CN106755470A

CN106755470A - A kind of method of probiotics species and content in utilization Q PCR detections mixing probiotics

Info

Publication number: CN106755470A
Application number: CN201710029477.6A
Authority: CN
Inventors: 于大海; 房学迅; 杨吉雨; 胡健微
Original assignee: Pu Sheng Tai Biotechnology Co ltd Of Jilin Province
Current assignee: Pu Sheng Tai Biotechnology Co ltd Of Jilin Province
Priority date: 2017-01-16
Filing date: 2017-01-16
Publication date: 2017-05-31

Abstract

The invention provides a kind of method for detecting thalline species and content in mixing probiotics.Methods described is to be inoculated into MRS lactic acid bacteria culture mediums and fermented by by mixing probiotics strain.Mixed fermentation bacterium solution genome is extracted, the special primer of contained various probiotics, carries out Q PCR in addition zymotic fluid（Quantitative fluorescent PCR）.The foundation of system is used to detect the quantitative PCR method of Lactobacillus salivarius, lactobacillus fermenti, Lactobacillus plantarum, Lactobacillus rhamnosus, bifidobacterium breve, lactobacillus paracasei, Lactobacillus casei, excrement intestines lactobacillus, bafillus natto, lactobacillus bulgaricus and gut flora total amount.Whether each bacterial strain grows outward during the method for the invention decapacitation quickly determines mixed fermentation system, moreover it is possible to quick and precisely detect the quantity of each probiotics in mixed fermentation liquid.After the method can be used for thalline mixed fermentation, growing state and increment to various thalline in zymotic fluid make effective evaluation.

Description

A kind of method of probiotics species and content in utilization Q-PCR detections mixing probiotics

Technical field

The present invention relates to field of microbial fermentation, and in particular to application quantitative fluorescent PCR (Q- after lactobacillus mixed fermentation PCR) the method that detection mixes probiotics species and content in probiotics.After the method can be used for thalline mixed fermentation, to hair The growing state and increment of various thalline make qualitative, quantitative effective evaluation in zymotic fluid.

Background technology

Probiotics is that a class is colonized in human body intestinal canal, reproductive system, can produce definite health efficacy so as to improve host Microecological balance, the general name of the microorganism of the work played an important role to health is widely used in food, health products and medicine In production.In recent years, the research of enteric microorganism is increasingly paid attention to by living nature, and sudden emergence becomes the research of hot topic Field.There is now a large amount of beneficial bacteriums offered in report enteric microorganism at aspects such as prevention and treatment disease, improvement gastroenteric environments There is remarkable effect.The most powerful product mainly composite reactive probiotics of the current function of studying in the world, it is widely used in Bioengineering, industrial or agricultural, food security and life and health field.Present invention research finds that compound lactobacillus ferment can be adjusted The balance of microorganism and the absorption of nutriment can also be promoted in enteron aisle.

Under normal conditions, the profitable strain and pernicious bacteria in our enteron aisles are in a state for relative equilibrium , they mutually restrict, interdependently coexist, and maintain the balance of human body Tiny ecosystem.Once intestinal flora disequilibrium, When i.e. pernicious bacteria is dominant, protein and other materials in food can be converted to harmful substance, and these materials can cause Some diseases occur, and most common is exactly constipation, diarrhoea etc..The generation of constipation is closely bound up with the pH value of colon, and beneficial bacterium leads to The pH value for crossing reduction colon is allowed to be partial to acidity, reduces the quantity of pernicious bacteria, the normal creepage of gastrointestinal functions of regulating intestinal canal, so as to improve Constipation.Diarrhoea is the extremely extreme symptom of excretion that intestinal flora disorder causes, and diarrhoea can cause dehydration and body electricity when serious Solution matter disorder etc., long-term chronic diarrhea also results in anaemia, hypovitaminosis, malnutrition, resistance decline etc..Probiotics Diarrhoea can rapidly be alleviated, acid is produced, an environment for being unfavorable for that harmful virus and bacterium are present is createed, suppress pathogenic microorganism Existence and growth；Close microbial film is formed in intestinal mucosa, stops pathogenic bacteria invasion.

