CN101851593B - Lactobacillus planetarium strain LP28 and application thereof in mitigation of anaphylactic reaction - Google Patents
Lactobacillus planetarium strain LP28 and application thereof in mitigation of anaphylactic reaction Download PDFInfo
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Abstract
The invention relates to a Lactobacillus planetarium strain LP28, which is preserved in China General Microbiological Culture Collection Center (CGMCC). The preservation number of the Lactobacillus planetarium strain LP28 is CGMCC No.3346. The Lactobacillus planetarium strain or metabolite thereof has excellent capability of mitigating an anaphylactic reaction and can be widely used for preparing food compounds or medical pharmaceutical compounds.
Description
Technical field
The present invention relates to a kind of anaphylactoid lactic bacilli strains that slows down.
Background technology
Anaphylaxis means immunity system to some kickbacks that does not have offensive foreign matter to produce, and between nearly many decades, patients such as anaphylactic disease such as bronchial allergy, allergic rhinitis and atopic dermatitis increase severely.Human immunity centrocyte T lymph corpuscle can be divided into two types according to the difference of the cytokine of secretion; The first type T lymph corpuscle (Th1) is mainly secreted interferon-gamma (Interferon-γ; IFN-γ); The main secretion of the second type T lymph corpuscle (Th2) be situated between white plain-4 (Interleukin-4, IL-4), (Interleukin-5 IL-5) waits cytohormone to be situated between white plain-5; Yet, have dynamic equilibrium relationship between first type and the second type T lymph corpuscle, if the effect of the first type T lymph corpuscle is faint, cause the anaphylaxis of human body easily.At present, the treatment of anaphylactic disease system is mostly treated to symptom with medicine, but with the pharmacological agent anaphylactic disease, the patient is easy to generate spinoff, makes the research institution of many countries urgently seek safer and more effective anaphylactic disease treat-ment.
The researcher finds; The bacterium of harmful bacterium and probiotic bacterium is counted the great dependency of having of ratio and anaphylactic disease in the intestines and stomach; The many persons of probiotic bacterium in the intestines and stomach, probiotic bacterium can be protected the digestive tube mucous membrane, promotes the Tegeline hyperplasia, produce natural antibody and make immunocyte infiltrate the digestive tube mucous membrane easily; And harmful many persons of bacterium in the intestines and stomach, harmful bacterium is destroyed the intestines and stomach mucous membrane easily, causes lymphopenia and the insufficient phenomenon of Tegeline kind, however the destruction of intestines and stomach mucous membrane can cause the antigen amount to increase, and then cause that sensitization produces anaphylactic disease; Be with, the human bacterial species that is contacted is enough to influence the trend that immunity system is grown, many researchers hope and can further control anaphylactoid generation by probiotics bacterial number and kind in the adjustment intestines and stomach.
Milk-acid bacteria is a probiotic bacterium the most common in the intestines and stomach, is the high mikrobe of a kind of security.Milk-acid bacteria has the abilities such as growth, adjusting immunoreation, removing superoxide and reducing cholesterol that suppress harmful bacterium.In addition, milk-acid bacteria contains pleasant fragrance matter, and can produce lactic acid or bacteriocin bacterinertness materials such as (bacteriocin), thereby is used in food such as processing fermented-milk widely.
Yet; Desire utilizes milk-acid bacteria to reach the purpose of treatment anaphylactic disease; The effect of present lactobacillus inoculation still can't be satisfactory, and the human stomach acidacidity is up to pH 2.5, and most lactic acid bacteria can't be survived in the environment of so acid; Make and to pass through the erosion of hydrochloric acid in gastric juice and choline by the milk-acid bacteria that is eaten for human body afterwards thalline is impaired or dead, can't effectively be attached to intestinal mucosa and excrete with ight soil.Enteron aisle is the immune organ of human body maximum, and most of milk-acid bacterias of being taken in by human body can't healthy surviving in the enteron aisle, promptly can't bring into play the function of milk-acid bacteria, and then the anaphylaxis that is difficult to effectively to improve immunity system and is caused.Therefore, filter out and to improve the first type T lymph corpuscle effect, and can resist the lactobacillus strain of hydrochloric acid in gastric juice and choline simultaneously, be the very urgent problem of treatment anaphylactic disease.
