CN108753650A - Enterococcus faecium and compound micro-ecological preparation prepared therefrom - Google Patents
Enterococcus faecium and compound micro-ecological preparation prepared therefrom Download PDFInfo
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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
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- 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
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- 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
Abstract
The present invention proposes a kind of enterococcus faecium and compound micro-ecological preparation prepared therefrom, belongs to lactic acid bacteria culture technique field, can filter out the significant manure enterococcin strain of probiotic and resistance as feed addictive, can effectively replace part antibiotic.Meanwhile it can be made into the compound micro-ecological preparation of survival rate height, long shelf-life with Bacillus acidi lactici compounding.The enterococcus faecium Enterococcus faecium that the technical solution provides are E.F-2, and deposit number is CCTCC No:M 2018314.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 and prepared therefrom compound micro-
Ecological agent.
Background technology
Enterococcus faecium belong to Streptococcaceae, enterococcus spp bacterium, Gram-positive, amphimicrobian.It is with excellent
Biological characteristics are commensal gut bacterium, can form dominant microflora in enteron aisle, since growth speed is fast, have preferably adherency
Power generates lactic acid and some antibacterial materials, and take advantage status in new born 2-3 days of many animals, to which increase has
The quantity of beneficial bacteria inhibits harmful bacteria, promotes intestinal health, adjusts the microecological balance of enteron aisle and can prevent diarrhea, is given birth to improving
Long 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 conversion ratio of feed can be improved in the probiotics preparation of bacterium composition, improves livestock products quality, reduces diarrhea rate and the death rate.Cause
This, enterococcus faecium has a wide range of applications in livestock and poultry breeding industry, and probiotics are because it is with environmental-friendly, without residual
The advantages that staying is increasingly being applied to Animal husbandry production, the sustainable development of protection and aquaculture to breeding ecological environment
Have 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 cannot meet the feed industry of modernization and the demand of livestock breeding industry, therefore, filter out it is probiotic and
The significant manure enterococcin strain of resistance becomes a big bottleneck problem of animal husbandry development.
Bacillus acidi lactici is the beneficial bacterium in a kind of enteron aisle, can generate many antibiotic complexes, such as bacteriocin, to inhibit have
The growth of evil bacterium.In addition, it can be attached on intestinal epithelial cell, the binding site of the epithelial cell of enteron aisle is occupied, and shape
At a kind of protective barrier, attachment of the pathogen on enteron aisle is prevented.Bacillus acidi lactici has heat resistance, stomach juice-resistant, bile tolerance, adherency
The excellent biological characteristics such as power, biocidal property and antibiotic resistance, moreover it is possible to enhance the immunity of animal body, improve food conversion
Rate improves livestock products quality, reduces diarrhea rate and the death rate.But not high survival rate after Bacillus acidi lactici freeze-drying is that current animal is used
One of higher strain of cost in probiotics product.
It is increasingly aggravated in addition, the lasting feeding of antibiotic results in the medicament residue etc. in the drug resistance of bacterium, livestock products.
Probiotics as a kind of nontoxic, noresidue, without the feed addictive of drug resistance, it has also become effective antibiotic substitute products it
One.Therefore, finding safe and efficient novel fodder additive partial alternative antibiotic becomes aquaculture urgent problem to be solved.
Invention content
The present invention proposes a kind of enterococcus faecium and probiotics prepared therefrom, and it is equal to filter out probiotic and resistance
Significant manure enterococcin strain is 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 E.F-2, and deposit number is CCTCC No:M 2018314.
The enterococcus faecium Enterococcus faecium described in technical solution are stated the present invention also provides more than one
E.F-2 is the compound micro-ecological preparation that one of primary raw material is prepared.
The preparation method of the present invention also provides a kind of as described in above-mentioned technical proposal compound micro-ecological preparation, including it is following
Step：
Enterococcus faecium Enterococcus faecium E.F-2 are fermented in high-efficiency fermenting culture medium, after fermentation
It is centrifuged under the conditions of rotating speed 10000r/min, obtains bacterium mud；
Fluid nutrient medium is added into the bacterium mud to be resuspended, the fluid nutrient medium and the volume ratio of former amount of fermentation are
1:50-1:200, then protective agent is added into the fluid nutrient medium of resuspension and is sufficiently mixed, the fluid nutrient medium and guarantor
The mass ratio for protecting agent is 100:8-100:15, it is then lyophilized, obtains enterococcus faecium bacterium powder；
By the Bacillus acidi lactici bacterium powder being lyophilized under lyophilisation condition identical as the enterococcus faecium bacterium powder and the first auxiliary material with quality
Than 1:1-1:3 ratio is sufficiently mixed, then with mass ratio 1:1:0-1:1:2 successively with enterococcus faecium bacterium powder and second
Auxiliary material is sufficiently mixed, and compound micro-ecological preparation is obtained.
