CN106822918A - A kind of surface modification liposome and its preparation method and application - Google Patents
A kind of surface modification liposome and its preparation method and application Download PDFInfo
- Publication number
- CN106822918A CN106822918A CN201710079967.7A CN201710079967A CN106822918A CN 106822918 A CN106822918 A CN 106822918A CN 201710079967 A CN201710079967 A CN 201710079967A CN 106822918 A CN106822918 A CN 106822918A
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- Prior art keywords
- liposome
- lactobacillus acidophilus
- layer protein
- surface modification
- layers
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Abstract
The invention discloses a kind of surface modification liposome and its preparation method and application, the liposome is soybean lecithin and cholesterol in mass ratio 7.5~2.5:1 mixing, and it is protein modified by lactobacillus acidophilus S layers.(1) present invention is modified surface of liposome using S layers of albumen of lactobacillus acidophilus, improves the stability and easy storage characteristics of liposome；(2) liposome of the present invention has tolerance to gastrointestinal proteases, it is ensured that the active ingredient for wherein embedding smoothly can reach target tissue and effectively play medicine effect.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of liposome and its preparation method and application, specially one kind is adopted
The liposome modified with lactobacillus acidophilus S-layer protein and its application in functional food and medicine field.
Background technology
Liposome as a kind of new pharmaceutical dosage form, due to its unique targeting, excellent controllable release property with
And as the focus of research the advantages of less drug dose.But although liposome can protect encapsulated medicine, it is in body
It is outer oxidizable, cause liposome membrane to rupture, content seepage, stability is poor, storage is difficult, in vivo also easily by some enzyme things
Matter is degraded, and is easily corroded by gastro-intestinal Fluid during by intestines and stomach, it is impossible to is reached target tissue and is effectively played medicine effect.In the last few years,
Improving the stability of liposome turns into exploitation focus.Using layer-by-layer polyelectrolyte is deposited in surface of liposome (such as
Poly-D-lysine, sodium alginate and shitosan etc.) formed layer protecting film can improve its storage-stable and digest stability, but
The relevant report of lactobacillus acidophilus S-layer protein modified liposome is not related to also.
S- layers of albumen be many bacteriums and Archaea cells wall surface coated one layer of lattice-like arrangement unimolecule egg
The macromolecular structure of white or glycoprotein subunit composition.The S- layers of presence of albumen is had been found that in many lactobacillus, for example：Switzerland's breast
Bacillus, lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus brevis etc..With non-covalent interaction between S- layers of protein surface subunit,
The S- layers of hydrogen bond of albumen is destroyed using high concentration denaturant or dissociation agent, S- layers of albumen is depolymerized to monomer and is dissolved out, and
After separation agent is removed, S- layers of protein protomer can be reassembled into the lattice structure of height rule in various matrix again, i.e.,
Generation self assembly effect.Therefore, the present invention will provide a kind of preparation and application of lactobacillus acidophilus S-layer protein modified liposome,
And evaluate the intestines and stomach stability of lactobacillus acidophilus S-layer protein modified liposome.
The content of the invention
The technical problem of solution：In order to overcome the defect of prior art, a kind of good stability is obtained, easily stored, prevented
A kind of liposome digested in intestines and stomach, there is provided surface modification liposome and its preparation method and application.
Technical scheme：A kind of surface modification liposome, the liposome is soybean lecithin and cholesterol in mass ratio 7.5
~2.5:1 mixing, and modified by lactobacillus acidophilus S-layer protein；Wherein, the quality of lactobacillus acidophilus S-layer protein and liposome
Than being 1:1.2~1.9.
