CN103275907A - Bacillus amyloliquefacien and preparation method and application thereof - Google Patents
Bacillus amyloliquefacien and preparation method and application thereof Download PDFInfo
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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
Technical field
The present invention relates to the animal feedstuff additive technical field, relate in particular to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6, the preparation method who also relates to a kind of bacillus amyloliquefaciens H6 also relates to the application of a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 in the preparation animal feedstuff additive.
Background technology
Completely forbidding the interpolation microbiotic in feed is global development trend: European Union formally forbade adding any microbiotic in feed in 2006; Korea S began to forbid the use of feeding antibiotic in 2010; Strict restriction has also been done to the microbiotic that adds in the feed by countries such as Japan and the U.S.; Stop using antibiotic feed additive, must select to substitute the substitute of antibiotic feed additive, unaffected with the efficient and the benefit that guarantee livestock industry production.In this case, being hopeful most as microbiotic surrogate fodder additives---the exploitation of microbial preparation causes thereupon that just various countries pay attention to.Study and use the history of livestock and poultry probiotics the earliest and can trace back to nineteen forty-seven, Meng Hade (Mollgaard) at first finds to use the lactobacillus piglet of feeding can effectively increase the body weight of piglet and improve the healthy of piglet.Yet the microbiotic research just of 20th century 50, the sixties, the gold period of producing and using, cause the probiotics research-and-development activity to be at low ebb, be not subjected to enough attention for a long period, up to the mid-80 situation reversing of essence arranged.American-European all states have developed many microbial preparations in succession in recent years, and basic design is with the normal microorganism member of people and animals, through artificial propagation, makes active bacteria formulation, and then makes it get back to original living environment, brings into play its natural physiological action.United States food and drug administration and U.S. feed management association just announced 43 kinds " can directly feed and be commonly referred to be safe microorganism (GRAS) " in 1989, and as the starting strain of probiotics, the various countries research staff has developed rich and varied product category on this basis.Although China is more late than developed country's starting to research and the application of microbial preparation, starting point is higher.Only in short several years, the development and application of microbial preparation has been brought into play effect energetically to promoting China's Developing of Animal Industry, and potentiality are very big.It uses and to make that not only 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 veterinary drugs such as antibacterials.Therefore, we can say that microbial preparation is the green food of livestock and poultry.On the other hand, from the Economic development situation of China, the development microbial preparation should be subjected to due attention.China Ministry of Agriculture has announced 16 kinds of microbial feed additive strains that can directly use in 2008, mainly based on milk-acid bacteria, streptococcus faecium, genus bacillus, yeast, sheet coccus and photosynthetic bacterium etc.; In addition, thermophilus streptococcus and Bacillus licheniformis also were 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 big, 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 extensively to be present in natural a kind of non-curing the disease property bacterium, in the process of growth of himself, can produce a series of meta-bolites, have the activity that suppresses bacterium and fungi widely, it is extensive use of the approval that security has also obtained the European food safety council (EFSA) in agriculture production.At present, domestic the replacement organic pesticide is only limited in its applied research, suppress biological controls such as plant pathogenic fungi and virus; And the degraded agricultural wastes, suppress in the water body fields such as environmental improvement such as algal grown.Studies show that, bacillus amyloliquefaciens can produce multiple hydrolase and antibiotic, antiviral protein isoreactivity material, the premium properties that utilizes this bacterial classification from field widely is necessary for people's production and service for life, further enrich China's microbial feed additive strain resource, satisfy 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 culture collection center on June 5th, 2013 and carries out preservation, classification name: bacillus amyloliquefaciens H6 Bacillus amyloliquefaciens H6; Deposit number: CCTCC NO.M2013249, address China Wuhan Wuhan University.This bacterium has the characteristics of good anti-bacterial effect, enzyme activity height, strong stress resistance, and starch, albumen and fiber are have very strong degradability.
A further object of the invention has been to provide a kind of preparation method of bacillus amyloliquefaciens H6 bacterium powder.The preparation method is simple, and easily row is suitable for scale operation, the bacterium powder that utilizes this method to make, and its bacillus amyloliquefaciens H6 bacterium powder yield is that powder yield is 40kg/m
3Fermented liquid, the 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 application in the preparation fodder additives, utilize the fodder additives of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the H6 preparation among the present invention, can obviously prevent intestinal tract disease, improve growth of animal characteristic and purifying aquaculture environment, and have safe, effective, with low cost, characteristics.
