CN106479924A - The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application - Google Patents
The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application Download PDFInfo
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- CN106479924A CN106479924A CN201610927003.9A CN201610927003A CN106479924A CN 106479924 A CN106479924 A CN 106479924A CN 201610927003 A CN201610927003 A CN 201610927003A CN 106479924 A CN106479924 A CN 106479924A
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Abstract
The invention discloses the preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application, Clostridium butyricum (Clostridium butyricum) LXKJ on March 24th, 1,2016 is preserved in China typical culture collection center, preserving number is CCTCC NO:M201613.Also disclosed the production method of seed culture medium, fermentative medium formula, condition of culture and its active bacteria formulation of this bacterial strain simultaneously.The Clostridium butyricum that the present invention provides is acidproof, alkaline-resisting, high temperature resistant, produce butanoic acid ability strong, all inhibited to animal pathogens such as escherichia coli, Salmonella, shigella, golden yellow Fructus Vitis viniferae bacillus, Listeria monocytogenes, bacillus perfringens, the diarrhoea that poultry can be prevented to be caused by escherichia coli, Salmonella, bacillus perfringens etc., improves intestinal microbial population balance, promotes growth of animals or poultry, alleviate prevention of sow constipation, improve laying hen egg size, improve laying hen eggshell quality, reduce 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
Application.
Background technology
Cultivate the expansion further of scale now with people, in order to improve culture benefit, various antibiotic are as growth-promoting
The a large amount of use of long agent has become as a kind of universal phenomenon, and its drawback increasingly highlights, such as drug residue, the report of pathogenic bacteria of drug-resistant
Road, unbalance, environmental pollution of microbial population of animal intestinal tract etc., have seriously threatened health and the food safety of the mankind.Microbial ecological agent
As antibiotic substitute products that are a kind of safe efficient, pollution-free and having no drug resistance progressively as diseases prevention, growth promotion formulation application
In aquaculture.
Microbial ecological agent is probiotics fermention and the biological system of microbial cells obtained from rear processing and its metabolite
Agent and active bacteria formulation.Therefore there is maintenance intestinal microecology balance, improve word material transformation efficiency, improve growth of animals or poultry performance, carry
High immunologic function, antioxidation and anti-cancer and improve environment, reduce the effect such as generation of harmful substance, are increasingly subject to aquaculture and are chased after
Hold in both hands.
Clostridium butyricum (Clostridium butyricum), it is to enter closely to control doctor by Chiba, Japan medical university palace first
Find first and report, therefore Clostridium butyricum is also designated as Clostridium Butyricum, Japan also becomes research Clostridium butyricum history simultaneously
Country the longest.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 paired, short chain, amphitrichous are movable, and thalline can form spore, and middle part enlarges into
Fusiformis, spore bias Cheng Ciduan life, no epispore or appendage；How rounded bacterium colony is, less, raised, milky, butanoic acid shuttle
Pseudomonas, in anaerobe, can produce butanoic acid, propanoic acid, acetic acid, can also produce hydrogen etc. simultaneously, studies have found that during liquid fermentation
, to the such as bacillus bifiduss of the probiotic bacteria in the animal intestinals such as pig, chicken, the growth of lactic acid bacteria etc. is numerous for the butanoic acid of Clostridium butyricum generation, acetic acid
Grow and there is good facilitation, and harmful intestinal tract bacteria such as escherichia coli, bacillus perfringens, salmonella typhi etc. are all had
There is good inhibiting effect, the butanoic acid that metabolism produces directly is utilized by intestinal fine hair, promotes gut epithelium histiocytic
Regeneration and reparation, the hydrogen that metabolism produces has repair to the oxidative damage of body liver, kidney organ, can strengthen machine simultaneously
Body function of detoxification.Therefore Clostridium butyricum is known as " intestinal health first bacterium ".
Patent CN201110116927, CN201110126498 etc. has 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 butanoic acid shuttle
The application of bacterium, but be not directed to various dose Clostridium butyricum to pig, laying hen, meat chicken production performance impact.
Content of the invention
The present invention from health pig intestinal separation screening one plant of Clostridium butyricum (Clostridium butyricum)LXKJ-
1, its activity is high, strong stress resistance, have found its growth suitable, the culture medium of breeding and condition of culture, fermentation gained culture simultaneously
Thing viable count is high, spore rate is high, and the medium component that this invention is used is simple, with low cost, is well suited for the scale of factory
Produce.Clostridium butyricum (Clostridium butyricum) LXKJ-1 is acidproof, alkaline-resisting, high temperature resistant, produces butanoic acid ability strong, to big
The animal pathogens such as enterobacteria, Salmonella, shigella, golden yellow Fructus Vitis viniferae bacillus, Listeria monocytogenes, bacillus perfringens
Diarrhoea all inhibited, that pig, chicken can be prevented to be caused by escherichia coli, Salmonella, bacillus perfringens etc., improves intestinal
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 is：Clostridium butyricum LXKJ-1Clostridium butyricumLXKJ-1, deposit number is：CCTCC NO:M201613, preservation date：On March 24th, 2016, preservation address
For：China, Wuhan, Wuhan University, depositary institution：China typical culture collection center.
A kind of clostridium butyricum active bacteria preparation of Clostridium butyricum LXKJ-1 preparation.
