CN106434427A - Lactic acid bacillus probiotic CGMCC NO.12421 and applicaiton to preparing lipid-lowering drugs - Google Patents
Lactic acid bacillus probiotic CGMCC NO.12421 and applicaiton to preparing lipid-lowering drugs Download PDFInfo
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Abstract
The invention discloses a lactic acid bacillus probiotic bacterial strain. The preservation number of the bacterial strain is CGMCC NO.12421, and the preservation date is May 6th, 2016. The sequence of the 16S rRNA of the bacterial strain is shown in SEQ ID NO:1. The invention further discloses application of the bacterial strain to preparing lipid-lowering drugs, food and health care products. The bacterial strain is harmless to animals and has the functions of regulating and lowering blood lipids.
Description
Technical field
The present invention relates to a kind of probio and application thereof, belong to microorganism field.
Background technology
Human serum cholesterol level is closely related with angiocardiopathies such as coronary heart disease, artery sclerosis, hyperlipidemias.High fat
Mass formed by blood stasis cardinal symptom shows that serum total cholesterol, LDL-C, triglycerides equal size are too high.How to control
Reducing human serum cholesterol levels has become sanatory major issue, and a lot of blood lipid-lowering medicines have good lipopenicillinase to imitate
Really, the side effect but caused by long-term taking also gets more and more studied confirmation, seeks new side effect little, can regulate again reduction
The research of blood fat becomes new demand.
Probio (Probiotics) be defined as " one class live microorganism, machine can be given with suitable quantity after being ingested
Body brings benefit ".Generally believe can should possess following condition as the bacterium of probiotic composition at present:(1) beneficial to host;
(2) free of toxic effects and pathogenic effects;(3) can survive in alimentary canal;(4) hydrochloric acid in gastric juice and cholate are adapted to;(5) can be at alimentary canal
Surface field planting;(6) useful lipid and metabolin can be produced;(7) activity can be kept in processing and storage;(8) have
Good organoleptic attribute.Within 1963, African tribes makes internal serum cholesterol content due to a large amount of edible lactobacillus-fermented goods
Relatively low discovery causes the concern that probio is reduced cholesterol effect by scientific circles.Reduce courage under Bacillus acidi lactici environment in vitro
The mechanism of sterol mainly includes co-precipitation mechanism, and thalline absorbs and metabolic mechanism, and film inlays mechanism.Have many passes both at home and abroad
In the document report of lactic acid bacteria norcholesterol effect, but concrete lactic acid bacteria screening index, the vivo efficacy of regulation blood fat and machine
Reason etc. is still not clear, and the effect of lactic acid bacteria norcholesterol is also difficult to reach gratifying effect.Animal and plant diet
Can promote that different gut floras grows.It is contemplated that separation screening has from the Qinghai Wild marmot ight soil of only feeding plant
There is the lactic bacteria strain of good norcholesterol function, and develop its application in preparing fat-reducing medicament further.
Content of the invention
Based on foregoing invention purpose, present invention firstly provides a kind of Bacillus acidi lactici probiotics strain, described probiotics bacterial
The deposit number of strain is CGMCC NO.12421, and preservation Classification And Nomenclature is lactobacillus reuteri (Lactobacillus
Reuteri), preservation date is on May 6th, 2016, and depositary institution is that China Committee for Culture Collection of Microorganisms is commonly micro-
Bio-Centers, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode:
100101.
Present invention also offers a kind of Bacillus acidi lactici probiotics strain, the sequence such as SEQ ID of the 16S rRNA of described bacterial strain
NO:Shown in 1.
Present invention also offers application in preparing fat-reducing medicament for the above-mentioned bacterial strains.
Present invention also offers application in preparing food and/or health products for the above-mentioned bacterial strains.
Present invention also offers the composition containing above-mentioned bacterial strains.
In a preferred technical scheme, described composition is prepared as capsule, freeze-dried powder or bacterium solution preparation.