To achieve these goals, the present invention takes following technical measures:
A kind of bacillus amyloliquefaciens obtains by the following steps screening:
1) gathers the health pig fresh excreta from Hubei Huang Po nature compost pig farm, with excrement sample aerobic enrichment culture muddiness in nutrient broth (NB) substratum, in 80 ℃ of water-baths, handle 15min, enrichment and heat screening are once so again, suitably be coated with flat board after the dilution, 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 the 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, identify by Physiology and biochemistry and 16s, 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 culture collection center on June 5th, 2013 and carries out preservation, classification name: bacillus amyloliquefaciens H6 Bacillus amyloliquefaciens H6 deposit number: CCTCC NO:M2013249, address China Wuhan Wuhan University.The 16Sr dna sequence dna is shown in the SEQ ID NO.1.
The H6 bacterial strain specifically has typical bacillus amyloliquefaciens physiological and biochemical property: this bacterial strain is subcircular at the bacterium colony that nutrient agar medium (NA) substratum breeding growth forms, 3-4.5mm, tarnish, central protuberantia, edge cognate shape, canescence, by microscopy G+, thalline is shaft-like, single arrangement, the gemma column, inferior end is given birth to, 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, anti-7%NaCl.28～32 ℃ of optimum temperutures, 50 ℃ of top temperatures.Alkali is produced in the litmus milk reaction, peptonizes reduction.Utilize nitrate.
The prescription of NB substratum (1L): extractum carnis 5g, peptone 10g, NaCl5g; NA adds agar 15g again.
A kind of preparation method of 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 is cultivated 7～10h for 37 ℃, and the OD value reaches about 1.0.The seed liquor of cultivating is inoculated amplification culture in the 50L fermentor tank by the inoculum size of 1% (V/V), fermention medium liquid amount 65%, real-time online is added defoamer in the culturing process, the control oxyty is 30%～50%, 32 ℃ of temperature controls are cultivated 10～12h, and the OD value reaches more than 4.0.In the seed fermentation liquid of the amplification culture inoculum size inoculation 500L ferment tank liquid by 10% (V/V), liquid amount is cultivated about 40h for 65%, 30 ℃, and froth breaking and control dissolved oxygen in real time reach 70%～80% to the gemma rate of formation.Put jar by the ceramic membrane micro-filtration, be concentrated into original volume 1/8～1/4 volume at gauge pressure 0.2～0.3MPa, spraying drying 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, the online silicon oil foam killer of adding.
The application of a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 in the preparation fodder additives: its application process is:
With bacillus amyloliquefaciens H6 viable bacteria powder or compound with other microbial inoculums, add in animal and fowl fodder or the drinking-water in 0.01%～0.1%(w/w) ratio, the security in heavy dose of animal is fed of bacillus amyloliquefaciens bacterium powder is good, thereby feed and to alleviate the excrement dirt to the pollution of environment for improving intestinal health, prevent constipation, prevent and treat bacterial diarrhea, promoting intestinal absorption to improve production and reproductive performance in the different steps of livestock and poultry cultivation production.
Compared with prior art, the present invention has the following advantages:
1. the invention provides the bacillus amyloliquefaciens of a kind of good anti-bacterial effect, enzyme activity height, strong stress resistance, scale operation preparation and effective the use partly replace at present to environment and the healthy antibiotic use that causes increasing threat at an easy rate.
2. bacillus amyloliquefaciens of the present invention has the obvious growth restraining effect to Gram-positive and the feminine gender bacterium of curing the disease, in animal and bird intestines, can suppress the harmful microbe growth, and have very strong starch, albumen and cellulose degradation ability, environmental resistance is strong; Good control diarrhoea is arranged in livestock and poultry cultivation is fed and promote growth result, and heavy dose of feeding can not produce detrimental action; Culture condition is simple, with low cost, has the excellent development application prospect.
Description of drawings
Fig. 1 is a kind of bacillus amyloliquefaciens H6 brood cell form synoptic diagram.
