on the survival rate of 2h between 1.2%~79.1% under the conditions of pH2.0, the tolerance gallbladder salinity of conventional bacterial strain
Ranging from 0.03%~0.3% (mass percent concentration)) it compares, can have preferable heat-resisting, stomach juice-resistant, resistance to courage simultaneously
The biological characteristics of salt are suitable for growing industrialization demand, are met the feed of modernization based on its many-sided advantage
The demand of industry and livestock breeding industry.
Sequence table
<110>Qingdao Weilan Biology Co., Ltd.
Qingdao is dynamic to protect national project Technical Research Center Co., Ltd
<120>A kind of enterococcus faecium, its fermentation process in high density and probiotics prepared therefrom
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1458
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aaggggggcg tgctatacat gcaagtcgta cgcttctttt tccaccggag cttgctccac 60
cggaaaaaga agagtggcga acgggtgagt aacacgtggg taacctgccc atcagaaggg 120
gataacactt ggaaacaggt gctaataccg tataacaatc gaaaccgcat ggttttgatt 180
tgaaaggcgc tttcgggtgt cgctgatgga tggacccgcg gtgcattagc tagttggtga 240
ggtaacggct caccaaggcc acgatgcata gccgacctga gagggtgatc ggccacattg 300
ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt cggcaatgga 360
cgaaagtctg accgagcaac gccgcgtgag tgaagaaggt tttcggatcg taaaactctg 420
ttgttagaga agaacaagga tgagagtaac tgttcatccc ttgacggtat ctaaccagaa 480
agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg 540
atttattggg cgtaaagcga gcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc 600
tcaaccgggg agggtcattg gaaactggga gacttgagtg cagaagagga gagtggaatt 660
ccatgtgtag cggtgaaatg cgtagatata tggaggaaca ccagtggcga aggcggctct 720
ctggtctgta actgacgctg aggctcgaaa gcgtggggag caaacaggat tagataccct 780
ggtagtccac gccgtaaacg atgagtgcta agtgttggag ggtttccgcc cttcagtgct 840
gcagctaacg cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg 900
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960
accttaccag gtcttgacat cctttgacca ctctagagat agagcttccc cttcgggggc 1020
aaagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaaccctta ttgttagttg ccatcattta gttgggcact ctagcaagac 1140
tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1200
tgggctacac acgtgctaca atgggaagta caacgagttg cgaagtcgcg aggctaagct 1260
aatctcttaa agcttctctc agttcggatt gcaggctgca actcgcctgc atgaagccgg 1320
aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca 1380
ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac ctttttggag 1440
ccagccgcct aatggaat 1458
Claims (9)
1. an Enterococcus faecalis, which is characterized in that the enterococcus faecium Enterococcus faecium are E13-5, and preservation is compiled
Number be CCTCC M2017191.
2. the high density fermentation culture side of enterococcus faecium Enterococcus faecium E13-5 according to claim 1
Method, which is characterized in that include the following steps：
Level-one produces seed culture：The inclined-plane for being inoculated in MRS culture mediums under the basic bacteria aseptic condition of enterococcus faecium is taken to carry out
Pure culture, test tube produce seed after 37 DEG C of -40 DEG C of stationary cultures 24-48 hours, microscopy qualification as level-one；
Two level produces seed culture：Level-one production seed inclined-plane is taken, sterile saline is added into strain inclined plane will be on inclined-plane
Bacterium colony wash down, be inoculated into secondary seed culture solution according to 1% inoculum concentration, every bottle of liquid amount is 300-400mL, 37
DEG C of -40 DEG C stationary cultures 14-16 hours, microscopy is without miscellaneous bacteria, and thalline is in division stage, after the assay was approved, is produced as two level
Seed；
Three-level produces seed culture：Two level production seed liquor is taken to be inoculated into three-level seed culture fluid according to the inoculum concentration of 5-10%
In, fermentation temperature is 37 DEG C -40 DEG C, and pH controls are cultivated 6-8 hours, tank in fermentation process in 7.0, speed of agitator 50r/min
Pressure is set as 0.05mpa, and when the content of sugar is less than 0.6%, and microscopy is without miscellaneous bacteria, transferred species carries out tank fermentation；
Upper tank fermentation：Three-level seed culture fluid is taken to be inoculated into fermented and cultured tank according to the inoculum concentration of 5-10%, fermentation temperature is
37 DEG C -40 DEG C, pH controls are 6.5, speed of agitator 150r/min, in fermentation process dissolved oxygen amount control 10% or more, according to
Dissolved oxygen increases tank pressure, tank pressure not more than 0.1mpa；Feed supplement is carried out using fed-batch mode when sugared content is less than 1.0%, when
Sugared content no longer reduces, bacterium turbidity is not further added by, and when microscopy is without miscellaneous bacteria, and fermented and cultured terminates.
