CN102335413B - Preparation method, product and application of lactobacillus whole peptidoglycan with effect for resisting beta-lactoglobulin hypersusceptibility - Google Patents
Preparation method, product and application of lactobacillus whole peptidoglycan with effect for resisting beta-lactoglobulin hypersusceptibility Download PDFInfo
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Abstract
The invention discloses a preparation method, a product and application of lactobacillus whole peptidoglycan with an effect for resisting beta-lactoglobulin hypersusceptibility and relates to a microemulsion preparation prepared from the lactobacillus whole peptidoglycan obtained in the invention. The microemulsion preparation is prepared by the following steps of: with the lactobacillus whole peptidoglycan as a main effective component, heating the whole peptidoglycan, an oil phase, a surfactant, a cosurfactant and water at 70DEG C by stirring to enable the whole peptidoglycan to be completely dissolved; and uniformly mixing to obtain a semitransparent emulsion product with the liquid drop diameter of 30-45nm. After the lactobacillus whole peptidoglycan microemulsion preparation is orally taken to enter the interior of the body, lymphocyte can be effectively stimulated to secrete IL-12 and IFN-gamma; the IL-4 level is reduced; excessive hyperfunction of Th2 cells is reversed; and the lactobacillus whole peptidoglycan microemulsion preparation plays an important role of inhibiting beta-lactoglobulin allergic inflammation mediated by IgE and has potential application values for developing an immunopotentiator with antiallergic performance and the like.
Description
Technical field
The present invention relates to a kind of preparation method and application of Peptidoglycan, be particularly related to a kind of preparation method of integrated peptidoglycan of lactobacillus, also relate to the microemulsion formulation being prepared into by this integrated peptidoglycan, its preparation method and the application in the immunostimulant of the anti-beta lactoglobulin allergy of preparation thereof.Belong to field of biological pharmacy.
Background technology
Lac Bovis seu Bubali is nutritious, is the important food proteins source of infant.But show according to the sick investigation of Foreign Epidemic, 2%～6% child produces anaphylaxis to Lac Bovis seu Bubali, has a strong impact on its absorbing guaranteed milk albumen.Beta lactoglobulin (β-lactglobulin) is regarded as one of topmost milk allergen albumen, beta lactoglobulin has the function of similar immunoglobulin to calf, but for baby, be main anaphylactogen, easily cause baby's anaphylaxis, beta lactoglobulin mainly brings out the I type allergy of specific IgE antibody mediation, can cause vomiting, suffer from abdominal pain and the gastrointestinal upset such as diarrhoea.And the irritated gastroenteropathy (as infant colitis) causing often causes growthretardation.How to reduce due to beta lactoglobulin milk allergy become modern milk product produce in problem anxious to be resolved.
Applicant obtained certain achievement in research aspect the unbalance alleviation beta lactoglobulin allergy of probiotic bacteria adjusting Th1/Th2 over 3 years, screen a strain has good therapeutic effect Lactobacillus johnsonii KLDS1.7032 to the beta lactoglobulin allergy of IgE mediation, and the antianaphylactic effect of this Lactobacillus johnsonii of new discovery is relevant with the integrity of cell wall structure, its whole cell peptidoglycan is the main component that suppresses irritated.
At present, it is more that the physiological function to integrated peptidoglycan and functional mechanism are studied, and less to the research of its product preparation method.Existing whole cell peptidoglycan adopts injection system administration substantially, exists the low and too high dose of oral administration biaavailability can cause the problem of untoward reaction.And microemulsion is the drug release carrier of a new type ideal.Have transparently, the feature such as stable absorption is perfect, targeting drug release, and improved curative effect of medication, reduces toxic and side effects.Clinical value improves day by day, has very large development prospect.Therefore, the lactobacillus integrated peptidoglycan of extraction is prepared into microemulsion formulation by the present invention, after its oral administration arrives in body, can spontaneous formation particle diameter be less than the O/W microemulsion of 100nm, because medicine is scattered in microemulsion with molecular forms, can obviously improve the release of medicine, stability increases, oral administration biaavailability is 3～4 times of ordinary preparation, for the suitability for industrialized production of Novel immune reinforcing agent has been established certain theoretical basis.
Summary of the invention
One of technical problem to be solved by this invention is to disclose the application of lactobacillus integrated peptidoglycan microemulsion in the immunostimulant of the anti-beta lactoglobulin allergy of preparation; Particularly be applied in the immunostimulant of beta lactoglobulin allergy of the anti-IgE mediation of preparation.