Fig. 2 is the growth curve chart of bacillus amyloliquefaciens H6 in fermentor tank.
Embodiment
Experimental technique and condition are ordinary method if no special instructions in the embodiment of the invention.
Embodiment 1:
Strains separation
Gather the health pig fresh excreta from the natural compost of Hubei Huang Po pig farm, with excrement sample aerobic enrichment culture muddiness in nutrient broth (NB) substratum, in 80 ℃ of water-baths, handle 15min, enrichment and heat screening are once so again, suitably be coated with flat board after the dilution, 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 the microscopy scalping.
The prescription of NB substratum (1L): extractum carnis 5g, peptone 10g, NaCl5g, agar 15g.
Multiple sieve and evaluation
Screen streptococcus aureus (G by Oxford cup bacteriostatic test
+), intestinal bacteria (G
-) wait the good genus bacillus of pathogenic bacterium growth-inhibiting performance, identify that by Physiology and biochemistry and 16s finishing screen is chosen a bacillus amyloliquefaciens, called after Bacillus amyloliquefaciens H6.This bacterium is sent to Chinese typical culture collection center on June 5th, 2013 and carries out preservation, classification name: bacillus amyloliquefaciens H6 Bacillus amyloliquefaciens H6 deposit number: CCTCC NO.M2013249, address China Wuhan Wuhan University.
The H6 bacterial strain specifically has typical bacillus amyloliquefaciens physiological and biochemical property: this bacterial strain is subcircular at the bacterium colony that NB substratum breeding growth forms, 3-4.5mm, tarnish, central protuberantia, edge cognate shape, canescence, by microscopy G+, thalline is shaft-like, single arrangement, the gemma column, inferior end is given birth to, 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, anti-7%NaCl.28～32 ℃ of optimum temperutures, 50 ℃ of top temperatures.Alkali is produced in the 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 ℃ of high-temperature steam sterilization 20min, be cooled to 40 ℃, to the seed liquor 1% of wherein inoculating cell age 10h, 32 ℃ of cultivations, the control mixing speed is kept dissolved oxygen (the DO value is more than 30%), the on-line automatic defoamer (silicone oil) of adding.The OD value is surveyed in every 2h sampling, and fermentation 24h is put jar, and plate count bacillus amyloliquefaciens H6 viable bacteria amount is 1.78 * 10
10Cfu/ml(Fig. 2).
Embodiment 3: the preparation of bacillus amyloliquefaciens bacterium powder
The bacterium liquid of embodiment 2 fermentations is removed big solid substance through the vibratory screening apparatus coarse filtration, be concentrated into 1/6 of original volume through 0.2 μ m ceramic membrane micro-filtration again, concentrated solution is directly used peristaltic pump people spray-drying tower, 180 ± 3 ℃ of inlet temperatures, temperature out 67～70, control charging flow velocity and heater switch, the holding temperature balance, calculating the bacterium powder yield after the rewinding 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), the H6 bacterial strain is recovered in the PA bottle, get the 5ul point and be connected on the flat board, cultivate.Whether observe casein and Mierocrystalline cellulose flat board has the hydrolysis ring, toward the dull and stereotyped point of starch connect bacterium around drip iodine liquid, whether have hydrolysis ring, and measure the hydrolysis ring diameter if observing.
Table 1H6 strains for degrading ability
| Degraded is dull and stereotyped | The starch flat board | The casein flat board | The Mierocrystalline cellulose flat board |
| Degraded circle (mm) | 26.6 | 27.53 | 20.18 |
Embodiment 5:H6 bacterial strain resistance detects
Hot tolerance: get the medium centrifugal that the H6 growth has been completed into the brood cell latter stage, suspending again after the 0.9% physiological saline washed twice, (all amount (1～2 * l0 live
9Cfu/m) in 75,80,85,90,95 ℃ of water-baths, handle 15min after, flowing water cooling is rapidly adopted dilution-plate method to measure bud then and is embraced survival rate.
