CN110438042A - One plant of bifidobacterium longum and its application - Google Patents
One plant of bifidobacterium longum and its application Download PDFInfo
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- CN110438042A CN110438042A CN201910664408.1A CN201910664408A CN110438042A CN 110438042 A CN110438042 A CN 110438042A CN 201910664408 A CN201910664408 A CN 201910664408A CN 110438042 A CN110438042 A CN 110438042A
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- bifidobacterium longum
- bifidobacterium
- intestinal flora
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The present invention relates to microorganism fields, specifically disclose one plant of bifidobacterium longum (Bifidobacterium longum) W68 and its application, the bacterium is isolated from the enteron aisle of Henan Xiayi County long lived elder, and the deposit number of the bifidobacterium longum is CGMCC No.15028.The bifidobacterium longum can effectively improve one of Bifidobacterium in enteron aisle, lactobacillus or a variety of quantity in the case where not changing intestinal flora total quantity substantially；Reduce one of Colibacter, enterococcus spp or a variety of quantity in enteron aisle；And for other floras, for example, the quantity of fusobacterium does not make significant difference then.It is thus been found that bifidobacterium longum of the present invention can effectively adjust intestinal flora, promote body Tiny ecosystem forward direction balance.
Description
Technical field
The present invention relates to microorganisms technical fields, more particularly relate to bifidobacterium longum in the application for adjusting intestinal flora.
Background technique
There are more than 1000 kinds of intestinal floras, sums about 10 in human body intestinal canal14A, weigh 1-1.5kg, encodes about 3,300,000
Gene is more than 150 times of human gene number, referred to as " the second genome " of the mankind.According to the relationship with human health, enteron aisle
Flora is divided into beneficial bacterium, harmful bacteria and neutral bacterium three categories.Beneficial bacterium (probiotics) is element necessary to human health, mainly
Including Bifidobacterium, lactic acid bacteria etc..Harmful bacteria refers in particular to those quantity once raised growth out of control, will cause a variety of diseases
Enterobacteriaceae mainly includes salmonella, staphylococcus aureus.Neutral bacterium has both the dual characteristics of beneficial bacterium and harmful bacteria, In
Harmless to body under normal circumstances, possible initiation disease out of control of rising in value, mainly includes Escherichia coli, enterococcus etc..
According to the definition that FAO (Food and Agriculture Organization of the United Nation) (FAO) World Health Organization (WTO) joint specialist group was provided in 2001,
Probiotics is " after taking in right amount, being beneficial to the microorganism of the work of its host health ".Generally, the effect of probiotics is
The growth for promoting beneficial bacterium, inhibiting pathogenic bacteria, maintains the balance of intestinal flora, beneficial to human health.Wherein such as lactic acid bacteria
(Lactobacillus) and Bifidobacterium (Bifidobacterium) is common probiotic strain.Probiotics has human body
Beneficial aspect, which is embodied in it, can prevent the generation of alimentary canal inflammation disease, facilitate the digestion to lactose and decompose, inhibit canceration and its
His damage of the mutagens to body, reduces the cholesterol in serum, and Helicobacter pylori Infection stimulates immune system to improve machine
The resistance etc. of body itself.The probiotics strain list that can be used for health food that the Ministry of Public Health, China in 2001 announces has: not tally
Bifidobacterium, bifidobacterium infantis, bifidobacterium longum, bifidobacterium breve bifidobacterium adolescentis, lactobacillus bulgaricus, acidophilus cream
Bacillus, L. casei casei, streptococcus thermophilus.Lactobacillus inoculation of the foreign countries for Yoghourt and microorganism formulation has: acidophilus
Lactobacillus, Lactobacillus rhamnosus Lactobacillus rogosae, lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus Jensenii, bifidobacterium breve, long pair
Discrimination bacillus bifidobacterium bifidum etc..At present using more in probiotics: lactic acid bacteria and Bifidobacterium.Its effect is mainly concentrated
The normal bowel microecological balance of body is being maintained and adjusted, and is thus generating a series of prebiotic effect.