Described immunostimulant can be further used in the medicine or food preparation of anti-beta lactoglobulin allergy.
Preferably, described lactobacillus integrated peptidoglycan extracts from about formula lactobacillus KLDS1.7032.
Described Lactobacillus johnsonii KLDS1.7032, for lactobacillus (Lactobacillus johnsonii), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCCNO.5050, preservation date is on July 8th, 2011.
Two of technical problem to be solved by this invention has been to provide a kind of method of preparing lactobacillus integrated peptidoglycan, and concrete comprises the following steps:
(1) cleaning of thalline and collection: after Lactobacillus johnsonii actication of culture is gone down to posterity, be inoculated in MRS fluid medium 37 ℃ of static cultivation 18h; After cultivation, be cooled to rapidly 4 ℃.At 5000g, 4 ℃ of centrifugal 20min, collect bacterial sediment; 4 ℃ of distilled water cyclic washings, to thalline white, are collected thalline, are placed in 100 ℃ of boiling water water bath deactivation 30min, obtain the dead bacterium mud of thermic;
(2) dephosphorization Teichaic acid: the mass fraction that the dead bacterium mud of thermic step (1) Suo Shu is suspended in to preheating is that in 10% trichloroacetic acid solution, boiling water bath 20min is with cracking thalline; After cooling, 5000g, 4 ℃ of centrifugal 15min, collecting precipitation;
(3) defat: concentration is 0.02mol/L, the sodium acetate solution that pH value is 4.6, with chloroform, methanol, three mixes for 4: 5: 10 by volume; The precipitation obtaining with mixed liquor dissolving step (2), the volume ratio of described mixed liquor is 15: 1 with precipitation volume ratio; Stirring at room temperature 18h, 8000g, 4 ℃ of centrifugal 20min, collecting precipitation;
(4) Deproteinization: the 0.1mol/L phosphate buffer that adds the pH 8.0 that contains 3mg/mL trypsin and 3mg/mL alkaline protease of 15 times of precipitation volumes in the precipitation that above-mentioned steps (3) obtains, 37 ℃ of water-bath vibration 12h, with 5000g, 4 ℃ of centrifugal 5min, precipitate with deionized water is washed 2 times;
(5) purify: the precipitation that step (4) obtains is through 0.01molL
-185～95 ℃ of processing 5min of sulfuric acid solution.Cooling immediately in ice-water bath, after 12000g, 4 ℃ of centrifugal 30min, abandon supernatant, precipitate with deionized water is continuously dialysis 7 days under 4 ℃ of conditions, and rearmounted 4 ℃ of lyophilization saves backup.
Three of technical problem to be solved by this invention has been to provide a kind of integrated peptidoglycan preparing according to above-described preparation method.
Four of technical problem to be solved by this invention has been to provide a kind of microemulsion formulation of anti-beta lactoglobulin allergy, and the above-described integrated peptidoglycan microemulsion that it is characterized in that comprising effective dose is as active component, also comprise oil phase, surfactant, cosurfactant and water.
Preferably, described surfactant is tween 80, and described cosurfactant is dehydrated alcohol, and described oil phase is the mixture of ethyl butyrate and vitamin E.
Preferred, described microemulsion formulation comprises each material of following weight fraction: above arbitrary described integrated peptidoglycan 3.00%, mass ratio is the ethyl butyrate of 7: 3 and the mixture 1.07%～13.59% of vitamin E composition, mass ratio is the tween 80 of 2: 8 and the mixture 2.10%～25.42% of dehydrated alcohol composition, and water 58.00%～89.00%.
Five of technical problem to be solved by this invention has been to provide the method for the microemulsion formulation of the anti-beta lactoglobulin allergy described in preparation, it is characterized in that being prepared by following methods: the mixture 2.10%～25.42% that the tween 80 that is 2: 8 by mass ratio forms with dehydrated alcohol is scattered in 58.00%～89.00% water and becomes water, mass ratio is that the ethyl butyrate of 7: 3 and the mixture 1.07%～13.59% of vitamin E are oil phase, described integrated peptidoglycan 3.00% adds in oil phase, biphasely be preheated to respectively 70 ℃, to after oil phase and water mixing, make thick breast through high-speed shearing emulsion machine at 6000rpm down cut 5min, add 6 times of the normal saline dilutions of 5 times of volumes, adopt again high pressure homogenizer homogenizing 6 times under the pressure of 70MPa, regulate pH to 6.5～6.8, flowing steam sterilization 15min, obtain microemulsion formulation.
