CN101028515B - Anti-preisz bacterium and gingival carbon dioxide fibrophilic bacterium mixed IgY and its use in treating ozostomia and gingiva - Google Patents
Anti-preisz bacterium and gingival carbon dioxide fibrophilic bacterium mixed IgY and its use in treating ozostomia and gingiva Download PDFInfo
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- CN101028515B CN101028515B CN2006101254975A CN200610125497A CN101028515B CN 101028515 B CN101028515 B CN 101028515B CN 2006101254975 A CN2006101254975 A CN 2006101254975A CN 200610125497 A CN200610125497 A CN 200610125497A CN 101028515 B CN101028515 B CN 101028515B
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- general salmonella
- capnocytophaga gingivalis
- igy
- gingivalis
- capnocytophaga
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract
An application of the IgY antibody and its products in the form of spray, drops, buccal lozenge, tooth-paste in preventing and treating foul breath, oral odor and gingival diseases, the process for preparing said antibody and its products, and its application n range are disclosed.
Description
Technical field
The present invention relates to general Salmonella in a kind of anti-centre and capnocytophaga gingivalis mixed IgY and the application in treatment halitosis and gingiva thereof, the preparation of preparation method for antibody and antibody preparation and the scope of application.
Background technology
At present there is not effectively to treat the appropriate method of halitosis, although halitosis and gingiva, periodontal are closely related, but the method for modal treatment halitosis is to use antibiotics class medicine treatment gingiva, periodontal earlier, indirect again treatment halitosis, but use antibiotics class Drug therapy halitosis that a lot of drawbacks are arranged.
China's gingiva, periodontal sickness rate account for more than 70% of population, are many with adult's gingivitis, periodontitis wherein.Gingivitis, patients with periodontitis red swelling of gingiva, blackout, take food and brush teeth easily hemorrhage, halitosis and halitosis, periodontal pocket formation, gingival atrophy, alveolar bone destroy, odontoseisis comes off, if untimely treatment can cause the loss of tooth forfeiture, gingivitis, periodontitis still are the focus and the bacterial origin of other systemic infection disease in addition.Surplus the suspicious cause of disease bacterium of gingivitis, periodontitis has ten more than the kind, but main suspicious cause of disease bacterium comprises Fusobacterium nucleatum, porphyromonas gingivalis, companion actinomycetes Actinobacillus, the treponema dentium of dwelling, streptococci, capnocytophaga gingivalis, middle general Salmonella etc.Patient's number of isolating capnocytophaga gingivalis from gingivitis, periodontitis focus is on the increase in recent years.
Gingivitis, periodontitis do not have ideal medicine, substantially all be antibiotics class medicine, as: metronidazole, for its nitre azoles, and surfactant, as hibitane etc., their unsatisfactory curative effect and certain side effect arranged, as: life-time service easily causes candida albicans infection, even peripheral neuritis etc. appears, also easily cause the generation of Resistant strain simultaneously.Therefore be badly in need of seeking new ways and means prevention and treatment halitosis, halitosis, gingivitis and periodontitis.
Comprise that from main suspicious cause of disease bacterium seeking the principal element that causes halitosis Fusobacterium nucleatum, porphyromonas gingivalis, companion actinomycetes Actinobacillus, the treponema dentium of dwelling, streptococci, capnocytophaga gingivalis, the middle general Salmonella is to suppress the key of halitosis.
There is the lot of documents report to show and uses specific IgY antibody caries prevention and treatment infant rotavirus diarrhoea clinically, prevention gallbladder cystic fibrosis patient charrin's disease, alleviate the microbial stomachache of helicobacter pylorus, these therapeutic effect have all obtained definite proof.
We think that in field of biology also there is certain misunderstanding in academia to specific IgY.A kind of typical misunderstanding is to think that certain IgY must suppress certain disease, for example thinks that not needing to do experiment just can infer anti-Fusobacterium nucleatum IgY and can specificity suppress Fusobacterium nucleatum, resisting porphyromonas bacterium IgY can specificity to suppress that porphyromonas gingivalis, anti-companion actinomycetes Actinobacillus IgY can specificity suppress to accompany the actinomycetes Actinobacillus all be unscientific.Above-mentioned inhibitory action data by experiment could proving effect.
Find that the experimental data of the IgY of the antigen immune of the kind that has does not have or do not have obvious suppression trend in the IgY of the numerous species that we study, briefly, not every IgY has the obvious treatment effect.For example there is not experimental data can prove that the IgY of HIV (human immunodeficiency virus) immunity can treat AIDS at present.
Because surplus the suspicious cause of disease bacterium of gingivitis, periodontitis has ten more than the kind, but main suspicious cause of disease bacterium comprises Fusobacterium nucleatum, porphyromonas gingivalis, companion actinomycetes Actinobacillus, the treponema dentium of dwelling, streptococci, capnocytophaga gingivalis, middle general Salmonella etc.When the IgY that explores the preparation correspondence was used for the treatment of gingiva, periodontal, we inferred that Fusobacterium nucleatum, porphyromonas gingivalis, companion actinomycetes Actinobacillus, the treponema dentium of dwelling, streptococci, capnocytophaga gingivalis, middle general Salmonella all might cause halitosis; But our prior unpredictable which bacterium in the middle of this is the principal element that causes halitosis, and which bacterium is the secondary cause that causes halitosis.
We think must have the clinical trial of suitable halitosis to determine which bacterium is the principal element that causes halitosis.Thereby instruct the medicine of preparation halitosis or the functional food of halitosis.
Present prior art does not have relevant prompting guidance technology personnel to find that anti-middle general Salmonella and capnocytophaga gingivalis mixed IgY are the keys that solves the treatment halitosis.
The technical staff can't learn that in advance capnocytophaga gingivalis, middle general Salmonella are the principal elements that causes halitosis in advance under the prerequisite of the experiment of not doing halitosis.
The technical staff can't be in advance under the prerequisite of the experiment of not doing anti-gingiva, learn in advance anti-in the middle of general Salmonella and capnocytophaga gingivalis mixed IgY than the better effects if of independent anti-capnocytophaga gingivalis IgY, the independent general Salmonella IgY in anti-centre.
