CN1398531A - Immunological milk with several health functions - Google Patents
Immunological milk with several health functions Download PDFInfo
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- CN1398531A CN1398531A CN 01126326 CN01126326A CN1398531A CN 1398531 A CN1398531 A CN 1398531A CN 01126326 CN01126326 CN 01126326 CN 01126326 A CN01126326 A CN 01126326A CN 1398531 A CN1398531 A CN 1398531A
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Abstract
The present invention relates to one kind of multifunctional disease preventing immunological milk and union immunological method. Subunit vaccine and deactivated pathogenic bacteria through special culture are used to inoculate milk to obtain specific pathogenic bacteria milk antibody. The selected bacterial includes pathogenic bacteria of chronic gastropathy, gastric cancer, gingivitis, periodontitis, pharyngitis, etc. The said method can obtain high specific antibody with high potency and the immunological milk is especially suitable for old people, infant and valetudinarian.
Description
The present invention relates to a kind of multi-functional diseases prevention immune milk food and combined immunization method.Pathogenic bacteria with subunit vaccine, specific culture inoculate milk cow after deactivation, obtain special pathogenic bacteria milk antibody.The institute.
Selected pathogenic bacteria are a large amount of popular chronic gastropathy, cancer of the stomach, gingivitis, periodontitis, tooth dental caries disease, sphagitis, halitosis, acnes.This method obtains that the antibody specificity is good, the height of tiring, easily promote.These immunity milk products especially are fit to old children.
Although there is a large amount of antibiotic to be used for tackling the human infection,, still there are many chronic infection diseases and discomfort now, tormenting a large amount of crowds.
The caries prevalence rate per capita of China is 40% at present, and wherein children's caries prevalence rate is 80%, and young people's caries prevalence rate is 60-70%, and the elderly's caries prevalence rate is 52%.The gingivitis of China's population and periodontitis illness rate are 70%, and pharyngo-laryngitis chronica disease is also very general.Modern study proves, chronic gastritis, gastric ulcer, duodenal ulcer etc. are main relevant with helicobacter pylori infections, cancer of the stomach, gastric lymphoma be first verified be the tumour relevant with bacterial infection.This bacterial infection is exactly the virulence helicobacter pylori infections.Sphagitis and last sense, halitosis, acne be common disease especially.These diseases have a strong impact on individual diet ability and are harmful to one's health then and the labour.These diseases are depended merely on treat-and-release and are difficult to finish, and should find out the effective measures of prevention and control.Discover that these frequently-occurring diseases all belong to bacterial infection disease.There are some bacterium of being everlasting in oral cavity and alimentary canal under the normal condition, and the disease pathogenic bacteria that have also are the bacterium of being everlasting, and each microorganism is in kind with quantitatively constitute the combination of certain physiology when health status.Certain pathogenic stronger germ excessively multiplies and causes disease when flora imbalance, or the infection of bacterium dystopy causes disease.Long-term broad-spectrum antibiotic and the chemical synthetic drug of using will destroy normal microecological balance simultaneously in the removing pathogenic bacteria.Still unmatchful so far these pathogenic bacteria have single-minded optionally medicine.
Immunological method is widely used in bacteriological detection at present owing to have high specificity.Aspect disease preventing and treating, tetanic control etc. is arranged.Above-mentioned some diseases all belongs to mucosal infections, and the control that immunological method is used for these diseases has certain advantage.About the existing nearly 40 years history of the research of immune diseases prevention.Result of study so far thinks that the passive immunity method is safer, is better than active immunity.Passive immunity is the pathogenic effects of direct using specific antibody with neutralization and antagonism specificity pathogenic bacteria.Owing to may avoid some unexpected side effect of active immunity, as can not the activating system immune response, can be because of system employs does not cause the heart cross reaction, therefore easily accepted by people and receive publicity day by day.From action effect, polyclonal antibody is better than monoclonal antibody.At first attempted with the how anti-immune anticaries research in the milk as (1987) such as Michalek.With 4 kinds of full bacterium antigen immunes of serotype of mutans streptococcus milk cow, the bacterium mouse is decided in the milk nursing that acquisition is contained specificity resistance chain bacteria antibody, and the result shows, the reduction of the active all conspicuousnesses of experimental group animal mutans streptococcus adhesion level, plaque index and carious tooth.About the preventing decayed tooth mechanism of passive immunity, infer at present monoclonal antibody and many anti-may be physicochemical property by influencing bacterium surface such as electric charge, surface free energy, hydrophobicity etc., then may play opsonic action in vivo and the adhesion of bacterium is exerted an influence.But (1996) such as Van Raamsdonk are discovered, not as good as how anti-, and in vitro study shows that monoclonal antibody acts on mutans streptococcus to monoclonal antibody to the influence of mutans streptococcus surface characteristic, and can not resemble manyly stimulates polymorphonuclear leukocyte that it is engulfed and lethal effect anti-.A large amount of at present production monoclonal antibodies are used for common oral liquid certain difficulty.
