CN102618461B - Lactobacillus plantarum capable of promoting secretion of SIgA (Secretory Immunoglobulin A) in intestinal canal and detecting method of lactobacillus plantarum - Google Patents
Lactobacillus plantarum capable of promoting secretion of SIgA (Secretory Immunoglobulin A) in intestinal canal and detecting method of lactobacillus plantarum Download PDFInfo
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Abstract
The invention discloses lactobacillus plantarum capable of promoting the secretion of SIgA (Secretory Immunoglobulin A) in an intestinal canal. The lactobacillus plantarum is characterized by being separated from koumiss samples collected in China and is collected at China General Microbiological Culture Collection Center of Microbiological Culture Management Committee, the collection number is CGMCC No. 5468, and the collection date is 18th, November 2011. The invention also discloses a detecting method of the lactobacillus plantarum.
Description
Technical field
The present invention relates to probiotic bacterium-Lactobacillus plantarum P8 that a strain can promote SIgA secretion in enteron aisle.
Background technology
Miscellaneous microorganism that living away from home in the gi tract of Healthy People, these microorganisms are called intestinal microflora, compared to the flora of passing by one's way, the most of microorganism for living away from home for a long time of intestinal microflora, intestinal microflora is huge, is approximately 10
14left and right, and formed by the bacterium of quite fixing, and be settled in regularly some privileged sites of health.Between intestinal microflora, between intestinal microflora and host and environment, all the time in dynamic balance state, form one and depend on each other for existence, the system of restriction mutually, thereby it is useful and harmless to human body when body defensive enginery is normal.Normal the gut flora has many important physiological functions, as the antagonistic action to the flora of passing by one's way, to host's immunization, toxin expelling effect, antitumor action, anti-aging effects and other trophism (as diabetes, hypertension, hyperlipidemia etc.), once but there is alteration of intestinal flora, will certainly upset these normal physiological functions.Applying in recent years probiotic bacterium improves alteration of intestinal flora and day by day causes extensive concern.
Secretory immunoglobulin A (SIgA) is the main antibody of the local anti-infectious immunity of body mucous membrane, You Cheng mucous membrane local antibody.It is the main component of body mucous membrane system of defense, and the double focusing IgA mainly being connected into by J chain and SC (secretory piece) are in conjunction with the rear mixture forming, and its molecular weight is about 400kD.People's digestive tube is especially in enteron aisle, a large amount of microorganism that surviving, and time have various pathogenic agent to enter enteric cavity with food, also there are the various food antigens that much do not digested, once these antigenic substances enter blood circulation will activation system immunity, to body, bring potential harm.This just determines that digestive tube must have some barriers when these materials of opposing, and the biological barrier that SIgA forms acts on eliminating antigen aspect and brought into play important effect.SIgA not only can assemble pathogenic agent potential and invasion to be made it to be easy to move its removing by wriggling and mucociliary, and SIgA-immunocomplex can be specifically with intestines PP in M Cell binding transported in pancreatic polypeptide (PP), with the PP cell of some amount as DC, T and bone-marrow-derived lymphocyte selective crosslinking, as being combined with DC and being ingested, can strengthen the immune process that does not have Inflammatory response to follow; Can play another kind of local immunity regulating effect with the SIgA of T Cell binding, cause IL-4 in mucous membrane, IL-10 and TGF-β cytokine to generate.As can be seen here, SIgA enters mucous membrane by PP may play very important immunoregulation effect, to protect the complete of gut barrier.At present SIgA value is because of the difference of detection method, and great disparity is very large, and in existing bibliographical information in the detection technique of SIgA, Elisa technology is the most fast and accurate.