Summary of the invention
The purpose of this invention is to provide the general Salmonella specific IgY in a kind of anti-centre, anti-capnocytophaga gingivalis, anti-middle general Salmonella and capnocytophaga gingivalis mixed IgY and the application in treatment halitosis and gingiva thereof, the preparation of preparation method for antibody and antibody preparation and the scope of application.
The international standard of the general Salmonella in centre that the present invention relates to is called Prevotella intermedia.
The international standard of the capnocytophaga gingivalis that the present invention relates to is called Capnocytophaga gingivalis.
Purpose of the present invention also provides a kind of method and the general Salmonella in a kind of anti-centre and capnocytophaga gingivalis mixing specific IgY extract and pure product that prepare anti-middle general Salmonella and capnocytophaga gingivalis mixing specific IgY, and by the preparation of this resistant to carbon dioxide Cytophaga, middle general Salmonella mixed IgY antibody extract and the preparation of pure product, and with the prevention of this preparation and treatment by capnocytophaga gingivalis, middle general Salmonella, and the microbial halitosis of the anaerobism that common antigenic determinant is arranged with it, halitosis, and gingivitis etc.
The objective of the invention is to realize by following technical proposal:
The application of general Salmonella specific IgY in the preparation of preparation prevention and treatment halitosis in the middle of anti-.
The application of anti-capnocytophaga gingivalis specific IgY in the preparation of preparation prevention and treatment halitosis.
The application of general Salmonella specific IgY in the preparation of preparation prevention and treatment gingiva in the middle of anti-.
The application of anti-capnocytophaga gingivalis specific IgY in the preparation of preparation prevention and treatment gingiva.
General Salmonella and capnocytophaga gingivalis mixings specific IgY in the middle of anti-, wherein: get anti-capnocytophaga gingivalis strain specific IgY30～60 parts, the anti-general Salmonella strain specific IgY40 in centre～70 parts mixes.
General Salmonella and the capnocytophaga gingivalis mixing specific IgY application in the preparation of preparation prevention and treatment halitosis in the middle of anti-.
The preparation method of general Salmonella and capnocytophaga gingivalis mixing specific IgY in the middle of anti-, wherein:
A bacterial species: capnocytophaga gingivalis and middle general Salmonella;
Source of antibacterial and cultivation: capnocytophaga gingivalis and middle general Salmonella, from the isolating capnocytophaga gingivalis of periodontal disease patient oral cavity periodontal pocket inflammation part, middle general Salmonella, and the capnocytophaga gingivalis ATCC 33624 of standard and middle general Salmonella ATCC 25261 bacterial strains, through the capnocytophaga gingivalis of BHI-S culture medium separation and Culture and amplification cultivation, middle general Salmonella, and confirm as capnocytophaga gingivalis, middle general Salmonella through evaluation;
The B immunogen preparing:
Get 30～60 parts of the fragments of capnocytophaga gingivalis thalline, the fragment of middle general Salmonella thalline is mixed into bacterium liquid for 40～70 parts;
Isopyknic bacterium liquid is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant capnocytophaga gingivalis, middle general Salmonella mixed immunity is former.
Perhaps isopyknic bacterium liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant capnocytophaga gingivalis, middle general Salmonella mixed immunity is former;
All contain antibacterial 2,000,000,000-6,000,000,000 during the former and freund 's incomplete adjuvant mixed immunity of every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former;
The C immunity:
Use syringe to give bird inlay with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity, every bird inlay injection 1.5ml-2.0ml immunogen, use the subcutaneous and intramuscular injection of the former 1.5ml-2.0ml of freund 's incomplete adjuvant mixed immunity to give bird inlay then week about, behind the booster immunization secondary; After this former 1ml of freund 's incomplete adjuvant mixed immunity is subcutaneous to give bird inlay booster immunization four times with intramuscular injection every re-using in eight weeks, front and back immune seven times altogether.
The preparation of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract in the middle of D is anti-:
Collect immunity the 30th day the immune hen egg in immunity back for the first time, the ethanol of use 70% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and distilled water by volume 1: the ratio adding distil water of 7-9, fully stir, and use 1N HCl to regulate acid-base value to pH5.0-5.2, place then 4 ℃ 12 hours.
Then with egg yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, with egg yolk diluent supernatant place molecular cut off be 100KD hollow fiber membrane ultrafiltration device ultrafiltration and concentration 5-10 doubly, the hibitane that adds 0.02%-2.0% then fully stirs, and use 1N NaOH to regulate acid-base value to pH7.0-7.5, place 4 ℃ 12 hours, after this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance, with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ-0.22 μ, promptly get resist in the middle of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract.
Technical scheme of the present invention can also be unfolded as follows:
General Salmonella and capnocytophaga gingivalis mixed IgY antibody extract in the middle of one species specificity is anti-is characterized in that this IgY antibody extract is to be produced by following method and step:
(1) bacterial origin and cultivation:
Source of antibacterial and cultivation: from the isolating capnocytophaga gingivalis of periodontal disease patient oral cavity periodontal pocket inflammation part, middle general Salmonella, and capnocytophaga gingivalis of standard (ATCC 33624) and middle general Salmonella bacterial strains such as (ATCC25261), through the capnocytophaga gingivalis of BHI-S culture medium separation and Culture and amplification cultivation, middle general Salmonella, and confirm as capnocytophaga gingivalis, middle general Salmonella through evaluation;
(2) immunogen preparing:
Get 30～60 parts of capnocytophaga gingivalis bacterial chips, middle general Salmonella bacterial chip is mixed into bacterium liquid for 40～70 parts;
Isopyknic bacterium liquid is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant capnocytophaga gingivalis, middle general Salmonella mixed immunity is former.
Isopyknic bacterium liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant capnocytophaga gingivalis, middle general Salmonella mixed immunity is former.