Preferably, the formula of the high-efficiency fermenting culture medium includes：Corn flour：30~50g/L, urea：3~6g/L,
Crystallize sodium acetate：7~10g/L, dipotassium hydrogen phosphate：2~4g/L, anhydrous magnesium sulfate：0.05~0.40g/L, manganese sulfate：0.04~
0.08g/L, diammonium hydrogen citrate：1~3g/L, Tween-80：0.8~1.2g/L.
Preferably, the formula of the high-efficiency fermenting culture medium includes：Corn flour：38.54g/L urea：4.57g/L knot
Brilliant sodium acetate：8.51g/L dipotassium hydrogen phosphate：3.0g/L, anhydrous magnesium sulfate：0.2g/L, manganese sulfate：0.06g/L, hydrogen citrate
Diammonium：2.0g/L, Tween-80：1.0g/L.
Preferably, the protective agent includes skimmed milk 5g/100mL, sucrose 1g/100mL and maltodextrin 5g/100mL.
Preferably, first auxiliary material is 30 POVIDONE K 30 BP/USP30, the second auxiliary material in terms of mass fraction including 95% lactose,
2.5% sodium chloride and 2.5% potassium chloride.
Preferably, will be successively lyophilized in the following conditions with the well-mixed bacterium mud of protective agent：In -40 DEG C of pre-freeze 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 20-25 hours, in 0 DEG C of lyophilization
8-10 hours, in 25 DEG C of parsing-desiccations 4-6 hours, finally close vacuum 2-4 hours.
Preferably, the fluid nutrient medium being added in the bacterium mud is 1 with former amount of fermentation volume ratio:100, the liquid of resuspension
Culture medium is 100 with protectant mass ratio:11, the mass ratio of the Bacillus acidi lactici bacterium powder and the first auxiliary material is 1:2, lactic acid bar
The mass ratio of the mixture of bacterium bacterium powder and the first auxiliary material, enterococcus faecium bacterium powder and the second auxiliary material is 1:1:1.
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, have preferable heat-resisting, stomach juice-resistant,
The biological characteristics of bile tolerance adapt to growing industrialization demand, and the feed industry and herding for meeting modernization are supported
Grow the demand of industry；
2, the present invention is to the optimal screening of freeze drying protectant and lyophilized technique so that enterococcus faecium bacterial strain after freeze drying
Survival rate may be up to 90% or more, the viable bacteria rate after -20 DEG C of storages 12 months still is able to 80% or more；
3, it after compound formulation is made using enterococcus faecium and Bacillus acidi lactici compounding in the present invention, can be formed to Bacillus acidi lactici preferably
Outer layer protection, improve the survival rate of Bacillus acidi lactici, its survival rate made to be increased to 80% or more by original 60% or more,
The storage life of compound micro-ecological preparation product is extended, known Bacillus acidi lactici survival rate in freeze-drying process is solved and substantially drops
It is low and extremely serious problem is lost during preservation.
Meanwhile the glucose that previous probiotics use has been abandoned using the compound micro-ecological preparation of the enterococcus faecium,
But lactose has been selected, it both ensure that the required energy of Strain survival, while lactose is more stablized compared with glucose, is not easy to inhale
Water so that preparation drying is not easy the moisture absorption；Efficiently avoid the growth and breeding of miscellaneous bacteria, the also adjustable crystallization behavior of lactose, shape
At micro crystal so that preparation is more stablized, and it is also just more permanent that bacterial strain is wrapped in its time-to-live in micro crystal.In addition, electric
The addition for solving matter i.e. two kind chlorate, also ensures that bacterial strain is not died because of dehydration.
4, the compound micro-ecological preparation of enterococcus faecium provided by the invention can both be added as solid and can also adopt
With the mode of liquid oral, and it can guarantee that the survival of bacterial strain is unaffected, especially the relatively low Bacillus acidi lactici of survival rate.
