CN106399154B - Bacillus acidi lactici probiotics CGMCC NO.12422 and preparing the application in fat-reducing medicament - Google Patents

Bacillus acidi lactici probiotics CGMCC NO.12422 and preparing the application in fat-reducing medicament Download PDF

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CN106399154B
CN106399154B CN201610757751.7A CN201610757751A CN106399154B CN 106399154 B CN106399154 B CN 106399154B CN 201610757751 A CN201610757751 A CN 201610757751A CN 106399154 B CN106399154 B CN 106399154B
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任志鸿
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National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
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Abstract

The invention discloses a kind of Bacillus acidi lactici probiotics strain, the deposit number of the bacterial strain is CGMCC NO.12422, and the deposit date is on Mays 6th, 2016.For the sequence of the 16S rRNA of the bacterial strain as shown in SEQ ID NO:2, the invention also discloses the bacterial strains to prepare the application in fat-reducing medicament, food and health care product, and the bacterial strain is harmless to animal, and has effects that adjust reduction blood lipid.

Description

Bacillus acidi lactici probiotics CGMCC NO.12422 and preparing the application in fat-reducing medicament
Technical field
The present invention relates to a kind of probiotics and its applications, belong to microorganism field.
Background technique
The cardiovascular diseases such as human serum cholesterol level and coronary heart disease, artery sclerosis, hyperlipidemia are closely related, high in fat Mass formed by blood stasis cardinal symptom shows that serum total cholesterol, low density lipoprotein cholesterol, triglycerides equal size are excessively high.How to control It reduces human serum cholesterol levels and has become sanatory major issue, many blood lipid-lowering medicines have good lipid-loweringing to imitate Fruit, but take caused side effect also more and more studied confirmations for a long time, seek new Small side effects, and reduction can be adjusted The research of blood lipid becomes new demand.
Probiotics (Probiotics) is defined as that " one kind microorganism living, can give machine after being ingested with suitable quantity Body brings benefit ".Generally believe that the bacterium that can be used as probiotic composition should have the following conditions at present: (1) beneficial to host； (2) free of toxic effects and pathogenic effects；(3) it can survive in alimentary canal；(4) gastric acid and cholate are adapted to；It (5) can be in alimentary canal Surface field planting；(6) useful lipid and metabolin can be generated；(7) activity is able to maintain in processing and storage；(8) have Good organoleptic attribute.African tribes in 1963 make internal serum cholesterol content due to large amounts of food lactobacillus-fermented product Lower discovery, which causes scientific circles, reduces the concern of cholesterol effect to probiotics.Bacillus acidi lactici reduces gallbladder under environment in vitro The mechanism of sterol mainly includes co-precipitation mechanism, and thallus absorbs and metabolic mechanism, film inlay mechanism.There are many close both at home and abroad In the document report of lactic acid bacteria norcholesterol effect, but specifically lactic acid bacteria screening index, the vivo efficacy and machine of adjusting blood lipid Reason etc. is still not clear, and the effect of lactic acid bacteria norcholesterol is also difficult to reach satisfactory effect.Animal and plant diet Different intestinal floras can be promoted to grow, the present invention is directed to from the Qinghai Wild marmot excrement of only feeding plant separation screening have There is the lactic bacteria strain of good norcholesterol function, and further develops it and preparing the application in fat-reducing medicament.
Summary of the invention
Based on foregoing invention purpose, present invention firstly provides a kind of Bacillus acidi lactici probiotics strain, the probiotics bacterials The deposit number of strain is CGMCC NO.12422, and the deposit date is on May 6th, 2016, preservation classification naming was lactobacillus plantarum (Lactobacillus plantarum), depositary institution are in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode: 100101.
The present invention also provides a kind of Bacillus acidi lactici probiotics strain, the sequence of the 16S rRNA of the bacterial strain such as SEQ ID Shown in NO:2.
The present invention also provides above-mentioned bacterial strains to prepare the application in fat-reducing medicament.
The present invention also provides above-mentioned bacterial strains to prepare the application in food and/or health care product.
The present invention also provides the composition containing above-mentioned bacterial strains, the deposit number of the bacterial strain is CGMCC NO.12422.
In a preferred technical solution, the composition is prepared as capsule, freeze-dried powder or bacterium solution preparation.
The present invention be isolate and purify to obtain the Bacillus acidi lactici with probiotic properties from wild marmot excrement, experiments have shown that Isolated lactic acid bacteria is harmless to animal, and has effects that adjust blood lipid through zoopery confirmation.CGMCC NO.12422 Bacterial strain can significantly reduce low-density lipoprotein, and total cholesterol, triglycerides, increasing high density lipoprotein and simvastatin have phase As effect of regulating blood fat.The rat sign for taking in CGMCC NO.12422 bacterial strain is normal, and intake surrounding takes stomach after terminating experiment Intestines liver and spleen does pathology no abnormality seen.Sterile tissue does Bacteria Detection and has no that lactic acid bacteria dystopy is colonized.
Detailed description of the invention
