CN102618459A - Lactobacillus plantarum P8 capable of regulating human intestinal flora and detection method of Lactobacillus plantarum P8 - Google Patents
Lactobacillus plantarum P8 capable of regulating human intestinal flora and detection method of Lactobacillus plantarum P8 Download PDFInfo
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Abstract
The invention relates to Lactobacillus plantarum P8 capable of regulating human intestinal floras, which is characterized in that the Lactobacillus plantarum P8 is separated from yoghourt made by hand by Inner Mongolia shepherds and is collected in the China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms on November 18, 2011, with the collection number being CGMCC No.5468. The invention additionally discloses a detection method of the Lactobacillus plantarum P8.
Description
Technical field
The present invention relates to probiotic bacterium-Lactobacillus plantarum P8 and detection method thereof that a strain can mediator's intestinal microflora.Using the RT-qPCR technology for detection finds: take this bacterial strain every day and can promote profitable strain genus bifidobacterium in the enteron aisle, regulate the intestinal microflora ratio thereby the growth of pula fusobacterium and unusual Pseudomonas reaches, improve the effect of intestinal microflora structure.
Background technology
Have about 4 in health adult's intestines; More than 400 kind of mikrobe; Wherein 99% mikrobe only is made up of 30～40 kinds of bacteriums, is mainly bacterioide, Bifidobacterium, clostridium, enterococcus bacteria, Eubacterium, fusobacterium, Peptostreptococcus, milk-acid bacteria and escherichia etc.The field planting of enteron aisle normal microflora forms with host's long-term evolution process at host's intestinal mucosa.Wherein a part is specific dominant population, and another part is general population.Source according to enteric microorganism can be divided into ancestral home bacterium, foreign nationality bacterium and symbiosis flora.They and host environment form and interdepend, the whole of mutual restriction.Under the normal physiological situation, mainly show as the microflora that is of value to the host, but under pathologic condition, also possibly show as host's harmful microorganism group.
Edible contain probiotic food and can reach the purpose of keeping gastrointestinal health, received people's attention.Kind surplus the probiotic bacterium that China's approval is at present used in food has 10 mainly is probiotic lactobacillus and bifidus bacillus.Research shows that probiotic bacterium has multiple physiological hygiene function and comprises the pair cell immunologic enhancement, handles lactose intolerance, reduces serum cholesterol, regulates the human intestinal microflora balance, promotes that gastrointestinal peristalsis prevents constipation, helps digest; Antianaphylaxis; Synthesizing amino acid and vitamin B complex; Suppress carcinogenic substance and other harmful microorganism; Promote the growth of probiotics in the body.Gi tract probiotic bacterium physiological function is still waiting further exploitation, and we can further understand the interaction at enteron aisle of probiotic bacterium and intestinal microflora through future studies.
The investigator has found that probiotic bacterium undertaking multiple important physical function, has the effect of keeping the human body microecological balance very close with healthy relation.Yet in probiotic bacterium numerous physiological function and effect, most important function remains its influence to human intestinal environment and intestinal function, " the whole intestines effect " that promptly it has often been said." whole intestines effect " is meant the food through selecting einnehmen function property probiotic bacterium and being made by these functional probiotic bacteriums; Can improve enteric microorganism and form, increase intestines peristalsis, reduce corrupt substance in the enteron aisle; Make defecation be in perfect condition, promote and safeguard intestinal health.
Summary of the invention
The purpose of this invention is to provide probiotic bacterium and detection method thereof that a strain can mediator's intestinal microflora
The object of the invention is realized through following technical scheme: the probiotic bacterium that a strain can mediator's intestinal microflora; It is characterized in that said probiotic bacterium (Lactobacillus plantarum P8) separates in the herdsman's home built sour milk of the Inner Mongol; Be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms; Preservation address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; The classification name: plant lactobacillus, preserving number: CGMCC No.5468, preservation date is: on November 18th, 2011.
