CN104531590B - One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11 - Google Patents
One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11 Download PDFInfo
- Publication number
- CN104531590B CN104531590B CN201510018144.4A CN201510018144A CN104531590B CN 104531590 B CN104531590 B CN 104531590B CN 201510018144 A CN201510018144 A CN 201510018144A CN 104531590 B CN104531590 B CN 104531590B
- Authority
- CN
- China
- Prior art keywords
- bifidobacterium
- bacterial strain
- bifidobacterium animalis
- norcholesterol
- oxytolerant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11, the bacterial strain are bifidobacterium animalis subspecies（Bifidobacterium animalis subsp. Lactis）, the bacterial strain pure culture is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 18th, 2014（CGMCC）, address is：No. 3 Institute of Microorganism, Academia Sinica of city of BeiJing, China Chaoyang District North Star West Road 1 institute, postcode 100101；Deposit number CGMCC NO.10224, abbreviation bifidobacterium animalis subspecies BZ11.Bifidobacterium animalis subspecies BZ11 not only norcholesterol ability it is high, it is acidproof by property, bile tolerance by property, oxytolerant is functional and has probiotic properties, it can be added to as probiotics in fermented dairy product and become functional food, bifidobacterium species resource is enriched, is advantageous to the exploitation of Bifidobacterium Bifidum health product.
Description
Technical field
The present invention relates to microorganism, more particularly to norcholesterol, oxytolerant Bifidobacterium.
Background technology
Cholesterol is widely present in animal tissue cell, is one of important component for forming cell, is played in human body
Important physiological action, it converts to form steroid hormone in vivo, is the precursor of synthesis of vitamin d and bile acid
Matter.Internal cholesterol mainly has two sources, when food, second, endogenous synthesis.Some people are due to unreasonable dietary, blood
Liquid cholesterol level exceedes normal index；And in vivo, cholesterol level is too high can cause the heart and brain such as atherosclerosis, coronary heart disease
Vascular diseases, serious threat to human health.It is reported that global cardiovascular and cerebrovascular disease causes the number of human death to account for total death
The 29% of number, is predicted according to the World Health Organization, and to the year two thousand thirty, cardiovascular and cerebrovascular disease will cause that human death's is main
Reason, and cholesterol in serum it is too high be trigger the cardiovascular and cerebrovascular diseases such as coronary heart disease, artery sclerosis, headstroke it is important because
Element.Studies have found that compared with the people for possessing normal lipid, the risk that the people of hypercholesterolemia suffers from a heart complaint is its normal person
3 times.Therefore, by reduce serum cholesterol level come to cardiovascular and cerebrovascular disease carry out preventing and treating be a kind of feasible research of actual effect
Direction, have potential application value and it is wide be the market space.
It is intended to reduce internal cholesterol, in addition to by reasonable diet, with biotransformation method, particularly with probiotics
Directly degraded is carried out to cholesterol has turned into the trend of research.Bifidobacterium is as a kind of probiotics in human body intestinal canal, its benefit
Raw effect, which has, to be maintained intestinal flora balance, reduces the prebiotic effect such as serum cholesterol, antitumor.And Guizhou Xiang pig is Guizhou Province
Characteristic resources, belong to miniature pig, its inheritance stability, organ structure and function are similar to human body.Therefore, screened from fragrant pig
Bifidobacterium be easy to be received and utilized by human body.
At present in Chinese patent database, it is many to be related to the patent application of Bifidobacterium, but is related to oxytolerant Bifidobacterium
Patent application only have Zhejiang University application No. ZL2009100965117《A kind of oxygen-resistant bifidobacteria》With Tianjin science and technology
No. ZL2011100894937 of university's application《A kind of oxygen-resistant acid-resistant Bifidobacterium longum》.Can the two Bifidobacteriums drop courage
Sterol is unknown.So far, it there is no the application part for being related to norcholesterol, oxytolerant animal bifidobacteria.
The content of the invention
The present invention is intended to provide one plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium, make it possible to as probiotics
Further deep development utilizes, and is added in fermented dairy product and becomes functional food, to reduce human cholesterol content,
Promote human health.
The fragrant pig source property norcholesterol of inventor's offer, oxytolerant Bifidobacterium, the bacterial strain is bifidobacterium animalis subspecies
(Bifidobacterium animalis subsp.Lactis), the pure culture of the bacterial strain are protected on December 18th, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) is hidden in, the centre address is：City of BeiJing, China
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode：100101；Deposit number is CGMCC
NO.10224, referred to as bifidobacterium animalis subspecies BZ11.The bacterial strain is molten out of Guizhou characteristic resources Mini-musk swine enteron aisle
Screen and isolate and purify to obtain in thing.
Above-mentioned bifidobacterium animalis subspecies BZ11 morphological feature and physiological and biochemical property bacterium are all close to Bifidobacterium
Category, its 16S rRNA gene order and bifidobacterium animalis subspecies (Bifidobacterium animalis
Subsp.Lactis 16S rRNA sequence homologies) are 100%, according to《Primary Jie Shi systematic bacteriologies handbook》Middle Bifidobacterium
The categorised regulation of category, bifidobacterium animalis subspecies BZ11 belong to actinomyces door (Actinobacteria), Actinomycetes
(Actinobacteria), actinomyces subclass (Actinobacteridae), Bifidobacterium mesh (Bifidoacteriales), double
Discrimination Bacteriaceae (Bifidobacteriaceae), Bifidobacterium (Bifidobacterium), bifidobacterium animalis subspecies
(Bifidobacteriumanimalis subsp.Lactis)。
Above-mentioned animal bifidobacteria BZ11 security is as follows：The bacterial strain is to benzyl penicillin, Piperacillin, Amoxicillin/rod
Acid, vancomycin, CTX, cephazoline, azithromycin, rifampin, the class antibiotic sensitive of furantoin 9, it is husky to ring third
Star, neomycin, SMZco, Ofloxacin, Norfloxacin and polymyxin B totally 6 class antibiotics resistance.Penicillin medicine
In, the bacterial strain is to ampicillin sensitive；In cephalo element class medicine, the bacterial strain is sensitive to cefoperazone；In Macrocyclolactone lactone kind medicine,
Bacterial strain BZ11 is to the mould sensitivity of acetyl spiral；In QNS, the bacterial strain is sensitive to woods mycin.It is and real by gavage mouse
Test, show bifidobacterium animalis subspecies BZ11 bacterial strains without acute toxicity, thus can with the security of tentative confirmation BZ11 bacterial strains,
It is contemplated that utilized as the further deep development of probiotics.And its drop courage in vivo is further demonstrated by gavage mouse experiment
Sterol acts on.