All contain antibacterial 2,000,000,000-6,000,000,000 during the former and freund 's incomplete adjuvant mixed immunity of every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former;
(3) immunity:
Use syringe to give bird inlay with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity, every bird inlay injection 1.5ml-2.0ml immunogen, use the subcutaneous and intramuscular injection of the former 1.5ml-2.0ml of freund 's incomplete adjuvant mixed immunity to give bird inlay then week about, behind the booster immunization secondary; After this former 1ml of freund 's incomplete adjuvant mixed immunity is subcutaneous to give bird inlay booster immunization four times with intramuscular injection every re-using in eight weeks, front and back immune seven times altogether.
(4) preparation of anti-middle general Salmonella and capnocytophaga gingivalis mixing specific IgY extract:
Collect immunity the 30th day the immune hen egg in immunity back for the first time, the ethanol of use 70% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and distilled water by volume 1: the ratio adding distil water of 7-9, fully stir, and use 1N HCl to regulate acid-base value to pH5.0-5.2, place then 4 ℃ 12 hours.
Then with egg yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, with egg yolk diluent supernatant place molecular cut off be 100KD hollow fiber membrane ultrafiltration device ultrafiltration and concentration 5-10 doubly, the hibitane that adds 0.02%-2.0% then fully stirs, and use 1N NaOH to regulate acid-base value to pH7.0-7.5, place 4 ℃ 12 hours, after this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance, with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ-0.22 μ, promptly get resist in the middle of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract;
The preparation method of general Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY also comprises the steps: in the middle of anti-
With the anti-capnocytophaga gingivalis after the weeding of grease metallic substance, general Salmonella mixed IgY antibody extract uses 1N NaOH to regulate acid-base value to pH5.5-5.7 in the middle of anti-, the saturation that adds sulphuric acid amine to 55%, fully stir, room temperature left standstill 4-12 hour, high speed centrifugation 10000rpm is 10 minutes then, get precipitation, to precipitate and use distilled water diluting to the original volume amount, the saturation that adds sulphuric acid amine to 33% again, fully stir, room temperature left standstill 4-12 hour, high speed centrifugation 10000rpm is 10 minutes then, gets precipitation, with the dissolved in distilled water post precipitation bag filter of packing into, use the distill water dialysis desalination, promptly get and resist capnocytophaga gingivalis, middle general Salmonella, mixed IgY pure antibody product;
Capnocytophaga gingivalis in the described immunogen preparing is meant the thalline of capnocytophaga gingivalis and the bacterial chip of capnocytophaga gingivalis, thalline of general Salmonella and the bacterial chip of middle general Salmonella in the middle of the general Salmonella in described centre is meant;
Antibacterial mixed proportion in the described immunogen preparing is 10～70 parts of capnocytophaga gingivalis bacterial chips, 30～90 parts of middle general Salmonella bacterial chips; The ratio of antibacterial and bacterial chip is 40～80 parts on an antibacterial, 20～60 parts of bacterial chips; Bacterial number is from 500,000,000 to every milliliter every milliliter 10,000,000,000;
Bird inlay in the described immunity comprises that raising under the conventional environment, cleaning ambient are raised down and the bird inlay of the various Different Chicken kinds that the SPF environment is raised down;
General Salmonella and capnocytophaga gingivalis mixing specific IgY also comprised the steps: to add pharmaceutically acceptable additives and make spray, instillation, buccal tablet and the toothpaste that contains anti-middle general Salmonella of a species specificity and capnocytophaga gingivalis mixing specific IgY in the middle of a described species specificity was anti-.
The general Salmonella in anti-centre of producing by method of the present invention and the purity of capnocytophaga gingivalis mixing specific IgY extract are measured through irreducibility SDS-PAGE, and the purity of IgY antibody reaches (〉=30%) more than 30%; And the general Salmonella in anti-centre that the method for process invention is extracted and the purity of capnocytophaga gingivalis mixing specific IgY purification product are measured through irreducibility SDS-PAGE, and the purity of IgY antibody reaches (〉=82%) more than 82%;
The general Salmonella in anti-centre of producing by method of the present invention and the biological value of capnocytophaga gingivalis mixing specific IgY extract, measure through ELISA (enzyme-linked immunosorbent assay) method, general Salmonella and capnocytophaga gingivalis mixing specific IgY were in conjunction with capnocytophaga gingivalis in the middle of specificity was anti-, the specificity that the biological value of middle general Salmonella hybrid antigen reaches in every milligram of albumen resists tiring more than or equal to 1: 4000 of middle general Salmonella and capnocytophaga gingivalis mixing specific IgY, promptly tire 〉=1: 4000/mg albumen, perhaps claim in the extract specificity in every milligram of albumen anti-in the middle of the ratio of general Salmonella and capnocytophaga gingivalis mixings specific IgY to live be 〉=1: 4000; And by the general Salmonella in anti-centre of method extraction of the present invention and the biological value of the pure product of capnocytophaga gingivalis mixing specific IgY, measure through ELISA (enzyme-linked immunosorbent assay) method, general Salmonella and capnocytophaga gingivalis mixing specific IgY were in conjunction with capnocytophaga gingivalis in the middle of specificity was anti-, the specificity that the biological value of middle general Salmonella hybrid antigen reaches in every milligram of albumen resists tiring more than or equal to 1: 16000 of middle general Salmonella and capnocytophaga gingivalis mixing specific IgY, promptly tire 〉=1: 16000/mg albumen claims that perhaps specificity resists the ratio work of general Salmonella in centre and capnocytophaga gingivalis mixing specific IgY to be 〉=1: 16000 in every milligram of albumen of pure product;
Parts such as the oral spray of general Salmonella in anti-centre of producing by method of the present invention and the preparation of capnocytophaga gingivalis mixing specific IgY, instillation, buccal tablet, toothpaste impose on oral cavity, gingiva, periodontal pocket etc. and locate, combine the growth that has suppressed antibacterial with the target bacteria surface antigen, repelled the existence of antibacterial, promoted phagocyte such as segmented cell and macrophage engulfing and remove antibacterial at inflammation part.There are not simultaneously the limitation and the side effect of systemic administration yet, have again that local application is rapid-action, dosage is few, good effect, do not absorb, do not destroy oral cavity, gingiva, periodontal tissue's normal flora ecological environment, do not cause effects such as local dysbacteriosis, also have no side effect, more do not have the local problems such as easily producing drug resistance of using of antibiotics.IgY antibody is the activating complement system not, does not produce non-specific binding with rheumatoid factor, fixing staphylococcal protein A not, and not with Protein G, and human cell Fc receptors bind, thereby be difficult for taking place allergy.General Salmonella and capnocytophaga gingivalis mixing specific IgY extract do not produce acute toxic reaction and chronic toxicity reaction in the middle of anti-, belong to actual nontoxic level; General Salmonella and capnocytophaga gingivalis mixing specific IgY extract three cause the test feminine gender in the middle of anti-, belong to no teratogenesis, carcinogenic, mutagenic security article.
