CN106479924B - The preparation and application of a kind of clostridium butyricum and clostridium butyricum active bacteria preparation - Google Patents
The preparation and application of a kind of clostridium butyricum and clostridium butyricum active bacteria preparation Download PDFInfo
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 206010061393 typhus Diseases 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses one plant of clostridium butyricum and clostridium butyricum active bacteria preparation production method, clostridium butyricum (Clostridium butyricum) LXKJ on March 24th, 1,2016 is preserved in China typical culture collection center, preserving number is CCTCC M 201613.The production method of the seed culture medium of the bacterial strain, fermentative medium formula, condition of culture and its active bacteria formulation is also disclosed simultaneously.Clostridium butyricum provided by the invention is acidproof, alkaline-resisting, high temperature resistant, it is strong to produce butyric acid ability, it is all inhibited to animal pathogens such as Escherichia coli, salmonella, Shigella, golden yellow grape bacillus, Listeria monocytogenes, C.perfringens, it can prevent livestock and poultry diarrhea as caused by Escherichia coli, salmonella, C.perfringens etc., improve intestinal flora balance, promote growth of animals or poultry, alleviate prevention of sow constipation, laying hen egg size is improved, improves laying hen eggshell quality, reduces feedstuff-egg ratio.
Description
Technical field
The invention belongs to field of microbial fermentation, be related to a kind of clostridium butyricum and clostridium butyricum active bacteria preparation production method and
Using.
Background technology
The further expansion of scale is cultivated now with people, in order to improve culture efficiency, various antibiotic are as growth-promoting
A large amount of uses of long agent have become a kind of universal phenomenon, and drawback is increasingly prominent, such as medicament residue, the report of pathogenic bacteria of drug-resistant
Road, the unbalance of microbial population of animal intestinal tract, environmental pollution etc., serious threat have arrived the health and food security of the mankind.Probiotics
As a kind of safe efficient, pollution-free and antibiotic substitute products for having no drug resistance gradually as diseases prevention, growth promotion formulation application
In aquaculture.
Probiotics are the biology systems of microbial cells obtained from probiotics fermention and post-processing and its metabolite
Agent and active bacteria formulation.Therefore with intestinal microecology balance, raising word material transformation efficiency is maintained, improve growth of animals or poultry performance, carry
High immune function, anti-oxidant and anti-cancer and improve environment, the effects that reducing the generation of harmful substance, be increasingly subject to aquaculture and chased after
It holds in both hands.
Clostridium butyricum (Clostridium butyricum), it is to be entered closely to control doctor by Chiba, Japan medical university palace for the first time
It finds and reports for the first time, therefore clostridium butyricum is also designated as Clostridium Butyricum, while Japan also becomes research clostridium butyricum history
Longest country.Clostridium butyricum (Clostridium butyricum) it is gram-positive bacteria, thalline is in straight or bending,
(0.5 ~ 1.7x2.4 ~ 7.6 μm), single or pairs of, short chain, amphitrichous is movable, and thalline can form gemma, and middle part enlarges into
Fusiformis, gemma bias Cheng Ciduan is raw, without epispore or appendage；How rounded bacterium colony is, smaller, protrusion, milky, butyric acid shuttle
Pseudomonas can generate butyric acid, propionic acid, acetic acid when anaerobic bacteria, liquid fermentation, while also be able to generate hydrogen etc., studies have found that
Butyric acid, the acetic acid of clostridium butyricum generation are numerous to the growth of such as Bifidobacterium of the probiotics in the animal intestinal tracts such as pig, chicken, lactic acid bacteria etc.
It grows with good facilitation, and all has to harmful intestinal tract bacteria such as Escherichia coli, C.perfringens, salmonella typhi etc.
There is good inhibiting effect, the butyric acid for being metabolized generation is directly utilized by enteron aisle villus, and it is histiocytic to promote gut epithelium
Regeneration and reparation, the hydrogen for being metabolized generation have repair, while can enhance machine to the oxidative damage of body liver, kidney organ
Body function of detoxification.Therefore clostridium butyricum is known as " the first bacterium of intestinal health ".
Patent CN201110116927, CN201110126498 etc. have been related to the production and processing method of clostridium butyricum, but
These method generally existing medium components are complicated, and fermentation costs are high, are not suitable for industrialized production problem.
CN201110454790, CN201310082656, CN201310083900, CN201410469885 etc. have been related to butyric acid shuttle
The application of bacterium, but it is not directed to influence of the various dose clostridium butyricum to pig, laying hen, meat chicken production performance.
Invention content
The present invention from one plant of clostridium butyricum of health pig enteron aisle separation screening (Clostridium butyricum)LXKJ-
1, activity is high, and resistance is strong, while has found suitable for the culture medium and condition of culture of growth, breeding, and fermentation gained is cultivated
Object viable count is high, gemma rate is high, and medium component used in the invention is simple, of low cost, is well suited for the scale of factory
Production.Clostridium butyricum (Clostridium butyricum) LXKJ-1 is acidproof, alkaline-resisting, high temperature resistant, production butyric acid ability is strong, to big
The animal pathogens such as enterobacteria, salmonella, Shigella, golden yellow grape bacillus, Listeria monocytogenes, C.perfringens
It is all inhibited, pig, the chicken diarrhea as caused by Escherichia coli, salmonella, C.perfringens etc. can be prevented, improve intestines
Road colony balance promotes pig, chicken growth, improves laying rate of laying hen and egg size, improve eggshell quality, reduce feedstuff-egg ratio.
The technical scheme is that：
A kind of clostridium butyricum, entitled LXKJ-1, specific name are：Clostridium butyricum LXKJ-1Clostridium butyricumLXKJ-1, deposit number are：CCTCC NO:M201613, preservation date：On March 24th, 2016, preservation address
For：China, Wuhan, Wuhan University, depositary institution：China typical culture collection center.
Clostridium butyricum active bacteria preparation prepared by a kind of clostridium butyricum LXKJ-1.