Finally, present invention also offers a kind of method separating from wild marmot ight soil and screening probio, described side
Method comprises the following steps:
(1) use bacteria culture media to be separately cultured bacterial strain from marmot stool sample, filter out the excellent bacterium of enduring organic acid
Strain;
(2) bacterial strain filtering out step (1) carries out 16S rRNA order-checking and biochemical identification, filters out and meets Bacillus acidi lactici
16S rRNA and the bacterial strain of biochemical character;
(3) bacterial strain filtering out step (2) carries out screening acidproof, bile tolerance;
(4) bacterial strain that preservation steps (3) filters out.
Preferably, the culture medium described in step (1) is MRS culture medium.
Preferably, the 16S rRNA order-checking described in step (2) is PCR PCR sequencing PCR.
In a preferred technical scheme, described method also includes step (5):The bacterial strain that step (4) screening is obtained
Carry out the screening step of external lipid-reducing function.
The present invention is the isolated and purified Bacillus acidi lactici obtaining having probiotic properties from wild marmot ight soil, and experiment proves
The lactic acid bacteria of isolated is harmless to animal, and has effect of regulation blood fat through zoopery confirmation.CGMCC NO.12421
Bacterial strain can significantly reduce low-density lipoprotein, T-CHOL, triglycerides, increasing high density lipoprotein, and simvastatin phase
As effect of regulating blood fat.The rat sign taking in lactic acid bacteria CGMCC NO.12421 bacterial strain is normal, takes in surrounding and terminates experiment
After take stomach and intestine liver spleen and do pathology no abnormality seen.Sterile tissue is done Bacteria Detection and is had no lactic acid bacteria dystopy field planting.
Brief description
Fig. 1 .CGMCC NO.12421 bacterial strain biochemical identification result figure;
Fig. 2 .CGMCC NO.12422 bacterial strain biochemical identification result figure;
Fig. 3. core gene group system evolution tree graph;
Fig. 4 .CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain cholate tolerance schematic diagram;
Fig. 5 .CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain external lipid-lowering effect schematic diagram
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantages of the present invention and feature will be with describe and
Apparent.But these embodiments are only exemplary, any restriction is not constituted to protection scope of the present invention.
Separation, screening and the qualification of embodiment 1. lactic acid bacteria
1. the separation of Bacillus acidi lactici
1) from protecting taking-up sample 100 μ L tube, join in the EP pipe of the aseptic PBS of prepackage 900 μ L, successively sample is entered
Row gradient dilution, marmot stool sample concentration dilution is to 10-6Times;
2) take different dilution sample 100 μ L to coat on MRS culture medium, put in incubator;
3) at 37 DEG C, 0.5%CO2Environment is cultivated 48h;
4) take out culture dish, with the bacterium colony of aseptic inoculation ring picking different shape feature, be forwarded to new MRS solid culture
Base is purified, 37 DEG C of Anaerobic culturel 48h, continuously switching 3 times, cultivates purifying bacterial strain in the liquid MRS of pH=3.5,
Screen acidproof, grow excellent bacterial strain can be used for experiment or freezing.
2. culture presevation
The MRS culture medium containing 25% glycerine for this use for laboratory carries out the freezing of bacterial classification as fungus preserving liquid, and method is as follows:
1) by guarantor's tube that capacity is 2mL through 121 DEG C, in case using after 15min autoclaving is processed;
2) Bacillus acidi lactici is on MRS solid medium continuously after switching 3 times, adds the aseptic guarantor of 1.5ml on culture dish
Bacterium solution;
3) with L rod, culture dish is scratched, make bacterium colony fully incorporate in fungus preserving liquid;
4) bacterium solution is transferred to protect in tube ,-80 DEG C of preservations after mixing.
3. bacterium colony outward appearance and thalli morphology are observed
Lactobacillus facultative anaerobe, under anaerobic well-grown, bacterium colony is creamy white, smooth surface;Having
Under the conditions of oxygen, Bacillus acidi lactici also can grow, and major part bacterial classification bacterium colony surface is rougher, and colony colour mostly is milky.See under mirror
Examining lactobacillus cell form is polymorphy, how in elongated rod-shaped, compared with thick bar shape, club shape etc., arrangement in paliform, chain
Deng.
4. the extraction of bacteria total DNA
By single colony inoculation on BHI culture medium, 37 DEG C of overnight in anaerobiosis are cultivated, and extract examination according to bacterial genomes DNA
Agent box (TIANGEN) specification operates, and extracts DNA.
