CN110951640A - Probiotic clostridium butyricum and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of microorganism application, and relates to probiotic clostridium butyricum and application thereof. The clostridium butyricum disclosed by the invention is separated from fresh chicken manure in an autotrophic chicken farm, and has a remarkable inhibiting effect on pathogenic escherichia coli and clostridium perfringens of bred animals. After the clostridium butyricum is subjected to anaerobic fermentation culture, a liquid microbial inoculum product can be prepared for animals to drink to play a probiotic role. The zymophyte liquid can be further dried and processed to prepare viable bacteria powder which is used as a solid microbial inoculum to be added into livestock and poultry feed for feeding animals. The strain is applied to livestock and poultry breeding, effectively improves the health condition of bred animals, improves the production performance, reduces the incidence rate of enteritis and diarrhea of livestock and poultry, reduces the use of antibiotics, and provides support for green breeding of livestock and poultry industry.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and relates to probiotic clostridium butyricum and application thereof.
Background
The microecological preparation as a novel antibiotic substitute has the advantages of no toxic or side effect, no drug resistance, no residue and the like, and is affirmed by the breeding industry. Aiming at building environment-friendly and ecological healthy livestock and poultry breeding, the clostridium butyricum with excellent performance is found through research and applied to livestock and poultry breeding, so that the growth performance of livestock and poultry is improved, the diarrhea rate is reduced, the use of antibiotics is reduced, the sustainable development of livestock and poultry breeding is promoted, and the coordinated development of livestock and poultry breeding and ecological environment is finally realized. In the process of breeding livestock and poultry, diarrhea diseases have high mortality, acute diarrhea of livestock and poultry is difficult to prevent by adopting a conventional method, and after the acute diarrhea occurs, conventional drug treatment often has hysteresis, so that the mortality of livestock and poultry is difficult to reduce.
The clostridium butyricum is a probiotic with strict anaerobic growth, can form clostridia on one hand, has excellent biological characteristics of stress resistance, high temperature resistance and the like, and can metabolize to generate various metabolites such as acetic acid, butyric acid and the like on the other hand, promote the proliferation of lactic acid bacteria and bifidobacteria and effectively inhibit the growth of intestinal pathogenic bacteria.
As a new microecological preparation, the clostridium butyricum has the advantages of acid production and spore production, and has obvious effect of preventing common diarrhea in livestock and poultry breeding. Good processing performance and outstanding feeding function, so that the clostridium butyricum gradually becomes an important member in the feed microecological preparation industry. The clostridium butyricum is applied to the large-scale livestock and poultry breeding, and has important effects of realizing green breeding and reducing antibiotic addition.
Disclosure of Invention
The invention provides a novel probiotic clostridium butyricum and application thereof, aiming at the problem of diarrhea in the traditional livestock and poultry breeding process.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
clostridium butyricum is separated from chicken manure, and the strain is named Clostridium butyricum (Clostridium butyricum) WL-53 which is preserved in China center for type culture Collection in 12 months 4 and 2019 with the preservation number of CCTCCNO: M2019252.
The preparation method of the strain comprises the following steps: 10 parts of fresh chicken manure in a chicken farm are collected, 25g of samples are weighed respectively, 225mL of sterile water is added to prepare suspension, the suspension is coated on an anaerobic bacteria agar plate, and anaerobic culture is carried out for 72 hours at 37 ℃. Selecting single colony to observe the thallus form one by one, screening out thallus which is detected as fusiform by microscopic examination, further marking and purifying for 3 times to obtain pure bacterial strains.
The preparation method of the strain seed liquid comprises the following steps: a single colony of Clostridium butyricum WL-53 is selected and inoculated in 50mL of fresh broth culture medium, the components of the single colony are 10g/L glucose, 10g/L peptone, 5g/L sodium chloride and 1g/L sodium thioglycollate, and the single colony is subjected to anaerobic culture at 37 ℃ for 48h to obtain seed liquid.
