CN102517227A - Enterococcus faecalis and applications and feed additive and leavening agent thereof - Google Patents
Enterococcus faecalis and applications and feed additive and leavening agent thereof Download PDFInfo
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- CN102517227A CN102517227A CN2011103782238A CN201110378223A CN102517227A CN 102517227 A CN102517227 A CN 102517227A CN 2011103782238 A CN2011103782238 A CN 2011103782238A CN 201110378223 A CN201110378223 A CN 201110378223A CN 102517227 A CN102517227 A CN 102517227A
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Abstract
The invention belongs to the biologically technical field and relates to Enterococcus faecalis and applications and a feed additive and a leavening agent thereof. The Enterococcus faecalis has a good stress resistance function and a good function of benefiting life, can be dried at high temperature under the protection of a protective agent, is also capable of tolerating pelleting temperature of 90 DEG C, has a stronger inhibitory action on escherichia coli k88 and k99 and Staphylococcus aureus, has very high capacity of producing acids and cellulase and is capable of reducing pH values and decomposing certain celluloses. The biological feed additive prepared by using the Enterococcus faecalis is capable of replacing part of antibiotics in livestock breeding and aquaculture, thereby, the use of the antibiotics is reduced, the immunity of animals is improved, the conversion rate of the feed is increased, and the culture benefits are increased. The Enterococcus faecalis also can be used for leavening bean meals, cottonseed meals, green grass and the like, and is capable of effectively preventing feeds from mildewing and increasing the use ratio of nutrients in the feeds.
Description
Technical field
The invention belongs to biological technical field, relate to a strain enterococcus faecalis (Enterococcus faecalis) and application and its fodder additives and starter.
Background technology
In China's aquaculture; Antibiotic excessive use; Caused drug residue and a large amount of drug tolerant bacteria to produce; Its result has seriously hindered the healthy and sustainable development of poultry, fowl, culture fishery, is food safety and human consumer's the healthy hidden danger of having buried, and the microbiotic substitute of therefore seeking safety, noresidue is extremely urgent.The biology feed additive that comprises probiotic bacterium is the antibiotic substitute of a kind of very good part, and probiotic strain is the important foundation of the biology feed additive that development quality is stable, effect is remarkable, cost performance is high.The key issue of the research and development of probiotic bacterium is separation and the screening of strain excellent, good probiotic bacterium treatment process.At present; There is following problem in the probiotic bacterium bacterial classification: 1. most probiotic bacterium is asporulate milk-acid bacteria; And belong to obligatory anaerobic bacteria more, very easily receive the influence of external environment factors such as temperature, oxygen, moisture and soda acid, usually the active relatively difficulty that keeps of bacterium; 2. the high temperature when most probiotic bacteriums are difficult to tolerate feed granulating in the feed course of processing; 3. the characteristic of bacterial classification itself is bad, produces enzyme, bacteriostasis is poor.
Summary of the invention:
The object of the present invention is to provide a strain strong stress resistance, benefit to give birth to the outstanding probiotic bacterium of function---enterococcus faecalis (Enterococcus faecalis); Deposit number is CGMCC No.5092; This bacterium has good resistance function and gives birth to function with benefit: anti-artificial hydrochloric acid in gastric juice, anti-bile fluid, high temperature resistant, intestinal bacteria K88, K99 and streptococcus aureus are had strong restraining effect, acid producing ability is strong, can mould fungus inhibition.
Another object of the present invention has provided the application of this enterococcus faecalis.
The present invention also provides fodder additives and the starter that comprises this enterococcus faecalis.
The objective of the invention is to realize through following technical scheme:
The present invention provides a strain enterococcus faecalis, and its deposit number is CGMCC No.5092.
Wherein, the 16s rRNA gene order of this bacterium is shown in SEQ ID NO:1.
Enterococcus faecalis of the present invention can be used for preparing fodder additives.Fodder additives is mainly used in poultry, fowl or the aquaculture feed, and like chicken, duck, pig, ox, sheep, fish etc., wherein the viable count of enterococcus faecalis is 10 in the fodder additives
9-10
10Cfu/g; After fodder additives made an addition to fowl poultry or aquaculture feed, the viable count of enterococcus faecalis was 10 in the feed
6-10
8Cfu/g.