Specific implementation mode
Below in conjunction with specific embodiment to enterococcus faecium provided by the present invention and probiotics prepared therefrom
It is clearly and completely described, described embodiments are only a part of the embodiments of the present invention, rather than whole implementation
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, 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, K2HPO4
2.0g/L, citric acid hydrogen diamine 2.0g/L, sodium acetate 5.0g/L, MgSO4 0.2g/L、MnSO40.04g/L, Tween-80
1.0g/L.) 100mL conical flasks in, then enrichment culture 8h draws 1mL and 9mL sterile salines and is configured to 1:10 dilutions
Liquid (is configured to 10-1Dilution), 3-5min is vibrated, dilution 1mL is accurately drawn with micropipettor and is sterilized to 9mL is filled
Physiological saline test tube in, with turbula shaker vibrate 1-2min, be configured to 10-2Dilution, carry out 10 successively-3-10-6It is dilute
It releases, selects 10-3-10-6Three dilutions draw 200 μ L and are coated on MRS culture mediums respectively, after 37 DEG C are inverted culture 48h, use
After oese picking single bacterium colony repeats passage pure culture 3 times on MRS slant mediums again, picking single bacterium colony to MRS
It cultivates in culture medium, 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
The features such as being cultivated, observing the edge of its bacterium colony, smoothness, surface gloss, viscosity, transparency, color, shape.Experiment
The results show that the bacterial strain is in well-grown on the MRS agar mediums of pH6.2-6.4, neat in edge is smooth, lustrous surface, glues
It is thick, it is opaque, 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 the bacterium
Strain is gram-positive cocci.
To the gram-positive cocci bacterial strain carry out Physiology and biochemistry identification, catalase test, nitrate reduction 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 arginine water
Solution experiment, 10 DEG C and 45 DEG C of growth tests and all kinds of sugar fermentating tests etc., and with《Common bacteria system identification handbook》With《Breast
The taxonomic identification of acetic bacterial》Control, qualification result are shown in Table 1, identify that the bacterial strain is enterococcus faecium.
1 Physiology and biochemistry qualification result of table
| Project | As a result | Project | As a result | Project | As a result |
| Catalase test | - | 10 DEG C of growth tests | + | Maltose | + |
| Nitrate reduction test | - | 45 DEG C of growth tests | + | Sucrose | + |
| Indole test | - | 6.5%NaCl growth tests | + | Lactose | + |
| Gelatin liquefaction test | - | PH4.5 growth tests | + | Sorbierite | + |
| Hydrogen sulfide production test | - | PH9.6 growth tests | + | Mannitol | + |
| Arginine hydrolysis experiment | + | Glucose aerogenesis | - | Trehalose | + |
To specifications the step of, requires, and examination is extracted using the bacterial genomes of TIANGEN Biotech (Beijing) Co., Ltd.
Agent box (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 pieces of the bacterial strain
Section, PCR reaction systems are 10 × Buffer 5.0 μ L, dNTP (10mmol/L) 1.0 μ L, Primer1 (20 μm of ol/L) 1.0 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 1min30s, totally 35 recycle；
Last 72 DEG C of extensions 5min.PCR carries out 1% agarose gel electrophoresis detection after reaction, to pcr amplification product, as a result shows
Show, the specific band of size about 1500bp is obtained through amplification.With the agar purchased from TIANGEN Biotech (Beijing) Co., Ltd.
Sugared gel reclaims kit, to specifications the step of require recycle and purify the purpose band, send raw work bioengineering (on
Sea) limited liability company's progress sequencing.
Compared with measured sequence is carried out Blastn similarity analysis with the 16S rRNA sequences in Genbank, as a result
The 16Sribosomal RNA gene sequences such as the bacterial strain and Enterococcus faecium strain 54-LR3/ZDHHM1
Homology reached 99%, sequence refers to sequence table.The bacterial strain was preserved in Chinese Typical Representative culture on May 28th, 2018
Object collection, deposit number are CCTCC No:M2018314, preservation place are the Wuhan Wuhan Universitys of China.