Summary of the invention
The present invention's purpose provides a kind of lactic bacterium strains, and it can promote the first type T lymph corpuscle effect, with the secretion interferon-gamma.
Another object of the present invention provides this a kind of lactic bacilli strains, and it can effectively resist hydrochloric acid in gastric juice and choline.
The present invention's a purpose again provides this strain lactic bacilli strains, and it can slow down anaphylaxis.
The present invention provides a kind of lactobacillus plantarum bacterial strain LP28 (Lactobacillus plantarum), and this strain lactic bacilli strains lies in and was deposited at Chinese common micro-organisms preservation administrative center on October 19th, 2009, deposits and is numbered CGMCCNo.3346.This strain lactic bacilli strains or its meta-bolites have the anaphylactoid ability of slowing down of excellence, and can be widely used in the preparation of food product consisting thing or pharmaceutical composition.
Lactobacillus plantarum strain LP28 according to the invention is determined as gram-positive microorganism through gramstaining, does not have a mobility, and outward appearance is a rod-short, and the righttest growing environment temperature is 37 ℃.
Table 1 is analyzed the result of LP28 to different glucide fermentation capacities for utilizing API KIT CHL 50 (available from bioMerieux), is judged to be lactobacillus plantarum Lactobacillus plantarum (various glucide abbreviations see also the bioMerieux operational manual in the table one).LP28 is through 16S rDNA sequencing result such as table two.
Table 1:LP28 is to the fermentation capacity (API KIT 50CH) of different glucide
| ? | ?24H | 48H | ? |
| 0 | ? | ? | 0 |
| GLY | ? | ? | 1 |
| ERY | ? | ? | 2 |
| DARA | ? | ? | 3 |
| LARA | ? | ? | 4 |
| RIB | ? | ? | 5 |
| DXYL | ? | ? | 6 |
| LZYL | ? | ? | 7 |
| ADO | ? | ? | 8 |
| MDX | ? | ? | 9 |
| GAL | ? | + | 10 |
| GLU | ? | + | 11 |
| FRU | ? | + | 12 |
| MNE | ? | + | 13 |
| SBE | ? | + | 14 |
| RHA | ? | ? | 15 |
| DUL | ? | ? | 16 |
| INO | ? | ? | 17 |
| MAN | ? | + | 18 |
| SOR | ? | + | 19 |
| MDM | ? | ? | 20 |
| MDG | ? | ? | 21 |
| NAG | ? | + | 22 |
| AMY | ? | + | 23 |
| ARB | ? | + | 24 |
| ESC | ? | + | 25 |
| ? | 24H | 48H | ? |
| SAL | ? | + | 26 |
| CEL | ? | + | 27 |
| MAL | ? | + | 28 |
| LAC | ? | + | 29 |
| MEL | ? | ? | 30 |
| SAC | ? | + | 31 |
| TRE | ? | + | 32 |
| INU | ? | ? | 33 |
| MLZ | ? | + | 34 |
| RAF | ? | + | 35 |
| AMD | ? | ? | 36 |
| GLYG | ? | ? | 37 |
| XLT | ? | ? | 38 |
| GEN | ? | + | 39 |
| TUR | ? | ? | 40 |
| LYX | ? | ? | 41 |
| TAG | ? | ? | 42 |
| DARL | ? | ? | 43 |
| LFUC | ? | ? | 44 |
| DARL | ? | ? | 45 |
| LARL | ? | ? | 46 |
| GNT | ? | + | 47 |
| 2KG | ? | ? | 48 |
| 5KG | ? | ? | 49 |
Table 2:LP28-16S rDNA sequence:
CGGCCGGGGGGGTGTCCTATACTGCAAGTCGAACGCGTTGACCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGA?GCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGACCTTACCAGTCTTGACATCTTGCGCAACCCTAGAGATAGGGCGTTTCCTCGGGACGCAATGACAGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTATTGA
Description of drawings
Fig. 1 be anti-two periods of artificial hydrochloric acid in gastric juice pH2.0 of lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 deposit viable count histogram as a result.
Fig. 2 be anti-two periods of artificial hydrochloric acid in gastric juice pH3.0 of lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 deposit viable count histogram as a result.
Fig. 3 is the anti-0.3% cholate test-results histogram of lactic bacterium strains LP28, LF22, LF44, LF46 and LH45.
Fig. 4 is the histogram as a result that lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 stimulating human periphery blood monocyte cell produce IFN-γ.