The detection method of probiotic bacterium that can mediator's intestinal microflora is characterized in that comprising the following steps:
(1) extraction of macro genome DNA in the faecal samples:
Adopt frozen-thawed-CTAB method: get 1.0g faecal samples carrying out washing treatment, in the 1.5mL centrifuge tube, place liquid nitrogen to freeze fully immediately the thalline of wash-out; Putting into 65 ℃ of water-baths after the taking-up melts; Multigelation 3 times adds 0.1mL 10%SDS and 10.0 μ L 10mg/mL Proteinase Ks, and 200r/min shakes 2h in 37 ℃ of constant temperature shaking tables; The centrifugal 10min of 12000g under the room temperature collects supernatant and is transferred in another centrifuge tube; Supernatant and isopyknic chloroform are in the centrifugal 10min of 12000g; The absorption supernatant is transferred to and carries out phenol chloroform extracting 2 times in another centrifuge tube, through the sodium-acetate of 0.1 times of volume, the total DNA of ice isopropanol precipitating of 1 times of volume; 70% washing with alcohol deposition is 2 times then, returns molten subsequent use;
(2) enteron aisle dominant microflora Auele Specific Primer design
To genus bifidobacterium, Bacteroidetes, synthetic four group-specific primerses of the Pseudomonas of adorning and unusual Pseudomonas design, thalline primer information sees the following form:
Amplification system:
Reaction system 50 μ L:10 * PCR Buffer 5 μ L, 25mmo1/L Mg
2+1.0 μ L, 2.5mmol/LdNTPs 4 μ L, each 2 μ L of 5mmol/L upstream and downstream primer, about dna profiling 100ng, 5U/ μ L Taq enzyme (Promega) 0.5uL, dd H
2O complements to 50 μ L;
Amplification condition:
95 ℃ of sex change 5min; Circulate 30 times: 95 ℃ of sex change 1min, annealing 45s, 72 ℃ are extended 1min, and 72 ℃ are extended 7min, 4 ℃ of preservations then;
(3) agarose gel electrophoresis
With PCR product voltage with 5V/cm in 1% sepharose, electrophoresis 20-30min, EB dyeing, ultraviolet is taken pictures, and detects expanding effect;
(4) preparation of outer standard substance
Application specific primer amplification genus bifidobacterium, Bacteroides, the specific fragment of pula fusobacterium and unusual Pseudomonas detects back glue and reclaims the purpose that fragment separately reaches purifying; Then the fragment with purifying is connected with the pMD-19T carrier; Be transformed in the competent escherichia coli cell after the connection; Select the sub-enzyme of positive colony and cut checking; Positive colony of verifying is extracted plasmid, calculate copy number after the mensuration concentration as of the quantitative use of outer standard substance for this Pseudomonas in the follow-up test unknown sample;
(5) quantity of each superiority bacteria spp in the RT-PCR test sample
At first using above-mentioned outer standard substance and make the standard amplification curve of each Pseudomonas, is foundation with the typical curve, with genus bifidobacterium in the Auele Specific Primer detection by quantitative sample of each Pseudomonas, Bacteroides, the quantity of pula fusobacterium and unusual Pseudomonas then;
The RT-PCR amplification system:
Reaction system 20 μ L:10 * PCR mix 10 μ L, each 0.4 μ L of 10mmol/L upstream and downstream primer, about dna profiling 100ng, dd H
2O complements to 20 μ L;
The RT-PCR amplification condition:
95 ℃ of sex change 20s; Circulate 40 times: 95 ℃ of sex change 5s, annealing 30s, 72 ℃ are extended 35s.
