CN104531590A - Fragrant pig derived cholesterol-assimilating and oxygen-resistant bifidobacterium BZll - Google Patents
Fragrant pig derived cholesterol-assimilating and oxygen-resistant bifidobacterium BZll Download PDFInfo
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Abstract
Provided is fragrant pig derived cholesterol-assimilating and oxygen-resistant bifidobacterium BZll. The fragrant pig derived cholesterol-assimilating and oxygen-resistant bifidobacterium BZll is Bifidobacterium animalis subsp.Lactis, the pure culture of the fragrant pig derived cholesterol-assimilating and oxygen-resistant bifidobacterium BZll is preserved at the China General Micro-biological Culture Collection Center (CGMCC), China Microbial Culture Preservation Management Committee, the address is Institute of Microbiology, Institution No.3 China Academy of Science, Western Beichen Road No.1 in Chaoyang District of Beijing in China, the postal code is 100101, the preservation number is CGMCC NO.10224, and the Bifidobacterium animalis subsp.Lactis BZll is called for short. The Bifidobacterium animalis subsp.Lactis BZll is high in cholesterol-assimilating capacity, acid resistance and cholate resistance, good in oxygen resisting performance, has probiotic property and can serve as probiotics to be added into a fermentative milk product to produce a functional food, bifidobacterium culture resources are enriched, and development of bifidobacterium health-care products is facilitated.
Description
Technical field
The present invention relates to microorganism, particularly decreasing cholesterol, oxytolerant bifidus bacillus.
Background technology
Cholesterol is extensively present in animal tissue cell, and be one of important component forming cell, in human body, play important physiological action, it transforms formation steroid hormone in vivo, is the precursor substance of synthesis of vitamin d and bile acide.Body inner cholesterol mainly contains two sources, and one is food, and two is endogenous synthesis.Some people is due to unreasonable dietary, and blood cholesterol level exceedes normal index; And body inner cholesterol too high levels can cause the cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, serious threat is to human health.It is reported, whole world cardiovascular and cerebrovascular diseases causes the number of human death to account for 29% of total death toll, predict according to the World Health Organization, to the year two thousand thirty, cardiovascular and cerebrovascular diseases will be the major cause causing human death, and cholesterol in serum too high be the important factor causing the cardiovascular and cerebrovascular diseases such as coronary heart disease, arteriosclerosis, cerebral apoplexy.Studies have found that compared with the people having normal lipid, the risk that the people of hypercholesterolemia suffers from a heart complaint is 3 times of its normal people.Therefore, be the feasible research direction of a kind of actual effect by reducing that serum cholesterol level prevents and treats cardiovascular and cerebrovascular diseases, have potential using value and wide be the market space.
For reducing body inner cholesterol, except by except reasonable diet, using biotransformation method, particularly using probiotic bacterium to carry out to cholesterol the trend that direct degraded has become research.Bifidus bacillus as the class probiotic bacterium in human intestinal, its prebiotic effect have maintain intestinal flora balance, reduce serum cholesterol, the prebiotic effect such as antitumor.And Guizhou Xiang Zhu Shi Guizhou Province characteristic resources, belong to miniature pig, its inheritance stability, organ structure is similar to human body with function.Therefore, the bifidus bacillus screened from fragrant pig is easy to be accepted by human body and utilize.
In current Chinese patent database, the patent application relating to bifidus bacillus is a lot, but relates to No. ZL2009100965117 " a kind of oxygen-resistant bifidobacteria " of the patent application Jin You Zhejiang University application of oxytolerant bifidus bacillus and No. ZL2011100894937 " a kind of oxygen-resistant acid-resistant Bifidobacterium longum " of University Of Science and Technology Of Tianjin's application.These two bifidus bacilluss can decreasing cholesterol unknown.Up to now, there is no the application part relating to decreasing cholesterol, oxytolerant animal bifidobacteria.
Summary of the invention
The present invention aims to provide a strain fragrant pig source property decreasing cholesterol, oxytolerant bifidus bacillus, make it likely to utilize as the further deep development of probiotic bacterium, add in cultured milk prod and become functional foodstuff, to reduce human cholesterol content, promote human health.
The fragrant pig source property decreasing cholesterol that contriver provides, oxytolerant bifidus bacillus, this bacterial strain is bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.Lactis), the pure growth of this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 18th, 2014, this centre address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica, postcode: 100101; Deposit number is CGMCC NO.10224, referred to as bifidobacterium animalis subspecies BZ11.This bacterial strain is that screening and separation and purification obtain from the enteron aisle Dissolve things inside of Guizhou characteristic resources Mini-musk swine.