The present invention also provides the fodder additives that comprises this enterococcus faecalis CGMCC No.5092, and the viable count that it is characterized in that enterococcus faecalis in the fodder additives is 10
9-10
10Cfu/g.
Enterococcus faecalis of the present invention also can be used for preparing starter.
Wherein, described starter is the starter that is used for dregs of beans, cotton dregs, green grass and/or corn straw, and the viable count that starter adds behind dregs of beans, cotton dregs, green grass and/or the corn straw is 10
6-10
7Cfu/g.
The present invention also provides the starter that comprises this enterococcus faecalis CGMCC No.5092, and the viable count that it is characterized in that enterococcus faecalis in the starter is 10
8-10
10Cfu/g.
Enterococcus faecalis of the present invention (Enterococcus faecalis) obtains for the applicant separates from Radix Polygalae Crotalarioidis, and its biological property is following: the bacterium colony rounding, and smooth surface, opaque, oyster white, the edge is smooth, gram-positive microorganism; Single thalline is spherical or ellipticity.Enterococcus faecalis of the present invention is through simulated gastric fluid, bile fluid and stable on heating screening; Again through enzymatic productivity screening and bacteriostasis screening; Can tolerate pH2.0,1% pepsic simulated gastric fluid can tolerate 0.3% artificial bile fluid; Can tolerate 85 ℃ pelleting temperature; Also can suppress pathogenic colon bacillus K88, K99 and streptococcus aureus have stronger product acid and the ability that suppresses mould in dregs of beans, cotton dregs, the corn straw.Enterococcus faecalis of the present invention (Enterococcus faecalis) applicant has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 26th, 2011; Be called for short CGMCC; The address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.5092.
Effective effect:
(1) strong stress resistance of enterococcus faecalis CGMCC No.5092 of the present invention can tolerate granulation high temperature, the tolerance simulated gastric fluid; Artificial cholate is therefore at the process feed granulating, behind the animal gastrointestinal tract environment; Also have a large amount of viable bacterias can get into the enteron aisle of animal smoothly; The performance benefit is given birth to function in enteron aisle, improves animal immunizing power, reduces aquaculture cost;
(2) enterococcus faecalis of the present invention can effectively suppress common pathogen intestinal bacteria K88, K99 and streptococcus aureus in the animal intestinal.
(3) the product acid that enterococcus faecalis of the present invention can be a large amount of can reduce growth and the breeding of the harmful bacterium of pH inhibition in the enteron aisle, also helps digestion simultaneously.
(4) enterococcus faecalis of the present invention can be used as biology feed additive with high density fermentation and is added in the drinking-water or feed of cultivated animals through cultivating, and improves animal intestinal health, promotes animal immunizing power, the production performance of increase animal.
(5) enterococcus faecalis of the present invention can also be handled dregs of beans, cotton dregs, the dish dregs of rice and/or corn straw as starter, and generation that can mould fungus inhibition increases the absorption of cultivated animals to nutritive ingredient in dregs of beans, cotton dregs, the dish dregs of rice or the corn straw.
Description of drawings
Fig. 1: Fig. 1 suppresses intestinal bacteria K88, the inhibition zone picture of K99 and staphylococcus aureus for enterococcus faecalis.
Fig. 2: the picture of enterococcus faecalis (CMGCC No.5092) cassiri
Through the following example the present invention will be described more specifically, and it should be understood that said embodiment only is for the present invention is described, rather than limit scope of the present invention by any way.
Embodiment:
The screening of embodiment 1 enterococcus faecalis CGMCC No.5092.