And (3) amplification culture: transferring 10% of the inoculum size into fresh broth culture medium, anaerobically culturing at 37 deg.C, stopping culturing when spore formation reaches above 90%, and making into liquid microbial inoculum.
The preparation method of the fungus powder comprises the following steps: firstly, sub-packaging anaerobically cultured clostridium butyricum WL-53 bacterial liquid into a sample bottle, refrigerating at 4 ℃, and keeping the bacterial liquid fresh; then, setting the centrifugation condition to be 4000rpm for 10min, centrifuging the bacterial liquid, removing the supernatant, and collecting the Clostridium butyricum WL-53 thallus for later use; and finally, selecting corn starch as a carrier, uniformly stirring and mixing the corn starch and the collected clostridium butyricum WL-53 thalli according to the ratio of 1:1, drying for 24 hours in a drying oven at 60 ℃, and crushing to obtain clostridium butyricum WL-53 bacterial powder.
The clostridium butyricum is applied to feed additives.
A microbial preparation comprising the above Clostridium butyricum.
The biological agent is liquid preparation or powder of Clostridium butyricum.
The microbial preparation is a liquid microbial inoculum comprising clostridium butyricum WL-53, and is added into the drinking of livestock and poultry according to the volume percentage of 0.001-1%.
The microbial preparation is bacterial powder comprising clostridium butyricum WL-53, and is added into the livestock and poultry feed according to the mass percentage of 0.01-1%.
The microecological preparation is applied to livestock and poultry breeding, so that the production performances such as daily gain, feed-weight ratio and the like are effectively improved, the diarrhea rate of livestock and poultry is reduced, and the economic benefit of breeding is improved.
Compared with the prior art, the invention has the advantages and positive effects that:
the clostridium butyricum disclosed by the invention is separated from fresh chicken manure in an autotrophic chicken farm, and has a remarkable inhibiting effect on pathogenic escherichia coli and clostridium perfringens of bred animals. After the clostridium butyricum is subjected to anaerobic fermentation culture, a liquid microbial inoculum product can be prepared for animals to drink to play a probiotic role. The zymophyte liquid can be further dried and processed to prepare viable bacteria powder which is used as a solid microbial inoculum to be added into livestock and poultry feed for feeding animals. The strain is applied to livestock and poultry breeding, effectively improves the health condition of bred animals, improves the production performance, reduces the incidence rate of enteritis and diarrhea of livestock and poultry, reduces the use of antibiotics, and provides support for green breeding of livestock and poultry industry.
Drawings
FIG. 1 is a diagram of the spore morphology of Clostridium butyricum WL-53.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
Example 1 isolation and identification of clostridium butyricum WL-53:
1. clostridium butyricum separation and purification
10 parts of fresh chicken manure in a chicken farm are collected, 25g of samples are weighed respectively, 225mL of sterile water is added to prepare suspension, the suspension is coated on an anaerobic bacteria agar plate, and anaerobic culture is carried out for 72 hours at 37 ℃. Selecting single colony to observe the thallus form one by one, screening out thallus which is detected as fusiform by microscopic examination, further marking and purifying for 3 times to obtain pure bacterial strains.
2. Identification of Clostridium butyricum
1) Physiological and biochemical characteristics:
culturing on anaerobic agar medium for 72 hr to obtain gray, opaque, smooth surface and colony of 1-3mm diameter; the spore is in a spindle shape and is positioned in the middle or at the end part; V-P assay (-), gelatin hydrolysis (-), indole assay (-), hydrolyzed starch (+), hydrolyzed casein (-).
2)16SrRNA gene assay:
extracting the genome DNA of the strain, amplifying a 16SrRNA sequence by utilizing a PCR technology, and sequencing to obtain a 1428bp fragment. Through BLAST comparison analysis, the similarity of the 16S rRNA sequence of the clostridium butyricum strain to the published 16S rRNA sequence of the clostridium butyricum strain is 99.99 percent, and the similarity of the 16S rRNA sequence of other strains is lower than 99 percent. The identification proves that the strain is clostridium butyricum, and the result is consistent with the biochemical identification result.
