CN102335413A - 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 method for preparing and application of Peptidoglycan; Be particularly related to a kind of method for preparing of integrated peptidoglycan of lactobacillus; Also relate to the microemulsion formulation that is prepared into by this integrated peptidoglycan, its preparation method and the application in the anti-beta lactoglobulin of preparation immunostimulant hypersensitive 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 that according to external Epidemiological study 2%～6% child produces anaphylaxis to Lac Bovis seu Bubali, have a strong impact on it and absorb guaranteed milk is proteic.(β-lactglobulin) is regarded as one of topmost milk allergen albumen to beta lactoglobulin; Beta lactoglobulin has the function of similar immunoglobulin to calf; But for the baby main anaphylactogen; Cause baby's anaphylaxis easily, beta lactoglobulin mainly brings out the I type allergy of specific IgE antibody mediation, can cause vomiting, suffer from abdominal pain and gastrointestinal upset such as diarrhoea.And the irritated gastroenteropathy (like infant colitis) that causes often causes growthretardation.How to reduce due to the beta lactoglobulin milk allergy become modern milk product produce in problem anxious to be solved.
The applicant obtained certain achievement in research aspect the unbalance alleviation beta lactoglobulin allergy of probiotic bacteria adjusting Th1/Th2 over 3 years; Screen the Lactobacillus johnsonii KLDS1.7032 that a strain has better curative effect 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, and its whole cell peptidoglycan is to suppress main component hypersensitive.
At present, more to the physiological function of integrated peptidoglycan and functional mechanism research, and less to the research of its product preparation method.Existing whole cell peptidoglycan adopts the injection system administration basically, exists oral administration biaavailability to hang down and reaches the problem that too high dose can cause untoward reaction.And microemulsion is the drug release carrier of a new type ideal.Have transparently, stable absorption is perfect, characteristics such as targeting drug release, and improved curative effect of medication, reduce toxic and side effects.Clinical value improves day by day, has very big development prospect.Therefore, the present invention is prepared into microemulsion formulation with the lactobacillus integrated peptidoglycan that extracts, after its oral administration arrives in the body; Can spontaneous formation particle diameter less than the O/W microemulsion of 100nm; Because medicine is scattered in the microemulsion with molecular forms, can obviously improve the release degree of medicine, stability increases; Oral administration biaavailability is 3～4 times of ordinary preparation, for the suitability for industrialized production of novel immunostimulant has been established the certain theory 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 anti-beta lactoglobulin of preparation immunostimulant hypersensitive; Particularly be applied to prepare in the beta lactoglobulin immunostimulant hypersensitive of anti-IgE mediation.
Described immunostimulant can be further used in anti-beta lactoglobulin medicine hypersensitive or the food preparation.
Preferably, described lactobacillus integrated peptidoglycan extracts from about formula lactobacillus KLDS1.7032.
Described Lactobacillus johnsonii KLDS1.7032; Be lactobacillus (Lactobacillus johnsonii); Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, address No. 3, Institute of Microorganism, Academia Sinica in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Its culture presevation is numbered: CGMCCNO.5050, preservation date are on July 8th, 2011.
Two of technical problem to be solved by this invention has provided a kind of method for preparing the lactobacillus integrated peptidoglycan, and concrete may further comprise the steps:
(1) cleaning of thalline and collection: after the Lactobacillus johnsonii actication of culture gone down to posterity, be inoculated in the MRS fluid medium 37 ℃ of static cultivation 18h; After finishing, cultivation is cooled to 4 ℃ rapidly.At 5000g, 4 ℃ of centrifugal 20min, collect bacterial sediment; 4 ℃ of distilled water cyclic washing to thalline whites are collected thalline, place 100 ℃ of boiling water water bath deactivation 30min, obtain the dead bacterium mud of thermic;
(2) remove LTA: the mass fraction that the dead bacterium mud of the described thermic of step (1) is suspended in preheating is that boiling water bath 20min is with the cracking thalline in the 10% trichloroacetic acid solution; After the cooling, 5000g, 4 ℃ of centrifugal 15min, collecting precipitation;
(3) defat: concentration is 0.02mol/L, and pH value is 4.6 sodium acetate solution, and with chloroform, methanol, the three mixed in 4: 5: 10 by volume; With the deposition that mixed liquor dissolving step (2) obtains, the volume ratio of said mixed liquor is 15: 1 with the precipitation volume ratio; Stirring at room 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 deposition 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 deposition that step (4) obtains is through 0.01molL
-1Sulfuric acid solution is handled 5min for 85～95 ℃.Cooling is immediately abandoned supernatant behind 12000g, 4 ℃ of centrifugal 30min in the ice-water bath, and precipitate with deionized water was dialysed 7 days under 4 ℃ of conditions continuously, and the rearmounted 4 ℃ of preservations of lyophilization are subsequent use.