Six of technical problem to be solved by this invention has been to provide the application of described microemulsion formulation in the irritated immunostimulant of the anti-beta lactoglobulin of preparation.
This research based on lactobacillus and suppress food anaphylaxis generation, development between be closely connected, by setting up BLG sensitized animal model, research gives Peptidoglycan microemulsion to the anaphylactoid intervention effect of sensitization animal pattern, after having illustrated that the Peptidoglycan microemulsion formulation oral administration of being made up of integrated peptidoglycan arrives in body, energy effective stimulus lymphocytic emiocytosis IL-12 and IFN-γ, reduce IL-4 level, reverse Th2 cell transition hyperfunction, in the irritated inflammation of the beta lactoglobulin that suppresses IgE mediation, brought into play important function.The aspect such as immunostimulant in exploitation with antiallergic performance has potential using value, simultaneously also for providing experimental basis for lactobacillus and thalline ingredients for preventing and treating food anaphylaxis thereof.
Compared with prior art, the invention has the advantages that: the lactobacillus integrated peptidoglycan micro emulsion composition of preparation, meet the microemulsion that body fluid can spontaneous formation Nano Particle, can obviously improve the dissolubility of medicine, increase medicine stability, improve the bioavailability of medicine; Lactobacillus integrated peptidoglycan preparation method is simple, stable in properties; The integrated peptidoglycan for preparing adopts microemulsion to make oral integrated peptidoglycan microemulsion as pharmaceutical carrier, and the aspect such as immunostimulant in exploitation with antiallergic performance has potential using value.
Accompanying drawing explanation
Fig. 1 is the sediments microscope inspection figure of Lactobacillus johnsonii;
Fig. 2 is the perspective Electronic Speculum figure of Lactobacillus johnsonii;
Fig. 3 Sekine extracts the perspective Electronic Speculum figure (A) of Peptidoglycan and the perspective Electronic Speculum figure (B) of improved method extraction Peptidoglycan of the present invention;
Fig. 4 is the impact of Peptidoglycan microemulsion on total IgE, BLG specific IgE and total IgG in BLG sensitized mice serum.1: blank microemulsion group; 2: sensitization group; 3: Peptidoglycan group; 4: Peptidoglycan microemulsion group
The specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
The preparation of embodiment 1 lactobacillus integrated peptidoglycan, integrated peptidoglycan microemulsion
1.1.1 experimental strain
Lactobacillus johnsonii KLDS1.7032 is obtained by screening in baby intestinal, Lactobacillus johnsonii KLDS1.7032, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered CGMCC 5050, and preservation date is on July 8th, 2011.
1.1.2 experiment reagent
Ketone, phenol, chloroform, glucose, cholesterol, lysozyme, Tris, sodium acetate, phenol, Triton X-100 in hydrochloric acid, concentrated sulphuric acid, phosphoric acid, sodium chloride, sodium hydroxide, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, dehydrated alcohol (food grade), ethyl butyrate, formaldehyde, methanol, acetone, hydrogen peroxide, perchloric acid, ascorbic acid, acetic acid, ether, acetic acid, SDS, trypsin, alkaline protease (being analytical pure, purchased from Dalian precious biological engineering company limited).Crystal violet, iodine liquid, husky yellow (being analytical pure, Sigma company).Tween 80, vitamin E (commercially available, food stage).(being analytical pure, purchased from Bo Erdi bio tech ltd, Nanjing) such as, glucosamine liquor-saturated to Dimethylaminobenzene first.Deionized water, laboratory self-control.
1.1.3 experimental apparatus
1.2 experimental techniques:
1.2.1 the extraction of lactobacillus integrated peptidoglycan
(1) cleaning of thalline and collection: after Lactobacillus johnsonii actication of culture is gone down to posterity, be inoculated in 1000mLMRS fluid medium 37 ℃ of static cultivation 18h by 3%; After cultivation, be cooled to rapidly 4 ℃.At 5000g, 4 ℃ of centrifugal 20min, collect bacterial sediment; 4 ℃ of distilled water cyclic washings, to thalline white, are collected thalline, are placed in 100 ℃ of boiling water water bath deactivation 30min, obtain the dead bacterium mud of thermic;
(2) dephosphorization Teichaic acid: the mass fraction that the dead bacterium mud of thermic step (1) Suo Shu is suspended in to 200mL preheating is that in 10% trichloroacetic acid solution, boiling water bath 20min is with cracking thalline; After cooling, 5000g, 4 ℃ of centrifugal 15min, collecting precipitation;
(3) defat: with concentration be 0.05mol/L acetic acid to regulate concentration be 0.02mol/L sodium acetate solution pH value to 4.6, after above-mentioned solution is mixed again with chloroform, methanol, three mixes for 4: 5: 10 by volume.The precipitation obtaining with mixed liquor dissolving step (2), volume ratio is 15: 1.Stirring at room temperature 18h, 8000g, 4 ℃ of centrifugal 20min, collecting precipitation.