A kind of clostridium butyricum active bacteria formulation preparation method, comprises the following steps：
1. by Clostridium butyricum (Clostridium butyricum) LXKJ-1 its be deposited in glycerol tube, freezing, by draw
Collimation method accesses slant medium, under anaerobic, 35～37 DEG C of culture 20～24h, wash lower spore with physiological saline solution and hang
Liquid, as original bacterium solution；
2. original bacterium solution is carried out amplification cultivation for strain, it comprises the following steps that：
The bacteria suspension of 1 volume is linked in the primary-seed medium of 20 volumes, in 1 L indigo plant lid reagent bottle, anaerobic condition
Under, 35～37 DEG C culture 8～12 h, as primary seed solution；
The primary seed solution of 1 volume is linked in the secondary seed medium of 10 volumes, in 20 L fermentation tanks, nitrogen environment
Under, 35～37 DEG C culture 8～12 h, as secondary seed solution；
Secondary seed solution is moved in the three grade fermemtation culture medium of 20 times of volumes, in 200 L fermentation tanks, under nitrogen environment, 35
～37 DEG C culture 20～24 h, that is, obtain Clostridium butyricum (Clostridium butyricum) LXKJ-1 culture；
3. microorganism collection is carried out to culture using supercentrifugal process, the bacterium mud being collected by centrifugation；
4. press 1:1～5 defatted milk powder suspension of ratio interpolation 5～20%, sucrose, Lactose, trehalose, maltodextrin, Pyrusussuriensiss
One of alcohol, glycerol or more than one combination, as freeze drying protectant, prepare lyophilizing thalline using freeze-drying；
5. using one of glucose, starch, stone powder, zeolite powder, bean cake powder, maize cob meal, wheatfeed or more than one
Combination is diluted to mycopowder as adjuvant, as needed, is 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%, 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.
Further, above-mentioned primary-seed medium is：Glucose 5～30 g/L, L-Cysteine 0.1～5 g/L,
Sodium thioglycolate 0.1～5 g/L, K2HPO40.1～5 g/L, yeast extract 1～10 g/L, soy peptone 5～20
G/L, NaCl 1～10 g/L, sodium alginate 1～10 g/L, pH 6.5～7.0；
Described secondary seed medium is：Peptone 10～40 g/L, soybean cake powder 1～5 g/L, glucose 10～30 g/L,
KNO30.5～2.0 g/L, K2HPO40.5～2.0 g/L, MgSO40.1～0.5 g/L, MnSO40.1～0.5 g/L, L-
Cysteine 0.1～1.0 g/L, sodium alginate 1.0～10 g/L, carbamide 1.0～5.0 g/L, CaCO31.0～10 g/
L, pH 6.5～7.0；
Described three grade fermemtation culture medium is：Semen Maydis powder 10～40 g/L, soybean cake powder 1～5 g/L, tryptone 10～40 g/L,
Glucose 10～40 g/L, NH4NO30.5～2.0 g/L, K2HPO4.5～2.0 g/L, MgSO40.1～0.5 g/L,
MnSO40.1～0.5 g/L, L-Cysteine 0.1～1.0 g/L, sodium alginate 1.0～10 g/L, carbamide 1.0～5.0
G/L, CaCO31.0～10 g/L, pH6.5～7.0；
Above all of culture medium is required for steam sterilization before use（121 DEG C, 30 min）, cool down stand-by.
A kind of above-mentioned Clostridium butyricum or above-mentioned Clostridium butyricum preparation answering in preparing animal and fowl fodder microbe additive
With.
Further, above-mentioned acid clostridium active bacteria formulation is used for ablactational baby pig and improves production performance and reduction diarrhea rate, and shows
Write and reduce escherichia coli and Salmonella quantity in caecum.
Further, above-mentioned clostridium butyricum active bacteria preparation is used for laying hen and improves production performance, Egg Quality, increases broiler caecum
The quantity of middle lactic acid bacteria and bacillus bifiduss and reduction escherichia coli quantity.
Further, above-mentioned clostridium butyricum active bacteria preparation is used for broiler and improves broiler survival rate, reduces diarrhea rate, promotes life
Long, increase the quantity of lactic acid bacteria and bacillus bifiduss and reduction escherichia coli quantity in broiler caecum.
The present invention from health pig intestinal separation screening one plant of Clostridium butyricum (Clostridium butyricum)LXKJ-
1, its activity is high, strong stress resistance, have found its growth suitable, the culture medium of breeding and condition of culture, fermentation gained culture simultaneously
Thing viable count is high, spore rate is high, and the medium component that this invention is used is simple, with low cost, is well suited for the scale of factory
Produce.Clostridium butyricum (Clostridium butyricum) LXKJ-1 is acidproof, alkaline-resisting, high temperature resistant, produces butanoic acid ability strong, to big
The animal pathogens such as enterobacteria, Salmonella, shigella, golden yellow Fructus Vitis viniferae bacillus, Listeria monocytogenes, bacillus perfringens
Diarrhoea all inhibited, that pig, chicken can be prevented to be caused by escherichia coli, Salmonella, bacillus perfringens etc., improves intestinal
Road colony balance, promotes pig, chicken growth, improves laying rate of laying hen and egg size, improve eggshell quality, reduce feedstuff-egg ratio.
The Clostridium butyricum that the present invention provides has acidproof, alkaline-resisting, resistant to elevated temperatures feature.PH value 1.0-4.0 remains to survive, pH
All can grow during value 5.0-12.0, optimum pH is 6.0-7.0；External 80 DEG C of dry heat treatment 10min, 90 DEG C of dry heat treatment
10min, 100 DEG C of dry heat treatment 5min spore survival rates 100%.