A kind of clostridium butyricum active bacteria formulation preparation method, includes the following steps：
1. by clostridium butyricum (Clostridium butyricum) LXKJ-1 its be deposited in glycerol tube, freezing, lead to
Method of scoring access slant medium is crossed, under anaerobic, 35~37 DEG C of cultures 20~for 24 hours, wash lower bud with sterile saline
Spore suspension, as original bacterium solution；
2. original bacterium solution is carried out amplification cultivation for strain, it is as follows：
The bacteria suspension of 1 volume is linked into the primary-seed medium of 20 volumes, in 1 L indigo plant lid reagent bottles, anaerobism
Under the conditions of, 35~37 DEG C culture 8~12 h, as primary seed solution；
The primary seed solution of 1 volume is linked into the secondary seed medium of 10 volumes, in 20 L fermentation tanks, nitrogen
Under environment, 35~37 DEG C culture 8~12 h, as secondary seed solution；
Secondary seed solution is moved into the three grade fermemtation culture medium of 20 times of volumes, in 200 L fermentation tanks, nitrogen environment
Under, 35~37 DEG C culture 20~24 h to get to clostridium butyricum (Clostridium butyricum) LXKJ-1 culture；
3. microorganism collection, the bacterium mud being collected by centrifugation are carried out to culture using supercentrifugal process；
4. by 1:The skimmed milk power suspension of 1~5 ratio addition 5~20%, sucrose, lactose, trehalose, maltodextrin, mountain
One or more kinds of combinations in pears alcohol, glycerine prepare freeze-drying thalline as freeze drying protectant using freeze-drying；
5. using glucose, starch, mountain flour, zeolite powder, bean cake powder, maize cob meal, one kind in wheatfeed or it is a kind of with
On combination bacterium powder is diluted as auxiliary material, as needed, be configured to the clostridium butyricum active bacteria preparation of different viable counts.
Further, above-mentioned slant medium is：Glucose 1.2%, L-cysteine 0.03%, sodium thioglycolate
0.03%, K2HPO4 0.2%, yeast extract 0.3%, soy peptone 0.5%, peptone 1%, tryptone 1%, NaCl
0.3%, sodium alginate 0.5%, agar 2%, pH 6.5~7.0.
Further, above-mentioned primary-seed medium is：5~30 g/L of glucose, 0.1~5 g/L of L-cysteine,
Sodium thioglycolate 0.1~5 g/L, K2HPO4 0.1~5 g/L, 1~10 g/L of yeast extract, soy peptone 5~20
1~10 g/L of g/L, NaCl, sodium alginate 1~10 g/L, pH 6.5~7.0；
The secondary seed medium is：10~40 g/L of peptone, 1~5 g/L of beancake powder, 10~30 g/ of glucose
L, KNO3 0.5~2.0 g/L, K2HPO4 0.5~2.0 g/L, MgSO40.1~0.5 g/L, MnSO40.1~0.5 g/L,
0.1~1.0 g/L of L-cysteine, 1.0~10 g/L of sodium alginate, urea 1.0~5.0 g/L, CaCO31.0~10
G/L, pH 6.5~7.0；
The three grade fermemtation culture medium is：10~40 g/L of corn flour, 1~5 g/L of beancake powder, tryptone 10~40
G/L, glucose 10~40 g/L, NH4NO3 0.5~2.0 g/L, K2HPO4.5~2.0 g/L, MgSO40.1~0.5 g/L,
MnSO40.1~0.5 g/L, 0.1~1.0 g/L of L-cysteine, 1.0~10 g/L of sodium alginate, urea 1.0~5.0
G/L, CaCO3 1.0~10 g/L, pH6.5~7.0；
Above all of culture medium is required for steam sterilizing before use（121 DEG C, 30 min）, cooling is for use.
A kind of above-mentioned clostridium butyricum or above-mentioned clostridium butyricum preparation answering in animal and fowl fodder microbe additive is prepared
With.
Further, above-mentioned sour clostridium active bacteria formulation improves production performance for weanling pig and reduces diarrhea rate, and show
Writing reduces Escherichia coli and salmonella quantity in caecum.
Further, above-mentioned clostridium butyricum active bacteria preparation improves production performance, Egg Quality for laying hen, increases broiler chicken caecum
Middle lactic acid bacteria and the quantity of Bifidobacterium and reduction Escherichia coli quantity.
Further, above-mentioned clostridium butyricum active bacteria preparation improves broiler chicken survival rate for broiler chicken, reduces diarrhea rate, promotes life
It is long, increase lactic acid bacteria and the quantity of Bifidobacterium and reduction Escherichia coli quantity in broiler chicken caecum.
The present invention from one plant of clostridium butyricum of health pig enteron aisle separation screening (Clostridium butyricum)LXKJ-
1, activity is high, and resistance is strong, while has found suitable for the culture medium and condition of culture of growth, breeding, and fermentation gained is cultivated
Object viable count is high, gemma rate is high, and medium component used in the invention is simple, of low cost, is well suited for the scale of factory
Production.Clostridium butyricum (Clostridium butyricum) LXKJ-1 is acidproof, alkaline-resisting, high temperature resistant, production butyric acid ability is strong, to big
The animal pathogens such as enterobacteria, salmonella, Shigella, golden yellow grape bacillus, Listeria monocytogenes, C.perfringens
It is all inhibited, pig, the chicken diarrhea as caused by Escherichia coli, salmonella, C.perfringens etc. can be prevented, improve intestines
Road colony balance promotes pig, chicken growth, improves laying rate of laying hen and egg size, improve eggshell quality, reduce feedstuff-egg ratio.
Clostridium butyricum provided by the invention has acidproof, alkaline-resisting, heat safe feature.PH value 1.0-4.0 remains to survive, pH
It can be grown during value 5.0-12.0, optimum pH 6.0-7.0；External 80 DEG C of dry heat treatment 10min, 90 DEG C of dry heat treatments
10min, 100 DEG C of dry heat treatment 5min gemma survival rates 100%.