Inventor provide bacterial strain be from the characteristic resources Mini-musk swine enteron aisle Dissolve things inside of Guizhou separation and preliminary screening provide
There is the Bifidobacterium of norcholesterol ability, and the bacterial strain of gained is carried out using norcholesterol rate, acid and bile salt tolerance by property as index
Further secondary screening, secondary screening go out one plant of removal rate of cholesterol it is high and it is acidproof by property, bile tolerance by the good Bifidobacterium bacterium of property
Strain, oxytolerant performance test, safety evaluatio experiment and gavage mouse experiment are then carried out, afterwards by carrying out form to the bacterial strain
, physiological and biochemical test identification and 16S rRNA Sequence Identifications, it is bifidobacterium animalis subspecies finally to identify this bacterial strain
(Bifidobacterium animalis subsp.Lactis)。
In order to verify the bacterial strain of the present invention, inventor has carried out following experiment：
(1) collection of sample is with isolating and purifying
Sampled from multiple positions of Guizhou Province's characteristic resources Mini-musk swine, including small intestine, large intestine and fresh Mini-musk swine excrement
Just in, number and record, and take back laboratory treatment rapidly.10g fresh sample is taken to be put into the sterile physiological salt equipped with 90mL
In water, after manually shaking up, it is coated in clean bench with liquid-transfering gun 100 μ l mixed liquors of absorption containing Li-Mupirocin's
On MRS flat boards, anaerobic culture box (anaerobic environment N is placed in2∶CO2∶H2=90: 5: in 5), 37 DEG C are cultivated 48h, then picking list
Colony inoculation is put into 37 DEG C, 20%CO on the PTYG flat boards containing X-gal2Two are cultivated in the CO2gas incubator of concentration
My god, observation colony colour, form simultaneously carry out Gram's staining, microscopy, record strain morphology feature.By doubtful bifidobacterium strain
After purifying is passed on 4 times on PTYG flat boards, glycerol stocks are in -80 DEG C of ultralow temperature refrigerator-freezer.
Above-mentioned MRS agar mediums composition is：Peptone 10g, beef extract 10g, dusty yeast 5g, glucose 20g, tween
801mL, K2HPO42g, sodium acetate 5g, dibasic ammonium citrate 2g, MgSO4·7H2O 0.58g, MnSO4·4H2O 0.25g, agar
20g；Preparation method is to dissolve by heating each composition in 1000mL distilled water, adjusts pH value to 6.5,121 DEG C of 15min that sterilize, standby.
In the above-mentioned MRS containing Li-Mupirocin, 100mLMRS agar mediums contain 5mg mupirocin lithium salts
(Li-Mupirocin)。
Above-mentioned PTYG Liquid Cultures based component is：Tryptone 5g, soy peptone 5g, dusty yeast 10g, glucose 10g,
Tween 80 1mL, salting liquid 40mL；Preparation method is to dissolve by heating each composition in 1000mL distilled water, adjusts pH value to 6.5,121 DEG C
Sterilize 15min, standby.
Salting liquid composition in above-mentioned culture medium is：Anhydrous CaCl20.2g,K2HPO41.0g,KH2PO41.0g,MgSO4·
7H2O 0.48g, Na2CO310g,NaCl 2g；Preparation method is by CaCl2And MgSO47H2O be blended in 300mL distilled water until
Dissolving, 500mL water is added, stirring is slowly added to other salts simultaneously, until dissolving；200mL distilled water is added, is store after mixing
It is stored in 4 DEG C, it is standby.
Above-mentioned PTYG agar mediums preparation method is：20g agar powders are added in every 1000mL fluid nutrient medium, heating is boiled
Boil, pour plate after 121 DEG C of sterilizing 15min is standby.
Above-mentioned PTYG (PTYG-X) culture medium preparation method containing X-gal is：In every 1000mL improvement PTYG culture mediums
X-gal 40mg are added, are 20mg/mL directly with sterilized water diluted concentration due to X-gal non-refractories, each PTYG flat boards apply
Cloth 40 μ L, it is standby.
(2) measure of removal rate of cholesterol
Bacterial strain obtained by primary dcreening operation is surveyed into its removal rate of cholesterol using o-phthalaldehyde method (OPA).
(3) preparation of bacteria suspension to be measured
The bacterial strain being deposited in -80 DEG C of ultralow temperature refrigerator-freezer is activated, after line purifies 3 times, is inoculated in the training of PTYG liquid
Support in base and carry out enrichment culture, then centrifuge bacteria suspension, collect bacterium mud, bacterial concentration is adjusted to 108CFU/mL, bacterium suspension
Band is used for external tolerance test.
(4) sour tolerance test
The sterilizing PTYG liquid that bacteria suspension to be measured is inoculated in into pH3.0 and pH7.0 respectively by 5% (volume ratio) inoculum concentration is trained
Support in base, stand 2h after mixing at 37 DEG C, carry out plate count.
(5) Bile salt resistance is tested
Bacteria suspension to be measured is inoculated in addition 0.3% (mass/volume) bovine bile respectively by the inoculum concentration of 5% (volume ratio)
Sterilizing PTYG fluid nutrient mediums and sterilizing PTYG Liquid Cultures without bovine bile in, 37 DEG C, train in CO2gas incubator
Support, count plate is carried out respectively at 0h and 24h.
