CN104117062A - Preparation method of composite IgY against periodontal disease pathogenic bacteria - Google Patents
Preparation method of composite IgY against periodontal disease pathogenic bacteria Download PDFInfo
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Abstract
The invention relates to a preparation method of a composite IgY against periodontal disease pathogenic bacteria. The method comprises the steps of: preparing antigen; injection; acquiring an immune egg; extracting and purifying antibodies: disinfecting the eggshell, and separating the egg yolk; diluting the egg yolk and filtering to obtain a filtrate; clarifying the flocculation; conducting ultrafiltration condensation on the filtrate to obtain an ultrafiltration concentrated liquid; adjusting the ultrafiltration concentrated liquid to a neutral state, measuring the total protein, and adding a protein protective agent; and conducting freeze-drying on the substance added with the protective agent, so as to obtain the composite IgY. The extraction and purification process in the invention use a non-shear process, and adopts a combined technology containing flocculant, protein protective agent and protein stabilizer according to the characteristics of IgY protein, so that the protein does not endure denaturation and keeps the activity in a series of treatement, and the potency of IgY for the corresponding antigen is enhanced. The preparation method is simple and refined, has low energy consumption, good safety and low comprehensive cost, and is applicable to industrialized batch production.
Description
Technical field
The present invention relates to bioengineering field, refer to especially a kind of preparation method of anti-periodontal disease pathogenic bacterium composite IgY.
Background technology
Periodontal disease is a kind of common and multiple oral disease, clinical main manifestations is gingivitis and the large class of periodontitis two, periodontitis, gingivitis adult child morbidity rate is more than 75%, the main pathogenic microorganism porphyromonas gingivalis (Pg generally acknowledging, ), companion unwrapping wire actinomycetes (Aa), good fortune Sai Shi bacillus (Tf), tartar treponema (Td), middle these microorganism secretion enzymes of Pu Shi bacillus (Pi), the inflammatory destruction that causes periodontal support tissue makes gingiva liquefaction, atrophy of the gum, causing odontoseisis comes off, also can cause cardiovascular related diseases, larger than dental caries harm, owing to not being that mortality disease people also ignore early stage prevention and treatment, Epidemiological study shows that periodontitis is the first reason that adult loses tooth, anaphase cost increases the weight of more greatly medical treatment burden.In existing clinical medicine, non-selectivity is for the medicine of the preventions of periodontal disease pathogenic bacterium and treatment periodontal disease, the general antibiotic therapy problem that easily develops immunity to drugs, and there is situation undesirable and Relapse rate recurrence in therapeutic effect.There is no at present and see the treatment of utilization yolk antibody and the clinical case of pre-preventing parodontitis.Anti-periodontal disease pathogenic bacterium composite IgY preparation is according to antigenic determinant principle, pathogen to be in optimized selection and to be combined, prepare immunizing antigen, injection bird inlay, after immunne response, can constantly in yolk, produce corresponding polyclone IgY antibody, an Ovum Gallus domesticus Flavus is containing more than the about 100mg of IgY, and oviposition period generally can reach more than 7 months.Make the large-tonnage suitability for industrialized production of low cost become possibility.Its feature protein content of yolk IgY antibody MW180000 dalton; Antibody activity is high, and 50ng/ml still has activity specific; There is the homogeneity that is similar to monoclonal antibody; Within 62.5 DEG C, 30 minutes, still have activity; Stablize pH4.0～11.0 o'clock.IgY antibody is taken from birds egg yolk and mammal and mankind's sibship and is thought that gap is large, between birds IgY and mammalian immune globulin, can not intersect serological reaction, do not activate mammiferous complement system, first show as the specific biological of its antigen activity stabilized, it is long-lasting, safety good, strong with antigen-binding, active high.The application of antibody is only for pathogenic bacterium, the Drug resistance problem that can avoid life-time service antibiotic to produce, avoid side effect and the stimulation of various ora, disinfectants, therefore, maintain preferably oral cavity microecological balance, apply compound anti-periodontal disease IgY, in child and the sick prevention of human adult periodontal, especially child and old people oral cavity health are had to good social meaning.
The defect that IgY preparation method for antibody exists is at present generally all culture of bacteria, the deactivation antibacterial that ins all sorts of ways, then prepare vaccine immunity hen, a little less than hen immunne response, in yolk, the level of tiring of the corresponding antigen of IgY is low; Centrifugal clarification separating liquid in IgY leaching process, shearing and destruction that the centrifugal shear force that centrifuge produces forms albumen have been reduced tiring and yield of IgY antibody; Direct lyophilizing or spray dry-cure by IgY common process leaching process have inevitably reduced IgY antibody activity, and the IgY raw material attribute that general industryization forms in producing, has limited IgY antibody application approach and scope.