Clostridium butyricum production butyric acid ability provided by the invention is strong, to Escherichia coli, salmonella, Shigella, golden yellow
The animal pathogens such as grape bacillus, Listeria monocytogenes, C.perfringens are all inhibited.
Description of the drawings
Fig. 1 clostridium butyricums (Clostridium butyricum) LXKJ-1 culture bacterium colony photo（The bacterial strain bacterium colony is in
Circle, milky, bacterium colony protrusion）；
Fig. 2 clostridium butyricums (Clostridium butyricum) (gemma is oval, gemma for the gemma photo of LXKJ-1
Wall thickness)；
Fig. 3 clostridium butyricums (Clostridium butyricum) LXKJ-1 taxonomy and phylogenetic tree；
Fig. 4 clostridium butyricums centrifuged supernatant is to the bacteriostasis comparison diagram of C.perfringens；
Clostridium butyricum in attached drawing 1, which is numbered, is：LXKJ-1, specific name are：Clostridium butyricum LXKJ-1ClostridiumbutyricumLXKJ-1, deposit number are：CCTCC NO:M201613, preservation date：March 24 in 2016
Day, preservation address is：China, Wuhan, Wuhan University, depositary institution：China typical culture collection center.
Specific embodiment
Below by specific embodiment detailed description come the present invention is furture elucidated, but be not to the present invention limit
System, only illustrates.
One clostridium butyricum of embodiment (Clostridium butyricum) LXKJ-1 separation and identification
Clostridium butyricum (Clostridium butyricum) LXKJ-1 is located away from the enteron aisle of health pig, it first will acquisition
Sample normal saline dilution after in 80 DEG C of 10 min of heating water bath, to kill non-Bacillus；Then it is inoculated in inclined-plane culture
On base, in 36 DEG C of anaerobic jar culture 24-30 h, Preliminary Identification is carried out to bacterial strain by colonial morphology and thalli morphology, is therefrom chosen
Select cultural characteristic, colonial morphology and thalli morphology meet clostridium butyricum feature bacterial strain continuously cross purifying culture three generations, point
Other marker number enters identifies and screens in next step.
Further determine that clostridium butyricum (Clostridium butyricum) LXKJ-1 taxonomy, the present invention adopts
Identification is classified to the bacterial strain with conventional sorting methods and molecular classification method.
1 morphologic observation culture medium
Glucose 1.2%, L-cysteine 0.03%, sodium thioglycolate 0.03%, K2HPO40.2%, yeast extract
0.3%, soy peptone 0.5%, peptone 1%, tryptone 1%, NaCl 0.3%, sodium alginate 0.5%, agar 2%,
PH 6.5~7.0
2 primers
Using bacterial universal primers, primer is synthesized by AudioCodes biotechnology (Wuhan) Co., Ltd.Its sequence is respectively：
27F (5 '-AGAGTTTGATCCTGGCTC-3 ') and 1492R (5 '-CGGCTACCTTGTTACGACTT-3 ')
3 test methods
3.1 morphological observation
By the microbionation culture of determining pure culture, its morphological feature is observed
Four zoning collimation method of tablet, obtains single bacterium colony, observes the size of its bacterium colony, color, shape, color and luster, transparency, densification
The features such as degree and edge, and record.
Bacterial cell form（Individual morphology）Observation
Gram's staining is carried out to bacterial strain to be checked, gram-positive bacteria body is interior containing special nucleoprotein magnesium salts and more
The compound of sugar, can be combined very firm with the compound of iodine and crystal violet, be not easy to decolourize, negative bacterium compound combination degree bottom,
It is poor to adsorb dyestuff, easily decolourizes.Observe its Gram's staining situation under an optical microscope, judge its for gram-positive bacteria or
Gram-negative bacteria.And the morphological feature of preliminary observation cell.
3.2 clostridium butyricums (Clostridium butyricum) LXKJ-1 physiological and biochemical property
3.2.1 API 20NE identification marks
3.2.1.1 the preparation of test bar
Take out culture box, it is small recessed to add in sterile water covering honeycomb in side in culture plate for strain number and dat recorder
In, prevent reagent strip in incubation from drying.Test bar is taken out from the package, is placed in culture plate.
3.2.1.2 the preparation of inoculum
The tablet of inoculation experiments bacterial strain is obtained with four zoning collimation methods, is scraped new fresh thalli to physiological saline with aseptic cotton carrier
In, until Maxwell concentration 0.5.
3.2.1.3 the inoculation of reagent strip
3.2.1.4 it is inoculated with brine bacteria suspension respectively from NO with same root suction pipe3 To PNPG developmental tubes.Test strips slightly towards
It leans forward and tubule inner edge liquid feeding body will be leaned against at the top of suction pipe, to avoid bubble is formed.
3.2.1.5 an ampoule bottle APIAUX culture mediums are opened and add in 200 microlitres of remaining physiological saline bacteria suspensions to ampoule
Bottle, careful mixing have avoided bubble generation.GUL to PAC pipes are filled it up with, 3 scribing line experiment GLU, ADH, URE are covered with mineral oil
Lid, in 29 DEG C of cultures.
3.2.2 NO3It measures：
Add a drop NIT1 and NIT2 to NO3It manages, after 5min, red represents positive, and because of nitrogen, there may be feminine genders, add 2-
3mgZn to NO3It manages, color is remained unchanged after 5min as the positive, is reddened as feminine gender.
1 bacterial strain LXKJ-1 physio-biochemical characteristics of table-enzyme activity, carbon assimilation
+：Positive reaction； -：Negative reaction； W：Weakly positive is reacted.
2 bacterial strain LXKJ-1 physio-biochemical characteristics of table-sour using carbon source production
+：Positive reaction； -：Negative reaction
3.3 clostridium butyricums (Clostridium butyricum) LXKJ-1 molecular biology identification
3.3.1 clostridium butyricum (Clostridium butyricum) LXKJ-1 genomic DNAs extraction use SDS-
CTAB methods.