(6) oxytolerant performance test
By by the inoculation of acid resistance and Bile salt resistance into PTYG culture mediums respectively in standard incubator and
20%CO2CO2gas incubator in cultivate 24h after, carry out plate count.Both are grown and goes to be contrasted to judge it
Oxygen resistence.
Tested by above-mentioned sampling, the primary dcreening operation isolated and purified and the norcholesterol rate with bacterial strain, tolerance are entered for index
Row secondary screening, finally give one plant of bacterial strain BZ11, its norcholesterol rate is high, reaches 38.52%, and it is acidproof by property and bile tolerance by property
It is good, and have preferable tolerance to oxygen.
(7) the safety evaluatio experiment of bacterial strain
A, drug sensitivity assay
After BZ11 bacterial strains are activated and purify for 3 generations in PTYG agar mediums, liquid PTYG culture medium enrichment cultures, so
Bacterium solution is centrifuged into 10min under the conditions of 5000r/min, 4 DEG C afterwards, after collecting bacterium mud washing, is configured to PBS
108CFU/mL bacteria suspension.Then 8 class, 19 kinds of susceptibility analoidses are chosen, medicine is carried out using K-B drug sensitive test papers agar diffusion method
Sensitive experiment.
B, acute toxicity testing
After BZ11 bacterial strains are activated, isolate and purify three times, inoculating strain carbon dioxide in PTYG fluid nutrient medium is trained
Support in case and cultivate 48h, by bacterium solution with 5000r/min, 10min is centrifuged under the conditions of 4 DEG C, collects bacterium mud, then use sterile phosphate
Buffer solution PBS washed once newly formed suspension of laying equal stress on, and bacterial concentration is adjusted into 3 × 1010CFU/mL.Then according to food security
Property toxicological evaluation program and method carry out acute toxicity testing.
(8) norcholesterol, test inside oxytolerant Bifidobacterium
Purified 3 times after strain to be tested BZ11 and the defrosting activation of control strain Infantile diarrhea, respectively with the inoculation of 5% (volume ratio)
Amount is inoculated in PTYG fluid nutrient mediums, after cultivating 24h in 37 DEG C of CO2gas incubator, with 5000r/min, 4 DEG C of bars
10min is centrifuged under part, collects bacterium mud.10 times of gradient dilutions are carried out with sterilizing PBS again, take 0.1mL to be put down in PTYG agar
It is coated with plate, carries out bacterium colony counting after 37 DEG C of 36~48h of culture, according to plate count result, respectively adjust BZ11 and Infantile diarrhea
To bacteria suspension to 3.0 × 109CFU/mL.Then mouse stomach experiment is carried out, was taken a blood sample at the 10th, 20 day, is surveyed total in its serum
Cholesterol level, content of triglyceride, HDL-C content, LDL-C content, and calculate
Its atherogenic index.To prevent Bifidobacterium is dead from influenceing result, daily fresh configuration bacterium solution, morning timing feeds and put
Put feed.
(9) identification of bacterial strain
Physiology and biochemistry identification and the identification of kind are carried out to the bacterial strain of gained, its physiological and biochemical property is understood, to effective
Ground application document, further research and application is instructed to play an important role.Therefore, physiology life is carried out to the bacterial strain BZ11 of the present invention
Change the identification of experimental identification and 16S rRNA sequences.
Morphological feature：By inoculation on PTYG flat boards, 37 DEG C of culture 48h, its colonial morphology is that bacterium colony is smaller, light
Cunning, dome, neat in edge, white or milky is opaque and soft texture, then show navy blue on PTYG-X flat boards
Bacterium colony.Its bacterium colony of picking carries out Gram's staining, and its Gram's staining is positive, and observes its cell shape under an optical microscope
State is in polymorphic shaft-like, meets bifidobacterium cells morphological feature.Its bio-chemical characteristics the results are shown in Table 1.
The cellular morphology of table 1 and Physicochemical test result
Note：+：The positive ,-：It is negative
Cellular morphology more than meets the feature of Bifidobacterium with Physicochemical test result, bacterial strain BZ11 features, just
Step identification category Bifidobacterium.
The bacterial strain of the present invention is by China General Microbiological culture presevation administrative center (China General
Microbiological Culture Collection Center, CGMCC) 16S rRNA Sequence Identifications are carried out, by measured by
16S rRNA gene orders log in http://blast.ncbi.nlm.nih.gov/Blast.cgi websites, by online
BLAST (Basic Local Alignment Search Tool), is searched 100 higher with the sequence homology altogether
Genetic fragment, and the 16S rRNA gene orders of the bacterial strain and bifidobacterium animalis subspecies (Bifidobacterium
Animalis subsp.Lactis) 16S rRNA sequence homologies reach 100%, choose 20 plants of higher bacterium of homology, should
With MEGA5.0 analysis software constructing system chadograms.Mapping is shown, in animal bifidobacteria BZ11 bacterial strains and Genbank
Bifidobacterium animalis subsp.lactis AD011strain AD011 and Bifidobacterium
Animalis subsp.lactis strain 4,121 two plants of bifidobacterium animalis subspecies of YIT belong to a minimum branch
In, show that the affiliation between them is nearest.
Therefore, according to its morphological feature, Physicochemical test result and according to the system constructed by 16S rRNA gene orders
Development tree analysis, according to《Primary Jie Shi systematic bacteriologies handbook》The categorised regulation of middle Bifidobacterium, bifidobacterium animalis are sub-
Kind BZ11 bacterial strains belong to actinomyces door (Actinobacteria), Actinomycetes (Actinobacteria), actinomyces subclass
(Actinobacteridae), Bifidobacterium mesh (Bifidoacteriales), bifidobacterium family
(Bifidobacteriaceae), Bifidobacterium (Bifidobacterium), bifidobacterium animalis subspecies
(Bifidobacterium animalis subsp.Lactis)。
Fragrant pig source property bifidobacterium animalis subspecies BZ11 (the Bifidobacterium animalis of the present invention
Subsp.Lactis), not only norcholesterol ability it is high, it is acidproof by property, bile tolerance by property, oxytolerant is functional and has prebiotic spy
Property, and can be added to as probiotics in fermented dairy product and become functional food, so as to enrich Bifidobacterium bacterium
Kind resource, the exploitation to Bifidobacterium Bifidum health product have positive effect.