It is strong, to escherichia coli, Salmonella, shigella, golden yellow that the Clostridium butyricum that the present invention provides produces butanoic acid ability
The animal pathogens such as Fructus Vitis viniferae bacillus, Listeria monocytogenes, bacillus perfringens are all inhibited.
Brief description
Fig. 1 Clostridium butyricum (Clostridium butyricum) LXKJ-1 culture bacterium colony photo（This bacterial strain bacterium colony is in
Circle, milky, bacterium colony projection）；
Fig. 2 Clostridium butyricum (Clostridium butyricum) LXKJ-1 spore photo (spore ovalize, spore wall
Thick)；
Fig. 3 Clostridium butyricum (Clostridium butyricum) taxonomy of LXKJ-1 and phylogenetic tree；
The bacteriostasis comparison diagram to bacillus perfringens for Fig. 4 Clostridium butyricum centrifuged supernatant；
Clostridium butyricum in accompanying drawing 1 is numbered and is：LXKJ-1, specific name is：Clostridium butyricum LXKJ-1Clostridium butyricumLXKJ-1, deposit number is：CCTCC NO:M201613, preservation date：On March 24th, 2016, preservation address
For：China, Wuhan, Wuhan University, depositary institution：China typical culture collection center.
Specific embodiment
To be further elucidated with the present invention below by the detailed description of specific embodiment, but to be not the limit to the present invention
System, only illustrates.
Embodiment one Clostridium butyricum (Clostridium butyricum) LXKJ-1 separation and identification
Clostridium butyricum (Clostridium butyricum) LXKJ-1 is located away from the intestinal of health pig, first will collection sample
In 80 DEG C of heating in water bath 10 min after product normal saline dilution, to kill non-sporeformer；Then it is inoculated in slant medium
On, cultivate 24-30 h in 36 DEG C of anaerobic jars, Preliminary Identification is carried out to bacterial strain by colonial morphology and thalli morphology, therefrom selects
Go out cultural characteristic, colonial morphology and thalli morphology and meet the bacterial strain of Clostridium butyricum feature continuously to rule purification culture three generations, respectively
Marker number enters next step identification and screens.
Further determine that Clostridium butyricum (Clostridium butyricum) LXKJ-1 taxonomy, the present invention adopts
This bacterial strain is classified identify 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) company limited.Its sequence is respectively： 27F
(5 '-AGAGTTTGATCCTGGCTC-3 ') and 1492R (5 '-CGGCTACCTTGTTACGACTT-3 ')
3 test methods
3.1 morphological observation
The microbionation culture of pure culture will be determined, observe its morphological characteristic
Flat board four zoning collimation method, obtains single bacterium colony, observe the size of its bacterium colony, color, shape, color and luster, transparency, consistency and
The features such as edge, and record.
Bacterial cell form（Individual morphology）Observation
Bacterial strain to be checked is carried out with Gram’s staining, in gram positive bacteria body, contains special nucleoprotein magnesium salt and polysaccharide
Complex, can be combined very firm with the complex of iodine and crystal violet, be difficult decolour, negative bacterium complex combination degree bottom, inhale
Attached dye is poor, easily decolours.Observe its Gram’s staining situation under an optical microscope, judge that it is gram positive bacteria or leather
Lan Shi negative bacterium.And the morphological characteristic of preliminary observation cell.
3.2 Clostridium butyricum (Clostridium butyricum) LXKJ-1 physiological and biochemical property
3.2.1 API 20NE identification mark
3.2.1.1 the preparation of test bar
Take out culture box, strain number and dat recorder in side, add in culture plate sterilized water cover honeycomb little recessed in, prevent
Only in incubation, reagent strip is dried.Take out test bar from the package, be placed in culture plate.
3.2.1.2 the preparation of inoculum
Obtain the flat board of inoculation experiments bacterial strain with four zoning collimation methods, scraped new fresh thalli to normal saline with aseptic cotton carrier, extremely
Maxwell concentration 0.5.
3.2.1.3 the inoculation of reagent strip
3.2.1.4 inoculate saline bacteria suspension respectively from NO with same suction pipe3To PNPG developmental tube.Test strips slightly forward
Incline and the top of suction pipe is leaned against tubule inner edge liquid feeding body, to avoid the formation of bubble.
3.2.1.5 open an ampoule bottle APIAUX culture medium and add 200 microlitres of remaining normal saline bacteria suspensions to ampoule
Bottle, carefully mixes, it is to avoid have bubble to produce.Fill it up with GUL to PAC pipe, 3 line experiment GLU, ADH, URE, covered with mineral oil
Lid, cultivates in 29 DEG C.
3.2.2 NO3Measure：
Plus a NIT1 and NIT2 to NO3Pipe, after 5min, red expression is positive, because nitrogen may produce feminine gender, plus 2-3mgZn
To NO3Pipe, keeps after 5min not changing color as the positive, reddens as feminine gender.
Table 1 bacterial strain LXKJ-1 physio-biochemical characteristics enzyme activity, carbon assimilation
+：Positive reaction； -：Negative reaction； W：Weakly positive is reacted.