5. the biochemical identification method of bacterial strain
This research uses " BioMerieux " bacterial system biochemical identification carton API50CHL that Mei Liai company of France produces.
Result:According to biochemical identification, it is thus achieved that a strain biochemical character is the bacterial strain of lactobacillus fermenti, and bacterial strain preserving number is
CGMCC NO.12421 (biochemical identification result is shown in Fig. 1), it is Lactobacillus plantarum that inventor obtains a strain biochemical character also simultaneously
Bacterial strain, its bacterial strain deposit number is CGMCC NO.12422 (biochemical identification result is shown in Fig. 2), and preservation date is May 6 in 2016
Day, preservation Classification And Nomenclature is Lactobacillus plantarum (Lactobacillus plantarum), and depositary institution is Chinese microorganism strain
Preservation administration committee common micro-organisms center, address is the micro-life of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Thing research institute, postcode:100101.
6. bacterial universal primers 16S rRNA PCR amplification
Bacterial 16 S rRNA identifies:Extracting bacterial genomes DNA, amplification Bacillus acidi lactici universal primer 16S rDNA PCR produces
Thing checks order, and sequence carries out BLAST comparison on NCBI, carries out Preliminary Identification.
The condition of the primer of this experiment bacterial 16 S rRNA PCR amplification used and PCR reaction is as follows, and experiment is all
PCR system with 50 μ L.By enterprising at NCBI to Bacillus acidi lactici universal primer 16S rRNA PCR primer sequencing result to be measured
Row BLAST comparison, identifies.Select representative sample PCR primer order-checking.Sequencing result completes according to the comparison of BLAST
Preliminary Identification to Bacillus acidi lactici.Result display CGMCC NO.12421 bacterial strain is lactobacillus reuteri (SEQ ID NO:1),
CGMCC NO.12422 bacterial strain is Lactobacillus plantarum (SEQ ID NO:2).
Universal primer 16S rRNA PCR amplification condition
Primer sequence:
27F, 5'-AGAGTTTGATCMTGGCTCAG-3'
1492R 5'-TACGGYTACCTTGTTACGACTT-3'
Reaction system (50 μ l)
Amplification condition
Inconsistent, further for the biochemical reaction of CGMCC NO.12421 bacterial strain and the qualification result of 16S rRNA sequence
After doing gene order-checking, systematic evolution tree (seeing Fig. 3) is done to core (core-pan) genome, can by systematic evolution tree
To find out the evolutionary relationship of CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain and other Bacillus acidi lacticis, it is thus identified that
The qualification result of 16S rRNA sequence, CGMCC NO.12421 bacterial strain is lactobacillus reuteri, rather than biochemical character is similar
Lactobacillus fermenti.
7. acidproof, the bile tolerance screening technique of bacterial strain
1) after the experimental strain growth of three generations to be activated is saturated, with 1 × 106Or 1 × 107The inoculum concentration of CFU/mL is inoculated in
In the MRS culture medium of pH=2.0, put into incubator;
2) after cultivating 2h in 37 DEG C of environment, carry out 10 times of gradient dilutions, drip with suitable concentration and plant in the training of MRS solid plate
Support in base, put into incubator;
3), after cultivating 48h through 37 DEG C, the bacterium colony on plate is counted, calculates the survival number of bacterium;
4) select the preferable inoculation of acidproof effect in the MRS culture medium containing 0.3% fel bovis salt;
5) after cultivating 4h in 37 DEG C of environment, carry out 10 times of gradient dilutions, drip with suitable concentration and plant in the training of MRS solid plate
Support in base, put into incubator;
6), after cultivating 48h through 37 DEG C, the bacterium colony on plate is counted, calculates the survival number of bacterium.
Result:This experimental simulation human body intestinal canal environment investigates the MRS culture medium at pH=2.0 and 0.3% cholate for the bacterial strain
In survival condition, evaluate bacterial strain to GI tolerance.Result shows that the acidproof of two strain bacterium and bile tolerance ability are good
Good, and the acid and bile salt tolerance function of rhamnose LGG bacterial strain is close to (seeing table 1).
