CN102533587A - Bacillus licheniformis and fungicide, feed additive and premix thereof - Google Patents
Bacillus licheniformis and fungicide, feed additive and premix thereof Download PDFInfo
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- CN102533587A CN102533587A CN2011103844446A CN201110384444A CN102533587A CN 102533587 A CN102533587 A CN 102533587A CN 2011103844446 A CN2011103844446 A CN 2011103844446A CN 201110384444 A CN201110384444 A CN 201110384444A CN 102533587 A CN102533587 A CN 102533587A
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- bacillus licheniformis
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- 230000000855 fungicidal effect Effects 0.000 title abstract 4
- 239000000417 fungicide Substances 0.000 title abstract 4
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of microorganism, in particular to a bacillus licheniformis and a fungicide, a feed additive and premix thereof. A bacillus licheniformis strain has the advantages of being strong in stress resistance, rapid in growth, high in spore generation rate after fermentation, strong in beneficial bacteria or antibacterial activity and the like. The bacillus licheniformis severing as the fungicide and the feed additive or the premix containing the bacillus licheniformis are safe, can partially replace antibiotics, eliminate drug residues, improve quality of livestock products, can improve animal production performance, enhance immunity or disease-resistant capacity, lower animal mortality rate, reduce diarrhea rate, and can further purify barn environment. The bacillus licheniformis and the fungicide, the feed additive and the premix of the bacillus licheniformis have high social benefit, economic benefit and ecological benefit.
Description
Technical field
The present invention relates to microbial technology field, relate to a bacillus licheniformis and microbial inoculum thereof, fodder additives and Preblend.
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 bacterium is the important foundation of the biology feed additive that development quality is stable, effect is remarkable, cost performance is high.But 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 that a strain characteristic is good, high-output stress-resistance, product enzyme, Bacillus licheniformis (Bacillus licheniformis) that bacteriostasis is strong.
Another object of the present invention provides microbial inoculum, fodder additives and the Preblend thereof that comprises this bacterial strain.
The objective of the invention is to realize through following technical scheme:
The present invention provides a bacillus licheniformis (Bacillus licheniformis), and its deposit number is CGMCC No.5094.
The present invention also provides the microbial inoculum that comprises this Bacillus licheniformis.
Wherein, the preparation method of this bacterial strain is following: Bacillus licheniformis carries out fermentation culture and obtains zymocyte liquid, and gained bacterium liquid mixes with protective material and carrier, and high temperature spray-drying promptly obtains microbial inoculum; Described the lichen bacillus ferments cultured method is following: by mass volume ratio is the inoculum size of 1%-10% inserts the 16-22h cell age in fermention medium said Bacillus licheniformis seed liquor; Fermentation condition is: 30-40 ℃; Rotating speed is 200-300rpm, cultivates 15-22h; The preparation of said fermention medium, its mass volume ratio is following: brown sugar 0.7-2.1%, Semen Maydis powder 0.5-1.5%, dregs of beans 1-3%, ammonium citrate 0.3-1.2%, mixing salt solution 0.4-0.8%, skimmer 0.05-0.1%, pH are 6.2-7.4.
Microbial inoculum of the present invention is mainly used in poultry, livestock and the aquatic products raising.
The present invention also provides the fodder additives that comprises this Bacillus licheniformis, and described fodder additives is mainly used in poultry, livestock and the aquatic products raising.
The present invention also provides the Preblend that comprises this Bacillus licheniformis.
Wherein, described Preblend is mainly used in poultry, livestock and the aquatic products raising, and the weight percent that Bacillus licheniformis is added in the Preblend is 0.5 ‰-5 ‰.