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Brief description of the drawings
Fig. 1 is bacterial strain BZ11 and the 16S rRNA sequential systems development chadogram of relevant bacteria species of the present invention, and Fig. 2 is bacterial strain
BZ11 colonial morphology figure, Fig. 3 are bacterial strain BZ11 Gram's staining schematic diagrames, and Fig. 4 is that the standard of Determination of Cholesterol Content is bent
Line chart, Fig. 5 are bacterial strain BZ11 to green grass or young crops-benzyl penicillin, ammonia-ampicillin, oxygen-Piperacillin, amp- Amoxicillin/clavulanic acid antibiotic
Drug susceptibility, Fig. 6 be bacterial strain BZ11 to must-cefoperazone, oxime-CTX, V- Cefazolins, Ah-azithromycin antibiosis
Plain drug susceptibility, Fig. 7 are bacterial strain BZ11 to second-acetyl spiral shell mycin, new-neomycin, multiple-SMZco, ten thousand-vancomycin
Antibiotic susceptibility, Fig. 8 be bacterial strain BZ11 to ring-Ciprofloxacin, piperazine-Ofloxacin, fluoro- Norfloxacin, gram-crin is mould
Plain antibiotic susceptibility, Fig. 9 are bacterial strain BZ11 to profit-rifampin, furan-furantoin, more-polymyxin B antibiolics
Thing sensitiveness.
Embodiment
Embodiment 1：Screening obtains animal pair with separation screening from the enteron aisle Dissolve things inside of Guizhou Province's characteristic resources Mini-musk swine
Discrimination bacillus BZ11：
(1) collection of sample：
Pig age is taken from the ecological park of Guiyang City, Guizhou Province less than the multiple positions samplings of the Mini-musk swine of 2 months, including it is small intestine, big
Intestines and fresh Mini-musk swine excrement, fresh Mini-musk swine sample is positioned in sterile sealing bag with sterilized tweezers, numbered
It is put into afterwards in the phase of refrigeration certainly equipped with ice bag, laboratory treatment is taken back in 1h；
(2) separation and purifying of Bifidobacterium
10g fresh sample is put into the sterile purified water equipped with 90mL and (includes bead), is carried out after manually shaking up
Gradient dilution.In superclean bench, its sample diluting liquid 0.1mL is taken to be added to the MRS (MUP- containing Li-Mupirocin
MRS) it is coated in plating medium, is put into anaerobic culture box (anaerobic environment N2∶CO2∶H2=90: 5: 5), 37 DEG C of trainings
Foster two days, then picking single bacterium colony is seeded on the PTYG containing X-gal (PTYG-X) flat board, is put into 37 DEG C, 20%CO2Concentration
CO2gas incubator in cultivate 2d.Pay attention to：Coating is diluted to from sample, it is desirable to which the plate exposure aerial time does not surpass
Cross 15min.
(3) colonial morphology and cell morphological characteristic observation
After sample treatment and culture, observe and record colonial morphology, color, and carry out Gram's staining, microscopy observation
And record.Select the smaller bacterium colony on MUP-MRS flat boards, smooth, dome, neat in edge, white or milky is opaque, quality
Soft single bacterium colony, and navy blue bacterium colony is showed on PTYG-X flat boards, after purifying is passed on four times, obtain purifying bacterium
Strain, numbers and records the form of bacterium colony.
(4) preservation of bacterial strain
By obtained inoculation in PTYG fluid nutrient mediums, after cultivating 48h in CO2gas incubator at 37 DEG C,
Take 2mL bacterium solutions to be mixed in 3mL50% glycerine in the sterilized clean centrifuge tubes of 10mL, finally reach its glycerol concentration
30%, it is positioned in -80 DEG C of ultralow temperature refrigerator-freezer and is freezed and preserved.
By first round primary dcreening operation, using colonial morphology and strain morphology as index, preliminary screening goes out the doubtful bifid bar of more than 70 strains
Bacteria strain, glycerol stocks are in -80 DEG C of ultralow temperature refrigerator-freezer.
(5) measure of norcholesterol rate：Its norcholesterol rate is surveyed using OPA (OPA) method, its specific method is such as
Under：
A, o-phthalaldehyde method (OPA) Specification Curve of Increasing
It is accurate draw cholesterol standard liquid it is each 0.02,0.05,0.10,0.12,0.15,0.20mL in clean tube,
Adding absolute ethyl alcohol makes its volume be 1mL, and OPA reagent 4mL are then added in each test tube, and room temperature places 10min.Again to it
In be slowly added to the 4mL concentrated sulfuric acids, shaken 20s, be sufficiently mixed after placing 10min under room temperature dark condition with oscillator immediately.It is empty
Compare in vain and cholesterol storing solution replaced with 1mL absolute ethyl alcohols, finally survey its light absorption value at 550nm, using cholesterol concentration as
Abscissa, absorbance are that ordinate is standard curve such as Fig. 1, and it is y=4.9264x- to calculate its equation of linear regression
0.0130, coefficient R2For 0.9992.