Foreign countries are more early for the research of probiotics prebiotic effect, and many reports show bacterial strain Lactobacillus rhamnosus LGG, newborn bar
Bacterium F19 and Bifidobacterium Bb-12 etc. all have the function of adjusting intestinal microecology.Yoichi Fukushima etc. has studied bifid
Effect of the bacillus Bb-12 to SPF suckling mice intestinal flora, as the result is shown: compared with the control group, bifidobacteria (log
Cfu/g) by increasing to 9.67 less than 3, enterobacteria quantity has micro increase.M.Alander etc. has studied probiotics (plant cream
5 plants of bacterium such as bacillus, Lactobacillus rhamnosus, Bifidobacterium) corrective action to human body intestinal canal Tiny ecosystem simulation system, research points out
Probiotic strain comprehensive function the result is that: simulation enteron aisle in Bifidobacterium and lactobacillus quantity have significant increase, enterobacteria number
Amount is significantly reduced, and enterococcus quantity has slight decrease trend during the experiment.Studies in China scholar was for prebiotic in recent years
The effect that bacterium adjusts intestinal microecology also expands many researchs.Yan Jiwen etc. has studied certain bifidobacteria viable bacteria granular preparation pair
The adjustment effect of mouse intestinal flora, experimental animal gavage basic, normal, high three dosage groups, Bifidobacterium, lactobacillus and enterobacteria
Quantity dramatically increase, enterococcus quantity is without significant changes.Xu causes far to wait scholars' studies have shown that lactobacillus plantarum ST-III pair
Mouse intestinal flora has certain regulating effect, and effective dose is 6 × 108Cfu/ml, experimental period, 15d, stopped administration 3-
Still there is remarkable result, the amount of indices bacterium is returned to level before stomach-filling after 5-7d after 5d.Human relations are always cherished the memory of etc. with inactivation bifid bar
Bacterium adjusts the antibiotic associated flora imbalance of mouse, it is believed that the Bifidobacterium of inactivation and its culture supernatant are to mouse intestinal physiology
Flora has certain adjustment effect.But also show that probiotics is poor to the adjustment effect kind of intestinal flora with the presence of document
It is different and less to the research of intestinal flora adjustment effect for bifidobacterium longum at present.
The quantitative detecting method of intestinal flora: bacterium separation, identification and quantification are for studying intestinal flora function in enteron aisle
Energy and body correlation are extremely important.There are commonly culture-based method and real-time quantitatives for enteric bacteria quantitative detecting method
PCR directly can qualitatively and quantitatively analyze the DNA of bacteria extracted from sample with Real-time quantitative PCR, with biography
When system cultural method is compared to having the characteristics that more time saving and energy saving, sensibility high specific is strong, easy to operate quick, and not tested
Between limit.
Therefore, the present invention by Bifidobacterium, lactobacillus, enterobacteria, enterococcus, fusobacterium specific primer, into
Row real-time quantitative polymerase chain reaction.Its response procedures is optimized, determines optimal response procedures, and suitable
Standard curve is made in template concentrations.Bifidobacterium longum (Bifidobacterium is detected with real-time quantitative PCR standard measure
Longum) influence of the W68 to mouse intestinal flora.
Summary of the invention
In view of above content, the object of the present invention is to provide the bifidobacterium longums that one plant can adjust intestinal flora function
(Bifidobacterium longum)W68.The bacterium is isolated from the enteron aisle of Henan Xiayi County long lived elder, and the bacterium is through Physiology and biochemistry
It is accredited as bifidobacterium longum (Bifidobacterium longum).
It is another object of the present invention to provide bifidobacterium longums as described above to adjust the application in intestinal flora.More
Specifically the answering in the food of preparation adjusting intestinal flora, health care product, drug, food supplement for above-mentioned bifidobacterium longum
With.
Bifidobacterium longum W68 provided by the invention can generate a large amount of bifidobacterium longum viable bacteria bodies, the training by culture
Feeding method has no special requirements, as long as bacterial strain can be made to be proliferated, such as can be according to 107The inoculum concentration of CFU/mL is double by the length
Discrimination bacillus is inoculated in Medium of Bifidobacterium, under anaerobic, after cultivating 5-72 hours at a temperature of 25-45 DEG C, obtains
Culture solution.The culture medium of the Bifidobacterium can be the culture medium of various suitable Bifidobacterium cultures well known in the art, example
It can be such as milk and/or " lactic acid bacteria --- Basic of Biology and application " (Yang Jiebin, light industry publishing house publish for 1996)
Described in lactic acid bacteria (MRS) culture medium.