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 the external lipid-lowering effect schematic diagram of CGMCC NO.12422 bacterial strain
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, does not constitute any restrictions to protection scope of the present invention.
Separation, screening and the identification of 1. lactic acid bacteria of embodiment
1. the separation of Bacillus acidi lactici
1) from protect tube in take out 100 μ L of sample, be added to prepackage the 900 sterile PBS of μ L EP pipe in, successively to sample into Row gradient dilution, marmot stool sample concentration dilution to 10-6Times；
2) it takes the 100 μ L of sample of different dilutions to be coated on MRS culture medium, is put into incubator；
3) at 37 DEG C, 0.5%CO248h is cultivated in environment；
4) it takes out culture dish and is forwarded to new MRS solid culture with the bacterium colony of aseptic inoculation ring picking different shape feature It being purified in base, 37 DEG C of Anaerobic culturel 48h, continuous switching 3 times cultivates purifying bacterial strain in the liquid MRS of pH=3.5, The excellent bacterial strain of screening enduring organic acid can be used for testing or freezing.
2. culture presevation
MRS culture medium of this use for laboratory containing 25% glycerol carries out the freezing of strain as fungus preserving liquid, the method is as follows:
1) by guarantor's tube that capacity is 2mL through 121 DEG C, in case using after the processing of 15min high pressure sterilization；
2) after Bacillus acidi lactici is continuously transferred 3 times on MRS solid medium, the sterile guarantor of 1.5ml is added on culture dish Bacterium solution；
3) culture dish is scratched with L stick, incorporates bacterium colony sufficiently in fungus preserving liquid；
4) bacterium solution is transferred to and is protected in tube, -80 DEG C of preservations after mixing.
3. bacterium colony appearance and thalli morphology observation
Lactobacillus facultative anaerobic bacteria, well-grown under anaerobic, bacterium colony is creamy white, surface is smooth；Having Under the conditions of oxygen, Bacillus acidi lactici can also be grown, and most of strain bacterium colony surface is rougher, and colony colour is mostly milky.It is seen under mirror Examining lactobacillus cell form is pleomorphism, and mostly in elongated rod-shaped, compared with thick bar shape, club-shaped etc., arrangement is in paliform, chain Deng.
4. the extraction of bacteria total DNA
Single bacterium colony is inoculated on BHI culture medium, 37 DEG C of overnight in anaerobiosis cultures, extracts and try according to bacterial genomes DNA The operation of agent box (TIANGEN) specification, extracts DNA.
5. the biochemical identification method of bacterial strain
" BioMerieux " bacterial system biochemical identification carton API50 that this research is produced using Mei Liai company, France CHL。
As a result: according to biochemical identification, obtaining the bacterial strain that one plant of biochemical character is lactobacillus plantarum, bacterial strain deposit number is CGMCC NO.12422 (biochemical identification result is shown in Fig. 2), it is lactobacillus fermenti that inventor, which goes back while obtaining one plant of biochemical character, Bacterial strain, bacterial strain deposit number be CGMCC NO.12421 (biochemical identification the result is shown in Figure 1), the deposit date is Mays 6 in 2016 Day, preservation classification naming is lactobacillus reuteri (Lactobacillus reuteri), and depositary institution is Chinese microorganism strain Preservation administration committee common micro-organisms center, address are the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Object research institute, postcode: 100101.
6. bacterial universal primers 16S rRNA PCR amplification
Bacterial 16 S rRNA identification: extracting bacterial genomes DNA, and amplification Bacillus acidi lactici universal primer 16S rDNA PCR is produced Object sequencing, sequence carry out BLAST comparison on NCBI, carry out Preliminary Identification.
The primer of this experiment bacterial 16 S rRNA PCR amplification used and the condition of PCR reaction are as described below, experiment All with the PCR system of 50 μ L.By to Bacillus acidi lactici universal primer 16S rRNA PCR product sequencing result to be measured in NCBI Upper progress BLAST comparison, identification.Select representative sample PCR product sequencing.Sequencing result is according to the comparison of BLAST Complete the Preliminary Identification to Bacillus acidi lactici.CGMCC NO.12421 bacterial strain is Lactobacillus rogosae (SEQ ID NO:1) as the result is shown, 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 biochemical reaction and 16S rRNA sequence for CGMCC NO.12421 bacterial strain is inconsistent, further After doing gene order-checking, systematic evolution tree (referring to Fig. 3) has been done to core (core-pan) genome, it 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 rogosae, rather than the similar hair 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, it is put into incubator；
2) after cultivating 2h in 37 DEG C of environment, 10 times of gradient dilutions are carried out, are trained with suitable concentration drop kind in MRS solid plate It supports in base, is put into incubator；
3) after 37 DEG C of culture 48h, the bacterium colony on plate is counted, the survival number of bacterium is calculated；
4) select the acidproof preferable strain inoculated of effect into the MRS culture medium containing 0.3% fel bovis salt；
5) after cultivating 4h in 37 DEG C of environment, 10 times of gradient dilutions are carried out, are trained with suitable concentration drop kind in MRS solid plate It supports in base, is put into incubator；
6) after 37 DEG C of culture 48h, the bacterium colony on plate is counted, the survival number of bacterium is calculated.