The strain is named Clostridium butyricum (Clostridium butyricum) WL-53, which is preserved in China center for type culture Collection in 2019 at 12.4.4.4.M, and the preservation number is CCTCC NO: M2019252.
Example 2: preparation of Clostridium butyricum WL-53 bacterial liquid
1. Preparing a seed solution: selecting a single colony of clostridium butyricum WL-53, inoculating the single colony in 50mL of fresh broth culture medium, wherein the components are 10g/L of glucose, 10g/L of peptone, 5g/L of sodium chloride and 1g/L of sodium thioglycolate, and carrying out anaerobic culture at 37 ℃ for 48h to obtain seed liquid;
2. and (3) amplification culture: transferring 10% of the inoculum size into fresh broth culture medium, anaerobically culturing at 37 deg.C, stopping culturing when spore formation reaches above 90%, and making into liquid microbial inoculum.
Example 3: preparation of clostridium butyricum WL-53 bacterial powder
1. Collecting bacterial liquid: sub-packaging the clostridium butyricum WL-53 bacterial liquid cultured in the anaerobic way in the embodiment 2 into a sample bottle, refrigerating at 4 ℃, and keeping the bacterial liquid fresh;
2. and (3) centrifuging thalli: setting the centrifugation condition to be 4000rpm for 10min, centrifuging the bacterial liquid, removing supernatant, and collecting the Clostridium butyricum WL-53 thallus for later use;
3. and (3) drying: according to the embodiment, corn starch is selected as a carrier, the corn starch and the collected Clostridium butyricum WL-53 thalli are uniformly stirred according to the proportion of 1:1, the mixture is dried for 24 hours in a drying oven at the temperature of 60 ℃, and the Clostridium butyricum WL-53 bacterial powder is obtained after crushing.
4. And (3) measuring the bacterial powder quantity: weighing 1g of clostridium butyricum WL-53 bacterial powder, adding 9mL of sterile water, oscillating and mixing uniformly, gradually diluting with 10 times of gradient of sterile water, counting the bacterial quantity by adopting an anaerobic agar pouring method, and counting the bacterial quantity of the bacterial powder to be 100 hundred million CFU/g.
The stability and heat resistance of the fungal powder obtained in this example were investigated, and the results are shown in the following table.
TABLE 1 Clostridium butyricum WL-53 bacterial powder storage stability test (25 ℃ C.)
| Storage time (moon) | Viable bacteria amount detection (CFU/g) | Survival rate of bacterial strain |
| 0 | 1.0×1010 | 100％ |
| 3 | 1.0×1010 | 100％ |
| 6 | 9.9×109 | 99％ |
| 9 | 9.6×109 | 96％ |
| 12 | 9.6×109 | 96％ |
| 15 | 9.4×109 | 94％ |
| 18 | 9.3×109 | 93％ |
| 24 | 9.0×109 | 90％ |
The clostridium butyricum prepared in the embodiment is put into an oven, and is respectively treated according to the test temperature and the drying time in the table 2, each test condition is repeated for 3 times, and the test data is counted. And inspecting the survival rate of the clostridium butyricum at different temperatures and drying times, and further obtaining heat resistance data of the clostridium butyricum WL-53. The viable bacteria determination adopts a dilution inverted plate method. The results are shown in Table 2.
TABLE 2 Heat resistance test of Clostridium butyricum WL-53 bacterial powder
As can be seen from the test results in tables 1 and 2, the Clostridium butyricum WL-53 prepared in this example has very good stability and heat resistance.