Table 2 bacterial strain LXKJ-1 physio-biochemical characteristics utilize carbon source to produce acid
+：Positive reaction； -：Negative reaction
3.3 Clostridium butyricum (Clostridium butyricum) LXKJ-1 molecular biology identification
3.3.1 Clostridium butyricum (Clostridium butyricum) LXKJ-1 genomic DNA extraction adopt SDS-CTAB method.
3.3.2 Clostridium butyricum (Clostridium butyricum) amplification of LXKJ-116S rDNA gene used
Primer is 27F (5'AGAGTTTGATCCTGGCTCAG), 1492R (5'TACGGCTACCTTGTTACGACTT), and sequence measurement is adopted
Use clone sequencing.PCR reaction system is 50 μ L: ddH2O 40.7 μ L, 10 × Buffer 5 μ L, dNTP Mixture
（10 μM）1 μ L, primer 2 7F（10 μmol/L）1 μ L, primer 1492R（10 μmol/L）1 μ L,TaqArchaeal dna polymerase 0.3 μ
L, template DNA 1 μ L.PCR amplification condition is：94 DEG C of 5 min, 95 DEG C of 30 s, 56 DEG C of 30s, 72 DEG C of 90 s, follow
Ring number of times 30 times；72 ℃ 8 min.Examining order entrusts Shanghai bioengineering Services Co., Ltd to complete.
3.3.3 the comparison analysis of sequencing result
In EZ-Biocloud, BLAST comparison is carried out to the sequencing result of bacterial strain, chooses representative strain, using adjacent method（NJ）Method
Tetraploid rice is carried out to this bacterial strain, simultaneously constructing system cladogram, then determine this bacterial strain by Phylogenetic analysis
Race relation.In conjunction with the cultural characteristic of bacterial strain, morphological characteristic and the evolutionary relationship to this bacterial strain etc., preliminary classification is carried out to bacterial strain
Identification.
4 result of the tests
4.1 Clostridium butyricum (Clostridium butyricum) LXKJ-1 morphological observations
4.2 Clostridium butyricum (Clostridium butyricum) LXKJ-1 molecular biology identification result
4.2.1 Clostridium butyricum (Clostridium butyricum) LXKJ-1 16S rDNA sequencing results use
DNAMAN5.2 software splice, determine this fragment by 1412 base compositions, obtain Clostridium butyricum (Clostridium butyricum) LXKJ-1 16S rDNA sequence (seeing below table).
4.2.2 homology cladogram builds
Clostridium butyricum (Clostridium butyricum) the 16S rDNA the sequencing results of LXKJ-1 show, bacterial strain butanoic acid
Clostridium (Clostridium butyricum) LXKJ-1 and bacterial strainClostridium butyricumDSM 10702 (T) phase
It is 99.15% like degree to the maximum, next to thatClostridium diolisDSM 5431(T)（97.37%）WithClostridium saccharoperbutylacetonicumN1-4(HMT)(T)（97.23%）, then build bacterium with MEGA 5.05 software NJ method
Strain Clostridium butyricum (Clostridium butyricum) LXKJ-1 phyletic evolution growth tree, tied according to 16S rDNA comparison result
Close morphological characteristic of bacterial strain etc. and determine that this bacterial strain is potential Clostridium butyricum new species（See accompanying drawing 3）.
Embodiment two Clostridium butyricum three grade fermemtation（200L fermentation tank）
（1）The preparation of primary seed solution：By the Clostridium butyricum preparing (Clostridium butyricum) LXKJ-1 is original
Bacterium solution is inoculated on primary-seed medium and carries out seed preparation, and first order seed, using blue lid bottle preparation, prepares volume 1L, culture
Base is as follows：Glucose 15 g/L, L-Cysteine 1 g/L, sodium thioglycolate 1 g/L, K2HPO42 g/L, yeast extract
5 g/L, soy peptone 20 g/L, NaCl 5 g/L, 36 DEG C of sodium alginate 5 g/L, pH 6.5～7.0 cultivation temperature, training
Foster time 24h.
（2）The preparation of secondary seed solution：By ready one-level Clostridium butyricum (Clostridium butyricum)
LXKJ-1 seed liquor is inoculated in 20L fermentation tank according to 5% inoculum concentration, and as secondary seed solution, secondary seed formula of liquid is two grades
Seed culture medium, concrete culture medium prescription is as follows：Peptone 20 g/L, soybean cake powder 2.5 g/L, glucose 15 g/L, NH4NO31
G/L, K2HPO41 g/L, MgSO40.1 g/L, MnSO40.1 g/L, L-Cysteine 0.5 g/L, sodium alginate 5 g/L,
Carbamide 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 grades of Clostridium butyricum (Clostridium butyricum) LXKJ-1 seed
Liquid is inoculated in 200L fermentation tank according to 10% inoculum concentration, is cultivated, and fermentation medium adopts fermentation medium, concrete culture
Based component is as follows：Semen Maydis powder 25 g/L, bean cake powder 15 g/L, tryptone 20 g/L, glucose 15 g/L, NH4NO31 g/L,
K2HPO41 g/L, MgSO40.1 g/L, MnSO40.1 g/L, CaCO31 g/L, L-Cysteine 0.5 g/L, alginic acid
Sodium 5 g/L, carbamide 5 g/L, pH 6.5～7.0,36 DEG C of cultivation temperature, incubation time 24h.
By the zymotic fluid viable count of above-mentioned three grade fermemtation gained up to 2.8*109More than cfu/mL, spore rate 95% with
On.