Three of technical problem to be solved by this invention has provided a kind of integrated peptidoglycan for preparing according to above-described method for preparing.
Four of technical problem to be solved by this invention has provided a kind of anti-beta lactoglobulin microemulsion formulation hypersensitive, 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 a tween 80, and described cosurfactant is a dehydrated alcohol, and described oil phase is the mixture of n-butyric acie ethyl ester and vitamin E.
Preferred; Described microemulsion formulation comprises each material of following weight fraction: more than arbitrary described integrated peptidoglycan 3.00%; Mass ratio is 7: 3 the n-butyric acie ethyl ester and the mixture 1.07%～13.59% of vitamin E composition; Mass ratio is 2: 8 the tween 80 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 provided the method for preparing described anti-beta lactoglobulin microemulsion formulation hypersensitive; It is characterized in that preparing: be that 2: 8 tween 80 is scattered in mixture 2.10%～25.42% that dehydrated alcohol is formed and becomes water in 58.00%～89.00% water with mass ratio by following method; Mass ratio is that 7: 3 the n-butyric acie ethyl ester and the mixture 1.07%～13.59% of vitamin E are oil phase; Described integrated peptidoglycan 3.00% adds in the oil phase; Biphasely be preheated to 70 ℃ respectively, oil phase and water are mixed after the high speed shear mulser makes thick breast at 6000rpm down cut 5min, add 6 times of the normal saline dilutions of 5 times of volumes; Adopt high pressure homogenizer homogenizing 6 times under the pressure of 70MPa again; Regulate pH to 6.5～6.8, flowing steam sterilization 15min promptly gets microemulsion formulation.
Six of technical problem to be solved by this invention has provided the application of described microemulsion formulation in the irritated immunostimulant of the anti-beta lactoglobulin of preparation.
This research is based on lactobacillus and suppress being closely connected between the generation, development of food anaphylaxis; Through setting up BLG sensitized animal model; Research gives the Peptidoglycan microemulsion to the anaphylactoid intervention effect of sensitization animal pattern; After having explained that the Peptidoglycan microemulsion formulation oral administration of being processed by integrated peptidoglycan arrives in the body, ability effective stimulus lymphocytic emiocytosis IL-12 and IFN-γ reduce the IL-4 level; It is hyperfunction to reverse the Th2 cell transition, in the irritated inflammation of the beta lactoglobulin that suppresses the IgE mediation, has brought into play important function.The aspects such as immunostimulant that have the antiallergic performance in exploitation have potential using value, simultaneously also for for lactobacillus and thalline ingredients for preventing and treating food anaphylaxis thereof experimental basis being provided.
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 nanoscale particle diameter, can obviously improve the dissolubility of medicine, increase medicine stability, improve bioavailability of medicament; Lactobacillus integrated peptidoglycan method for preparing is simple, stable in properties; The integrated peptidoglycan for preparing adopts microemulsion to process oral integrated peptidoglycan microemulsion as pharmaceutical carrier, and the aspects such as immunostimulant that have the antiallergic performance in exploitation have potential using value.