In the acidified milk sample being combined to many bacterium at present the qualitative and quantitative analysis aspect of lactobacillus lack science, rationally, it is fast The detection method of speed.Traditional Physiology and biochemistry method is easily influenceed detection to imitate by factors such as Selective agar medium, condition of culture, strain properties Rate is restricted.Viable lactic acid bacteria quantity is to evaluate the important indicator of biodiasmin combinations quality, and the method for plate culture count is micro- A kind of most common in biology, most standard viable count method, but cumbersome time and effort consumings.In molecular Biological Detection In method, quantitative fluorescent PCR technology more because with it is quick, can quantify, sensitivity is high, it is reproducible, life or death bacterium can be detected The features such as become one of important molecular biology method in intestinal microecology research.

Using fluorescence quantitative PCR detection mix probiotics, quantitative fluorescent PCR technology because have quickly, can quantify, it is sensitive Degree is high, reproducible, become intestinal microecology research the features such as life or death bacterium can be detected in important molecular biology method One of.Although existing both at home and abroad much set up the report with application on quantitative PCR detection method, setting up for system is various The research of gut flora quantitative PCR detecting method is less.Therefore, it is purpose base that the present invention is studied with enteric bacteria 16S rRNA Cause, introduces the SYBR-Green I fluorescent dye smaller on PCR reaction influences, it is intended to which the foundation of system is used to detect saliva breast Bacillus, lactobacillus fermenti, Lactobacillus plantarum, Lactobacillus rhamnosus, bifidobacterium breve, lactobacillus paracasei, Lactobacillus casei, excrement The quantitative PCR method of intestines lactobacillus, bafillus natto, lactobacillus bulgaricus and gut flora total amount, to be intestines The research of road flora provides detection means that is quick, special, can quantifying, is the related wholesomeness evaluation of microorganism, prebiotic Bacterium is developed and other researchs relevant with gut flora provide technical support.

The content of the invention

In order to solve the problems, such as prior art, it is an object of the invention to provide strain in one kind detection mixing probiotics The method of type and content.

In order to realize the object of the invention, the invention provides one kind using types of spawn in Q-PCR detection mixing probiotics With the method for content, methods described is：Mixing probiotics is fermented in MRS culture mediums, Q-PCR detections are carried out.

Preceding method concrete technical scheme is comprised the following steps：

（1）MRS culture mediums are configured：Precise glucose, peptone, beef extract, yeast extract, sodium acetate, dipotassium hydrogen phosphate, The medicines such as diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, Tween-80, stirring to dissolving, 115 DEG C of 30 min of sterilizing；

（2）The fermented and cultured of probiotics：Each strain is individually cultivated into obtain seed liquor, by seed liquor etc. than adding to sterilizing after mixing In MRS culture mediums；

（3）The design of primer：According to DNA specific genes fragment design primer in every kind of probiotics 16S rRNA；

（4）The extraction of genome：Mixed Microbes genome is extracted using Easy Bacteria Genomic DNA Kit

（5）Quantitative fluorescent PCR：The parameters such as fluorescence threshold, PCR conditions are set, and measurement obtains Ct values, is converted into viable count.

Present invention accurate detection probiotics in probiotics is mixed with utilization fluorescence quantifying PCR method.Design specificity is drawn Thing, can accurately detect growth and the viable count of probiotics, so that probiotics micro-ecological formulation is prepared for after provides quantitative Foundation.

Brief description of the drawings

Fig. 1：Lactobacillus plantarum, Lactobacillus casei, lactobacillus bulgaricus, excrement intestines lactobacillus, lactobacillus fermenti, saliva breast Bacillus, the zymogenic viable count of bafillus natto single bacterium.