The morphological specificity of above-mentioned bifidobacterium animalis subspecies BZ11 and physiological and biochemical property bacterium are all close to genus bifidobacterium, the 16S rRNA sequence homology of its 16S rRNA gene order and bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.Lactis) is 100%, according to the categorised regulation of genus bifidobacterium in " uncle Jie Shi systematic bacteriology handbook ", bifidobacterium animalis subspecies BZ11 belongs to actinomycetes door (Actinobacteria), Actinomycetes (Actinobacteria), actinomycetes subclass (Actinobacteridae), bifidus bacillus order (Bifidoacteriales), bifidobacterium family (Bifidobacteriaceae), genus bifidobacterium (Bifidobacterium), bifidobacterium animalis subspecies (Bifidobacteriumanimalis subsp.Lactis).
The security of above-mentioned animal bifidobacteria BZ11 is as follows: this bacterial strain to penicillin G, piperacillin, amoxycilline Trihydrate bp/clavulanic acid, vancomycin, cefotaxime, Kefzol, Azythromycin, Rifampin, furadantin 9 class antibiotic sensitive, to Ciprofloxacin, Liu Suanyan NEOMYCIN SULPHATE, trimethoprim-sulfamethoxazole, Ofloxacine USP 23, norfloxicin and PXB totally 6 class antibiotics resistances.In penicillin medicine, this bacterial strain is to ampicillin sensitive; In cephalo element class medicine, this bacterial strain is responsive to cefoperazone; In Macrocyclolactone lactone kind medicine, bacterial strain BZ11 is to the mould sensitivity of acetyl spiral; In quinolones, this bacterial strain is responsive to woods mycin.And by gavage mouse experiment, show that bifidobacterium animalis subspecies BZ11 bacterial strain is without acute toxicity, and therefore can the security of tentative confirmation BZ11 bacterial strain, the further deep development of probiotic bacterium can be considered as and utilize.And demonstrate decreasing cholesterol effect in its body further by gavage mouse experiment.
The bacterial strain that contriver provides from the characteristic resources Mini-musk swine enteron aisle Dissolve things inside of Guizhou, is separated also preliminary screening go out to have the bifidus bacillus of decreasing cholesterol ability, and to the bacterial strain of gained with decreasing cholesterol rate, acid and bile salt tolerance is that index carries out further multiple sieve by property, sift out again a strain removal rate of cholesterol high and acidproof by property, the bifidobacterium strains that bile tolerance is subject to property all good, then oxytolerant performance test is carried out, safety evaluation experiment and gavage mouse experiment, afterwards by carrying out morphology to this bacterial strain, physiological and biochemical test qualification and 16S rRNA Sequence Identification, finally identifying this bacterial strain is bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.Lactis).
In order to verify bacterial strain of the present invention, inventors performed following experiment:
(1) collection of sample and separation and purification
From multiple positions sampling of Guizhou Province's characteristic resources Mini-musk swine, comprise in small intestine, large intestine and fresh Mini-musk swine ight soil, number and record, and take back laboratory treatment rapidly.The fresh sample of getting 10g puts into the stroke-physiological saline solution that 90mL is housed, and after manually shaking up, in clean bench, draw 100 μ l mixed solutions with liquid-transfering gun coat on the MRS flat board containing Li-Mupirocin, (anaerobic environment is N to be placed in anaerobic culture box
2: CO
2: H
2=90: 5: 5), in, cultivate 48h for 37 DEG C, then picking list colony inoculation is on the PTYG flat board containing X-gal, puts into 37 DEG C, 20%CO
2cultivate two days in the CO2gas incubator of concentration, observe colony colour, form carry out gramstaining, microscopy, record strain morphology feature.Gone down to posterity by doubtful bifidobacterium strains purifying on PTYG flat board after 4 times, glycerol stocks is in the very low temperature refrigerator-freezer of-80 DEG C.
Above-mentioned MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast powder 5g, glucose 20g, tween 80 1mL, K
2hPO
42g, sodium-acetate 5g, dibasic ammonium citrate 2g, MgSO
47H
2o 0.58g, MnSO
44H
2o 0.25g, agar 20g; Method for making is by each composition heating for dissolving in 1000mL distilled water, adjust pH to 6.5, and 121 DEG C of sterilizing 15min are for subsequent use.
Above-mentioned containing in the MRS of Li-Mupirocin, 100mLMRS nutrient agar contains the mupirocin lithium salts (Li-Mupirocin) of 5mg.
Above-mentioned PTYG liquid culture based component is: Tryptones 5g, soy peptone 5g, yeast powder 10g, glucose 10g, tween 80 1mL, salts solution 40mL; Method for making is by each composition heating for dissolving in 1000mL distilled water, adjust pH to 6.5, and 121 DEG C of sterilizing 15min are for subsequent use.
Salts solution composition in above-mentioned substratum is: anhydrous CaCl
20.2g, K
2hPO
41.0g, KH
2pO
41.0g, MgSO
47H
2o 0.48g, Na
2cO
310g, NaCl 2g; Method for making is by CaCl
2and MgSO47H
2o is blended in until dissolve in 300mL distilled water, and add 500mL water, stirring slowly adds other salts simultaneously, until dissolve; Add 200mL distilled water again, after mixing, be stored in 4 DEG C, for subsequent use.
