CN116103198B - Lactobacillus reuteri MC1 as chicken source, screening method and application thereof - Google Patents
Lactobacillus reuteri MC1 as chicken source, screening method and application thereof Download PDFInfo
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- CN116103198B CN116103198B CN202211706709.4A CN202211706709A CN116103198B CN 116103198 B CN116103198 B CN 116103198B CN 202211706709 A CN202211706709 A CN 202211706709A CN 116103198 B CN116103198 B CN 116103198B
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- lactobacillus reuteri
- chicken
- chickens
- intestinal
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Classifications
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses chicken source lactobacillus reuteri MC1, a screening method and application thereof, wherein the strain is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of North Star Xili No. 1, 3 in the Korean region of Beijing, a preservation date of 2022, 11 months and 02 days, a preservation number of CGMCC No.26013 and a Latin name of Lactobacillus reuteri; the strain is obtained by separating and screening adult healthy chicken cecum. The lactobacillus reuteri MC1 provided by the invention has a certain tolerance to gastric juice, intestinal juice and bile salt, can be adhered to intestinal epithelial cells, remodels intestinal flora, and the secreted metabolite inhibits adhesion and colonization of intestinal harmful bacteria, so that the immune organ index of broiler chickens can be improved, and the immune organ index of the broiler chickens can be improved, and is used for improving enteritis and diarrhea of the chickens and improving immunity of the chickens.
Description
Technical Field
The invention belongs to the field of probiotics for livestock, relates to poultry home bacteria, in particular to chicken-origin lactobacillus reuteri MC1, a screening method and application thereof.
Background
Lactobacillus reuteri has strong adhesion capability to intestinal mucosa, can improve intestinal flora distribution, antagonize harmful bacteria colonization, and can inhibit growth of harmful bacteria, and can effectively improve body resistance.
Clostridium welchii is also known as clostridium perfringens, and the disease is an acute non-contact bacterial infectious disease. The bacterium is not pathogenic under the condition of normal organism resistance; however, when the body resistance is reduced or the causative factor exists, the disease can rapidly become a cause of the disease, resulting in the onset of the animal. Adult healthy chickens which are mainly harmful to 14-40 days old are characterized by necrosis of small intestinal mucosa, and the sick chickens have poor appetite, increased appetite, emaciation and dehydration, and die within 1-2 weeks.
At present, antibiotics are mostly adopted for treating enteritis, and penicillin and streptomycin can be used for mixing or gentamicin intramuscular injection; when the diseases of the chickens are more, oxytetracycline and metronidazole can be added into daily ration or amoxicillin and clavulanate A and metronidazole can be added into drinking water, but the use of antibiotics is limited because the antibiotics medicine is used for a long time, so that the drug resistance of bacteria is enhanced and remains, thereby affecting the food safety and the like. The probiotic preparation can effectively improve the balance of animal intestinal flora, inhibit the colonization and growth of clostridium perfringens in intestinal tracts, improve the immune function of organisms and solve the problems of enteritis and diarrhea of chicken flocks.
Disclosure of Invention
The invention aims to provide chicken lactobacillus reuteri MC1 which is used as a probiotic for poultry, and can improve enteritis of chickens and improve immunity of the chickens.
Another object of the present invention is to provide a method for screening Lactobacillus reuteri MC1 of chicken origin.
It is also an object of the present invention to provide the use of the above Lactobacillus reuteri MC1 of chicken origin.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The chicken source lactobacillus reuteri MC1 is preserved in China general microbiological culture Collection center (China Committee for culture Collection) with a preservation address of North Star Xiyu No. 1, no. 3, a preservation date of 2022, 11 months and 02 days, a preservation number of CGMCC No.26013 and a Latin name of Lactobacillus reuteri.
As a limitation, the 16SrRNA gene sequence of the lactobacillus reuteri MC1 of chicken origin is as follows:
TGCTCAGGATGAACGCCGGCGGTGTGCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCATGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCC.
the invention also provides a screening method of the chicken lactobacillus reuteri MC1, which comprises the following steps in sequence:
S1, taking the content of cecum of adult healthy chickens under the aseptic condition;
S2, inoculating the content into a solid agar culture medium, and culturing for 24-72 hours under anaerobic conditions at 30-40 ℃;
S3, selecting single colony on the solid agar culture medium, inoculating the single colony to the MRS agar culture medium, and placing the single colony in a bacterial incubator at 30-40 ℃ for static culture for 24-72 hours to obtain a pure culture;
S4, inoculating single bacterial colony of the pure culture into a liquid culture medium, and carrying out shake culture for 24-72 hours at the temperature of 30-40 ℃ after the inoculated liquid culture to obtain the chicken source lactobacillus reuteri MC1.