Summary of the invention
The present invention proposes a kind of preparation method of anti-periodontal disease pathogenic bacterium composite IgY, has solved the problem that in prior art, activity is low, the activity keeping phase is short in the process of preparation IgY antibody.
Technical scheme of the present invention is achieved in that
A preparation method for anti-periodontal disease pathogenic bacterium composite IgY, comprising:
(1) antigen preparation:
With companion unwrapping wire actinomycetes (Aa), porphyromonas gingivalis (Pg,), Streptococcus mutans (S.mutans change of serum C type) as antigen bacteria expand bacterium, concentrated, by the dense preparation of certain bacterium bacterium liquid, do not adopt the processing method of general inactivation antigen bacterium to prepare Freund's complete adjuvant and/or incomplete Freund's adjuvant is made vaccine;
(2) injection: laying poultry is injected with above-mentioned vaccine;
(3) obtain immune egg: search the raw immune egg of described poultry;
(4) the extraction purification of antibody:
By eggshell sterilization, beat eggs and isolate egg yolk; Then dilute egg yolk and filter, obtaining filtrate;
(5) flocculate and clarify:
In above-mentioned filtrate, adding concentration is the protein stabiliser of 2-5%; Add again a kind of flocculating agent; Sedimentation 20-30h, siphon supernatant filtered through gauze; Wherein often add flocculating agent one time, all will be thread and under agitation slowly add;
(6) ultrafiltration and concentration:
Filtrate after step (5) is filtered is carried out ultrafiltration and concentration, obtains ultrafiltration and concentration liquid; Regulate described ultrafiltration and concentration liquid to neutral, survey Tot Prot.Ultrafiltration and concentration liquid store method: add protein stabiliser ,-(20-30) DEG C preservation; This step makes the aqueous goods of IgY can directly be used as raw material, as for liquid, mastic application product;
(7) lyophilization:
Step (6) concentrated solution is added to protein protective agent, and lyophilization or spraying are drying to obtain IgY powder.
As preferred technical scheme, described protein stabiliser is the mixture of glycerol, tween 20, Plronic188 and carboxymethyl cellulose.
As preferred technical scheme, described protein protective agent is that trehalose is appropriate, glycerol 0.1-0.2WT%, tween 80 1-2WT%, PVP
k301-2WT% and Plronic1880.2-0.3WT%.
As preferred technical scheme, described injection is specific as follows: get order in 25 weeks by bird inlay injection 1ml vaccine; Head exempts from, with Freund's complete adjuvant, to inject latter 7 days, and immunity incomplete Freund's adjuvant for the second time, injects on the 14th day for the third time.
As preferred technical scheme, the extraction purification concrete steps of described antibody comprise:
Method one; Water dilution HAC adjusts pH value method: egg yolk is blended to homogenize, add 9 times of purified water by egg yolk amount, be made into the diluent of 1:10; Adjust PH, add the HAC of 1mol/L by 0.773% of diluent total amount, regulating diluent PH is 5.0～5.2; After the diluent preparing is added a cover and sealed, in-20 DEG C of refrigerators, make to freeze completely, at 4～6 DEG C, thaw, filter, obtain clear filtrate;
Method two: HAC-N
aaC buffer dilution method
Examination pH value, by 9 times of sterile pure water waters of egg yolk, adds anhydrous sodium acetate (AR) dissolving of 0.1mol/L to stir evenly, pour egg yolk into and fully stir evenly, consider except vitellinae membrana with gauze, under agitation regulate PH to 5.18～5.24. with 1mol/LHAC, record HAC consumption, so that accurate quantitative analysis HAC-N
aaC buffer solution ph 5.2 additions;
Dilute in batches, get 9 times of sterile pure water of yolk amount, in residue water, press 10 times of amount weight of egg yolk, the 1mol/LHAC-N of ratio while adding examination to adjust pH value
aaC, fully dissolves and stirs evenly, and under agitation the yolk liquid of dilution is slowly poured into and stirred evenly; Survey pH value 5.18～5.24; Seal shady and cool place's sedimentation 12-24h.