1, primary dcreening operation
Each 5g of sample such as sample thief pig manure, cow dung and chicken manure are soaked in aseptic 0.85% saline water of 20ml, and sample is broken up in concussion; Make the abundant and contact with normal saline of sample, 70 ℃ of thermostat water baths left standstill 3 minutes, got supernatant; Centrifugal 10 minutes of 5000rpm, supernatant discarded.Bacterial sediment after centrifugal is added in the 10ml simulated gastric fluid, and 37 ℃ leave standstill 2h, centrifugal then, behind the collection thalline, bacterial sediment are added in the artificial cholate of 10ml, and 37 ℃ leave standstill 4h, collect thalline, and the LB substratum is coated in dilution.After 37 ℃ of incubators are cultivated 12-24h, bacterium colony is carried out purifies and separates.Bacterium to obtaining preserves, and further multiple sieve.
The compound method of saline water is: 0.85% sodium chloride solution, and subsequent use behind the autoclaving.
The compound method of simulated gastric fluid is: 1% stomach en-, and 0.85% sodium-chlor, with concentrated hydrochloric acid adjust pH to 2.0, subsequent use after the biofilter filtration sterilization.
The compound method of artificial cholate is: in liquid meat soup, add 0.3% pig cholate (analytical pure), and subsequent use behind the autoclaving.
The making method of LB solid medium is: peptone 10g, and yeast extract paste 5g, sodium-chlor 10g, agar 1.5%, pH7.0, subsequent use behind the autoclaving.
2, multiple sieve
(1) will configure, the LB substratum temperature of the bacterium of having gone out is cooled to 45 ℃, adds intestinal bacteria K88, K99 and the streptococcus aureus (every milliliter of substratum adds 1 microlitre bacterium liquid) of incubated overnight, the concussion mixing; Pour aseptic flat board into, after level was solidified, each flat board utilized four Oxford cups to punch; Add 200 μ l bacterium liquid to be measured in every Oxford cup; Build the ware lid, carefully move to 37 ℃ of incubators, plate is just being put and is being left standstill cultivation.After cultivating 20h, open the ware lid, remove the Oxford cup, use the kind of calliper antibacterial circle diameter.
The Molecular Identification of embodiment 2 enterococcus faecalis
Utilize the universal primer of bacterium that the bacterial strain that screens is carried out the PCR evaluation: to press DNA of bacteria and extract test kit process specifications extraction template DNA.Use the upstream primer 5 '-AGAGTT TGA TCC TGG CTC AG-3 ' of 16S rRNA conserved sequence, see SEQ ID No.2 and downstream primer 5 '-GGT TACCTT GTT ACG ACT T-3, see SEQ ID No.3 amplification bacterium 16s rRNA gene fragment.Obtain this Pseudomonas in enterococcus faecalis through order-checking, the sequencing result of amplified fragments is seen SEQ ID No.1.
The checking of embodiment 3 enterococcus faecalis resistance
(1) high thermal resistance
The enterococcus faecalis of cultivating 12h is carried out centrifugal collection, insert carry out in the saline water resuspended after, sampling coating, meter viable count; Saline water, is cooled off rapidly after ten minutes in heating in 70 ℃ the water-bath again, carry out plate count again, remaining viable count is 85.3%.
(2) tolerance of simulated gastric fluid
Get the bacterium liquid that 0.5ml cultivates the 12h enterococcus faecalis, be dissolved in the 4.5ml saline water, with colony counting method meter viable count.Get quantitative above-mentioned bacterium liquid again and add in the artificial bile salt culture-medium of 4.5ml, 37 ℃ leave standstill 2h, and the remaining viable count of colony counting method meter, remaining viable count are 92.2%.
(3) tolerance of artificial bile fluid
Get the bacterium liquid that 0.5ml cultivates the 12h enterococcus faecalis, be dissolved in the 4.5ml saline water, with colony counting method meter viable count.Then get the above-mentioned bacterium liquid of 0.5ml in addition and add in the artificial bile fluid substratum of 4.5ml, 37 ℃ leave standstill 2h, and the remaining viable count of the method for plate culture count meter, remaining viable count are 86.2%.
