CN106497840A - A kind of method of directional separation immune regulative intestinal lactobacilluss - Google Patents
A kind of method of directional separation immune regulative intestinal lactobacilluss Download PDFInfo
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Abstract
The invention discloses a kind of method of directional separation immune regulative intestinal lactobacilluss, belongs to biologic product technology field.The present invention processes animal or people's saliva, intestinal contentses or fecal bacteria suspension with biotinylated anti-IgA antibody, then sorted using the nanometer magnetic bead of streptavidin, the intestinal lactobacilluss with immunoloregulation function are obtained after enrichment culture and Selective Separation.Compared with traditional method, the method can quickly, directional separation immune regulative probioticss, the bacterial strain of acquisition is through further checking acid resistance, after resisting biliar and adhesiveness, can be used for feedstuff, bread and cheese or functional food.
Description
Technical field
The present invention relates to a kind of method of directional separation immune regulative intestinal lactobacilluss, belongs to biologic product technology neck
Domain.
Background technology
Lactobacilluss are the important members of probiotic bacteria, are widely present in human body and animal gastrointestinal tract.Home and abroad is a large amount of
Test confirms internal multiple lactobacilluss scalable hosts to function of immune system, and this is their infection prevention disease, improvement
Food anaphylaxiss, anticancer, firm gut barrier, improve immunological adjuvant performance and ease up the basis of the effects such as solution enteritiies.Which is made
With mechanism, directly immune cell activated is relevant with secretory immune instrumentality.
A large amount of researchs for separating intestinal immune modulability lactobacilluss are carried out both at home and abroad.Separation screening intestinal immune is adjusted
Section property lactobacilluss existing forefathers are screened using conventional traditional method, are carried out spread plate with MRS solid mediums and are obtained
To a large amount of bacterial strains, Gram’s staining is then carried out, microscopy is discarded gram negative strain, tested by catalase afterwards
With bacillus perfringens stormy fermentation test, discard catalase positive and bacillus perfringens, most at last Gram-positive,
Negative catalase and fix tentatively as lactobacilluss without spore is shaft-like, be finally the specific PCR of lactobacilluss and finally determine and sift out
Bacterium is lactobacilluss.The method has certain limitation, and the bacterium that sieves bacterium cycle length and sift out differs and is set to intestinal immune modulability
Lactobacilluss, the uncertainty of traditional method cause the uncertainty of screening operation.In addition, this culture method method detection limit is
10cfu/g.
To sum up, intestinal immune modulability lactobacilluss are carried out screening and are obtained using the traditional separation method of experience directiveness
's.A large amount of intestinal lactobacillus strains are obtained first with selective medium separation, which is then investigated respectively and is adhered to intestinal bacteria
Ability, acid resistance and external evoked cytokine activity.This separation method workload is very big, needs a large amount of human and material resources,
The active bacterial strain screening cycle is also very long.But the activity in vivo difference that many Physiological in vitro show similar bacterial strain is very big
[Ibnouzekri et al., 2003], more increased the complexity of screening operation.
Content of the invention
In order to solve the above problems, the present invention establishes the side that a kind of targeting separates high-immunity regulation activity lactobacillus strain
Method.The quick of the present invention, the method for separating high immune regulative lactobacilluss from human or animal's intestinal contents or feces of orientation,
Based on the advantage of immunomagnetic bead technique, it is easy to the characteristics of combining using biotin and Streptavidin, fast and effeciently from feces
The middle IgA that separates wraps up lactobacilluss, then recycles special media isolated strains, and is carried out soon with lactobacilluss specific PCR
Fast Preliminary Identification.