3.3.2 clostridium butyricum (Clostridium butyricum) LXKJ-116S rDNA genes amplification used in
Primer is 27F (5'AGAGTTTGATCCTGGCTCAG), and 1492R (5'TACGGCTACCTTGTTACGACTT), sequencing approach is adopted
Use clone sequencing.PCR reaction systems are 50 μ L: ddH240.7 5 μ L, dNTP Mixture of μ L, 10 × Buffer of O
（10 μM）1 μ L, primer 2 7F（10 μmol/L）1 μ L, primer 1492R（10 μmol/L）1 μ L,Taq0.3 μ of archaeal dna polymerase
L, 1 μ L of template DNA.PCR amplification conditions are：94 DEG C of 5 min, 95 DEG C of 30 s, 56 DEG C of 30s, 72 DEG C of 90 s are followed
Ring number 30 times；72 ℃ 8 min.Examining order commission Shanghai bioengineering Services Co., Ltd is completed.
3.3.3 the comparison analysis of sequencing result
BLAST comparisons are carried out in EZ-Biocloud to the sequencing result of bacterial strain, representative strain are chosen, using adjacent method
（NJ）Method carries out tetraploid rice to the bacterial strain, while builds systematic evolution tree, then determines this by Phylogenetic analysis
The race relation of bacterial strain.Bacterial strain is carried out with reference to the cultural characteristic of bacterial strain, morphological feature and evolutionary relationship to the bacterial strain etc. just
Walk taxonomic identification.
4 result of the tests
4.1 clostridium butyricums (Clostridium butyricum) LXKJ-1 morphological observations
4.2 clostridium butyricums (Clostridium butyricum) LXKJ-1 molecular biology identification result
4.2.1 clostridium butyricum (Clostridium butyricum) LXKJ-1 16S rDNA sequencing results use
DNAMAN5.2 softwares splice, and determine the segment by 1412 base compositions, obtain clostridium butyricum (Clostridium butyricum) LXKJ-1 16S rDNA sequences (seeing below table).
4.2.2 homologous chadogram structure
Clostridium butyricum (Clostridium butyricum) the 16S rDNA the sequencing results of LXKJ-1 show bacterial strain
Clostridium butyricum (Clostridium butyricum) LXKJ-1 and bacterial strainClostridium butyricum DSM 10702
(T) similarity is up to 99.15%, secondlyClostridium diolisDSM 5431(T)（97.37%）WithClostridium saccharoperbutylacetonicumN1-4(HMT)(T)（97.23%）, then it is soft with MEGA 5.05
Part NJ methods structure bacterial strain clostridium butyricum (Clostridium butyricum) LXKJ-1 phyletic evolutions development tree, according to 16S
Morphological feature of rDNA comparison result combination bacterial strains etc. determines that the bacterial strain is potential clostridium butyricum new species（See attached drawing 3）.
Two clostridium butyricum three grade fermemtation of embodiment（200L fermentation tanks）
（1）The preparation of primary seed solution：By the clostridium butyricum prepared (Clostridium butyricum) LXKJ-1
Original bacterium solution is inoculated into progress seed preparation on primary-seed medium, and first order seed is prepared using blue lid bottle, prepares volume 1L,
Culture medium is as follows：15 g/L of glucose, 1 g/L of L-cysteine, sodium thioglycolate 1 g/L, K2HPO4 2 g/L, yeast
Soak 5 g/L of powder, soy peptone 6.5~7.0 cultivation temperature 36 of 5 g/L of 20 g/L, NaCl, sodium alginate 5 g/L, pH
DEG C, incubation time is for 24 hours.
（2）The preparation of secondary seed solution：By ready level-one clostridium butyricum (Clostridium butyricum)
LXKJ-1 seed liquors are inoculated in 20L fermentation tanks according to 5% inoculum concentration, and as secondary seed solution, secondary seed formula of liquid is two level
Seed culture medium, specific culture medium prescription are as follows：20 g/L of peptone, 2.5 g/L of beancake powder, glucose 15 g/L, NH4NO3 1
G/L, K2HPO41 g/L, MgSO40.1 g/L, MnSO40.1 g/L, 0.5 g/L of L-cysteine, 5 g/L of sodium alginate,
Urea 5 g/L, CaCO31 g/L, pH 6.5~7.0,36 DEG C of cultivation temperature, incubation time 8h.
（3）Three grade fermemtation：By ready two level clostridium butyricum (Clostridium butyricum) LXKJ-1 seeds
Liquid is inoculated in 200L fermentation tanks according to 10% inoculum concentration, is cultivated, and fermentation medium uses fermentation medium, specific to cultivate
Based component is as follows：25 g/L of corn flour, 15 g/L of bean cake powder, 20 g/L of tryptone, glucose 15 g/L, NH4NO3 1 g/L,
K2HPO4 1 g/L, MgSO40.1 g/L, MnSO40.1 g/L, CaCO31 g/L, 0.5 g/L of L-cysteine, alginic acid
5 g/L of sodium, 5 g/L of urea, pH 6.5~7.0,36 DEG C of cultivation temperature, incubation time is for 24 hours.
Zymotic fluid viable count as obtained by above-mentioned three grade fermemtation is up to 2.8*109More than cfu/mL, gemma rate 95% with
On.
The preparation of bacterium powder and active bacteria formulation is lyophilized in three clostridium butyricum of embodiment
（1）Thalline were collected by centrifugation：By the clostridium butyricum of above-mentioned gained (Clostridium butyricum) LXKJ-1
Zymotic fluid is centrifuged using tube centrifuge, centrifugal speed 12000rpm, charging rate 200L/h.
（2）The freeze-drying of thalline：By bacterium mud obtained by above-mentioned centrifugation according to weight ratio 1:3, add in 20% defatted milk after sterilizing
Powder suspension and 10% trehalose, mixing carry out vacuum freeze drying, and freeze-drying time is for 24 hours.
（3）Bacterium powder is lyophilized：It is crushed after thalline freeze-drying, freeze-drying bacterium powder is made.After measured using above method production gained
Bacterium powder viable bacteria viable count reaches as high as 1.0*1011More than cfu/g.