Fig. 5 is the histogram as a result that lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 stimulating human periphery blood monocyte cell produce IL-4.
Fig. 6 is the histogram as a result that lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 stimulating human periphery blood monocyte cell produce Interferon-r/IL-4 ratio.
Embodiment
For let address other purpose, characteristic and advantage on the present invention can be more obviously understandable, the special preferred embodiment of lifting the present invention of hereinafter, and cooperate appended graphicly elaborates as follows:
The applicant filters out a strain and has lactobacillus plantarum bacterial strain LP28 (the Lactobacillus plantarum that excellence is slowed down anaphylaxis through extensive and deep experiment; Deposit numbering: CGMCC No.3346).The screening process of this strain lactic bacilli strains is following:
1, by screening lactic bacterium strains in the sample
The first step of the present invention's screening milk-acid bacteria: get the traditional market dried bean curd as milk-acid bacteria screening source sample.Sample is inoculated in the 10wt% skim-milk nutrient solution, carries out 37 ℃ earlier, and 16～20 hours collection is grown cultivation (Enrichment culture), and filters out the bacterial strain that produces the curdled milk phenomenon.。
Second step of the present invention's screening lactic bacterium strains: get collection and grow and cultivate sample later and carry out serial dilution and plate loop method, and cultivated 2 to 3 days down, to pick out the single bacterium colony of different bacterium phases in 37 ℃ of anaerobic conditions.
The third step of the present invention's screening lactic bacterium strains: this is had single bacterium colony carry out gramstaining and catalase reaction test; And filter out that gramstaining is positive and number strain bacterial strain that the catalase reaction is negative; Be the lactic bacterium strains of preliminary screening; And the lactic bacterium strains that this preliminary screening goes out carried out the evaluation of bacterial classification; In several strains of lactic acid bacteria bacterial strains that this preliminary screening goes out; Include a strain lactobacillus plantarum bacterial strain (Lactobacillus plantarum), three strain fermentation lactobacillus strains (Lactobacillus fermentum) and a strain Switzerland lactobacillus spp (Lactobacillus helveticus), the applicant is respectively with this five strains of lactic acid bacteria bacterial strain called after LP28, LF22, LF44, LF46 and LH45.
2, acid resistance bile tolerance test
The present invention carries out acid resistance and the test of anti-choline respectively with this five strains of lactic acid bacteria bacterial strain (LP28, LF22, LF44, LF46 and LH45).
The first step of this acid resisting test: (Sharpe Difco) in the nutrient solution, and cultivates several hours to produce the respectively MRS nutrient solution of this lactic bacterium strains under 37 ℃ for deMan, Rogosa in MRS with this five strains of lactic acid bacteria inoculation.
Second step of this acid resisting test: the MRS nutrient solution of getting each a certain amount of strains of lactic acid bacteria bacterial strain; With 5000rpm, 5 minutes centrifugal after; And get to cover behind the thalline with aseptic PBS flushing centrifuging and taking and be dissolved in aseptic PBS, making an addition to respectively in the simulated gastric fluid, the acidity of this simulated gastric fluid is pH2.0 and pH3.0; And place 37 ℃ to cultivate two hours, and each hour analyzed a bacterium number.
The acid resisting test pH2.0 of this five strains of lactic acid bacteria bacterial strain (LP28, LF22, LF44, LF46 and LH45) and the result of pH3.0 are respectively shown in the 1st figure and the 2nd figure; Can know by figure; Carry out anti-artificial hydrochloric acid in gastric juice pH2.0 after two hours; Has the highest viable count (＞106Log CFU/mL) of depositing with lactic bacilli strains LP28, LF22 and LF46; Show and carry out two hours results of anti-artificial hydrochloric acid in gastric juice pH3.0; Lactic bacilli strains LP28, LF22 have the highest deposit viable count (＞109LogCFU/mL), The above results shows that milk-acid bacteria LP28 and LF22 have good acidproof ability, anti-two periods of artificial hydrochloric acid in gastric juice pH2.0 of those lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 deposit the viable count result as shown in the 1st schemes; Anti-two periods of artificial hydrochloric acid in gastric juice pH3.0 of those milk-acid bacterias deposits the viable count result shown in the 2nd figure in addition.
