CN106399154A - Lactobacillus probiotics CGMCC NO. 12422 and application of lactobacillus probiotics in preparing lipid-lowering drug - Google Patents
Lactobacillus probiotics CGMCC NO. 12422 and application of lactobacillus probiotics in preparing lipid-lowering drug Download PDFInfo
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Abstract
The invention discloses a lactobacillus probiotic strain. The preservation number of the strain is CGMCC NO. 12422, and the preservation date is May, 6th, 2016. The 16S rRNA sequence of the strain is shown as SEQ ID NO: 2. The invention also discloses an application of the strain in preparing a lipid-lowering drug, food and a healthcare product. The strain is harmless to animals and has effects of regulating and lowering blood fat.
Description
Technical field
The present invention relates to a kind of probiotic bacteria and its application, belong to microorganism field.
Background technology
Human serum cholesterol level is closely related with cardiovascular disease such as coronary heart disease, arteriosclerosis, hyperlipemias, high fat
Mass formed by blood stasis cardinal symptom shows that serum total cholesterol, low-density lipoprotein cholesterol, triglyceride 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 lipid-loweringing effect
Really, the studied confirmation but the side effect caused by long-term taking also gets more and more, seeks new Small side effects, can adjust reduction again
The research of blood fat becomes new demand.
Probiotic bacteria (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 antibacterial of probiotic composition at present：(1) beneficial to host；
(2) free of toxic effects and pathogenic effects；(3) can survive in digestive tract；(4) gastric acid and cholate are adapted to；(5) can be in digestive tract
Surface is colonized；(6) useful lipid and metabolite can be produced；(7) activity can be kept in processing and storage；(8) have
Good organoleptic attribute.African tribes make internal serum cholesterol content due to eating lactic acid bacteria fermentation product in a large number within 1963
Relatively low discovery causes the concern that scientific circles reduce cholesterol effect to probiotic bacteria.Lactobacilluss reduce gallbladder in vitro under environment
The mechanism of sterin mainly includes co-precipitation mechanism, and thalline absorbs and metabolic mechanism, and film inlays mechanism.There are many passes both at home and abroad
In the document report of lactic acid bacteria cholesterol reducing effect, but specific lactic acid bacteria screening index, the vivo efficacy adjusting blood fat and machine
Reason etc. is still not clear, and the gratifying effect of the effect of lactic acid bacteria cholesterol reducing also difficult to reach.Animal and plant diet
Different intestinal microbial populations can be promoted to grow, it is contemplated that separation screening tool from the Qinghai Wild marmot feces of only feeding plant
There is the lactic bacteria strain of good cholesterol reducing 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 lactobacilluss probiotics strain, described probiotics bacterial
The deposit number of strain is CGMCC NO.12422, and preservation date is on May 6th, 2016, and preservation Classification And Nomenclature is Lactobacillus plantarum
(Lactobacillus plantarum), depositary institution is in China Committee for Culture Collection of Microorganisms's common micro-organismss
The heart, 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 lactobacilluss probiotics strain, the sequence such as SEQ ID of the 16S rRNA of described bacterial strain
NO：Shown in 2.
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 product for the above-mentioned bacterial strains.
Present invention also offers the compositionss containing above-mentioned bacterial strains, the preserving number of described bacterial strain is CGMCC NO.12422.
In a preferred technical scheme, described compositionss are prepared as capsule, lyophilized powder or bacterium solution preparation.
The present invention is to isolate and purify the lactobacilluss obtaining thering is probiotic properties from wild marmot feces, and experiment proves
Separate the lactic acid bacteria obtaining harmless to animal, and have functions that to adjust blood fat through zoopery confirmation.CGMCC NO.12422
Bacterial strain can significantly reduce low density lipoprotein, LDL, T-CHOL, triglyceride, high density lipoprotein increasing, and simvastatin phase
As effect of regulating blood fat.The rat sign taking in CGMCC NO.12422 bacterial strain is normal, takes in after surrounding terminates experiment and takes stomach
Intestinal liver spleen does pathology no abnormality seen.Sterile tissue is done Bacteria Detection and is had no that lactic acid bacteria dystopy is colonized.