The result: more than several experiments show that the resistance of the enterococcus faecalis that from Radix Polygalae Crotalarioidis, is separated among the present invention is stronger, can tolerate higher temperature; The tolerance simulated gastric fluid; Therefore artificial cholate uses as fodder additives, can guarantee a large amount of thalline enteron aisle through animal smoothly; In enteron aisle, survive, bring into play its benefit and give birth to function.
The benefit of embodiment 4 enterococcus faecalis (CGMCC No.5092) is given birth to function
(1) bacteria resistance function checking
The pathogenic colon bacillus K88 of incubated overnight, K99 and streptococcus aureus bacterium liquid are counted with colony counting method.In intestinal bacteria K88, K99 and the streptococcus aureus of 3 pipe 5ml, add 5ml enterococcus faecalis bacterium liquid respectively.Cultivate 4h for 37 ℃, the method for plate culture count calculates the viable count of remaining intestinal bacteria K88, K99 and streptococcus aureus.The viable count of remaining pathogenic colon bacillus K88, K99 and streptococcus aureus is 24.7%, 30.2% and 31.8%.
(2) checking of mould fungus inhibition in dregs of beans or Semen Maydis powder
Get the bacterium liquid 20ml of the enterococcus faecalis of cultivating 12h; Carry out uniform mixing with 30g Semen Maydis powder or dregs of beans; Blank assay is that 20ml sterilized water and 30g Semen Maydis powder or dregs of beans carry out intimate mixing, at 3d, 5d, 8d and 10d the corn of two bottles of the insides or the growing state of dregs of beans and mould is observed respectively then.Show that through experiment after 5 days, not adding in Semen Maydis powder and the dregs of beans of enterococcus faecalis bacterium liquid all has the mould filament length to go out, and it is still fresh to add the color of Semen Maydis powder and dregs of beans of enterococcus faecalis bacterium liquid, decomposes to some extent but do not have the perhaps appearance of mould mycelia of mould bacterial plaque.
The preparation of embodiment 5 enterococcus faecalis (CGMCC No.592) fodder additives
(1) high density fermentation of enterococcus faecalis
The fermention medium of using during the fermentation enterococcus faecalis is grouped into by following one-tenth: brown sugar 18g/L, and peptone 4g/L, Carnis Bovis seu Bubali cream 3g/L, yeast soaks powder, 0.8/L, bitter salt 0.4g/L, manganous sulfate 0.005g/L, pH are 7.2.
Fermentation condition is: leavening temperature is 37 ℃, and fermentation time is 12h, and the pH value is 7.2, and rotating speed is 100rpm, and the initial inoculation amount is 2%.After the fermentation ends, obtain 7.3 * 10
9The bacterium liquid of cfu/ml.
(2) preparation of enterococcus faecalis (CMGCC No.5092) fodder additives
Enterococcus faecalis is carried out high temperature spray-drying, and its protective material composition is: XG 550, and trehalose, maltodextrin, 180 ℃ of air intakes, 70 ℃ of air-out, it is good to obtain free-running property, and soluble in water, viable count is 4 * 10
10The enterococcus faecalis bacterium powder of cfu/g.
The effect of embodiment 6 enterococcus faecalis (CGMCC No.5092) in the cassiri process
With Semen Maydis powder and the water abundant mixing of ratio with 1: 0.5, with 1% inoculum size inoculation enterococcus faecalis CGMCC No.5092 bacterium powder, addition is 10
7/ g, leavening temperature are 37 ℃, and fermentation time is 3d.Fermenting container is the Erlenmeyer flask of 250mL, then inserts in the incubator and ferments.Blank is not for inoculating the Semen Maydis powder of enterococcus faecalis bacterium powder and the mixture of water (1: 0.5), and contrast 2 is the Semen Maydis powder of 1% inoculum size inoculation subtilis bacterium powder and the mixture of water (1: 0.5).