（4）The preparation of clostridium butyricum active bacteria preparation：By above-mentioned gained bacterium powder using glucose, starch, mountain flour, zeolite powder,
One or more than one kinds of conduct diluents in bean cake powder, maize cob meal or wheatfeed are prepared into viable count not less than 2.0*
108The clostridium butyricum active bacteria preparation of cfu/g.
In conclusion the present invention provides one plant of clostridium butyricum (Clostridium butyricum) LXKJ-1 and should
Seed culture medium, fermentative medium formula, condition of culture and its and the use three grade fermemtation method production clostridium butyricum work of bacterial strain
The method of bacteria preparation, medium component used in this method is simple, of low cost, high financial profit, is well suited for the rule of factory
Modelling produces, and fermentation gained culture viable count, gemma rate are high.It is very steady during storage since gemma is a kind of hypopus
It is fixed, and with good heat resistance, therefore it is strong with stability using the clostridium butyricum active bacteria preparation of above method production, it is resistance to
The advantages of storage.
Example IV clostridium butyricum high temperature resistant is tested
Clostridium butyricum active bacteria preparation is individually placed to 80 DEG C of processing 10min, 20min, 30min, 90 DEG C of processing in baking oven
5min, 10min, 15min, 100 DEG C of processing 5min, 10min, 15min, 105 DEG C of processing 5min, 10min, 15min each locate
Reason take 3 it is parallel, processing terminate room temperature cooling after measure viable count respectively.The average value of 3 parallel results is taken as most terminating
Fruit, to investigate the influence of different temperatures, different high-temperature process times to clostridium butyricum.Using the dilution progress of falling flat band method viable count
It measures.It the results are shown in Table 3.
3 clostridium butyricum active bacteria heat-resistance test of table
Result above can be seen that clostridium butyricum LXKJ-1 in vitro 80 DEG C processing 10min, 90 DEG C processing 10min,
100 DEG C of processing 5min gemma survival rates are still 100%.Illustrate that the gemma that the bacterial strain is formed has good heat resistance, Ke Yiyou
Effect protects thalline not influenced by feed granulating process high temperature, can be used as feed addictive.
Five clostridium butyricum of embodiment produces butyric acid and acetic acid ability
Clostridium butyricum is subjected to tablet culture, is inoculated into fermentation medium, is cultivated under 37 DEG C of anaerobic conditions for 24 hours, centrifugation
Supernatant is taken, butyric acid and acetic acid content are measured using gas chromatography.Testing conditions：By the injection of 1 μ L gas phase samples equipped with FID
19091 N-213 capillary columns of hydrogen flame ionization detector and HP-Innowax（30 m×0.32 mm ×0.5 um）Gas
Phase chromatography（Agilent Technologist, 7890A GC System）, carrier gas is helium, and flow velocity is 1.8 mL/min, point
Flow ratio 40:1, temperature program：90 DEG C of 0.5 min of maintenance, are promoted to 110 DEG C with the speed of 10 DEG C/min, then with 5 DEG C/min
Speed be raised to 170 DEG C, finally with the speed of 20 DEG C/min to 210 DEG C.275 DEG C of injector and detector temperature.
Measurement result：Butyric acid content is 8.12 g/L, and acetic acid content is 1.37 g/L.Show that clostridium butyricum LXKJ-1 is produced
Butyric acid ability is stronger.
Six clostridium butyricum extracorporeal bacteria inhibitor test of embodiment
Clostridium butyricum active bacteria preparation is subjected to tablet culture, is inoculated into fluid nutrient medium, is cultivated under 37 DEG C of anaerobic conditions
48h is using salmonella, Escherichia coli, Shigella, staphylococcus aureus, Listeria monocytogenes, C.perfringens as target
Bacterium is marked, the bacteriostatic activity of clostridium butyricum LXKJ-1 zymotic fluids is measured using cylinder-plate method, the results are shown in Table 4.
4 clostridium butyricum bacteriostatic test effect of table
The result shows that clostridium butyricum is husky to salmonella typhi YF, Bacterium enteritidis S (ATCC13076), mouse typhus
Door Salmonella zjc (ATCC14028), S. pullonum C79-13, shigella flexneri zjc, shigella flexneri 268, dysentery
Disease Shigella 269, Shigella sonnei Z, e. coli bl21, swine escherichia coli（Wild strain）, chicken colibacillosis（It is wild
Bacterial strain）, staphylococcus aureus JN, Listeria monocytogenes Lis, Listeria monocytogenes Lu and C.perfringens all have it is fine
Bacteriostatic activity.
Influence of seven clostridium butyricum of embodiment to Production Performance of Weaning Pigs and diarrhea rate
1 test method
1.1 test material：Clostridium butyricum（Contain clostridium butyricum active bacteria >=2.0 × 10 per g of formulation8cfu/g）.
Experimental animal：35 ± 1 ages in days Du × big × long three way cross piglet.
Experimental design：Experiment is designed using single-factor.The basically identical sodium selenite 364 of 35 ± 1 age in days weight is chosen,
It is randomly divided into 5 groups, every group of 4 repetitions, often repeatedly 18（Wherein there are four repeat 19）.Control group fed basal diet, 4
Test group distinguishes basal diet clostridium butyricum additive amount and lives for the g/t clostridium butyricums of 250g/t, 500 g/t, 1000 g/t, 2000
Bacteria preparation.Experimental period is 30 d.
Feeding management
During experiment, daily 07:00、14:00 and 21:00 feeding, feeding capacity is to there is a small amount of remaining material to be advisable in hopper, entirely
Phase is freely eaten and drinking-water, and epidemic prevention and feeding management carry out according to a conventional method.Daily 15:00 carries out diarrhea statistics, simultaneously
Observe the health status of piglet, record illness, extremely naughty piglet head number and its reason.