Above-mentioned PTYG nutrient agar method for making is: in the liquid nutrient medium of every 1000mL, add 20g agar powder, heated and boiled, and pour plate after 121 DEG C of sterilizing 15min is for subsequent use.
Above-mentioned PTYG (PTYG-X) the substratum method for making containing X-gal is: in the improvement PTYG substratum of every 1000mL, add X-gal 40mg, due to X-gal non-refractory, direct sterilized water weaker concn is 20mg/mL, each PTYG flat board coating 40 μ L, for subsequent use.
(2) mensuration of removal rate of cholesterol
O-phthalaldehyde method (OPA) is adopted to survey its removal rate of cholesterol the bacterial strain of primary dcreening operation gained.
(3) preparation of bacteria suspension to be measured
By the bacterial strain activation be deposited in the very low temperature refrigerator-freezer of-80 DEG C, after line purifying 3 times, be inoculated in PTYG liquid nutrient medium and carry out enrichment culture, then that bacteria suspension is centrifugal, collect bacterium mud, bacterial concentration is adjusted to 10
8cFU/mL, bacterium suspension band is used for external tolerance test.
(4) sour tolerance test
Bacteria suspension to be measured is inoculated in the sterilizing PTYG liquid nutrient medium of pH3.0 and pH7.0 respectively by 5% (volume ratio) inoculum size, leaves standstill 2h at mixing latter 37 DEG C, carry out plate count.
(5) Bile salt resistance experiment
Bacteria suspension to be measured is inoculated in the sterilizing PTYG liquid nutrient medium of interpolation 0.3% (mass/volume) bovine bile respectively by the inoculum size of 5% (volume ratio) and does not contain in the sterilizing PTYG liquid culture of bovine bile, 37 DEG C, cultivate in CO2gas incubator, carry out live bacterial count respectively at 0h and 24h.
(6) oxytolerant performance test
By the inoculation through acid resistance and Bile salt resistance in PTYG substratum respectively at standard incubator and 20%CO
2cO2gas incubator in cultivate 24h after, carry out plate count.Go to contrast to judge its oxygen resistence to both growths.
By the primary dcreening operation experiment of above-mentioned sampling, separation and purification and with the decreasing cholesterol rate of bacterial strain, tolerance is that index carries out multiple sieve, finally obtains a strain bacterial strain BZ11, its decreasing cholesterol rate is high, reach 38.52%, and acidproof property and the bile tolerance of being subject to is subject to property good, and has better tolerance to oxygen.
(7) the safety evaluation experiment of bacterial strain
A, drug sensitivity assay
By the activation of BZ11 bacterial strain and in PTYG nutrient agar after purifying 3 generation, liquid PTYG substratum enrichment culture, then by bacterium liquid at 5000r/min, centrifugal 10min under 4 DEG C of conditions, after collecting the washing of bacterium mud, is configured to 10 with PBS damping fluid
8the bacteria suspension of CFU/mL.Then choose 8 class, 19 kinds of susceptibility analoidses, adopt K-B drug sensitive test paper agar diffusion method to carry out drug sensitivity assay.
B, acute toxicity test
After BZ11 bacterial strain is activated, separation and purification three times, inoculating strain cultivates 48h in CO2gas incubator in the liquid nutrient medium of PTYG, by bacterium liquid with 5000r/min, centrifugal 10min under 4 DEG C of conditions, collect bacterium mud, then wash with sterile phosphate buffer PBS newly formed suspension of once laying equal stress on, bacterial concentration is adjusted to 3 × 10
10cFU/mL.Then acute toxicity test is carried out according to toxicological evaluation of food safety procedure and method.
(8) experiment in vivo of decreasing cholesterol, oxytolerant bifidus bacillus
Purifying 3 times after test strains BZ11 and control strain Infantile diarrhea thaw and activate, be inoculated in PTYG liquid nutrient medium with the inoculum size of 5% (volume ratio) respectively, cultivate 24h in the CO2gas incubator of 37 DEG C after, with centrifugal 10min under 5000r/min, 4 DEG C of conditions, collect bacterium mud.Carry out 10 times of gradient dilutions with sterilizing PBS damping fluid again, get 0.1mL and be coated with on PTYG agar plate, carry out enumeration after 37 DEG C of cultivation 36 ~ 48h, according to plate count result, respectively BZ11 and Infantile diarrhea are adjusted to bacteria suspension to 3.0 × 10
9cFU/mL.Then carry out mouse stomach experiment, took a blood sample the 10th, 20 day time, survey total cholesterol level in its serum, content of triglyceride, high density lipoprotein cholesterol content, low density lipoprotein cholesterol content, and calculate its atherogenic index.For preventing bifidus bacillus death from affecting result, fresh configuration every day bacterium liquid, timing in the morning is filled with and is fed and placement feed.