Result: compare with blank; After Semen Maydis powder ferments through enterococcus faecalis; Its color, viscosity and local flavor all obviously improve (Fig. 2), wherein inoculate the cassiri of enterococcus faecalis CGMCC No.5092 and color, viscosity and the local flavor of the cassiri of inoculation subtilis and improve more obvious.Continue to place not cassiri and cassiri in room temperature and observe not fermented maize pruinescence mould contamination after one week, the fermented maize powder is contaminated.After placing for two weeks; The Semen Maydis powder of subtilis and enterococcus faecalis fermentation is not still by mould contamination; But its local flavor of Semen Maydis powder of enterococcus faecalis fermentation is better, explains that enterococcus faecalis CGMCC No.5092 has the effect of the fungal infection of preventing, and effect is better than the subtilis effect.
Embodiment 7 enterococcus faecalis fodder additivess are to the influence of laying hen production performance, egg product matter, death rate and antibody horizontal
With identical 9000 of the blue laying hens in later stage sea of laying eggs of age in days, divide three groups, one group 3000 of every buildings divide three repetitions, 1000 of each repetitions.The grouping situation is following:
The A group, blank group, the laying hens essential ration of feeding; The B group is added microbiotic (duomycin) 80g/t in laying hens essential ration; The C group is added enterococcus faecalis fodder additives 0.18% in laying hens essential ration.
Preceding 7 days is preliminary trial period, and back 28 days is trial period, and presses above-mentioned grouping revision test 3 times, measures NH in the hen house
3, H
2Indexs such as S concentration, broken rate of eggshell, Hough unit, laying rate, egg size, death rate, eggshell smooth finish, antibody horizontal.The result lists in table (1)
The influence of table 1 pair laying hen production performance, protein, death rate
State: egg light smooth finish with+how much represent that the smooth finish of representing good more more.
From table, can find out; Though it is good not as the microbiotic group to have added the indexs such as egg size, laying rate of enterococcus faecalis fodder additives one group; But significantly reduced hen house obnoxious flavoures such as ammonia, hydrogen sulfide, reduce death rate, antibody horizontal also is greatly improved; Good economic benefit and ecological benefits are arranged, also help to reduce the drug residue problem simultaneously, improved the security of food.
Claims (8)
1. a strain enterococcus faecalis (Enterococcus faecalis), deposit number is CGMCC No.5092.
2. the described enterococcus faecalis of claim 1 is characterized in that this bacterium 16sRNA gene order is shown in SEQ ID No:1.
3. the application of the described enterococcus faecalis of claim 1 in the preparation fodder additives.
4. the application of the described enterococcus faecalis of claim 3 in the preparation fodder additives is characterized in that described fodder additives is mainly used in fowl poultry or the aquaculture feed, and the viable count of enterococcus faecalis is 10 in the feed
6-10
8Cfu/g.
5. one kind comprises the fodder additives of weighing 1 described enterococcus faecalis, and the viable count that it is characterized in that enterococcus faecalis in the fodder additives is 10
9-10
10Cfu/g.
6. the application of the described enterococcus faecalis of claim 1 in the preparation starter.
7. the application of the described enterococcus faecalis of claim 6 in the preparation starter; It is characterized in that described starter is the starter that is used for dregs of beans, cotton dregs, green grass and/or corn straw, the viable count that starter adds behind dregs of beans, cotton dregs, green grass and/or the corn straw is 10
6-10
7Cfu/g.
