CN113151110A - Bacillus licheniformis for preventing and treating animal diarrhea and microecological preparation thereof - Google Patents
Bacillus licheniformis for preventing and treating animal diarrhea and microecological preparation thereof Download PDFInfo
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- CN113151110A CN113151110A CN202110547331.7A CN202110547331A CN113151110A CN 113151110 A CN113151110 A CN 113151110A CN 202110547331 A CN202110547331 A CN 202110547331A CN 113151110 A CN113151110 A CN 113151110A
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- bacillus licheniformis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a bacillus licheniformis for preventing and treating animal diarrhea and a microecological preparation thereof, wherein the preservation number of the strain preservation is CGMCC NO. 20892; the formulation contains an extract of a culture of the bacillus licheniformis bacteria; the invention has the advantages that: the prevention and treatment efficiency is high, the action condition is mild, the concentrated product does not destroy the nutrient components in the feed, the extract can be used for biodegradation detoxification of the feed, the diarrhea of animals can be effectively prevented and treated, and the prevention and treatment rate can reach more than 90%.
Description
Technical Field
The invention relates to bacillus licheniformis, in particular to bacillus licheniformis for preventing and treating animal diarrhea and a microecological preparation thereof, belonging to the field of agricultural biology.
Background
Animal diarrhea is caused by multiple pathogens and multiple factors, usually causes are complex, and clinical diagnosis and treatment are difficult. Can lead to the loss of a large amount of electrolytes, dehydration, malnutrition and other symptoms, and even death of animals. When antibiotics are used for treating herbivorous animals such as cattle, sheep, rabbits, and the like, gastrointestinal flora disorder is easily caused. The microbial ecological agent mainly containing bacillus can improve the disease resistance of animals, improve the utilization rate of feed, promote the growth of animals and overcome some defects of antibiotics. Bacillus licheniformisBacillus licheniformisB2.0118 can produce enzymes such as protease and amylase, and nutrients such as amino acids and vitamins, and has no toxic or side effect.
Disclosure of Invention
The invention aims to provide the bacillus licheniformis and the microecological preparation thereof, which can prevent and treat animal diarrhea, and have the advantages of high prevention and treatment efficiency and mild action condition, the concentrated product does not destroy the nutrient components in the feed, the extract can be used for biodegradation detoxification of the feed, the animal diarrhea can be effectively prevented and treated, and the prevention and treatment rate can reach more than 90%.
The technical scheme of the invention is as follows:
the bacillus licheniformis for preventing and treating animal diarrhea is deposited in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No. 3, North Chen Xilu No. 1, of the rising area in Beijing, and the preservation number is CGMCC NO. 20892.
The liquid culture medium formula of the bacillus licheniformis comprises the following components in percentage by weight: 10 per mill sodium chloride, 10 per mill tryptone and 5 per mill yeast powder, and the culture conditions are as follows: ventilating and fermenting at 37 deg.C and pH 7.4 to obtain Bacillus licheniformis liquid culture; the formula weight fraction of the solid culture medium is as follows: 90% of bran, 5% of peptone and 5% of yeast powder, wherein the water content is 60%; the culture conditions were: ventilating and fermenting at 37 ℃ to obtain the bacillus licheniformis solid culture.
A microecological preparation for preventing and treating diarrhea in animals comprises extract of the culture of Bacillus licheniformis.
The preparation method of the extract comprises the following steps: after culturing the solid culture of Bacillus licheniformis at 32 deg.C for 5 days, leaching with warm water at 30 deg.C for 24 hr, adsorbing with carrier, and air drying at low temperature.
The carrier comprises one or more of wheat bran, corncob powder, yeast cell wall, corn starch, rice bran, glucose, zeolite powder, bentonite and montmorillonite.
The content of the extract in the microecological preparation is 20-60% by weight.
The mass ratio of the bacillus licheniformis fermentation extract to the carrier is as follows: 20-60: 80-40.