The invention has the beneficial effects as follows: Lactobacillus plantarum P8 separates in the herdsman's home built sour milk of the Inner Mongol, is the probiotic bacterium of a strain through systematic study.It has immunomodulatory properties, and has been used to the suitability for industrialized production of starter and milk-product, and as living vaccine treatment widely is being provided medically.The present invention collects the volunteer and takes Lactobacillus plantarum P8 tablet, takes the content of other superiority bacteria spp in this bacterial strain different steps enteron aisle through mensuration and probes into adjusting and the improvement of Lactobacillus plantarum P8 to the quantity and the ratio of people's enteron aisle autochthonous flora.
Below in conjunction with accompanying drawing and embodiment the present invention is done further explanation.
Description of drawings
Fig. 1 is the Auele Specific Primer electrophorogram;
The sub-plasmid electrophorogram of the positive clone of Fig. 2;
Fig. 3 is the standard amplification curve diagram of each Pseudomonas;
Fig. 4 is the influence figure of probiotic bacterium Lactobacillus plantarum P8 to bifidus bacillus quantity in the enteron aisle;
Fig. 5 is the influence figure of Lactobacillus plantarum P8 to pula clostridium, unusual Pseudomonas and bacterioide quantity in the enteron aisle.
Embodiment
One strain can mediator's intestinal microflora probiotic bacterium; Said probiotic bacterium (Lactobacillus plantarum P8) separates in the herdsman's home built sour milk of the Inner Mongol; 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.
The detection method of probiotic bacterium that can mediator's intestinal microflora is characterized in that comprising the following steps:
1. the collection of test sample
33 healthy volunteers (specifying information is seen table 1) are collected in this test altogether, are divided into three groups, young group (Ygroup, 11 people, the male sex 5 people, women 6 people), and 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 in food Lactobacillus plantarum P8 tablet every day after meal, and every contains Lactobacillus plantarum P8 viable bacteria 2 * 10
10CFU takes continuously after 28 days and cuts out.Respectively at the 0th day, 14 days, 28 days, and volunteer's ight soil is gathered in cut out back 7 days (continuous 35 days), 14 days (continuous 42 days), 28 days (continuous 56 days).Put into the liquid nitrogen quick-frozen behind the sample collecting rapidly, and in 48 hours, accomplish the extraction of faecal samples macro genome DNA.
2. the extraction of macro genome DNA in the faecal samples:
Adopt frozen-thawed-CTAB method: get 1.0g faecal samples carrying out washing treatment, in the 1.5mL centrifuge tube, place liquid nitrogen to freeze fully immediately the thalline of wash-out; Put into 65 ℃ of water-baths after the taking-up and melt (about 5min); Multigelation 3 times adds 0.1mL 10%SDS and 10.0 μ L 10mg/mL Proteinase Ks, and 200r/min shakes 2h in 37 ℃ of constant temperature shaking tables; The centrifugal 10min of 12000g under the room temperature collects supernatant and is transferred in another centrifuge tube.Supernatant and isopyknic chloroform are in the centrifugal 10min of 12000g (in order to make deposition, water and organic phase layering); The absorption supernatant is transferred to and carries out phenol chloroform extracting 2 times in another centrifuge tube; Sodium-acetate through 0.1 times of volume; The total DNA of ice isopropanol precipitating of 1 times of volume, 70% washing with alcohol deposition is 2 times then, returns molten subsequent use.
3. the preparation of outer standard substance
Application specific primer amplification genus bifidobacterium, Bacteroides, the specific fragment of pula fusobacterium and unusual Pseudomonas (is seen Fig. 1; Annotate: among Fig. 1, the unusual Pseudomonas of A, B pula fusobacterium; The C Bacteroides; D genus bifidobacterium, M DL2000 marker), detect back glue and reclaim the purpose that fragment separately reaches purifying.Then the fragment with purifying is connected with the pMD-19T carrier, is transformed into after the connection in the competent escherichia coli cell, selects the sub-enzyme of positive colony and cuts checking; With the positive colony verified extract plasmid (see Fig. 2, annotate: among Fig. 2, the unusual Pseudomonas of A; B pula fusobacterium, C Bacteroides, D genus bifidobacterium; M DL2000 marker), calculate copy number after the mensuration concentration as of the quantitative use of outer standard substance for this Pseudomonas in the follow-up test unknown sample.