B, the measure of Bifidobacterium cholesterol
The bacterial strain of preservation at -80 DEG C is quickly removed into activation, and after the flat lining outs of PTYG purify 3 times, is inoculated into liquid
After carrying out enrichment culture 48h under the same terms in body PTYG culture mediums, with 5000r/min, 10min is centrifuged under the conditions of 4 DEG C, is collected
Thalline.Then with PBS cushioning liquid dilution thalline, bacterium solution to be measured is adjusted to 3 × 10810% (v/v) inoculum concentration is pressed after CFU/mL
It is inoculated in the cholesterol PTYG culture mediums that cholesterol level is 0.1mg/mL, anaerobic environment, 37 DEG C of culture 48h.Bacterium solution with
10000r/min, 20min is centrifuged under the conditions of 4 DEG C, retain supernatant liquor and wash tears bacterium as prepare liquid, while with PBS cushioning liquid
Twice, washing lotion is incorporated to supernatant prepare liquid to mud, and for determining cholesterol level, sky is used as using nonvaccinated cholesterol PTYG culture mediums
White control group.Then its Bifidobacterium norcholesterol rate is surveyed using OPA (OPA) method.
Norcholesterol rate S is calculated as follows：
S=(1-A/B) × 100%
Note：S-norcholesterol rate, %
Cholesterol concentration in A-fermented supernatant fluid to be measured, mg/mL
Cholesterol concentration in B-nonvaccinated cholesterol PTYG culture mediums, mg/mL
Through first time secondary screening, go out bacterial strain of 7 plants of norcholesterol rates higher than 30% by index screening of norcholesterol rate, carry out
The secondary screening of next step.
(6) preparation of suspension to be measured
The bacterial strain of preservation at -80 DEG C is quickly removed into activation, and purified 3 times in the flat lining outs of PTYG, is inoculated into liquid
After carrying out enrichment culture 48h under the same terms in PTYG culture mediums, bacterium solution is centrifuged into 10min under the conditions of 5000r/min, 4 DEG C,
Bacterium mud is collected, then tears once seriously ill newly formed suspension is washed with sterile phosphate buffer salt PBS, bacterial concentration is adjusted to
108CFU/mL, suspension band are used for external tolerance test.
(7) sour tolerance test
The sterilizing PTYG liquid that bacteria suspension to be measured is inoculated in into pH3.0 and pH7.0 respectively by 5% (volume ratio) inoculum concentration is trained
Support in base, mix and stand 2h after 37 DEG C of constant temperature, take 1mL to be added to mass fraction containing 9mL as 0.84% sterile saline
In test tube, mixed with quick vortex mixer, 10 times of continuation, which is successively decreased, is diluted to suitable gradient.0.1mL is respectively taken to be coated on PTYG flat boards
37 DEG C, 48h is cultivated in CO2gas incubator, carries out count plate, each flat board does 3 parallel repetitions.According to below equation
Calculate survival rate N.
N=N0/N1× 100%
In formula, N-bacterial strain acid resistance survival rate
N0- pH3.0PTYG cultivates 2h survival bacterium number
N1- pH3.0PTYG cultivates 2h survival bacterium number
(8) Bile salt resistance is tested
Bacteria suspension to be measured is inoculated in addition 0.3% (the ratio between quality and volume) respectively by the inoculum concentration of 5% (volume ratio)
In the sterilizing PTYG fluid nutrient mediums of bovine bile and the sterilizing PTYG Liquid Cultures without bovine bile, 37 DEG C of CO2gas incubators
Middle culture, count plate is carried out respectively at 0h and 24h.Each flat board does 3 parallel repetitions.
(9) oxytolerant performance test
By by the inoculation of acid resistance and Bile salt resistance into PTYG culture mediums respectively in standard incubator and
20%CO2CO2gas incubator in cultivate 24h after, carry out plate count.Its oxytolerant performance Y is evaluated according to below equation.
Y=N3/N2× 100%
In formula, Y-bacterial strain patience survival rate
N3The viable count after 24h is cultivated in-standard incubator
N2- 20%CO2CO2gas incubator in cultivate 24h after viable count
As a result show, the viable count cultivated in standard incubator after 24h is CO212.4% in incubator, show compared with
Good oxygen resistence, and the growth performance of bacterial strain is good.
Through second of secondary screening, secondary screening is carried out as index by property using acid and bile salt tolerance, one plant of bacterial strain BZ11 drop courage is filtered out and consolidates
Alcohol ability it is high and it is acidproof reached 38.52% by the good bacterial strain of property, its norcholesterol rate by property, bile tolerance, in the condition of pH value 3.0
Under survival rate reach 98.65%, its survival rate reaches more than 90% in the PTYG culture mediums containing 0.3% cholate, and
Its oxytolerant performance is good.
(10) the safety evaluatio experiment of bacterial strain
A, drug sensitivity test
The bacterial strain of preservation at -80 DEG C is quickly removed into activation, and after the flat lining outs of PTYG purify 3 times, is inoculated into liquid
After carrying out enrichment culture 48h under the same terms in body PTYG culture mediums, bacterium solution is centrifuged under the conditions of 5000r/min, 4 DEG C
10min, after collecting bacterium mud washing, 10 are configured to PBS8CFU/mL bacteria suspension.Then K-B drug sensitive test paper fine jades are used
Fat diffusion method carries out drug sensitivity test.
Take the PTYG-F agar mediums for 50 DEG C or so of bacterium solution 0.5mL and 10mL of examination to mix rapidly, pour into ready
In the sterilizing flat board of 10mL agar shop fixtures.After culture medium cooled and solidified, standard drug sensitive test paper is placed with, is put into 37 DEG C of titanium dioxide
In carbon incubator, 20h or so is rear to be measured and records the diameter of inhibition zone.
This experiment have selected 8 major class, 19 kinds of antibacterials, be penicillins respectively according to different pharmacological actions：Including
Benzyl penicillin (penicillin), ampicillin (ampicillin), Piperacillin (piperacillin) and beta-lactam suppress
The compound of agent：Amoxicillin/clavulanic acid (amoxicillin/clavulanic), glycopeptide class：Including vancomycin
(vancomycin) and Ciprofloxacin (ciprofloxacin), cephalo-type：Including CTX (cefotaxime), cephalo azoles
Quinoline (cefoazolin) and cefoperazone (cefoperazon), macrolides：Including azithromycin (azithromycin) and
Acetyl spiral is mould (acetylspiramycin), aminoglycoside：Neomycin (neomycin), sulfamido：SMZco
(sulfomethorxazole), quinolones, including Ofloxacin (ofloaxacin), Norfloxacin (norfloxacin) and
Clindamycin (clindamycin) and other classes：Including rifampin (rifampin), furantoin (nitrofurantoin)
With polymyxin B (polymyxin B), drug sensitivity assay is carried out to strain to be tested, according to the World Health Organization in 1977
Regulation, when testing gram-positive bacteria, Quality Control bacterium staphylococcus aureus (Staphylococcus aureus) need to be used
The NCCLS standards that ATCC25923 bacterial strains provide as reference culture reference, result judgement with reference to WHO.