A kind of preparation method of surface modification liposome, comprises the steps of：
(1) lactobacillus acidophilus S-layer protein is slightly carried：Collect the bacterium that lactobacillus acidophilus grows to logarithmic phase in MRS culture mediums
Body, it is suspended to mix with acidity LiCl solution, be incubated afterwards, supernatant is collected by centrifugation；
(2) lactobacillus acidophilus S-layer protein purifying：Step (1) is slightly carried using Sephadex G-75 gel permeation chromatographies
The S- layers of albumen for taking is isolated and purified；
(3) lactobacillus acidophilus S-layer protein identification：Using SDS-PAGE (dodecyl sodium sulfate polyacrylamide gel electricity
Swimming) authentication step (2) S- layers of molecular weight of albumen after purification, MALDI-TOF-MS is (during substance assistant laser desorpted ionized flight
Between mass spectrum) identification the S- layers of sequence of albumen；
(4) liposome preparation：Soybean lecithin and cholesterol are dissolved in ether, wherein soybean lecithin and cholesterol matter
Amount sum is 8~19 with the ratio range of ether volume:1；Being placed in round-bottomed flask carries out shape in decompression rotary evaporation to flask
Into thin film, to phosphate buffer is added in flask, uniform liposome is obtained through aquation, ultrasound；
(5) lactobacillus acidophilus S-layer protein modified liposome：S- layers of albumen and step after step (2) is isolated and purified
(4) liposome is dissolved in phosphate buffer respectively, and protein concentration is 100 μ g/mL, and concentration of liposomes is 120~190 μ g/
mL；After the two is mixed, 15~35 DEG C, 50~100r/min constant-temperature incubations 2~3 hours；13000r/min, 4 DEG C of 10 points of centrifugations
Clock, abandoning supernatant will be precipitated with phosphate buffer and disperseed again, obtain lactobacillus acidophilus S-layer protein modified liposome；
(6) lactobacillus acidophilus S-layer protein modified liposome proterties observation：After being modified using transmission electron microscope observation
Liposome, nano particle size ZETA potentiometers determine particle diameter and current potential；
(7) lactobacillus acidophilus S-layer protein modified liposome intestines and stomach Detection of Stability：Prepare embedding FITC-RLSFNP's
Lactobacillus acidophilus S-layer protein modified liposome, is respectively placed in SGF/intestinal juice, and fluorescence intensity is determined after incubation.
A kind of preparation method of surface modification liposome according to claim 2, it is characterised in that the acidity
The concentration of LiCl solution is 5mol/L, and pH=2.0, bacterium solution OD600 are 1.0, and bacteria suspension is 35 with acidity LiCl liquor capacities ratio:
1,37 DEG C of incubation 30min.
Preferably, soybean lecithin and cholesterol quality sum and the ratio of ether volume are 9.6 in step (4):1, phosphorus
Acid buffer pH=7.4,37 DEG C of aquations 1 hour.
Preferably, protein concentration is 100 μ g/mL in step (5), 22 DEG C, 50r/min constant-temperature incubations 2.5 hours.
Application of the described surface modification liposome in functional food.
Application of the described surface modification liposome in medicine.
Principle of the invention is：Lactobacillus S- layers of albumen after isolating and purifying has self assembly effect, you can various
The lattice structure of height rule is reassembled into matrix, according to this principle, the invention provides a kind of lactobacillus acidophilus S-layer
The method of protein modified liposome.
Beneficial effect：(1) present invention is modified surface of liposome using lactobacillus acidophilus S-layer protein, improves lipid
The stability of body and easy storage characteristics；(2) liposome of the present invention has tolerance to gastrointestinal proteases, it is ensured that wherein embed
Active ingredient smoothly can reach target tissue and effectively play medicine effect.
Brief description of the drawings
Fig. 1 is lactobacillus acidophilus S-layer protein gel permeation chromatography figure；
Fig. 2 is lactobacillus acidophilus S-layer protein SDS-PAGE；
Wherein, M is protein markers, and 1 is lactobacillus acidophilus S-layer protein after purification；
Fig. 3 is liposome transmission electron microscope picture；
Fig. 4 is lactobacillus acidophilus S-layer protein modified liposome transmission electron microscope picture；
Fig. 5 is liposomal particle size distribution map；
Fig. 6 is the liposomal particle size figure that lactobacillus acidophilus S-layer protein modification has embedded RLSFNP；
Fig. 7 is stability diagram in the front and rear simulation gastro-intestinal Fluid of lactobacillus acidophilus S-layer protein modified liposome modification.
Specific embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of the invention and essence, the modification and replacement made to the inventive method, step or condition belong to the present invention
Scope.If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.This
Term used in invention, unless otherwise specified, the implication being typically generally understood that with those of ordinary skill in the art.