(4) Deproteinization: the trypsin and alkaline protease phosphate buffer (3mg/mL trypsin and the 3mg/mL alkaline protease that add 15 times of precipitation volumes in the precipitation that above-mentioned steps (3) obtains; 0.1mol/L phosphate buffer; PH 8.0), 37 ℃ of water-bath vibration 12h, with 5000g, 4 ℃ of centrifugal 5min, precipitate with deionized water is washed 2 times.
(5) purify: the precipitation that step (3) obtains is through 0.01molL
-195 ℃ of processing 5min of sulfuric acid solution 10mL.Cooling immediately in ice-water bath, after 12000g, 4 ℃ of centrifugal 30min, abandon supernatant, precipitate with deionized water continuous dialysis 7d under 4 ℃ of conditions, rearmounted 4 ℃ of lyophilization saves backup.
1.2.2 the chemical identification of Peptidoglycan extract
(1) lactobacillus integrated peptidoglycan morphological examination: adopt microscope (key lab of the dairy science Ministry of Education of Northeast Agricultural University) and transmission electron microscope (Northeast Agricultural University's life center) to observe.
(2) lactobacillus integrated peptidoglycan chemical constituent is analyzed
I Morgan-Elson colorimetric method for determining aminohexose: get a certain amount of integrated peptidoglycan sample and use through 105 ℃ of 6mol/L hydrochloric acid hydrolysis 10hrs, be neutralized to pH7.0 with 2mol/L sodium carbonate.Get 10mL band plug scale test tube, add sample liquid 2mL, add 1mL second phthalein acetone reagent, shake up, boiling water bath 30min, taking-up frozen water is cooling, adds 2mL dehydrated alcohol, 1mL paradime thylaminobenzaldehyde reagent, shakes up, mend to 10mL with dehydrated alcohol, in 60 ℃ of water bath heat preservations 1 hour, cooling, in 30min, under 530nm, measure light absorption value.Take 100 μ g/mL glucosamine as standard solution drawing standard curve, calculate a content according to standard curve and sample concentration with method.
Ii egg micro-Kjeldahl is measured protein: adopt GB GB/T5009.5-850 to measure protein content.
The mensuration of iii neutral sugar: precision takes 105 ℃ of glucose 10mg that are dried to constant weight, puts in 100mL volumetric flask, dissolves and mixes with distilled water, and standardize solution becomes the standard solution of 100mg/mL.Get respectively in this standard solution 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL to 10mL test tube with ground stopper, add distilled water supply volume to 1mL as standard pipe, get distilled water 1mL as blank pipe, get and detect sample 1mL as detector tube, add 1.4mL 5% phenol to shake up to each pipe respectively, concentrated sulphuric acid 5mL shakes up, put 100 ℃ of water-bath 25min, detect each pipe absorbance at 490nm place, calculating and drawing standard curve are also extrapolated the sugared content in sample as follows: polyoses content (%)=C × D × V/W × 100
Wherein: C is concentration of glucose in sample (μ g/mL), the extension rate that D is sample, V is sample volume (mL), W is example weight (mg)
The mensuration of the total fat of iv:
Drawing standard curve: after cholesterol titer and ethanol are shaken up, get respectively 0.2mL, add concentrated sulphuric acid to mix rear 100 ℃ of water-bath 10min and make hydrolysis of lipid, after being cooled to room temperature, add developer, room temperature leaves standstill 20min, measures light absorption in 525nm place, get two pipe meansigma methodss, take standard cholesterol level as abscissa, absorbance value is vertical coordinate, drawing standard curve.
Total fat is measured: program is the same, records absorbance value, checks in total lipid content from standard curve.