(9) qualification of bacterial strain
The bacterial strain of gained is carried out to the qualification of Physiology and biochemistry qualification and kind, understand its physiological and biochemical property, to effectively applying document, instructing further investigation and application to play an important role.Therefore, bacterial strain BZ11 of the present invention is carried out to the qualification of bio-chemical characteristics qualification and 16S rRNA sequence.
Morphological specificity: by inoculation on PTYG flat board, cultivate 48h for 37 DEG C, its colonial morphology is that bacterium colony is less, smooth, dome, neat in edge, white or oyster white are opaque and quality soft, and PTYG-X flat board then presents navy blue bacterium colony.Its bacterium colony of picking carries out gramstaining, and its gramstaining is positive, and to observe its cellular form be under an optical microscope polymorphic shaft-like, meets bifidobacterium cells morphological specificity.Its bio-chemical characteristics the results are shown in Table 1.
Table 1 cellular form and Physicochemical test result
Note :+: positive ,-: negative
According to above cellular form with Physicochemical test result, bacterial strain BZ11 feature meets the feature of genus bifidobacterium, and preliminary evaluation belongs to genus bifidobacterium.
Bacterial strain of the present invention is by China General Microbiological culture presevation administrative center (China General Microbiological Culture Col lection Center, CGMCC) 16S rRNA Sequence Identification is carried out, measured 16S rRNA gene order is logged in http://blast.ncbi.nlm.nih.gov/Blast.cgi website, by online BLAST (Basic Local Al ignment Search Tool), search 100 gene fragments higher with this sequence homology altogether, and the 16S rRNA sequence homology of the 16S rRNA gene order of this bacterial strain and bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.Lactis) reaches 100%, choose the 20 strain bacterium that homology is higher, application MEGA5.0 analysis software constructing system evolutionary tree.Mapping display, in animal bifidobacteria BZ11 bacterial strain and Genbank, Bifidobacterium animalis subsp.lactis AD011strain AD011 and Bifidobacterium animalis subsp.lactis strain YIT 4121 liang of strain bifidobacterium animalis subspecies belong in a minimum branch, show that the sibship between them is nearest.
Therefore, according to its morphological specificity, Physicochemical test result and the phylogenetic tree analysis constructed by 16S rRNA gene order, according to the categorised regulation of genus bifidobacterium in " uncle Jie Shi systematic bacteriology handbook ", bifidobacterium animalis subspecies BZ11 bacterial strain belongs to actinomycetes door (Actinobacteria), Actinomycetes (Actinobacteria), actinomycetes subclass (Actinobacteridae), bifidus bacillus order (Bifidoacteriales), bifidobacterium family (Bifidobacteriaceae), genus bifidobacterium (Bifidobacterium), bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.Lactis).
Fragrant pig source property bifidobacterium animalis subspecies BZ11 (Bifidobacterium animalis subsp.Lactis) of the present invention, not only decreasing cholesterol ability high, acidproof by property, bile tolerance by property, oxytolerant is functional and have probiotic properties, and can add in cultured milk prod as probiotic bacterium and become functional foodstuff, thus enriched bifidobacterium species resource, to the exploitation of bifidus bacillus healthcare product, there is positive effect.
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Accompanying drawing explanation
Fig. 1 is that the 16S rRNA sequential system of bacterial strain BZ11 of the present invention and relevant bacteria species grows evolutionary tree, Fig. 2 is the colonial morphology figure of bacterial strain BZ11, Fig. 3 is the gramstaining schematic diagram of bacterial strain BZ11, Fig. 4 is the canonical plotting of Determination of Cholesterol Content, Fig. 5 is that bacterial strain BZ11 is to green grass or young crops-penicillin G, ammonia-Ampicillin Trihydrate, oxygen-piperacillin, amp-amoxycilline Trihydrate bp/clavulanic acid antibiotic susceptibility, Fig. 6 be bacterial strain BZ11 to must-cefoperazone, oxime-cefotaxime, V-Cephazolin, Ah-Azythromycin antibiotic susceptibility, Fig. 7 is that bacterial strain BZ11 is to second-acetyl spiral shell mycin, newly-Liu Suanyan NEOMYCIN SULPHATE, multiple-trimethoprim-sulfamethoxazole, ten thousand-vancomycin antibiotic drug susceptibility, Fig. 8 is that bacterial strain BZ11 is to ring-Ciprofloxacin, piperazine-Ofloxacine USP 23, fluoro-norfloxicin, gram-clindamycin antibiotic susceptibility, Fig. 9 is that bacterial strain BZ11 is to profit-Rifampin, furan-furadantin, many-PXB antibiotic susceptibility.