Example 4: clostridium butyricum WL-53 ability to antagonize intestinal pathogens
Clostridium butyricum WL-53 belongs to strict anaerobe, and is metabolized to generate a plurality of organic acids including acetic acid, butyric acid and the like in the growth process of the clostridium butyricum, so that the growth and the propagation of intestinal pathogenic microorganisms can be effectively inhibited.
1. Preparation of indicator plates: the pathogenic bacteria used in this example were E.coli (serotype O1) isolated from the intestinal tract of the sick chicken and a standard strain of Clostridium perfringens ATCC13124 purchased, cultured in nutrient broth to logarithmic phase, added to nutrient agar in a ratio of 0.1%, poured into 90mm petri dishes, coagulated and then used
The puncher punches holes on the culture medium;
2. preparation of WL-53 supernatant: subpackaging Clostridium butyricum WL-53 bacterial solution cultured at 37 ℃ in a sterile centrifuge tube, setting the centrifugation condition to be 4000rpm for 10min, and collecting supernatant after centrifugation is finished;
3. bacteriostasis: adding 20 mu L of the supernatant obtained in the step 2 into a culture medium hole, placing the culture medium hole in an incubator at 37 ℃, and culturing overnight;
4. measuring the bacteriostatic zone: the results are shown in Table 3, and the larger the diameter of the inhibition zone is, the more remarkable the inhibition effect of Clostridium butyricum WL-53 on intestinal pathogenic bacteria is.
TABLE 3 inhibition of pathogenic bacteria by Clostridium butyricum
Example 5: influence of clostridium butyricum WL-53 on broiler chicken production performance
Purchasing 1000 healthy white feather broilers of 1 day age, randomly dividing the broilers into two groups, namely a control group and an experimental group, repeating 10 treatment groups for each treatment group, repeating 50 chickens for each treatment group, wherein the production test period is 35 days, the control group is only fed with common commodity daily ration and tap water drinking water, the experimental group is fed with common commodity daily ration and 0.005% clostridium butyricum WL-53 liquid microbial inoculum drinking water, measuring the average daily gain, the feed-weight ratio and the diarrhea rate of the broilers of each group in the test process, collecting fresh chicken manure of each group on the 28 th day, inspecting the change conditions of lactic acid bacteria, coliform bacteria and clostridium perfringens in the manure, and the experimental result is shown in a table 4:
TABLE 4 statistical analysis of broiler productivity
Through statistical analysis, as can be seen from table 4, the differences of the broiler feed weight ratios of the experimental group and the control group are not significant, and the differences of the average daily gain and diarrhea rate of the experimental group and the control group are significant (P is less than 0.05) in the whole period. In the test indexes, the average daily gain and diarrhea rate of the experimental group are obviously superior to those of the control group. By adding 0.005% of clostridium butyricum WL-53 into drinking water in broiler breeding, the average daily gain of broilers is remarkably improved, the diarrhea rate is reduced, loss of farmers is reduced, and economic benefit is increased.
Table 528 day old broiler manure flora detection condition
In the feeding experiment, the intestinal flora of 28-day-old broiler chickens is nearly stable, so that fresh excrement on the 28 th day is selected for flora detection and analysis, and the experimental analysis of the table 5 shows that 1) the bacterial quantity of the probiotic lactobacillus experimental group is remarkably higher than that of a control group (P is less than 0.05), 2) the bacterial quantities of the intestinal pathogenic bacteria coliform and clostridium perfringens are far lower than that of the control group (P is less than 0.05), which indicates that in the feeding process, clostridium butyricum WL-53 is added into drinking water of the experimental group, so that the probiotic proliferation of the intestinal tracts of the broiler chickens is remarkably promoted, and the intestinal pathogenic bacteria are remarkably inhibited and are better than that of the control group. Clostridium butyricum WL-53 is added in the feeding process of the broiler chickens, so that the intestinal flora structure of the broiler chickens is improved.
