CN103275907B - Bacillus amyloliquefacien and preparation method and application thereof - Google Patents

Bacillus amyloliquefacien and preparation method and application thereof Download PDF

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CN103275907B
CN103275907B CN201310240706.0A CN201310240706A CN103275907B CN 103275907 B CN103275907 B CN 103275907B CN 201310240706 A CN201310240706 A CN 201310240706A CN 103275907 B CN103275907 B CN 103275907B
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bacillus amyloliquefaciens
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徐高原
王杨波
周明光
金建云
于福祥
李芳�
蔡浩
金梅林
陈焕春
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WUHAN KEQIAN BIOLOGICAL CO., LTD.
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WUHAN KEQIAN ANIMAL BIOLOGICAL PRODUCTS CO Ltd
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Abstract

The invention discloses a bacillus amyloliquefacien and a preparation method and application thereof. According to the bacillus amyloliquefacien and the preparation method and the application thereof, the culture collection number of the bacillus amyloliquefaciens H6 is CCTCC No: M2013249; the bacillus amyloliquefaciens has obvious growth inhibition effect for gram-positive bacteria and gram-negative bacteria, strong degradation ability for starch, albumen, and cellulose, strong environment tolerance, and excellent effect for preventing diarrhea and promoting growing, and can not produce adverse effect with large doses of feeding; and the rear condition is simple, the cost is low, and the development and application prospect is excellent.

Description

A kind of bacillus amyloliquefaciens and preparation method and application
Technical field
The present invention relates to animal feedstuff additive technical field, relate in particular to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6, also relate to the preparation method of a kind of bacillus amyloliquefaciens H6, also relate to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 in the application of preparing in animal feedstuff additive.
Background technology
In feed, completely forbidding interpolation microbiotic was global development trend: European Union formally forbade adding in feed any microbiotic in 2006; Korea S started to forbid the use of feeding antibiotic in 2010; Strict restriction has also been done to the microbiotic adding in feed by the countries such as Japan and the U.S.; Stop using antibiotic feed additive, must select can substitute antibiotics fodder additives substitute, unaffected to ensure efficiency and benefit that livestock industry produces.In this case, being hopeful most as microbiotic surrogate fodder additives---the exploitation of microbial preparation just causes that various countries pay attention to thereupon.Study the earliest and apply the history of livestock and poultry probiotics and can trace back to nineteen forty-seven, Meng Hade (Mollgaard) first finds to use the lactobacillus piglet of feeding can effectively increase the body weight of piglet and improve the healthy of piglet.But the gold period of 20th century 50, the sixties of microbiotic research just, production and use, cause probiotics research-and-development activity at low ebb, be not given sufficient attention for a long period, until the mid-80 situation has just had the torsion of essence.American-European all states have developed many microbial preparations in succession in recent years, and basic conception is with the normal microorganism member of people and animals, through artificial propagation, make active bacteria formulation, and then make it get back to original living environment, bring into play its natural physiological action.United States food and drug administration and U.S. feed management association has just announced 43 kinds " can Direct-fed and be commonly referred to be safe microorganism (GRAS) " for 1989, and, as the starting strain of probiotics, various countries research staff has developed rich and varied product category on this basis.Although research and the application of China to microbial preparation is more late than developed country's starting, starting point is higher.Only, in short several years, the development and application of microbial preparation is to promoting the development of China's livestock industry to bring into play effect energetically, and potentiality are very large.Its application not only makes that animal growth is accelerated, disease reduces, the price of deed increases, and the quality of animal products might as well, avoided in animal body residual of the veterinary drugs such as antibacterials.Therefore, can say, microbial preparation is the green food of livestock and poultry.On the other hand, from the Economic Development Status of China, development microbial preparation should be subject to due attention.The Ministry of Agriculture of China has announced 16 kinds of microbial feed additive strains that can directly use in 2008, mainly taking milk-acid bacteria, streptococcus faecium, genus bacillus, yeast, sheet coccus and photosynthetic bacterium etc. as main; In addition, thermophilus streptococcus and Bacillus licheniformis were also in the protection period of new feed additive.The animal specific microbiotic of China is of less types, and researchs and develops that new for animals antibiotic difficulty is large, cost is high, the cycle is long, by contrast, the research of microbial preparation is relative simple with production, use safelyr, meet the national conditions of China, and have huge application market.