Index determining
Each repetition piglet head number, diarrhea are recorded daily and extremely washes in a pan situation, count a feed intake and diarrhea rate, meter weekly
Calculate average daily feed intake；It weighs on the day of off-test, counts full period feed intake, calculate full period feedstuff-meat ratio, grice diarrhoea rate is washed in a pan with dead
Rate.
Cecum microorganisms measure：Escherichia coli, the variation of salmonella, Bacillus acidi lactici, bifidobacteria.
Result of the test
Influence and its cecum microorganisms situation of the clostridium butyricum to Production Performance of Weaning Pigs and diarrhea rate are shown in Table 5, table 6.
Influence of 5 clostridium butyricum of table to Production Performance of Weaning Pigs and diarrhea rate
Influence of 6 clostridium butyricum of table to weanling pig cecum flora
The result shows that 0.025-0.2% clostridium butyricums are added in daily ration can significantly reduce the diarrhea rate of weanling pig, production performance is improved,
And significantly reduce Escherichia coli and salmonella quantity in caecum（Table 5 and 6）.
Influence of nine clostridium butyricum of embodiment to performance in layers, Egg Quality and cecum microorganisms
1 test method
1.1 material：Clostridium butyricum LXKJ-1（Contain clostridium butyricum active bacteria >=2.0 × 10 per g of formulation8cfu/g）
1.2 experimental animal：Red No. 1 laying hen in 46 week old capital
1.3 experimental design：Experiment is designed using single-factor.Choose 46 basically identical week old commercial generation eggs of 960 weight
Chicken is randomly divided into 5 groups, and every group of 6 repetitions are each to repeat 32.Control group fed basal diet, 4 test groups are respectively in base
Clostridium butyricum preparation additive amount is 125 g/t, 250 g/t, 500 g/t, 1000 g/t in plinth daily ration.14 days preliminary trial periods, formally
56 days phases.
Testing index
Egg laying performance：Average daily laying rate, average egg weight, daily output egg size, feed intake, feedstuff-egg ratio etc..
Egg quality：Hangh unit, shell thickness, eggshell strength.
Hangh unit：Hu=100lg（H-1.7W0.37+ 0.76) it is eggshell weight/g that, wherein H, which is dense albumen height/mm, W,.
Cecum microorganisms measure：Escherichia coli, the variation of lactic acid bacteria, bifidobacteria.
Result of the test
Influences of the 2.1 clostridium butyricum LXKJ-1 to performance in layers
Clostridium butyricum the results are shown in Table 7 to performance in layers influence.
Influence of 7 clostridium butyricum of table to performance in layers
Influence of 2.2 clostridium butyricums to laying hen Egg Quality
Clostridium butyricum is shown in Table 8 to the influence of laying hen egg quality
Influence of 8 clostridium butyricum of table to laying hen Egg Quality
2. influence of 3 clostridium butyricums to laying hen cecum microorganisms
Influence of the clostridium butyricum to laying hen intestines microorganism is shown in Table 9
Influence of 9 clostridium butyricum of table to laying hen intestines microorganism（Unit：lgcfu/g）
The result shows that 0.0125-0.1% clostridium butyricums are added in daily ration can effectively improve performance in layers, improve egg
Shell quality, while have improvement trend to flora in enteron aisle（Table 7,8,9）.
Ten clostridium butyricum of embodiment is to the growth-promoting effect of broiler chicken
1 test method
1.1 material：Clostridium butyricum LXKJ-1（Contain clostridium butyricum active bacteria >=2.0 × 10 per g of formulation8cfu/g）
1.2 experimental design：
It chooses 1 age in days, 10,000 plumage AA broiler chicken to be tested, is divided into control group and test group.Control group feeds basal diet,
Test group clostridium butyricum preparation additive amount in basal diet is 125 g/t, 250 g/t, 500 g/t, 1000 g/t, is tested
42 days periods.
Testing index
Growth performance measures：In first day on-test, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weekend when, every group is selected at random
50 broiler chicken are weighed, and are averaged, and calculate each group broiler chicken weight.Record experiment early period and the feed consumption rate in later stage, calculate meat
Chicken average daily gain, average daily gain and feedstuff-meat ratio.
Diarrhea rate and the death rate：Each group diarrhea number of elements and dead number of elements, calculate diarrhea rate and death during record feeding
Rate.
The measure of Microflora in excrement：Each group takes the fresh excrement samples of 1.0 g respectively, dilute using 10 times of gradient dilution methods
It releases to 10-8, selects continuous three dilutions, be respectively coated on the differential mediums tablet such as EMB, MRS, BBL, inoculum concentration
For 100 μ L/9cm tablets, each dilution is 3 parallel, 37 DEG C of constant temperature incubation 24-48 h.It predominantly detects in excrement
Escherichia coli, lactic acid bacteria, Bifidobacterium content.
Result of the test
Influence of 2.1 clostridium butyricums to meat chicken growth performance
Influence of 10 clostridium butyricum of table to AA meat chicken growth performances
Influence of 2.1 clostridium butyricums to broiler chicken cecum microorganisms
Influence of 11 clostridium butyricum of table to laying hen intestines microorganism（Unit：lgcfu/g）
The result shows that 0.0125%~0.1% clostridium butyricum preparation is added in daily ration is remarkably improved broiler chicken survival rate, promote
Into growth, weightening is improved, while reduce diarrhea rate（Table 10）, increase the quantity of lactic acid bacteria and Bifidobacterium in broiler chicken caecum,
Reduce Escherichia coli quantity（Table 11）.
The present invention from one plant of clostridium butyricum of health pig enteron aisle separation screening (Clostridium butyricum)LXKJ-
1, activity is high, and resistance is strong, while has found suitable for the culture medium and condition of culture of growth, breeding, and fermentation gained is cultivated
Object viable count is high, gemma rate is high, and medium component used in the invention is simple, of low cost, is well suited for the scale of factory
Production.Clostridium butyricum (Clostridium butyricum) LXKJ-1 is acidproof, alkaline-resisting, high temperature resistant, production butyric acid ability is strong, to big
The animal pathogens such as enterobacteria, salmonella, Shigella, golden yellow grape bacillus, Listeria monocytogenes, C.perfringens
It is all inhibited, pig, the chicken diarrhea as caused by Escherichia coli, salmonella, C.perfringens etc. can be prevented, improve intestines
Road colony balance promotes pig, chicken growth, improves laying rate of laying hen and egg size, improve eggshell quality, reduce feedstuff-egg ratio.