Embodiment 1
A kind of surface modification liposome, the liposome is soybean lecithin and cholesterol in mass ratio 5:1 mixing, and by
Lactobacillus acidophilus S-layer protein is modified；Wherein, lactobacillus acidophilus S-layer protein and the mass ratio of liposome are 1:1.44.
A kind of preparation method of surface modification liposome, comprises the steps of：
(1) lactobacillus acidophilus S-layer protein is slightly carried
Lactobacillus acidophilus (Lactobacillus acidophilus) CICC6074 (Chinese industrial Microbiological Culture Collections
Administrative center) with liquid MRS medium cultures to logarithmic phase is grown, bacterial sediment is collected by centrifugation, thalline is adjusted with physiological saline
It is 1.0 to OD600, mixes with acid LiCl solution, acid LiCl concentration is 5mol/L, pH=2.0, bacteria suspension and acidity LiCl
Liquor capacity ratio is 35:1, bacteria suspension mix with acidity LiCl solution after incubation temperature be 37 DEG C, the time is 30min, then from
The heart (8000r/min, 20min) discards precipitation, and it is the thick lactobacillus acidophilus S-layer protein for carrying to collect supernatant；
(2) lactobacillus acidophilus S-layer protein purifying
The lactobacillus acidophilus S-layer protein crude extract that step (1) is extracted is through Sephadex G-75 gel-filtration chromatographies
Isolated and purified.Chromatographic column is balanced with 0.025mol/L Tris-HCl buffer solutions (pH=9.5), and applied sample amount is column volume
1%-2%, elution speed is 1mL/min, and eluent is 0.025mol/L Tris-HCl buffer solutions (pH=9.5), at 280nm
Detection absorbance, collects eluting peak, sees Fig. 1, there is two absworption peaks, collects identified with SDS-PAGE respectively, and discovery peak 1 is S-
Layer absorbing proteins peak, dialysis 18 hours or so in molecular cut off 14KDa bag filters is placed in after collecting peak 1, and dialysis medium is double
Water is steamed, the lactobacillus acidophilus S-layer protein of purifying is after dialyzate freeze-drying；
(3) lactobacillus acidophilus S-layer protein identification
Using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) authentication step (2) S- layers after purification
Molecular weight of albumen：SDS-PAGE using 10% separation gel and 5% concentration glue, take in step (2) through Sephadex G-75 gels
S- layers of albumen of chromatography mixes in equal volume with sample-loading buffer, boiling water bath 5min, is 10uL per hole applied sample amount, concentrates glue
80V constant pressure 30min, separation gel 100V constant pressure 3h, electrophoresis are dyeed after terminating with coomassie brilliant blue R_250, and electrophoretogram is shown in Fig. 2, can
See lactobacillus acidophilus S-layer protein molecular weight about 46kDa；
MALDI-TOF-MS (Matrix-assisted laser desorption ionization) identifies the S- layers of sequence of albumen：SDS-
The single lactobacillus acidophilus band that PAGE electrophoresis is obtained cuts down, and band sequentially passes through washing, decolouring, dehydration, alkylation, water
Wash, be dehydrated, the step such as digestion, finally extract peptide fragment, peptide fragment sample lysate (0.1% formic acid, 2% acetonitrile) dissolves, fully
Vibration is vortexed, 13200r/min, and 4 DEG C are centrifuged 10 minutes, and supernatant is transferred in loading pipe, carries out Mass Spectrometric Identification.By NCBI numbers
According to library searching, it is determined that surveyed albumen source is in lactobacillus acidophilus S-layer protein, table 1 is lactobacillus acidophilus S-layer protein in database
Sequence (SEQ ID NO:1), plus horizontal line amino acid matches sequence to be surveyed albumen with lactobacillus acidophilus S-layer protein in database,
C-terminal amino acid sequence reaches 38% with homologous protein coverage rate, and the protein molecular weight is about 46.5kDa, with electrophoresis result one
Cause, albumen theory isoelectric point is 9.59；
The lactobacillus acidophilus S-layer protein amino acid sequence of table 1
(4) liposome preparation
With hexapeptide Arg-Leu-Ser-Phe-Asn-Pro (RLSFNP) (Sangon Biotech (Shanghai) Co., Ltd.