Embodiment
Embodiment 1: screening and separation screening obtain animal bifidobacteria BZ11 from the enteron aisle Dissolve things inside of Guizhou Province's characteristic resources Mini-musk swine:
(1) collection of sample:
Mini-musk swine multiple positions sampling that pig is less than 2 months age is got from the ecological park of Guiyang City, Guizhou Province, comprise small intestine, large intestine and fresh Mini-musk swine ight soil, with sterilized tweezers, fresh Mini-musk swine sample is positioned in sterile seal bag, put into the refrigeration certainly that ice bag is housed after numbering and mutually, in 1h, take back laboratory treatment;
(2) separation of bifidus bacillus and purifying
The fresh sample of 10g is put into the sterile purified water (including granulated glass sphere) that 90mL is housed, after manually shaking up, carry out gradient dilution.In Bechtop, get in MRS (MUP-MRS) plate culture medium that its sample diluting liquid 0.1mL joins containing Li-Mupirocin and be coated with, (anaerobic environment is N to put into anaerobic culture box
2: CO
2: H
2=90: 5: 5), cultivate two days for 37 DEG C, then picking list colony inoculation is on PTYG (PTYG-X) flat board containing X-gal, puts into 37 DEG C, 20%CO
22d is cultivated in the CO2gas incubator of concentration.Attention: from diluted sample to coating, requires that the plate exposure aerial time is no more than 15min.
(3) colonial morphology and cell morphological characteristic are observed
In sample preparation and after cultivating, observe and record colonial morphology, color, and carrying out gramstaining, microscopy is observed and record.Select the single bacterium colony that bacterium colony on MUP-MRS flat board is less, smooth, dome, neat in edge, white or oyster white are opaque, quality is soft, and on PTYG-X flat board, present navy blue bacterium colony, purifying goes down to posterity after four times, obtains purifying bacterial strain, numbers and records the form of bacterium colony.
(4) preservation of bacterial strain
By the inoculation that obtains in PTYG liquid nutrient medium, after cultivating 48h at 37 DEG C in CO2gas incubator, get 2mL bacterium liquid and 3mL50% glycerine is mixed in the sterilized clean centrifuge tube of 10mL, finally make its glycerol concentration reach 30%, be positioned in the very low temperature refrigerator-freezer of-80 DEG C and carry out freezing and preserving.
Through first round primary dcreening operation, with colonial morphology and strain morphology for index, preliminary screening goes out the doubtful bifidobacterium strains of more than 70 strain, and glycerol stocks is in the very low temperature refrigerator-freezer of-80 DEG C.
(5) mensuration of decreasing cholesterol rate: adopt o-phthalaldehyde(OPA) (OPA) method to survey its decreasing cholesterol rate, its concrete grammar is as follows:
A, o-phthalaldehyde method (OPA) Specification Curve of Increasing
Accurate absorption cholesterol standardized solution is each 0.02,0.05,0.10,0.12,0.15,0.20mL in clean tube, then adds dehydrated alcohol and makes its volume be 1mL, then in each test tube, add OPA reagent 4mL, room temperature places 10min.Slowly add the 4mL vitriol oil wherein again, to shake 20s with vibrator immediately, fully under room temperature dark condition, place 10min after mixing.Blank replaces cholesterol storing solution with 1mL dehydrated alcohol, finally survey its light absorption value at 550nm place, take cholesterol concentration as X-coordinate, absorbance is that ordinate zou does typical curve as Fig. 1, calculating its equation of linear regression is y=4.9264x-0.0130, coefficient R
2be 0.9992.
The mensuration of b, bifidus bacillus cholesterol
The bacterial strain fast fetching of preservation at-80 DEG C is gone out activation, and after the flat lining out purifying of PTYG 3 times, is inoculated into after carrying out enrichment culture 48h under the same terms in liquid PTYG substratum, with 5000r/min, centrifugal 10min under 4 DEG C of conditions, collects thalline.Then with PBS buffered soln dilution thalline, bacterium liquid to be measured is adjusted to 3 × 10
8being inoculated in cholesterol level by 10% (v/v) inoculum size after CFU/mL is in the cholesterol PTYG substratum of 0.1mg/mL, anaerobic environment, cultivates 48h for 37 DEG C.Bacterium liquid is with 10000r/min, and centrifugal 20min under 4 DEG C of conditions, retains supernatant liquid as liquid to be measured, wash tears bacterium mud twice with PBS buffered soln simultaneously, washing lotion is incorporated to supernatant liquid to be measured, for measuring cholesterol level, using nonvaccinated cholesterol PTYG substratum as blank group.Then o-phthalaldehyde(OPA) (OPA) method is adopted to survey its bifidus bacillus decreasing cholesterol rate.