Embodiment three Clostridium butyricum lyophilizing mycopowder and the preparation of active bacteria formulation
（1）Thalline is collected by centrifugation：By the Clostridium butyricum of above-mentioned gained (Clostridium butyricum) LXKJ-1 fermentation
Liquid is centrifuged using tube centrifuge, and centrifugal speed is 12000rpm, and charging rate is 200L/h.
（2）The lyophilizing of thalline：Above-mentioned centrifugation gained bacterium mud is compared 1 according to weight:3, add the defatted milk of 20% after sterilizing
Powder suspension and 10% trehalose, mix, carry out vacuum lyophilization, freeze-drying time 24h.
（3）Lyophilizing mycopowder：Pulverize after thalline lyophilizing, prepared lyophilizing mycopowder.Produce gained using said method after measured
Mycopowder 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 mycopowder adopt glucose, starch, stone powder, zeolite powder,
One of bean cake powder, maize cob meal or wheatfeed or more than one as diluent be prepared into viable count be not less than 2.0*
108The clostridium butyricum active bacteria preparation of cfu/g.
In sum, the invention provides one plant of Clostridium butyricum (Clostridium butyricum) LXKJ-1 and should
The seed culture medium of bacterial strain, fermentative medium formula, condition of culture and its and using three grade fermemtation method produce Clostridium butyricum live
The method of bacteria preparation, the medium component that the method is used is simple, and with low cost, high financial profit is well suited for the rule of factory
Modelling produces, and fermentation gained culture viable count, spore rate are high.Because spore is a kind of hypopuss, very steady during depositing
Fixed, and there is good thermostability, therefore stability is had by force using the clostridium butyricum active bacteria preparation that above method produces, resistance to
The advantage of storage.
Test that example IV Clostridium butyricum is high temperature resistant
Clostridium butyricum active bacteria preparation is individually placed to 80 DEG C of process 10min, 20min, 30min in baking oven, 90 DEG C of process
5min, 10min, 15min, 100 DEG C process 5min, 10min, 15min, and 105 DEG C process 5min, 10min, 15min, at each
Reason take 3 parallel, process terminate room temperature cooling after measure viable count respectively.Take the meansigma methodss of 3 parallel results as terminating most
Really, different temperatures, the different impact to Clostridium butyricum for the high-temperature process time are investigated.Using dilution, flat band method carries out viable count
Measure.The results are shown in Table 3.
Table 3 clostridium butyricum active bacteria heat-resistance test
Result above can be seen that Clostridium butyricum LXKJ-1 in vitro 80 DEG C process 10min, 90 DEG C process 10min, 100
DEG C process 5min spore survival rate still be 100%.Illustrate that the spore that this bacterial strain is formed has good thermostability, can be effective
Protect thalline not affected by feed granulating process high temperature, can use as feed additive.
Embodiment five Clostridium butyricum produces butanoic acid and acetic acid ability
Clostridium butyricum is carried out flat board culture, is inoculated in fermentation medium, cultivate 24h under 37 DEG C of anaerobic conditions, in centrifuging and taking
Clear liquid, measures butanoic acid and acetic acid content using gas chromatography.Testing conditions：1 μ L gas phase sample injection is furnished with FID hydrogen fire
Flame ion detector and HP-Innowax 19091 N-213 capillary column（30 m×0.32 mm ×0.5 um）Gas phase color
Spectrum（Agilent Technologist, 7890A GC System）, carrier gas is helium, and its flow velocity is 1.8 mL/min, split ratio
40:1, heating schedule：90 DEG C of maintenance 0.5 min, rise to 110 DEG C, then the speed with 5 DEG C/min with the speed of 10 DEG C/min
Degree is raised to 170 DEG C, finally with the speed of 20 DEG C/min to 210 DEG C.Injector and 275 DEG C of detector temperature.
Measurement result：Butanoic acid content is 8.12 g/L, and acetic acid content is 1.37 g/L.Show that Clostridium butyricum LXKJ-1 produces
Butanoic acid ability is stronger.
Embodiment six Clostridium butyricum extracorporeal bacteria inhibitor test
Clostridium butyricum active bacteria preparation is carried out flat board culture, is inoculated in fluid medium, under 37 DEG C of anaerobic conditions, cultivate 48h
With Salmonella, escherichia coli, shigella, staphylococcus aureuses, Listeria monocytogenes, bacillus perfringens as target
Bacterium, is measured the bacteriostatic activity of Clostridium butyricum LXKJ-1 fermentation liquid, the results are shown in Table 4 using cup-plate method.
Table 4 Clostridium butyricum bacteriostatic test effect
Result shows, Clostridium butyricum is to salmonella typhi YF, Salmonella enteritidis S (ATCC13076), Salmonella typhimurium
Bacterium zjc (ATCC14028), S. pullonum C79-13, shigella flexneri zjc, shigella flexneri 268, dysentery will
Hayes bacterium 269, Shigella sonnei Z, e. coli bl21, swine escherichia coli（Wild strain）, chicken colibacillosis（Wild mushroom
Strain）, staphylococcus aureuses JN, Listeria monocytogenes Lis, Listeria monocytogenes Lu and bacillus perfringens all have well
Bacteriostatic activity.
The impact to Production Performance of Weaning Pigs and diarrhea rate for embodiment seven Clostridium butyricum
1 test method
1.1 test material：Clostridium butyricum（Every g of formulation contains clostridium butyricum active bacteria >=2.0 × 108cfu/g）.
