CN103087963A - Method for screening probiotics - Google Patents
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- CN103087963A CN103087963A CN201310037320XA CN201310037320A CN103087963A CN 103087963 A CN103087963 A CN 103087963A CN 201310037320X A CN201310037320X A CN 201310037320XA CN 201310037320 A CN201310037320 A CN 201310037320A CN 103087963 A CN103087963 A CN 103087963A
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Abstract
The invention discloses a method for screening probiotics. The method comprises the following steps of: (1) carrying out a primary culture on spleen tissues of a pig or a domestic animal; (2) attacking toxicity of the tissues through colon bacillus K88, and testing variations of levels of an interleukin 8 and an interleukin 4; (3) respectively processing the tissues through candidate probiotic strains, and testing variations of levels of two interleukins; (4) comparing the variations to select probiotic strains which are similar in variation degrees of the two interleukins but opposite in effect due to treatment of the colon bacillus K88; and (5) through the colon bacillus K88 animal toxicity attacking test, observing protecting effects of primarily selected probiotic strains, and finally determining an efficient probiotic strain. The method for screening the probiotic disclosed by the invention has the remarkable advantages that: the method combining in vitro immunology experiment and in vivo toxicity attacking experiment is high in accuracy, strong in pertinency and time-saving; and the screened probiotics can effectively replace antibiotic, and greatly reduce diseases caused by colon bacillus so as to promote the health of the animal.
Description
Technical field
The invention belongs to the animal and veterinary technical field, relate in particular to the method for screening probiotic bacterium.
Background technology
Probiotic bacterium refers to bring into play beneficial effect by improving host's intestinal microflora eubiosis, reach the active bacteria formulation that improves host's (humans and animals) health level and state of health, have the normal function that can help to keep enteron aisle, have to promote dietetic alimentation, the effect that stimulates body immune system, improves immunizing power.Probiotic bacterium ensureing human and livestock health or preventing and treating and bringing into play more and more important effect aspect some disease, and screening different sources and the probiotic bacterium bacterial classification with specific function become the focus of domestic and international research.China still lacks probiotic bacterium gordian technique and the product of original innovation at present, has formed the dual monopolization of external product and technology on industry and market, has limited the sound development of China's probiotic bacterium industry.
The primary gordian technique of probiotic bacterium exploitation is the screening of bacterial classification.Biology and functional character that the bacterial classification that screening method is based on that adopts as prior art has especially stick characteristic, survival property, functional, workability etc., these characteristics all depend on the characteristic of bacterial classification itself, therefore, and from what approach, how to filter out this bacterial strain be crucial.Current, use traditional phenotypic evaluation method and 16S rDNA sequence homology analysis authentication method is the universal method of bacterium, these class methods can filter out to have preferably sticks by force characteristic, survival property is high, workability is strong bacterial strain, but can not Effective selection go out to be of value to the bacterial strain of animal health, as preventing the bacterial strain of animal large intestine bacillosis.
Summary of the invention
The purpose of this invention is to provide a kind of method of screening probiotic bacterium.
Technical problem to be solved by this invention is to filter out the high efficient strain that can resist the intestinal bacteria negative interaction from the probiotic bacteriums such as lactobacillus, lactococcus, streptococcus, enterococcus spp, yeast, lichengermium and bacillus subtilis.
A kind of method of screening probiotic bacterium of the present invention realizes by the following method:
The first step is separated pig or poultry spleen, carries out tissue in primary culture;
Second step is got the spleen tissue of a part of former culture, and K88 attacks poison to it with intestinal bacteria, measures the variation of interleukin 8 and interleukin-4 level;
The 3rd goes on foot, and gets the spleen tissue of a part of former culture, respectively it is processed with a plurality of probiotic strains of candidate, measures the variation of interleukin 8 and interleukin-4 level;
The 4th step, by relatively a plurality of probiotic strains of candidate and the variation that intestinal bacteria K88 processes rear interleukin 8 and interleukin-4 level, pick out and process 2 kinds of causing the interleukin-intensity of variation is similar with intestinal bacteria K88 but probiotic strain that effect is opposite, tentatively determine effectively probiotic strain;
The 5th step, attack the poison experiment by intestinal bacteria K88 animal, the protection effect of the probiotic strain of observation preliminary screening is finally determined efficient probiotic strain.
The present invention is applicable to can be used for the screening of the probiotic bacteriums such as lactobacillus, lactococcus, streptococcus, enterococcus spp, yeast, lichengermium and bacillus subtilis of domestic animal, poultry, Mouse and rat etc.