Acidproof, the bile tolerance situation of the alternative bacterial strain of table 1
The lipid-reducing function evaluation of embodiment the 2nd, lactic acid bacteria
1. lipid-reducing function in-vitro evaluation
(1) configuration of culture medium containing cholesterol
1) the MRS culture medium containing 0.1% cholate:Weigh cholate powder by 0.1%, join the MRS liquid that 400ml configures
In body culture medium, through 121 DEG C after fully mixing, 15min high pressure steam sterilization is in case using.
2) MRS CHOL (high cholesterol MRS culture medium):Accurately weigh cholesterol 0.5g, use a small amount of anhydrous alcohol solution
And be settled to 50mL, final concentration 10.0mg/mL, with the filtering with microporous membrane that aperture is 0.22 μm degerming after, add according to the amount of 5%
Enter in sterilized MRS fluid nutrient medium (or the MRS fluid nutrient medium containing 0.1% cholate), make courage in MRS fluid nutrient medium
The final concentration of 500 μ g/mL of sterol.
(2) DAOS method cholesterol detection content
1) principle
Hydrogen peroxide can be produced while cholesterol is oxidized.These hydrogen peroxide can make reagent in DAOS and 4-amino
Antipyrine aoxidizes, and by peroxidase (HRP) condensation, and produce a kind of blue pigment.By comparing with standard items
Right, so that the total cholesterol level in sample can be carried out quantitatively by absorbance.
2) cholesterin detection reagent box (Cholesterol E-test, Wako company, Japan) operating procedure:
1.1) dilution of standard items:Cholesterol standards needs to dilute 7 concentration, and concentration is from high to low, half-and-half dilute step by step
Release.Taking the EP pipe of 7 1.5mL, numbering 1-7, elder generation is toward the ultra-pure water respectively adding 50 μ L in 2-7 pipe.No. 1 Guan Weiyong cholesterol standard
Product stoste 100 μ L (2000 μ g/mL), after concussion mixes, takes 50 μ L and is added in No. 2 pipes, and the rest may be inferred.
1.2) sample-adding product take and have diluted standard items and test solution 2 μ L is accurately added at the bottom of 96 orifice plate plates, if multiple hole.
1.3) add the every hole of developer to add the chromogenic reagent solution that 150 μ L configure and (1 bottle of developer dry powder is joined
In 150mL buffer solution, fully dissolve and mix), under the conditions of 37 DEG C, lucifuge places 5min.
1.4), after terminating 5min, 96 orifice plates are taken out, uses punctures bubble, prevent from affecting OD value.
1.5) reading is put into ELIASA and is read OD value at 600nm.
(3) result:After cultivating 24h in the culture medium containing cholesterol, relatively seeing of the removal rate of cholesterol of three kinds of bacterial strains
Fig. 4.In Fig. 4,1 is LGG, and 2 is CGMCC NO.12421 bacterial strain, and 3 is CGMCC NO.12422 bacterial strain, and result shows CGMCC
NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain, under the conditions of without cholate, reduce cholesterol function and LGG are close.Containing
Under the conditions of cholate, Lactobacillus plantarum CGMCC NO.12422 bacterial strain norcholesterol ability is higher than another two bacterial strain.
2 lipid-reducing function interior evaluatings
Divide four by female sd inbred rats and 40 about 20 grams of BALB/c female rats of 40 about 200 grams of body weight respectively at random
Group, gives high lipid food (cholesterol 1%, lard 10%, 0.2 cholate, 10% yolk powder) and within five weeks, terminates experiment detection blood totally
Clear every blood lipid level, one week later group per os of high fat feeding gives 109CFU CGMCC NO.12421 Bacillus acidi lactici bacterium solution, one
Group gives CGMCC NO.12422 bacterial strain, and one group gives physiological saline, and one group terminates the last fortnight in experiment and gives 20mg/kg body weight
Simvastatin, observe the prevention intervention effect that hyperlipoidemia is occurred by medicine or lactic acid bacteria.
