CN102533587B - 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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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, cause 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, for food safety and human consumer's the healthy hidden danger of having buried, the Substitutes For Antibiotic of therefore finding 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 the Isolation and screening of strain excellent, good probiotic bacterium treatment process.At present, there is following problem in probiotic bacterium bacterial classification: 1. most probiotic bacterium is asporulate milk-acid bacteria, and many genus obligatory anaerobic bacterias, are very easily subject to the impact of the external environment factors such as temperature, oxygen, moisture and soda acid, and bacterium activity keeping is more difficult conventionally; 2. 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 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 is to provide the microbial inoculum, fodder additives and the Preblend thereof that comprise this bacterial strain.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a bacillus licheniformis (Bacillus licheniformis), 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 as follows: 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 obtains microbial inoculum; The method that described the lichen bacillus ferments is cultivated is as follows: the inoculum size that is 1%-10% by mass volume ratio is to the described Bacillus licheniformis seed liquor that accesses 16-22h cell age in fermention medium, fermentation condition is: 30-40 DEG C, rotating speed is 200-300rpm, cultivates 15-22h; The preparation of described fermention medium, its mass volume ratio is as follows: 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%, defoamer 0.05-0.1%, pH is 6.2-7.4.
Microbial inoculum of the present invention is mainly used in poultry, livestock and 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 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 aquatic products raising, and the weight percent that in Preblend, Bacillus licheniformis is added is 0.5 ‰-5 ‰.
Bacillus licheniformis of the present invention (Bacillus licheniformis) separates from Roll road for applicant, obtains through directed primary dcreening operation and multiple sieve.Its vegetative cell is shaft-like, and its gemma is oval or long tubular, is middle life or the life of inferior end, and sporangiocyst slightly expands.Isolated or be short chain, rod end semicircle.In 12～16h, can form bacterium colony, bacterium colony is circular, or irregular, and edge is hair-like, and bacteria colony white is opaque, corrugationless.Gram-positive.Applicant has carried out the experiments such as acid resistance, bile tolerance, high thermal resistance, fermentation character and bacteriostatic activity to this bacillus licheniformis, this bacillus licheniformis has the features such as strong to contrary environmental resistance, adhesivity strong, growth fast, biomass is large, bacteriostatic activity is good.Bacillus licheniformis of the present invention (Bacillus licheniformis) applicant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2011, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, 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, fermented liquid biomass is large, have the advantages such as bacteriostatic activity.Bacterial strain of the present invention utilizes zymotechnique of the present invention, and the Bacillus licheniformis obtaining is as microbial inoculum, fodder additives or the Preblend that comprises Bacillus licheniformis of the present invention, safety, viable count is high, can part substitute antibiotics, eliminate drug residue, improve 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 animal house environment.The present invention has very high social benefit, economic benefit and ecological benefits.
Further illustrate content of the present invention by following examples, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, some technique means are well known to those skilled in the art.
Embodiment
Acid resistance, bile tolerance, high thermal resistance, fermentation character and the bacteriostatic activity experiment of embodiment 1 Bacillus licheniformis CGMCC No.5094
Utilize BPY culture medium culturing Bacillus licheniformis of the present invention, the Bacillus licheniformis CGMCC No.5094 obtaining has been carried out to acid resistance, bile tolerance, high thermal resistance and bacteriostatic activity experiment.Bacillus licheniformis acid resistance, resistance to cholate, antagonistic property are respectively as shown in table 1-4.
Table 1 Bacillus licheniformis acid resistance experimental result
Table 2 Bacillus licheniformis bile tolerance experimental result
Table 3 Bacillus licheniformis is to pathogenic bacteria inhibition experimental result
The resistance to stomach en-experimental result of table 4 Bacillus licheniformis
Result is as 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 the many merits such as good bacteriostatic activity.
The safety experiment of embodiment 2 Bacillus licheniformis CGMCC No.5094
Mouse and AA broiler chicken, as animal pattern, are tested the security of Bacillus licheniformis.
Prepare 40 of 18-22g mouse, male and female half and half, is divided into four treatment group at random, and 10 every group, raise and train after 7 days, 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 organizes Escherichia Coli Injection (10
7cFU/ml) 1ml, the lichen bacillus ferments liquid of D group injection 0.5ml and the intestinal bacteria of 0.5mL.Gavage its toxicity symptom of rear observation, see whether occur expiratory dyspnea, tic, limb paralysis etc.Prepare 80 AA broiler chicken, be divided at random four groups, 20 every group, after brooding one week, in feed, admix the lichen bacillus ferments liquid/or escherichia coli fermented broth, concentration and each group of processing are the same.Mouse and broiler chicken safety testing the results are shown in Table 5 and table 6.