Lactobacilluss can enter small intestinal peyer's patcheses (PP) by M cells.PP is that the most important antigen of small intestinal gathers position, interior
There is a large amount of macrophages, dendritic cell, T cell and B cell in portion.Dendritic cell absorb lactobacilluss, are stimulated maturation, will induction
B cell becomes product IgA plasma cells.B cell can produce two kinds of IgA, and one kind is to combine most of intestinal bacteria with weak force
Natural IgA, another are the IgA for combining immune induction bacterium with high-affinity.The immunoregulation effect of lactobacilluss has substantially
Bacterial strain dependency, high activity bacterial strain can enter PP induction high-affinity IgA and produce, therefore, the stronger intestinal of immunostimulatory activity
Road lactobacilluss its cell surfaces is all wrapped up by high-affinity IgA.Lactobacilluss can be also promoted to enter PP successive inductions after IgA parcels
Immunoreation, it is also possible to strengthen its anti-inflammatory activity.
The method of the directional separation immune regulative intestinal lactobacilluss of the present invention, successively includes：(1) thalline suspension is prepared
Liquid；(2) lowlenthal serum or bovine serum albumin are added in suspension, ice bath is for a period of time；It is subsequently adding biotin labeling
Rabbit-anti people IgA ice baths for a period of time；Streptavidin MagneSphere is added, ice bath is for a period of time；Last magnetic absorption is combined with
The Streptavidin MagneSphere of thalline, washing obtain thalline；(3) by thalline enrichment culture obtained in the previous step；(4) identification enrichment
The bacterial strain that cultivates, the bacterial strain for being accredited as lactobacilluss are the lactobacillus strain of high-immunity regulation activity.
In one embodiment of the invention, the thallus suspension liquid is prepared by saliva, intestinal contents or feces
Bacterial suspension.
In one embodiment of the invention, the thallus suspension liquid, when sample is solid-state or semisolid, is by sample
Product are added in the buffer of sterilizing, and whirlpool is mixed, and low-speed centrifugal takes supernatant and through cell filtering net process, the liquid being filtrated to get
Then body is resuspended in buffer through multiple high speed centrifugation, washing, obtains the thallus suspension liquid of mixed cell；When sample is liquid
During body, directly by the buffer of sample addition sterilizing, whirlpool is mixed, and high speed centrifugation takes precipitation, through repeatedly washing, is centrifuged, so
After be resuspended in buffer and obtain thallus suspension liquid.
In one embodiment of the invention, the low-speed centrifugal is in 800～1200rpm；The high speed centrifugation is
In 8000～12000rpm.
In one embodiment of the invention, the buffer is peptone buffer agent.
In one embodiment of the invention, the effect of the lowlenthal serum or bovine serum albumin is that closing is non-specific
Property combine.
In one embodiment of the invention, the addition of the lowlenthal serum or bovine serum albumin is in bacteria suspension
Final concentration be respectively 10%, 0.05～0.5%.
In one embodiment of the invention, the addition of the Streptavidin MagneSphere is 0.1-1.0mg/mL.
In one embodiment of the invention, the preparation of the Streptavidin MagneSphere is first to prepare amination nano magnetic
Pearl, is then connected with glutaraldehyde, and the amination nanometer magnetic bead of upper for connection glutaraldehyde is reacted in buffer with Streptavidin,
The nanometer magnetic bead of streptavidin is adsorbed in reaction using externally-applied magnetic field after terminating, cleaning obtains Streptavidin MagneSphere.
In one embodiment of the invention, the preparation of the Streptavidin MagneSphere is specifically：
1. amination nanometer magnetic bead：Experimental ware used is overnight soaked in chloroazotic acid, is cleaned and is dried, to being placed with rotor
60mL ethylene glycol, 13g hexamethylene diamines, 4g anhydrous sodium acetates, 2g FeCl is added in round-bottomed flask3·6H2O, in 50 DEG C of oil baths to molten
Liquid is uniform, and liquid is transferred in the autoclave with tetrafluoroethene liner, reacts 6h in 198 DEG C, after being cooled to room temperature
It is transferred in beaker, is rinsed after multiple black solid repeatedly using dehydrated alcohol and deionized water, by black solid in 50 DEG C of bars
10h is dried under part, and the black solid for then drying obtains amination nanometer magnetic bead using agate mortar grind into powder.