The present invention can further separate the viable bacteria body of the bifidobacterium longum in above-mentioned culture solution, the separation method without
Especially limitation, if can be enriched with thallus from culture solution, such as can by it is well known in the art centrifugation and/or filter
Method realizes that the condition of the centrifugation and the filtering can be well known condition, and the present invention repeats no more.
According to the present invention, the dead thallus preparation method industry routine, for example, the viable bacteria body after above-mentioned culture can be added
Thermal killed can also be radiated lethal.The lethal condition of the heating may include: 65-85 DEG C of temperature, time 0.5-1.5
Hour.
According to the present invention, the viable bacteria containing the bifidobacterium longum in the food, health care product, drug, food supplement
Body and/or dead thallus are as active constituent.Preferably, body containing viable bacteria is as active constituent.Make when using viable bacteria body and dead thallus
When for active constituent, the quantity of viable bacteria body is higher than the quantity of dead thallus.
According to the present invention, by above-mentioned bifidobacterium longum be added to food, health care product, drug, in food supplement, and to a
Body is eaten, and the object of the invention can be realized, and plays the role of adjusting intestinal flora.Preferably, with every gram or every milliliter institute
On the basis of stating food, health care product, drug, food supplement, the content of the bifidobacterium longum is 106-1011CFU/g or/ml,
More preferably 108-1010CFU/g or/ml.
CFU (Colony-Forming Units, Colony Forming Unit) refers to viable bacteria number.When viable bacteria is cultivated and counts, by
Single thallus or the pockets of multiple thallus of aggregation breed in cultured on solid medium and are formed by colony, and referred to as bacterium colony forms list
Position expresses the quantity of viable bacteria with it.
The present invention also provides a kind of food, health care product, drug, food supplement, the drug can be according to administration
Approach is different and is prepared into different form, for example, can be prepared into powder, tablet, granule, capsule, solution, emulsion, mixing
The forms such as suspension may also include pharmaceutically acceptable adjuvant in the drug, and those skilled in the art can be according to different
The different adjuvant of dosage form selection, the present invention are no longer described in detail.
The food includes any type of food, such as juice product, bean product, dairy products etc..Food can also root
According to the different and different of edible object.Also containing conventional additive, such as flavors and fragrances, stabilization in the food
Agent, thickener, preservative etc. and nutrition fortifier, such as minerals, vitamin etc..
The utility model has the advantages that
Bifidobacterium longum of the present invention can be improved effectively in the case where not changing intestinal flora total quantity substantially
One of Bifidobacterium, lactobacillus or a variety of quantity in enteron aisle；Reduce Colibacter in enteron aisle, in enterococcus spp
One or more quantity；And for other floras, for example, the quantity of fusobacterium does not make significant difference then.It is thus been found that this hair
The bright bifidobacterium longum can effectively adjust intestinal flora, promote body Tiny ecosystem forward direction balance.
The preservation of strain:
Strain bifidobacterium longum (Bifidobacterium longum) W68 of the invention, was protected on December 7th, 2017
Ensconce China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica, postcode: 100101) (depositary institution is abbreviated as CGMCC), deposit number is
CGMCC No.15028。
Specific embodiment
Further illustrate that the present invention, following embodiment are the preferable embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments, therefore the scope of protection of present invention is not limited to
In described.
The present embodiment, by Bifidobacterium, lactobacillus, Escherichia coli, enterococcus, fusobacterium specific primer, into
Row real-time quantitative polymerase chain reaction.Its response procedures is optimized, determines optimal response procedures, and suitable
Standard curve is made in template concentrations.Then, with real-time quantitative PCR standard measure detection mouse in stomach-filling tested material bifidobacterium longum
After W68 in excrement Bifidobacterium, lactobacillus, Escherichia coli, enterococcus, fusobacterium amount, evaluate bifidobacterium longum W68 to small
The influence of mouse intestinal flora.Experimental procedure are as follows: strain separating identifies --- drafting of standard curve --- standard of tested material
Standby --- experimental animal grouping and processing --- point of the extraction of fecal specimens DNA --- real-time quantitative PCR --- experimental result
Analysis evaluation.
Test consumptive material, equipment source:
(1) experimental strain: bifidobacterium longum W68, deposit number are CGMCC No.15028.It is isolated from Henan Xiayi county magistrate
The enteron aisle of longevity old man.