3. high density fermentation cultural method according to claim 2, which is characterized in that in terms of mass fraction, the two level
The formula of seed culture fluid includes 1% glucose, 1% peptone, 1% yeast extract and 4% trace salt, pH7.0；The three-level
The formula of seed culture fluid includes 2% glucose, 1.5% peptone, 1.5% yeast extract and 4% trace salt, pH7.0.
4. high density fermentation cultural method according to claim 2, which is characterized in that in terms of mass fraction, upper tank fermentation
When the fermented and cultured formula of liquid that uses include 2% glucose, 2% peptone, 2% yeast extract and 4% trace salt, pH7.0.
5. high density fermentation cultural method according to claim 2, which is characterized in that feed supplement parameter control when upper tank fermentation
It is made as：Sugared content control range is 0.5-1.0%, and the rotating speed of peristaltic pump is 120-150rpm, and the feed supplement period is 100s, and aperture is
50-60s。
6. one kind is prepared into using enterococcus faecium Enterococcus faecium E13-5 described in claim 1 as primary raw material
The probiotics arrived.
7. the preparation method of probiotics according to claim 6, which is characterized in that include the following steps：
Take the enterococcus faecium Enterococcus faecium E13-5 of predetermined amount carried out under the conditions of rotating speed 10000r/min from
The heart obtains bacterium mud, by bacterium mud and protective agent with mass ratio 1:3 ratio is sufficiently mixed, and is then lyophilized, and will freeze-drying
Bacterium mud afterwards is thoroughly mixed with auxiliary material, obtains probiotics.
8. preparation method according to claim 7, which is characterized in that in terms of mass fraction, the protective agent includes 5% sugarcane
Sugar, 4% sorbierite, 3% skimmed milk power, 1% gelatin and 0.5%Vc, the auxiliary material include 49% starch, 20% lactose, 15%
Dextrin, 10% PVP K30,5% microcrystalline cellulose and 1% chlorate.
9. preparation method according to claim 7, which is characterized in that will be with the well-mixed bacterium mud of protective agent in following item
Part is lyophilized successively：In -40 DEG C of pre-freezes 3-4 hours, it is evacuated to 20Pa or so at -40 DEG C to -45 DEG C or less, is risen at 5 DEG C
Dry 20-24 hour of China, in 0 DEG C of lyophilization 8-10 hours, in 25 DEG C of parsing-desiccations 4-6 hours, finally closing vacuum 2-4 was small
When.
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| CN112481174A (en) * | 2020-12-28 | 2021-03-12 | 桂林南药股份有限公司 | Fermentation method of enterococcus faecium |
| CN113583905A (en) * | 2019-11-29 | 2021-11-02 | 天津市天合力药物研发有限公司 | Preparation method and application of enterococcus faecium microbial inoculum |
| CN114317344A (en) * | 2021-12-28 | 2022-04-12 | 武汉科缘生物发展有限责任公司 | Enterococcus faecium, application thereof, composition thereof and fermentation culture method of enterococcus faecium |
| CN114350543A (en) * | 2021-12-01 | 2022-04-15 | 云南省微生物发酵工程研究中心有限公司 | Feed-grade enterococcus faecium preparation and production method thereof |
| CN116200302A (en) * | 2023-01-10 | 2023-06-02 | 太原市威尔潞威科技发展有限公司 | Enterococcus faecium LV-434, microbial agent, and preparation methods and applications thereof |
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| CN113583905A (en) * | 2019-11-29 | 2021-11-02 | 天津市天合力药物研发有限公司 | Preparation method and application of enterococcus faecium microbial inoculum |
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| CN116200302A (en) * | 2023-01-10 | 2023-06-02 | 太原市威尔潞威科技发展有限公司 | Enterococcus faecium LV-434, microbial agent, and preparation methods and applications thereof |
| CN116200302B (en) * | 2023-01-10 | 2023-10-27 | 太原市威尔潞威科技发展有限公司 | Enterococcus faecium LV-434, microbial agent, and preparation methods and applications thereof |
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