2. glutaraldehyde connection：2mg amination magnetic beads are weighed in centrifuge tube, glutaraldehyde/PBS buffer solution (volumes are added
Than for 5%) 2mL, reacting 2h in 37 DEG C of shaking tables, holding magnetic bead using the magnetic force of magnet, clean repeatedly 5 with PBS buffer solution
More than secondary, unconjugated glutaraldehyde is removed.
3. Streptavidin MagneSphere：In the connection in the amination magnetic bead of glutaraldehyde plus 2mL PBS buffer solution, Ran Houzai
The Streptavidin 250uL of 1mg/mL is added, and 12h is reacted in 37 DEG C of shaking tables, is cleaned using externally-applied magnetic field PBS repeatedly
More than 5 times, unconjugated Streptavidin is removed, collect precipitation, that is, obtain the nanometer magnetic bead of streptavidin.Magnetic bead is cleaned
After be placed in 4 DEG C of environment and save backup.
In one embodiment of the invention, the time of the ice bath is 10-30min.
In one embodiment of the invention, step (3) specifically：
1. 5% lowlenthal serum, ice, in the thallus suspension liquid that mixed cell is suspended in that peptone buffer agent is obtained, are added
Bath 15min；Add the rabbit-anti people IgA, ice bath 20min of biotin labeling；It is subsequently adding Streptavidin MagneSphere, ice bath 20min.
2. antibacterial is combined with magnet or magnetic bead sorting device absorption magnetic bead, outwell supernatant；Peptone buffer agent is used afterwards
3 thalline of cleaning.
3. finally the thalline that Streptavidin MagneSphere adsorbs is resuspended in peptone buffer agent.
In one embodiment of the invention, the enrichment culture, is first enrichment culture 6-8h on enrichment medium,
Then candidate strain is obtained in the upper line of lactobacilluss specific isolation culture medium (LAMVAB), purification repeatedly.
In one embodiment of the invention, the formula of the enrichment medium：Arabinose 20mM；Ribose 20mM；
MgSO41mM；MnSO40.1mM；FeSO45uM.
In one embodiment of the invention, the formula of the lactobacilluss specific isolation culture medium：Solution A：MRS is trained
Foster base 250mL, Cysteine Hydrochloride 0.0125g, bromocresol green 0.0012g adjust pH to 5.0 ± 0.1；Solution B：10g agar is molten
In 250mL deionized waters；Solution C：5mL Lyphocin (Fujisawa)s, 2mg/mL, sterilized water are prepared；Equivalent A, 121 DEG C of B solution are gone out
Bacterium 15min, B are cooled to 50 DEG C, and A is cold to be gone to room temperature to add solution C, mix, then A, B solution are mixed, and are down flat plate.
In one embodiment of the invention, the identification is to enter performing PCR identification using Lactobacillus specific primer.
In one embodiment of the invention, described identification specifically：Candidate strain genomic DNA is extracted, using breast
Bacillus Genus-specific primers carry out PCR, and the primer is for (sequence is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2)：
LbLMA1-rev (5 '-CTCAAAACTAAACAAAGTTTC-3 ') and R16-1 (5 '-
CTTGTACACACCGCCCGTCA-3’)；The bacterial strain containing 200～500bp distinctive products for obtaining is high immunomodulating and lives
The lactobacillus strain of property.
Advantages of the present invention：
Prepared by the inventive method, preparation, intestinal contents including immunomagnetic beadses or fecal bacteria suspension, magnetic bead is combined
Reaction and magnetic bead sorting, Enrichment of bacteria and selectivity culture, PCR methods lactobacilluss are identified.The inventive method is based on high activity bacterial strain
The IgA of secretion inducing enters enteric cavity and may specifically bind the bacterial strain, affine magnetic bead and the fecal bacteria for combining IgA using targeting
Suspension is acted on and is cleaned.In experimentation, the natural IgA combined with antibacterial and magnetic bead in combination in cleaning process just
Can be washed off, only retain the magnetic bead combined with high-affinity IgA, by magnetic bead sorting, and enrichment culture, so as to sharp separation is lived
Property bacterial strain.Meanwhile, in separation method, the addition of lowlenthal serum or bovine serum albumin, Streptavidin MagneSphere
Addition, ice bath time etc. optimize, and further increase screening efficiency, shorten the screening cycle.