Description of drawings
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 influence of Peptidoglycan microemulsion to total IgE, BLG specific IgE and total IgG in the BLG sensitized mice serum.1: blank microemulsion group; 2: the sensitization group; 3: the Peptidoglycan group; 4: Peptidoglycan microemulsion group
The specific embodiment
Below in conjunction with specific embodiment the present invention is further specified, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present 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 the baby intestinal; Lactobacillus johnsonii KLDS1.7032; Be deposited in China Committee for Culture Collection of Microorganisms 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), n-butyric acie ethyl ester, formaldehyde, methanol, acetone, hydrogen peroxide, perchloric acid, ascorbic acid, acetic acid, ether, the acetic acid; SDS; Trypsin, alkaline protease (being analytical pure) available from the precious biological engineering company limited in Dalian.Crystal violet, iodine liquid, husky yellow (being analytical pure, Sigma company).Tween 80, vitamin E (commercially available, food stage).(being analytical pure) such as, glucosamine liquor-saturated available from Nanjing Bo Erdi bio tech ltd to the Dimethylaminobenzene first.Deionized water, the laboratory self-control.
1.1.3 experimental apparatus
1.2 experimental technique:
1.2.1 the extraction of lactobacillus integrated peptidoglycan
(1) cleaning of thalline and collection: after the Lactobacillus johnsonii actication of culture gone down to posterity, be inoculated in the 1000mLMRS fluid medium 37 ℃ of static cultivation 18h by 3%; After finishing, cultivation is cooled to 4 ℃ rapidly.At 5000g, 4 ℃ of centrifugal 20min, collect bacterial sediment; 4 ℃ of distilled water cyclic washing to thalline whites are collected thalline, place 100 ℃ of boiling water water bath deactivation 30min, obtain the dead bacterium mud of thermic;
(2) remove LTA: the mass fraction that the dead bacterium mud of the described thermic of step (1) is suspended in the 200mL preheating is that boiling water bath 20min is with the cracking thalline in the 10% trichloroacetic acid solution; After the cooling, 5000g, 4 ℃ of centrifugal 15min, collecting precipitation;
(3) defat: use concentration to regulate concentration and be 0.02mol/L sodium acetate solution pH value to 4.6 as 0.05mol/L acetic acid, after above-mentioned solution is mixed again with chloroform, methanol, the three mixed in 4: 5: 10 by volume.With the deposition that mixed liquor dissolving step (2) obtains, volume ratio is 15: 1.Stirring at room 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 deposition 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 deposition that step (3) obtains is through 0.01molL
-1Sulfuric acid solution 10mL handles 5min for 95 ℃.Cooling is immediately abandoned supernatant behind 12000g, 4 ℃ of centrifugal 30min in the ice-water bath, and precipitate with deionized water is continuous dialysis 7d under 4 ℃ of conditions, and the rearmounted 4 ℃ of preservations of lyophilization are subsequent use.
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 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, be neutralized to pH7.0 with the 2mol/L sodium carbonate through 105 ℃ of hydrolysis 10hrs of 6mol/L hydrochloric acid.Get 10mL band plug scale test tube, add appearance liquid 2mL, add 1mL second phthalein acetone reagent; Shake up, boiling water bath 30min takes out the frozen water cooling; Add 2mL dehydrated alcohol, 1mL paradime thylaminobenzaldehyde reagent, shake up, mend to 10mL with dehydrated alcohol; In 60 ℃ of water bath heat preservations 1 hour, cooling, mensuration light absorption value 530nm under in 30min.Is standard solution drawing standard curve with method with 100 μ g/mL glucosamine, calculates a content according to standard curve and sample concentration.
Ii egg trace Kjeldahl is measured protein: adopt GB GB/T5009.5-850 to measure protein content.
The mensuration of iii neutral sugar: precision takes by weighing 105 ℃ of glucose 10mg that are dried to constant weight, puts in the 100mL volumetric flask, uses the dissolved in distilled water mixing, 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 the 10mL test tube with ground stopper; Add distilled water supply volume to 1mL as standard pipe; Get distilled water 1mL as the blank pipe, get test sample 1mL, add 1.4mL 5% phenol to each pipe respectively and shake up as detector tube; Concentrated sulphuric acid 5mL shakes up; Put 100 ℃ of water-bath 25min, detect at the 490nm place and respectively manage absorbance, calculate and drawing standard curve and go out the sugared content in the sample: polyoses content (%)=C * D * V/W * 100 by following formula to calculating
Wherein: C is concentration of glucose in the sample (μ g/mL), and D is the dilution of sample multiple, and V is sample volume (mL), and W is example weight (mg)
The mensuration of the total fat of iv:
The drawing standard curve: after cholesterol titer and ethanol shaken up, get 0.2mL respectively, add the concentrated sulphuric acid mixing after 100 ℃ of water-bath 10min make hydrolysis of lipid; Add developer after being cooled to room temperature, room temperature leaves standstill 20min, measures light absorption in the 525nm place; Get two pipe meansigma methodss; With the standard cholesterol level is abscissa, and absorbance value is a vertical coordinate, the 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/T14965-1994.Get a certain amount of Peptidoglycan extract,, measure aminoacid respectively with the 835-50 of Hitachi type amino-acid analyzer and form with 110 ℃ of vacuum hydrolysis 20hrs of 6mol/L hydrochloric acid.