The amino acid whose mensuration of v: assay method adopts standard GB/T/T14965-1994.Get a certain amount of Peptidoglycan extract, with 110 ℃ of vacuum hydrolysis 20hrs of 6mol/L hydrochloric acid, measure respectively aminoacid with the 835-50 of Hitachi type amino-acid analyzer and form.
Vi trace amounts of phosphorus method is surveyed nucleic acid: be phosphorous organic compound according to nucleic acid, its phosphorus content is 9.5%, therefore, can calculate by the content of organophosphor total phosphorus one Phos the content of nucleic acid.
(3) lysozyme solubility test
Take Peptidoglycan extract, be made into 1mg/mL solution with the phosphate buffer of pH6.2, add egg white lysozyme 200 μ g/mL, visible spectrophotometer is surveyed A
450after, 37 ℃, 120r/min shakes processing, surveys A respectively at different time
450.
(4) calculating of extraction lactobacillus integrated peptidoglycan yield
This test is extracted Peptidoglycan with producing mycopowder with three batches, calculates the yield of Sekine method and improved method extraction Lactobacillus johnsonii Peptidoglycan.
1.2.3 the discrimination method of the preparation of Peptidoglycan microemulsion and type thereof
The formation condition of Peptidoglycan microemulsion is studied in this experiment by pseudo-ternary phase diagram, and size to prepared Peptidoglycan microemulsion, distribution, preliminary physicochemical property, microemulsion form and system type etc. are studied.
(1) preparation of Peptidoglycan microemulsion: take tween 80 2.348g, dehydrated alcohol 9.392g, be scattered in 79g water and become water, take 4.382g ethyl butyrate, vitamin E 1.878g is mixed into solution as oil phase, embodiment 1.2.1 saves the integrated peptidoglycan 3g preparing and adds in oil phase, biphasely be preheated to respectively 70 ℃, to after oil phase and water mixing, make slightly breast of 100g through high-speed shearing emulsion machine at 6000rpm down cut 5min, add 6 times of the normal saline dilutions of 5 times of volumes, adopt again high pressure homogenizer homogenizing 6 times under the pressure of 70MPa, regulate pH to 6.5～6.8, flowing steam sterilization 15min, obtain microemulsion formulation.
(2) discrimination method of microemulsion
Character: the transparent or solution of opalescence slightly, free of birefringence phenomenon under polarizing microscope.
Centrifuging: adopt 10000r/min centrifugal 15min, observe its whether layering and whether remain clear and bright, as still maintained the clear and bright microemulsion that is judged to.
Staining: utilize oil-soluble dyes tonyred and water-soluble dye serge blue red or blue diffusion speed in microemulsion to judge the type of microemulsion, if red diffusion is w/o type microemulsion in blueness fast; Otherwise be O/W type.
Index of refraction: by 20 ℃ of mensuration of document specifies method constant temperature
Particle diameter: take a morsel and leave standstill the microemulsion sample of 1 week, be diluted to sample moisture content with deionized water and be about 98%, use vortex mixer mix homogeneously, again through 0.22 μ m filtering with microporous membrane, cross the sample cell of the sample injection nano particle size distribution instrument after film, design temperature is the index of refraction of 25 ℃ and various oil phases, carries out particle size determination, each sample parallel assay 3 times.
1.3 experimental result
1.3.1 Peptidoglycan visual examination:
Gained Lactobacillus peptidoglycan is that milky outward appearance is flocculence distribution, is evenly distributed sedimentation easily occurs in water.Sediments microscope inspection figure as shown in Figure 1, perspective Electronic Speculum figure as shown in Figure 2, this test is by groping extraction process, contrast Sekine extracts Peptidoglycan method, determine the improved method of optimum extraction Lactobacillus johnsonii Peptidoglycan, due to mild condition, and in whole purge process, do not use any physics and chemical method to destroy the framing structure of cell wall, therefore separate and obtained the complete Peptidoglycan of cytoskeleton, the cytoskeleton extracting by transmission electron microscope observing presents bag shaped structure, as shown in Figure 3.
1.3.2 the main component of Lactobacillus johnsonii Peptidoglycan
This experiment utilizes the existing improved method of groping to extract Lactobacillus johnsonii Peptidoglycan, and its main component is protein and neutral sugar, and content is respectively 55% and 40%, is secondly fat, aminohexose, nucleic acid.Tradition Sekine method is extracted Lactobacillus johnsonii Peptidoglycan, and its main component is protein and neutral sugar, and content is respectively 58% and 42%, is secondly fat, aminohexose, nucleic acid.The improved method of surveying extract Lactobacillus johnsonii Peptidoglycan every composition and compare with traditional Sekine method extraction, its quality does not reduce.