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 systematic evolution tree figure；
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
Specific embodiment
To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
Apparent.But these embodiments are only exemplary, any restriction is not constituted to protection scope of the present invention.
Separation, screening and the identification of embodiment 1. lactic acid bacteria
1. the separation of lactobacilluss
1) from protecting taking-up specimen 100 μ L tube, it is added in the EP pipe of the prepackage aseptic PBS of 900 μ L, successively sample is entered
Row gradient dilution, marmot stool sample concentration dilution to 10-6Times；
2) take different dilution sample 100 μ L to coat in MRS culture medium, put in incubator；
3) at 37 DEG C, 0.5%CO248h is cultivated in environment；
4) take out culture dish, with the bacterium colony of aseptic inoculation ring picking different shape feature, be forwarded to new MRS solid culture
Carry out purification, 37 DEG C of Anaerobic culturel 48h, continuously switching 3 times, purification bacterial strain is cultivated in the liquid MRS of pH=3.5 in base,
The excellent bacterial strain of screening enduring organic acid can be used for experiment or freezing.
2. culture presevation
The MRS culture medium containing 25% glycerol for this use for laboratory carries out the freezing of strain as fungus preserving liquid, and method is as follows：
1) by guarantor's tube for 2mL for the capacity through 121 DEG C, in case using after 15min autoclaving is processed；
2), after lactobacilluss continuous switching 3 times on MRS solid medium, the aseptic guarantor of 1.5ml is added on culture dish
Bacterium solution；
3) with L rod, culture dish is scratched, so that bacterium colony is fully incorporated in fungus preserving liquid；
4) bacterium solution is transferred in guarantor's tube, -80 DEG C of preservations after mixing.
3. bacterium colony outward appearance and thalli morphology are observed
Lactobacilluss facultative anaerobe, well-grown under anaerobic, bacterium colony is creamy white, smooth surface；Having
Under the conditions of oxygen, lactobacilluss also can grow, and most of strain bacterium colony surface is rougher, and colony colour mostly is milky.See under mirror
Examining lactobacillus cell form is pleomorphism, and how in elongated rod-shaped, compared with thick bar shape, club shape etc., arrangement is in paliform, chain
Deng.
4. the extraction of bacteria total DNA
Single bacterium colony is inoculated in BHI culture medium, 37 DEG C of overnight in anaerobiosis cultures, according to the examination of bacterial genomes DNA extraction
Agent box (TIANGEN) description operates, and extracts DNA.
5. the biochemical identification method of bacterial strain
" BioMerieux " bacterial system biochemical identification carton API50 that this research is produced using French Mei Liai company
CHL.
Result：According to biochemical identification, obtain the bacterial strain that one plant of biochemical character is Lactobacillus plantarum, bacterial strain preserving number is
CGMCC NO.12422 (biochemical identification result is shown in Fig. 2), it is Lactobacillus fermenti that inventor also obtains one plant of biochemical character simultaneously
Bacterial strain, bacterial strain deposit number be CGMCC NO.12421 (biochemical identification result is shown in Fig. 1), preservation date be May 6 in 2016
Day, preservation Classification And Nomenclature is Lactobacillus reuteri (Lactobacillus reuteri), and depositary institution is Chinese microorganism strain
Preservation administration committee common micro-organisms center, address is the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Thing institute, postcode：100101.
6. bacterial universal primers 16S rRNA PCR amplification
Bacterial 16 S rRNA identifies：Extract bacterial genomes DNA, amplification lactobacilluss universal primer 16S rDNA PCR produces
Thing is sequenced, and sequence carries out BLAST comparison on NCBI, carries out Preliminary Identification.