Experimental animal：35 ± 1 age in days Du × big × long three way cross piglet.
EXPERIMENTAL DESIGN：Test is using single-factor design.Choose the basically identical sodium selenite 364 of 35 ± 1 age in days body weight,
It is randomly divided into 5 groups, every group 4 repetitions, often repeat 18（Four are wherein had to repeat 19）.Control group fed basal diet, 4
Basal diet Clostridium butyricum addition is that 250g/t, 500 g/t, 1000 g/t, 2000 g/t Clostridium butyricum is lived to test group respectively
Bacteria preparation.Experimental period is 30 d.
Feeding and management
During test, daily 07:00、14:00 and 21:00 feeding, there to be a small amount of remaining material to be advisable in hopper, full period is certainly for feeding capacity
By searching for food and drinking water, epidemic prevention and feeding and management are carried out according to a conventional method.Daily 15:00 carries out diarrhoea statistics, observes simultaneously
The health status of piglet, record ill, extremely naughty piglet head number and its reason.
Index determining
Record each repetition piglet head number, diarrhoea daily and extremely wash in a pan situation, count a feed intake and diarrhea rate weekly, calculate day
All feed intakes；The off-test same day weighs, and counts full period feed intake, calculates full period feedstuff-meat ratio, piglet diarrhea rate and death rate.
Cecum microorganisms measure：Escherichia coli, Salmonella, lactobacilluss, the change of bifidobacteria.
Result of the test
The impact to Production Performance of Weaning Pigs and diarrhea rate for the Clostridium butyricum and its cecum microorganisms situation are shown in Table 5, table 6.
The impact to Production Performance of Weaning Pigs and diarrhea rate for table 5 Clostridium butyricum
The impact to ablactational baby pig cecum flora for table 6 Clostridium butyricum
Result shows, adds, in daily ration, the diarrhea rate that 0.025-0.2% Clostridium butyricum can significantly reduce ablactational baby pig, improves and produces
Performance, and significantly reduce escherichia coli and Salmonella quantity in caecum（Table 5 and 6）.
The impact to performance in layers, Egg Quality and cecum microorganisms for embodiment nine Clostridium butyricum
1 test method
1.1 material：Clostridium butyricum LXKJ-1（Every g of formulation contains clostridium butyricum active bacteria >=2.0 × 108cfu/g）
1.2 experimental animal：Red No. 1 laying hen in 46 week old capital
1.3 EXPERIMENTAL DESIGN：Test is using single-factor design.Choose 46 basically identical week old commercial generation laying hens of 960 body weight,
It is randomly divided into 5 groups, every group 6 repetitions, each repeats 32.Control group fed basal diet, 4 test group are respectively in basic day
In grain, Clostridium butyricum preparation addition is 125 g/t, 250 g/t, 500 g/t, 1000 g/t.14 days preliminary trial periods, the formal phase 56
My god.
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), wherein H is dense albumen height/mm, and W is eggshell weight/g.
Cecum microorganisms measure：Escherichia coli, lactic acid bacteria, the change of bifidobacteria.
Result of the test
The impact to performance in layers for the 2.1 Clostridium butyricum LXKJ-1
Clostridium butyricum the results are shown in Table 7 to performance in layers impact.
The impact to performance in layers for table 7 Clostridium butyricum
The impact to laying hen Egg Quality for 2.2 Clostridium butyricum
Clostridium butyricum is shown in Table 8 to the impact of laying hen egg quality
The impact to laying hen Egg Quality for table 8 Clostridium butyricum
2. the impact to laying hen cecum microorganisms for 3 Clostridium butyricum
The impact to laying hen intestinal microorganism for the Clostridium butyricum is shown in Table 9
The impact to laying hen intestinal microorganism for table 9 Clostridium butyricum（Unit：lgcfu/g）
Result shows, adding 0.0125-0.1% Clostridium butyricum in daily ration can effectively improve performance in layers, improve eggshell product
Matter, has improvement trend to flora in intestinal simultaneously（Table 7,8,9）.
The growth-promoting effect to broiler for embodiment ten Clostridium butyricum
1 test method
1.1 material：Clostridium butyricum LXKJ-1（Every g of formulation contains clostridium butyricum active bacteria >=2.0 × 108cfu/g）
1.2 EXPERIMENTAL DESIGN：
Choose 1 age in days 10,000 plumage AA broiler to be tested, be divided into matched group and test group.Matched group feeds basal diet, test
Group Clostridium butyricum preparation addition in basal diet is 125 g/t, 250 g/t, 500 g/t, 1000 g/t, the test period
42 days.
Testing index
Growth performance measures：First day on-test, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weekend when, every group is selected 50 at random
Broiler is weighed, and averages, and calculates each group broiler weight.Record test early stage and the feed consumption rate in later stage, calculate broiler and put down
All daily gain, average daily gain and feedstuff-meat ratios.
Diarrhea rate and mortality rate：During record feeding, each group diarrhoea number of elements and dead number of elements, calculate diarrhea rate and death
Rate.
The mensure of Microflora in feces：Each group takes 1.0 g fresh excrement sample respectively, dilute using 10 times of gradient dilution methods
Release to 10-8, select continuous three dilution factors, be respectively coated on the differential medium flat board such as EMB, MRS, BBL, inoculum concentration
For 100 μ L/9cm flat boards, each dilution factor is 3 parallel, 37 DEG C of constant temperature culture 24-48 h.Predominantly detect in feces
Escherichia coli, lactic acid bacteria, the content of bacillus bifiduss.