B, acute toxicity test
After BZ11 bacterial strains are activated, isolate and purify three times, inoculating strain carbon dioxide in PTYG fluid nutrient medium is trained
Support in case and cultivate 48h, by bacterium solution with 5000r/min, 10min is centrifuged under the conditions of 4 DEG C, collects bacterium mud, then use sterile phosphate
Buffer solution PBS washed once newly formed suspension of laying equal stress on, and bacterial concentration is adjusted into 3 × 1010CFU/mL.Then according to food security
Property toxicological evaluation program and method carry out acute toxicity testing.
The Kunming kind hero mouse 20 of healthy adult is selected by body weight requirement, weight differences are not notable between mouse.Adapt to dynamic
Thing room environment (relative humidity：50 ± 5%, temperature：20 DEG C~25 DEG C) after 7d, mouse is divided into 2 groups, every group immediately by only weighing
10, i.e. experimental group and blank control group.Experimental animal is contaminated with BZ11 bacterium solutions, empty stomach 12h fasting for solids and liquids mouse is entered
The gavage volume of row 0.4mL/20g body weight；Blank control group mouse, with same dose gavage pure water.After mouse contamination, one
Its general state, changes of weight, poisoning symptom and death condition are observed in all, puts to death mouse within the 8th day.Latter stage is tested again to dynamic
Thing is weighed, and dead animal and the execution animal that expires are autopsied, general pathology is visually observed and changes situation.Experiment
Overall process and observed content do detailed record.
By carrying out safety experiment evaluation experimental to bacterial strain, the results showed that the bacterial strain BZ11 for screening to obtain is without acute poison
Property, it was demonstrated that security preliminary bacterial strain BZ11, it may be considered that utilized as the further deep development of probiotics.
(11) norcholesterol, test inside oxytolerant Bifidobacterium
Purified 3 times after strain to be tested BZ11 and the defrosting activation of control strain Infantile diarrhea, respectively with the inoculation of 5% (volume ratio)
Amount is inoculated in PTYG fluid nutrient mediums, and 24h is cultivated in 37 DEG C of CO2gas incubator, by bacterium solution in 5000r/min, 4
10min is centrifuged under the conditions of DEG C, collects bacterium mud.10 times of gradient dilutions are carried out with sterilizing PBS again, take 0.1mL in PTYG fine jades
Be coated with fat flat board, bacterium colony counting carried out after 37 DEG C of 36~48h of culture, according to plate count result, respectively by BZ11 bacterial strains and
It is 3.0 × 10 that Infantile diarrhea, which is adjusted to as bacterial concentration,9CFU/mL bacteria suspension.To prevent the death of Bifidobacterium from influenceing result,
Daily fresh configuration bacterium solution, morning timing are fed with placing feed.
The health Kunming mouse 48 of 4 weeks, male and female half and half, purchased from Guiyang Medical College.Mouse is randomly divided into 4 groups, often
Group 12, male and female half and half.(the relative humidity in Animal House environment：50 ± 5%, temperature：20 DEG C~25 DEG C, daily 12h illumination,
12h is dark, and sanitation and hygiene environment is good) free water feed, after normal diet normally raises 7d, weighed, pressed one by one
Average weight is divided into 4 groups, every group 12 without significant difference again between each group.Experimental result see the table below：
Note：A groups represent Normal group, and B groups represent hyperlipidemia model group, and C groups represent in Infantile diarrhea experiment that D groups represent
BZ11 experimental groups.
Feed formula and collocation method：Basal feed is purchased from Guiyang Medical College, reference method, and the formula of high lipid food is：
Lard 12%, cholesterol 1%, bovine bile 0.5%, basal feed 86.5%.Specific feed making step is as follows：(1) will be common
Feed is crushed with pulverizer, and it is well mixed to add bovine bile etc.；(2) heat lard in pot to boil, while add cholesterol to make
Dissolving；(3) lard of liquid is added in the normal diet of mixing, mixed with glass bar, then progressively add suitable quantity of water, carried out
Kneading is shaped as cylindric feed, is careful not to plus water causes excessively soft not easy-formation too much.(4) 4 DEG C are put in after making
Refrigerator in preservation it is standby, be put into microwave stove heat 5 seconds or so during feeding, taking-up.
Body weight determination one by one was carried out to experiment mice in the 10th, 20 day after gavage mouse experiment starts, observed between group
Whether body weight is variant.And every group is selected 10 mouse and carries out blood specimen collection at random, and blood-sample withdrawal is had altogether twice in experimentation, point
It is not respectively once to be taken a blood sample at the 10th, 20 day, each group mouse needs 12h fasting for solids and liquids before blood sampling every time.First time Blood collection is
The blood sampling of retroorbital venous clump, second is to pluck eyeball blood sampling, and blood sampling volume is 0.2mL/.All mouse cervical vertebras after second of blood sampling
Dislocation is put to death.