1.3.3 the extraction yield of Lactobacillus johnsonii Peptidoglycan
The main component that confirms the extract of purification procedures gained by lysozyme solubility test is strictly Peptidoglycan.Extract Peptidoglycan by the Lactobacillus johnsonii mycopowder by three batches of different batches suitability for industrialized production, adopt relatively improved method and Sekine method extraction integrated peptidoglycan yield of mass ratio (whole WPG quality/starter bacteria opaque amount).Result shows that the yield of improved method is 18%, and traditional Sekine method only can obtain 3% extraction ratio.
1.3.4 the discriminating of the preparation of Peptidoglycan microemulsion and type thereof
Selecting food stage tween 80 is surfactant, and selecting the mixture of ethyl butyrate and vitamin E is oil phase.Employing ethanol is cosurfactant.Investigate acquired results by phasor, weigh in proportion a certain amount of Peptidoglycan, surfactant, cosurfactant and oil phase mix homogeneously, drip distilled water, stirring and evenly mixing obtains the translucent product emulsion of liquid-drop diameter at 30～45nm.
Obtain Peptidoglycan microemulsion after centrifugal, solution is all without layering.Orange, transparent, the visible opalescence of outward appearance, there is certain fluidity.
The diffusion velocity of this experimental result demonstration methylene blue in microemulsion, obviously faster than Sudan IV, can judge that Peptidoglycan microemulsion is as O/w type microemulsion.
The Peptidoglycan microemulsion of 3 batches of preparations is placed 3 months in 37 ℃ of baking ovens, observed the indexs such as character, layering, assay and show that indices is without significant change, the having good stability of said preparation.Under transmission electron microscope, be spheroidal, be evenly distributed, mean diameter is (39.6 ± 3.3) nm.
The oral integrated peptidoglycan microemulsion of embodiment 2 is to the anaphylactoid intervention effect of sensitization animal pattern
The integrated peptidoglycan microemulsion that embodiment 1 is prepared and Peptidoglycan are for following experimentation:
2.1 experiment materials:
2.1.1 laboratory animal
Female clean level BALB/c mouse, 6～8 week age, purchased from Harbin tumour hospital Experimental Animal Center.Feeding environment temperature (23 ± 2) ℃, humidity 50%～75%, standard mouse feed is fed.
2.1.2 main agents
The Peptidoglycan extract that embodiment 1 prepares, the Peptidoglycan microemulsion that embodiment 1 prepares, IL-12, IFN-γ, IL-4ELISA test kit (R & D company of the U.S.), total IgE test kit (bethyl company of the U.S.), sheep anti mouse IgE ELIAS secondary antibody (AbD Serotec company of Britain), sheep anti-mouse igg ELIAS secondary antibody (Chinese Zhong Shan Golden Bridge company), RPMI1640 culture fluid (GIBCO company of the U.S.), hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological engineering material company), lymphocyte separation medium (Salarbio company), Lac Bovis seu Bubali beta lactoglobulin mark product and Freund's complete adjuvant (Sigma company) etc.
2.1.3 instrument and equipment
2.2 experimental technique
2.2.1 gavage the preparation of integrated peptidoglycan and the microemulsion thereof of use
Gavage the preparation of the integrated peptidoglycan of use: take the integrated peptidoglycan 4g that in embodiment 1,1.2.1 prepares, add 20g physiological saline solution (containing the sodium-chloride water solution of 0.9% (W/V)) to dissolve, dilute 6 times of uses, make suspension.
Gavage the preparation of the integrated peptidoglycan microemulsion of use: get the integrated peptidoglycan microemulsion preparing of 1.2.3 in embodiment 1 and gavage use.
Gavage the preparation with blank microemulsion: take tween 80 2.348g, dehydrated alcohol 9.392g, be scattered in 82g water and become water, take 4.382g ethyl butyrate, vitamin E 1.878g is mixed into solution as oil phase, biphasely be preheated to respectively 70 ℃, to after oil phase and water mixing, make thick newborn 100g through high-speed shearing emulsion machine at 6000rpm down cut 5min, add 6 times of the normal saline dilutions of 5 times of volumes, adopt again high pressure homogenizer homogenizing 6 times under the pressure of 70MPa, regulate pH to 6.5～6.8, flowing steam sterilization 15min, obtains blank microemulsion formulation.