The specific embodiment:
Describe implementation of the present invention in detail below in conjunction with embodiment.
Embodiment 1:
General Salmonella and capnocytophaga gingivalis mixing specific IgY in the middle of anti-, wherein: get capnocytophaga gingivalis strain specific IgY30～60 parts, middle general Salmonella strain specific IgY40～70 parts mixes.
General Salmonella and the capnocytophaga gingivalis mixing specific IgY application in the preparation of preparation prevention and treatment halitosis and gingiva in the middle of anti-.
The preparation method of general Salmonella and capnocytophaga gingivalis mixing specific IgY in the middle of anti-, wherein:
A bacterial species: capnocytophaga gingivalis and middle general Salmonella;
Source of antibacterial and cultivation: capnocytophaga gingivalis and middle general Salmonella, from the isolating capnocytophaga gingivalis of periodontal disease patient oral cavity periodontal pocket inflammation part, middle general Salmonella, and the capnocytophaga gingivalis ATCC 33624 of standard and middle general Salmonella ATCC 25261 bacterial strains, through the capnocytophaga gingivalis of BHI-S culture medium separation and Culture and amplification cultivation, middle general Salmonella, and confirm as capnocytophaga gingivalis, middle general Salmonella through evaluation;
The B immunogen preparing:
Get 30～60 parts of the fragments of capnocytophaga gingivalis thalline, the fragment of middle general Salmonella thalline is mixed into bacterium liquid for 40～70 parts;
Isopyknic bacterium liquid is mixed with isopyknic Fu Shi Freund's complete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil Fu Shi Freund's complete adjuvant capnocytophaga gingivalis, middle general Salmonella mixed immunity is former.
Perhaps isopyknic bacterium liquid is mixed with isopyknic freund 's incomplete adjuvant, use the high speed agitator stirring and emulsifying, finally make Water-In-Oil freund 's incomplete adjuvant capnocytophaga gingivalis, middle general Salmonella mixed immunity is former;
All contain antibacterial 2,000,000,000-6,000,000,000 during the former and freund 's incomplete adjuvant mixed immunity of every milliliter of Fu Shi Freund's complete adjuvant mixed immunity is former;
The C immunity:
Use syringe to give bird inlay with the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity, every bird inlay injection 1.5ml-2.0ml immunogen, use the subcutaneous and intramuscular injection of the former 1.5ml-2.0ml of freund 's incomplete adjuvant mixed immunity to give bird inlay then week about, behind the booster immunization secondary; After this former 1ml of freund 's incomplete adjuvant mixed immunity is subcutaneous to give bird inlay booster immunization four times with intramuscular injection every re-using in eight weeks, front and back immune seven times altogether.
The preparation of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract in the middle of D is anti-:
Collect immunity the 30th day the immune hen egg in immunity back for the first time, the ethanol of use 70% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and distilled water by volume 1: the ratio adding distil water of 7-9, fully stir, and use 1N HCl to regulate acid-base value to pH5.0-5.2, place then 4 ℃ 12 hours.
Then with egg yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, with egg yolk diluent supernatant place molecular cut off be 100KD hollow fiber membrane ultrafiltration device ultrafiltration and concentration 5-10 doubly, the hibitane that adds 0.02%-2.0% then fully stirs, and use 1N NaOH to regulate acid-base value to pH7.0-7.5, place 4 ℃ 12 hours, after this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance, with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ-0.22 μ, promptly get resist in the middle of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract.
Embodiment 2: the fragment of getting the capnocytophaga gingivalis thalline separately is as bacterium liquid; Prepare anti-capnocytophaga gingivalis specific IgY extract separately.All the other preparations are with reference to embodiment 1.
Embodiment 3: the fragment of general Salmonella thalline is as bacterium liquid in the middle of getting separately; General Salmonella specific IgY extract in the middle of preparation resists separately.All the other preparations are with reference to embodiment 1.
Embodiment 4: get the anti-centre general Salmonella specific IgY extract of embodiment 2,3 gained, anti-capnocytophaga gingivalis specific IgY extract.By anti-capnocytophaga gingivalis strain specific IgY30 part, anti-middle general Salmonella strain specific IgY70 part mixes.
All the other preparations are with reference to embodiment 1.
Embodiment 5: get the anti-centre general Salmonella specific IgY extract of embodiment 2,3 gained, anti-capnocytophaga gingivalis specific IgY extract.By anti-capnocytophaga gingivalis strain specific IgY40 part, anti-middle general Salmonella strain specific IgY60 part mixes.
Embodiment 6: get the anti-centre general Salmonella specific IgY extract of embodiment 2,3 gained, anti-capnocytophaga gingivalis specific IgY extract.By anti-capnocytophaga gingivalis strain specific IgY35 part, anti-middle general Salmonella strain specific IgY65 part mixes.
Embodiment 7:
General Salmonella and capnocytophaga gingivalis mixing specific IgY extract production process flow process in the middle of preparation 500 grams are anti-:
(1) antibacterial culturing:
Capnocytophaga gingivalis of standard (ATCC 33624) and middle general Salmonella bacterial strains such as (ATCC 25261) are through the capnocytophaga gingivalis of BHI-S culture medium separation and Culture and amplification cultivation, middle general Salmonella, and confirm as capnocytophaga gingivalis, middle general Salmonella, and collect antibacterial through evaluation;
(2) immunogen preparing:
With above-mentioned thalline through the carbolic acid inactivation treatment.In addition,, the antibacterial thalline is pulverized be cracked into fragment tiny and/or molecule antibacterial thalline Ultrasonic Pulverization 30 minutes with the ultrasonic grinding machine.