Synthesis) it is liposomal encapsulated substrate, the envelop rate with RLSFNP liposomes is tested as evaluation index using single factor test and response surface
Determine the optimal case of liposome preparation.Investigation factor is the mass ratio of soybean lecithin and RLSFNP, soybean lecithin and courage
The mass ratio of sterol, the volume ratio of organic phase and water phase, ultrasonic time, PBS pH.
The measure of liposome encapsulation：Take RLSFNP liposomes obtained in 5mL to load in bag filter, bag filter two tightens
(bag filter that PBS will be equipped with liposome floods completely) is placed in the beaker for filling 100mLPBS afterwards, is dialysed 4 hours at 4 DEG C.
Appropriate dialyzate is taken after 4 hours, light absorption value is determined according to the method described in RLSFNP assays, calculated free
The content of RLSFNP.Liposome encapsulation=(total-M of M dissociate) ,/M was always × 100%.Wherein M is always for the RLSFNP for initially weighing is total
Amount, it is the content of free RLSFNP for calculating that M dissociates.
The optimum process for determining RLSFNP liposome preparations is：120mg soybean lecithins are weighed to be dissolved with 24mg cholesterol
In 15mL ether (organic phase), weigh 15mg RLSFNP and be dissolved in 5mLPBS (pH=7.4), be water phase, then water is mutually used
Needle applicator is all in injection organic phase.Lysate is placed in 50mL round-bottomed flasks, decompression rotation is carried out in fume hood and is steamed
Hair, removes organic solvent, and originally vacuum is maintained at 0.05kPa or so, and vacuum is maintained at a 0.03kPa left sides when being nearly spin-dried for
The right side, when lipid forms thin film in bottle, stops revolving.10mL phosphate buffers (pH=is added in round-bottomed flask
7.4) round-bottomed flask, is placed in 37 DEG C of aquations, the time is 1h, helps the film layer on wall to come off.By the suspension after aquation
Ultrasound is carried out under conditions of ice-water bath, ultrasonic power is 400w, and the time is 5min, well mixed liposome is obtained, herein
Under the conditions of prepare the actual envelop rate of liposome be 67.5 ± 0.8%.The preparation of blank liposome and RLSFNP liposome preparations
It is identical, the difference is that to be not added with the 5mLPBS (pH=7.4) of RLSFNP for water phase.
(5) lactobacillus acidophilus S-layer protein modified liposome：By lactobacillus acidophilus S-layer protein purified product in step (2)
It is dissolved in phosphate buffer (pH=7.4), the mass concentration of lactobacillus acidophilus S-layer protein is 100 μ g/mL, by 10uL steps
(4) liposome prepared in is dissolved in 1mL PBSs (pH=7.4), then S- layers of albumen after dissolving is added dilute
In liposome after releasing, on constant-temperature table mix, time 2.5h, 22 DEG C of temperature, rotating speed 50r/min, then freeze at a high speed from
The heart, rotating speed is 13000r/min, and 4 DEG C of centrifugation 10min, abandoning supernatant will be precipitated again with phosphate buffer (pH=7.4)
Dispersion, obtains lactobacillus acidophilus S-layer protein modified liposome；
(6) lactobacillus acidophilus S-layer protein modified liposome proterties observation
Liposome after being modified using transmission electron microscope observation：Using negative staining, the fat that two drippings of drop are got ready
Plastid or S- layers of protein modified liposome are online in special purpose copper, dry naturally, then carry out negative staining with 2.5% phosphotungstic acid, natural
Volatilize, make particle that deposition is concentrated on copper mesh, with transmission electron microscope observation and take a picture.Fig. 3 is the transmission electron microscope of liposome
Figure, liposome is spherical structure, and form more rounding, liposomal dispersion is more uniform, and Fig. 4 is repaiied for lactobacillus acidophilus S-layer protein
Adorn the transmission electron microscope picture of liposome, it is seen that surface of liposome is gloomy, liposome adhesion, there is the profile of indistinct white liposome outer ring