Decreasing cholesterol rate S calculates as follows:
S＝(1-A/B)×100％
Note: S-decreasing cholesterol rate, %
Cholesterol concentration in A-fermented supernatant fluid to be measured, mg/mL
Cholesterol concentration in B-nonvaccinated cholesterol PTYG substratum, mg/mL
Through first time multiple sieve, go out 7 strain decreasing cholesterol rates higher than the bacterial strain of 30% with decreasing cholesterol rate for index screening, carry out next step multiple sieve.
(6) preparation of suspension to be measured
The bacterial strain fast fetching of preservation at-80 DEG C is gone out activation, and the flat lining out purifying of PTYG 3 times, be inoculated into after carrying out enrichment culture 48h under the same terms in liquid PTYG substratum, by bacterium liquid at 5000r/min, centrifugal 10min under 4 DEG C of conditions, collect bacterium mud, then wash tears once seriously ill newly formed suspension with sterile phosphate buffer salt PBS, bacterial concentration is adjusted to 10
8cFU/mL, suspension band is used for external tolerance test.
(7) sour tolerance test
Bacteria suspension to be measured is inoculated in the sterilizing PTYG liquid nutrient medium of pH3.0 and pH7.0 by 5% (volume ratio) inoculum size respectively, 2h is left standstill in 37 DEG C of constant temperature after mixing, getting 1mL, to join containing 9mL massfraction be in the test tube of 0.84% stroke-physiological saline solution, mix with quick vortex mixer, continue 10 times and successively decrease and be diluted to suitable gradient.Respectively get 0.1mL and coat dull and stereotyped upper 37 DEG C of PTYG, cultivate 48h in CO2gas incubator, carry out live bacterial count, each flat board does 3 parallel repetitions.Survival rate N is gone out according to following formulae discovery.
N＝N
0/N
1×100％
In formula, N-bacterial strain acid resistance survival rate
N
0-pH3.0PTYG cultivation 2h deposits viable count
N
1-pH3.0PTYG cultivation 2h deposits viable count
(8) Bile salt resistance experiment
By bacteria suspension to be measured by the inoculum size of 5% (volume ratio) be inoculated in respectively interpolation 0.3% ratio of volume (quality with) bovine bile sterilizing PTYG liquid nutrient medium and not containing in the sterilizing PTYG liquid culture of bovine bile, cultivate in 37 DEG C of CO2gas incubator, carry out live bacterial count respectively at 0h and 24h.Each flat board does 3 parallel repetitions.
(9) oxytolerant performance test
By the inoculation through acid resistance and Bile salt resistance in PTYG substratum respectively at standard incubator and 20%CO
2cO2gas incubator in cultivate 24h after, carry out plate count.Its oxytolerant performance Y is evaluated according to following formula.
Y＝N
3/N
2×100％
In formula, the heat resistance survival rate of Y-bacterial strain
N
3the viable count after 24h is cultivated in-standard incubator
N
2-20%CO
2cO2gas incubator in cultivate the viable count after 24h
Result shows, cultivating the viable count after 24h in standard incubator is CO
2in incubator 12.4%, shows good oxygen resistence, and the growth performance of bacterial strain is good.
Through the multiple sieve of second time, with acid and bile salt tolerance by property for index carries out multiple sieve, the strain bacterial strain BZ11 decreasing cholesterol ability that filters out high and acidproof by property, bile tolerance by the good bacterial strain of property, its decreasing cholesterol rate reaches 38.52%, survival rate under pH value 3.0 condition is to 98.65%, in the PTYG substratum containing 0.3% cholate, its survival rate is to more than 90%, and its oxytolerant performance is good.
(10) the safety evaluation experiment of bacterial strain
A, drug sensitivity test
The bacterial strain fast fetching of preservation at-80 DEG C is gone out activation, and after the flat lining out purifying of PTYG 3 times, be inoculated into after carrying out enrichment culture 48h under the same terms in liquid PTYG substratum, by bacterium liquid at 5000r/min, centrifugal 10min under 4 DEG C of conditions, after collecting the washing of bacterium mud, be configured to 10 with PBS damping fluid
8the bacteria suspension of CFU/mL.Then K-B drug sensitive test paper agar diffusion method is adopted to carry out drug sensitivity test.
The PTYG-F nutrient agar got for examination bacterium liquid 0.5mL and 10mL about 50 DEG C mixes rapidly, pours in the sterilizing flat board of ready 10mL agar shop fixtures.After substratum cooled and solidified, be placed with standard drug sensitive test paper, put into the CO2gas incubator of 37 DEG C, about 20h measures afterwards and records the diameter of inhibition zone.