(2) positive control strain (Bifidobacterium animalis ssp.Lactis BB12, animal bifidobacteria
BB12) it is purchased from Denmark Chr.Hansen company.
(3) experimental animal: SPF grades Balb/c male mice 60, three week old, 18-22g is purchased from Guangdong Province experimental animal
The heart.
(4) animal feed: no Bifidobacterium mouse feed is purchased from Shanghai Slac Experimental Animal Co., Ltd., feeding
It is preceding to sterilize via radiation.
(5) DNA QIAquick Gel Extraction Kit is purchased from Zemo company, and PCR kit for fluorescence quantitative is purchased from Takara company, RNAlater
Purchased from the great biotech firm in Shanghai.
(6) anaerobism dilution: weighing 6.0g Na2HPO412H2O, 4.5g KH2PO4,0.5g cysteine hydrochloride,
800mL deionized water is added in 0.5g Tween-80, is sufficiently stirred, and adjusts pH to 7.4, then plus deionized water constant volume to 1L.
(7) it 50 times of TAE of agarose gel electrophoresis buffer: weighs 242g Tris and 57.1mL glacial acetic acid, 100mL is added
0.5M EDTA adjusts pH=8.0, with ultrapure water constant volume to 1L.
(8) MRS fluid nutrient medium: peptone 8-12g, beef extract 8-12g, yeast extract 4-6g, diammonium hydrogen citrate 1.8-
2.2g, glucose 18-22g, Tween 80 0.8-1.2mL, sodium acetate 4.5-5.5g, dipotassium hydrogen phosphate 1.8-2.2g, magnesium sulfate
0.56-0.6g, manganese sulfate 0.24-0.26g add distilled water to 1000mL, pH6.2-6.6.
(9) reagent used below, culture medium composition and experiment method etc. in case of no particular description,
For reagent commonly used in the art, culture medium composition and experiment method.
1, the separation identification of strain
This research is carried out external using traditional spread plate method separating lactic acid bacterium from the long lived elder excrement of Henan Xiayi County
Experiment sieving has the bacterial strain of stronger relax bowel and defecation characteristic, is then screened by simulating the environment of human gastrointestinal tract with resistance to
The bacterial strain of acid, bile tolerance, and its biocidal property and drug resistance are studied, it is most excellent prebiotic by the way that the prebiotic performance of acquisition is comprehensively compared
Bacteria strain.
Physiology and biochemistry qualification result shows that bacterial strain W68 is Gram-positive, catalase, catalase, motility, nitre
Hydrochlorate reduction is feminine gender, does not form the bacterial strain of gemma, tentatively judges bacterial strain W68 for the bacterial strain of lactic acid bacteria.
Bacterial strain 16S rDNA sequencing identification display: using NCBI-BLAST to the 16S rDNA sequence of bacterial strain of the present invention with
Accession sequence carries out homology alignment, the 16S rDNA sequence and bifidobacterium longum of bacterial strain of the present invention in GenBank
(Bifidobacterium Longum) sequence similarity is up to 99% or more.According to 16S rDNA sequence, bacterial strain of the present invention is reflected
It is set to bifidobacterium longum (Bifidobacterium Longum).
2, the drafting of standard curve:
(1) reaction system of real-time quantitative PCR determines: 10 μM of upstream and downstream of the excrement genomic DNA of 1 μ L, 0.4 μ L are drawn
Object, the SYBR Premix Ex Taq of 10 μ L, the ROX of 0.4 μ L, the sterile water of 7.8 μ L；Response procedures are as follows: (95 DEG C of initial denaturation
React 30s), it is denaturalized (95 DEG C of reaction 5s), annealing (annealing temperature in table 1 reacts 30s) extends (72 DEG C of reaction 30s), PCR
40-45 circulation of reaction.
(2) above-mentioned quantitative PCR reaction system and condition are pressed, it is anti-to carry out quantitative PCR with specific primer respective in table 1
It answers, using the logarithm of the copy Particle density of DNA in each gradient dilution liquid as abscissa, using Ct value as ordinate, makes different in table 1
The standard curve of target bacteria group.Calculate the amplification efficiency of primer, formula are as follows: amplification efficiency=10(1/ slope)-1.The standard made
Curve correlation coefficient is 0.99 or more.