As one limitation, in the step S2, the solid medium is trypticase soy agar medium, MRS agar medium, ferric sulfite agar medium, LB liquid medium or MRS broth medium containing 1-3% calf serum.
As another limitation, in the step S2, the anaerobic condition is defined by a volume ratio of 10:5: 85H 2、CO2 and N 2.
As a third limitation, in the step S4, the liquid medium is MRS broth.
The invention also provides application of the chicken lactobacillus reuteri MC1 in preparation of a preparation for improving chicken enteritis.
The invention also provides application of the chicken lactobacillus reuteri MC1 in preparation of a preparation for improving chicken immunity.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
The chicken lactobacillus reuteri MC1 is a home bacterium, has strong adaptability, can be adhered to intestinal epithelial cells, improves intestinal flora, secretes antibacterial substances such as reuterin, and the like, can inhibit harmful bacteria, secretes other metabolism-stimulated organism to produce mucosa-related immune factors, improves organism immunity, and can improve enteritis and diarrhea effects of chicken flocks.
The method is suitable for separating and screening chicken lactobacillus reuteri MC1, and the screened chicken lactobacillus reuteri MC1 is suitable for improving enteritis and diarrhea of chicken flocks and improving chicken immunity.
Drawings
FIG. 1 is an electrophoresis pattern of the 16S rRNA sequence of Lactobacillus reuteri MC1 of chicken origin in example 1 of the present invention, wherein the electrophoresis conditions were 3. Mu.L sample+1.0% agarose gel, marker band composition: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp,750bp band concentration is 60ng/3 mu L, the other band concentrations are 30ng/3 mu L, and the electrophoresis direction is from top to bottom;
FIG. 2 is a colony morphology diagram of Lactobacillus reuteri MC1 of chicken origin in MRS medium in example 2 of the present invention;
FIG. 3 is a gram stain form (1000X) of Lactobacillus reuteri MC1 of chicken origin in example 7 of the present invention;
FIG. 4 is a plate diagram of the zone of inhibition of Clostridium perfringens by Lactobacillus reuteri MC1 of chicken origin in example 8 of the present invention.
Detailed Description
The invention will now be described in further detail by way of specific examples, which are to be understood as illustrative only and not limiting.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following examples: tryptone soy agar medium (TSA medium), MRS agar medium, MRS broth medium, brain heart infusion medium (BHI), nutrient agar medium (NA), ferric sulfite agar medium, available from beijing obbo biotechnology limited;
tryptone, yeast powder was purchased from OXOI D company;
sodium chloride, available from Tianjin Fengshou chemical reagent technology Co., ltd;
biochemical test tube, gram staining solution, drug sensitive paper purchased from Hangzhou beach and biological reagent limited company;
Calf serum, purchased from Thermo company.
Example 1 Lactobacillus reuteri MC1 as chicken origin
1. Strain information
The chicken source lactobacillus reuteri MC1 is separated and screened from the cecum content of adult healthy chickens, and the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year 2022, wherein the preservation address is the number 3 of the West-Lou 1, the Korean region North Star in Beijing city, the preservation number is CGMCCNO.26013, and the Latin name is Lactobacillus reuteri.