Bacillus licheniformis of the present invention (Bacillus licheniformis) separates from the Roll road for the applicant, obtains through directed primary dcreening operation and multiple sieve.Its vegetative cell is shaft-like, and its gemma is oval or grows tubular, is middle life or the life of inferior end, and sporangiocyst expands slightly.Isolated or be short chain, rod end is semicircle.In 12～16h, can form bacterium colony, bacterium colony is circular, or irregular, and the edge is hair-like, and bacteria colony white is opaque, corrugationless.Gram-positive.The applicant has carried out experiments such as acid resistance, bile tolerance, high thermal resistance, fermentation character and bacteriostatic activity to this bacillus licheniformis, this bacillus licheniformis has characteristics such as strong to contrary environmental resistance, that adhesivity strong, the growth fast, living weight is big, bacteriostatic activity is good.Bacillus licheniformis of the present invention (Bacillus licheniformis) 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.5094.
That Bacillus licheniformis CGMCC No.5094 of the present invention has is strong to contrary environmental resistance, growth fast, the fermented liquid living weight is big, advantage such as bacteriostatic activity is arranged.Bacterial strain of the present invention utilizes zymotechnique of the present invention, and the Bacillus licheniformis that obtains is as microbial inoculum, fodder additives or comprise the Preblend of Bacillus licheniformis of the present invention, safety; Viable count is high; Can partly substitute microbiotic, eliminate drug residue, improve the livestock product quality; Can improve breeding performonce fo animals, improve immunizing power or resistance against diseases, reduction animal death rate, reduce diarrhea rate; Can also purify the animal house environment.The present invention has very high social benefit, economic benefit and ecological benefits.
Further specify content of the present invention through following examples, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that some technique means are well known to those skilled in the art among the embodiment.
Embodiment
The acid resistance of embodiment 1 Bacillus licheniformis CGMCC No.5094, bile tolerance, high thermal resistance, fermentation character and bacteriostatic activity experiment
Utilize BPY culture medium culturing Bacillus licheniformis of the present invention, the Bacillus licheniformis CGMCC No.5094 that obtains has been carried out acid resistance, bile tolerance, high thermal resistance and bacteriostatic activity experiment.Bacillus licheniformis acid resistance, anti-cholate, antagonistic property are respectively shown in table 1-4.
Table 1 Bacillus licheniformis acid resistance experimental result
Table 2 Bacillus licheniformis bile tolerance property experimental result
Table 3 Bacillus licheniformis is to pathogenic bacteria inhibition experimental result
Table 4 Bacillus licheniformis is to the acid experimental result of Pepsin
The result is shown in form, and Bacillus licheniformis of the present invention is strong to contrary environmental resistance, growth is fast, high thermal resistance is good and have good numerous advantages such as bacteriostatic activity.
The safety experiment of embodiment 2 Bacillus licheniformis CGMCC No.5094
Mouse and AA fryer are tested the security of Bacillus licheniformis as animal pattern.
Prepare 40 of 18-22g mouse, public female half and half, be divided into four treatment group at random, 10 every group, raise and train 7 days after, adopt the mode of abdominal injection to carry out challenge test, 21 days trial periods.Test is divided into 4 treatment group, control group injecting normal saline 1ml, B group injection the lichen bacillus ferments liquid (10
8Cfu/ml) 1ml, C group injection intestinal bacteria (10
7CFU/ml) 1ml, the lichen bacillus ferments liquid of D group injection 0.5ml and the intestinal bacteria of 0.5mL.Irritate the clothes back and observe its toxicity symptom, see expiratory dyspnea, tic, limb paralysis etc. whether occur.Prepare 80 AA fryer, be divided into four groups at random, 20 every group, the lichen bacillus ferments liquid/or escherichia coli fermented broth is admixed in the week back of brooding in feed, and concentration is handled the same with each group.Mouse and fryer safety testing result see table 5 and table 6.
Table 5 Bacillus licheniformis is to mouse safety experiment result
Table 6 Bacillus licheniformis is to fryer safety testing result
Experimental result can find out that Bacillus licheniformis all is safe to mouse and fryer among the present invention, and to no any toxic actions such as mouse, fryer, all are tried mouse and fryer is all strong alive, and the situation of D group totally is better than the C group.Dissect from 5 mouse of each group picked at random, 5 fryer, the B group is compared with control group, and each internal organs is not seen difference, and D group organ disease obviously is weaker than the C group.This shows that Bacillus licheniformis can alleviate the detrimentally affect that intestinal bacteria bring for mouse and fryer, reduced mortality ratio to a certain extent.