The condition of this experiment primer of bacterial 16 S rRNA PCR amplification used and PCR reaction is as described below, experiment
All with the PCR system of 50 μ L.By to lactobacilluss universal primer 16S rRNA PCR primer sequencing result to be measured in NCBI
On carry out BLAST comparison, identification.Select representative sample PCR primer sequencing.Sequencing result is according to the comparison of BLAST
Complete the Preliminary Identification to lactobacilluss.Result display CGMCC NO.12421 bacterial strain is Lactobacillus rogosae (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
The qualification result of the biochemical reaction for CGMCC NO.12421 bacterial strain and 16S rRNA sequence is inconsistent, further
After doing gene order-checking, systematic evolution tree (referring to Fig. 3) is done to core (core-pan) genome, can by systematic evolution tree
Finding out the evolutionary relationship of CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain and other lactobacilluss it is thus identified that
The qualification result of 16S rRNA sequence, CGMCC NO.12421 bacterial strain is Lactobacillus rogosae, rather than similar the sending out of biochemical character
Kefir milk bacillus.
7. acidproof, the bile tolerance screening technique of bacterial strain
1) after the experimental strain growth saturation of three generations to be activated, 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 culture 2h in 37 DEG C of environment, carry out 10 times of gradient dilutions, dripped with suitable concentration and plant in the training of MRS solid plate
In foster base, put into incubator；
3) after 37 DEG C of culture 48h, the bacterium colony on plate is counted, calculates the survival number of antibacterial；
4) select the preferable inoculation of acidproof effect in the MRS culture medium containing 0.3% Fel Bovis seu Bubali salt；
5) after culture 4h in 37 DEG C of environment, carry out 10 times of gradient dilutions, dripped with suitable concentration and plant in the training of MRS solid plate
In foster base, put into incubator；
6) after 37 DEG C of culture 48h, the bacterium colony on plate is counted, calculates the survival number of antibacterial.
Result：This experimental simulation human body intestinal canal environment is investigating the MRS culture medium in pH=2.0 and 0.3% cholate for the bacterial strain
In survival condition, to evaluate bacterial strain to gastrointestinal tolerance.Result shows that the acidproof of two plants of bacterium and bile tolerance ability are good
Good, and the acid and bile salt tolerance function of rhamnose LGG bacterial strain is close to (referring to table 1).
Acidproof, the bile tolerance situation of the alternative bacterial strain of table 1
Embodiment two, the lipid-reducing function evaluation of lactic acid bacteria
1. lipid-reducing function in-vitro evaluation
(1) contain the configuration of cholesterol culture medium
1) contain the MRS culture medium of 0.1% cholate：Weigh cholate powder by 0.1%, be added to 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 use.
2) MRS CHOL (hypercholesterolemia MRS culture medium)：Accurately weigh cholesterol 0.5g, use a small amount of anhydrous alcohol solution
And it is settled to 50mL, final concentration 10.0mg/mL, after the filtering with microporous membrane being 0.22 μm with aperture is degerming, the amount according to 5% adds
Enter in sterilized MRS fluid medium (or the MRS fluid medium containing 0.1% cholate), make gallbladder in MRS fluid medium
The final concentration of 500 μ g/mL of sterin.
(2) DAOS method cholesterol detection content
1) principle
Hydrogen peroxide can be produced while cholesterol is oxidized.These hydrogen peroxide can make DAOS the and 4- amino in reagent
Phenazone aoxidizes, and is condensed by peroxidase (HRP), and produces a kind of blue pigment.By being compared with standard substance
Right, so that the total cholesterol level in sample can carry out quantitation by absorbance.
2) cholesterin detection reagent box (Cholesterol E-test, Wako company, Japan) operating procedure：
1.1) dilution of standard substance：Cholesterol standards need to dilute 7 concentration, and concentration is from high to low, half-and-half dilute step by step
Release.The EP taking 7 1.5mL manages, numbering 1-7, first respectively adds the ultra-pure water of 50 μ L toward in 2-7 pipe.No. 1 Guan Weiyong cholesterol standard
Product stock solution 100 μ L (2000 μ g/mL), after concussion mixes, takes 50 μ L to be added in No. 2 pipes, the rest may be inferred.