Vi trace amounts of phosphorus method is surveyed nucleic acid: according to nucleic acid is phosphorated organic compound, and its phosphorus content is 9.5%, therefore, can calculate the content of nucleic acid through the content of organophosphor total phosphorus one Phos.
(3) lysozyme solubility test
Take by weighing the 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 that Sekine method and improved method extract the Lactobacillus johnsonii Peptidoglycan.
1.2.3 the discrimination method of preparation of Peptidoglycan microemulsion and type thereof
The formation condition of pseudo-ternary phase diagram method research Peptidoglycan microemulsion is passed through in this experiment, and size, distribution, preliminary physicochemical property, microemulsion form and the system type etc. of prepared Peptidoglycan microemulsion are studied.
(1) preparation of Peptidoglycan microemulsion: take by weighing tween 80 2.348g, dehydrated alcohol 9.392g is scattered in the 79g water and becomes water; Take by weighing 4.382g n-butyric acie ethyl ester, vitamin E 1.878g is mixed into solution as oil phase, and the integrated peptidoglycan 3g that embodiment 1.2.1 restraining obtains fully adds in the oil phase; Biphasely be preheated to 70 ℃ respectively, with oil phase with water mixes after the high speed shear mulser makes slightly breast of 100g at 6000rpm down cut 5min, add 6 times of the normal saline dilutions of 5 times of volumes; Adopt high pressure homogenizer homogenizing 6 times under the pressure of 70MPa again; Regulate pH to 6.5～6.8, flowing steam sterilization 15min promptly gets microemulsion formulation.
(2) discrimination method of microemulsion
Character: transparent or have a little opalescent solution, free of birefringence phenomenon under the polarizing microscope.
Centrifuging: adopt the centrifugal 15min of 10000r/min, observe its whether layering and whether keep clear and bright, as still keeping 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 then is the w/o type microemulsion in blueness fast; Otherwise be the 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 in 1 week; Be diluted to sample moisture content with deionized water and be about 98%, use the vortex mixer mix homogeneously, again through 0.22 μ m filtering with microporous membrane; Cross the sample cell that sample behind the film injects the nano particle size distribution instrument; Design temperature is 25 ℃ of indexs of refraction with 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 distributes for the milky outward appearance is flocculence, and in water, being evenly distributed is prone to take place sedimentation.Sediments microscope inspection figure is as shown in Figure 1; Electronic Speculum figure is as shown in Figure 2 for perspective, and this test is through groping contrast Sekine extraction Peptidoglycan method to extraction process; Confirmed the improved method of optimum extraction Lactobacillus johnsonii Peptidoglycan; Since mild condition, and do not use any physics and chemical method to destroy the framing structure of cell wall in the whole purge process, therefore separate having obtained the complete Peptidoglycan of cytoskeleton; Present bag shaped structure through the cytoskeleton that transmission electron microscope observing extracted, 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 the Lactobacillus johnsonii Peptidoglycan, and its main component is protein and neutral sugar, and content is respectively 55% and 40%, is fat, aminohexose, nucleic acid secondly.Tradition Sekine method is extracted the Lactobacillus johnsonii Peptidoglycan, and its main component is protein and neutral sugar, and content is respectively 58% and 42%, is fat, aminohexose, nucleic acid secondly.The improved method of surveying extract Lactobacillus johnsonii Peptidoglycan each item composition and traditional Sekine method extract phase relatively, 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 through the lysozyme solubility test is strictly Peptidoglycan.Lactobacillus johnsonii mycopowder through with three batches of different batches suitability for industrialized production extracts Peptidoglycan, adopts mass ratio (whole WPG quality/starter bacteria opaque amount) relatively improved method and Sekine method extraction integrated peptidoglycan yield.The 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 preparation of Peptidoglycan microemulsion and type thereof
Selecting the food stage tween 80 is surfactant, and selecting the mixture of n-butyric acie ethyl ester and vitamin E is oil phase.Employing ethanol is cosurfactant.Investigate the gained result by phasor, weigh a certain amount of Peptidoglycan, surfactant, cosurfactant and oil phase mix homogeneously in proportion, drip distilled water, stirring and evenly mixing promptly gets the translucent product emulsion of liquid-drop diameter at 30～45nm.