Table 5 Bacillus licheniformis is to mouse safety experiment result
Table 6 Bacillus licheniformis is to broiler chicken safety testing result
Experimental result can find out, in the present invention, Bacillus licheniformis is all safe to mouse and broiler chicken, and to mouse, broiler chicken etc., without any toxic action, all tested mouse and broiler chicken be strong living all, and the situation of D group is totally better than C group.From each group choose at random 5 mouse, 5 broiler chicken are dissected, B group is compared with control group, each internal organs have no difference, D group organ disease is obviously weaker than C group.Bacillus licheniformis can be alleviated the detrimentally affect that intestinal bacteria bring to mouse and broiler chicken as can be seen here, has reduced to a certain extent mortality ratio.
The fermentation of embodiment 3 Bacillus licheniformis and the preparation of microbial inoculum
The high density fermentation of Bacillus licheniformis: being formulated as of fermention medium used: 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), defoamer 0.05% (V/V), be made in proportion starter, then regulating pH is 6.8.Through 121 DEG C of high-temperature steam sterilization 20min, while being cooled to 37 DEG C, inoculate wherein the seed liquor 1.5% of cell age 15h, at 30 DEG C, keep rotating speed 200rpm to stir.Cultivation 15h is fermentation termination, puts tank, and obtaining Bacillus licheniformis viable count is 4.86 × 10
10cFU/mL.
The dry protective material of spray (glycerine 0.8%, Sodium Glutamate 0.2%, W-Gum 19%) will be added in fermented liquid, then through the dry Bacillus licheniformis powder that obtains of high temperature spray, viable count is about 38,900,000,000/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, distilled water 1000ml.
The fermentation of embodiment 4 Bacillus licheniformis and the preparation of microbial inoculum
Being formulated as of fermention medium used: 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), defoamer 0.08% (V/V), be made in proportion starter, then regulating pH is 7.2.Through 121 DEG C of high-temperature steam sterilization 20min, while being cooled to 37 DEG C, inoculate wherein the seed liquor 3.5% of cell age 20h, at 37 DEG C, keep rotating speed 250rpm to stir, being cultured to 18h is fermentation termination, puts tank, obtaining Bacillus licheniformis viable count is 7.69 × 10
10cFU/mL.
To in fermented liquid, add the dry protective material of spray (glycerine 0.8%, Sodium Glutamate 0.2%, W-Gum 19%), then obtain Bacillus licheniformis powder through high temperature spray-drying, 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, distilled water 1000ml.
The fermentation of embodiment 5 Bacillus licheniformis and the preparation of microbial inoculum
Being formulated as of fermention medium used: 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), defoamer 0.1% (V/V), be made in proportion starter, then regulating pH is 7.4.Through 121 DEG C of high-temperature steam sterilization 20min, while being cooled to 40 DEG C, inoculate wherein the seed liquor 3.5% of cell age 22h, at 40 DEG C, keep rotating speed 300rpm to stir, being cultured to 22h is fermentation termination, puts tank, obtaining Bacillus licheniformis viable count is 5.87 × 10
10cFU/mL.
The dry protective material of spray (glycerine 0.8%, Sodium Glutamate 0.2%, W-Gum 19%) will be added in fermented liquid, then through the dry Bacillus licheniformis powder that obtains of high temperature spray, viable count is about 46,200,000,000/g.
The composition of wherein said mixing salt solution is: anhydrous CaCl
20.2g, KH
2pO
41.0g, MgSO
4.7H
2o0.48g, NaHCO
310.0g, NaCl 2.0g, distilled water 1000ml.
The application of embodiment 6 Bacillus licheniformis in laying hen produces
Blue grey laying hen 600 plumages in sea of selecting 360-388 age in days, are divided into two treatment group: blank group is not added any composition; Test group is added 0.1% the embodiment of the present invention 3 bacillus licheniformis agents.6 repetitions of each processing, 50 chickens of each repetition.After raising, butcher the changing conditions of different intestinal segment Bacillus licheniformis and other milk-acid bacteria total amounts, intestinal bacteria and total aerobic bacteria in observation analysis laying hen enteron aisle.Test-results is listed in table 7.