As preferred technical scheme, the interpolation of the flocculating agent material in described step (5) is as follows:
First adding concentration is that chitosan to the final concentration of 1-2% is 0.06%-0.07%
Adding concentration is that sodium alginate to the final concentration of 1-2% is 0.04%-0.06;
Adding concentration is that calcium chloride to the final concentration of 8-10% is 0.02%-0.06 again;
Finally adding concentration is that 8-10% sodium tripolyphosphate to final concentration is 0.04%0.06.
As preferred technical scheme, described step (7) yolk antibody lyophilized powder moisture is < 50%.
The bioactivity of yolk antibody prepared by the present invention, uses enzyme-labeled immunity adsorption test, record the corresponding antigen of anti-periodontal disease pathogenic bacteria composite IgY tire and be respectively:
To porphyromonas gingivalis (Pg) antigen 1: 128000
To accompanying unwrapping wire actinomycetes (Aa) antigen 1: 128000
To Streptococcus mutans antigen 1: 256000
Electrophoresis test, adopts SDS-PAGE method to record anti-periodontal disease pathogenic bacteria composite IgY content 40-60%, protein value 90-98%.
Application aspect: compound anti-periodontal disease pathogenic bacterium IgY makes oral spray and the class dosage form of gargling as active component, in formula for a product, apply protective agent, stabilizing agent perfect prescription make it have good dispersion and can not produce protein coacervation activity decreased; Prescription takes into full account that IgY class preparation protein active up till now keeps the problem that the phase do not grow, for this reason and the prescription of design and the selection of technique make this preparation keep its specific biological activity to antigen to have reliable stability, its long-lasting and stability shows corresponding the tiring of antigen of 2-4 at normal temperatures and is not less than 1:128000
Basic prescription, can derive other demand formulas on this basis:
In the present invention, extraction and purification process uses non-shearing technique, adopted flocculating agent, protein protective agent, protein stabiliser combination technique according to IgY protein characteristic, made in a series of processing, albumen is several without degeneration, keep protein active, improved IgY tiring for corresponding antigens; The simple and direct refine of preparation method, energy consumption low-security is good, and integrated cost is low, is suitable for industrialized mass production.
In the present invention, added protein protective agent, protein stabiliser, makes to spray dry technique and freeze drying process IgY antibody not damaged, and albumen is several without degeneration, has kept higher activity to tire; In the dosage form application aspect taking IgY antibody as raw material; general IgY antibody preparation 6-8 month activity of guaranteeing the quality tired and only had below 1:32; in pharmaceutical solutions, added stabilizing agent, protective agent, extraordinary oil-water interfaces repellents improved IgY antibody in carrier stability, 2-3 preparation is tired and is also kept 1:128 to 1:256.
The preparation method of anti-periodontal disease pathogenic bacterium composite IgY of the present invention, has good industrial chain combination, and technological process is environmentally friendly, and the few integrated cost of power consumption is low.Be applicable to industrialized mass production.Its manufactured goods safety is good, can be used as the antibody raw material of liquid dosage form, powder preparation type, cream class and gel-like goods.
Brief description of the drawings
In order to be illustrated more clearly in embodiment of the present invention or technical scheme of the prior art, to the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described below, apparently, accompanying drawing in the following describes is only embodiments more of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1: IgY electrophoretogram;
In figure:
A: standard BSA
B: anti-periodontal disease pathogenic bacterium composite IgY adds adjuvant concentrated solution lyophilized powder
C: anti-periodontal disease pathogenic bacterium composite IgY freeze thawing concentrated solution lyophilized powder
D: anti-periodontal disease pathogenic bacterium composite IgY adds the concentrated spray powder of adjuvant
E: hen's egg-white lysozyme.
Detailed description of the invention
To the technical scheme in the embodiment of the present invention be clearly and completely described below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Anti-periodontal disease pathogenic bacterium composite IgY provides a kind of safe and effective periodontal disease method of preventing and treating, known porphyromonas gingivalis (Pg,), companion unwrapping wire actinomycetes (Aa), good fortune Sai Shi bacillus (Tf), tartar treponema (Td), middle Pu Shi bacillus (Pi) be the Main Pathogenic Bacteria of periodontal disease, the processing method of antigen bacteria deactivation is taked in general antigen preparation, this makes immunne response not strong, and the yolk IgY of extraction tires not high.This method employing is exempted from deactivation and is prepared the injection of Kang Yuan Give chicken, and far away according to animal species people and birds blood relationship, chicken house chicken group can not produce colony's death of stress and chicken group, can improve the immunne response of chicken, improves tiring of the middle IgY antibody of laying eggs.
