CN103087963B - Method for screening probiotics - Google Patents
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- CN103087963B CN103087963B CN201310037320.XA CN201310037320A CN103087963B CN 103087963 B CN103087963 B CN 103087963B CN 201310037320 A CN201310037320 A CN 201310037320A CN 103087963 B CN103087963 B CN 103087963B
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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 animal and veterinary technical field, particularly relate to the method for screening probiotic bacterium.
Background technology
Probiotic bacterium refers to by improving the host intestine flora eubiosis and plays beneficial effect, reach the active bacteria formulation improving host's (humans and animals) health level and state of health, there is the normal function that can help to maintain enteron aisle, have the effect promoting dietetic alimentation, stimulate body immune system, improve immunizing power.Probiotic bacterium is at guarantee human and livestock health or prevent and treat in some disease and play more and more important effect, and screening different sources and the probiotic bacterium bacterial classification with specific function become the focus of research both at home and abroad.Current China still lacks probiotic bacterium gordian technique and the product of original innovation, industry and market defines the dual monopolization of external product and technology, limits the sound development of China's probiotic bacterium industry.
The primary gordian technique of probiotic bacterium exploitation is bacteria selection.As the screening method that prior art adopts be based on the biology and functional character that bacterial classification has, especially adhesion properties, survival property, functional, workability etc., these characteristics all depend on the characteristic of bacterial classification itself, and therefore, from what approach, how to filter out this bacterial strain be crucial.Current, apply the universal method that traditional phenotypic evaluation method and 16S rDNA sequence homology analysis authentication method are bacteriums, these class methods can filter out preferably has strong adhesion properties, survival property is high, workability is strong bacterial strain, but can not go out be of value to the bacterial strain of animal health, as the bacterial strain of animal large intestine bacillosis can be prevented by Effective selection.
Summary of the invention
The object of this invention is to provide a kind of method of screening probiotic bacterium.
Technical problem to be solved by this invention filters out the high efficient strain can resisting 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, gets the spleen tissue of a part of original cuiture, attacks poison with E.coli K88 to it, measures the change of interleukin 8 and interleukin-4 level;
3rd step, gets the spleen tissue of a part of original cuiture, processes respectively with multiple probiotic strains of candidate to it, measures the change of interleukin 8 and interleukin-4 level;
4th step, by comparing the change of interleukin 8 and interleukin-4 level after multiple probiotic strain of candidate and E.coli K88 process, pick out but probiotic strain that effect contrary similar with 2 kinds of interleukin-intensity of variations that E.coli K88 process causes, tentatively determining may effective probiotic strain;
5th step, by E.coli K88 animal challenge viral dosage, the protected effect of the probiotic strain of observation preliminary screening, finally determines efficient probiotic strain.
The present invention is applicable to can be used for the screening of the lactobacillus of domestic animal, poultry, Mouse and rat etc., lactococcus, streptococcus, enterococcus spp, yeast, the probiotic bacterium such as lichengermium and bacillus subtilis.
Outstanding advantages of the present invention is: adopt ion vitro immunization to test the method accuracy combined with challenge viral dosage in body high, with strong points and save time, the probiotic bacterium screened can effective substitute antibiotics, reduce the disease that intestinal bacteria cause, promote animal health.
Embodiment
Embodiment 1
By fresh pig manure normal saline dilution, streak culture on selective medium, then all kinds of bacteria purification is cultivated, and the probiotic strain (lactobacillus, streptococcus, enterococcus spp) being selected candidate by bacterium micro biochemical reaction tubes is for subsequent use.Asepticly get 21 age in days piglet spleens, shred, grinding homogenate, then 150 order copper mesh filter, centrifugal collecting cell.By erythrocyte cracked liquid and the process of GKN buffered soln, centrifugally spleen cell can be obtained.With RPMI1640 nutritive medium re-suspended cell, adjustment 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 E.coli K88 or candidate's probiotic strain (lactobacillus, streptococcus, enterococcus spp) Dual culture 2 hours, collecting cell, detects spleen cell interleukin 4 and Interleukin-8 Expression level.Pick out the object changing contrary probiotic strain (lactobacillus, suis, in table 1.1) with E.coli K88 treatment group interleukin 4 and interleukin 8 and screen as in vitro tests.
The various candidate probiotic bacterium of table 1.1 is on the impact of piglet spleen cell interleukin-level
Note: the different lowercase alphabet of colleague's shoulder mark shows significant difference (P < 0.05).Below roughly the same.
By the lactobacillus of in-vitro screening and suis (by 1 × 10
9individual/kg feed) to add in mixed feed, the weanling pig (90,21 ages in days) that body weight of feeding is consistent with sex, after 21 days, carries out challenge test (E.coli K88,1 × 10 to piglet
9individual/head).Detect piglet blood interleukin 4 and Interleukin 8 (the results are shown in Table 1.2).Determine probiotic strain piglet to protected effect.
Table 1.2 microbial forage additive is situated between on weanling pig blood leucocyte the impact of plain level
Result shows, compared with blank group, lactobacillus combination chain coccus group has been significantly increased the level of interleukin 4, reduces Interleukin 8 (P < 0.05), improves the immunological competence of piglet.