Bifidobacterium, lactobacillus, enterobacteria, enterococcus, the standard curve of fusobacterium and formula show knot in EXCEL
Fruit: Bifidobacterium calibration curve equation is y=-3.3124x+37.032, coefficient R2=0.9954；The standard of lactobacillus is bent
Line equation is y=-3.4617x+33.899, coefficient R2=0.9982；The calibration curve equation of enterobacteria is y=-
3.3229x+38.478 coefficient R2=0.9967；Enterococcal calibration curve equation is y=-3.4414x+35.612, R2
=0.9978；The calibration curve equation of fusobacterium is y=-3.4514x+35.612, R2=0.9982.Related coefficient reaches
0.99 or more, it can be used as the standard detected in the future.
3, the preparation (preparation example) of tested material:
(1) preparation of bifidobacterium longum bacteria suspension: strain is inoculated in MRS liquid with the inoculum concentration of 1% (volumetric concentration) and trains
It supports in base, 37 DEG C of Anaerobic culturel 12h, later, culture solution is centrifuged 15min in 6000g, abandons supernatant, the sterile life of bacterial sediment
Reason salt water washing 1 time, 6000g is centrifuged 15min and takes 10 with sterile saline gradient dilution again-6、10-7With 10-8Three ladders
It spends each 1mL progress plate culture and is finally configured to 1 × 10 after carrying out bacterium colony counting after 37 DEG C of Anaerobic culturel 48h6CFU/mL、1
×108CFU/mL and 1 × 1010CFU/mL bacteria suspension, matching while using.It is well known to those skilled in the art, it include one in the bacteria suspension
The dead thallus of fixed number amount bifidobacterium longum.
(2) preparation of positive control bacteria suspension: 1 × 10 is configured according to the preparation method of (1)8The animal bifid bar of CFU/mL
The bacteria suspension C2 of bacterium BB12.
(3) experiment mice is randomly divided into 5 groups, every group 12 by weight.20 ± 2 DEG C of animal house temperature, humidity 40-50%,
Feed and drinking-water are freely taken in 12h illumination/dark cycle, spare after adaptive feeding 1 week.
4, the packet transaction of experimental animal
Experiment mice is randomly divided into 5 groups, respectively W68 model low dose group (106Cfu/ml bacteria suspension), model middle dosage
Group (108Cfu/ml bacteria suspension), model high dose group (1010Cfu/ml bacteria suspension).Positive controls (stomach-filling 108Cfu/ml's
The bacteria suspension of animal bifidobacteria BB12), blank control group (stomach-filling physiological saline), every group 12, each group mouse during test
Given low, continuous gavage 14d are calculated according to 5mL/kg mouse weight daily, while the daily feed of 2g being pulverized in a manner of stomach-filling
It is fed for mouse, the stomach-filling phase is 14d.Mouse freely ingests, drinks water during stomach-filling, monitors weight and food ration daily.
5, bacterial genomic DNA samples are collected in enteron aisle
Before stomach-filling bifidobacterium longum (0 day) and after stomach-filling 14 days, with equipped with 1mL anaerobism dilution and sterile glass beads
Centrifuge tube collect mouse fresh excreta 0.1g, with vortex oscillator homogeneous it is complete after, using phenol chloroform method extract excrement in
Bacterium.DNA agarose gel electrophoresis detection is carried out with 1% agarose.Testing result performance is good, bacterium in all fecal specimens
It organizes DNA successfully to propose, and without obvious degradation phenomenon.
6, in excrement intestinal flora content measurement
Real-time quantitative is carried out to the fecal specimens bacterium group DNA obtained in step 4 respectively using the specific primer in table 1
PCR experiment.The reaction system of real-time quantitative PCR: the excrement genomic DNA of 1 μ L, 10 μM of upstream and downstream primers of 0.4 μ L, 10 μ L's
SYBR Premix Ex Taq, the ROX of 0.4 μ L, the sterile water of 7.8 μ L；Response procedures are as follows: initial denaturation (95 DEG C of reaction 30s),
It is denaturalized (95 DEG C of reaction 5s), annealing (annealing temperature in table 1 reacts 30s) extends (72 DEG C of reaction 30s), PCR reacts 40-45
A circulation.Pcr amplification product is subjected to the agarose gel electrophoresis that concentration is 2%, with aseptic operation knife by single target item
Band is cut, and in clean 5mL centrifuge tube, carries out DNA recycling using Zemo plastic recovery kit and by specification.Gel extraction
Standard DNA template measure respective DNA concentration using Qubit Assays after, 10 times of dilutions, 8 gradients are as quantitative PCR
Standard sample.