Capnocytophaga gingivalis thalline, middle general Salmonella thalline and capnocytophaga gingivalis body fragment, 50: 50 quantity parts of middle general Salmonella bacterial chip are mixed, wherein the ratio of antibacterial thalline and bacterial chip is 60 parts of antibacterial thalline, 40 parts of bacterial chips.
The bacterium liquid of 400ml is mixed with the Fu Shi Freund's complete adjuvant of 400ml, use the high speed agitator stirring and emulsifying, make the Water-In-Oil freunds complete adjuvant immunogen.The preparation of freunds incomplete adjuvant immunogen: the bacterium liquid of 400ml is mixed with the freund 's incomplete adjuvant of 400ml, use the high speed agitator stirring and emulsifying, make the Water-In-Oil freunds incomplete adjuvant immunogen.Contain antibacterial 2,500,000,000 in every milliliter of immunogen;
(3) immune bird inlay:
Use syringe the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to be given the bird inlay in 28 weeks, every injection 2.0ml immunogen, freunds incomplete adjuvant immunogen 2.0ml is subcutaneous to give bird inlay with intramuscular injection every using in a week then, behind the booster immunization secondary, after this freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization totally four times with intramuscular injection every re-using in eight weeks, front and back immune seven times altogether, immune hen adds up to 300;
(4) preparation of anti-middle general Salmonella and capnocytophaga gingivalis mixing specific IgY extract:
Collect immunity the 50th～70 day the immune hen egg in immunity back for the first time, the ethanol of use 70% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and 1: 9 by volume ratio adding distil water of distilled water, fully stir, and use 1N HCl to regulate acid-base value to pH5.0, place then 4 ℃ 12 hours;
Then with egg yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, supernatant is placed 10 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of molecular cut off 100KD, adding 1.0% hibitane then fully stirs, and use 1N NaOH to regulate acid-base value to pH7.0, and place 4 ℃ 12 hours.After this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance, with the sterilization filtering with microporous membrane degerming of supernatant through 0.45 μ-0.22 μ, promptly get resist in the middle of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract.General Salmonella and capnocytophaga gingivalis mixing specific IgY extract are measured through ELISA (enzyme-linked immunosorbent assay) method in the middle of anti-, tiring of specific IgY antibody in the extract in every milligram of albumen is 1: 4000/mg albumen claims perhaps in the extract that the ratio work of the specific IgY antibody in every milligram of albumen is 1: 4000.In IgY antibody extract, the purity of IgY antibody is determined as 33.5% through irreducibility SDS-PAGE.
Embodiment 8:
The general Salmonella and the pure product technological process of production of capnocytophaga gingivalis mixing specific IgY in the middle of preparation 500 grams are anti-:
(1) antibacterial culturing:
Capnocytophaga gingivalis of standard (ATCC 33624) and middle general Salmonella bacterial strains such as (ATCC 25261) are through the capnocytophaga gingivalis of BHI-S culture medium separation and Culture and amplification cultivation, middle general Salmonella, and confirm as capnocytophaga gingivalis, middle general Salmonella, and collect antibacterial through evaluation;
(2) immunogen preparing:
With above-mentioned thalline through the carbolic acid inactivation treatment.In addition,, the antibacterial thalline is pulverized be cracked into fragment tiny and/or molecule antibacterial thalline Ultrasonic Pulverization 30 minutes with the ultrasonic grinding machine.
Capnocytophaga gingivalis thalline, middle general Salmonella thalline and capnocytophaga gingivalis body fragment, 60: 40 quantity parts of middle general Salmonella bacterial chip are mixed, wherein the ratio of antibacterial thalline and bacterial chip is 50 parts of antibacterial thalline, 50 parts of bacterial chips.
The bacterium liquid of 300ml is mixed with the Fu Shi Freund's complete adjuvant of 300ml, use the high speed agitator stirring and emulsifying, make the Water-In-Oil freunds complete adjuvant immunogen.The preparation of freunds incomplete adjuvant immunogen: the bacterium liquid of 300ml is mixed with the freund 's incomplete adjuvant of 300ml, use the high speed agitator stirring and emulsifying, make the Water-In-Oil freunds incomplete adjuvant immunogen.Contain antibacterial 5,000,000,000 in every milliliter of immunogen;
(3) immune bird inlay:
Use syringe the former subcutaneous and intramuscular injection of Fu Shi Freund's complete adjuvant mixed immunity to be given the bird inlay in 26 weeks, every injection 1.5ml immunogen, freunds incomplete adjuvant immunogen 1.5ml is subcutaneous to give bird inlay with intramuscular injection every using in a week then, behind the booster immunization secondary, after this freunds incomplete adjuvant immunogen 1ml is subcutaneous to give bird inlay booster immunization totally four times with intramuscular injection every re-using in eight weeks, front and back immune seven times altogether, immune hen adds up to 300;
(4) preparation of anti-middle general Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY:
Collect immunity the 60th～80 day the immune hen egg in immunity back for the first time, the ethanol of use 70% carries out the egg surface sterilization, open eggshell and get egg yolk, remove membrane of yolk and get egg yolk liquid, with egg yolk liquid and 1: 9 by volume ratio adding distil water of distilled water, fully stir, and use 1N HCl to regulate acid-base value to pH5.05, place then 4 ℃ 12 hours.
Then with egg yolk diluent high speed centrifugation 10000rpm, 30 minutes, get supernatant, supernatant is placed 10 times of the hollow fiber membrane ultrafiltration device ultrafiltration and concentration of molecular cut off 100KD, adding 2.0% hibitane then fully stirs, and use 1N NaOH to regulate acid-base value to pH7.03, and place 4 ℃ 12 hours.After this high speed centrifugation 10000rpm, 15 minutes, get supernatant, the weeding of grease metallic substance, promptly get resist in the middle of general Salmonella and capnocytophaga gingivalis mixing specific IgY extract.