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is to be extensively present in non-the curing the disease property bacterium of natural one, in the process of growth of himself, can produce a series of meta-bolites, have the activity of anti-bacteria and fungi widely, it is widely used security and has also obtained the accreditation of the European food safety council (EFSA) in agriculture production.At present, domestic its applied research is only limited to replacement organic pesticide, suppress plant pathogenic fungi and virus waits biological control; And degraded agricultural wastes, suppress the fields such as environmental improvement such as Measures of Algae in Water Body growth.Research shows, bacillus amyloliquefaciens can produce multiple hydrolase and antibacterial, antiviral protein isoreactivity material, it is necessary utilizing from field widely production and the service for life that the premium properties of this bacterial classification is people, further enrich China's microbial feed additive strain resource, meet the needs of the more and more higher green cultivation of social environmentally friendlyization and resources conservation requirement.
Summary of the invention
The object of the present invention is to provide a kind of bacillus amyloliquefaciens, called after bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6, this bacterium is sent to Chinese Typical Representative culture collection center on June 5th, 2013 and carries out preservation, Classification And Nomenclature: bacillus amyloliquefaciens H6 Bacillus amyloliquefaciens H6; Deposit number: CCTCC NO.M2013249, address Wuhan, China Wuhan University.This bacterium has that good anti-bacterial effect, enzyme activity are high, the feature of strong stress resistance, and starch, albumen and fiber are have to very strong degradability.
A further object of the invention has been to provide a kind of preparation method of bacillus amyloliquefaciens H6 bacterium powder.Preparation method is simple, and Yi Hang is suitable for scale operation, the bacterium powder that utilizes the method to make, and its bacillus amyloliquefaciens H6 bacterium powder yield is that powder yield is 40kg/m 3fermented liquid, viable bacteria amount reaches 4.7 × 10 11cfu/g.
A further object of the invention has been to provide a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 in the application of preparing in fodder additives, the fodder additives that utilizes bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 in the present invention to prepare, obviously prevention of intestinal tract disease, improve growth of animal characteristic and purifying aquaculture environment, and have safe, effective, with low cost, feature.
To achieve these goals, the present invention takes following technical measures:
A kind of bacillus amyloliquefaciens, is screened and is obtained by following steps:
1) gather health pig fresh excreta from Hubei Huang Po nature compost pig farm, by excrement sample aerobic enrichment culture muddiness in nutrient broth (NB) substratum, in 80 DEG C of water-baths, process 15min, enrichment and heat screening are once so again, suitably after dilution, be coated with dull and stereotyped, according to the single bacterium colony of colonial morphology picking typical case (bacterium colony subcircular, 3-4.5mm, tarnish, central authorities' protuberantia, edge cognate shape, canescence), go out genus bacillus by microscopy scalping.
2) screen streptococcus aureus (G by Oxford cup bacteriostatic test +), intestinal bacteria (G -) the good genus bacillus of pathogenic bacterium growth-inhibiting performance, by Physiology and biochemistry and 16s qualification, finishing screen is chosen a bacillus amyloliquefaciens, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6(is hereinafter to be referred as H6 bacterial strain or H6), this bacterium is sent to Chinese Typical Representative culture collection center on June 5th, 2013 and carries out preservation, Classification And Nomenclature: bacillus amyloliquefaciens H6 Bacillus amyloliquefaciens H6 deposit number: CCTCC NO:M2013249, address Wuhan, China Wuhan University.16Sr DNA sequence dna is shown in SEQ ID NO.1.