The fermentation of embodiment 3 Bacillus licheniformis and the preparation of microbial inoculum
The high density fermentation of Bacillus licheniformis: being formulated as of used fermention medium: brown sugar 0.7% (m/V), Semen Maydis powder 0.5% (m/V); Dregs of beans 1% (m/V); Ammonium citrate 0.3% (m/V), mixing salt solution 0.4% (V/V), skimmer 0.05% (V/V); Be made into starter in proportion, regulating pH then is 6.8.Through 121 ℃ of high-temperature steam sterilization 20min, when being cooled to 37 ℃,,, keep rotating speed 200rpm to stir at 30 ℃ to the seed liquor of wherein inoculating cell age 15h 1.5%.Cultivation 15h is a fermentation termination, puts jar, and obtaining the Bacillus licheniformis viable count is 4.86 * 10
10CFU/mL.
With adding spray dried protective material (glycerine 0.8%, Sodium Glutamate 0.2%, W-Gum 19%) in the fermented liquid, through the dried Bacillus licheniformis bacterium powder that obtains of high temperature spray, viable count is about 38,900,000,000/g again.
The composition of wherein said mixing salt solution is: anhydrous CaCl
20.2g, KH
2PO
41.0g, MgSO
4.7H
2O 0.48g, NaHCO
310.0g, NaCl 2.0g, zero(ppm) water 1000ml.
The fermentation of embodiment 4 Bacillus licheniformis and the preparation of microbial inoculum
Being formulated as of used fermention medium: brown sugar 1.8% (m/V), Semen Maydis powder 1% (m/V); Dregs of beans 2.4% (m/V); Ammonium citrate 0.8% (m/V), mixing salt solution 0.6% (V/V), skimmer 0.08% (V/V) are made into starter in proportion, and regulating pH then is 7.2.Through 121 ℃ of high-temperature steam sterilization 20min, when being cooled to 37 ℃,,, keep rotating speed 250rpm to stir at 37 ℃ to the seed liquor of wherein inoculating cell age 20h 3.5%, being cultured to 18h is fermentation termination, puts jar, obtaining the Bacillus licheniformis viable count is 7.69 * 10
10CFU/mL.
With adding spray dried protective material (glycerine 0.8%, Sodium Glutamate 0.2%, W-Gum 19%) in the fermented liquid, obtain Bacillus licheniformis bacterium powder through high temperature spray-drying again, viable count is about 56,500,000,000/g.
The composition of wherein said mixing salt solution is: anhydrous CaCl
20.2g, KH
2PO
41.0g, MgSO
4.7H
2O 0.48g, NaHCO
310.0g, NaCl 2.0g, zero(ppm) water 1000ml.
The fermentation of embodiment 5 Bacillus licheniformis and the preparation of microbial inoculum
Being formulated as of used fermention medium: brown sugar 2.1% (m/V), Semen Maydis powder 1.5% (m/V); Dregs of beans 3% (m/V); Ammonium citrate 1.2% (m/V), mixing salt solution 0.8% (V/V), skimmer 0.1% (V/V) are made into starter in proportion, and regulating pH then is 7.4.Through 121 ℃ of high-temperature steam sterilization 20min, when being cooled to 40 ℃,,, keep rotating speed 300rpm to stir at 40 ℃ to the seed liquor of wherein inoculating cell age 22h 3.5%, being cultured to 22h is fermentation termination, puts jar, obtaining the Bacillus licheniformis viable count is 5.87 * 10
10CFU/mL.
With adding spray dried protective material (glycerine 0.8%, Sodium Glutamate 0.2%, W-Gum 19%) in the fermented liquid, through the dried Bacillus licheniformis bacterium powder that obtains of high temperature spray, viable count is about 46,200,000,000/g again.