Compared with conventional lactobacilluss screening, there is situation by cell surface high-affinity IgA and separate, screen in the present invention
Lactobacilluss, greatly improve immunomodulating bacterium separation selectivity, also substantially shorten the bacterial strain screening cycle.
Description of the drawings
Fig. 1 is the flow chart that directional separation high-affinity IgA combines lactobacilluss；
Fig. 2 is the lactobacilluss specific PCR electrophoresis result figure that the IgA screened from feces wraps up lactobacilluss；
Fig. 3 is the lactobacilluss specific PCR electrophoresis result figure that the IgA screened from saliva wraps up lactobacilluss；
Immune regulative lactobacilluss inducing mouse peritoneal macrophage Raw264.7 cytokines of the Fig. 4 for separate sources
The generation of TNF-α (a), IL-6 (b), IL-12 (c) and IL-10 (d)；Wherein, LPS represents lipopolysaccharide；Magnetic bead in F1 generation table feces
The lactobacilluss of absorption；FS1 represents the lactobacilluss after magnetic bead adsorbs in supernatant；T1 represents the lactobacilluss of magnetic bead absorption in saliva；
TS1 represents the lactobacilluss after magnetic bead adsorbs in supernatant；Wherein * represents that there were significant differences compared with blank group, P<0.05；* tables
Show<0.01；# represents that there were significant differences compared with LPS negative control groups, P<
0.05；## is represented pole significant difference, P compared with LPS negative control groups<0.01；
Fig. 5 is TNF-α/IL-10 (e), IL-6/IL-10 (f) and IL-12/IL-10 (g)；Wherein, LPS represents lipopolysaccharide；
The bacterium of magnetic bead absorption in F1 generation table feces；FS1 represents the bacterium after magnetic bead adsorbs in supernatant；T1 represents magnetic bead absorption in saliva
Bacterium；TS1：Bacterium after magnetic bead absorption in supernatant；Wherein * represents that there were significant differences compared with blank group, P<0.05；* represent with
Blank group has compared pole significant difference, P<0.01；# represents that there were significant differences compared with LPS negative control groups, P<0.05；## tables
Show<0.01.
Specific embodiment
Below in conjunction with case study on implementation, the present invention is further described；Following embodiments are illustrative, are not limited
, it is impossible to protection scope of the present invention is limited with following embodiments.
Embodiment 1：The preparation of Streptavidin MagneSphere
Streptavidin MagneSphere is prepared in accordance with the following methods
(1) amidized Fe3O4The preparation of nanometer magnetic bead
1. round-bottomed flask, rotor and reactor inner bag are all overnight soaked in chloroazotic acid, are then cleaned up with liquid detergent,
It is put in baking oven after deionized water cleaning again and dries.
2. 30mL ethylene glycol is added in the round-bottomed flask for be placed with rotor, add 6.5g 1,6- hexamethylene diamines to add 2.0g
Anhydrous sodium acetate, finally plus 1.0g FeCl3·6H2O.
3. by round-bottomed flask, in 50 DEG C, oil bath adds reflux, and the general 0.5h-1h of magnetic agitation.React to flask
Interior solution is uniform.
4. the solution in round-bottomed flask is carefully transferred in the autoclave inner bag of tetrafluoroethene liner, and will be anti-
Answer kettle to tighten, be placed on reaction 6h under 198 DEG C of high temperature, after terminating, naturally cool to room temperature (cooling overnight).
5. liquid in reactor is stirred evenly with Glass rod, carefully pours in 250mL beakers, repeatedly rushed with dehydrated alcohol
Reactor is washed, cleanout fluid is also poured in beaker in the lump.