H6 bacterial strain specifically has typical bacillus amyloliquefaciens physiological and biochemical property: the bacterium colony that this bacterial strain is bred growth formation on nutrient agar medium (NA) substratum is subcircular, 3-4.5mm, tarnish, central protuberantia, edge cognate shape, canescence, by microscopy G+, thalline is shaft-like, single arrangement, gemma column, inferior end is raw, and sporangiocyst does not expand.Physiological and biochemical property is: gelatine liquefication.Glucose fermentation, fructose, sucrose, wood sugar and maltose, nonfermented semi-lactosi, pectinose and trehalose.The V.P positive, the M.R. positive, H 2s is weak positive, urease negative, and the catalase positive, aerobic, resistance to 7%NaCl.28~32 DEG C of optimum temperutures, 50 DEG C of top temperatures.Alkali is produced in litmus milk reaction, peptonizes reduction.Utilize nitrate.
The formula (1L) of NB substratum: extractum carnis 5g, peptone 10g, NaCl5g; NA adds agar 15g again.
A preparation method for bacillus amyloliquefaciens H6 bacterium powder, its step is as follows:
With transfering loop picking bacillus amyloliquefaciens inclined-plane seed, inoculation NB shake-flask seed substratum, cultivates 7~10h for 37 DEG C, and OD value reaches 1.0 left and right.The seed liquor of cultivating is inoculated to amplification culture in 50L fermentor tank by the inoculum size of 1% (V/V), fermention medium liquid amount 65%, in culturing process, real-time online is added defoamer, control oxyty 30%~50%, 32 DEG C of temperature controls, cultivate 10~12h, and OD value reaches more than 4.0.By the seed fermentation liquid of amplification culture, by the inoculum size inoculation 500L ferment tank liquid of 10% (V/V), 65%, 30 DEG C of liquid amount is cultivated 40h left and right, and froth breaking and control dissolved oxygen in real time reaches 70%~80% to spore forming rate.Put after tank through microfiltration of ceramic membrane, be concentrated into original volume 1/8~1/4 volume at gauge pressure 0.2~0.3MPa, spraying is dry, obtains bacillus amyloliquefaciens bacterium powder.
Fermentative medium formula (1L): Semen Maydis powder 10~50g, bean cake powder 10~50g, yeast extract paste 5~20g, peptone 5~20g, CaCO31~5g, NaCl1~5g, MgSO41~5g, KH 2pO41~5g, polyethers 0.5~3ml, add silicon oil foam killer online.
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 is in the application of preparing in fodder additives: its application process is:
By bacillus amyloliquefaciens H6 viable bacteria powder or compound with other microbial inoculums, in 0.01%~0.1%(w/w) ratio add to animal and fowl fodder or drinking-water in, the security in heavy dose of detoxification of bacillus amyloliquefaciens bacterium powder is good, thereby the different steps of producing at livestock and poultry cultivation is fed and can, for improving intestinal health, prevent constipation, prevent and treat bacterial diarrhea, promoting intestinal absorption to improve production and reproductive performance, be alleviated the pollution of excrement dirt to environment.
Compared with prior art, the present invention has the following advantages:
1. the invention provides that a kind of good anti-bacterial effect, enzyme activity are high, the bacillus amyloliquefaciens of strong stress resistance, scale operation preparation and effectively using at an easy rate, part replaces at present to environment and the healthy antibiotic use that causes increasing threat.
2. bacillus amyloliquefaciens of the present invention has obvious growth-inhibiting effect to Gram-positive and the feminine gender bacterium of curing the disease, in animal and bird intestines, can suppress harmful microbe growth, and have very strong starch, albumen and cellulose degradation ability, environmental resistance is strong; In livestock and poultry cultivation is fed, have good control diarrhoea and promote growth result, and heavy dose of feeding can not produce detrimental action; Culture condition is simple, with low cost, has good development prospect.
Brief description of the drawings
Fig. 1 is a kind of bacillus amyloliquefaciens H6 brood cell form schematic diagram.