4.RT-qPCR genus bifidobacterium in the test sample, Bacteroides, the quantity of pula fusobacterium and unusual Pseudomonas
At first using above-mentioned outer standard substance and make the standard amplification curve (see figure 3) of each Pseudomonas, is foundation with the typical curve, with genus bifidobacterium in the Auele Specific Primer detection by quantitative sample of each Pseudomonas, Bacteroides, the quantity of pula fusobacterium and unusual Pseudomonas then.
The RT-PCR amplification system:
Reaction system 20 μ L:10 * PCR mix 10 μ L, each 0.4 μ L of 10mmol/L upstream and downstream primer, about dna profiling 100ng, dd H
2O complements to 20 μ L.
The RT-PCR amplification condition:
95 ℃ of sex change 20s; Circulate 40 times: 95 ℃ of sex change 5s, annealing 30s, 72 ℃ are extended 35s.
Genus bifidobacterium in the different times sample, Bacteroides, the quantitative result of pula fusobacterium and unusual Pseudomonas are seen Fig. 4, Fig. 5 respectively.Can find out that from Fig. 4 and Fig. 5 using the species specificity primer carries out quantitative analysis to the dominant microflora in volunteer's enteron aisle of taking probiotic bacterium Lactobacillus plantarum P8; Analytical results shows that using bifidus bacillus kind Auele Specific Primer carries out quantitative examination to the Bifidobacterium in volunteer's enteron aisle of taking probiotic bacterium Lactobacillus plantarum P8; Analytical results shows takes behind this bacterial strain that the average quantity of Bifidobacterium significantly increases in volunteer's enteron aisle; And after cutting out this bacterial strain; The quantity of Bifidobacterium does not fall after rise, still maintains (9.47 ± 0.26Log on the higher order of magnitude
10CFU/g).Explain that probiotic bacterium Lactobacillus plantarum P8 can promote the growth of Bifidobacterium in the enteron aisle.Find the lower (8.78 ± 0.33Log of Bifidobacterium quantity in old volunteer's enteron aisle through measuring
10CFU/g); After taking Lactobacillus plantarum P8; The interior Bifidobacterium quantity increase of enteron aisle is (p＜0.01) extremely significantly, even cut out this bacterial strain after 28 days, Bifidobacterium quantity still can reach 9.36 ± 0.35Log in the elderly's enteron aisle
10CFU/g explains that probiotic bacterium Lactobacillus plantarum P8 is bigger to the elderly's influence power.Bibliographical information; Seriously lack the pula clostridium in the cd patient enteron aisle; And interior this bacterial classification of volunteer's enteron aisle significantly increases (Fig. 5) after taking probiotic bacterium Lactobacillus plantarum P8, explains that probiotic bacterium Lactobacillu splantarum P8 has excellent prevention and result of treatment to Crohn's disease.
Claims (2)
1. the probiotic bacterium that a strain can mediator's intestinal microflora; It is characterized in that said probiotic bacterium (Lactobacillus plantarum P8) separates in the herdsman's home built sour milk of the Inner Mongol; 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.