Example 6: influence of clostridium butyricum WL-53 on weaned pig breeding
Selecting 180 25-day old white binary weaned piglets, randomly dividing into 3 treatment groups, wherein each treatment group has 6 repetitions, 10 pigs are repeated, and each treatment group has half of a male and a female, wherein a control group is used for feeding common piglet daily ration, a treatment group 1 is used for feeding common piglet daily ration added with 0.01% clostridium butyricum WL-53 bacterial powder, a treatment group 2 is used for feeding common piglet daily ration added with 0.05% clostridium butyricum WL-53 bacterial powder, the growth performance and diarrhea condition of the weaned piglets within four weeks after 25 days are inspected, and the experimental results are shown in table 6:
TABLE 6 statistical analysis of piglet growth performance
The average daily gain, the feed-weight ratio and the diarrhea rate of the weaned piglets were determined by the experiment, and the statistical analysis results in table 6 show that the differences of the treatment groups 1 and 2 in the average daily gain, the feed-weight ratio and the diarrhea rate are significant (P is less than 0.05) compared with the control group. Treatment groups 1 and 2 were compared and the differences were not significant. The results show that the growth performance of the weaned piglets is remarkably improved after the treatment groups 1 and 2 are added with clostridium butyricum WL-53. Therefore, from comprehensive evaluation of feeding cost and growth performance, the treatment group 1 adds 0.01% of clostridium butyricum WL-53 bacterial powder in the common piglet diet, so that the requirement of weaned piglet cultivation can be met, and the effects of improving the growth performance of the weaned piglets and reducing the diarrhea rate are achieved.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
SEQUENCE LISTING
<110> Shandong blue Biotech Co., Ltd
<120> probiotic clostridium butyricum and application thereof
<130>1
<160>1
<170>PatentIn version 3.5
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<211>1428
<212>DNA
<213> Artificial sequence
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tgagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgagc 60
gatgaagctc cttcgggagt ggattagcgg cggacgggtg agtaacacgt gggtaacctg 120
cctcatagag gggaatagcc tttcgaaagg aagattaata ccgcataaga ttgtagtacc 180
gcatggtaca gcaattaaag gagtaatccg ctatgagatg gacccgcgtc gcattagcta 240
gttggtgagg taacggctca ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg 300
ccacattggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 360
acaatggggg aaaccctgat gcagcaacgc cgcgtgagtg atgacggtct tcggattgta 420
aagctctgtc tttagggacg ataatgacgg tacctaagga ggaagccacg gctaactacg 480
tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggatttact gggcgtaaag 540
ggagcgtagg tggatattta agtgggatgt gaaatacccg ggcttaacct gggtgctgca 600
ttccaaactg gatatctaga gtgcaggaga ggaaaggaga attcctagtg tagcggtgaa 660
atgcgtagag attaggaaga ataccagtgg cgaaggcgcc tttctggact gtaactgaca 720
ctgaggctcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 780
acgatgaata ctaggtgtag gggttgtcat gacctctgtg ccgccgctaa cgcattaagt 840
attccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca 900
caagcagcgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc tagacttgac 960
atctcctgaa ttactctgta atggaggaag ccacttcggt ggcaggaaga caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttattgttag ttgctaccat ttagttgagc actctagcga gactgcccgg gttaaccggg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg tctagggcta cacacgtgct 1200
acaatggtcg gtacaatgag atgcaacctc gcgagagtga gcaaaactat aaaaccgatc 1260
tcagttcgga ttgtaggctg aaactcgcct acatgaagct ggagttgcta gtaatcgcga 1320
atcagaatgt cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga 1380
gagttggcaa tacccaaagt tcgtgagcta accgcaagga ggcagcga 1428
Claims (6)
1. The clostridium butyricum is characterized in that the clostridium butyricum is named as clostridium butyricum（Clostridium butyricum）WL-53 is separated from chicken manure, the preservation number is CCTCC NO: M2019252, the preservation date is 12 months 04 in 2019, and the preservation place is the China center for type culture Collection.