The composition of wherein said mixing salt solution is: anhydrous CaCl
20.2g, KH
2PO
41.0g, MgSO
4.7H
2O 0.48g, NaHCO
310.0g, NaCl 2.0g, zero(ppm) water 1000ml.
The application of embodiment 6 Bacillus licheniformis in laying hen produces
Select extra large blue grey laying hen 600 plumages of 360-388 age in days for use, be divided into two treatment group: the blank group is not added any composition; Test group is added 0.1% the embodiment of the invention 3 bacillus licheniformis agents.Each handles 6 repetitions, and each repeats 50 chickens.Butcher the changing conditions of different intestinal segment Bacillus licheniformis and other milk-acid bacteria total amounts, intestinal bacteria and total aerobic bacteria in the observation analysis laying hen enteron aisle after the raising.Test-results is listed in table 7.
Table 7 laying hen intestinal microflora separates count results [log10 (bacterium number/g content)]
Annotate: colleague's sample room is all to be indicated different capitalization persons and is difference extremely significantly (p＜0.01); Colleague's sample room is all to be indicated different lowercase persons and reaches level of signification (p＜0.05) for difference.
Can find out from last table result: add 0.1% active bacillus licheniformis agent; Bacillus licheniformis in the crop and lactic acid bacterium number summation have increased more than 10 times than blank group; The Bacillus licheniformis at other positions and lactic acid bacterium number summation also are significantly higher than the blank group, and intestinal bacteria also significantly descend with total aerobic bacteria quantity and are inhibited.
The application of embodiment 7 Bacillus licheniformis in fryer is produced
Select 300 of the fryer that the public mother of 1-28 age in days raises together with for use, be divided into 3 processing at random, be i.e. blank group, microbiotic control group (duomycin 100g/t) and 0.15% embodiment of the invention, 3 bacillus licheniformis agents, 100 of each processing.Brood after 1 week, each processing is divided into five repetitions, and each repeats 20.Butcher the changing conditions of different intestinal segment lichens, lactic acid summation, intestinal bacteria, Salmonellas and total aerobic bacteria in the observation analysis fryer enteron aisle (being the bacterium phase change) after the raising.Test-results is listed in table 8.
Table 8 fryer intestinal microflora separates count results [log10 (bacterium number/s content)]
Annotate: colleague's sample room is all to be indicated different capitalization persons and is difference extremely significantly (p＜0.01); Colleague's sample room is all to be indicated different lowercase persons and reaches level of signification (p＜0.05) for difference.
Can find out from last table result; Adding bacillus licheniformis agent of the present invention can make the quantity of Bacillus licheniformis and lactobacillus spp in the ileum increase more than 10 times than blank group; And the concentration that has reduced intestinal bacteria and Salmonellas is more than 10 times, and also there is similar result at other positions.Simultaneously, the diarrhea rate that adds 0.2% bacillus licheniformis agent group extremely significantly is lower than blank group and microbiotic control group (p＜0.01).
Test-results shows, bacillus licheniformis agent provided by the invention be applied to can obviously to promote when the fryer laying hen produces with the stimulating gastrointestinal road in the growth of probiotics; And can significantly suppress the growth and breeding of intestinal bacteria and other aerobic bacterias (Salmonellas).Regulate the intestinal microflora balance, improve the fryer disease resistance, diarrhea rate descends greatly.
The effect of embodiment 3 bacillus licheniformis agents in piglet, broiler chicken and grass carp are cultured
Microbial inoculum among the embodiment 3 is applied to the breed of piglet, broiler chicken and grass carp respectively, all is made as blank group, microbiotic combination Bacillus licheniformis test group, every group of 3 repetitions.Wherein, the blank group basal diet of feeding; The microbiotic group is added duomycin (80g/t) in basal diet; The Bacillus licheniformis experimental group is added the microbial inoculum of embodiment 3, and concrete addition is as shown in table 9.