2. Molecular biological identification of Lactobacillus reuteri MC1
Taking chicken source lactobacillus reuteri MC1 to carry out molecular biological identification, and finally determining the chicken source lactobacillus reuteri through DNA extraction, PCR amplification and 16SrDNA gene sequencing at NCBI website b1 ast;
An electrophoretogram of the 16S rRNA sequence of Lactobacillus reuteri MC1 of chicken origin is shown in FIG. 1; the electrophoresis conditions were 3. Mu.L sample+1.0% agarose gel, marker band composition: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp,750bp band concentration is 60ng/3 mu L, the other band concentrations are 30ng/3 mu L, and the electrophoresis direction is from top to bottom; the 16SrRNA sequence is as follows:
TGCTCAGGATGAACGCCGGCGGTGTGCTAATACATGCAAGTCGTACGCACTGGCCCAACTGATT
GATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTA
ACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACAT
GGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGT
AAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTG
AGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGG
AGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTG
AGAGTAACTGTTCATGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGC
GGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAG
GTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAG
AGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGC
GGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGG
TAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACG
CATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCA
CAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCG
CTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTC
GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGT
TGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCC
CCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGAGTAAG
CTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGC
TAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC
ATGGGAGTTTGTAACGCCC。
example 2 isolation and purification method of Lactobacillus reuteri MC1 as chicken source
This example is a screening method of lactobacillus reuteri MC1 of chicken origin according to example 1, comprising the following steps in order:
S1, taking the content in the cecum of an adult healthy chicken under the aseptic condition, and placing the content in an aseptic anaerobic incubator for standby;
s2, inoculating the content into a trypticase soy agar medium (TSA medium) containing 1.5% calf serum, and standing and culturing in an anaerobic incubator at 37 ℃ for 48 hours;
The anaerobic environment in the anaerobic incubator is composed of the following components in volume ratio of 10:5: 85H 2、CO2 and N 2;
s3, selecting single colony on the TSA agar culture medium, inoculating the single colony to the MRS agar culture medium, and standing and culturing the single colony in a bacterial incubator at 37 ℃ for 48 hours to obtain a pure culture, wherein the pure culture is shown in FIG. 2;
S4, inoculating single bacterial colony of the pure culture into an MRS broth culture medium, performing shaking culture for 48 hours at 37 ℃ after the inoculated MRS broth culture, and obtaining the chicken source lactobacillus reuteri MC1 and preserving.
EXAMPLES 3-6 screening method of chicken-origin Lactobacillus reuteri MC1
Examples 3 to 6 are screening methods of Lactobacillus reuteri MC1 of chicken origin, which are the same as example 2 except that the partial condition parameters in the screening process are different, and the specific details are shown in Table 1:
table 1 screening of a list of different Condition parameters of Lactobacillus reuteri MC1 of chicken origin
Example 7 bacteriological characterization of Lactobacillus reuteri MC1 of chicken origin
1. Basic features
This example is a basic bacteriological feature of lactobacillus reuteri MC1 of chicken origin, and gram staining of lactobacillus reuteri MC1 of chicken origin is performed, as a result of which see fig. 3 (1000×);
As can be seen from fig. 1, lactobacillus reuteri MC1 of chicken origin is a gram positive bacterium; the basic characteristics of lactobacillus reuteri MC1 of chicken origin are shown in table 2 below:
TABLE 2 essential characteristics of Lactobacillus reuteri MC1 of chicken origin
| Experimental items | Gram staining | PH4.0 growth | Cell morphology | Sucrose | Trehalose | Xylose |
| Experimental results | Positive and negative | + | Curvularia with rounded ends | + | - | - |
2. Growth characteristics
The chicken lactobacillus reuteri MC1 is a facultative anaerobe, has good acid resistance, high growth rate and fast acid production rate, and has an optimal growth temperature of 36-40 ℃, and different strains can have easy dyeing property and no acid resistance.
The chicken lactobacillus reuteri MC1 in example 1 is inoculated into glucose, lactose, sucrose, fructose, maltose, ribose, salicin, sorbitol, catalase, xylose, mannitol and motile biochemical test tubes respectively, and the test tubes are placed in a bacterial incubator at 37 ℃ for culturing for 48 hours, so that biochemical experiment results are observed and recorded.
Individual colonies were mixed with 20 μl of 3.0% hydrogen peroxide (contact enzyme biochemical test reagent) and observed for the generation of bubbles (positive for bubble generation), and biochemical characteristics were as shown in table 3 below:
TABLE 3 Biochemical Properties of Lactobacillus reuteri MC1 from chicken source
| Test item | Results | Test item | Results |
| Glucose | + | Salicin | - |
| Lactose and lactose | + | Sorbitol | - |
| Sucrose | + | Catalase enzyme | - |
| Fructose | + | Xylose | - |
| Maltose | + | Mannitol (mannitol) | - |
| Ribose | + | Motility of | - |
Note that: +: positive is indicated; -: negative is indicated
It can be seen from Table 3 that the chicken source Lactobacillus reuteri MC1 utilizes most of the saccharides, does not produce catalase, and has no motility (growth along the inoculation line in semi-solid biochemical tubes, i.e.no motility).