For S- layers in vain, it is seen that surface of liposome is covered by lactobacillus acidophilus S-layer protein；
Nano particle size ZETA potentiometers determine particle diameter and current potential：Particle diameter and electricity are carried out with nanometer laser granularity potentiometric analyzer
The measure of position, compares the particle diameter before and after liposome modification, and accompanying drawing 5 is the grain size distribution of liposome, and particle diameter is averagely left in 199nm
The right side, size distribution is more uniform, and accompanying drawing 6 is the grain size distribution of lactobacillus acidophilus S-layer protein, and particle diameter is averagely in 240nm, grain
The raising explanation lactobacillus acidophilus S-layer protein in footpath is successfully modified on liposome.In addition, liposome Zeta potential for-
31.2mV, lactobacillus acidophilus S-layer protein modified liposome Zeta potential is -12.8mV, and the change of current potential also show acidophilus breast
Bacillus S- layers of albumen there occurs self assembly on the surface of liposome；
(7) lactobacillus acidophilus S-layer protein modified liposome intestines and stomach Detection of Stability：Prepare embedding FITC-RLSFNP's
Lactobacillus acidophilus S-layer protein modified liposome, is respectively placed in SGF/intestinal juice, fluorescence intensity is determined after incubation, specifically
Operation is as follows：
(raw work is given birth to mark hexapeptide Arg-Leu-Ser-Phe-Asn-Pro (RLSFNP) with fluorescein isothiocynate (FITC)
Thing engineering (Shanghai) limited company) synthesis, FITC-RLSFNP liposomes are prepared according to step 4, difference is to take
It is water phase that 15mg FITC-RLSFNP are dissolved in 5mL phosphate buffers (pH=7.4), and subsequent step is with (4)；S- layers of albumen
The same step of the preparation method (5) of FITC-RLSFNP liposomes is modified, except that lactobacillus acidophilus S-layer protein is same
FITC-RLSFNP liposomes are incubated；
Prepare artificial gastro-intestinal Fluid：Watery hydrochloric acid 16.4mL, plus distilled water 800mL and pepsin 10g are taken, after shaking up, plus is steamed
Distilled water is diluted to 1000mL as pepsin solutions；Potassium dihydrogen phosphate 6.8g is taken, plus distilled water 500mL dissolves it, uses
0.1mol/L sodium hydroxide solutions adjust pH value to 6.8, separately take pancreatin 10g, plus distillation appropriate amount of water dissolves it, and two liquid are mixed
Afterwards, plus distilled water diluting is trypsin solution to 1000mL.
The protein modified FITC-RLSFNP liposomes of 1mLFITC-RLSFNP liposomes and 1mLS- layers are respectively placed in 4mL
In intestinal juice/gastric juice, slowly shaken at 37 DEG C, 200 μ L solution are taken respectively at 15min, 30min, 1h, 2h, 4h, add 200 μ L
0.1M HCL/NaOH terminating reactions, by the mixed liquor high speed centrifugation after terminating reaction, 13000rpm/min, 10min, after centrifugation
Abandoning supernatant, is demulsified with 5%Triton X-100, the FITC-RLSFNP that release embedding is not revealed, and is then centrifuged again
(13000rpm/min, 4 DEG C), is centrifuged 10min, takes 50 μ L of supernatant liquid in the orifice plate of black 96, and being placed in ELIASA, to determine its relative
Fluorescence intensity, so as to compare the stability of liposome and S- layers of protein modified liposome in Imitative gastroenteric environments.
Fig. 7 is that RLSFNP liposomes and S- layers of protein modified RLSFNP liposome place a period of time in artificial gastro-intestinal Fluid
Fluorescence intensity change situation afterwards.As can be seen from Figure, S- layers it is protein modified after liposome fluorescence intensity apparently higher than liposome
Fluorescence intensity, illustrate that lactobacillus acidophilus S-layer protein serves a certain degree of protective effect to liposome, enhance lipid
The intestines and stomach stability of body.