And (2) determining the gene sequence of the 16SrDNA of the bacillus licheniformis strain: AAAATCCTGTCCACTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAGCCTCGCGGCTTCGCTGCCCTTTGTTCTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTTGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTTTTCCCTAACAACAGAGTTTTACGATCCGAAAACCTTCATCAATCACGGGGGGGTGGTCCGTCAGACTTTTGTCCCTTGGGGAAGATTCCCTAATGGTGCCTTCCGTAGGAGTTTGGGCCGTGTTTCAGTTCCCGGGTGGCCGATCACCCTTTCAGGTTGGGTACGCATTGTTGCCTTGGTGAGCCGTTACCTCACCAAATAGGTAATGCGCCGCGGGTCCATTTGTAAGTGGTAGGTAAAAGCCACCTTTTATAATTGAACCATGCGGTTCAATCAAGCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTGCATGTATAGCACGCCGCAATCTCCG are provided.
The invention relates to a preparation for preventing and treating animal diarrhea, and the action objects of the preparation comprise feed and preparation raw materials thereof.
The invention has the beneficial effects that: the prevention and treatment efficiency is high, the action condition is mild, the concentrated product does not destroy the nutrient components in the feed, the extract can be used for biodegradation detoxification of the feed, the diarrhea of animals can be effectively prevented and treated, and the prevention and treatment rate can reach more than 90%.
The invention is further illustrated by the following figures and examples.
Drawings
FIG. 1 is a spore diagram of a Bacillus licheniformis strain for preventing and treating animal diarrhea under a 1600X microscope;
FIG. 2 is a diagram showing that Bacillus licheniformis has obvious inhibition zones on Staphylococcus aureus, Escherichia coli and Salmonella in pigs according to the embodiment of the invention.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Example 1
The bacillus licheniformis for preventing and treating animal diarrhea is characterized in that the liquid culture medium formula of the bacillus licheniformis comprises the following components in parts by weight: 10 per mill sodium chloride, 10 per mill tryptone and 5 per mill yeast powder, and the culture conditions are as follows: ventilating and fermenting at 37 deg.C and pH 7.4 to obtain Bacillus licheniformis liquid culture; the formula weight fraction of the solid culture medium is as follows: 90% of bran, 5% of peptone and 5% of yeast powder, wherein the water content is 60%; the culture conditions were: ventilating and fermenting at 37 ℃ to obtain the bacillus licheniformis solid culture.
A microecological preparation for preventing and treating diarrhea in animals comprises extract of the culture of Bacillus licheniformis.
The preparation method of the extract comprises the following steps: after culturing the solid culture of Bacillus licheniformis at 32 deg.C for 5 days, leaching with warm water at 30 deg.C for 24 hr, adsorbing with carrier, and air drying at low temperature.
The carrier comprises one or more of wheat bran, corncob powder, yeast cell wall, corn starch, rice bran, glucose, zeolite powder, bentonite and montmorillonite.
The content of the extract in the microecological preparation is 20-60% by weight.
The mass ratio of the bacillus licheniformis fermentation extract to the carrier is as follows: 20-60: 80-40.
The Bacillus licheniformis strain is isolated from soil in corn fields.
Samples in the soil of the corn field are collected and diluted by 10 times for screening experiments. The diluted solution was spread on an LB plate and cultured at 37 ℃ for 36 hr. Each single colony on the plate was transferred to a blank LB plate to obtain a pure culture strain. Culturing the purified strain in liquid LB culture medium for 36hr, and detecting its ability to prevent and treat animal diarrhea.
Example 2 prevention and treatment of diarrhea in animals with Bacillus licheniformis
Test animal and test daily ration
The strain purified in example 1 was activated in liquid LB medium for 22hr to obtain a culture of the strain of the present invention. 100 weaned piglets of 25 days old are selected in the test, and the average weight is 7.37 +/-0.02 kg. Group 1 was a blank control group and was fed basal diet. And 2 groups are test groups, and the test groups are fed with test daily ration, and the test groups are added according to 200ml/t of bacillus licheniformis on the basis of basic daily ration, wherein the viable count of the bacillus licheniformis is 2000 hundred million CFU/g. The diarrhea condition of each group of feed is recorded in the whole process of the test, the diarrhea condition of each group of feed is weighed on an empty stomach in the morning of the first day and the 21 st day of the test, the residual feed amount is weighed, and the average daily gain and the feed-meat ratio (F/G) of each group are calculated.
Second, data analysis
Statistical software SPSS 21.0 software was used to perform one-factor analysis of variance and correlation analysis on the data. As can be seen from the test results, the average initial weights of the blank group and the Bacillus licheniformis group were not significantly different at the initial stage of the test (P > 0.05). At the end of the test (21 st day), the body weight of the spore group is obviously higher than that of the blank control group, the average total weight gain and the average daily weight gain are respectively improved by 12.33 percent and 14.89 percent, and the difference is obvious (P is less than 0.05).