Obtain the Peptidoglycan microemulsion after centrifugal, solution does not all have layering.Outward appearance is orange, transparent, visible opalescence, have certain flowability.
The diffusion velocity of this experimental result demonstration methylene blue in microemulsion can judge that obviously faster than Sudan IV the Peptidoglycan microemulsion is an O/w type microemulsion.
The Peptidoglycan microemulsion of 3 batches of preparations was placed 3 months in 37 ℃ of baking ovens, observed indexs such as character, layering, assay and show that each item index do not have significant change, the having good stability of said preparation.Be spheroidal under the transmission electron microscope, be evenly distributed, mean diameter is (39.6 ± 3.3) nm.
Integrated peptidoglycan microemulsion and Peptidoglycan that embodiment 1 is prepared are used for following experimentation:
2.1 experiment material:
2.1.1 laboratory animal
Female cleaning level BALB/c mouse, 6～8 ages in week are available from Harbin tumour hospital Experimental Animal Center.Feeding environment temperature (23 ± 2) ℃, humidity 50%～75%, the 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 (U.S. R&D company); Total IgE test kit (U.S. bethyl company); Sheep anti mouse IgE ELIAS secondary antibody (Britain AbD Serotec company); Sheep anti-mouse igg ELIAS secondary antibody (company of China fir Golden Bridge in the China); RPMI1640 culture fluid (U.S. GIBCO company); Hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological engineering material company); Lymphocyte separation medium (Salarbio company); Lac Bovis seu Bubali beta lactoglobulin mark article and Freund's complete adjuvant (Sigma company) etc.
2.1.3 instrument and equipment
2.2 experimental technique
2.2.1 the integrated peptidoglycan that filling is taken and the preparation of microemulsion thereof
The preparation of the integrated peptidoglycan that filling is taken: take by weighing the integrated peptidoglycan 4g that 1.2.1 prepares among the embodiment 1, add 20g physiological saline solution (sodium-chloride water solution that contains 0.9% (W/V)) dissolving, promptly dilute 6 times of uses, make suspension.
The preparation of the integrated peptidoglycan microemulsion that filling is taken: the integrated peptidoglycan microemulsion for preparing of getting 1.2.3 among the embodiment 1 is irritated the clothes use.
The preparation of blank microemulsion is taken in filling: take by weighing tween 80 2.348g, dehydrated alcohol 9.392g is scattered in the 82g water and becomes water; Take by weighing 4.382g n-butyric acie ethyl ester, vitamin E 1.878g is mixed into solution as oil phase, biphasely is preheated to 70 ℃ respectively; Oil phase and water are mixed after the high speed shear mulser makes thick newborn 100g at 6000rpm down cut 5min; High pressure homogenizer homogenizing 6 times under the pressure of 70MPa is adopted in 6 times of normal saline dilutions that add 5 times of volumes again, regulates pH to 6.5～6.8; Flowing steam sterilization 15min promptly gets blank microemulsion formulation.
2.2.2 animal model is set up:
Mice is bought the back adaptability and fed random packet (8 every group), i.e. blank control group, sensitization group, Peptidoglycan group and Peptidoglycan microemulsion group 4 days.From the 1st day, Peptidoglycan group and Peptidoglycan microemulsion group mice were only irritated clothes integrated peptidoglycan and microemulsion 0.1mL/ thereof respectively, and 3 times weekly, in totally 4 weeks, blank control group is irritated clothes 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 day, blank control group mouse peritoneal injection equivalent normal saline.In the 30th day of experiment, promptly detect index behind the last lumbar injection 48h.