6. beaker is lifted down by black magnetic granule dispersion (about 5min) in beaker from ultrasonic pond using ultrasound
It is placed on Magnet, is collected using Magneto separate, black particle will be attracted on a glass bottom, pour out the solution in beaker with Magnet,
Remaining liquid is sucked out with liquid-transfering gun.
7. 40mL deionized waters and washes of absolute alcohol is used alternatingly, the black magnetic granule in beaker is divided using ultrasound
Dissipate (about 5min), beaker is lifted down from ultrasonic pond and be placed on Magnet, collected using Magneto separate, black particle will be inhaled
At cup bottom, the solution in beaker is poured out with Magnet, remaining liquid is sucked out with liquid-transfering gun.
8. repeat step 7 is twice.
10h is dried under the conditions of the black solid for obtaining is placed on 50 DEG C.
9. grind into powder in the black solid particle agate mortar for drying, that is, obtain amidized Fe3O4Nano magnetic
Pearl, is placed in 4 DEG C and stores for future use.
(2) connect glutaraldehyde in amination magnetic bead
The above-mentioned amination magnetic bead 2mg for making is weighed in centrifuge tube, (volume ratio is to add glutaraldehyde/PBS buffer solution
5%) 2mL, in 37 DEG C of shaking tables react 2h, hold magnetic bead using the magnetic force of magnet, with PBS buffer solution clean repeatedly 5 times with
On, remove unconjugated glutaraldehyde.
(3) Streptavidin MagneSphere
Add 2mLPBS buffer solution in the amination magnetic bead of glutaraldehyde in connection, the strepto- for then adding 1mg/mL is affine
Plain 250uL, is reacted 12h in 37 DEG C of shaking tables, is cleaned repeatedly more than 5 times using externally-applied magnetic field PBS, is removed unconjugated
Streptavidin, collects precipitation, that is, obtains the nanometer magnetic bead of streptavidin.It is placed in 4 DEG C of environment after magnetic bead cleaning and protects
Deposit standby.
Embodiment 2：Using Streptavidin MagneSphere technology from human faecal mass separation screening immunomodulating lactobacilluss
The embodiment be using Streptavidin MagneSphere technology from human faecal mass separation screening immunomodulating lactobacilluss, Fig. 1
For the flow chart that directional separation high-affinity IgA combines lactobacilluss.
Specifically include following steps：
Using Streptavidin MagneSphere method, from feces, separation screening IgA wraps up lactobacilluss：
1. the feces of 1g Healthy Peoples are weighed, adds the peptone buffer agent of 10mL sterilizings, whirlpool to mix 5min.
2. 5min being centrifuged under 1000rpm, taking supernatant, big fragment and supernatant are by 200 aseptic aim cells
Filter screen filtration is in a new pipe.
3. the centrifuge tube for obtaining for filtering is centrifuged 5min under 10000rpm, outwells supernatant.Add the peptone of 5mL
Buffer, piping and druming are mixed, and be centrifuged 5min, outwell supernatant, repeat once under 10000rpm.
4. again precipitation is resuspended in 5mL peptone buffer agents, adds 5% lowlenthal serum, ice bath 15min.Add
The rabbit-anti people IgA of biotin labeling, ice bath 20min.Then (i.e. strepto- is affine to enter the nano immune magnetic bead of marked by streptavidin
Biscuit porcelain pearl), ice bath 20min.
5. adsorbed in tube bottom with magnet, supernatant is poured in a new sterilizing pipe.Afterwards with sterilizing
Peptone buffer agent cleans 3 thalline.
6. finally the thalline that immunomagnetic beadses adsorb is resuspended in peptone buffer agent, carries out the experiment of next step.