The effect of table 9 bacillus licheniformis agent in piglet, broiler chicken and grass carp are cultured
Can find out that from table 9 result piglet, add Bacillus licheniformis preparation of the present invention in broiler chicken and the finishing period grass carp daily ration, each item index obviously is better than microbiotic control group and blank group.Explain that Bacillus licheniformis preparation provided by the invention can effectively prevent and treat livestock and poultry and aquatic animal disease, significantly improve production performance, improve animal house environment and aquatic animal water quality, explain that it has good economic benefit and ecological benefits.
Claims (7)
1. a bacillus licheniformis (Bacillus licheniformis), deposit number is CGMCC No.5094.
2. microbial inoculum that comprises the described Bacillus licheniformis of claim 1.
3. weigh the microbial inoculum of 2 described Bacillus licheniformis, it is characterized in that its preparation method is following: Bacillus licheniformis carries out fermentation culture and obtains zymocyte liquid, and gained bacterium liquid mixes with protective material and carrier, and high temperature spray-drying promptly obtains microbial inoculum; Described the lichen bacillus ferments cultured method is following: by mass volume ratio is the inoculum size of 1%-10% inserts the 16-22h cell age in fermention medium said Bacillus licheniformis seed liquor; Fermentation condition is: 30-40 ℃; Rotating speed is 200-300rpm, cultivates 15-22h; The preparation of said fermention medium, its mass volume ratio is following: brown sugar 0.7-2.1%, Semen Maydis powder 0.5-1.5%, dregs of beans 1-3%, ammonium citrate 0.3-1.2%, mixing salt solution 0.4-0.8%, skimmer 0.05-0.1%, pH are 6.2-7.4.
4. weigh the microbial inoculum of 3 described Bacillus licheniformis, it is characterized in that described microbial inoculum is the microbial inoculum that is applied in poultry, livestock and the aquatic products raising.
5. a fodder additives that comprises the described Bacillus licheniformis of claim 1 is characterized in that described fodder additives is the fodder additives that is applied in poultry, livestock and the aquatic products raising.
6. Preblend that comprises the described Bacillus licheniformis of claim 1.
7. weigh the Preblend of 6 described Bacillus licheniformis, it is characterized in that described Preblend is the Preblend that is applied in poultry, livestock and the aquatic products raising, the weight percent that Bacillus licheniformis is added in the Preblend is 0.5 ‰-5 ‰.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110384444.6A CN102533587B (en) | 2011-11-28 | 2011-11-28 | Bacillus licheniformis and fungicide, feed additive and premix thereof |
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Cited By (12)
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| CN102876614A (en) * | 2012-10-16 | 2013-01-16 | 北京龙科方舟生物工程技术有限公司 | Bacillus licheniformis and application of bacillus licheniformis |
| CN102994426A (en) * | 2012-11-26 | 2013-03-27 | 青岛蔚蓝生物集团有限公司 | Bacillus licheniformis and applications thereof |
| CN103845365A (en) * | 2014-01-03 | 2014-06-11 | 青岛东海药业有限公司 | Bacillus licheniformis probiotics for abalone and sea cucumber aquaculture |
| CN105219682A (en) * | 2015-11-03 | 2016-01-06 | 山东省农业科学院生物技术研究中心 | A kind of Bacillus licheniformis of substitute antibiotics fodder additives and application thereof |
| TWI552682B (en) * | 2014-08-29 | 2016-10-11 | 生百興業有限公司 | A feed additive for decreasing intestinal pathogens infections in poultry and its manufacturing method |