Embodiment 2
By fresh chicken manure normal saline dilution, streak culture on selective medium, then all kinds of bacteria purification is cultivated, and the probiotic strain (lactobacillus, streptococcus, enterococcus spp) being selected candidate by bacterium micro biochemical reaction tubes is for subsequent use.Asepticly get 6 age in days chick spleens, shred, grinding homogenate, then 100 order net filtrations, centrifugal collecting cell liquid, adds lymphocyte separation medium, leukocytic cream in the middle of collected by centrifugation, and with RPMI1640 nutritive medium re-suspended cell, adjustment 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 E.coli K88 or probiotic bacterium (lactobacillus, streptococcus, enterococcus spp) Dual culture 2 hours, collecting cell, detects spleen cell interleukin 4 and Interleukin-8 Expression level.Pick out the object changing contrary probiotic strain (lactobacillus and suis, in table 2.1) with E.coli K88 treatment group interleukin 4 and interleukin 8 and screen as in vitro tests.
The various candidate probiotic bacterium of table 2.1 is on the impact of broiler chicken spleen cell interleukin-level
By the lactobacillus of in-vitro screening and suis (1 × 10
8individual/kg feed) add in mixed feed, Cobb broiler chicken of feeding (300,1 age in days, male and female half and half), after 42 days, carries out challenge test (E.coli K88,1 × 10 to broiler chicken
9individual/only).Detect broiler chicken blood leucocyte Jie element-4 and Interleukin 8 (the results are shown in Table 2.2).Determine probiotic strain broiler chicken to protected effect.
Table 2.2 microbial forage additive is situated between on broiler chicken blood leucocyte the impact of plain level
Result shows, compared with blank group, lactobacillus combination chain coccus group has been significantly increased the level of interleukin 4, reduces Interleukin 8 (P < 0.05), improves the immunological competence of broiler chicken.
Claims (1)
1. screen a method for probiotic bacterium, it is characterized in that comprising the following steps:
(1) separating animal's spleen, carries out tissue in primary culture;
(2) get the spleen tissue of a part of original cuiture, with RPMI1640 nutritive medium re-suspended cell, adjustment cell concn is l × 10
6individual/ml, and inoculate 2 × 10
6individual/porocyte is cultivated after 48 hours in 6 orifice plates, adds 1 × 10
8then individual E.coli K88 Dual culture measures the change of interleukin 8 and interleukin 4 level in spleen cell to attack poison to it in 2 hours;
(3) get the spleen tissue of a part of original cuiture, with RPMI1640 nutritive medium re-suspended cell, adjustment cell concn is l × 10
6individual/ml, and inoculate 2 × 10
6individual/porocyte is cultivated after 48 hours in 6 orifice plates, adds 1 × 10
8the probiotic strain of individual candidate processes it, then measures the change of interleukin 8 and interleukin 4 level in spleen cell;
(4) by comparing the change of interleukin 8 and interleukin 4 level after the probiotic strain of candidate and E.coli K88 process, pick out but probiotic strain that effect contrary similar with 2 kinds of interleukin-intensity of variations that E.coli K88 process causes, tentatively determining may effective probiotic strain, wherein said effect be on the contrary improve interleukin 4 level and reduce Interleukin 8;
(5) probiotic strain of in-vitro screening is added in mixed feed, the animal that body weight of feeding is consistent with sex, and with E.coli K88 to animal with 1 × 10
9individual/head carries out challenge test, detects animal blood interleukin 4 and Interleukin 8;
(6) observe the protected effect of probiotic strain of preliminary screening, final select to significantly improve interleukin 4 level and reduce the efficient probiotic strain of Interleukin 8;
Wherein said probiotic strain is selected from streptococcus, and described animal is selected from piglet or chick.
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| CN103087963B true CN103087963B (en) | 2015-03-04 |
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Families Citing this family (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 |
| 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 |
| 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 |
| CN114717168B (en) * | 2022-06-08 | 2022-09-20 | 微康益生菌(苏州)股份有限公司 | Screening method of functional probiotics based on inhibition of lipopolysaccharide production |
| CN117757892B (en) * | 2024-02-22 | 2024-06-11 | 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102438637A (en) * | 2009-03-05 | 2012-05-02 | 益生菌股份公司 | Bacteria strains having a high anti-inflammatory activity |
| CN102884174A (en) * | 2010-03-26 | 2013-01-16 | 株式会社明治 | Method for screening intestinal immunity suppression agents |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102438637A (en) * | 2009-03-05 | 2012-05-02 | 益生菌股份公司 | Bacteria strains having a high anti-inflammatory activity |
| CN102884174A (en) * | 2010-03-26 | 2013-01-16 | 株式会社明治 | Method for screening intestinal immunity suppression agents |
Non-Patent Citations (5)
| Title |
|---|
| Marianna Roselli 等.Probiotic bacteria Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG protect intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli K88.《British Journal of Nutrition》.2006,第95卷(第6期),1177-1184. * |
| Marjorie E. Koenen 等.Development and validation of a new in vitro assay for selection of probiotic bacteria that express immune-stimulating properties in chickens in vivo.《FEMS Immunology and Medical Microbiology》.2006,第40卷(第2期),119-127. * |
| rance:In vitro and in vivo considerations.《Clinical Nutrition》.2006,第25卷(第6期),994-1003. * |
| Sophie Drouault-Holowacz 等.Anti-inflammatory potential of the probiotic dietary supplement Lactibiane Tole´ * |
| 张赛群 等.产肠毒素性大肠杆菌K88的致病特性及其益生菌的筛选.《中国兽医科学》.2008,第38卷(第5期),386-392. * |
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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 |