Probiotic bacterium (Probiotic), come from Greek " useful to life ", refer to be colonizated in enteron aisle and bring into play beneficial effect by improving host's intestinal microflora eubiosis, reach and produce definite health efficacy and improve the active bacteria formulation of host health level and the general name of meta-bolites thereof, as far back as eighties of last century, first (1907) famous bacteriologist Nobel Laureate plum Cheney husband of section proposes the hypothesis that probiotic bacterium can be promoted longevity.Up to now, the probiotic bacterium that scientist finds can be divided into three major types substantially, comprising Bacterium lacticum class, bifidus bacillus class and gram-positive cocci (as streptococcus faecium, galactococcus, intermediary suis etc.), wherein Bacterium lacticum class mainly contains Lactobacterium acidophilum, lactobacterium casei, plant lactobacillus etc.And should meet following condition as probiotic bacterium: the normal member that should be normal microflora in enteron aisle; Probiotic bacterium must have survival ability, and can carry out scale operation; Using and duration of storage, should keep existing state and stable; Can field planting in the ecosystem in intestines and possess higher survival rate; Must produce beneficial effect to host.In recent years, more and more on the research report of intestinal microflora impact about probiotic strain, new research means also constantly presents, and probiotic bacterium has become the study hotspot of probiotic bacterium and association area on the impact of intestinal microflora.Probiotic bacterium has many wonderful effects, but these different effect height depend on " specificity of bacterial strain ".That is to say, by a large amount of and long-term clinical research confirmation certain specific probiotic strain there is certain clear and definite function, but and the different strains that does not mean that other probiotic bacteriums of the same race also must have same or similar function, based on this understanding, this experiment studies confirm that the impact of bacterial strain Lactobacillus plantarumP8 on intestinal microflora from many aspects, wherein, by the mensuration to SIgA, indirect reaction Lactobacillus plantarum P8 be beneficial to this fact of secretion of SIgA.
Summary of the invention
The object of this invention is to provide probiotic bacterium and detection method thereof that a strain can promote SIgA secretion in enteron aisle.
Object of the present invention is achieved through the following technical solutions: a strain can promote the secretion of SIgA in enteron aisle probiotic bacterium, it is characterized in that the separated koumiss sample from gathering of described probiotic bacterium (Lactobacillus plantarum P8) within Chinese territory, be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, Classification And Nomenclature: plant lactobacillus, preserving number: CGMCC No.5468, preservation date is: on November 18th, 2011.
Can promote the secretion of SIgA in enteron aisle the detection method of probiotic bacterium, it is characterized in that comprising the following steps:
(1) faecal samples pre-treatment: take 1.0g faecal samples, add 9.0ml PBS (PH7.4), with vibrator, sample is fully shaken even, centrifugal 20min left and right then, rotary speed 2000-3000r/min, carefully collects supernatant;
(2), dilution and the application of sample of standard substance: on the coated plate of enzyme mark, establish 10 holes, standard substance hole, in first, second hole, add respectively standard substance 100 μ l, then in first, second hole, add standard substance diluent 50 μ l, mix; Then from the first hole, the second hole, respectively get 100 μ l and be added to respectively the 3rd hole and the 4th hole, then add respectively standard substance diluent 50 μ l in the 3rd, the 4th hole, mix; Then in the 3rd hole and the 4th hole, first respectively get 50 μ l and discard, respectively get 50 μ l and be added to respectively in the 5th, the 6th hole, then add respectively standard substance diluent 50ul in the 5th, the 6th hole, mix; After mixing, from the 5th, the 6th hole, respectively get that 50 μ l are added to respectively the 7th, in octal, again the 7th, add respectively standard substance diluent 50 μ l in octal, after mixing from the 7th, get respectively 50 μ l octal and be added in the 9th, the tenth hole, in the 90 hole, add respectively standard substance diluent 50 μ l again, after mixing, from the 90 hole, respectively get 50 μ l and discard.(after dilution, each hole application of sample amount is all 50 μ l, and concentration is respectively 24 μ g/ml, 16 μ g/ml, 8 μ g/ml, 4 μ g/ml, 2 μ g/ml);
(3) application of sample: establish respectively blank well and testing sample hole, wherein blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical, on the coated plate of enzyme mark, in testing sample hole, first add sample diluting liquid 40 μ l, and then adding testing sample 10 μ l, the final extent of dilution of sample is 5 times; Application of sample is added on bottom, enzyme plate hole by sample, does not touch hole wall as far as possible, rocks and mixes gently;
(4) incubation: with the rearmounted 37 ℃ of incubation 30min of shrouding film shrouding;
(5) dosing: by 20 times of 30(48T) times concentrated cleaning solution is with standby after 30 times of dilutions of distilled water;
(6) washing: carefully take shrouding film off, discard liquid, dry, washings is filled it up with in every hole, discards after standing 30s, so repeats 5 times, pats dry;
(7) enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
(8) incubation: establish respectively blank well and testing sample hole, wherein blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical, on the coated plate of enzyme mark, in testing sample hole, first add sample diluting liquid 40 μ l, and then adding testing sample 10 μ l, the final extent of dilution of sample is 5 times; Application of sample is added on bottom, enzyme plate hole by sample, does not touch hole wall as far as possible, rocks and mixes gently;
(9) washing: by 20 times of 30(48T) times concentrated cleaning solution is with standby after 30 times of dilutions of distilled water;
(10) colour developing: every hole first adds developer A50 μ l, then adds developer B50 μ l, and light shaking mixes, 37 ℃ of lucifuge colour developing 15min;
(11) stop: every hole adds stop buffer 50 μ l termination reaction, the now blue vertical yellow that turns of liquid color;
(12) measure: with blank air-conditioning zero, the absorbancy that 450nm wavelength is sequentially measured each hole is OD value, and mensuration should be carried out with interior by 15min after adding stop buffer;
(13) calculate: the concentration of standard substance of take is X-coordinate, OD value is ordinate zou drawing standard curve, and according to the concentration of standard substance and OD value, calculate the linear regression equation of typical curve, by the OD value substitution equation of sample, calculate sample concentration, be multiplied by again extension rate, be the actual concentrations of sample.