| CN107043724A (en) * | 2017-04-13 | 2017-08-15 | 中国农业科学院特产研究所 | A kind of bacillus licheniformis and its separation method and application |
| CN108841760A (en) * | 2018-07-13 | 2018-11-20 | 中国科学院水生生物研究所 | One plant can improve Growth of Grass Carps Ctenopharyngodon Idellus performance, immunocompetence and the bacillus licheniformis FA6 and its application that adjust intestinal microflora |
| CN109536418A (en) * | 2018-12-28 | 2019-03-29 | 佛山市艳晖生物科技有限公司 | A kind of microbial bacterial agent and its application in aquaculture |
| CN111909868A (en) * | 2020-07-24 | 2020-11-10 | 天津开发区坤禾生物技术有限公司 | Composite microbial inoculum for preventing and treating anorexia dead fish of grass carps as well as preparation method and application thereof |
| CN112760251A (en) * | 2021-01-05 | 2021-05-07 | 安徽农业大学 | Compound bacillus probiotics and preparation and application thereof |
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| CN102876614B (en) * | 2012-10-16 | 2013-11-06 | 北京龙科方舟生物工程技术有限公司 | Bacillus licheniformis and application of bacillus licheniformis |
| CN102876614A (en) * | 2012-10-16 | 2013-01-16 | 北京龙科方舟生物工程技术有限公司 | Bacillus licheniformis and application of bacillus licheniformis |
| CN102994426A (en) * | 2012-11-26 | 2013-03-27 | 青岛蔚蓝生物集团有限公司 | Bacillus licheniformis and applications thereof |
| CN102994426B (en) * | 2012-11-26 | 2014-03-12 | 山东蔚蓝生物科技有限公司 | Bacillus licheniformis and applications thereof |
| CN103845365A (en) * | 2014-01-03 | 2014-06-11 | 青岛东海药业有限公司 | Bacillus licheniformis probiotics for abalone and sea cucumber aquaculture |
| TWI552682B (en) * | 2014-08-29 | 2016-10-11 | 生百興業有限公司 | A feed additive for decreasing intestinal pathogens infections in poultry and its manufacturing method |
| CN105219682B (en) * | 2015-11-03 | 2018-07-10 | 山东省农业科学院生物技术研究中心 | A kind of bacillus licheniformis of substitute antibiotics feed addictive and its application |
| CN105219682A (en) * | 2015-11-03 | 2016-01-06 | 山东省农业科学院生物技术研究中心 | A kind of Bacillus licheniformis of substitute antibiotics fodder additives and application thereof |
| CN107043724A (en) * | 2017-04-13 | 2017-08-15 | 中国农业科学院特产研究所 | A kind of bacillus licheniformis and its separation method and application |
| CN107043724B (en) * | 2017-04-13 | 2020-04-28 | 中国农业科学院特产研究所 | Bacillus licheniformis and separation method and application thereof |
| CN108841760A (en) * | 2018-07-13 | 2018-11-20 | 中国科学院水生生物研究所 | One plant can improve Growth of Grass Carps Ctenopharyngodon Idellus performance, immunocompetence and the bacillus licheniformis FA6 and its application that adjust intestinal microflora |
| CN108841760B (en) * | 2018-07-13 | 2021-10-08 | 中国科学院水生生物研究所 | Bacillus licheniformis FA6 capable of improving growth performance and immunity of grass carp and regulating intestinal flora structure and application thereof |
| CN109536418A (en) * | 2018-12-28 | 2019-03-29 | 佛山市艳晖生物科技有限公司 | A kind of microbial bacterial agent and its application in aquaculture |
| CN111909868A (en) * | 2020-07-24 | 2020-11-10 | 天津开发区坤禾生物技术有限公司 | Composite microbial inoculum for preventing and treating anorexia dead fish of grass carps as well as preparation method and application thereof |
| CN112760251A (en) * | 2021-01-05 | 2021-05-07 | 安徽农业大学 | Compound bacillus probiotics and preparation and application thereof |
| CN113786377A (en) * | 2021-08-31 | 2021-12-14 | 武汉中博绿亚生物科技有限公司 | Composition for repairing skin micro-ecological balance, nursing bath lotion and preparation method thereof |
| CN116121109A (en) * | 2022-10-26 | 2023-05-16 | 武汉合缘绿色生物股份有限公司 | Bacillus licheniformis strain and application thereof |
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