IgA parcel bacterium enrichment culture 6-8h on enrichment medium obtained in previous step, then special in LAMVAB
Streak culture, 37 DEG C of culture 48h are carried out on different in nature culture medium flat plate, are subsequently selected on each flat board with specific, bacterium
4 plants of different bacterium of the form that falls, are then inoculated into this 4 plants of bacterium in MRS fluid mediums respectively, 37 DEG C of culture 24h, then
Line purification is carried out on LAMVAB special media flat boards, single bacterium colony culture is chosen, such purification 3 times obtains tens plants of bacterium.
The basis of enrichment medium is：Arabinose 20mM；Ribose 20mM；MgSO41mM；MnSO40.1mM；
FeSO45uM.
After MRS fluid medium amplification culture, bacterial strain antibacterial group DNA extraction is extracted：
Lactobacilluss specific PCR：
For proving whether above-mentioned detached bacterial strain is sIgA parcel lactobacilluss, using the DNA of purification bacterial strain obtained above
Carry out lactobacilluss specific PCR.
PCR reaction systems 25uL：10buffer-MgCl2 2.5uL；MgCl2 2uL；dNTP 0.5uL；Forward primer
0.5uL；Downstream primer 0.5uL；Template DNA 3uL；Taq enzyme 0.5uL；DEPC water 13.5uL.
PCR amplification programs are：94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 60s, 30
Circulation；72 DEG C of extension 15min, are cooled to 12 DEG C.
After PCR terminates, in 1% agarose gel electrophoresiies, the bacterial strain for not running out of band is rejected, so as to obtain from feces
The sIgA parcel lactobacilluss of screening.
Embodiment 3：Using Streptavidin MagneSphere technology from saliva separation screening immunomodulating lactobacilluss
The embodiment is that from saliva, separation screening IgA wraps up lactobacilluss using Streptavidin MagneSphere technology.
A kind of method for separating IgA parcel lactobacilluss based on Streptavidin MagneSphere technology specifically includes following steps：
The preparation of Streptavidin MagneSphere：In the same manner as in Example 1.
Using Streptavidin MagneSphere method, from saliva, separation screening IgA wraps up lactobacilluss：
1. the saliva of 1mL Healthy Peoples is drawn, adds the peptone buffer agent of 9mL sterilizings, whirlpool to mix 5min.
2. 5min is centrifuged under 10000rpm after mixing, outwell supernatant.The peptone buffer agent of 5mL sterilizings is added, is blown
Beat and mix, 5min is centrifuged under 10000rpm, outwells supernatant, repeat once.
4. again precipitation is resuspended in 5mL peptone buffer agents, adds 5% lowlenthal serum, ice bath 15min.Add
The rabbit-anti people IgA of biotin labeling, ice bath 20min.Then the nano immune magnetic bead of marked by streptavidin, ice bath 20min are entered.
5. adsorbed in tube bottom with magnet, supernatant is poured in a new sterilizing pipe.Afterwards with sterilizing
Peptone buffer agent cleans 3 thalline.
6. finally the thalline that immunomagnetic beadses adsorb is resuspended in peptone buffer agent, carries out the experiment of next step.
IgA parcel bacterium enrichment culture 6-8h in enrichment medium obtained in previous step, then special in LAMVAB
Streak culture, 37 DEG C of culture 48h are carried out on different in nature culture medium flat plate, are subsequently selected on each flat board with specific, bacterium
4 plants of different bacterium of the form that falls, are then inoculated into this 4 plants of bacterium in MRS fluid mediums respectively, 37 DEG C of culture 24h, then
Line purification is carried out on LAMVAB special media flat boards, single bacterium colony culture is chosen, such purification 3 times obtains tens plants of bacterium.
After MRS fluid medium amplification culture, a part preserves thalline in 40% Freezing Glycerine, and a part is used for extracting
Bacterial genomes DNA.
Purification bacterial strain antibacterial group DNA extraction：
Using Ezup pillar bacterial genomes DNA extraction agent box extraction purification strain gene group DNAs.
Lactobacilluss specific PCR：
For proving whether above-mentioned detached bacterial strain is IgA parcel lactobacilluss, entered using the DNA of purification bacterial strain obtained above
Row lactobacilluss specific PCR.