Result of the test
The impact to meat chicken growth performance for 2.1 Clostridium butyricum
The impact to AA meat chicken growth performance for table 10 Clostridium butyricum
The impact to broiler cecum microorganisms for 2.1 Clostridium butyricum
The impact to laying hen intestinal microorganism for table 11 Clostridium butyricum（Unit：lgcfu/g）
Result shows, adds 0.0125%～0.1% Clostridium butyricum preparation and is remarkably improved broiler survival rate, promote life in daily ration
Long, improve weightening, reduce diarrhea rate simultaneously（Table 10）, increase the quantity of lactic acid bacteria and bacillus bifiduss in broiler caecum, reduce
Escherichia coli quantity（Table 11）.
The present invention from health pig intestinal separation screening one plant of Clostridium butyricum (Clostridium butyricum)LXKJ-
1, its activity is high, strong stress resistance, have found its growth suitable, the culture medium of breeding and condition of culture, fermentation gained culture simultaneously
Thing viable count is high, spore rate is high, and the medium component that this invention is used is simple, with low cost, is well suited for the scale of factory
Produce.Clostridium butyricum (Clostridium butyricum) LXKJ-1 is acidproof, alkaline-resisting, high temperature resistant, produces butanoic acid ability strong, to big
The animal pathogens such as enterobacteria, Salmonella, shigella, golden yellow Fructus Vitis viniferae bacillus, Listeria monocytogenes, bacillus perfringens
Diarrhoea all inhibited, that pig, chicken can be prevented to be caused by escherichia coli, Salmonella, bacillus perfringens etc., improves intestinal
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
agtgcggcagcttaccatgcagtcgagcgatgaagctccttcgggagtggattagcggcggacgggtgagtaa
cacgtgggtaacctgcctcatagaggggaatagcctttcgaaaggaagattaataccgcataagattgtagtaccgc
atggtacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggtgaggtaacggctc
accaaggcgacgatgcgtagccgacctgagagggtgatcggccacattgggactgagacacggcccagactcctacg
ggaggcagcagtggggaatattgcacaatgggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcg
gattgtaaagctctgtctttagggacgataatgacggtacctaaggaggaagccacggctaactacgtgccagcagc
cgcggtaatacgtaggtggcaagcgttgtccggatttactgggcgtaaagggagcgtaggtggatatttaagtggga
tgtgaaatacccgggcttaacctgggtgctgcattccaaactggatatctagagtgcaggagaggaaaggagaattc
ctagtgtagcggtgaaatgcgtagagattaggaagaataccagtggcgaaggcgcctttctggactgtaactgacac
tgaggctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatactaggtgt
aggggttgtcatgacctctgtgccgccgctaacgcattaagtattccgcctggggagtacggtcgcaagattaaaac
tcaaaggaattgacgggggcccgcacaagcagcggagcatgtggtttaattcgaagcaacgcgaagaaccttaccta
gacttgacatctcctgaattactctgtaatggaggaagccacttcggtggcaggaagacaggtggtgcatggttgtc
gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgttagttgctaccatttagtt
gagcactctagcgagactgcccgggttaaccgggaggaaggtggggatgacgtcaaatcatcatgccccttatgtct
agggctacacacgtgctacaatggtcggtacaatgagatgcaacctcgcgagagtgagcaaaactataaaaccgatc
tcagttcggattgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatgtcgcggtg
aatacgttcccgggccttgtacacaccgcccgtcacaccatgagagttggcaatacccaaagttcgtgagctaaccg
caaggaggcagcgacctaagtagtagagtt
agtgcggcagcttaccatgcagtcgagcgatgaagctccttcgggagtggattagcggcggacgggtgagtaa
cacgtgggtaacctgcctcatagaggggaatagcctttcgaaaggaagattaataccgcataagattgtagtaccgc
atggtacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggtgaggtaacggctc
accaaggcgacgatgcgtagccgacctgagagggtgatcggccacattgggactgagacacggcccagactcctacg
ggaggcagcagtggggaatattgcacaatgggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcg
gattgtaaagctctgtctttagggacgataatgacggtacctaaggaggaagccacggctaactacgtgccagcagc
cgcggtaatacgtaggtggcaagcgttgtccggatttactgggcgtaaagggagcgtaggtggatatttaagtggga
tgtgaaatacccgggcttaacctgggtgctgcattccaaactggatatctagagtgcaggagaggaaaggagaattc
ctagtgtagcggtgaaatgcgtagagattaggaagaataccagtggcgaaggcgcctttctggactgtaactgacac
tgaggctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatactaggtgt
aggggttgtcatgacctctgtgccgccgctaacgcattaagtattccgcctggggagtacggtcgcaagattaaaac
tcaaaggaattgacgggggcccgcacaagcagcggagcatgtggtttaattcgaagcaacgcgaagaaccttaccta
gacttgacatctcctgaattactctgtaatggaggaagccacttcggtggcaggaagacaggtggtgcatggttgtc
gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgttagttgctaccatttagtt
gagcactctagcgagactgcccgggttaaccgggaggaaggtggggatgacgtcaaatcatcatgccccttatgtct
agggctacacacgtgctacaatggtcggtacaatgagatgcaacctcgcgagagtgagcaaaactataaaaccgatc
tcagttcggattgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatgtcgcggtg
aatacgttcccgggccttgtacacaccgcccgtcacaccatgagagttggcaatacccaaagttcgtgagctaaccg
caaggaggcagcgacctaagtagtagagtt
Claims (9)
1. a kind of Clostridium butyricum it is characterised in that：Entitled LXKJ-1, specific name is：Clostridium butyricum LXKJ-1Clostrid
Ium butyricum LXKJ-1, deposit number is：CCTCC NO:M201613, preservation date：On March 24th, 2016, preservation
Address is：China, Wuhan, Wuhan University, depositary institution：China typical culture collection center.