2.2.2 Animal Model:
Mice is bought rear adaptability and feeds 4 days, random packet (8 every group), blank group, sensitization group, Peptidoglycan group and Peptidoglycan microemulsion group.From the 1st day, Peptidoglycan group and Peptidoglycan microemulsion group mice only gavaged respectively integrated peptidoglycan and microemulsion 0.1mL/ thereof, and 3 times weekly, totally 4 weeks, blank group gavaged with the blank microemulsion of equivalent simultaneously.Sensitization group, Peptidoglycan group and Peptidoglycan microemulsion group mice gave the anaphylactogen (1mL Freund adjuvant+1mL 1mg/mL BLG) of lumbar injection 0.1mL 0.5mg/mL at the 7th, 21,28 days, blank group mouse peritoneal injection equivalent normal saline.In the 30th day of experiment, after last lumbar injection 48h, detect index.
2.2.3ELISA method detects cytokine
Asepticly get each group of mouse spleen, after grinding on 200 eye mesh screens with glass nook closing member, join in RPMI1640 culture fluid and make single cell suspension.Add lymphocyte separation medium to carry out centrifugal, isolate lymphocyte, then with RPMI1640 culture fluid washing (4000r/min) 2 times.Adjusting cell density is 2 × 106/mL.Cell is joined in 96 well culture plates with the RPMI1640 culture medium and the BLG (final concentration 1g/L) that contain 100mL/L calf serum, and every hole cumulative volume 200 μ L, establish 3 repetitions (n=3) for every group.At 37 ℃, under 50mL/LCO2 incubator saturated humidity condition, cultivate after 48h centrifugal collection supernatant.Operate in strict accordance with each ELISA test kit description, measure absorbance (A) value in each hole at wavelength 450nm place by microplate reader, check in IL-12, IFN-γ, the IL-4 concentration of each sample from corresponding standard curve.
2.2.4ELISA method detects antibody horizontal
Each group mice is extractd eyeball blood-letting, and centrifugal rear absorption serum detects total IgE, BLG specific IgE and total IgG concentration in mice serum in strict accordance with each antibody kit description.
2.3 experimental result
2.3.1 the impact of integrated peptidoglycan microemulsion on sensitized mice Th1/Th2 cell balance
Because the ratio of Th1, Th2 cell is closely related with the secretion level of IFN-γ and IL-4, probiotic bacteria Peptidoglycan is by correcting the unbalance inflammatory reaction of alleviating due to allergy of Th1/Th2 cell, therefore the impact of lactobacillus peptide polysaccharide microemulsion on lymphocyte Th1/Th2 balance studied in this experiment take IFN-γ/IL-4 ratio as representative.As shown in table 1, compared with sensitization group, the IL-4 mass concentration of Peptidoglycan microemulsion group mice significantly decline (P < 0.05), and IFN-γ secretion value obviously increases (P < 0.05), its IFN-γ/IL-4 ratio is significantly higher than blank microemulsion group and sensitization group (P < 0.05).
The affect pg/mL of table 1 Peptidoglycan microemulsion on BLG sensitized mice Th1/Th2 emiocytosis,
n=3)
One factor analysis of variance, significant difference:
ap < 0.05vs sensitization group;
bthe blank group of P < 0.05vs
2.3.2 integrated peptidoglycan microemulsion is on the antibody secreted impact of sensitized mice
As shown in Figure 2, BLG can the total IgE of inducing mouse, BLG specific IgE and total IgG level raise, and is significantly higher than blank group (P < 0.05).With after Peptidoglycan microemulsion intervention sensitized mice, can effectively suppress the secretion (P < 0.05) of total IgE, BLG specific IgE and total IgG, and not remarkable (P > 0.05) of difference compared with blank group, result as shown in Figure 4.