TABLE 10.02% Effect of Bacillus licheniformis on weight gain of weaned piglets (kg/head)
As can be seen from Table 2, the feed consumption of the Bacillus licheniformis group is higher than that of the control group, but the difference is not significant (P > 0.05), while the average weight gain of the Bacillus licheniformis group is higher than that of the control group, the feed-meat ratio is obviously lower than that of the control group, the spore group reduces the feed-meat ratio by 15.49%, and the difference is very significant (P < 0.01). Test results show that the bacillus licheniformis group can obviously reduce the occurrence of diarrhea of piglets, the blank group has 12 diarrhea in total, and the diarrhea rate is 23.08%; the bacillus licheniformis group has 3 diarrhea, the diarrhea rate is 5.77%, the diarrhea rate of the spore group is reduced by 17.31% compared with the blank group, and the difference is very obvious (P < 0.01).
TABLE 20.02 influence of Bacillus licheniformis on weaned pig feed intake and feed-meat ratio
As a result: the Bacillus licheniformis can reduce intestinal diseases. In the test process, the diarrhea of the blank group of piglets is mainly concentrated in the initial stage of the test, because the weaned piglets are just transferred into a nursing house and are not suitable for various living environments, and obvious weaning and reloading stresses are presented. And the bacillus licheniformis group has less diarrhea, and the bacillus licheniformis can improve the immunity and has a certain anti-stress effect.
Example 3: antagonistic ability of Bacillus licheniformis on staphylococcus aureus, Escherichia coli and salmonella of pig
Test sample and treatment
The strain purified in example 1 was activated in liquid LB medium for 36hr to obtain a culture of the strain of the present invention. And (3) expanding and culturing the Escherichia coli, the staphylococcus aureus and the salmonella of the pig in a liquid LB culture medium. The liquid LB culture medium formula is tryptone 1%, NaCl 1%, yeast extract 0.5%, agar 1.5%, pH 7.4.
Second, antagonistic experiment
Respectively sucking 1000ul of strain staphylococcus aureus, pig escherichia coli and salmonella liquid by using a pipette gun, respectively adding the strain staphylococcus aureus, pig escherichia coli and salmonella liquid into a sterile LB culture medium with the temperature reduced to 40 ℃, shaking, uniformly mixing, subpackaging into a flat plate, standing, cooling and solidifying. Two for each strain. A sterile punch punched a hole in the coagulation plate and a Bacillus licheniformis pipetting gun added 20ul of the hole. Culturing in 40 deg.C incubator for 20h, and observing whether antibacterial zone appears around the perforation.
Three, result in
The bacillus licheniformis has obvious inhibition zone on staphylococcus aureus, colibacillus of pigs and salmonella. The diameter of the bacteriostatic zone is 6mm-13 mm.
Sequence listing
<110> science and technology development Co., Ltd
<120> Bacillus licheniformis for preventing and treating animal diarrhea and microecological preparation thereof
<141> 2021-05-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1459
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 1
aaaatcctgt ccacttcggc ggctggctcc aaaaggttac ctcaccgact tcgggtgtta 60
caaactctcg tggtgtgacg ggcggtgtgt acaaggcccg ggaacgtatt caccgcggca 120
tgctgatccg cgattactag cgattccagc ttcacgcagt cgagttgcag actgcgatcc 180
gaactgagaa cagatttgtg ggattggctt agcctcgcgg cttcgctgcc ctttgttctg 240
cccattgtag cacgtgtgta gcccaggtca taaggggcat gatgatttga cgtcatcccc 300
accttcctcc ggtttgtcac cggcagtcac cttagagtgc ccaactgaat gctggcaact 360
aagatcaagg gttgcgctcg ttgcgggact taacccaaca tctcacgaca cgagctgacg 420
acaaccatgc accacctgtc actctgcccc cgaaggggaa gccctatctc tagggttgtc 480
agaggatgtc aagacctggt aaggttcttc gcgttgcttc gaattaaacc acatgctcca 540
ccgcttgtgc gggcccccgt caattccttt gagtttcagt cttgcgaccg tactccccag 600
gcggagtgct taatgcgttt gctgcagcac taaagggcgg aaaccctcta acacttagca 660