Fig. 2 is the growth curve chart of bacillus amyloliquefaciens H6 in fermentor tank.
Embodiment
In the embodiment of the present invention, experimental technique and condition if no special instructions, are ordinary method.
Embodiment 1:
Strains separation
Gather health pig fresh excreta from the natural compost of Hubei Huang Po pig farm, by excrement sample aerobic enrichment culture muddiness in nutrient broth (NB) substratum, in 80 DEG C of water-baths, process 15min, enrichment and heat screening are once so again, suitably after dilution, be coated with dull and stereotyped, according to the single bacterium colony of colonial morphology picking typical case (bacterium colony subcircular, 3-4.5mm, tarnish, central authorities' protuberantia, edge cognate shape, canescence), go out genus bacillus by microscopy scalping.
The formula (1L) of NB substratum: extractum carnis 5g, peptone 10g, NaCl5g, agar 15g.
Multiple sieve and qualification
Screen streptococcus aureus (G by Oxford cup bacteriostatic test +), intestinal bacteria (G -) etc. the good genus bacillus of pathogenic bacterium growth-inhibiting performance, by Physiology and biochemistry and 16s qualification, finishing screen is chosen a bacillus amyloliquefaciens, called after Bacillus amyloliquefaciens H6.This bacterium is sent to Chinese Typical Representative culture collection center on June 5th, 2013 and carries out preservation, Classification And Nomenclature: bacillus amyloliquefaciens H6 Bacillus amyloliquefaciens H6 deposit number: CCTCC NO.M2013249, address Wuhan, China Wuhan University.
H6 bacterial strain specifically has typical bacillus amyloliquefaciens physiological and biochemical property: the bacterium colony that this bacterial strain is bred growth formation on NB substratum is subcircular, 3-4.5mm, tarnish, central protuberantia, edge cognate shape, canescence, by microscopy G+, thalline is shaft-like, single arrangement, gemma column, inferior end is raw, and sporangiocyst does not expand (Fig. 1).Physiological and biochemical property is: gelatine liquefication.Glucose fermentation, fructose, sucrose, wood sugar and maltose, nonfermented semi-lactosi, pectinose and trehalose.The V.P positive, the M.R. positive, H 2s is weak positive, urease negative, and the catalase positive, aerobic, resistance to 7%NaCl.28~32 DEG C of optimum temperutures, 50 DEG C of top temperatures.Alkali is produced in litmus milk reaction, peptonizes reduction.Utilize nitrate.
Embodiment 2: the amplification culture of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6
Press Semen Maydis powder 15g/L, bean cake powder 20g/L, yeast extract paste 10g/L, peptone 10g/L, CaCO 33g/L, NaCl5g/L, MgSO 42g/L, KH 2pO 43g/L, polyethers 2ml/L, with 65%(V/V) liquid amount preparation fermented liquid, through 121 DEG C of high-temperature steam sterilization 20min, be cooled to 40 DEG C, inoculate wherein the seed liquor 1% of cell age 10h, 32 DEG C of cultivations, control mixing speed and maintain dissolved oxygen (DO value is more than 30%), the on-line automatic defoamer (silicone oil) of adding.OD value is surveyed in every 2h sampling, and fermentation 24h is put tank, and plate count bacillus amyloliquefaciens H6 viable bacteria amount is 1.78 × 10 10cfu/ml(Fig. 2).
Embodiment 3: bacillus amyloliquefaciens bacterium powder preparation
The bacterium liquid that embodiment 2 is fermented is removed large solid substance through vibratory screening apparatus coarse filtration, be concentrated into 1/6 of original volume through 0.2 μ m microfiltration of ceramic membrane again, concentrated solution is directly used peristaltic pump people spray-drying tower, 180 ± 3 DEG C of inlet temperatures, temperature out 67~70, control charging flow velocity and heater switch, holding temperature balance, after rewinding, calculating bacterium powder yield is 40kg/m 3fermented liquid, plate count bacterium powder bacterium amount is 4.7 × 10 11cfu/g.