2. Use of clostridium butyricum according to claim 1 in a feed additive.
3. A microbial preparation comprising the clostridium butyricum of claim 1.
4. The biological agent according to claim 3, wherein the biological agent is a liquid preparation or a powder of Clostridium butyricum according to claim 1.
5. The use of the biological agent of claim 4 for reducing the feed weight ratio and diarrhea rate of livestock and poultry, characterized in that the application method is that the liquid preparation of clostridium butyricum is added into the drinking water of livestock and poultry according to the proportion of 0.001-1 percent by volume.
6. The biological agent of claim 4 is applied to reduce the feed weight ratio and diarrhea rate of livestock and poultry, and is characterized in that the application method is that the clostridium butyricum bacterial powder is added into the livestock and poultry feed according to the proportion of 0.01-1% by mass.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111500521A (en) * | 2020-06-11 | 2020-08-07 | 扬州大学 | Method for preparing clostridium butyricum preparation rich in selenium by using inorganic selenium and application of clostridium butyricum preparation |
| CN112655827A (en) * | 2020-12-21 | 2021-04-16 | 广东溢多利生物科技股份有限公司 | Clostridium butyricum preparation special for ruminants and preparation method and application thereof |
| CN112843257A (en) * | 2021-01-25 | 2021-05-28 | 广东省农业科学院农业生物基因研究中心 | Test method for preventing salmonella pullorum by using high-activity probiotics and application |
| CN113151113A (en) * | 2021-05-26 | 2021-07-23 | 山东农业大学 | Clostridium butyricum with strong bacteriostatic ability and application thereof |
| CN113999787A (en) * | 2021-08-30 | 2022-02-01 | 西安海吉焦生物科技研究院有限公司 | Clostridium butyricum, microbial inoculum, preparation method and application of microbial inoculum and feed |
| CN116286480A (en) * | 2023-02-07 | 2023-06-23 | 湖南湘农动物药业有限公司 | Screening and separating method for clostridium butyricum and application thereof |
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| CN104611273A (en) * | 2015-02-11 | 2015-05-13 | 成都与康科技有限公司 | Novel strain Clostridium butyricum UCN-12 and composition and application thereof |
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| CN111500521A (en) * | 2020-06-11 | 2020-08-07 | 扬州大学 | Method for preparing clostridium butyricum preparation rich in selenium by using inorganic selenium and application of clostridium butyricum preparation |
| CN112655827A (en) * | 2020-12-21 | 2021-04-16 | 广东溢多利生物科技股份有限公司 | Clostridium butyricum preparation special for ruminants and preparation method and application thereof |
| CN112843257A (en) * | 2021-01-25 | 2021-05-28 | 广东省农业科学院农业生物基因研究中心 | Test method for preventing salmonella pullorum by using high-activity probiotics and application |
| CN113151113A (en) * | 2021-05-26 | 2021-07-23 | 山东农业大学 | Clostridium butyricum with strong bacteriostatic ability and application thereof |
| CN113151113B (en) * | 2021-05-26 | 2022-07-22 | 山东农业大学 | Clostridium butyricum with strong bacteriostatic ability and application thereof |
| CN113999787A (en) * | 2021-08-30 | 2022-02-01 | 西安海吉焦生物科技研究院有限公司 | Clostridium butyricum, microbial inoculum, preparation method and application of microbial inoculum and feed |
| CN116286480A (en) * | 2023-02-07 | 2023-06-23 | 湖南湘农动物药业有限公司 | Screening and separating method for clostridium butyricum and application thereof |
| CN116536205A (en) * | 2023-05-09 | 2023-08-04 | 轩凯生物科技(滁州)有限公司 | Clostridium butyricum composite microbial inoculum and application thereof |
| CN116536205B (en) * | 2023-05-09 | 2024-06-07 | 轩凯生物科技(滁州)有限公司 | Clostridium butyricum composite microbial inoculum and application thereof |
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