As a result: this experiment simulates human body intestinal canal environment to investigate bacterial strain in the MRS culture medium of pH=2.0 and 0.3% cholate In survival condition, to evaluate bacterial strain to the tolerance of gastrointestinal tract.The acidproof and bile tolerance ability of two plants of bacterium is good as the result is shown Good and rhamnose LGG bacterial strain acid and bile salt tolerance function is close to (referring to table 1).
Acidproof, the bile tolerance situation of the alternative bacterial strain of table 1
The lipid-reducing function evaluation of embodiment two, lactic acid bacteria
1. lipid-reducing function in-vitro evaluation
(1) configuration of the culture medium containing cholesterol
1) contain the MRS culture medium of 0.1% cholate: weighing cholate powder by 0.1%, be added to the configured MRS liquid of 400ml In body culture medium, through 121 DEG C after mixing well, 15min high pressure steam sterilization is in case use.
2) MRS-CHOL (high cholesterol MRS culture medium): accurately weighing cholesterol 0.5g, is dissolved with a small amount of dehydrated alcohol And it is settled to 50mL, final concentration 10.0mg/mL adds after the filtering with microporous membrane degerming for being 0.22 μm with aperture according to 5% amount Enter in sterilized MRS fluid nutrient medium (or MRS fluid nutrient medium containing 0.1% cholate), makes gallbladder in MRS fluid nutrient medium The final concentration of 500 μ g/mL of sterol.
(2) DAOS method cholesterol detection content
1) principle
Cholesterol can generate hydrogen peroxide while being oxidized.These hydrogen peroxide can make DAOS the and 4- amino in reagent Antipyrine oxidation, and condensed by peroxidase (HRP), and generate a kind of blue pigment.By being compared with standard items It is right, so that the total cholesterol level in sample be allow to be quantified by absorbance.
2) cholesterin detection reagent box (Cholesterol E-test, Wako company, Japan) operating procedure:
1.1) dilution of standard items: cholesterol standards need to dilute 7 concentration, and concentration is from high to low, half-and-half dilute step by step It releases.The EP of 7 1.5mL is taken to manage, number 1-7 first respectively adds the ultrapure water of 50 μ L into 2-7 pipe.No. 1 Guan Weiyong cholesterol standard 100 μ L of product stoste (2000 μ g/mL) takes 50 μ L to be added in No. 2 pipes after concussion mixes, and so on.
1.2) product are loaded --- it takes and has diluted standard items and 2 μ L of test solution is accurately added to 96 orifice plate board bottoms, if multiple holes.
1.3) plus color developing agent --- every hole adds the 150 configured chromogenic reagent solutions of μ L (to be added to 1 bottle of color developing agent dry powder In 150mL buffer, sufficiently dissolution is mixed), avoid light place 5min under the conditions of 37 DEG C.
1.4) terminate --- after 5min, 96 orifice plates are taken out, are pricked a bubble with syringe needle, prevents from influencing OD value.
1.5) read --- it is put into microplate reader and reads OD value at 600nm.
(3) result: after cultivating for 24 hours in the culture medium containing cholesterol, the comparison of the removal rate of cholesterol of three kinds of bacterial strains is shown in Fig. 4.In Fig. 4,1 is LGG, and 2 be CGMCC NO.12421 bacterial strain, and 3 be CGMCC NO.12422 bacterial strain, as the result is shown CGMCC NO.12421 bacterial strain and CGMCC NO.12422 bacterial strain reduce cholesterol function and LGG are close under the conditions of no cholate.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
By random point four respectively of the female sd inbred rats of 40 about 200 grams of weight and 40 about 20 grams of BALB/c female rats Group gives high lipid food (cholesterol 1%, lard 10%, 0.2 cholate, the 10% yolk powder) experiment of termination in totally five weeks detection blood Clear items blood lipid level, feeding high in fat one group of orally administration 10 after a week9CFU CGMCC NO.12421 Bacillus acidi lactici bacterium solution, one Group gives CGMCC NO.12422 bacterial strain, and one group is given physiological saline, and one group terminates the last fortnight in experiment and gives 20mg/kg weight Simvastatin, observe the prevention intervention effect that hyperlipoidemia occurs for drug or lactic acid bacteria.
Fig. 5 is lipid-lowering effect histogram of the experimental strain to rat model, wherein 1 is CGMCC NO.12422 bacterial strain group, 2 be CGMCC NO.12421 bacterial strain group, and 3 be simvastatin group, and control group is control with PBS.From fig. 5, it can be seen that CGMCC NO.12421 bacterial strain and the low-density lipoprotein (LDL) of CGMCC NO.12422 bacterial strain group be average be respectively 3.534mmol/L and 3.341mmol/L, substantially less than model control group 3.969mmol/L (p < 0.05).CGMCC NO.12421 bacterial strain and CGMCC It is 1.542mmol/L and 1.5761mmol/L respectively that the high density degree lipoprotein (HDL) of NO.12422 bacterial strain group is average, 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 substantially less than model control group 5.774mmol/ L(p<0.05).CGMCC NO.12421 bacterial strain and the triglyceride (TC) of CGMCC NO.12422 bacterial strain group are averagely respectively 3.47mmol/L and 3.288mmol/L, substantially less than model control group 3.853mmol/L (p < 0.05).Above-mentioned bacterial strains have Total cholesterol is significantly reduced, triglycerides, increasing high density lipoprotein and simvastatin have similar reduction blood lipid effect.
The rat for taking in two kinds of lactic acid bacterias is acted normally, and intake surrounding takes stomach and intestine liver and spleen to do pathology to have no different after terminating experiment Often.Sterile tissue does Bacteria Detection and has no that lactic acid bacteria dystopy is colonized.