2. the detection method of probiotic bacterium that can mediator's intestinal microflora is characterized in that comprising the following steps:
(1) extraction of macro genome DNA in the faecal samples:
Adopt frozen-thawed-CTAB method: get 1.0g faecal samples carrying out washing treatment, in the 1.5mL centrifuge tube, place liquid nitrogen to freeze fully immediately the thalline of wash-out; Putting into 65 ℃ of water-baths after the taking-up melts; Multigelation 3 times adds 0.1mL 10%SDS and 10.0 μ L 10mg/mL Proteinase Ks, and 200r/min shakes 2h in 37 ℃ of constant temperature shaking tables; The centrifugal 10min of 12000g under the room temperature collects supernatant and is transferred in another centrifuge tube; Supernatant and isopyknic chloroform are in the centrifugal 10min of 12000g; The absorption supernatant is transferred to and carries out phenol chloroform extracting 2 times in another centrifuge tube, through the sodium-acetate of 0.1 times of volume, the total DNA of ice isopropanol precipitating of 1 times of volume; 70% washing with alcohol deposition is 2 times then, returns molten subsequent use;
(2) enteron aisle dominant microflora Auele Specific Primer design
To genus bifidobacterium, Bacteroidetes, synthetic four group-specific primerses of the Pseudomonas of adorning and unusual Pseudomonas design, thalline primer information sees the following form:
Amplification system:
Reaction system 50 μ L:10 * PCR Buffer 5 μ L, 25mmo1/L Mg
2+1.0 μ L, 2.5mmol/LdNTPs 4 μ L, each 2 μ L of 5mmol/L upstream and downstream primer, about dna profiling 100ng, 5U/ μ L Taq enzyme (Promega) 0.5uL, dd H
2O complements to 50 μ L;
Amplification condition:
95 ℃ of sex change 5min; Circulate 30 times: 95 ℃ of sex change 1min, annealing 45s, 72 ℃ are extended 1min, and 72 ℃ are extended 7min, 4 ℃ of preservations then;
(3) agarose gel electrophoresis
With PCR product voltage with 5V/cm in 1% sepharose, electrophoresis 20-30min, EB dyeing, ultraviolet is taken pictures, and detects expanding effect;
(4) preparation of outer standard substance
Application specific primer amplification genus bifidobacterium, Bacteroides, the specific fragment of pula fusobacterium and unusual Pseudomonas detects back glue and reclaims the purpose that fragment separately reaches purifying; Then the fragment with purifying is connected with the pMD-19T carrier; Be transformed in the competent escherichia coli cell after the connection; Select the sub-enzyme of positive colony and cut checking; Positive colony of verifying is extracted plasmid, calculate copy number after the mensuration concentration as of the quantitative use of outer standard substance for this Pseudomonas in the follow-up test unknown sample;
(5) quantity of each superiority bacteria spp in the RT-PCR test sample
At first using above-mentioned outer standard substance and make the standard amplification curve of each Pseudomonas, is foundation with the typical curve, with genus bifidobacterium in the Auele Specific Primer detection by quantitative sample of each Pseudomonas, Bacteroides, the quantity of pula fusobacterium and unusual Pseudomonas then;
The RT-PCR amplification system:
Reaction system 20 μ L:10 * PCR mix 10 μ L, each 0.4 μ L of 10mmol/L upstream and downstream primer, about dna profiling 100ng, dd H
2O complements to 20 μ L;
The RT-PCR amplification condition:
95 ℃ of sex change 20s; Circulate 40 times: 95 ℃ of sex change 5s, annealing 30s, 72 ℃ are extended 35s.
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| CN102864096A (en) * | 2012-04-18 | 2013-01-09 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum and preparation method thereof for high-density culture and freeze-drying bacteria powder |
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| CN107480474A (en) * | 2017-08-01 | 2017-12-15 | 山东师范大学 | Grader modeling evaluation method of calibration and system based on gut flora abundance |
| CN108949630A (en) * | 2018-07-27 | 2018-12-07 | 内蒙古农业大学 | A kind of koumiss leavening and its preparation method and application |
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| CN109652571A (en) * | 2019-01-25 | 2019-04-19 | 山东大学 | A set of fast qualitative, six kinds of enteric microorganism of quantitative detection primer sets and its application |
| CN110699468A (en) * | 2019-10-29 | 2020-01-17 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting human intestinal bacteria |
| CN110699468B (en) * | 2019-10-29 | 2023-05-02 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting human intestinal bacteria |
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