Peptidoglycan composite micro-emulsion is compared with its suspension, and anti-allergic effects strengthens, and may be because microemulsion can increase dissolving and the absorption of Peptidoglycan component, and microemulsion particle diameter is little, specific surface area is large, absorption affinity is strong.Emulsifying agent is wherein the amphiphatic molecule with hydrophilic group and hydrophobic group, very similar with biomembranous mechanism, the microemulsion that is adsorbed on cell surface is easy to merge with biomembrane, be that microemulsion assists it to stride across cell membrane as pharmaceutical carrier, drug concentration raises relatively, greatly improved the utilization rate of Peptidoglycan component, thereby action effect obviously increases.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (4)
1. prepare a method for lactobacillus integrated peptidoglycan, it is characterized in that comprising the following steps:
(1) cleaning of thalline and collection: after Lactobacillus johnsonii KLDS1.7032 actication of culture is gone down to posterity, be inoculated in MRS fluid medium, 37 ℃ of static cultivation 18h, are cooled to rapidly 4 ℃ after cultivation; At 5000g, 4 ℃ of centrifugal 20min, collect bacterial sediment; 4 ℃ of distilled water cyclic washings, to thalline white, are collected thalline, are placed in 100 ℃ of boiling water water bath deactivation 30min, obtain the dead bacterium mud of thermic;
Described Lactobacillus johnsonii KLDS1.7032, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered: CGMCC5050;
(2) dephosphorization Teichaic acid: the mass fraction that the dead bacterium mud of thermic step (1) Suo Shu is suspended in to preheating is that in 10% trichloroacetic acid solution, boiling water bath 20min is with cracking thalline; After cooling, 5000g, 4 ℃ of centrifugal 15min, collecting precipitation;
(3) defat: concentration is 0.02mol/L, the sodium acetate solution that pH value is 4.6, with chloroform, methanol, three by volume 4 ︰ 5 ︰ 10 mixes; The precipitation obtaining with mixed liquor dissolving step (2), the volume of described mixed liquor is 15 ︰ 1 with precipitation volume ratio; Stirring at room temperature 18h, 8000g, 4 ℃ of centrifugal 20min, collecting precipitation;
(4) Deproteinization: the 0.1mol/L phosphate buffer that adds the pH8.0 that contains 3mg/mL trypsin and 3mg/mL alkaline protease of 15 times of precipitation volumes in the precipitation that above-mentioned steps (3) obtains, 37 ℃ of water-bath vibration 12h, with 5000g, 4 ℃ of centrifugal 5min, precipitate with deionized water is washed 2 times;
(5) purify: the precipitation that step (4) obtains is through 0.01molL
-185～95 ℃ of sulfuric acid solutions are processed 5min, cooling immediately in ice-water bath, after 12000g, 4 ℃ of centrifugal 30min, abandon supernatant, and precipitate with deionized water is dialysis 7 days continuously under 4 ℃ of conditions, and rearmounted 4 ℃ of lyophilization saves backup.
2. the lactobacillus integrated peptidoglycan preparing according to preparation method claimed in claim 1.
3. the microemulsion formulation of an anti-beta lactoglobulin allergy, it is characterized in that being prepared by following methods: according to weight fraction meter, the tween 80 that is 2: 8 by mass ratio is scattered in 58.00%～89.00% water and becomes water with the mixture 2.10%～25.42% of dehydrated alcohol composition, mass ratio is that the ethyl butyrate of 7: 3 and the mixture 1.07%～13.59% of vitamin E are oil phase, integrated peptidoglycan claimed in claim 2 3.00% adds in oil phase, biphasely be preheated to respectively 70 ℃, to after oil phase and water mixing, make thick breast through high-speed shearing emulsion machine at 6000rpm down cut 5min, add 6 times of the normal saline dilutions of 5 times of volumes, adopt again high pressure homogenizer homogenizing 6 times under the pressure of 70MPa, regulate pH to 6.5～6.8, flowing steam sterilization 15min, obtain microemulsion formulation.
4. the application of microemulsion formulation claimed in claim 3 in the irritated immunostimulant of the anti-beta lactoglobulin of preparation.
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| CN104987424A (en) * | 2014-12-31 | 2015-10-21 | 临沂格瑞食品有限公司 | Milk polysaccharide and preparation method thereof |
| CN106243238A (en) * | 2016-09-07 | 2016-12-21 | 辽宁大学 | A kind of method extracting probiotic cell wall skeleton polysaccharide |
| CN108396042B (en) * | 2018-03-05 | 2021-10-26 | 郑州安图生物工程股份有限公司 | Preparation method of small-molecule soluble peptidoglycan |
| CN115624183B (en) * | 2022-09-30 | 2024-04-09 | 浙江李子园食品股份有限公司 | Histidine preparation for relieving cow milk allergy and application thereof |
| CN115669542B (en) * | 2022-11-04 | 2023-08-25 | 西北农林科技大学 | Plant tissue culture detoxification method |
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