PCR reaction is carried out to 5 groups of stool in mice sample gene group DNA, obtains Ct value.By standard curve, sample is obtained
In the flora copy Particle density, according to sampling when stool quality, be converted into the copy in every gram of fecal specimens containing the flora
Number logarithm (lgDNA copy number/gram excrement).Statistic analysis result is shown in Table 2.
7, experimental data statisticallys analyze
Experimental data indicates that test data is taken statistics analysis using 13.0 Data Analysis Software of SPSS using x ± s
Processing, p > 0.05 indicate that difference is not significant；P < 0.05 indicates significant difference；P < 0.01 indicates that difference is extremely significant.
1 primer sequence of table and return of goods temperature
Influence (Mean, log/g excrement) of the 2 bifidobacterium longum W68 of table to mouse intestinal flora
As can be seen from Table 2, total bacteria log average value is 11.88 in normal mouse enteron aisle before stomach-filling.Blank control group
Extend with the stomach-filling time, total bacteria amount rises 0.01 order of magnitude, but with before stomach-filling without significant difference, therefore normal
In the case of, the metabolic function of mouse itself does not influence total bacteria.In 5 groups of experiments, high dose group is prolonged with the stomach-filling time
Long, total bacteria variation is maximum, improves 0.05 order of magnitude, but statistically analyze, the variation is not formed statistically
Difference.Therefore, total bacteria in mouse intestinal is influenced after the stomach-filling Bifidobacterium W68 not significant.
As can be seen from Table 2, Bifidobacterium logarithmic mean value is 6.96 in normal mouse enteron aisle before stomach-filling.Blank control group
Extend with the stomach-filling time, bifidobacteria has dropped 0.07 order of magnitude (in terms of logarithm, the same below), aobvious with nothing equal before stomach-filling
Difference is write, therefore under normal circumstances, the metabolic function of mouse itself does not influence Bifidobacterium number.After intragastric administration on mice
After 14d, 0.95,1.29,1.31 order of magnitude has been respectively increased in basic, normal, high three dosage groups bifidobacteria, is had extremely significant
It improves (p < 0.01)；The order of magnitude that wherein middle and high dosage group improves is apparently higher than low dose group, but high dose group and middle dosage
Without significant difference between group.Compared with positive control animals Bifidobacterium BB12, although positive control animals Bifidobacterium BB12
Also Bifidobacterium in mouse intestinal is improved into 0.97 order of magnitude, is slightly better than low dose group, effect is far from middle and high dosage group
Significantly.
As can be seen from Table 2, lactobacillus logarithmic mean value is 9.32 in normal mouse enteron aisle before stomach-filling.Blank control group with
The stomach-filling time extends, and lactobacillus quantity does not change, therefore under normal circumstances, the metabolic function of mouse itself is to lactobacillus number
Do not influence.After intragastric administration on mice after 14d, basic, normal, high three dosage groups bifidobacteria has been respectively increased 0.17,0.6,
0.76 order of magnitude has extremely significant raising (p < 0.01)；The order of magnitude that wherein middle and high dosage group improves is apparently higher than low dose
Amount group, but without significant difference between high dose group and middle dose group.Compared with positive control animals Bifidobacterium BB12, although positive
Property control-animal Bifidobacterium BB12 Bifidobacterium in mouse intestinal is also improved into 0.24 order of magnitude, slightly be better than low dosage
Group, the effect middle and high dosage group that is far from are significant.
As can be seen from Table 2, enterococcus logarithmic mean value is 7.0 in normal mouse enteron aisle before stomach-filling.Blank control group with
The stomach-filling time extends, and enterococcus quantity improves before 0.08 order of magnitude, with stomach-filling without significant difference, therefore in normal condition
Under, the metabolic function of mouse itself does not influence enterococcus number.After intragastric administration on mice 14d, in addition to low dose group, middle and high dose
Amount group enterococcus quantity reduces 0.27,0.15 order of magnitude respectively, there is extremely significant reduction (p < 0.01), but middle and high dosage
No significant difference between group.Compared with positive control animals Bifidobacterium BB12, although positive control animals Bifidobacterium BB12
Also enterococcus in mouse intestinal is reduced into 0.08 order of magnitude, is slightly better than low dose group, the effect middle and high dosage group that is far from is aobvious
It writes.