The survival rate of table 2H6 bacterial strain under treatment of different temperature
| Temperature (℃) | 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, adds the 1g stomach en-in every 100mL hydrochloric acid soln, and the fully dissolving back filtering with microporous membrane degerming of 0.22um obtains simulated gastric fluid.It is centrifugal to get the culture that H6 growth has been completed into the brood cell latter stage, suspends again after the 0.9% physiological saline washed twice, with 10
7The cfu/ml final concentration is inoculated in the simulated gastric fluid, the concussion mixing, and 37 ℃ of water-baths are hatched, and carry out live bacterial count every the 0.5h sampling.
Table 3H6 bacterial strain is handled the viable bacteria quantitative changeization 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 |
Preparation is extra adds 1.0%, 2.0%, 3.0%, 4.0%, 5.0%(w/v) 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, behind 37 ℃ of cultivation 12h, measure the nutrient solution turbidity and change, judge the thalli growth situation, see Table 1.
Table 4: different concns pig cholate is to the influence of H6 growth
Annotate: ++ 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 fully melts, be poured in the culture dish, every ware 15ml(lower floor), treat that it solidifies.In addition, the NB substratum that melts being cooled to sneak into about 50 ℃ pharmacosensitive test bacteria culture fluid 5ml is added to and waits to solidify (upper strata) on the substratum that has solidified.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), the Oxford cup generally can be adorned 200 microlitres, does not make it excessive.Fill it up with rearmounted 37 ℃ and cultivated 16-18 hour, observations, inhibition zone is directly measured with chi and just can.
Table 5H6 is to the antibacterial circle diameter of different pathogenic bacterium
| Pathogenic bacterium | Intestinal bacteria | Pasteurellosis bacillus | The white dysentery Salmonellas | Streptococcus aureus |
| Inhibition zone (mm) | 19.32 | 15.71 | 16.01 | 16.84 |
Embodiment 7: the mouse proof test
Kunming mice about 60 body weight 20g is divided into 3 groups at random, 20 every group, male and female half and half.Wherein do blank for one group, experimental group one every by 1 * 10
9Cfu/kg mouse body weight (100 times of a routine doses) continuous week gavages the H6 fermented liquid, the bacteria suspension (1 * 10 of experimental group two disposable celiacs injection 0.2ml
9Cfu/ml).Observe a week, growth and the death condition of mouse respectively organized in record.All drying, cleaning, draughty indoor finishing, mouse is death condition to all mouse feeding experiments, and two days body heavy phases are to lighter behind abdominal injection for experimental group two mouse, and it is normal that recover the back, and experimental group one mouse growing state is best.
Table 6: mouse experiment weighing results
| The experiment grouping | Initial weight (g) | Weighed in 3 days (g) | Weighed in 7 days (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: the 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 filling every day once, and continuous irrigation 7 days was observed 21 days.Free choice feeding, every day, record spirit and the situation of searching for food were weighed when experiment finishes.Gavaging continuously in heavy dose through more than 20 days experimental observations does not down all have abnormal response, search for food and growing state normal, and the bacillus group has significantly promotes growth effect.
Table 7: piglet gavages various dose H6 to the influence of weightening finish
| Group | First 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: the 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 previous month to wean for every of experimental group, by pig body weight 2 * 10
7The bacterium of cfu/kg amount is mixed H6 bacterium powder feeding in the 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 (about 7 kilograms of mean body weights), divide 2 groups, every group 20, the control group fed basal diet, experimental group is admixed H6 bacterium powder basal diet respectively and is fed (the 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 usefulness, fed 35 days, and free choice feeding, the record daily ingestion amount is weighed weekly on an empty stomach.Performance assessment criteria: diarrhea rate, feedstuff-meat ratio.
Table 8: anti-diarrhoea and weightening finish experimental result
Embodiment 11: the effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) H6 microbial inoculum in fattening piglet, broiler chicken and grass carp breed.
Use the bacterium powder of embodiment 3 preparations, be applied to fatten the breed of piglet, broiler chicken and grass carp respectively, all establish blank group, microbiotic group and H6 experimental group, every group of 3 repetitions, experimental period is one month.Wherein, the blank group basal diet of feeding; The 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, the H6 experimental group is added the microbial inoculum of experimental example, and addition is 100g/t.
The growth-promoting effect of table 9 bacillus amyloliquefaciens H6 in fattening piglet, broiler chicken and grass carp breed
SEQUENCE LISTING
＜110〉Wuhan Keqian Animal Biological Products Co., Ltd.