The saturation that the general Salmonella in anti-centre after the weeding of grease metallic substance and capnocytophaga gingivalis mixing specific IgY extract is added sulphuric acid amine to 55%, fully stir, room temperature left standstill 4 hours, high speed centrifugation 10000rpm is 10 minutes then, get precipitation, to precipitate and use distilled water diluting to the original volume amount, the saturation that adds sulphuric acid amine to 33% again, fully stir, room temperature left standstill 4 hours, and high speed centrifugation 10000rpm is 10 minutes then, get precipitation, with the bag filter of packing into after the distilled water diluting dissolving, use the distill water dialysis desalination, promptly get and resist general Salmonella in centre and the pure product of capnocytophaga gingivalis mixing specific IgY.General Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY are measured through the ELISA method in the middle of anti-, in the middle of anti-in general Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY the tiring of specific IgY antibody in every milligram of albumen be 1: 16000/mg albumen, perhaps claim anti-in the middle of in general Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY, the specific activity of the specific IgY antibody in every milligram of albumen is 1: 16000.In IgY pure antibody product, the purity of IgY antibody is determined as 93.6% through irreducibility SDS-PAGE.
Embodiment 9:
The oral spray of general Salmonella and capnocytophaga gingivalis mixing specific IgY in the middle of anti-:
General Salmonella and capnocytophaga gingivalis mixing specific IgY extract 0.20% in the middle of anti-
Mannitol 5.0%
Hibitane 0.001%
Distilled water is supplemented to 100% and is total to 100ml.
The 60ml distilled water is added in the beaker, add mentioned component more successively, at last distilled water is supplemented to 100ml, the sterilization filtering with microporous membrane degerming through 0.22, sterile filling is gone in the jar of disinfectant spray.
All the other repeat embodiment 5,6,7 respectively.
Embodiment 10:
The instillation of general Salmonella and capnocytophaga gingivalis mixing specific IgY in the middle of anti-:
General Salmonella and capnocytophaga gingivalis mixing specific IgY extract 0.30% in the middle of anti-
Mannitol 5.0%
Chlorhexidine 0.002%
Distilled water is supplemented to 100% and is total to 100ml.
The 60ml distilled water is added in the beaker, add mentioned component more successively, at last distilled water is supplemented to 100ml, through the sterilization filtering with microporous membrane degerming of 0.22 μ, sterile filling is gone in the bottle of disinfectant nasal drop.
All the other repeat embodiment 4,5,6 respectively.
Embodiment 11:
The buccal tablet of general Salmonella and capnocytophaga gingivalis mixing specific IgY in the middle of anti-:
General Salmonella and capnocytophaga gingivalis mixing specific IgY extract 1.0% in the middle of anti-
Carboxymethyl cellulose 0.15%
Magnesium stearate 0.10%
Sorbitol is supplemented to 100% and is total to 100g.
Above-mentioned buccal tablet medicine low temperature is granulated, be pressed into difform buccal tablet then, every 0.5g.
All the other repeat embodiment 4,5,6 respectively.
Embodiment 12:
The spray and the instillation of general Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY in the middle of anti-:
General Salmonella and the pure product 0.1% of capnocytophaga gingivalis mixing specific IgY in the middle of anti-
Mannitol 5.0%
Hibitane 0.002%
Distilled water is supplemented to 100% and is total to 100ml.
The 60ml distilled water is added in the beaker, add mentioned component more successively, at last distilled water is supplemented to 100ml, through the sterilization filtering with microporous membrane degerming of 0.22 μ, sterile filling is gone in the jar of disinfectant spray and instillation.
All the other repeat embodiment 4,5,6 respectively.
Embodiment 13:
The buccal tablet of general Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY in the middle of anti-:
General Salmonella and the pure product 0.15% of capnocytophaga gingivalis mixing specific IgY in the middle of anti-
Carboxymethyl cellulose 0.15%
Magnesium stearate 0.10%
Sorbitol is supplemented to 100% and is total to 100g.
Above-mentioned buccal tablet medicine low temperature is granulated, be pressed into difform buccal tablet then, every 0.5g.
All the other repeat embodiment 4,5,6 respectively.
Experimental example 1:
The immunogen of two kinds of bacteria combination such as capnocytophaga gingivalis, middle general Salmonella is by method immunity bird inlay of the present invention, get the egg of 30-40 days the immune hen in immunity back for the first time, and by method of the present invention prepare anti-in the middle of the extract of general Salmonella and capnocytophaga gingivalis mixings specific IgY, use the ELISA method and measure anti-middle general Salmonella and capnocytophaga gingivalis mixing specific IgY in conjunction with different antigenic biological values (seeing Table 1):
Tiring of table 1 capnocytophaga gingivalis, anti-middle general Salmonella mixed IgY antibody extract
| Mixed IgY antibody | Detect antigen | The IgY antibodies is tired |
| Anti-capnocytophaga gingivalis, middle general Salmonella antibody | Capnocytophaga gingivalis | ＞1: 2000/mg albumen |
| Middle general Salmonella | ＞1: 2000/mg albumen | |
| Capnocytophaga gingivalis, middle general Salmonella hybrid antigen | ＞1: 4000/mg albumen |
Can be clear that according to above-mentioned experimental result: (1). capnocytophaga gingivalis, two kinds of bacterium thalline of middle general Salmonella+bacterial chip immunogen immune bird inlay can produce the specific IgY antibody of anti-two kinds of antibacterials respectively; (2). generation to the specific IgY antibody of various antibacterials tire not with immune bird inlay the time every kind of antibacterial dosage of using change, that is: be not that tiring of the big bacteriogenic IgY antibody of immunizing dose is also high, this is relevant with immunogenicity of antigens; (3). the hybrid antigen of capnocytophaga gingivalis, middle general Salmonella mixed IgY antibody and capnocytophaga gingivalis, middle general Salmonella can both react its superposition of having tired; (4). by above-mentioned experiment as seen, general Salmonella in a kind of anti-centre and capnocytophaga gingivalis mixing specific IgY can be treated by above-mentioned bacterial halitosis, halitosis and gingivitis etc.; (5). also can treat by any bacterial halitosis, halitosis and gingivitis etc. in the above-mentioned antibacterial simultaneously with general Salmonella in a kind of anti-centre and capnocytophaga gingivalis mixing specific IgY; (6). can also expand to thus by bacterial halitosis, halitosis and the gingivitis etc. of common antigenic determinant being arranged, can use the treatment of general Salmonella in anti-centre of the present invention and capnocytophaga gingivalis mixing specific IgY with above-mentioned antibacterial.