As can be seen from Table 2, Escherichia coli logarithmic mean value is 7.0 in normal mouse enteron aisle before stomach-filling.Blank control group
Extend with the stomach-filling time, Escherichia coli quantity improves before 0.06 order of magnitude, with stomach-filling without significant difference, therefore normal
In the case of, the metabolic function of mouse itself does not influence coliform count.It is three doses basic, normal, high after intragastric administration on mice after 14d
Amount group Escherichia coli quantity reduces 0.12,0.68,0.72 order of magnitude respectively, there is extremely significant reduction (p < 0.01), but
No significant difference between middle and high dosage group.Compared with positive control animals Bifidobacterium BB12, although positive control animals bifid
Quantity enterococcal in mouse intestinal is also reduced 0.21 order of magnitude by bacillus BB12, is slightly better than low dose group, and effect does not have far
There is middle and high dosage group significant.
As can be seen from Table 2, fusobacterium logarithmic mean value has reached 9.03 in normal mouse enteron aisle before stomach-filling.Blank control
Group extends with the stomach-filling time, and total Clostridium counts rise 0.02, but with before stomach-filling without significant difference, therefore in normal condition
Under, the metabolic function of mouse itself does not influence fusobacterium.In 5 groups of experiments, high dose group extends with the stomach-filling time, total thin
Bacterium number variation is maximum, reduces 0.06 order of magnitude, but statistically analyze, the not formed statistically difference of the variation.Cause
This, influences fusobacterium number in mouse intestinal after the stomach-filling Bifidobacterium W68 not significant.
Comprehensive analysis, Bifidobacterium and lactobacillus are generally acknowledged beneficial bacteria of intestinal tract, and the increase of quantity anticipates to body health
Justice is great.If enterococcus and Escherichia coli quantity are excessively easy to cause a series of enteron aisle related symptoms such as diarrhea, dehydration in enteron aisle.
Experimental result is shown, after bifidobacterium longum W68 stomach-filling mouse 14d, while not changing enteron aisle total bacteria count, and Bifidobacterium
Quantity significantly increases, and lactobacillus digital display writes raising, enterococcus and Escherichia coli quantity and significantly reduces, and influences on fusobacterium unknown
It is aobvious.I.e. the bifidobacterium longum can effectively adjust intestinal flora, maintain the positive balance of intestinal microecology.
The foregoing describe the preferred embodiment of the present invention, but that present invention is not limited to the embodiments described above is specific thin
Section can carry out various simple variants to technical solution of the present invention, belong to this hair within the scope of the technical concept of the present invention
Bright protected range.
Claims (6)
1. the bifidobacterium longum of one plant of adjusting intestinal flora, which is characterized in that the bifidobacterium longum is named as W68, the bacterium
The deposit number of strain is CGMCC No.15028.
2. bifidobacterium longum described in claim 1 is adjusting the application in intestinal flora.
3. bifidobacterium longum according to claim 2 is adjusting the application in intestinal flora, which is characterized in that preparation is adjusted
Food, health care product, drug, the food supplement of intestinal flora.
4. bifidobacterium longum according to claim 3 is adjusting the application in intestinal flora, which is characterized in that the food
The viable bacteria body and/or dead thallus that product, health care product, drug, food supplement include bifidobacterium longum are as active constituent.
5. bifidobacterium longum according to claim 4 adjust intestinal flora in application, which is characterized in that with every gram or
Food described in every milliliter, health care product, drug, food supplement total weight be the additive amount of benchmark bifidobacterium longum be 106-
1011CFU/g or 106-1011CFU/ml。
6. bifidobacterium longum according to claim 5 adjust intestinal flora in application, which is characterized in that with every gram or
Food described in every milliliter, health care product, drug, food supplement total weight on the basis of the additive amount of the bifidobacterium longum be
108-1010CFU/g or 108-1010CFU/ml。
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Application publication date: 20191112 |