Effect of the present invention is: the separated koumiss sample from gathering within Chinese territory of Lactobncillus plantarum P8 is that a strain is through the probiotic bacterium of systematic study.It can suppress intestinal tract infections and alleviate the symptom of IBS, thereby plays a protective role, simultaneously; its energy biologically active secretory molecule, challenge, can be used as living vaccine in medically widespread use; due to these characteristics, it has been used to the suitability for industrialized production of starter and milk-product.This studies to take SIgA content in Lactobacillus plantarum P8 rear intestinal is research index, and assessing this probiotic bacterium affects intestinal mucosa SIgA.
The present invention is by taking probiotic bacterium Lactobacillus plantarum P8 tablet to experimenter, application Elisa technology detects the amount of SIgA in different times experimenter enteron aisle, Elisa result shows: take the secretion that this bacterial strain can promote intestinal epithelial cell SIgA every day, assembling pathogenic agent potential and invasion makes it to be easy to move its removing by wriggling and mucociliary, and SIgA-immunocomplex by pancreatic polypeptide can be specifically with intestines pancreatic polypeptide in M Cell binding transported in pancreatic polypeptide, with the pancreatic polypeptide cell of some amount as DC, T and bone-marrow-derived lymphocyte selective crosslinking, as being combined with DC and being ingested, can strengthen the immune process that does not have Inflammatory response to follow.SIgA also can play another kind of local immunity regulating effect with T Cell binding, causes IL-4 in mucous membrane, IL-10 and the generation of TGF-β cytokine to enter mucous membrane and may play very important immunoregulation effect, thereby protected the complete of gut barrier.
Below in conjunction with drawings and Examples, the present invention is described further.
Accompanying drawing explanation
Fig. 1 is bile acid determination canonical plotting;
Fig. 2 probiotic bacterium Lactobacillus plantarum P8 is on volunteer SIgA secretion level impact figure.
Embodiment
One strain can promote the secretion of SIgA in enteron aisle probiotic bacterium, the separated koumiss sample from gathering within Chinese territory of described probiotic bacterium (Lactobacillus plantarum P8), be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number: CGMCC No.5468, preservation date is: on November 18th, 2011.
Can promote the secretion of SIgA in enteron aisle the detection method of probiotic bacterium, it is characterized in that comprising the following steps:
1. the collection of test sample
33 healthy volunteers (specifying information is in Table 1) are collected in this test altogether, are divided into three groups, and young group (Ygroup, 11 people, the male sex 5 people, women 6 people), in age 25-29 year, the mean age is 26.2 ± 1.2 years old; Middle age group (M group, 12 people, the male sex 6 people, women 6 people), age 48-53 year, the mean age is 50.9 ± 1.4; Old group (E group, 10 people, the male sex 5 people, women 5 people), age 71-80 year, the mean age is 75.1 ± 3.3 years old.M-F is 16:17.Each volunteer chews 3, food Lactobacillus plantarum P8 tablet every day after meal, and every contains Lactobacillus plantatum P8 viable bacteria 2 * 1010CFU, takes continuously after 28 days and cut out.Respectively at the 0th day, 14 days, 28 days, and cut out latter 7 days (continuous 35 days), 14 days (continuous 42 days), 28 days (continuous 56 days) gather volunteer's ight soil.After sample collecting, should test as early as possible, if can not test at once, sample can be put in to-20 ℃ of preservations, and avoid multigelation.