2.2.3ELISA method detects cytokine
Aseptic getting respectively organized mouse spleen,, after grinding on 200 eye mesh screens, joins in the RPMI1640 culture fluid and processes single cell suspension with the glass nook closing member.The adding lymphocyte separation medium carries out centrifugal, isolates lymphocyte, reuse RPMI1640 culture fluid washing (4000r/min) 2 times.Transferring 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 the 100mL/L calf serum, and cumulative volume 200 μ L in every hole establish 3 repetitions (n=3) for every group.At 37 ℃, cultivate 48h under the 50mL/LCO2 incubator saturated humidity condition after, centrifugal collection supernatant.Operate in strict accordance with each ELISA test kit description, with absorbance (A) value in each hole of ELIASA mensuration, check in IL-12, IFN-γ, the IL-4 concentration of each sample from corresponding standard curve at wavelength 450nm place.
2.2.4ELISA method detects antibody horizontal
Each is organized mice and extracts the eyeball blood-letting, and serum is drawn in centrifugal back, detects total IgE, BLG specific IgE and total IgG concentration in the mice serum in strict accordance with each antibody kit description.
2.3 experimental result
2.3.1 the integrated peptidoglycan microemulsion is to the influence of 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; The probiotic bacteria Peptidoglycan is through correcting the unbalance inflammatory reaction of alleviating due to the allergy of Th1/Th2 cell, so this experiment is that representative is studied lactobacillus peptide polysaccharide microemulsion to the equilibrated influence of lymphocyte Th1/Th2 with IFN-γ/IL-4 ratio.As shown in table 1, to compare with the sensitization group, the IL-4 mass concentration of Peptidoglycan microemulsion group mice significantly descends (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).
Table 1 Peptidoglycan microemulsion is to the pg/mL that influences of BLG sensitized mice Th1/Th2 emiocytosis,
n=3)
One factor analysis of variance, significant difference:
aP＜0.05vs sensitization group;
bP＜0.05vs blank control group
2.3.2 the integrated peptidoglycan microemulsion is to the antibody secreted influence 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 control group (P＜0.05).Behind Peptidoglycan microemulsion intervention sensitized mice, can effectively suppress the secretion (P＜0.05) of total IgE, BLG specific IgE and total IgG, and compare difference not remarkable (P＞0.05) with blank control group, the result is as shown in Figure 4.
The Peptidoglycan composite micro-emulsion is compared with its suspension, and anti-allergic effects strengthens, and possibly be because microemulsion can increase the dissolving and the absorption of Peptidoglycan component, and the microemulsion particle diameter is little, specific surface area is big, absorption affinity is strong.Emulsifying agent wherein is 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, and promptly microemulsion assists it to stride across cell membrane as pharmaceutical carrier, and drug level raises relatively in the cell; Improved the utilization rate of Peptidoglycan component greatly, thereby action effect increases obviously.
The above is merely the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in spirit that claim of the present invention limited and scope, can carry out many changes to it, revise, even equivalence change, but all will fall in protection scope of the present invention.
Claims (10)
1. the application of lactobacillus integrated peptidoglycan in the irritated immunostimulant of the anti-beta lactoglobulin of preparation.
2. application as claimed in claim 1 is characterized in that it being that described lactobacillus integrated peptidoglycan is applied to prepare in the beta lactoglobulin immunostimulant hypersensitive of anti-IgE mediation.
3. application as claimed in claim 2; It is characterized in that described integrated peptidoglycan extracts from Lactobacillus johnsonii KLDS1.7032; Described Lactobacillus johnsonii KLDS1.7032; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, its culture presevation is numbered: CGMCC 5050.