Fig. 5 is the lipid-lowering effect block diagram to rat model for the experimental strain, and wherein, 1 is CGMCC NO.12422 bacterial strain group,
2 is CGMCC NO.12421 bacterial strain group, and 3 is simvastatin group, and control group is with PBS for comparison.From fig. 5, it can be seen that CGMCC
The low-density lipoprotein (LDL) of NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain group be respectively averagely 3.534mmol/L and
3.341mmol/L, all substantially less than model control group 3.969mmol/L (p<0.05).CGMCC NO.12421 bacterial strain and CGMCC
High density degree lipoprotein (HDL) of NO.12422 bacterial strain group is averagely 1.542mmol/L and 1.5761mmol/L respectively, all significantly
Higher than model control group 1.115mmol/L (p<0.05).CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain group
Serum total cholesterol (TG) is 4.387mmol/L and 4.390mmol/L respectively) it is all substantially less than model control group 5.774mmol/
L(p<0.05).The triglyceride (TC) of CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain group is respectively averagely
3.47mmol/L and 3.288mmol/L, all substantially less than model control group 3.853mmol/L (p<0.05).Above-mentioned bacterial strains has
Significantly reducing T-CHOL, triglycerides, increasing high density lipoprotein, with simvastatin has similar reduction blood fat effect.
The rat taking in two kinds of lactic acid bacterias is acted normally, and takes in and takes stomach and intestine liver spleen after surrounding terminates experiment and do pathology and have no different
Often.Sterile tissue is done Bacteria Detection and is had no lactic acid bacteria dystopy field planting.
Claims (10)
1. a Bacillus acidi lactici probiotics strain, the preserving number of described bacterial strain is CGMCC NO.12421, and preservation date is 2016
On May 6, in.
2. a Bacillus acidi lactici probiotics strain, it is characterised in that the sequence such as SEQ ID NO of the 16S rRNA of described bacterial strain:1
Shown in.
3. application in preparing fat-reducing medicament for the bacterial strain according to claim 1 and 2.
4. application in preparing food and/or health products for the bacterial strain according to claim 1 and 2.
5. the composition containing the bacterial strain described in claim 1 or 2.
6. composition according to claim 5, described composition is prepared as capsule, freeze-dried powder or bacterium solution preparation.
7. the method separating and screening excellent probio from wild marmot ight soil, said method comprising the steps of:
(1) use bacteria culture media to be separately cultured bacterial strain from marmot stool sample, filter out the excellent bacterial strain of enduring organic acid;
(2) bacterial strain filtering out step (1) carries out 16S rRNA order-checking and biochemical identification, filters out and meets Bacillus acidi lactici 16S
RRNA and the bacterial strain of biochemical character;
(3) bacterial strain filtering out step (2) carries out screening acidproof, bile tolerance;
(4) bacterial strain that preservation steps (3) filters out.
8. method according to claim 7, it is characterised in that the culture medium described in step (1) is MRS culture medium.
9. method according to claim 7, it is characterised in that the 16S rRNA order-checking described in step (2) is PCR order-checking
Method.
10. method according to claim 7, it is characterised in that described method also includes step (5):Step (4) is screened
The bacterial strain obtaining carries out the screening step of external lipid-reducing function.
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CN108210525A (en) * | 2018-02-08 | 2018-06-29 | 中国疾病预防控制中心传染病预防控制所 | Applications of the Bacillus acidi lactici probiotics CGMCC NO.12422 in slimming medicine is prepared |
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CN110272842A (en) * | 2019-06-25 | 2019-09-24 | 吉林农业大学 | One plant of lactobacillus plantarum LP104 with fat reducing and weight reducing function |
CN110272842B (en) * | 2019-06-25 | 2021-09-24 | 吉林农业大学 | Lactobacillus plantarum LP104 with weight-losing and lipid-lowering functions |
CN111000246A (en) * | 2019-12-27 | 2020-04-14 | 汤臣倍健股份有限公司 | Probiotic dietary fiber composition for assisting in reducing triglyceride, application thereof and health-care product |
CN113755396A (en) * | 2021-10-11 | 2021-12-07 | 中国疾病预防控制中心传染病预防控制所 | Lactobacillus gasseri RW2014 and application thereof in preparation of lipid-lowering drugs |
CN114344344A (en) * | 2022-01-05 | 2022-04-15 | 东北农业大学 | Application of lactobacillus reuteri in relieving high-fat diet induced obesity of mice and compound containing lactobacillus reuteri |
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