On IgY antibody extracts, use uncentrifugaled clarification, shearing and the destruction of having reduced centrifuge antagonist, tire after therefore extracting very high; After IgY antibody extracts, added protein protective agent, protein stabiliser, makes IgY antibody not damaged after lyophilizing, and albumen is several without degeneration, has kept higher activity to tire, and has improved IgY antibody recovery rate.
Active protection technology: anti-periodontal disease pathogenic bacterium composite IgY dosage form prepared by this technology has good high efficiency; stability and long-lasting; the liquid dosage form of the design has been added in solution; stabilizing agent; protective agent; dispersant; overcome the general IgY liquid formulation activity keeping time short; protein coacervation and attached wall phenomenon; strengthen IgY antibody activity keeping, improved the stability of IgY antibody, guarantee the quality in preparation 2 years to 4 years its 1:128 to 1:256(that tire in addition of general IgY antibody do not add the above-mentioned adjuvant only 1:8-1:16 that tires)
Embodiment 1
(1) antigen preparation
Porphyromonas gingivalis, companion's unwrapping wire actinomycetes, Streptococcus mutans respectively fluid medium anaerobism increase bacterium cultivation 48h, and collection bacterium, makes bacterium dense: 2 × 10
9mono-20 DEG C of cfu/ml, add complete Freund's adjuvant (CFA): with antigen bacteria liquid 1:1 emulsifying (head exempts from), and full Freund adjuvant (IFA) emulsifying of later toing many or too much for use.
(2) injection
First immunisation complete Freund's adjuvant CFA, the later booster immunization full Freund adjuvant IFA that toos many or too much for use.200 blue brown laying hens in sea of immunity, vaccine total amount 200ml.Vaccine prepares rear every chicken and divides 4 intramuscular injection by 1ml.
After first immunisation, cross 7 days row immune for the second time, two exempt from after 7 days row 3 after exempting from.The 4th immunity is tired and is injected when requiring again after February.
(3) obtain immune egg
After immunity for the first time, within the 21st day, start to pick up egg, and detect and tire.After immunity egg is collected, 4 DEG C of storages.(4) the extraction purification of antibody
Method one water dilution HAC adjusts pH value method: the separation egg yolk of beating eggs, egg yolk is blended to homogenize, and add 9 times of pure water by egg yolk amount, be made into the diluent of 1:10; Adjust PH, add the HAC of 1mol/L, regulating diluent PH is 5.0～5.2.After the diluent preparing is added a cover and sealed, in-20 DEG C of refrigerators, make to freeze completely, at 4～6 DEG C, thaw, can make layering completely easily filter.Obtain clear filtrate.
Method two HAC-N
aaC buffer dilution method
Egg yolk is blended to homogenize, with 1～2 times of sterile pure water be blended into stir evenly for subsequent use.
Add acetic acid-anhydrous sodium acetate (AR) dissolving of 0.1mol/L to stir evenly, pour egg yolk liquid into and fully stir evenly, consider except vitellinae membrana with gauze, under agitation regulate PH to 5.18～5.24 with 1mol/LHAC.The lower sedimentation of 4 degree is spent the night.
Filter siphon supernatant liquid filtering.
(5) flocculate and clarify (refining)
In above-mentioned filtrate, first add the chitosan (CTS) of 0.06%-0.07% fully to stir evenly, add again 0.04% sodium alginate (SA), then add 0.02% calcium chloride, finally add 0.04% sodium tripolyphosphate (TPP), often add adjuvant one time, all to be thread and under agitation slowly add, specifically see right part.
(6) ultrafiltration and concentration
Mi Libo for equipment (Millipore) company ultrafiltration system, Biomax improvement polysulfone composite membrane heap (molecular cut off: 100kDa) ultrafiltration and concentration makes to be concentrated into original volume at approximately 1/20 o'clock, regulates its pH value to neutral (about PH7.0) with 4% sodium hydroxide; Add protein stabiliser: glycerol 2%, tween 20 0.5%, Plronic188 0.5%, carboxymethyl cellulose 0.04% (g/ml) fully mixes, and deposits for-20 DEG C.
Concentrated solution is dried pre-treatment
Ultrafiltration and concentration liquid adds protein protective agent preparation:
For example, it is 5.71% that concentrated solution 2000g(steam seasoning is surveyed total protein), add
(7) lyophilization
Carry out lyophilization by general freeze-dry process method, make lyophilized powder moisture 5% left and right.