4. method for preparing the described integrated peptidoglycan of claim 3 is characterized in that may further comprise the steps:
(1) cleaning of thalline and collection: after Lactobacillus johnsonii KLDS1.7032 actication of culture gone down to posterity, be inoculated in the MRS fluid medium, 37 ℃ of static cultivation 18h cultivate and are cooled to 4 ℃ rapidly after finishing; At 5000g, 4 ℃ of centrifugal 20min, collect bacterial sediment; 4 ℃ of distilled water cyclic washing to thalline whites are collected thalline, place 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 common micro-organisms center, and its culture presevation is numbered: CGMCC 5050;
(2) remove LTA: the mass fraction that the dead bacterium mud of the described thermic of step (1) is suspended in preheating is that boiling water bath 20min is with the cracking thalline in the 10% trichloroacetic acid solution; After the cooling, 5000g, 4 ℃ of centrifugal 15min, collecting precipitation;
(3) defat: concentration is 0.02mol/L, and pH value is 4.6 sodium acetate solution, and with chloroform, methanol, the three mixed in 4: 5: 10 by volume; With the deposition that mixed liquor dissolving step (2) obtains, the volume of said mixed liquor is 15: 1 with the precipitation volume ratio; Stirring at room 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 deposition 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 deposition that step (4) obtains is through 0.01molL
-1Sulfuric acid solution is handled 5min for 85～95 ℃, and cooling is immediately abandoned supernatant behind 12000g, 4 ℃ of centrifugal 30min in the ice-water bath, and precipitate with deionized water was dialysed 7 days under 4 ℃ of conditions continuously, and the rearmounted 4 ℃ of preservations of lyophilization are subsequent use.
5. the lactobacillus integrated peptidoglycan for preparing according to the described method for preparing of claim 4.
6. anti-beta lactoglobulin microemulsion formulation hypersensitive, claim 3 or the 5 described integrated peptidoglycans that it is characterized in that comprising effective dose are as active component, also comprise oil phase, surfactant, cosurfactant and water.
7. microemulsion formulation as claimed in claim 6 is characterized in that described surfactant is a tween 80, and described cosurfactant is a dehydrated alcohol, and described oil phase is the mixture of n-butyric acie ethyl ester and vitamin E.
8. microemulsion formulation as claimed in claim 7; It is characterized in that microemulsion formulation comprises each material of following weight fraction: claim 3 or 5 described integrated peptidoglycans 3.00%; Mass ratio is 7: 3 the n-butyric acie ethyl ester and the mixture 1.07%～13.59% of vitamin E composition, and mass ratio is 2: 8 the tween 80 and the mixture 2.10%～25.42% and the water 58.00%～89.00% of dehydrated alcohol composition.
9. microemulsion formulation as claimed in claim 8; It is characterized in that preparing: be that 2: 8 tween 80 is scattered in mixture 2.10%～25.42% that dehydrated alcohol is formed and becomes water in 58.00%～89.00% water with mass ratio by following method; Mass ratio is that the mixture 1.07%～13.59% of 7: 3 n-butyric acie ethyl ester and vitamin E is oil phase, and claim 3 or 5 described integrated peptidoglycans 3.00% add in the oil phases, biphasely is preheated to 70 ℃ respectively; Oil phase and water are mixed after the high speed shear mulser makes thick breast at 6000rpm down cut 5min; High pressure homogenizer homogenizing 6 times under the pressure of 70MPa is adopted in 6 times of normal saline dilutions that add 5 times of volumes again, regulates pH to 6.5～6.8; Flowing steam sterilization 15min promptly gets microemulsion formulation.
10. the application of each described microemulsion formulation of claim 6-9 in the irritated immunostimulant of the anti-beta lactoglobulin of preparation.
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| CN104430924A (en) * | 2014-11-21 | 2015-03-25 | 东北农业大学 | Preparation method for soft soybean cheese containing active probiotics |
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| CN108396042A (en) * | 2018-03-05 | 2018-08-14 | 郑州安图生物工程股份有限公司 | A kind of preparation method of small soluble molecules peptide glycan |
| 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 |
| CN115624183A (en) * | 2022-09-30 | 2023-01-20 | 浙江李子园食品股份有限公司 | Histidine preparation for relieving cow milk allergy and application thereof |
| 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 |
| CN115669542A (en) * | 2022-11-04 | 2023-02-03 | 西北农林科技大学 | Plant tissue culture detoxification method |
| 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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