Embodiment 2
Present embodiments provide the enterococcus faecium Enterococcusfaecium obtained to above-described embodiment separation identification
Probiotic and resistance the performance detection of E.F-2, wherein, stomach juice-resistant heat-resisting to its, bile tolerance biological characteristics testing result
As shown in table 2：
2 enterococcus faecium of table is heat-resisting, stomach juice-resistant, bile tolerance testing result
| Viable count | Survival rate (based on 100%) | |
| Physiological saline | 7.8×109cfu | -- |
| 60 DEG C of effect 10min | 7.7×109cfu | 98.72% |
| 60 DEG C of effect 30min | 7.3×109cfu | 93.59% |
| Simulated gastric fluid (pH2.0) acts on 2.5h | 5.8×109cfu | 74.36% |
| Simulated intestinal fluid (0.3%) acts on 2.5h | 6.1×109cfu | 78.21% |
By the testing result of table 2, 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 managing 10min is only 1.68 × 109cfu/9.40×109Cfu=1.24%, Zhang Honggang and Li Li separated dung intestines
Coccus acts on the survival rate of 2h between 1.2%~79.1% under the conditions of pH2.0, the tolerance gallbladder salinity range of conventional bacterial strain
For 0.03%~0.3% (mass percent concentration)) it compares, there can be the life of preferable heat-resisting, stomach juice-resistant, bile tolerance simultaneously
Object characteristic is suitable for growing industrialization demand based on its many-sided advantage, meet modernization feed industry and
The demand of livestock breeding industry.
Embodiment 3
It present embodiments provides with the enterococcus faecium Enterococcus faeciumE.F-2 of above-described embodiment separation identification
It is specific as follows for the preparation process for the compound micro-ecological preparation that primary raw material is prepared：
Take the enterococcus faecium Enterococcus faecium E.F-2 of predetermined amount in high-efficiency fermenting culture medium (corn flour：
38.54g/L urea：4.57g/L crystallizes sodium acetate：8.51g/L dipotassium hydrogen phosphate：3.0g/L, anhydrous magnesium sulfate：0.2g/L,
Manganese sulfate：0.06g/L, diammonium hydrogen citrate：2.0g/L, Tween-80：It ferments in 1.0g/L), in rotating speed after fermentation
It is centrifuged under the conditions of 10000r/min, obtains bacterium mud；Fluid nutrient medium is added into the bacterium mud to be resuspended, the liquid
The volume ratio of culture medium and former amount of fermentation is 1:50-1:200, then protective agent is added into the fluid nutrient medium of resuspension and carries out fully
Ground mixes, and the fluid nutrient medium is 100 with protectant mass ratio:8-100:15, so it is lyophilized successively in the following conditions：
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 20-25 hours,
In 0 DEG C of lyophilization 8-10 hours,
In 25 DEG C of parsing-desiccations 4-6 hours, finally closes vacuum 2-4 hours, obtain enterococcus faecium bacterium powder.
By the Bacillus acidi lactici bacterium powder being lyophilized under lyophilisation condition identical as enterococcus faecium bacterium powder, (described first is auxiliary with the first auxiliary material
Material is PVP K30) with mass ratio 1:1-1:3 ratio is sufficiently mixed, then with mass ratio 1:1:0-1:1:2 successively with
Enterococcus faecium bacterium powder and the second auxiliary material (the second auxiliary material in terms of mass fraction containing 95% lactose (crossing 200 mesh sieve), 5% sodium chloride,
5% potassium chloride) it is sufficiently mixed, obtain compound micro-ecological preparation.
Pass through the optimal screening to freeze drying protectant and lyophilized technique in the present embodiment so that the Enterococcus faecalis is freezing
The survival rate of bacterial strain may be up to 90% or more after dry, and 12 months viable bacteria rates of -20 DEG C of storages are still 80% or more.Enterococcus faecium and breast
After acidfast bacilli compounding support compound formulation, preferable outer layer protection is formed to Bacillus acidi lactici, improves the survival rate of Bacillus acidi lactici,
So that its survival rate has been increased to 80% or more by original 60% or more, extend the storage life of compound micro-ecological preparation product,
It solves known Bacillus acidi lactici survival rate in freeze-drying process to be greatly reduced and lose during preserving extremely serious
Problem.