＜120〉a kind of bacillus amyloliquefaciens and preparation method and application
＜130〉a kind of 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 described bacillus amyloliquefaciens bacterium of claim 1 powder, its step is as follows:
1) with transfering loop picking bacillus amyloliquefaciens inclined-plane seed, inoculation NB shake-flask seed substratum is cultivated 7 ~ 10h for 37 ℃, and the OD value reaches 1.0;
2) seed liquor of cultivating is inoculated amplification culture in the 50L fermentor tank by 1% inoculum size, fermention medium liquid amount 65%, real-time online is added defoamer in the culturing process, and the control oxyty is 30% ~ 50%, and 32 ℃ of temperature controls are cultivated 10 ~ 12h, and the OD value reaches 4.0;
3) with the seed fermentation liquid of amplification culture by in 10% the inoculum size inoculation 500L ferment tank liquid, liquid amount is cultivated 40h for 65%, 30 ℃, froth breaking and control dissolved oxygen in real time reach 70% ~ 80% to the gemma rate of formation;
4) put jar by the ceramic membrane micro-filtration, be concentrated into original volume 1/8 ~ 1/4 volume about gauge pressure 0.2 ~ 0.3MPa, spraying drying obtains bacillus amyloliquefaciens bacterium powder.
3. the described bacterium powder of the described bacillus amyloliquefaciens of claim 1 or claim 2 is in the application in the preparation animal feedstuff additive.
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Publications (2)
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667142A (en) * | 2013-12-11 | 2014-03-26 | 黄冈师范学院 | Microorganism derived from Chinese chestnuts and application thereof as well as microbial agent preparation method |
| CN104611276A (en) * | 2015-02-12 | 2015-05-13 | 陈建军 | Bacillus amyloliquefaciens MBRC1 strain and application thereof |
| CN104957373A (en) * | 2015-06-10 | 2015-10-07 | 山东省农业科学院畜牧兽医研究所 | Method for reducing heavy metal emission in livestock and poultry manure |
| CN105056224A (en) * | 2015-07-15 | 2015-11-18 | 山东仙普爱瑞科技股份有限公司 | Method for improving immunity of rabbits by combining bursin and vaccine |
| CN106688869A (en) * | 2016-12-28 | 2017-05-24 | 江苏省海洋水产研究所 | Method for preventing and treating yellow leaf blotch of shell conchocelis of porphyra yezoensis and culture apparatus |
| CN107115365A (en) * | 2017-05-09 | 2017-09-01 | 黑龙江省兽医科学研究所 | One kind preventing and treating diarrhea of pigs biological agent and preparation method thereof |
| CN107586742A (en) * | 2017-10-18 | 2018-01-16 | 中国科学院天津工业生物技术研究所 | The bacillus amyloliquefaciens of one plant height production levulan and its application |
| CN107988118A (en) * | 2017-12-28 | 2018-05-04 | 四川龙蟒福生科技有限责任公司 | The fermentation medium and fermentation process of a kind of bacillus |
| CN108642022A (en) * | 2018-05-15 | 2018-10-12 | 祺兴生物工程技术（北京）有限公司 | Multidimensional complex enzyme microbial inoculum and the preparation method and application thereof |
| CN109897795A (en) * | 2018-12-26 | 2019-06-18 | 佛山市艳晖生物科技有限公司 | A kind of microbial bacterial agent and its application on anti-treat constipation |
| CN109939138A (en) * | 2019-04-11 | 2019-06-28 | 武汉科缘生物发展有限责任公司 | A kind of Chinese herbal microecological preparation and its application of anti-Eimeria species |
| CN109999060A (en) * | 2019-04-29 | 2019-07-12 | 华中农业大学 | A kind of composite probiotics preparations of anti-S. pullonum infection and application |
| CN110144307A (en) * | 2019-05-13 | 2019-08-20 | 华中农业大学 | A kind of lactobacillus gasseri of anti-enterohemorrhagic escherichia coli and fowl composite probiotics preparations and the application of preparation |