PCR reaction systems 25uL：10buffer-MgCl2 2.5uL；MgCl2 2uL；dNTP 0.5uL；Forward primer
0.5uL；Downstream primer 0.5uL；Template DNA 3uL；Taq enzyme 0.5uL；DEPC water 13.5uL.
PCR amplification programs are：94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 64 DEG C of annealing 30s, 72 DEG C of extension 60s, 30
Circulation；72 DEG C of extension 15min, are cooled to 12 DEG C.
After PCR terminates, in 1% agarose gel electrophoresiies, the bacterial strain for not running out of band is rejected, so as to obtain from saliva
The sIgA parcel lactobacilluss of separation screening.
Fig. 2-3 is the electrophoretogram after the lactobacilluss specific PCR of the IgA parcel lactobacilluss screened from feces and saliva.
F6, F7, F21 do not have band as shown in Figure 2, and remaining is just IgA parcel lactobacilluss；T2 does not run out of band as shown in Figure 3, remaining
Just wrap up lactobacilluss for IgA.
Embodiment 4：Cell experiment
The lactobacilluss obtained from feces and saliva that above-described embodiment 2-3 is obtained are carried out cell experiment, checking is obtained
The ability that produces of the external evoked cytokine of lactobacilluss.
Supernatant bacterial strain after Turnover of Mouse Peritoneal Macrophages Raw264.7 and the above-mentioned antibacterial for filtering out and magnetic bead absorption
Co-cultured, experiment is divided into following four groups：
(1) blank group：Add 100uL PBS；
(2) experimental group：It is 1 × 10 to add 100uL concentration5(CFU) F1 (lactobacilluss of magnetic bead absorption in feces) of/mL,
F1 supernatant (lactobacilluss after magnetic bead absorption in supernatant), T1 (lactobacilluss of magnetic bead absorption in saliva), T1 supernatant (magnetic beads
Lactobacilluss after absorption in supernatant)；
(3) positive controls：It is 1 × 10 to add 100uL concentration5(CFU) the L.rhamnosus GG (LGG) of/mL are (positive
Control)；
(4) negative control group：1μg/mL LPS；The concentration of Turnover of Mouse Peritoneal Macrophages Raw264.7 is adjusted to 5 × 105
(CFU)/mL.
37 DEG C, 5%CO2Cell culture incubator culture 12h, is collected by centrifugation supernatant, will in strict accordance with ELISA kit description
Ask, determine the level of IL-6, TNF-α, IL-12 and IL-10 in Turnover of Mouse Peritoneal Macrophages Raw264.7.
By can be seen that in Fig. 4 and Fig. 5 that LPS can significantly raise the expression of the inflammatory cytokines such as IL-6, TNF-α and IL-12
The secretory volume of amount, IL-6, TNF-α and IL-12 LPS groups compared with blank group reach pole significant difference (P<0.01)；But from Fig. 4
In to be obtained F1 and T1 low compared with the expression of the inflammatory cytokines such as the IL-6 of FS1 and TS1, TNF-α and IL-12, but to IL-10
Expression do not have an impact, preliminary identification immune regulative lactobacilluss immunomodulating performance in the present invention is described；Equally, exist
In Fig. 5, the ratio of TNF-α/IL-10 is raised and represents the uncoordinated of cytokine secretion, can cause inflammatory reaction, LPS, FS1 and
TNF-α/the IL-10 of TS1 is significantly higher than blank group, and TNF-α/IL-10 LPS, FS1 and TS1 groups compared with blank group reach aobvious
Write difference (P<0.05).Above-mentioned conclusion is based on, the inventive method can obtain intestinal immune modulability lactobacilluss with separation screening.
Although the present invention is disclosed as above with preferred embodiment, which is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore protection model of the invention
Enclosing should be by being defined that claims are defined.