8. one kind comprises the starter of weighing 1 described enterococcus faecalis, and the viable count that it is characterized in that enterococcus faecalis in the starter is 10
8-10
10Cfu/g.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167785A (en) * | 2016-08-29 | 2016-11-30 | 内蒙古工业大学 | A kind of E. Faecium strains and application thereof |
| CN110241043A (en) * | 2019-06-10 | 2019-09-17 | 大连理工大学 | The bacterial strain of one plant height temperature fermenting lactic acid and the method for producing lactic acid |
| CN110771723A (en) * | 2019-11-18 | 2020-02-11 | 北京大北农科技集团股份有限公司 | Cassava residue biological feed and fermentation method and application thereof |
| CN111607540A (en) * | 2020-06-10 | 2020-09-01 | 江西省科学院生物资源研究所 | Forage grass digestant and preparation method and application thereof |
| CN113215019A (en) * | 2021-02-04 | 2021-08-06 | 郑州大学 | Enterococcus faecalis and application thereof |
| WO2022063296A1 (en) * | 2020-09-28 | 2022-03-31 | 天津博菲德科技有限公司 | Feed additive for improving oxygen obtaining ability of poultry and use thereof |
| CN114424800A (en) * | 2022-04-06 | 2022-05-03 | 北京挑战生物技术有限公司 | Feed additive for improving intestinal health index of broiler chickens and application thereof |
Citations (1)
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| CN102031235A (en) * | 2010-11-09 | 2011-04-27 | 中国农业大学 | Enterococcus faecium ANSE228 and application thereof |
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2011
- 2011-11-24 CN CN201110378223.8A patent/CN102517227B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031235A (en) * | 2010-11-09 | 2011-04-27 | 中国农业大学 | Enterococcus faecium ANSE228 and application thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167785A (en) * | 2016-08-29 | 2016-11-30 | 内蒙古工业大学 | A kind of E. Faecium strains and application thereof |
| CN106167785B (en) * | 2016-08-29 | 2019-05-28 | 内蒙古工业大学 | A kind of E. Faecium strains and its application |
| CN110241043A (en) * | 2019-06-10 | 2019-09-17 | 大连理工大学 | The bacterial strain of one plant height temperature fermenting lactic acid and the method for producing lactic acid |
| CN110241043B (en) * | 2019-06-10 | 2021-12-21 | 大连理工大学 | Strain for producing lactic acid through high-temperature fermentation and method for producing lactic acid |
| CN110771723A (en) * | 2019-11-18 | 2020-02-11 | 北京大北农科技集团股份有限公司 | Cassava residue biological feed and fermentation method and application thereof |
| CN111607540A (en) * | 2020-06-10 | 2020-09-01 | 江西省科学院生物资源研究所 | Forage grass digestant and preparation method and application thereof |
| WO2022063296A1 (en) * | 2020-09-28 | 2022-03-31 | 天津博菲德科技有限公司 | Feed additive for improving oxygen obtaining ability of poultry and use thereof |
| CN113215019A (en) * | 2021-02-04 | 2021-08-06 | 郑州大学 | Enterococcus faecalis and application thereof |
| CN114424800A (en) * | 2022-04-06 | 2022-05-03 | 北京挑战生物技术有限公司 | Feed additive for improving intestinal health index of broiler chickens and application thereof |
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Effective date of registration: 20160114 Address after: 100080 Beijing City, Haidian District Zhongguancun street, No. 27 Zhongguancun building 14 DaBeiNong group Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: INNER MONGOLIA EVER SPRING FEED Co.,Ltd. Patentee after: Beijing Kegao Dabeinong Feedstuff Co.,Ltd. Patentee after: JILIN DABEINONG AGRICULTURE ANIMAL HUSBANDRY TECHNOLOGY Co.,Ltd. Address before: 100080 Beijing City, Haidian District Zhongguancun street, No. 27 Zhongguancun building 14 DaBeiNong group Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee before: INNER MONGOLIA EVER SPRING FEED Co.,Ltd. Patentee before: Beijing Kegao Dabeinong Feedstuff Co.,Ltd. |
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Address after: 100080 Beijing Haidian District Zhongguancun Street 27 Zhongguancun building 14 level big north agriculture group Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: INNER MONGOLIA EVER SPRING FEED Co.,Ltd. Patentee after: Beijing Kegao dabainong Biotechnology Co.,Ltd. Patentee after: JILIN DABEINONG AGRICULTURE ANIMAL HUSBANDRY TECHNOLOGY Co.,Ltd. Address before: 100080 Beijing Haidian District Zhongguancun Street 27 Zhongguancun building 14 level big north agriculture group Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee before: INNER MONGOLIA EVER SPRING FEED Co.,Ltd. Patentee before: Beijing Kegao Dabeinong Feedstuff Co.,Ltd. Patentee before: JILIN DABEINONG AGRICULTURE ANIMAL HUSBANDRY TECHNOLOGY Co.,Ltd. |