Embodiment 4:H6 bacterial strain enzymic activity detects
Be ready to starch, casein and Mierocrystalline cellulose agar plate (above three kinds of substratum are the rich commercially available culture medium in sea, commercially available Qingdao), H6 bacterial strain is recovered in PA bottle, get 5ul point and be connected to flat board above, cultivate.Whether have hydrolysis ring, the surrounding that connects bacterium toward the dull and stereotyped point of starch drips iodine liquid if observing casein and Mierocrystalline cellulose flat board, observe and whether have hydrolysis ring, and measure hydrolysis ring diameter.
Table 1H6 strains for degrading ability
Degraded is dull and stereotyped Starch flat board Casein plate Mierocrystalline cellulose flat board
Degraded circle (mm) 26.6 27.53 20.18
Embodiment 5:H6 bacterial strain resistance detects
Hot tolerance: get H6 growth and be completed into latter stage brood cell's medium centrifugal, (Eddy diffusion after 0.9% physiological saline washed twice is lived all amount (1~2 × l0 9cfu/m) in 75,80,85,90,95 DEG C of water-baths, process after 15min, flowing water is cooling rapidly, then adopts dilution-plate method to measure bud and embraces survival rate.
The survival rate of table 2H6 bacterial strain under treatment of different temperature
Temperature (DEG C) 75 80 85 90 95
Survival rate (%) 90.5 86.7 73.5 54.8 37.2
Get massfraction and be 10% hydrochloric acid 16.4mL adding distil water dilution, making pH value is 2.0, in every 100mL hydrochloric acid soln, adds 1g stomach en-, uses the filtering with microporous membrane degerming of 0.22um after fully dissolving, and obtains simulated gastric fluid.Get the culture that H6 growth has been completed into brood cell latter stage centrifugal, Eddy diffusion after 0.9% physiological saline washed twice, with 10 7cfu/ml final concentration is inoculated in simulated gastric fluid, and concussion mixes, and 37 DEG C of water-baths are hatched, and carries out live bacterial count every 0.5h sampling.
Table 3H6 bacterial strain is processed viable bacteria quantitative change under simulation hydrochloric acid in gastric juice condition
Treatment time (h) 0 0.5 1 1.5 2
Bacterium amount (cfu/ml) 1.2*10 7 10.3*10 6 8.2*10 6 7.6*10 6 5.9*10 6
Extra 1.0%, 2.0%, 3.0%, 4.0%, the 5.0%(w/v that adds of preparation) the NB substratum of pig cholate.Get 1ml H6 bacterium liquid (1~2 × l0 8cfu/mL), be inoculated in the different dense high bile salt concentiration NB substratum of 9mL, cultivate after 12h for 37 DEG C, measure nutrient solution turbidity and change, judge thalli growth situation, in table 1.
Table 4: the impact of different concns pig cholate on H6 growth
Note: ++ represent well-grown ,+representative growth is general, and-representative can not be grown.
Embodiment 6:H6 bacterial strain bacteriostasis detects
Sterilized NB nutrient agar is heated to completely and is melted, be poured in culture dish Mei Min 15ml(lower floor), treat that it solidifies.In addition, the NB substratum of thawing being cooled to 50 DEG C of left and right sneak into pharmacosensitive test bacteria culture fluid 5ml is added to and on the substratum having solidified, waits to solidify (upper strata).Directly vertically put cup (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm in Oxford with aseptic technique in media surface, the two ends of pipe are smooth), pressurization gently, make it contact tight with substratum, in cup, add sample to be checked (fermented liquid), Oxford cup generally can fill 200 microlitres, does not make it excessive.Fill it up with rearmounted 37 DEG C and cultivate 16-18 hour, observations, inhibition zone is directly measured and just can with chi.