Outstanding advantages of the present invention is: the method accuracy that adopts the experiment of external immunological experiment and body inside fire attack poison to combine is high, with strong points and save time, the probiotic bacterium of screening is substitute antibiotics effectively, reduce the disease that intestinal bacteria cause, promote animal health.
Embodiment
Embodiment 1
With fresh pig manure normal saline dilution, streak culture on selective medium, then all kinds of bacteria purifications are cultivated, and the probiotic strain (lactobacillus, streptococcus, enterococcus spp) of selecting the candidate by bacterium micro biochemical reaction tubes is standby.The aseptic 21 age in days piglet spleens of getting shred, and grind homogenate, and then 150 order copper mesh filter, centrifugal collecting cell.Process by erythrocyte cracked liquid and GKN buffered soln, centrifugally can obtain spleen cell.With RPMI1640 nutritive medium re-suspended cell, adjusting cell concn is 1 * 10
6Individual/ml.Inoculation 2 * 10
6Individual/porocyte is cultivated after 48 hours in 6 orifice plates, adds 1 * 10
8Individual intestinal bacteria K88 or candidate's probiotic strain (lactobacillus, streptococcus, enterococcus spp) were cultivated 2 hours altogether, and collecting cell detects spleen cell interleukin 4 and Interleukin-8 Expression level.Pick out with intestinal bacteria K88 treatment group interleukin 4 and interleukin 8 and change opposite probiotic strain (lactobacillus, suis see Table 1.1) as the object of in vitro tests screening.
The impact of the various candidate probiotic bacteriums of table 1.1 on piglet spleen cell interleukin-level
Annotate: the different lowercase alphabet differentials of colleague's shoulder mark different significantly (P＜0.05).Below roughly the same.
Lactobacillus and the suis of in-vitro screening (are pressed 1 * 10
9Individual/kg feed) add in mixed feed, the weanling pig that the body weight of feeding is consistent with sex (90,21 ages in days) is after 21 days, and piglet is carried out challenge test (intestinal bacteria K88,1 * 10
9Individual/head).Detect piglet blood interleukin 4 and Interleukin 8 (the results are shown in Table 1.2).Determine piglet is had the probiotic strain of protection effect.
The impact of table 1.2 microbial forage additive on the plain level of weanling pig blood leucocyte Jie
Result shows, compares with the blank group, and lactobacillus combination chain coccus is organized the level of the interleukin 4 that has been significantly increased, and has reduced Interleukin 8 (P＜0.05), has improved the immunological competence of piglet.
Embodiment 2
With fresh chicken manure normal saline dilution, streak culture on selective medium, then all kinds of bacteria purifications are cultivated, and the probiotic strain (lactobacillus, streptococcus, enterococcus spp) of selecting the candidate by bacterium micro biochemical reaction tubes is standby.The aseptic 6 age in days chick spleens of getting shred, and grind homogenate, 100 order net filtrations then, and centrifugal collecting cell liquid adds lymphocyte separation medium, leukocytic cream in the middle of centrifugal collection, with RPMI1640 nutritive medium re-suspended cell, adjusting cell concn is 1 * 10
6Individual/ml.Inoculation 2 * 10
6Individual/porocyte is cultivated after 48 hours in 6 orifice plates, adds 1 * 10
8Individual intestinal bacteria K88 or probiotic bacterium (lactobacillus, streptococcus, enterococcus spp) were cultivated 2 hours altogether, and collecting cell detects spleen cell interleukin 4 and Interleukin-8 Expression level.Pick out with intestinal bacteria K88 treatment group interleukin 4 and interleukin 8 and change opposite probiotic strain (lactobacillus and suis see Table 2.1) as the object of in vitro tests screening.
The impact of the various candidate probiotic bacteriums of table 2.1 on broiler chicken spleen cell interleukin-level
Lactobacillus and suis (1 * 10 with in-vitro screening
8Individual/kg feed) add in mixed feed, the Cobb broiler chicken of feeding (300,1 age in days, male and female half and half) is after 42 days, and broiler chicken is carried out challenge test (intestinal bacteria K88,1 * 10
9Individual/only).Detect broiler chicken blood leucocyte Jie element-4 and Interleukin 8 (the results are shown in Table 2.2).Determine broiler chicken is had the probiotic strain of protection effect.