<110>Hubei green snow bio tech ltd
<120>One plant of clostridium butyricum and clostridium butyricum active bacteria preparation and application
<141>2016-10-28 <160>1 <210> 1 <211>The length of sequence<212>DNA
<213>Clostridium butyricum<400>Nucleotide sequence
agtgcggcagcttaccatgcagtcgagcgatgaagctccttcgggagtggattagcggcggacgggtgagtaacacg
tgggtaacctgcctcatagaggggaatagcctttcgaaaggaagattaataccgcataagattgtagtaccgcatgg
tacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggtgaggtaacggctcacca
aggcgacgatgcgtagccgacctgagagggtgatcggccacattgggactgagacacggcccagactcctacgggag
gcagcagtggggaatattgcacaatgggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcggatt
gtaaagctctgtctttagggacgataatgacggtacctaaggaggaagccacggctaactacgtgccagcagccgcg
gtaatacgtaggtggcaagcgttgtccggatttactgggcgtaaagggagcgtaggtggatatttaagtgggatgtg
aaatacccgggcttaacctgggtgctgcattccaaactggatatctagagtgcaggagaggaaaggagaattcctag
tgtagcggtgaaatgcgtagagattaggaagaataccagtggcgaaggcgcctttctggactgtaactgacactgag
gctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatactaggtgtaggg
gttgtcatgacctctgtgccgccgctaacgcattaagtattccgcctggggagtacggtcgcaagattaaaactcaa
aggaattgacgggggcccgcacaagcagcggagcatgtggtttaattcgaagcaacgcgaagaaccttacctagact
tgacatctcctgaattactctgtaatggaggaagccacttcggtggcaggaagacaggtggtgcatggttgtcgtca
gctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgttagttgctaccatttagttgagc
actctagcgagactgcccgggttaaccgggaggaaggtggggatgacgtcaaatcatcatgccccttatgtctaggg
ctacacacgtgctacaatggtcggtacaatgagatgcaacctcgcgagagtgagcaaaactataaaaccgatctcag
ttcggattgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatgtcgcggtgaata
cgttcccgggccttgtacacaccgcccgtcacaccatgagagttggcaatacccaaagttcgtgagctaaccgcaag
gaggcagcgacctaagtagtagagtt
agtgcggcagcttaccatgcagtcgagcgatgaagctccttcgggagtggattagcggcggacgggtgagtaacacg
tgggtaacctgcctcatagaggggaatagcctttcgaaaggaagattaataccgcataagattgtagtaccgcatgg
tacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggtgaggtaacggctcacca
aggcgacgatgcgtagccgacctgagagggtgatcggccacattgggactgagacacggcccagactcctacgggag
gcagcagtggggaatattgcacaatgggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcggatt
gtaaagctctgtctttagggacgataatgacggtacctaaggaggaagccacggctaactacgtgccagcagccgcg
gtaatacgtaggtggcaagcgttgtccggatttactgggcgtaaagggagcgtaggtggatatttaagtgggatgtg
aaatacccgggcttaacctgggtgctgcattccaaactggatatctagagtgcaggagaggaaaggagaattcctag
tgtagcggtgaaatgcgtagagattaggaagaataccagtggcgaaggcgcctttctggactgtaactgacactgag
gctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatactaggtgtaggg
gttgtcatgacctctgtgccgccgctaacgcattaagtattccgcctggggagtacggtcgcaagattaaaactcaa
aggaattgacgggggcccgcacaagcagcggagcatgtggtttaattcgaagcaacgcgaagaaccttacctagact
tgacatctcctgaattactctgtaatggaggaagccacttcggtggcaggaagacaggtggtgcatggttgtcgtca
gctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgttagttgctaccatttagttgagc
actctagcgagactgcccgggttaaccgggaggaaggtggggatgacgtcaaatcatcatgccccttatgtctaggg
ctacacacgtgctacaatggtcggtacaatgagatgcaacctcgcgagagtgagcaaaactataaaaccgatctcag
ttcggattgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatgtcgcggtgaata
cgttcccgggccttgtacacaccgcccgtcacaccatgagagttggcaatacccaaagttcgtgagctaaccgcaag
gaggcagcgacctaagtagtagagtt
Claims (7)
1. clostridium butyricum active bacteria preparation prepared by a kind of clostridium butyricum LXKJ-1, the clostridium butyricum, entitled LXKJ-1, classification
It is entitled：Clostridium butyricum LXKJ-1（Clostridium butyricum）LXKJ-1, deposit number are：CCTCC NO:
M201613, preservation date：On March 24th, 2016, preservation address are：China, Wuhan, Wuhan University, depositary institution：Chinese allusion quotation
Type culture collection；
It is characterized in that, it is prepared by method comprising the following steps：
1. by clostridium butyricum (Clostridium butyricum) LXKJ-1 is deposited in glycerol tube, freezing passes through scribing line
Method accesses slant medium, under anaerobic, 35~37 DEG C of cultures 20~and for 24 hours, wash lower gemma with sterile saline
Suspension, as original bacterium solution；
2. original bacterium solution is carried out amplification cultivation for strain, it is as follows：The bacteria suspension of 1 volume is linked into 20 volumes
Primary-seed medium in, in 1L indigo plant lid reagent bottles, under anaerobic condition, 35~37 DEG C culture 8~12 h, as level-one
Seed liquor；The primary seed solution of 1 volume is linked into the secondary seed medium of 10 volumes, in 20L fermentation tanks, nitrogen ring
Under border, 35~37 DEG C culture 8~12h, as secondary seed solution；The three grade fermemtation that secondary seed solution is moved into 20 times of volumes is trained
It supports in base, in 200L fermentation tanks, under nitrogen environment, 35~37 DEG C of cultures 20~for 24 hours to get to clostridium butyricum
(Clostridium butyricum) LXK J-1 culture；
3. microorganism collection, the bacterium mud being collected by centrifugation are carried out to culture using supercentrifugal process；
4. by 1:Skimmed milk power suspension, sucrose, lactose, trehalose, maltodextrin, the sorb of 1~5 ratio addition 5~20%