Meanwhile the compound micro-ecological preparation of the enterococcus faecium has abandoned the glucose that previous probiotics use, but
Lactose has been selected, both ensure that the required energy of Strain survival, while lactose is more stablized than glucose, it is not hygroscopic, make
It obtains preparation drying and is not easy the moisture absorption, efficiently avoid the growth and breeding of miscellaneous bacteria, the also adjustable crystallization behavior of lactose is formed fine
Crystal makes preparation more stablize, and it is also just more permanent that bacterial strain is wrapped in its time-to-live in micro crystal.In addition, electrolyte i.e. two
The addition of kind chlorate, ensure that bacterial strain is not died because of dehydration.Lactose can be transformed into lactic acid, vinegar by the lactic acid bacterias such as enterococcus faecium
Acid so that the pH value of enteron aisle declines, these organic acids can stimulate intestines peristalsis, has whole intestines effect, further improves compound
The function and effect of probiotics.
PVP K30 in people with pharmaceutically there is relatively broad application, for the international three big medicinal new accessories advocated it
One, it may be used as adhesive, cosolvent, dispersant, the stabilizer of enzyme and heat-sensitive drug also acts as cryopreservative, gather dimension
The use of ketone K30 so that the compound micro-ecological preparation of the enterococcus faecium can be both added as solid, and liquid also can be used
Oral mode, while also assuring that the survival of bacterial strain is unaffected.
4 animal experiment of embodiment
There is yellowish-white dysentery symptom of diarrhea in the piglet of 2 nest of Weifang Shouguang pig farm totally 12 7-10 ages in days, and baby pig diarrhea disappears
Thin, buttocks is stained with the substances such as loose stool, and spirit is not good, and hair color is not bright, feeds by Bacillus acidi lactici (viable bacteria >=1010) and dung intestines cfu
Coccus (viable bacteria >=1010Cfu the compound micro-ecological preparation) being prepared, piglets 5 days by a definite date, on-test and at the end of scale
Piglet litter weight is taken, every piglet average growth rate is calculated, carries out (1 point of stools scored daily：Watery stool, 2 points：Loose stools, 3 points：It is soft
Just, 4 points：Molding soft stool, 5 points：Normally), daily diarrhea rate is recorded, the final death rate is calculated, detailed data is shown in Table 3.
3 piglet of table feeds the diarrhea and weightening, death condition of compound micro-ecological preparation
From the data in table 3, it can be seen that by the embodiment of the present invention provided by Bacillus acidi lactici (viable bacteria >=1010) and dung intestines ball cfu
Bacterium (viable bacteria >=1010Cfu compound micro-ecological preparation can improve livestock products quality made of) compounding, and reduce diarrhea rate and death
Rate, enhances the immunity of animal body, and can effectively replace antibiotic is used for livestock breeding industry.
Sequence table
<110>Qingdao Weilan Biology Co., Ltd.
Qingdao is dynamic to protect national project Technical Research Center Co., Ltd
<120>Enterococcus faecium and compound micro-ecological preparation prepared therefrom
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1452
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggggtggggg gtgctataca tgcaagtcgt acgcttcttt ttccaccgga gcttgctcca 60
ccggaaaaag aagagtggcg aacgggtgag taacacgtgg gtaacctgcc catcagaagg 120
ggataacact tggaaacagg tgctaatacc gtataacaat cgaaaccgca tggttttgat 180
ttgaaaggcg ctttcgggtg tcgctgatgg atggacccgc ggtgcattag ctagttggtg 240
aggtaacggc tcaccaaggc cacgatgcat agccgacctg agagggtgat cggccacatt 300
gggactgaga cacggcccaa actcctacgg gaggcagcag tagggaatct tcggcaatgg 360
acgaaagtct gaccgagcaa cgccgcgtga gtgaagaagg ttttcggatc gtaaaactct 420
gttgttagag aagaacaagg atgagagtaa ctgttcatcc cttgacggta tctaaccaga 480
aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg 540
gatttattgg gcgtaaagcg agcgcaggcg gtttcttaag tctgatgtga aagcccccgg 600
ctcaaccggg gagggtcatt ggaaactggg agacttgagt gcagaagagg agagtggaat 660
tccatgtgta gcggtgaaat gcgtagatat atggaggaac accagtggcg aaggcggctc 720
tctggtctgt aactgacgct gaggctcgaa agcgtgggga gcaaacagga ttagataccc 780
tggtagtcca cgccgtaaac gatgagtgct aagtgttgga gggtttccgc ccttcagtgc 840
tgcagctaac gcattaagca ctccgcctgg ggagtacgac cgcaaggttg aaactcaaag 900
gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag 960
aaccttacca ggtcttgaca tcctttgacc actctagaga tagagcttcc ccttcggggg 1020
caaagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080
ccgcaacgag cgcaaccctt attgttagtt gccatcattt agttgggcac tctagcaaga 1140
ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac 1200
ctgggctaca cacgtgctac aatgggaagt acaacgagtt gcgaagtcgc gaggctaagc 1260
taatctctta aagcttctct cagttcggat tgcaggctgc aactcgcctg catgaagccg 1320
gaatcgctag taatcgcgga tcagcacgcc gcggtgaata cgttcccggg ccttgtacac 1380
accgcccgtc acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttggag 1440
ccagccggcc ca 1452
Claims (9)
1. an Enterococcus faecalis, which is characterized in that the enterococcus faecium Enterococcus faecium are E.F-2, and preservation is compiled
Number be CCTCC No:M 2018314.