Claims (5)

1. the deposit number of a kind of Bacillus acidi lactici probiotics strain, the bacterial strain is CGMCC NO.12422, the deposit date is 2016 On May 6, in.
2. bacterial strain according to claim 1 is preparing the application in fat-reducing medicament.
3. bacterial strain according to claim 1 is preparing the application in food and/or health care product.
4. a kind of composition containing bacterial strain described in claim 1.
5. composition according to claim 4, the composition is prepared as capsule, freeze-dried powder or bacterium solution preparation.
CN201610757751.7A 2016-08-29 2016-08-29 Bacillus acidi lactici probiotics CGMCC NO.12422 and preparing the application in fat-reducing medicament Active CN106399154B (en)

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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
CN110205261B (en) * 2019-05-10 2020-08-04 湖南菲勒生物技术有限公司 Application of breast milk-derived lactobacillus reuteri in reducing blood fat 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
CN113755396B (en) * 2021-10-11 2023-03-14 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
CN114292795B (en) * 2022-01-20 2023-07-25 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 preparation of lipid-lowering drugs
CN114561320B (en) * 2022-01-20 2023-07-21 中国疾病预防控制中心传染病预防控制所 Application of lactobacillus probiotics CGMCC No.1.13855 in preparing medicines for treating liver diseases
EP4407023A1 (en) * 2022-11-30 2024-07-31 Aiage Life Science Corporation Ltd. Lactobacillus reuteri for prolonging lifespan, resisting aging and reducing fat, and product thereof and use thereof

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CN105567586A (en) * 2015-12-21 2016-05-11 南昌大学 Lactobacillus plantarum having anti-diabetic function and application thereof

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