The pharmacological action that this experimental basis is different, have selected 8 large classes, 19 kinds of antibacterials, penicillins respectively: comprise penicillin G (penicillin), Ampicillin Trihydrate (ampicillin), the mixture of piperacillin (piperacillin) and beta-lactam inhibitor: amoxycilline Trihydrate bp/clavulanic acid (amoxicillin/clavulanic), glycopeptide class: comprise vancomycin (vancomycin) and Ciprofloxacin (ciprofloxacin), cephalosporin: comprise cefotaxime (cefotaxime), Kefzol (cefoazolin) and cefoperazone (cefoperazon), Macrolide: comprise Azythromycin (azithromycin) and acetyl spiral mould (acetylspiramycin), aminoglycoside: Liu Suanyan NEOMYCIN SULPHATE (neomycin), sulfamido: trimethoprim-sulfamethoxazole (sulfomethorxazole), quinolones, comprise Ofloxacine USP 23 (ofloaxacin), norfloxicin (norfloxacin) and clindamycin (clindamycin) and other classes: comprise Rifampin (rifampin), furadantin (nitrofurantoin) and PXB (polymyxin B), drug sensitivity assay has been carried out to test strains, according to World Health Organization's regulation in 1977, during test gram-positive microorganism, need with Quality Control bacterium streptococcus aureus (Staphylococcus aureus) ATCC25923 bacterial strain as reference culture reference, result judges the NCCLS standard provided with reference to WHO.
B, acute toxicity test
After BZ11 bacterial strain is activated, separation and purification three times, inoculating strain cultivates 48h in CO2gas incubator in the liquid nutrient medium of PTYG, by bacterium liquid with 5000r/min, centrifugal 10min under 4 DEG C of conditions, collect bacterium mud, then wash with sterile phosphate buffer PBS newly formed suspension of once laying equal stress on, bacterial concentration is adjusted to 3 × 10
10cFU/mL.Then acute toxicity test is carried out according to toxicological evaluation of food safety procedure and method.
Select the male mouse 20 of the Kunming kind of healthy adult by body weight requirement, between mouse, weight differences is not remarkable.After adapting to Animal House environment (relative humidity: 50 ± 5%, temperature: 20 DEG C ~ 25 DEG C) 7d, mouse, by only weighing, is divided into 2 groups immediately, often organizes 10, i.e. experimental group and blank group.With BZ11 bacterium liquid, laboratory animal is contaminated, the mouse of fasting taboo water empty stomach 12h is carried out to the gavage volume of 0.4mL/20g body weight; Blank group mouse, with same dose gavage pure water.After mouse contamination, in one week, observe its general state, body weight change, toxicity symptom and death condition, within the 8th day, put to death mouse.Experiment is weighed to animal latter stage again, and carry out necrotomy to dead animal and the execution animal that expires, visual inspection general pathology changes situation.Experiment whole process and observed content do detailed record.
By carrying out safety experiment evaluation experimental to bacterial strain, result shows to screen the bacterial strain BZ11 that obtains without acute toxicity, confirms the security that bacterial strain BZ11 is preliminary, can be considered as the further deep development of probiotic bacterium and utilize.
(11) in vivo test of decreasing cholesterol, oxytolerant bifidus bacillus
Purifying 3 times after test strains BZ11 and control strain Infantile diarrhea thaw and activate, be inoculated in PTYG liquid nutrient medium with the inoculum size of 5% (volume ratio) respectively, 24h is cultivated in the CO2gas incubator of 37 DEG C, by bacterium liquid at 5000r/min, centrifugal 10min under 4 DEG C of conditions, collects bacterium mud.Carry out 10 times of gradient dilutions with sterilizing PBS damping fluid again, get 0.1mL and be coated with on PTYG agar plate, carry out enumeration after 37 DEG C of cultivation 36 ~ 48h, according to plate count result, respectively BZ11 bacterial strain and Infantile diarrhea are adjusted to as bacterial concentration is 3.0 × 10
9the bacteria suspension of CFU/mL.For preventing the death of bifidus bacillus from affecting result, fresh configuration every day bacterium liquid, timing in the morning is filled with and is fed and place feed.
The Kunming mouse 48 of healthy 4 weeks, male and female half and half, purchased from Guiyang Medical College.Mouse is divided into 4 groups at random, often organizes 12, male and female half and half.(relative humidity: 50 ± 5% in Animal House environment, temperature: 20 DEG C ~ 25 DEG C, 12h illumination every day, 12h is dark, and sanitation and hygiene environment is good) feed of freely drinking water, after normal diet normally raises 7d, weigh one by one, again be divided into 4 groups by mean body weight between each group without significant difference, often organize 12.Experimental result sees the following form:
Note: A group represents Normal group, B group represents hyperlipidemia model group, and C group represents that in Infantile diarrhea experiment, D group represents BZ11 experimental group.