The impact of table 2.2 microbial forage additive on the plain level of broiler chicken blood leucocyte Jie
Result shows, compares with the blank group, and lactobacillus combination chain coccus is organized the level of the interleukin 4 that has been significantly increased, and has reduced Interleukin 8 (P＜0.05), has improved the immunological competence of broiler chicken.
Claims (3)
1. method of screening probiotic bacterium is characterized in that comprising the following steps:
(1) separate pig or poultry spleen, carry out tissue in primary culture;
(2) get the spleen tissue of a part of former culture, K88 attacks poison to it with intestinal bacteria, measures the variation of interleukin 8 and interleukin-4 level;
(3) get the spleen tissue of a part of former culture, respectively it is processed with a plurality of probiotic strains of candidate, measure the variation of interleukin 8 and interleukin-4 level;
(4) by relatively a plurality of probiotic strains of candidate and the variation that intestinal bacteria K88 processes rear interleukin 8 and interleukin-4 level, pick out and process 2 kinds of causing the interleukin-intensity of variation is similar with intestinal bacteria K88 but probiotic strain that effect is opposite, tentatively determine effectively probiotic strain;
(5) attack the poison experiment by intestinal bacteria K88 animal, the protection effect of the probiotic strain of observation preliminary screening is finally determined efficient probiotic strain.
2. a kind of method of screening probiotic bacterium according to claim 1, it is characterized in that: described probiotic bacterium comprises lactobacillus or lactococcus or streptococcus or enterococcus spp or yeast or lichengermium and bacillus subtilis.
3. a kind of method of screening probiotic bacterium according to claim 1 is characterized in that: the animal that described animal is attacked in the poison experiment comprises livestock or poultry or mouse or rat.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020037531A1 (en) * | 2018-08-22 | 2020-02-27 | 江南大学 | Cell level-based method for screening probiotics that achieve antidepressant function by means of 5-hydroxytryptophan related-pathway |
| CN111118109A (en) * | 2020-01-03 | 2020-05-08 | 中国科学院亚热带农业生态研究所 | Method for evaluating protein nutrition state of live pig individual by using blood biochemical indexes |
| WO2021087890A1 (en) * | 2019-11-07 | 2021-05-14 | 特安康股份有限公司 | Method for building personalized probiotics database, and use thereof in screening of probiotics |
| CN114717168A (en) * | 2022-06-08 | 2022-07-08 | 微康益生菌(苏州)股份有限公司 | Screening method of functional probiotics based on inhibition of lipopolysaccharide production |
| CN117757892A (en) * | 2024-02-22 | 2024-03-26 | 潍坊华卓生物科技有限公司 | reverse screening method and application of functional probiotics for preventing newcastle disease virus |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020037531A1 (en) * | 2018-08-22 | 2020-02-27 | 江南大学 | Cell level-based method for screening probiotics that achieve antidepressant function by means of 5-hydroxytryptophan related-pathway |
| WO2021087890A1 (en) * | 2019-11-07 | 2021-05-14 | 特安康股份有限公司 | Method for building personalized probiotics database, and use thereof in screening of probiotics |
| CN111118109A (en) * | 2020-01-03 | 2020-05-08 | 中国科学院亚热带农业生态研究所 | Method for evaluating protein nutrition state of live pig individual by using blood biochemical indexes |
| CN111118109B (en) * | 2020-01-03 | 2023-08-01 | 中国科学院亚热带农业生态研究所 | Method for evaluating protein nutrition status of individual live pigs by using blood biochemical indexes |
| CN114717168A (en) * | 2022-06-08 | 2022-07-08 | 微康益生菌(苏州)股份有限公司 | Screening method of functional probiotics based on inhibition of lipopolysaccharide production |
| CN117757892A (en) * | 2024-02-22 | 2024-03-26 | 潍坊华卓生物科技有限公司 | reverse screening method and application of functional probiotics for preventing newcastle disease virus |
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| CN103087963B (en) | 2015-03-04 |
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Address after: 430023 Wuhan, East and West Lake District, evergreen garden, South Road, college, No. 68 Patentee after: WUHAN POLYTECHNIC University Address before: 430023 Wuhan, Hubei Evergreen Garden Road, No. 68 Patentee before: Wuhan Polytechnic University |
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Effective date of registration: 20180323 Address after: 330800 Jiangxi city of Yichun province high security industrial project area eight Town Patentee after: JIANGXI HUANONG HENGQING AGRICULTURE AND ANIMAL HUSBANDRY CO.,LTD. Address before: 430023 Wuhan, East and West Lake District, evergreen garden, South Road, college, No. 68 Patentee before: Wuhan Polytechnic University |
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