Table 1 volunteer information
2. faecal samples pre-treatment: take 1.0g faecal samples, add 9.0ml PBS (PH7.4), with vibrator, sample is fully shaken even, centrifugal 20min left and right (2000-3000r/min) then.Carefully collect supernatant.
3. the dilution of standard substance and application of sample: on the coated plate of enzyme mark, establish 10 holes, standard substance hole, in first, second hole, add respectively standard substance 100 μ l, then in first, second hole, add standard substance diluent 50 μ l, mix; Then from the first hole, the second hole, respectively get 100 μ l and be added to respectively the 3rd hole and the 4th hole, then add respectively standard substance diluent 50 μ l in the 3rd, the 4th hole, mix; Then in the 3rd hole and the 4th hole, first respectively get 50 μ l and discard, respectively get 50 μ l and be added to respectively in the 5th, the 6th hole, then add respectively standard substance diluent 50ul in the 5th, the 6th hole, mix; After mixing, from the 5th, the 6th hole, respectively get that 50 μ l are added to respectively the 7th, in octal, again the 7th, add respectively standard substance diluent 50 μ l in octal, after mixing from the 7th, get respectively 50 μ l octal and be added in the 9th, the tenth hole, in the 90 hole, add respectively standard substance diluent 50 μ l again, after mixing, from the 90 hole, respectively get 50 μ l and discard.(after dilution, each hole application of sample amount is all 50 μ l, and concentration is respectively 24 μ g/ml, 16 μ g/ml, 8 μ g/ml, 4 μ g/ml, 2 μ g/ml).
4. application of sample: establish respectively blank well (blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole.On the coated plate of enzyme mark, in testing sample hole, first add sample diluting liquid 40 μ l, and then to add the final extent of dilution of testing sample 10 μ l(sample be 5 times).Application of sample is added on bottom, enzyme plate hole by sample, does not touch hole wall as far as possible, rocks and mixes gently.
5. incubation: with the rearmounted 37 ℃ of incubation 30min of shrouding film shrouding.
6. dosing: by 20 times of 30(48T) times concentrated cleaning solution is with standby after 30 times of dilutions of distilled water.
7. washing: carefully take shrouding film off, discard liquid, dry, washings is filled it up with in every hole, discards after standing 30s, so repeats 5 times, pats dry.
8. enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
9. incubation: operation is with 3.
10. washing: operation is with 5.
11. colour developings: every hole first adds developer A50 μ l, then adds developer B50 μ l, and light shaking mixes, 37 ℃ of lucifuge colour developing 15min.
12. stop: every hole adds stop buffer 50 μ l, termination reaction (the now blue vertical yellow that turns).
13. measure: with blank air-conditioning zero, 450nm wavelength is sequentially measured the absorbancy (OD value) in each hole.Mensuration should be carried out with interior by 15min after adding stop buffer.
14. calculate: the concentration of standard substance of take is X-coordinate, OD value is ordinate zou drawing standard curve, and according to the concentration of standard substance and OD value, calculate the linear regression equation of typical curve, by the OD value substitution equation of sample, calculate sample concentration, be multiplied by again extension rate, be the actual concentrations of sample.
15. results: canonical plotting is shown in Fig. 1.
SIgA quantitative result: by the measurement result of SIgA in all age group volunteer enteron aisle as Fig. 2 (note: " * * * " represents conspicuous level P<0.0001).Before not taking probiotic bacterium Lactobacillus plantarum P8, the old volunteer of SIgA> middle age volunteer > of youth volunteer's intestinal epithelial cell secretion.Take after Lactobacillus plantarum P8, in youth volunteer's ight soil, SIgA content is not remarkable.And middle aged volunteer and old volunteer took this probiotic bacterium after 14 days, in ight soil, SIgA content significantly increases, but along with taking the growth of this probiotic bacterium time, in ight soil, SIgA content does not continue to increase, but fall after rise to some extent, finally return to the level while not taking.
Claims (1)
1. a strain can promote plant lactobacillus (Lactobacillus plantarum) P8 of SIgA secretion in enteron aisle, it is characterized in that, strains separation is from the koumiss sample gathering within Chinese territory, and preserving number is CGMCC No.5468, and preservation date is: on November 18th, 2011.
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