1.2) sample-adding product take and have diluted standard substance and test solution 2 μ L is accurately added to 96 orifice plate plate bottoms, if multiple holes.
1.3) plus the every hole of developer adds the chromogenic reagent solution that 150 μ L configure and (is added to 1 bottle of developer dry powder
In 150mL buffer, fully dissolving mixes), avoid light place 5min under the conditions of 37 DEG C.
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 microplate reader and is read OD value at 600nm.
(3) result：After culture 24h in the culture medium containing cholesterol, relatively the 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 no cholate, reduce cholesterol function and LGG are close.Containing
Under the conditions of cholate, Lactobacillus plantarum CGMCC NO.12422 bacterial strain cholesterol reducing ability is higher than another two bacterial strain.
2 lipid-reducing function interior evaluatings
The female sd inbred rats of 40 about 200 grams of body weight and 40 about 20 grams of BALB/c female rats are divided four respectively at random
Group, gives high lipid food (cholesterol 1%, Adeps Sus domestica 10%, 0.2 cholate, 10% yolk powder) and terminates within five weeks experiment detection blood totally
Every clearly blood lipid level, one week later group orally administration 10 of high fat feeding9CFU CGMCC NO.12421 lactobacilluss bacterium solution, one
Group gives CGMCC NO.12422 bacterial strain, and one group gives normal saline, and one group terminates the last fortnight in experiment and gives 20mg/kg body weight
Simvastatin, observe the prevention intervention effect that medicine or lactic acid bacteria occur to hyperlipoidemia.
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 matched group is with PBS for comparison.From fig. 5, it can be seen that CGMCC
The low density lipoprotein, LDL (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) all it is 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 have
Significantly reducing T-CHOL, triglyceride, high density lipoprotein increasing, and simvastatin has similar reduction blood fat effect.
The rat taking in two kinds of lactic acid bacterias is acted normally, and absorption surrounding takes gastrointestinal liver spleen to do pathology after terminating experiment and has no different
Often.Sterile tissue is done Bacteria Detection and is had no that lactic acid bacteria dystopy is colonized.
Claims (6)
1. a kind of lactobacilluss probiotics strain, the preserving number of described bacterial strain is CGMCC NO.12422, and preservation date is 2016
On May 6, in.
2. a kind of lactobacilluss probiotics strain is it is characterised in that the sequence such as SEQ ID NO of the 16S rRNA of described bacterial strain：2
Shown.
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 product for the bacterial strain according to claim 1 and 2.
5. a kind of compositionss containing the bacterial strain described in claim 1 or 2.
6. compositionss according to claim 5, described compositionss are prepared as capsule, lyophilized powder or bacterium solution preparation.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107981360A (en) * | 2017-11-24 | 2018-05-04 | 华东理工大学 | A kind of application of lactobacillus plantarum X7021 |
| 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 |
| 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 |
| WO2020228144A1 (en) * | 2019-05-10 | 2020-11-19 | 湖南菲勒生物技术有限公司 | Application of breast milk-derived lactobacillus reuteri in lowering lipid and regulating lipid metabolism rhythm |
| 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 |
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| CN107981360A (en) * | 2017-11-24 | 2018-05-04 | 华东理工大学 | A kind of application of lactobacillus plantarum X7021 |
| 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 |
| WO2020228144A1 (en) * | 2019-05-10 | 2020-11-19 | 湖南菲勒生物技术有限公司 | Application of breast milk-derived lactobacillus reuteri in lowering lipid and regulating lipid metabolism rhythm |
| 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 |
| CN114292795A (en) * | 2022-01-20 | 2022-04-08 | 中国疾病预防控制中心传染病预防控制所 | Lactobacillus probiotics CGMCC No.1.13855 and application thereof in preparing lipid-lowering drugs |
| CN114561320A (en) * | 2022-01-20 | 2022-05-31 | 中国疾病预防控制中心传染病预防控制所 | Application of lactobacillus probiotic CGMCC No.1.13855 in preparation of liver disease treatment drug |
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