Experimental example 2:
Capnocytophaga gingivalis, the former immune bird inlay of method of the present invention of pressing of middle general Salmonella mixed immunity, get the egg of 80-100 days the immune hen in immunity back for the first time, and by method of the present invention prepare anti-in the middle of the pure product of general Salmonella and capnocytophaga gingivalis mixings specific IgY, use the ELISA method and measure anti-middle general Salmonella and capnocytophaga gingivalis mixing specific IgY in conjunction with different antigenic biological values (seeing Table 2):
General Salmonella and the pure product of capnocytophaga gingivalis mixing specific IgY tired in the middle of table 2 was anti-
| Mixed IgY antibody | Detect antigen | The IgY antibodies is tired |
| The antibody of anti-capnocytophaga gingivalis, middle general Salmonella thalline+fragment | Capnocytophaga gingivalis | ＞1: 8000/mg albumen |
| Middle general Salmonella | ＞1: 8000/mg albumen | |
| Capnocytophaga gingivalis, middle general Salmonella thalline hybrid antigen | ＞1: 16000/mg albumen |
Can be clear that according to above-mentioned experimental result: (1). by method of the present invention prepare anti-in the middle of the pure product of general Salmonella and capnocytophaga gingivalis mixings specific IgY than preparing the specificity of extract of anti-general Salmonella in centre and capnocytophaga gingivalis mixing specific IgY in conjunction with capnocytophaga gingivalis and middle general Salmonella hybrid antigen by method of the present invention in the experimental example 1, and the antigenic separately biological values of antibacterial such as capnocytophaga gingivalis, middle general Salmonella all want high, are to increase in proportion substantially; (2). capnocytophaga gingivalis, the former immune bird inlay of middle general Salmonella mixed immunity can produce the specific IgY antibody of anti-two kinds of antibacterials respectively; (3). generation to the specific IgY antibody of various antibacterials tire not with immune bird inlay the time every kind of antibacterial dosage of using change, that is: it is also high to be not that the big antibacterial of immunizing dose produces tiring of IgY antibody, and this is relevant with immunogenicity of antigens; (4). the hybrid antigen of two kinds of bacterium such as general Salmonella and capnocytophaga gingivalis mixing specific IgY and capnocytophaga gingivalis, middle general Salmonella can both react in the middle of anti-, and total tiring of specific IgY antibody also increased its superposition of having tired; (5). by above-mentioned experiment as seen, general Salmonella in a kind of anti-centre and capnocytophaga gingivalis mixing specific IgY can be treated by above-mentioned bacterial halitosis, halitosis and gingivitis etc.; (6). also can treat by any bacterial halitosis, halitosis and gingivitis etc. in the above-mentioned antibacterial simultaneously with general Salmonella in a kind of anti-centre and capnocytophaga gingivalis mixing specific IgY; (7). can also expand to thus by bacterial halitosis, halitosis and the gingivitis etc. of common antigenic determinant being arranged, can use prevention of general Salmonella in anti-centre of the present invention and capnocytophaga gingivalis mixing specific IgY and treatment with above-mentioned antibacterial.
Experimental example 3:
The capnocytophaga gingivalis of preparing by method of the present invention, thalline and the capnocytophaga gingivalis of middle general Salmonella, two kinds of immunogen immune bird inlays such as thalline+bacterial chip of middle general Salmonella, and prepare anti-capnocytophaga gingivalis respectively, middle general Salmonella thalline mixed IgY antibody extract and anti-capnocytophaga gingivalis, middle general Salmonella thalline+bacterial chip mixed IgY antibody extract, use the ELISA method and measure their anti-capnocytophaga gingivalises, middle general Salmonella hybrid antigen in conjunction with biological value, the results are shown in Table 4:
The different antigenic biological values of the inductive hen specificity of the different immunogens of table 4 mixed IgY antibodies
Can be clear that according to above-mentioned experimental result: under identical immune condition, prepare capnocytophaga gingivalis by method of the present invention, middle general Salmonella thalline and anti-capnocytophaga gingivalis, two kinds of different immunogen immune laying hens such as middle general Salmonella thalline+bacterial chip, and prepare the anti-capnocytophaga gingivalis of the egg of the 80-100 days immune hens in immunity back for the first time, middle general Salmonella thalline mixed IgY antibody extract and anti-capnocytophaga gingivalis, middle general Salmonella thalline+two kinds of IgY antibody such as bacterial chip mixed IgY antibody extract.These two kinds of IgY antibody react with capnocytophaga gingivalis, middle general Salmonella hybrid antigen respectively, these two kinds of IgY antibody biologys, the biology of wherein anti-capnocytophaga gingivalis, middle general Salmonella thalline+bacterial chip mixed IgY antibody was higher in conjunction with tiring in conjunction with tiring certain difference as a result.These presentation of results capnocytophaga gingivalises, the immunogenic immune effect of middle general Salmonella thalline+bacterial chip are better than simple capnocytophaga gingivalis, the immunogenic immune effect of middle general Salmonella thalline.