By the blood sample collection of collection in the 2mL sterile centrifugation tubes with anti-coagulants, after 37 DEG C of water-bath 30min, 3000r/
Min centrifuges 10min, carefully takes upper serum.Consolidate with reference to kit operation instruction and using total courage in ELIASA measure serum
Alcohol (TC), triglycerides (TG), HDL-C (HDL-C) and LDL-C (LDL-C) contain
Amount.And atherogenic index (Atherogenic index, AI) is calculated, formula is as follows：
AI=LDL-C/HDL-C
Compared with control strain Infantile diarrhea, body weight influence of the BZ11 bacterial strains on high fat mouse is bigger, can more reduce mouse blood
The content of T-CHOL (TC) content, reduction Triglycerides in Serum (TG) in clear, suppress low-density lipoprotein courage in serum
Sterol (LDL-C) content, improve HDL-C (HDL-C) content, reduce artery sclerosis (AI) index.
(12) primary dcreening operation and the secondary screening more than are tested, finally filter out bacterial strain BZ11 norcholesterol abilities it is high and it is acidproof by
Property, bile tolerance are by property, oxytolerant bacterial strain of good performance, by carrying out bio-chemical characteristics identification and 16S rRNA gene orders
Identification.
Claims (1)
1. one plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11, it is characterised in that the bacterial strain is bifidobacterium animalis
Subspecies Bifidobacterium animalis subsp.Lactis, the pure culture of the bacterial strain is on December 18th, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC is preserved in, the centre address is：City of BeiJing, China
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode：100101；Deposit number is CGMCC
NO.10224, referred to as bifidobacterium animalis subspecies BZ11；
The bifidobacterium animalis subspecies BZ11 has security：The bacterial strain is to benzyl penicillin, Piperacillin, Amoxicillin/rod
Acid, vancomycin, CTX, cephazoline, azithromycin, rifampin, the class antibiotic sensitive of furantoin 9, it is husky to ring third
Star, neomycin, SMZco, Ofloxacin, Norfloxacin and polymyxin B totally 6 class antibiotics resistance；Penicillin medicine
In, the bacterial strain is to ampicillin sensitive；In cephalo element class medicine, the bacterial strain is sensitive to cefoperazone；In Macrocyclolactone lactone kind medicine,
The bacterial strain is to the mould sensitivity of acetyl spiral；In QNS, the bacterial strain is sensitive to clindamycin；It is and real by gavage mouse
Test, show bifidobacterium animalis subspecies BZ11 bacterial strains without acute toxicity, therefore the tentative confirmation security of the bacterial strain, and lead to
Cross gavage mouse experiment and further demonstrate its internal norcholesterol effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510018144.4A CN104531590B (en) | 2015-01-14 | 2015-01-14 | One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510018144.4A CN104531590B (en) | 2015-01-14 | 2015-01-14 | One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104531590A CN104531590A (en) | 2015-04-22 |
| CN104531590B true CN104531590B (en) | 2017-12-08 |
Family
ID=52847225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510018144.4A Active CN104531590B (en) | 2015-01-14 | 2015-01-14 | One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531590B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928215A (en) * | 2015-06-23 | 2015-09-23 | 贵州大学 | Fermentation medium cholesterol-lowering and oxygen-resistant animal bifidobacteria yoghurt subspecies BZ11 |
| CN106167775B (en) * | 2016-07-06 | 2019-11-12 | 贵州大学 | The fermentation process in high density of oxytolerant domesticated animal Bifidobacterium lactis spp BZ11 |
| CN108570429A (en) * | 2018-05-08 | 2018-09-25 | 西藏高原之宝牦牛乳业股份有限公司 | A kind of animal bifidobacteria and preparation method thereof |
| TWI709408B (en) * | 2018-08-16 | 2020-11-11 | 葡萄王生技股份有限公司 | A bifidobacterium lactis gkk2, a composition comprising thereof and its use for improving allergic asthma |
| KR101998067B1 (en) * | 2019-03-29 | 2019-09-27 | (주)지에프씨생명과학 | BIFIDOBACTERIUM SP. GFC-B09 and Fermented Product Manufactured Using Thereof |
| CN110577919B (en) * | 2019-10-14 | 2022-12-23 | 江西仁仁健康产业有限公司 | Heat-resistant animal bifidobacterium lactis subspecies separated from infant feces |
| CN110923166A (en) * | 2019-12-11 | 2020-03-27 | 石家庄君乐宝乳业有限公司 | Bifidobacterium animalis subsp lactis JMCC0025, and separation and purification method and application thereof |
| CN111567809A (en) * | 2020-05-26 | 2020-08-25 | 奥克斯路株式会社 | Bifidobacterium animalis BZ11 composite microbial agent with functions of improving immunity and regulating intestinal tract |
| CN111440750B (en) * | 2020-05-28 | 2021-12-07 | 江西仁仁健康产业有限公司 | Bifidobacterium animalis subsp lactis with functions of relieving lactose intolerance and reducing triglyceride and application thereof |
| CN114686393B (en) * | 2020-12-31 | 2023-09-05 | 杭州远大生物制药有限公司 | Bifidobacterium animalis subspecies lactis and composition thereof |
| CN113564078B (en) * | 2021-07-29 | 2023-10-20 | 微康益生菌(苏州)股份有限公司 | Bifidobacterium lactis BLa80 for reducing cholesterol and application thereof |
| CN114606157B (en) * | 2022-03-01 | 2023-07-14 | 湖北省微工生物技术研究有限公司 | Bifidobacterium microbial preparation and application thereof in kefir preparation |
| CN114574407B (en) * | 2022-05-06 | 2022-11-01 | 微康益生菌(苏州)股份有限公司 | Bifidobacterium animalis subsp lactis WKB99 and application thereof in preparation of product for improving metabolic syndrome and product |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101257910A (en) * | 2005-09-08 | 2008-09-03 | 株式会社益力多本社 | Cholesterol absorption inhibitor |