Example 8 inhibition of clostridium perfringens by lactobacillus reuteri MC1 of chicken origin
The example is an experiment of the inhibition of clostridium perfringens by lactobacillus reuteri MC1 of chicken origin, and the specific method is as follows:
S1, streaking and inoculating the chicken source lactobacillus reuteri MC1 in the embodiment 2 to an MRS culture medium, placing the inoculated culture medium into a bacterial incubator at 37 ℃ for culturing for 24-48 hours, picking single bacterial colony of the chicken source lactobacillus reuteri MC1, inoculating the single bacterial colony to the MRS culture medium, and performing shake culture in a shaking incubator at 37 ℃ for 24 hours;
S2, taking clostridium perfringens strains stored at the temperature of minus 75 ℃, streaking and inoculating to a blood agar flat plate culture medium, culturing for 24-48 hours in an anaerobic environment at the temperature of 37 ℃, selecting single bacterial colonies and inoculating to a TSB culture medium, and standing and culturing for 24 hours in the anaerobic environment at the temperature of 37 ℃;
s3, taking a stationary culture solution of the clostridium perfringens strain for 10 times, coating 100 mu L of liquid to an NA flat plate culture medium, punching three circular holes on the flat plate, dripping 100 mu L of chicken source lactobacillus reuteri MC1 culture solution into each hole, translating the flat plate into an anaerobic culture box (the liquid cannot overflow in the translation process), culturing for 24-48 hours in a 37 ℃ environment, and observing a bacteriostasis result, wherein the bacteriostasis result is shown in figure 4.
As can be seen from fig. 4: lactobacillus reuteri MC1 has a certain bacteriostatic effect on clostridium perfringens.
Example 9 application of Lactobacillus reuteri MC1 as chicken source in broiler chicken raising
The embodiment is an experiment for influencing immunity of broiler chickens by chicken lactobacillus reuteri MC1, and the specific experimental method is as follows:
(1) Test animals: 400 AA broilers which are healthy at 1 day old and have insignificant weight difference are randomly divided into 2 treatment groups, and 200 broilers in each group are fed with complete compound pellet feed;
Wherein group I is a control group and is free to drink water; i I groups are experimental groups, and 0.1: 0.1m l of the bacterial liquid of the chicken source lactobacillus reuteri cultured in the example 7 is drunk every day, and the rest time is free to drink water; each treatment group was set with 5 replicates of 40 chickens per replicate group.
(2) The test method comprises the following steps: sterilizing the henhouse, the hencoop and the surrounding environment before the test, and keeping constant illumination and mechanical ventilation in the test process;
the test adopts a cage raising mode, the chicken house adopts an automatic temperature control heating device, the test broiler freely feeds, and the test period is 42 days;
after the test, the animals were fasted for 12 hours, and 10 broilers were slaughtered for each repetition to calculate spleen index, thymus and bursa index. The calculation formula is as follows:
immune organ index = immune organ fresh weight (g)/pre-mortal live weight (kg);
(3) Test results: as shown in table 4.
TABLE 4 influence of Lactobacillus reuteri of chicken origin on immune organs of broilers
| Immune organ index (%) | Group I | Group II |
| Spleen | 9.89±0.42a | 11.78±0.31b |
| Thymus gland | 25.47±2.14a | 32.51±3.45b |
| Fabricius bursa | 21.91±2.04a | 29.10±2.32b |
Note that: the same row of superscript lower case letters differ significantly (P < 0.05).
The main immune organs of the poultry are spleen, thymus and bursa of Fabricius, and the development of each immune organ reflects the immunity of the organism. As can be seen from table 4, the spleen index, thymus index, bursa index were all significantly higher in the test group compared to the control group; thus, the test group taking the chicken lactobacillus reuteri MC1 has stronger immunity than the control group.
Claims (3)
1. A lactobacillus reuteri (Lactobacillus reuteri) MC1 of chicken origin, characterized in that: the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of North Star Xili No. 1, no. 3, a preservation date of 2022, 11 months and a preservation number of CGMCC No.26013.
2. Use of lactobacillus reuteri MC1 of chicken origin according to claim 1 for the preparation of a medicament for improving enteritis in chickens.
3. Use of lactobacillus reuteri MC1 of chicken origin according to claim 1 for the preparation of a medicament for improving chicken immunity.
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