SEQUENCE LISTING
<110>Nanjing Normal University
<120>A kind of surface modification liposome and its preparation method and application
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 444
<212> PRT
<213>Lactobacillus acidophilus
<400> 1
Met Lys Lys Asn Leu Arg Ile Val Ser Ala Ala Ala Ala Ala Leu Leu
1 5 10 15
Ala Val Ala Pro Val Ala Ala Ser Ala Val Ser Thr Val Ser Ala Ala
20 25 30
Thr Thr Ile Asn Ala Ser Ser Ser Ala Ile Asn Thr Asn Thr Asn Ala
35 40 45
Lys Tyr Asp Val Asp Val Thr Pro Ser Val Ser Ala Val Ala Ala Asn
50 55 60
Thr Ala Asn Asn Thr Pro Ala Ile Ala Gly Asn Leu Thr Gly Thr Ile
65 70 75 80
Ser Ala Ser Tyr Asn Gly Lys Thr Tyr Thr Ala Asn Leu Lys Ala Asp
85 90 95
Thr Glu Asn Ala Thr Ile Thr Ala Ala Gly Ser Thr Thr Ala Val Lys
100 105 110
Pro Ala Glu Leu Ala Ala Gly Val Ala Tyr Thr Val Thr Val Asn Asp
115 120 125
Val Ser Phe Asn Phe Gly Ser Glu Asn Ala Gly Lys Thr Val Thr Leu
130 135 140
Gly Ser Ala Asn Ser Asn Val Lys Phe Thr Gly Thr Asn Ser Asp Asn
145 150 155 160
Gln Thr Glu Thr Asn Val Ser Thr Leu Lys Val Lys Leu Asp Gln Asn
165 170 175
Gly Val Ala Ser Leu Thr Asn Val Ser Ile Ala Asn Val Tyr Ala Ile
180 185 190
Asn Thr Thr Asp Asn Ser Asn Val Asn Phe Tyr Asp Val Thr Ser Gly
195 200 205
Ala Thr Val Ile Asn Gly Ala Val Ser Val Asn Ala Asp Asn Gln Gly
210 215 220
Gln Val Asn Val Ala Asn Val Val Ala Ala Ile Asn Ser Lys Tyr Phe
225 230 235 240
Ala Ala Gln Tyr Ala Asp Lys Lys Leu Asn Thr Arg Thr Ala Asn Thr
245 250 255
Glu Asp Ala Ile Lys Ala Ala Leu Lys Asp Gln Lys Ile Asp Val Asn
260 265 270
Ser Val Gly Tyr Phe Lys Ala Pro His Thr Phe Thr Val Asn Val Lys
275 280 285
Ala Thr Ser Asn Thr Asn Gly Lys Ser Ala Thr Leu Pro Val Val Val
290 295 300
Thr Val Pro Asn Val Ala Glu Pro Thr Val Ala Ser Val Ser Lys Arg
305 310 315 320
Ile Met His Asn Ala Tyr Tyr Tyr Asp Lys Asp Ala Lys Arg Val Gly
325 330 335
Thr Asp Ser Val Lys Arg Tyr Asn Ser Val Ser Val Leu Pro Asn Thr
340 345 350
Thr Thr Ile Asn Gly Lys Thr Tyr Tyr Gln Val Val Glu Asn Gly Lys
355 360 365
Ala Val Asp Lys Tyr Ile Asn Ala Ala Asn Ile Asp Gly Thr Lys Arg
370 375 380
Thr Leu Lys His Asn Ala Tyr Val Tyr Ala Ser Ser Lys Lys Arg Ala
385 390 395 400
Asn Lys Val Val Leu Lys Lys Gly Glu Val Val Thr Thr Tyr Gly Ala
405 410 415
Ser Tyr Thr Phe Lys Asn Gly Gln Lys Tyr Tyr Lys Ile Gly Asp Asn
420 425 430
Thr Asp Lys Thr Tyr Val Lys Val Ala Asn Phe Arg
435 440
Claims (7)
1. a kind of surface modification liposome, it is characterised in that the liposome is soybean lecithin and cholesterol in mass ratio 7.5
~2.5:1 mixing, and modified by lactobacillus acidophilus S-layer protein；Wherein, the quality of lactobacillus acidophilus S-layer protein and liposome
Than being 1:1.2~1.9.