2. by the clostridium butyricum active bacteria preparation of the Clostridium butyricum LXKJ-1 preparation described in claim 1.
3. as claimed in claim 2 a kind of clostridium butyricum active bacteria formulation preparation method it is characterised in that comprising the following steps：
1. by Clostridium butyricum (Clostridium butyricum) LXKJ-1 its be deposited in glycerol tube, freezing, by draw
Collimation method accesses slant medium, under anaerobic, 35～37 DEG C of culture 20～24h, wash lower spore with physiological saline solution and hang
Liquid, as original bacterium solution；
2. original bacterium solution is carried out amplification cultivation for strain, it comprises the following steps that：
The bacteria suspension of 1 volume is linked in the primary-seed medium of 20 volumes, in 1 L indigo plant lid reagent bottle, anaerobic condition
Under, 35～37 DEG C culture 8～12 h, as primary seed solution；
The primary seed solution of 1 volume is linked in the secondary seed medium of 10 volumes, in 20 L fermentation tanks, nitrogen environment
Under, 35～37 DEG C culture 8～12 h, as secondary seed solution；
Secondary seed solution is moved in the three grade fermemtation culture medium of 20 times of volumes, in 200 L fermentation tanks, under nitrogen environment, 35
～37 DEG C culture 20～24 h, that is, obtain Clostridium butyricum (Clostridium butyricum) LXK J-1 culture；
3. microorganism collection is carried out to culture using supercentrifugal process, the bacterium mud being collected by centrifugation；
4. press 1:1～5 defatted milk powder suspension of ratio interpolation 5～20%, sucrose, Lactose, trehalose, maltodextrin, Pyrusussuriensiss
One of alcohol, glycerol or more than one combination, as freeze drying protectant, prepare lyophilizing thalline using freeze-drying；
5. using one of glucose, starch, stone powder, zeolite powder, bean cake powder, maize cob meal, wheatfeed or more than one
Combination is diluted to mycopowder as adjuvant, as needed, is configured to the clostridium butyricum active bacteria preparation of different viable counts.
4. preparation method as claimed in claim 3 is it is characterised in that described 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%, albumen
Peptone 1%, tryptone 1%, NaCl 0.3%, sodium alginate 0.5%, agar 2%, pH 6.5～7.0.
5. the preparation method as described in claim 3 or 4 it is characterised in that
Described primary-seed medium is：Glucose 5～30 g/L, L-Cysteine 0.1～5 g/L, sodium thioglycolate
0.1～5 g/L, K2HPO40.1～5 g/L, yeast extract 1～10 g/L, soy peptone 5～20 g/L, NaCl 1～
10 g/L, sodium alginate 1～10 g/L, pH 6.5～7.0；
Described secondary seed medium is：Peptone 10～40 g/L, soybean cake powder 1～5 g/L, glucose 10～30 g/L,
KNO30.5～2.0 g/L, K2HPO40.5～2.0 g/L, MgSO40.1～0.5 g/L, MnSO40.1～0.5 g/L, L-
Cysteine 0.1～1.0 g/L, sodium alginate 1.0～10 g/L, carbamide 1.0～5.0 g/L, CaCO31.0～10 g/
L, pH 6.5～7.0；
Described three grade fermemtation culture medium is：Semen Maydis powder 10～40 g/L, soybean cake powder 1～5 g/L, tryptone 10～40 g/L,
Glucose 10～40 g/L, NH4NO30.5～2.0 g/L, K2HPO4.5～2.0 g/L, MgSO40.1～0.5 g/L,
MnSO40.1～0.5 g/L, L-Cysteine 0.1～1.0 g/L, sodium alginate 1.0～10 g/L, carbamide 1.0～5.0
G/L, CaCO31.0～10 g/L, pH6.5～7.0；
Above all of culture medium is required for steam sterilization before use（121 DEG C, 30 min）, cool down stand-by.
6. a kind of Clostridium butyricum or Clostridium butyricum preparation as described in claim 2 as described in claim 1 is raised in preparation poultry
Application in material microbe additive.
7. application as claimed in claim 6 improves life it is characterised in that described clostridium butyricum active bacteria preparation is used for ablactational baby pig
Produce performance and reduce diarrhea rate.
8. application as claimed in claim 6 improves productivity it is characterised in that described clostridium butyricum active bacteria preparation is used for laying hen
Energy, Egg Quality, increase the quantity of lactic acid bacteria and bacillus bifiduss and reduction escherichia coli quantity in broiler caecum.
9. application as claimed in claim 6 improves broiler one-tenth it is characterised in that described clostridium butyricum active bacteria preparation is used for broiler
Motility rate reduces diarrhea rate, increases the quantity of lactic acid bacteria and bacillus bifiduss and reduction escherichia coli quantity in broiler caecum.
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