<110>Southern Yangtze University
<120>A kind of method of directional separation immune regulative intestinal lactobacilluss
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
ctcaaaacta aacaaagttt c 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cttgtacaca ccgcccgtca 20
Claims (10)
1. a kind of method of directional separation immune regulative intestinal lactobacilluss, it is characterised in that methods described successively includes：(1)
Prepare thallus suspension liquid；(2) lowlenthal serum or bovine serum albumin ice bath are added for a period of time in suspension；It is subsequently adding
The rabbit-anti people IgA ice baths of biotin labeling are for a period of time；Streptavidin MagneSphere is added, ice bath is for a period of time；Last magnetic
Absorption is combined with the Streptavidin MagneSphere of thalline, and washing obtains thalline；(3) by thalline enrichment culture obtained in the previous step；
(4) bacterial strain that identification enrichment culture is arrived, the bacterial strain for being accredited as lactobacilluss are the lactobacillus strain of high-immunity regulation activity.
2. method according to claim 1, it is characterised in that the thallus suspension liquid is saliva, intestinal contents or excrement
Just the bacterial suspension for preparing.
3. method according to claim 1, it is characterised in that the lowlenthal serum or bovine serum albumin addition are
10%, 0.05～0.5% is respectively in the final concentration of bacteria suspension.
4. method according to claim 1, it is characterised in that the addition 0.1-1.0mg/ of the Streptavidin MagneSphere
mL.
5. method according to claim 1, it is characterised in that the time of the ice bath is 10-30min.
6. method according to claim 1, it is characterised in that the buffer is peptone buffer agent.
7. method according to claim 1, it is characterised in that the thallus suspension liquid, when sample is solid-state or semisolid
When, it is to add sample in the buffer of sterilizing, whirlpool is mixed, and low-speed centrifugal takes supernatant and through cell filtering net process, mistake
Then the liquid that filter is obtained is resuspended in buffer, obtains the thallus suspension liquid of mixed cell through multiple high speed centrifugation, washing；
When sample is liquid, directly sample is added in the buffer of sterilizing, whirlpool is mixed, and high speed centrifugation takes precipitation, through repeatedly washing
Wash, be centrifuged, be then resuspended in buffer and obtain thallus suspension liquid.
8. the lactobacillus strain for obtaining according to the arbitrary methods described of claim 1～7.
9. application of the arbitrary methods described of claim 1～7 in feedstuff, bread and cheese or functional food field.
10. application of the lactobacillus strain described in claim 8 in feedstuff, bread and cheese or functional food field.
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| CN108456644A (en) * | 2018-06-08 | 2018-08-28 | 保睿集团有限公司 | High vigor high-purity human body intestines bacterium capsule preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575582A (en) * | 2008-05-08 | 2009-11-11 | 景岳生物科技股份有限公司 | Lactobacillus separation strains with anti-inflammatory activity and application thereof |
| CN102115721A (en) * | 2008-05-08 | 2011-07-06 | 景岳生物科技股份有限公司 | Lactobacillus isolated strains with anti-inflammatory activity and use thereof |
| CN102488717A (en) * | 2006-06-05 | 2012-06-13 | 生命大地女神有限公司 | Use of selected lactic acid bacteria for reducing infantile colic |
-
2016
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488717A (en) * | 2006-06-05 | 2012-06-13 | 生命大地女神有限公司 | Use of selected lactic acid bacteria for reducing infantile colic |
| CN101575582A (en) * | 2008-05-08 | 2009-11-11 | 景岳生物科技股份有限公司 | Lactobacillus separation strains with anti-inflammatory activity and application thereof |
| CN102115721A (en) * | 2008-05-08 | 2011-07-06 | 景岳生物科技股份有限公司 | Lactobacillus isolated strains with anti-inflammatory activity and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| HANNE R. CHRISTENSEN等: "Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells", 《THE JOURNAL OF IMMUNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456644A (en) * | 2018-06-08 | 2018-08-28 | 保睿集团有限公司 | High vigor high-purity human body intestines bacterium capsule preparation method thereof |
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