The antibacterial circle diameter of table 5H6 to different pathogenic bacterium
Pathogenic bacterium Intestinal bacteria Pasteurellosis bacillus White dysentery Salmonellas Streptococcus aureus
Inhibition zone (mm) 19.32 15.71 16.01 16.84
Embodiment 7: mouse proof test
The kunming mice of 60 body weight 20g left and right is divided into 3 groups at random, 20 every group, male and female half and half.Wherein one group is done blank, experimental group one every by 1 × 10 9cfu/kg Mouse Weight (100 times of routine doses) gavages H6 fermented liquid for continuous one week, the bacteria suspension (1 × 10 of experimental group two disposable celiac injection 0.2ml 9cfu/ml).Observe one week, record growth and the death condition of each group of mouse.All mouse feeding experiments are all dry, clean, draughty indoor completing, and mouse is death condition, and experimental group two mouse two days bodies after abdominal injection are heavy relatively light, and rear recovery is normal, and experimental group one mouse growth situation is best.
Table 6: mouse experiment weighing results
Experiment grouping Initial weight (g) Within 3 days, weigh (g) Within 7 days, weigh (g)
Control group 19.3±2.04 20.15±2.37 23.29±2.25
Experimental group one 20.4±0.35 22.4±0.70 25.30±0.15
Experimental group two 19.5±2.21 19.8±1.80 23.89±2.28
Embodiment 8: piglet experiment:
Choose body weight close, with 28 of batch 26 age in days piglets, be divided into 4 groups, 7 every group, one group is control group, every of experimental group A is by percentage of liveweight 10 7the bacterium amount of cfu/kg is mixed water and is gavaged, and every of experimental group B presses percentage of liveweight 10 8the bacterium amount of cfu/kg is mixed water and is gavaged, and every of experimental group C presses percentage of liveweight 10 9the bacterium amount of cfu/kg is mixed water and is gavaged experimental group and fill with once every day, and continuous irrigation 7 days, observes 21 days.Free choice feeding, every day entry spirit and the situation of searching for food, weigh when experiment finishes.Gavage continuously the lower abnormal response that all do not have through the experimental observations of more than 20 days in heavy dose, search for food and growing state normal, and bacillus group has obvious growth-promoting effect.
Table 7: piglet gavages the impact of various dose H6 on weightening finish
Group Just body weight kg The heavy kg of opisthosoma Weightening finish kg Rate of body weight gain
Blank group 7.20 11.93 4.73 65.6%
Experimental group A 7.08 12.91 5.83 82.3%
Experimental group B 7.13 13.87 6.74 94.5%
Experimental group C 6.71 12.62 5.91 88.1%
Embodiment 9: pregnant sow experiment
Choose 20 of same batch of pregnant sows, be divided into 2 groups, 10 every group, one group is control group, and another group is produced and arrived wean in previous month for every of experimental group, by pig body weight 2 × 10 7the bacterium amount of cfu/kg is mixed H6 bacterium powder to feeding in feed diet, observes sow spirit and ight soil situation every day, claims sucking pig initial weight and farrowing survival rate postpartum.Observe experimental group prevention of sow constipation and the condition of production and obviously improve, the average sucking pig initial weight of control group is 1.35 kilograms, and litter size is 9.4, and the average sucking pig initial weight of experimental group is 1.42 kilograms, and litter size is 10.1.
Embodiment 10:
Choose body weight close, with 40 of batch 26 age in days piglets (7 kilograms of left and right of mean body weight), divide 2 groups, every group 20, control group fed basal diet, experimental group is admixed H6 bacterium powder respectively basal diet and is fed (spice ratio is 0.03% weight ratio), and every is eaten the about percentage of liveweight of bacterium amount pig day is 2*10 7cfu/kg, now mixes existing use, feeds 35 days, and free choice feeding, records daily ingestion amount, weighs on an empty stomach weekly.Performance assessment criteria: diarrhea rate, feedstuff-meat ratio.
Table 8: anti-diarrhea and weightening finish experimental result
Embodiment 11: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 microbial inoculum is at the effect of fattening in piglet, broiler chicken and grass carp cultivation.