Feed formulation and collocation method: basal feed is purchased from Guiyang Medical College, reference method, the formula of high lipid food is: lard 12%, cholesterol 1%, bovine bile 0.5%, basal feed 86.5%.Concrete feed making step is as follows: normal diet pulverizer is pulverized by (1), adds bovine bile etc. and mixes; (2) in pot, heat lard to boil, add cholesterol simultaneously and make it to dissolve; (3) join in the normal diet of mixing by the lard of liquid state, with glass stick mixing, more progressively add suitable quantity of water, carry out kneading and be shaped as cylindric feed, noting does not add water causes too softness not easy-formation too much.(4) be put in preservation in the refrigerator of 4 DEG C after making for subsequent use, be put into microwave-oven-heating during feeding about 5 seconds, take out.
Within the 10th, 20 day after gavage mouse experiment starts, carry out body weight determination one by one to experiment mice, whether the body weight of observing between group is variant.And often group random choose 10 mouse carry out blood specimen collection, having blood-sample withdrawal twice altogether in experimentation, is respectively once took a blood sample at the 10th, 20 day respectively, and respectively organizing mouse before each blood sampling needs fasting to prohibit water 12h.First time, Blood collection was the blood sampling of retroorbital venous clump, and second time is for plucking eyeball blood sampling, and blood sampling volume is 0.2mL/.After second time blood sampling, all mouse cervical dislocation are put to death.
By the blood sample collection of collection in the 2mL sterile centrifugation tube with antithrombotics, after 37 DEG C of water-bath 30min, the centrifugal 10min of 3000r/min, carefully gets upper serum.Reference reagent box operation instruction and employing microplate reader measure the content of total cholesterol (TC), triglyceride level (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in serum.And calculate atherogenic index (Atherogenic index, AI), formula is as follows:
AI＝LDL-C/HDL-C
Compared with control strain Infantile diarrhea, the body weight impact of BZ11 bacterial strain on high fat mouse is larger, more can reduce total cholesterol (TC) content in mice serum, reduces the content of Triglycerides in Serum (TG), suppresses low density lipoprotein cholesterol (LDL-C) content in serum, improves high density lipoprotein cholesterol (HDL-C) content, reduce arteriosclerosis (AI) index.
(12) by above primary dcreening operation with sieve experiment again, finishing screen select bacterial strain BZ11 decreasing cholesterol ability high and acidproof by property, bile tolerance by property, oxytolerant bacterial strain of good performance, by carry out bio-chemical characteristics qualification and 16S rRNA gene order qualification.
Claims (3)
1. a strain fragrant pig source property decreasing cholesterol, oxytolerant bifidus bacillus BZ11, it is characterized in that this bacterial strain is bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.Lactis), the pure growth of this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 18th, 2014, this centre address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica, postcode: 100101; Deposit number is CGMCC NO.10224, referred to as bifidobacterium animalis subspecies BZ11.
2. fragrant pig source as claimed in claim 1 property decreasing cholesterol, oxytolerant bifidus bacillus BZ11, it is characterized in that the morphological specificity of described bifidobacterium animalis subspecies BZ11 and physiological and biochemical property bacterium are all close to genus bifidobacterium, its 16S rRNA gene order and bifidobacterium animalis subspecies (
bifidobacterium animalis subsp.
lactis ) 16S rRNA sequence homology be 100%, according to the categorised regulation of genus bifidobacterium in " uncle Jie Shi systematic bacteriology handbook ", bifidobacterium animalis subspecies BZ11 belongs to actinomycetes door (Actinobacteria), Actinomycetes (Actinobacteria), actinomycetes subclass (Actinobacteridae), bifidus bacillus order (Bifidoacteriales), bifidobacterium family (Bifidobacteriaceae), genus bifidobacterium (Bifidobacterium), bifidobacterium animalis subspecies (
bifidobacterium animalis subsp.
lactis ).
3. the fragrant pig source property decreasing cholesterol as described in one of claim 1 and 2, oxytolerant bifidus bacillus BZ11, it is characterized in that described bifidobacterium animalis subspecies BZ11 has security: this bacterial strain to penicillin G, piperacillin, amoxycilline Trihydrate bp/clavulanic acid, vancomycin, cefotaxime, Kefzol, Azythromycin, Rifampin, furadantin 9 class antibiotic sensitive, to Ciprofloxacin, Liu Suanyan NEOMYCIN SULPHATE, trimethoprim-sulfamethoxazole, Ofloxacine USP 23, norfloxicin and PXB totally 6 class antibiotics resistances; In penicillin medicine, this bacterial strain is to ampicillin sensitive; In cephalo element class medicine, this bacterial strain is responsive to cefoperazone; In Macrocyclolactone lactone kind medicine, this bacterial strain is to the mould sensitivity of acetyl spiral; In quinolones, this bacterial strain is responsive to woods mycin;
And by gavage mouse experiment, show that bifidobacterium animalis subspecies BZ11 bacterial strain is without acute toxicity, the therefore tentative confirmation security of this bacterial strain, and demonstrate decreasing cholesterol effect in its body further by gavage mouse experiment.
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