2. the preparation method of a kind of surface modification liposome described in claim 1, it is characterised in that comprise the steps of：
(1) lactobacillus acidophilus S-layer protein is slightly carried：The thalline that lactobacillus acidophilus grows to logarithmic phase in MRS culture mediums is collected,
Mix with acidity LiCl solution, be incubated after suspended, supernatant is collected by centrifugation；
(2) lactobacillus acidophilus S-layer protein purifying：Using Sephadex G-75 gel permeation chromatographies to step (1) coarse extraction
S- layers of albumen is isolated and purified；
(3) lactobacillus acidophilus S-layer protein identification：Using SDS-PAGE authentication steps (2) S- layers of molecular weight of albumen after purification,
MALDI-TOF-MS identifies the S- layers of sequence of albumen；
(4) liposome preparation：Soybean lecithin and cholesterol are dissolved in ether, wherein soybean lecithin and cholesterol quality it
And with the ratio range of ether volume be 8~19:1；Being placed in round-bottomed flask carries out forming one in decompression rotary evaporation to flask
Layer film, to phosphate buffer is added in flask, uniform liposome is obtained through aquation, ultrasound；
(5) lactobacillus acidophilus S-layer protein modified liposome：S- layers of albumen and step (4) after step (2) is isolated and purified
Liposome is dissolved in phosphate buffer respectively, and protein concentration is 100 μ g/mL, and concentration of liposomes is 120~190 μ g/mL；Will
After the two mixing, 50~100r/min constant-temperature incubations 2~3 hours；13000r/min, 4 DEG C be centrifuged 10 minutes, abandoning supernatant,
To be precipitated with phosphate buffer and disperseed again, obtain lactobacillus acidophilus S-layer protein modified liposome；
(6) lactobacillus acidophilus S-layer protein modified liposome proterties observation：Fat after being modified using transmission electron microscope observation
Plastide morphology, nano particle size ZETA potentiometers determine particle diameter and current potential；
(7) lactobacillus acidophilus S-layer protein modified liposome intestines and stomach Detection of Stability：Prepare the acidophilus of embedding FITC-RLSFNP
Lactobacillus S- layers of protein modified liposome, is respectively placed in SGF/intestinal juice, and fluorescence intensity is determined after incubation.
3. a kind of preparation method of surface modification liposome according to claim 2, it is characterised in that the acid LiCl
The concentration of solution is 5mol/L, and pH=2.0, bacterium solution OD600 are 1.0, and bacteria suspension is 35 with acidity LiCl liquor capacities ratio:1,37
DEG C be incubated 30min.
4. the preparation method of a kind of surface modification liposome according to claim 2, it is characterised in that big in step (4)
Beans lecithin and cholesterol quality sum and the ratio of ether volume are 9.6:1；Phosphate buffer pH=7.4,37 DEG C of aquations 1 are small
When.
5. a kind of preparation method of surface modification liposome according to claim 2, it is characterised in that egg in step (5)
White concentration is 100 μ g/mL, 22 DEG C, 50r/min constant-temperature incubations 2.5 hours.
6. application of the surface modification liposome described in claim 1 in functional food.
7. application of the surface modification liposome described in claim 1 in medicine.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN117298072A (en) * | 2023-09-19 | 2023-12-29 | 苏州弘森药业股份有限公司 | Compound aztreonam inhalant and preparation process thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107899021A (en) * | 2017-11-06 | 2018-04-13 | 扬州大学 | A kind of chitosan microball, preparation and the application of surface modification S layers of albumen |
| CN108272822A (en) * | 2018-01-18 | 2018-07-13 | 南京师范大学 | It is a kind of breast polar lipid extracting method and its application |
| CN108272822B (en) * | 2018-01-18 | 2021-04-13 | 南京师范大学 | Extraction method and application of milk polar lipid |
| CN111449187A (en) * | 2020-05-09 | 2020-07-28 | 扬州大学 | Lactobacillus buchneri S-layer protein modified carvacrol/β -cyclodextrin liposome and preparation method and antibacterial application thereof |
| CN112957266A (en) * | 2021-02-23 | 2021-06-15 | 青岛农业大学 | Peanut oil body membrane protein modified liposome and preparation method thereof |
| CN115006350A (en) * | 2022-05-14 | 2022-09-06 | 南京师范大学 | Construction of milk fat plastid and application of milk fat plastid in macrophage immunocompetence regulation |
| CN115006350B (en) * | 2022-05-14 | 2023-05-30 | 南京师范大学 | Construction of milk fat plastid and application thereof in macrophage immunocompetence regulation |
| CN117298072A (en) * | 2023-09-19 | 2023-12-29 | 苏州弘森药业股份有限公司 | Compound aztreonam inhalant and preparation process thereof |
| CN117298072B (en) * | 2023-09-19 | 2024-04-16 | 苏州弘森药业股份有限公司 | Compound aztreonam inhalant and preparation process thereof |
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