Table 7 laying hen intestinal microflora separates count results [log10 (bacterium number/g content)]
Note: colleague's sample room is all indicates different capitalization persons for extremely significantly (p < 0.01) of difference; Colleague's sample room is all to be indicated different lowercase persons and reaches conspicuous level (p < 0.05) for difference.
Can find out from upper table result: add 0.1% active bacillus licheniformis agent, Bacillus licheniformis in 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 are also significantly higher than blank group, and intestinal bacteria and total aerobic bacteria quantity also significantly decline and is inhibited.
The application of embodiment 7 Bacillus licheniformis in broiler production
Select 300 of the broiler chicken that 1-28 age in days male and female raises together with, be divided at random 3 processing, i.e. blank group, microbiotic control group (duomycin 100g/t) and 0.15% embodiment of the present invention 3 bacillus licheniformis agents, 100 of each processing.Brood after 1 week, each processing is divided into five repetitions, 20 of each repetitions.After raising, butcher the changing conditions (being bacterium phase change) of different intestinal segment lichens, lactic acid summation, intestinal bacteria, Salmonellas and total aerobic bacteria in observation analysis Intestine of Broiler.Test-results is listed in table 8.
Table 8 Intestine of Broiler flora separates count results [log10 (bacterium number/g content)]
Note: colleague's sample room is all indicates different capitalization persons for extremely significantly (p < 0.01) of difference; Colleague's sample room is all to be indicated different lowercase persons and reaches conspicuous level (p < 0.05) for difference.
Can find out from upper table result, adding bacillus licheniformis agent of the present invention can make the quantity of Bacillus licheniformis and lactobacillus in ileum increase more than 10 times than blank group, and the concentration that has reduced intestinal bacteria and Salmonellas is more than 10 times, also there is similar result at other positions.Meanwhile, the diarrhea rate that adds 0.2% bacillus licheniformis agent group is extremely significantly lower than blank group and microbiotic control group (p < 0.01).
Test-results shows, bacillus licheniformis agent provided by the invention be applied to when broiler chicken laying hen produces, can obviously promote with stimulating gastrointestinal road in the growth of probiotics; And can significantly suppress the growth and breeding of intestinal bacteria and other aerobic bacterias (Salmonellas).Regulating intestinal canal colony balance, improves broiler chicken disease resistance, and diarrhea rate declines greatly.
The effect of embodiment 8 bacillus licheniformis agents in piglet, broiler chicken and grass carp cultivation
Microbial inoculum in embodiment 3, is applied to respectively the cultivation of piglet, broiler chicken and grass carp, is all made as blank group, antibiotic combinations Bacillus licheniformis test group, every group of 3 repetitions.Wherein, the blank group basal diet of feeding; Microbiotic group is added duomycin (80g/t) in basal diet; 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 cultivation
As can be seen from Table 9, piglet, in broiler chicken and finishing period grass carp daily ration, add Bacillus licheniformis preparation of the present invention, indices is significantly better than microbiotic control group and blank group.Illustrate 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, illustrate that it has good economic benefit and ecological benefits.
Claims (2)
1. the bacterial preparation process of Bacillus licheniformis CGMCC No.5094, it is: Bacillus licheniformis CGMCC No.5094 is carried out to fermentation culture and obtain zymocyte liquid, gained bacterium liquid mixes with protective material and carrier, and high temperature spray-drying obtains microbial inoculum; The method that described the lichen bacillus ferments is cultivated is as follows: the inoculum size that is 1%-10% by mass volume ratio is to the described Bacillus licheniformis seed liquor that accesses 16-22h cell age in fermention medium, fermentation condition is: 30-40 DEG C, rotating speed is 200-300rpm, cultivates 15-22h; The preparation of described fermention medium, its mass volume ratio is as follows: 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 %, defoamer 0.05-0.1%, pH is 6.2-7.4, and 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, distilled water 1000ml.
2. the application of Bacillus licheniformis CGMCC No.5094 microbial inoculum in laying hen and grass carp cultivation.
3. a Preblend that comprises Bacillus licheniformis CGMCC No.5094, it is characterized in that, described Preblend is the Preblend being applied in laying hen and grass carp raising, and the weight percent that in Preblend, Bacillus licheniformis CGMCC No.5094 adds is 0.5 ‰-5 ‰.
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Families Citing this family (11)
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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 |
| 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 |
| 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 |
| 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 |
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