The first step of this bile tolerance test: this five strains of lactic acid bacteria bacterial strain (LP28, LF22, LF44, LF46 and LH45) is inoculated in respectively in the MRS nutrient solution, and cultivates several hours down to produce the respectively MRS and the nutrient solution of this lactic bacterium strains in 37 ℃.
Second step of this bile tolerance test: the MRS nutrient solution of getting each a certain amount of strains of lactic acid bacteria bacterial strain respectively; Make an addition to respectively in the MRS nutrient solution that contains 0.3% cholate (0.3%ox gall); With as experimental group; And the nutrient solution of getting each strains of lactic acid bacteria bacterial strain adds and not to add in the MRS nutrient solution of 0.3% cholate, with as control group; After placing 37 ℃ to cultivate several hours experimental group and the control group, analyze it and deposit viable count.The result of the bile tolerance test of this five strains of lactic acid bacteria bacterial strain (LP28, LF22, LF44, LF46 and LH45) is shown in the 3rd figure; Can know by figure; After carrying out bile tolerance number hour; Have the highest viable count of depositing with lactic bacterium strains LP28, LF22, LF44 and LF46, this five strains of lactic acid bacteria bacterial strain lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 bile tolerance deposit the viable count result as the 3rd figure shown in:
The present invention's the result that five strains of lactic acid bacteria bacterial strains are carried out acidproof and anti-choline test; System filters out the lactic bacterium strains LP28 and the LF22 of the highest anti-simulated gastric fluid of two strain tools and bile tolerance, and gets those lactic bacterium strains LP28, LF22, LF44, LF46 and LH45 and carry out the immuno-stimulating test.
3, immuno-stimulating test
The present invention system utilizes cell experiment assessment milk-acid bacteria to lower the ability of allergic immune response.It is to carry out with milk-acid bacteria stimulating human periphery blood monocyte cell (human peripheral blood mononuclear cells; HPBMC) immuno-stimulating test; And (Enzyme-linked immunosorbent assay ELISA) detects the output of the secreted cytohormone IL-4 of the secreted cytohormone IFN-γ of the first type T lymph corpuscle and the second type T lymph corpuscle to link the adsorption analysis method with ferment immunity.
The first step of this immuno-stimulating test: be the human peripheral blood lymph corpuscle of preparation, it is the first type T lymph corpuscle.Get dense liquid of human leukocyte and phosphoric acid buffer (Phosphate Buffered Saline, PBS) uniform mixing; This mixed solution that mixes is isolated human peripheral blood mononuclear cell with cell gradient centrifugation mode.
Second step of this immuno-stimulating test: be with this mankind's periphery blood mononuclear cell and Hanks balanced salt solution (Hanks balanced salt solution; HBSS) after uniform mixing cleans; Insert the RPMI-1640 substratum (available from GIBCO company; PIN No.11875) in, utilizes trypan blue staining method (Trypan blue) to calculate human peripheral blood monocyte cell number, and utilize the RPMI-1640 substratum to be adjusted into 2 * 106Cells/ml by periphery blood monocyte cell number; Divide and coil in 24 porose discs, cultivate altogether with different concns lactic acid thalline.Negative control group is that (Phytohemagglutinin PHA) joins 2 * 10 for phytohaemagglutinin with 10 μ g/ml
6The human peripheral blood monocyte cell uniform mixing of Cells/ml is done comparison with other lactic acid thalline stimulating human peripheral blood monocyte.
The third step of this immuno-stimulating test: being that irritation cell is plain produces.It is to get this five strains of lactic acid bacteria bacterial strain (LP28, LF22, LF44, LF46 and LH45) as experimental group; And get the human peripheral blood mononuclear cell (PBMC) that do not add any milk-acid bacteria as control group and the phytohaemagglutinin (PHA) of inducing the generation of IFN-γ as the control group; Each nutrient solution and enchylema were cultivated tens of hours with the suitable proportion uniform mixing; Get experimental group, control group and control group culture supernatant liquid and carry out ferment immunity binding adsorption analysis, to detect the output of cytohormone IFN-γ and IL-4.The result that each lactic bacterium strains stimulating human periphery blood monocyte cell generation IFN-γ, IL-4 reach ratio between the two is respectively shown in the 4th figure, the 5th figure and the 6th figure, and the value of the peripheral blood monocyte of this experimental group and control group stimulating human cell generation IFN-γ is shown in the 4th figure again:
Can know by the 4th figure; But this lactic bacterium strains LP28 stimulating human periphery blood monocyte cell produces more IFN-γ; And then the effect that can improve the first type T lymph corpuscle, the dynamic relationship between balance first type and the second type T lymph corpuscle can further be slowed down the mankind's anaphylaxis.