| CN112322542A (en) * | 2020-11-17 | 2021-02-05 | 青岛普罗百世生物科技有限公司 | Bacillus amyloliquefaciens and influence thereof on soil nutrient content and enzyme activity |
| CN114107128A (en) * | 2021-12-15 | 2022-03-01 | 四川汇邦环保科技有限公司 | Microbial agent for degrading colistin sulfate and application thereof |
| CN115820482A (en) * | 2022-11-11 | 2023-03-21 | 云南农业大学 | Bacillus amyloliquefaciens YN-BA2 and microbial inoculum and application thereof |
| CN116491577A (en) * | 2023-05-08 | 2023-07-28 | 宁夏大学 | High-activity probiotic composite microecological preparation and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667142A (en) * | 2013-12-11 | 2014-03-26 | 黄冈师范学院 | Microorganism derived from Chinese chestnuts and application thereof as well as microbial agent preparation method |
| CN103667142B (en) * | 2013-12-11 | 2015-03-04 | 黄冈师范学院 | Microorganism derived from Chinese chestnuts and application thereof as well as microbial agent preparation method |
| CN104611276A (en) * | 2015-02-12 | 2015-05-13 | 陈建军 | Bacillus amyloliquefaciens MBRC1 strain and application thereof |
| CN104611276B (en) * | 2015-02-12 | 2017-06-13 | 陈建军 | A kind of Bacillus amyloliquefaciens MBRC1 bacterial strains and its application |
| CN104957373A (en) * | 2015-06-10 | 2015-10-07 | 山东省农业科学院畜牧兽医研究所 | Method for reducing heavy metal emission in livestock and poultry manure |
| CN105056224A (en) * | 2015-07-15 | 2015-11-18 | 山东仙普爱瑞科技股份有限公司 | Method for improving immunity of rabbits by combining bursin and vaccine |
| CN106688869A (en) * | 2016-12-28 | 2017-05-24 | 江苏省海洋水产研究所 | Method for preventing and treating yellow leaf blotch of shell conchocelis of porphyra yezoensis and culture apparatus |
| CN107115365A (en) * | 2017-05-09 | 2017-09-01 | 黑龙江省兽医科学研究所 | One kind preventing and treating diarrhea of pigs biological agent and preparation method thereof |
| CN107586742A (en) * | 2017-10-18 | 2018-01-16 | 中国科学院天津工业生物技术研究所 | The bacillus amyloliquefaciens of one plant height production levulan and its application |
| CN107988118A (en) * | 2017-12-28 | 2018-05-04 | 四川龙蟒福生科技有限责任公司 | The fermentation medium and fermentation process of a kind of bacillus |
| CN108642022A (en) * | 2018-05-15 | 2018-10-12 | 祺兴生物工程技术（北京）有限公司 | Multidimensional complex enzyme microbial inoculum and the preparation method and application thereof |
| CN109897795A (en) * | 2018-12-26 | 2019-06-18 | 佛山市艳晖生物科技有限公司 | A kind of microbial bacterial agent and its application on anti-treat constipation |
| CN109939138A (en) * | 2019-04-11 | 2019-06-28 | 武汉科缘生物发展有限责任公司 | A kind of Chinese herbal microecological preparation and its application of anti-Eimeria species |
| CN109999060A (en) * | 2019-04-29 | 2019-07-12 | 华中农业大学 | A kind of composite probiotics preparations of anti-S. pullonum infection and application |
| CN110144307A (en) * | 2019-05-13 | 2019-08-20 | 华中农业大学 | A kind of lactobacillus gasseri of anti-enterohemorrhagic escherichia coli and fowl composite probiotics preparations and the application of preparation |
| CN112322542A (en) * | 2020-11-17 | 2021-02-05 | 青岛普罗百世生物科技有限公司 | Bacillus amyloliquefaciens and influence thereof on soil nutrient content and enzyme activity |
| CN114107128A (en) * | 2021-12-15 | 2022-03-01 | 四川汇邦环保科技有限公司 | Microbial agent for degrading colistin sulfate and application thereof |
| CN115820482A (en) * | 2022-11-11 | 2023-03-21 | 云南农业大学 | Bacillus amyloliquefaciens YN-BA2 and microbial inoculum and application thereof |
| CN116491577A (en) * | 2023-05-08 | 2023-07-28 | 宁夏大学 | High-activity probiotic composite microecological preparation and preparation method and application thereof |
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