Use the bacterium powder prepared of embodiment 3, be applied to respectively the cultivation of fattening piglet, broiler chicken and grass carp, all establish blank group, microbiotic group and H6 experimental group, every group of 3 repetitions, experiment periods is one month.Wherein, the blank group basal diet of feeding; Microbiotic group is added basal diet and is added bacillus skin zinc (effective content 50g/t) and colistin sulfate mouth (effective content 10g/t) in basal diet, and H6 experimental group is added the microbial inoculum of experimental example, and addition is 100g/t.
Table 9 bacillus amyloliquefaciens H6 is at the growth-promoting effect of fattening in piglet, broiler chicken and grass carp cultivation
SEQUENCE LISTING
<110> Wuhan Keqian Animal Biological Products Co., Ltd.
<120> bacillus amyloliquefaciens and preparation method and application
<130> bacillus amyloliquefaciens and preparation method and application
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 1400
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 1
tgaagcccga aagggagctt gctccctgat gttagcggcg gacgggtgag taacacgtgg 60
gtaacctgcc tgtaagactg ggataactcc gggaaaccgg ggctaatacc ggatggttgt 120
ttgaaccgca tggttcagac ataaaaggtg gcttcggcta ccacttacag atggacccgc 180
ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt agccgacctg 240
agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 300
tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg 360
ttttcggatc gtaaagctct gttgttaggg aagaacaagt gccgttcaaa tagggcggca 420
ccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 480
gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa 540
gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat tggaaactgg ggaacttgag 600
tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa 660
caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg 720
agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag 780
ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg 840
tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 900
ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcctctgac aatcctagag 960
ataggacgtc cccttcgggg gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt 1020
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt 1080
cagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1140
aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacag aacaaagggc 1200
agcgaaaccg cgaggttaag ccaatcccac aaatctgttc tcagttcgga tcgcagtctg 1260
caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1320
acgttcccgg gccttgtaca caccgcctcg tcacaccacg agagtttgta acacccgaag 1380
tcggtgatag tatataagtc 1400

Claims (3)

1. a bacillus amyloliquefaciens, is characterized in that: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6, CCTCC NO:M2013249.
2. the preparation method of the bacterium powder of bacillus amyloliquefaciens described in claim 1, its step is as follows:
1) with transfering loop picking bacillus amyloliquefaciens inclined-plane seed, inoculation NB shake-flask seed substratum, cultivates 7~10h for 37 DEG C, and O D value reaches 1.0;
The formula of described NB shake-flask seed substratum 1L is: extractum carnis 5g, peptone 10g, NaCl 5g;
2) seed liquor of cultivating is inoculated to amplification culture in 50L fermentor tank by 1% inoculum size, fermention medium liquid amount 65%, in culturing process, real-time online is added defoamer, control oxyty 30%~50%, 32 DEG C of temperature controls, cultivate 10~12h, and O D value reaches 4.0;
3) by the seed fermentation liquid of amplification culture by 10% inoculum size inoculation 500L ferment tank liquid, 65%, 30 DEG C of liquid amount is cultivated 40h, froth breaking and control dissolved oxygen in real time reaches 70%~80% to spore forming rate;
4) put after tank through microfiltration of ceramic membrane, be concentrated into original volume 1/8~1/4 volume at gauge pressure 0.2~0.3MPa, spraying is dry, obtains bacillus amyloliquefaciens bacterium powder.
3. bacillus amyloliquefaciens claimed in claim 1 or bacterium powder claimed in claim 2 are in the application of preparing in animal feedstuff additive.
CN201310240706.0A 2013-06-18 2013-06-18 Bacillus amyloliquefacien and preparation method and application thereof Active CN103275907B (en)

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CN104611276B (en) * 2015-02-12 2017-06-13 陈建军 A kind of Bacillus amyloliquefaciens MBRC1 bacterial strains and its application
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