| CN101503667A (en) * | 2009-03-05 | 2009-08-12 | 浙江大学 | Oxygen-resistant bifidobacteria |
| CN102191192A (en) * | 2010-12-21 | 2011-09-21 | 北京博锦元生物科技有限公司 | Animal Bifidobacterium and use method thereof |
| CN102206599A (en) * | 2011-04-11 | 2011-10-05 | 天津科技大学 | Oxygen-resistant acid-resistant Bifidobacterium longum |
| CN102488004A (en) * | 2011-12-01 | 2012-06-13 | 杭州娃哈哈集团有限公司 | Norcholesterol 0-fat fermented milk and preparation method thereof |
-
2015
- 2015-01-14 CN CN201510018144.4A patent/CN104531590B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101257910A (en) * | 2005-09-08 | 2008-09-03 | 株式会社益力多本社 | Cholesterol absorption inhibitor |
| CN101503667A (en) * | 2009-03-05 | 2009-08-12 | 浙江大学 | Oxygen-resistant bifidobacteria |
| CN102191192A (en) * | 2010-12-21 | 2011-09-21 | 北京博锦元生物科技有限公司 | Animal Bifidobacterium and use method thereof |
| CN102206599A (en) * | 2011-04-11 | 2011-10-05 | 天津科技大学 | Oxygen-resistant acid-resistant Bifidobacterium longum |
| CN102488004A (en) * | 2011-12-01 | 2012-06-13 | 杭州娃哈哈集团有限公司 | Norcholesterol 0-fat fermented milk and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Screening of Cholesterol-lowering Bifidobacterium from Guizhou Xiang Pigs, and Evaluation of Its Tolerance to Oxygen, Acid, and Bile;Rujiao Zhang等;《Korean J. Food Sci. An.》;20160228;第36卷(第1期);37-43 * |
| 动物双歧杆菌乳亚种BZ11的耐氧驯化及降胆固醇性能的研究;王猛等;《食品与发酵工业》;20160303;第42卷(第3期);1-7 * |
| 降胆固醇乳酸菌的筛选及对大鼠血脂的影响;杨琴等;《食品科学》;20111231;第32卷(第21期);223-228 * |
| 降胆固醇双歧杆菌的研究进展;何腊平等;《中国食品添加剂》;20131231(第4期);188-192 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104531590A (en) | 2015-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104531590B (en) | One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ11 | |
| CN104560829B (en) | One plant of fragrant pig source property norcholesterol, oxytolerant Bifidobacterium BZ25 | |
| CN104073455B (en) | One strain has the plant lactobacillus reducing cholesterol ability | |
| CN110317757B (en) | Lactobacillus plantarum HJ-S2 with cholesterol-reducing and selenium-rich effects and application thereof | |
| Orrhage et al. | Effect of supplements with lactic acid bacteria and oligofructose on the intestinal microflora during administration of cefpodoxime proxetil | |
| CN102031229B (en) | Lactobacillus plantarum with gastrointestinal adsorption capacity and cholesterol removing capacity | |
| CN110577912B (en) | Lactobacillus gasseri and application thereof in preparing fermented milk | |
| CN109810912A (en) | One lactobacillus plantarum LH-511 and its application | |
| CN110106119B (en) | Lactobacillus rhamnosus M9 separated from breast milk and application thereof | |
| CN106883995A (en) | Pediococcus acidilactici JQII-5 bacterial strains and application thereof | |
| CN107287133A (en) | One lactobacillus plantarum LLY 606 and its application | |
| CN112812999B (en) | Lactobacillus plantarum SLB01 with inhibition effect on enterobacter cloacae and derivative product and application thereof | |
| CN109810913A (en) | One plant of Lactobacillus rhamnosus ASD-9 and its application | |
| CN110373367A (en) | One kind having the active lactobacillus plantarum of degradation of pesticide, preparation method and application | |
| CN115927045B (en) | Lactobacillus salivarius 069 with cholesterol reducing and liver injury relieving functions caused by hyperlipidemia and application thereof | |
| CN110023484A (en) | A kind of small bifidobacterium catenulatum of vacation and its cultural method and application | |
| CN109456909A (en) | One plant has the Lactobacillus helveticus for reducing cholesterol ability | |
| CN116814510B (en) | Lactobacillus rhamnosus OPB41 capable of preventing or improving Alzheimer's disease and application thereof | |
| CN102590196B (en) | The detection method of antibacterial medicine residue in a kind of Rapid Screening animal foodstuff sample | |
| CN106754441A (en) | With reduction cholesterol, solution fat Asia sieve saccharomycete of triglycerides effect and application | |
| CN104789488A (en) | Lactobacillus rhamnosus having cholesterol lowering effect, and uses thereof | |
| CN109541205B (en) | Method for detecting lactic acid bacteria in probiotics | |
| TW201100086A (en) | Lactobacillus plantarum BB9 with gastrointestinal tract adhering ability and cholesterol eliminating capability | |
| CN116970512A (en) | Lactobacillus plantarum, and culture method and application thereof | |
| Mangwana | The in vitro faecal evaluation of prebiotic effects of rooibos phenolic compounds on the gut microbiota of vervet monkeys (Chlorocebus pygerythrus) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200327 Address after: Room 501, building 3, zone 3, No. 188, nansihuan West Road, Fengtai District, Beijing 100070 Patentee after: BEIJING ZHONGJING FENGCHUANG TECHNOLOGY Co.,Ltd. Address before: 550025 Guiyang Road, Huaxi District, Guizhou Province, Guizhou University (South Campus) Patentee before: Guizhou University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210108 Address after: 135099 No.1, building B, Laodong Jiayuan, Meihekou City, Tonghua City, Jilin Province Patentee after: Baizhi Health Technology (China) Co., Ltd Address before: Room 501, building 3, area 3, 188 South 4th Ring Road West, Fengtai District, Beijing 100070 Patentee before: BEIJING ZHONGJING FENGCHUANG TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211130 Address after: 135000 No. 3399, Nanhuan East Road, Meihekou City, Tonghua City, Jilin Province Patentee after: Naian Pharmaceutical (China) Co.,Ltd. Address before: 135099 No.1, building B, Laodong Jiayuan, Meihekou City, Tonghua City, Jilin Province Patentee before: Baizhi Health Technology (China) Co., Ltd |
|
| TR01 | Transfer of patent right |