Experimental example 4:
Biological value is 1: 32000/mg, purity of protein is 93.1% the anti-middle general Salmonella (P.i of specificity, ATCC 25261) IgY antibody respectively with capnocytophaga gingivalis (Cg, ATCC 33624), actinobacillus actinomycetem comitans (Aa, ATCC 29523), Fusobacterium nucleatum (Fn, ATCC 10953), immunological cross-reaction takes place in porphyromonas gingivalis (Pg, ATCC 33277), the results are shown in Table 10:
General Salmonella IgY antibody and other oral cavity anaerobium cross reaction biological value in the middle of table 10 is anti-
| Antibacterial | General Salmonella IgY is in conjunction with tiring in the middle of anti- |
| Capnocytophaga gingivalis | 1: 1000/mg albumen |
| Fusobacterium nucleatum | 1: 1000/mg albumen |
| Porphyromonas gingivalis | 1: 100/mg albumen |
| Companion actinomycetes Actinobacillus | 1: 8000/mg albumen |
Can be clear that according to above-mentioned experimental result: behind method immunity bird inlay of the present invention, and prepare the pure product of the anti-centre general Salmonella IgY antibody of the egg of the 30-40 days immune hens in immunity back for the first time by method of the present invention, its can with capnocytophaga gingivalis, and other oral cavity anaerobium generation immunological cross-reaction.This proves clearly common antigenic determinant between these oral cavity anaerobiums, identical antigenic determinant is many more, and cross reaction is just strong more.Therefore, anti-middle general Salmonella and capnocytophaga gingivalis mixing specific IgY preparation can prevent and treat bacterial halitosis, halitosis and the gingivitis etc. that have common antigenic determinant by above-mentioned.
Experimental example 5:
The clinical trial of halitosis
One, rule
1. checking of the effect of the halitosis product that carries out first or the effect checking that contains the halitosis product of novel active composition should be confirmed by more than one separate clinical trials.
2. when product contains the composition of the halitosis by clinical verification, can confirm its effectiveness by the equivalence that proves this product and the clinical product of having verified.The product of test group and matched group (positive control) should contain the same active component of same concentrations.In addition, matched group also should comprise blank or placebo.As fruit product is a kind of instrument of mechanical removal, and matched group should be the daily cleaning of brushing teeth of naturalness.
3. contain in the time of might influencing the active excipient of halitosis (non-active ingredient), the effect of product should be verified by clinical trial.
4. the applicant need provide the biological activity and the stability of active component in the material proof prescription.
Two, research design
Generally should adopt blind method, layering, the parallel test of two units or cross matching method.
Three, duration of trial
Should determine by the effect intensity of being examined product.Can be a few hours to several weeks.
Four, object of study:
The experimenter be meet include in and exclusion standard suffer from mouthful masculinity and femininity of source property halitosis.
General every group of crowd's quantity of finishing the test overall process should be more than or equal to 12 people.
Five, check index or employing index
Can adopt following two kinds of evaluation methodologys simultaneously:
1. sensory evaluation (being the olfactory sensation scoring): the implication of utilizing the index evaluation experimenter that effectively keeps the score by well-trained appraiser.Before the test evaluator is proofreaied and correct experiment.
Examiner and person under inspection's distance are about 10 centimetres, place a shielded partitions between the two.The person under inspection uses a mouthful outwards expiration, and the examiner marks respectively by subjective feeling, can adopt the grade of 0-5, and standards of grading are as follows: 0: discover less than abnormal smells from the patient; 1: can perceive abnormal smells from the patient reluctantly, not much, if any; 2: slight but can clearly discover; 3: moderate odor; 4: the severe stink; 5: extremely strong insupportable stench.The average of getting examiner scoring as a result of.
2. apparatus measures: the effective analytical tool that adopts assessment mainly to cause smelly factor is measured (as implication measuring instrument Halimeter)
The implication measuring instrument utilizes electrochemical principle, and the concentration of volatile sulfur compounds in the gas of the oral cavity form with numerical value is showed, and can be used as the index of weighing patient's implication level.Earlier with the surrounding air zeroing, measure after advising the experimenter to remain silent three minutes before using, the experimenter use nasal respiration during measurement, mouthful parts a little, and the gas collection mouth of pipe is placed on 0.5cm place, back 1/3rd intersections top in the back, the numerical value on reading displayed is shielded.Each triplicate, results averaged checked.
Six, effect assessment:
Press the medical statistics method, to the variation and the instrument readings Change in Mean situation of test group and the forward and backward implication value of matched group difference statistical test mean; And the relatively variation of each time period, each index between two groups.
General Salmonella and capnocytophaga gingivalis mixing specific IgY suppressed the experiment effect of halitosis in the middle of table 11 was anti-:
Using method: get 1 milliliter of the solution of (1), (2), (3), (4), (5), (6), sparging the halitosis rank is in level Four (severe stink) patient's the oral cavity, and rinsing the mouth three minutes, after one month course of treatment, is estimated therapeutic effect at every day three times.
Patient's number: 72 people are divided into six groups, every group 12 people at random.
| Antibacterial | Halitosis number rank (4 grades) before using | Use back halitosis number (0 grade) (1 grade) (2 grades) (3 grades) (4 grades) (5 grades) | |||||
| (1) group | 12 | 4 | 7 | 1 | 0 | 0 | 0 |
| (2) group | 12 | 0 | 6 | 5 | 1 | 0 | 0 |
| (3) group | 12 | 1 | 9 | 2 | 0 | 0 | 0 |
| (4) group | 12 | 0 | 0 | 2 | 6 | 4 | 0 |
| (5) group | 12 | 0 | 0 | 4 | 7 | 1 | 0 |
| (6) group | 12 | 0 | 0 | 0 | 1 | 11 | 0 |
(1) group: get capnocytophaga gingivalis strain specific IgY35 part, middle general Salmonella strain specific IgY65 part is mixed, and adds 100,000 parts of distilled water and forms.
(2) group: get capnocytophaga gingivalis strain specific IgY100 part, add 100,000 parts of distilled water and form.
(3) group: general Salmonella strain specific IgY100 part in the middle of getting adds 100,000 parts of distilled water and forms.
(4) group: get dwell 100 parts of treponema dentium specific IgYs of Treponemas denticola, add 100,000 parts of distilled water and form.
(5) group: get 100 parts of Mutans streptococci streptococci specific IgYs, add 100,000 parts of distilled water and form.
(6) group: distilled water.
Claims (2)
1. general Salmonella and capnocytophaga gingivalis mixings specific IgY in the middle of anti-is characterized in that: get anti-capnocytophaga gingivalis strain specific IgY30～60 parts, the anti-general Salmonella strain specific IgY40 in centre～70 parts mixes.
2. claim 1 general Salmonella in described anti-centre and the capnocytophaga gingivalis mixing specific IgY application in the preparation of preparation prevention and treatment halitosis and gingiva.
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