One or more kinds of combinations in alcohol, glycerine prepare freeze-drying thalline as freeze drying protectant using freeze-drying；
5. use the one or more in glucose, starch, mountain flour, zeolite powder, bean cake powder, maize cob meal, wheatfeed
Combination is diluted bacterium powder as auxiliary material, as needed, is configured to the clostridium butyricum active bacteria preparation of different viable counts；
The primary-seed medium is：Glucose 5~30 g/L, 0.1~5g/L of L-cysteine, sodium thioglycolate 0.1
~5g/L, K2HPO40.1~5 g/L, 1~10g/L of yeast extract, soy peptone 5~20g/L, NaCl1~10g/L, seaweed
Sour 1~10g/L of sodium, pH 6.5~7.0；
The secondary seed medium is：10~40g/L of peptone, 1~5g/L of beancake powder, glucose 10~30g/L, KNO3
0.5~2.0 g/L, K2HPO40.5~2.0g/L, MgSO40.1~0.5g/L, MnSO40.1~0.5g/L, L-cysteine
0.1~1.0g/L, 1.0~10g/L of sodium alginate, urea 1.0~5.0g/L, CaCO31.0~10g/L, pH6.5~7.0；
The three grade fermemtation culture medium is：10~40g/L of corn flour, 1~5g/L of beancake powder, 10~40g/L of tryptone, grape
Sugar 10~40g/L, NH4NO30.5~2.0g/L, K2HPO40.5~2.0 g/L, MgSO40.1~0.5 g/L, MnSO4 0.1
~0.5 g/L, L-cysteine 0.1~1.0 g/L, 1.0~10g/L of sodium alginate, urea 1.0~5.0 g/L, CaCO3
1.0~10g/L, pH6.5~7.0；Above all of culture medium is required for 121 DEG C, 30 min of steam sterilizing before use, cold
It is for use.
2. active bacteria formulation as described in claim 1, which is characterized in that the slant medium is：Glucose 1.2%, L- half
Cystine 0.03%, sodium thioglycolate 0.03%, K2HPO40.2%, yeast extract 0.3%, soy peptone 0.5%, peptone
1%, tryptone 1%, NaCl0.3%, sodium alginate 0.5%, agar 2%, pH 6.5~7.0.
3. a kind of method for preparing clostridium butyricum active bacteria preparation described in claims 1 or 2, includes the following steps：
1. by clostridium butyricum (Clostridium butyricum) LXKJ-1 its be deposited in glycerol tube, freezing, by draw
Line
Method accesses slant medium, under anaerobic, 35~37 DEG C of cultures 20~and for 24 hours, wash lower gemma with sterile saline
Suspension, as original bacterium solution；
2. original bacterium solution is carried out amplification cultivation for strain, it is as follows：The bacteria suspension of 1 volume is linked into 20 volumes
Primary-seed medium in, in 1L indigo plant lid reagent bottles, under anaerobic condition, 35~37 DEG C culture 8~12 h, as level-one
Seed liquor；The primary seed solution of 1 volume is linked into the secondary seed medium of 10 volumes, in 20L fermentation tanks, nitrogen ring
Under border, 35~37 DEG C culture 8~12h, as secondary seed solution；The three grade fermemtation that secondary seed solution is moved into 20 times of volumes is trained
It supports in base, in 200L fermentation tanks, under nitrogen environment, 35~37 DEG C of cultures 20~for 24 hours to get to clostridium butyricum
(Clostridium butyricum) LXK J-1 culture；
3. microorganism collection, the bacterium mud being collected by centrifugation are carried out to culture using supercentrifugal process；
4. by 1:Skimmed milk power suspension, sucrose, lactose, trehalose, maltodextrin, the sorb of 1~5 ratio addition 5~20%
One or more kinds of combinations in alcohol, glycerine prepare freeze-drying thalline as freeze drying protectant using freeze-drying；
5. use the one or more in glucose, starch, mountain flour, zeolite powder, bean cake powder, maize cob meal, wheatfeed
Combination is diluted bacterium powder as auxiliary material, as needed, is configured to the clostridium butyricum active bacteria preparation of different viable counts.
4. a kind of clostridium butyricum active bacteria preparation as described in claim 1 answering in animal and fowl fodder microbe additive is prepared
With.
5. application as claimed in claim 4, which is characterized in that the clostridium butyricum active bacteria preparation improves life for weanling pig
It produces performance and reduces diarrhea rate.
6. application as claimed in claim 4, which is characterized in that the clostridium butyricum active bacteria preparation improves productivity for laying hen
Energy, Egg Quality increase lactic acid bacteria and the quantity of Bifidobacterium and reduction Escherichia coli quantity in laying hen caecum.
7. application as claimed in claim 4, which is characterized in that the clostridium butyricum active bacteria preparation for broiler chicken improve broiler chicken into
Motility rate reduces diarrhea rate, increases lactic acid bacteria and the quantity of Bifidobacterium and reduction Escherichia coli quantity in broiler chicken caecum.
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| CN103205375A (en) * | 2013-03-15 | 2013-07-17 | 厦门和美科盛生物技术有限公司 | Quality clostridium butyricum for breeding egg-laying poultry |
| CN103205373A (en) * | 2013-03-15 | 2013-07-17 | 厦门和美科盛生物技术有限公司 | Quality clostridium butyricum for breeding piglets |
| CN104277999A (en) * | 2014-09-15 | 2015-01-14 | 中国农业大学 | Novel multiple-effect Clostridium butyricum and application thereof in aspects of enhancing oxidation resistance of animals and improving meat quality |
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