2. a kind of make by one of primary raw material of enterococcus faecium Enterococcus faecium E.F-2 described in claim 1
Standby obtained compound micro-ecological preparation.
3. the preparation method of compound micro-ecological preparation according to claim 2, which is characterized in that include the following steps：
Enterococcus faecium Enterococcus faecium E.F-2 are fermented in high-efficiency fermenting culture medium, are being turned after fermentation
It is centrifuged under the conditions of fast 10000r/min, obtains bacterium mud；
Fluid nutrient medium is added into the bacterium mud to be resuspended, the volume ratio of the fluid nutrient medium and former amount of fermentation is 1:
50-1:200, then protective agent is added into the fluid nutrient medium of resuspension and is sufficiently mixed, the fluid nutrient medium and protection
The mass ratio of agent is 100:8-100:15, it is then lyophilized, obtains enterococcus faecium bacterium powder；
By the Bacillus acidi lactici bacterium powder being lyophilized under lyophilisation condition identical as the enterococcus faecium bacterium powder and the first auxiliary material with mass ratio 1:
1-1:3 ratio is sufficiently mixed, then with mass ratio 1:1:0-1:1:2 successively with enterococcus faecium bacterium powder and the second auxiliary material
It is sufficiently mixed, obtains compound micro-ecological preparation.
4. preparation method according to claim 3, which is characterized in that the formula of the high-efficiency fermenting culture medium includes：It is beautiful
Rice flour：30~50g/L, urea：3~6g/L crystallizes sodium acetate：7~10g/L, dipotassium hydrogen phosphate：2~4g/L, anhydrous slufuric acid
Magnesium：0.05~0.40g/L, manganese sulfate：0.04~0.08g/L, diammonium hydrogen citrate：1~3g/L, Tween-80：0.8~1.2g/
L。
5. preparation method according to claim 4, which is characterized in that the formula of the high-efficiency fermenting culture medium includes：It is beautiful
Rice flour：38.54g/L urea：4.57g/L crystallizes sodium acetate：8.51g/L dipotassium hydrogen phosphate：3.0g/L, anhydrous magnesium sulfate：
0.2g/L, manganese sulfate：0.06g/L, diammonium hydrogen citrate：2.0g/L, Tween-80：1.0g/L.
6. preparation method according to claim 3, which is characterized in that the protective agent includes skimmed milk 5g/100mL, sugarcane
Sugared 1g/100mL and maltodextrin 5g/100mL.
7. preparation method according to claim 3, which is characterized in that first auxiliary material is PVP K30, the second auxiliary material
Including 95% lactose, 2.5% sodium chloride and 2.5% potassium chloride in terms of mass fraction.
8. preparation method according to claim 3, 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 20-25 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.
9. preparation method according to claim 3, which is characterized in that the fluid nutrient medium being added in the bacterium mud with it is primary
Ferment amount volume ratio is 1:100, the fluid nutrient medium of resuspension is 100 with protectant mass ratio:11, the Bacillus acidi lactici bacterium powder with
The mass ratio of first auxiliary material is 1:2, the mixture of Bacillus acidi lactici bacterium powder and the first auxiliary material, enterococcus faecium bacterium powder and the second auxiliary material
Mass ratio is 1:1:1.
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| CN113265352A (en) * | 2021-05-11 | 2021-08-17 | 塔里木大学 | Preparation method and application of enterococcus faecium powder |
| CN113265352B (en) * | 2021-05-11 | 2023-08-25 | 塔里木大学 | Preparation method and application of enterococcus faecium powder |
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