Experimental group and control group stimulating human periphery blood monocyte cell produce the value of IL-4 shown in the 5th figure: IL-4 is the cytohormone of the mastocyte in the activation Th2 immunity system; Can combine with intravital IgE; Transmitting irritation cell through message discharges chemical substance such as allied organization's amine and causes inflammation; It is irritated that this type of chemical substance causes the tissue of health different sites to produce, and suppresses the differentiation of Th1 cell so IL-4 can help the differentiation of Th2 cell.Can know that by the 5th figure and last table this lactic bacterium strains LP28 can significantly reduce the concentration that human peripheral blood monocyte cell produces IL-4, helps the balance between Th1 and Th2 immunoreation.
Experimental group and control group stimulating human periphery blood monocyte cell produce the ratio of IFN-γ and IL-4 shown in the 6th figure:
As stated; The present invention system is by stimulating the first type T lymph corpuscle (Th1) to secrete a large amount of interferon-gammas (Interferon-γ) to the useful the present invention three strains of lactic acid bacteria bacterial strain LP28 of organism; And reduce by the secretion of second type T lymph corpuscle (Th1) IL-4; And then can slow down anaphylaxis, the meta-bolites of lactic bacterium strains LP28 of the present invention also capable of using is widely used in this meta-bolites the processing of food product consisting thing or pharmaceutical composition; Form or therapeutical agent to make food, the pharmacy that can slow down anaphylaxis, wherein this food system can be milk-product such as fermented-milk.
Though the present invention has utilized above-mentioned preferred embodiment to disclose; Right its is not in order to limit the present invention; Anyly have the knack of this art; Within spirit that does not break away from the present invention and scope, when can doing various changes and modification, so the claim person of defining that the present invention's protection domain attaches after looking is as the criterion.
Claims (4)
1. a lactobacillus plantarum bacterial strain LP28 (Lactobacillus plantarum), it is deposited at Chinese common micro-organisms preservation administrative center, deposits and is numbered CGMCC No.3346.
2. claim 1 said lactobacillus plantarum bacterial strain LP28 and meta-bolites thereof slow down the application in the uneven anaphylactoid medicine that causes because of the dynamic relationship between first type and the second type T lymph corpuscle in preparation.
3. claim 1 said lactobacillus plantarum bacterial strain LP28 and meta-bolites thereof slow down the Application in Food because of the uneven anaphylaxis that causes of the dynamic relationship between first type and the second type T lymph corpuscle in preparation.
4. slow down Application in Food like claim 3 said lactobacillus plantarum bacterial strain LP28 and meta-bolites thereof in preparation, it is characterized in that said food is fermented-milk because of the uneven anaphylaxis that causes of the dynamic relationship between first type and the second type T lymph corpuscle.
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| CN107245468A (en) * | 2017-08-14 | 2017-10-13 | 青岛农业大学 | A kind of lactobacillus preparation for promoting pot marigold to bloom |
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| CN103173379A (en) * | 2011-12-22 | 2013-06-26 | 熊津食品有限公司 | Novel microbe for red ginseng fermentation, red ginseng fermentation liquid using the microbe and red ginseng fermentation drink |
| CN104171429A (en) * | 2014-08-14 | 2014-12-03 | 生合生物科技股份有限公司 | Use of lactobacillus plantarum LP28 |
| TWI662902B (en) * | 2017-06-05 | 2019-06-21 | 生合生物科技股份有限公司 | Improving dissolved oxygen in aquaculture ponds with a mixture of three lactic acid bacteria strains |
| CN107873841B (en) * | 2017-11-21 | 2021-03-26 | 山东世俱先创生物科技有限公司 | Lactic acid bacteria beverage and production process thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107245468A (en) * | 2017-08-14 | 2017-10-13 | 青岛农业大学 | A kind of lactobacillus preparation for promoting pot marigold to bloom |
| CN107245468B (en) * | 2017-08-14 | 2019-12-03 | 青岛农业大学 | A kind of lactobacillus preparation for promoting pot marigold to bloom |
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