ctcatcgttt acggcgtgga ctaccagggt atctaatcct gttcgctccc cacgctttcg 720
cgcctcagcg tcagttacag accagagagt cgccttcgcc actggtgttc ctccacatct 780
ctacgcattt caccgctaca cgtggaattc cactctcctc ttctgcactc aagttcccca 840
gtttccaatg accctccccg gttgagccgg gggctttcac atcagactta agaaaccgcc 900
tgcgcgcgct ttacgcccaa taattcccgg acaacgcttg ccacctacgt attaccgcgg 960
ctgctggcac gtagttagcc gtggctttct ggttaggtac cgtcaaggta ccgccctatt 1020
cgaacggtac ttgtttttcc ctaacaacag agttttacga tccgaaaacc ttcatcaatc 1080
acgggggggt ggtccgtcag acttttgtcc cttggggaag attccctaat ggtgccttcc 1140
gtaggagttt gggccgtgtt tcagttcccg ggtggccgat caccctttca ggttgggtac 1200
gcattgttgc cttggtgagc cgttacctca ccaaataggt aatgcgccgc gggtccattt 1260
gtaagtggta ggtaaaagcc accttttata attgaaccat gcggttcaat caagcatccg 1320
gtattagccc cggtttcccg gagttatccc agtcttacag gcaggttacc cacgtgttac 1380
tcacccgtcc gccgctaaca tcagggagca agctcccatc tgtccgctcg actgcatgta 1440
tagcacgccg caatctccg 1459
Claims (7)
1. The bacillus licheniformis for preventing and treating animal diarrhea is characterized in that: the preservation number of the strain preservation is CGMCC NO. 20892.
2. The bacillus licheniformis for controlling diarrhea in animals according to claim 1, characterized in that: the liquid culture medium formula of the bacillus licheniformis comprises the following components in percentage by weight: 10 per mill sodium chloride, 10 per mill tryptone and 5 per mill yeast powder, and the culture conditions are as follows: ventilating and fermenting at 37 deg.C and pH 7.4 to obtain Bacillus licheniformis liquid culture; the formula weight fraction of the solid culture medium is as follows: 90% of bran, 5% of peptone and 5% of yeast powder, wherein the water content is 60%; the culture conditions were: ventilating and fermenting at 37 ℃ to obtain the bacillus licheniformis solid culture.
3. A probiotic for the control of diarrhea in animals, said formulation comprising an extract of a culture of bacillus licheniformis according to claim 1 or 2.
4. The microecological formulation according to claim 3, wherein the probiotic comprises: the preparation method of the extract comprises the following steps: after culturing the solid culture of Bacillus licheniformis at 32 deg.C for 5 days, leaching with warm water at 30 deg.C for 24 hr, adsorbing with carrier, and air drying at low temperature.
5. The microecological formulation according to claim 4, wherein the ratio of: the carrier comprises one or more of wheat bran, corncob powder, yeast cell wall, corn starch, rice bran, glucose, zeolite powder, bentonite and montmorillonite.
6. The microecological formulation according to claim 3, wherein the probiotic comprises: the content of the extract in the microecological preparation is 20-60% by weight.
7. The microecological formulation according to claim 3, wherein the probiotic comprises: the mass ratio of the bacillus licheniformis fermentation extract to the carrier is as follows: 20: 80 or 30: 70 or 40: 60 or 50: 60 or 60: 40.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2019027073A1 (en) * | 2017-08-04 | 2019-02-07 | 씨제이제일제당(주) | Feedstuff additive containing bacillus subtilis and bacillus licheniformis, feedstuff composition containing same additive, and preparation method for same feedstuff additive |
| CN110055186A (en) * | 2019-02-20 | 2019-07-26 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | It is a kind of degrade vomitoxin microorganism formulation and application |
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- 2021-05-19 CN CN202110547331.7A patent/CN113151110B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2019027073A1 (en) * | 2017-08-04 | 2019-02-07 | 씨제이제일제당(주) | Feedstuff additive containing bacillus subtilis and bacillus licheniformis, feedstuff composition containing same additive, and preparation method for same feedstuff additive |
| CN110055186A (en) * | 2019-02-20 | 2019-07-26 | 山东省花生研究所（山东省农业科学院花生工程技术研究中心） | It is a kind of degrade vomitoxin microorganism formulation and application |
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