Sample: ultrafiltration and concentration liquid; Add adjuvant concentrated solution.Character comparison
ELISA detects and protein electrophoresis detects
ELISA detects
1. antigen breaking cellular wall.
2 is antigen coated: be added in 96 orifice plates by 1:4 dilution proportion bacterium liquid with coating buffer, with after preservative film sealing, 4 DEG C are spent the night.
3. add confining liquid (now with the current)
5. the preparation of testing sample Ovum Gallus domesticus Flavus IgY (proportional diluted)
6. application of sample: add dilution specimen to be measured to the ELISA Plate being coated with
7. adding two resists: add the ELIAS secondary antibody working solution preservative film having prepared and seal, put 37 DEG C of water-baths and place 40min.
8. add substrate solution (now with the current, to observe color): add the substrate solution (now with the current) having prepared, with preservative film sealing, be placed in immediately 37 DEG C of water-baths and place 15min,
9. add stop buffer (cessation reaction): add sulphuric acid stop buffer cessation reaction
10. Ultraviolet Detector photometry density
11 result of the tests are observed and are analyzed
SDS-PAGE detects
15%GEL
Referring to Fig. 1.
Embodiment 2
The anti-periodontal disease composite IgY of preparing with embodiment 1 is made a kind of spray as primary raw material.
Make 1000ml
Formula
Compound method:
1, take PVP
k30, xylitol is dissolved in 800ml distilled water, adds anti-periodontal disease composite IgY to stir stand-by
2, in ethanol, add Mentholum, Mint Essence stirring and dissolving glycerol adding, Tween 80 mix homogeneously again, under agitation slowly this liquid is added in above-mentioned liquid and stirred evenly.
3, filter and add purified water to amount of preparation.Adjust pH8.5 sealing for subsequent use.
4,, filling apparatus cleaning and sterilizing, sterile working, filling and sealing is made the spray of every bottle of 20ml, and 50 bottles of lot number stamps are inserted in outer package.
Embodiment 3
Make a kind of oral nursing liquid as primary raw material with the anti-periodontal disease composite IgY that embodiment 1 is prepared.Make 4000ml
Formula:
Compound method:
Method for making: 1, take PVP
k30, xylitol is dissolved in 3800ml distilled water, adds anti-periodontal pathogenic bacteria composite IgY to stir stand-by.
2, in ethanol, add Mentholum, stirring and dissolving glycerol adding, Tween 80, Mint Essence, jasmine flower essence mix homogeneously again, under agitation slowly this liquid is added in 1 liquid and stirred evenly.
3, filter and add purified water to amount of preparation.Adjust pH8.5 sealing for subsequent use.
4, filling apparatus cleaning and sterilizing, sterile working, filling and sealing is made the spray of every bottle of 200ml, and 20 bottles of lot number stamps, pack packing crates into.
Claims (10)
1. a preparation method for anti-periodontal disease pathogenic bacterium composite IgY, comprising:
(1) antigen preparation:
Preparation with companion unwrapping wire actinomycetes, porphyromonas gingivalis, Streptococcus mutans as antigen bacteria, ultrasonication processing reaches bacterin preparation concentration, prepares Freund's complete adjuvant and incomplete Freund's adjuvant;
(2) injection: laying poultry is injected with above-mentioned vaccine;
(3) obtain immune egg: search the raw immune egg of described poultry;
(4) the extraction purification of antibody:
By eggshell sterilization, beat eggs and isolate egg yolk; Then dilute egg yolk and filter, obtaining filtrate;
(5) flocculate and clarify:
In above-mentioned filtrate, adding concentration is the protein stabiliser of 2-5%; Add again a kind of flocculating agent; Sedimentation 20-30h, siphon supernatant filtered through gauze; Wherein often add flocculating agent one time, all will be thread and under agitation slowly add;
(6) ultrafiltration and concentration:
Filtrate after step (5) is filtered is carried out ultrafiltration and concentration, obtains ultrafiltration and concentration liquid; Regulate described ultrafiltration and concentration liquid to neutral, survey Tot Prot, ultrafiltration and concentration liquid is preserved: add protein stabiliser ,-(20-30) DEG C preservation;
(7) lyophilization:
Step (6) concentrated solution is added to protein protective agent, and lyophilization or spraying are drying to obtain IgY powder.
2. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 1, is characterized in that, described protein stabiliser is the mixture of glycerol, tween 20, Plronic188 and carboxymethyl cellulose.
3. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 1 and 2, is characterized in that, described protein protective agent is trehalose 2-6WT%, glycerol 0.1-0.2WT%, tween 80 1-2WT%, PVP
k301-2WT% and Plronic188 0.2-0.3WT%.
4. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 3, is characterized in that, described injection is specific as follows: get order in 25 weeks by bird inlay injection 1ml vaccine; Head exempts from, with Freund's complete adjuvant, to inject latter 7 days, and immunity incomplete Freund's adjuvant for the second time, injects on the 14th day for the third time.
5. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 3, is characterized in that, the extraction purification concrete steps of described antibody comprise:
Method one; Water dilution HAC adjusts pH value method: egg yolk is blended to homogenize, add 9 times of purified water by egg yolk amount, be made into the diluent of 1:10; Adjust PH, add the HAC of 1mol/L by 0.773% of diluent total amount, regulating diluent PH is 5.0～5.2; After the diluent preparing is added a cover and sealed, in-20 DEG C of refrigerators, make to freeze completely, at 4～6 DEG C, thaw, filter, obtain clear filtrate;
Method two: HAC-N
aaC buffer dilution method
Examination pH value, by 9 times of sterile pure water waters of egg yolk, adds anhydrous sodium acetate (AR) dissolving of 0.1mol/L to stir evenly, and pours egg yolk into and fully stirs evenly, and considers except vitellinae membrana with gauze, under agitation regulates PH to 5.18～5.24. with 1mol/LHAC, records HAC consumption;
Dilute in batches, get 9 times of sterile pure water of yolk amount, in residue water, press 10 times of amount weight of egg yolk, the 1mol/LHAC-N of ratio while adding examination to adjust pH value
aaC, fully dissolves and stirs evenly, and under agitation the yolk liquid of dilution is slowly poured into and stirred evenly; Survey pH value 5.18～5.24; Seal shady and cool place's sedimentation 12-24h.
6. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 3, is characterized in that, the interpolation of the flocculating agent material in described step (5) is as follows:
First adding concentration is that chitosan to the final concentration of 1-2% is 0.06%-0.07%
Adding concentration is that sodium alginate to the final concentration of 1-2% is 0.04%-0.06%;
Adding concentration is that calcium chloride to the final concentration of 8-10% is 0.02%-0.06% again;
Finally adding concentration is that 8-10% sodium tripolyphosphate to final concentration is 0.04%-0.06%.
7. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 6, is characterized in that, described step (7) yolk antibody lyophilized powder moisture is < 50%.
8. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 6, is characterized in that, the anti-periodontal disease antigen composite IgY obtaining in described step (7) as active component for making mouthspray dosage form or the oral cavity class dosage form of gargling.
9. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 8, is characterized in that, the gargle formula of class dosage form of described mouthspray dosage form or oral cavity comprises:
10. the preparation method of a kind of anti-periodontal disease pathogenic bacterium composite IgY according to claim 8, is characterized in that, the formula of described oral nursing liquid comprises:
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| CN106380518A (en) * | 2016-08-30 | 2017-02-08 | 安徽安科生物工程（集团）股份有限公司 | Extraction technology of egg yolk immunoglobulins |
| CN108042387A (en) * | 2017-12-11 | 2018-05-18 | 陕西瑞凯生物科技有限公司 | A kind of antibacterial mouthwash of bioprotein and preparation method thereof |
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| CN109908346A (en) * | 2018-08-24 | 2019-06-21 | 江苏润洁生物科技有限公司 | Product and the application for preventing and treating the pathogenic Yolk antibody composition of helicobacter pylori, porphyromonas gingivalis and Streptococcus mutans and its preparation |
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| CN110357955A (en) * | 2019-06-24 | 2019-10-22 | 桂林三金药业股份有限公司 | A kind of compound special yolk antibody and its preparation method and application |
| US11319382B1 (en) | 2021-06-28 | 2022-05-03 | King Abdulaziz University | Methods for producing and using IgY antibodies targeting the middle east respiratory syndrome coronavirus spike protein to treat or prevent MERS-CoV infection |
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| CN110237251B (en) * | 2019-06-24 | 2023-05-12 | 桂林三金药业股份有限公司 | Composite specificity egg yolk antibody oral spray and preparation method thereof |
| US11319382B1 (en) | 2021-06-28 | 2022-05-03 | King Abdulaziz University | Methods for producing and using IgY antibodies targeting the middle east respiratory syndrome coronavirus spike protein to treat or prevent MERS-CoV infection |
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