Fig. 2:Lactobacillus plantarum, Lactobacillus casei, lactobacillus bulgaricus, excrement intestines lactobacillus, lactobacillus fermenti, saliva Result after lactobacillus, bafillus natto PCR.

Fig. 3:The result of fluorescence quantifying PCR method detection mixing probiotics.

Specific embodiment

Below by way of the specific embodiment of example forms, the present invention is described in further detail.

Embodiment 1:

（1）Mix the fermentation of probiotics：Precisely weigh composition in MRS culture mediums（Glucose, peptone, beef extract, yeast extract, Sodium acetate, dipotassium hydrogen phosphate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, Tween-80）115 DEG C of 30 min of sterilizing.By each bacterium Plant and individually cultivate to obtain seed liquor, after the ratio such as seed liquor is mixed, according to 3%（V:V）Inoculation ratio be inoculated into MRS fermentation mediums In, ferment 12 hours；

（2）Genome is extracted：The fermentation liquid that step (1) is prepared takes out 1ml, and 12000 turns/min is centrifuged 1 minute, abandons Supernatant.First with the ethanol of 500 μ L 70% that thalline is resuspended, ice bath 20min adds lysozyme resuspended, 37 DEG C of 60 min of incubation, plus Enter protease, 55 DEG C of incubation 15min add RnaseA to stand 2 min, add the μ L of Binding Buffer 400 to mix, add To in centrifugal column, efflux is discarded, successively washed twice with Wash Buffer and Clean Buffer, use Elution Buffer eluted dnas.The genome of extraction is detected with 1% agarose gel electrophoresis；

（3）The design of primer：The 16SrDNA sequences of various thalline are obtained by website NCBI, is found according to BLAET comparing results Go out the specific fragment of each probiotics, corresponding primer is designed using software Primer5；

（4）The foundation of standard curve：By taking Lactobacillus rhamnosus as an example, using Lactobacillus rhamnosus reference culture, according to 1）Middle institute Cultural method training status bacterial strain is stated, according to 2）The middle specific method for extracting genome, obtains Lactobacillus rhamnosus genome.Make Enter performing PCR with Lactobacillus rhamnosus special primer, obtain a large amount of Lactobacillus rhamnosus specific DNA fragments, 2% Ago-Gel electricity Swimming detection PCR stripe sizes, and accurate cutting rod band, by glue reclaim obtaining the PCR bands of high-purity.Use NANODROP 1000^TMUV/VIS Spectrophotometer determine glue reclaim production concentration.Gradient dilution is carried out to the glue reclaim product, Each gradient after dilution carries out quantitative fluorescent PCR simultaneously, sets up with log (DNA copy number/μ L) as abscissa, Ct values are sat to indulge Target standard curve；Copy number computing formula is as follows：Copy number=【6.02×10²³× glue reclaim production concentration（g/μL）× template Volume（μL）】/【660 × PCR fragment base number（bp）】

（5）Fluorescence quantitative PCR detection：Mixutre genome is diluted 10⁵Times, quantitative fluorescent PCR reaction system is the μ L of template 1,10 μ The μ L of M sense primers 0.4,10 μM of the μ L of anti-sense primer 0.4, superReal PreMix plus10 μ L, RoXReference Dye 0.4μL、ddH2O 7.8μL.The μ L of PCR volumes 20.Quantitative fluorescent PCR：94 DEG C of predegenerations are denatured 5 seconds, 60 DEG C of annealing for 30 seconds, 94 DEG C Extend 10 seconds within 34 seconds, 72 DEG C, denaturation annealing extends circulation 40 times, obtains Ct values, and viable bacteria is obtained according to standard curve and positive control Number.

Claims

1. application of a kind of detection method in mixing probiotics species and content context of detection.

2. application in the way of fluorescence quantifying PCR method is to detect composite probiotics ferment liquid such as in claim 1.

3. such as in claim 1 fluorescence quantifying PCR method detecting the various solid-liquid shapes of compound probiotic granule tablet The mode application of formula.