Chronic gastritis, gastric ulcer, duodenal ulcer, cancer of the stomach, gastric lymphoma etc. are main relevant with helicobacter pylori infections.The campylobacter jejuni main parasitic is in domestic animals and fowls enteron aisle and ight soil, in milk and the water.After human body intestinal canal is infectd, can give birth to diarrhoeal diseases.The sick main and initial bacterium of dental caries is a mutans streptococcus.The sticking at first collection of this bacterium is at facing or dental groove, and decomposing sucrose produces glucan, and sticking again collection bacterium becomes bacterial plaque, secretion lactic acid corrosion dentine cavitation.Dental caries patient mutans streptococcus is too bred.The periodontosis for example main pathogenic bacteria of periodontitis, gingivitis is porphyromonas gingivalis, nuclear Fusobacterium, actinomyces viscosus, actinobacillus actinomycetem comitans, actinomyces naeslundii etc.They discharge virulence factor and cause inflammation.Cavity and periodontitis can cause pulpitis and develop into bacteremia even conditioned pathogen property endocarditis.The product thiamines bacterium that halitosis is human body be everlasting in the oral cavity when busy, tired, ill, hypoimmunity for example undue breeding such as helicobacter pylori, nuclear Fusobacterium, porphyromonas gingivalis institute causes.Acne is most of youthful worries.Especially in south.Discovering recently, mainly is that facial sebaceous gland duct grows propionibacterium acnes, and secretion lipase decomposes sebum, and producing propionic acid stimulates due to the narrow and inflammatory cell inflammation of mouth of pipe hyperplasia.These mainly are some facultative anaerobes.Secondary MRSE even staphylococcus aureus breeding and infection can increase the weight of the state of an illness.In sphagitis and the last sense mixed infection mainly is streptococcus, pneumococcus, MRSE and staphylococcus aureus, all produces toxin.Above bacterium is often to antibiotics resistance and repeated infection.The exploitation specific antibody helps preventing and treating this class disease.These thalline are united the special antibiotic milk of making vaccine and are not seen production as yet.
Milk daily life consumption is huge, and antibody activity preservation easily in milk, so milk is the ideal carrier based on the disease prevention and cure health care preparation of antibody.Because above-mentioned disease incidence is very high, the application of commodity has huge market.Up to the present, Shang Weijian makes the report of the immune milk of anti-multiple chronic disease of combined vaccine production and discomfort about using above-mentioned infection specific strain and subunit.
Multiple pathogen, multiple antigen mix stimulation, may occur each other disturbing, and influence the raising of tiring each other.
The thalline of the sick pathogenic bacteria mutans streptococcus of dental caries is made the milk that the antigen immune milk cow obtains resistance chain bacteria antibody, produces the rheumatism factor because of doubting, and is not applied to the preventing decayed tooth disease so far.The undope mutans streptococcus surface antigen subunit vaccine of human heart common antigen of production can be produced the milk of the no rheumatism factor.Subunit vaccine is made immunogene, exists to be difficult to improve to tire, and obtains the technical difficulty of productive milk products.
Thalline and subunit vaccine use in conjunction can produce mutual interference.Explore the inoculation method of above-mentioned vaccine compatibility.
The present invention overcomes above-mentioned technical difficulty exactly and captures an above-mentioned difficult problem, the disease prevention and cure health-care milk of development, the productive All-in-One of exploitation.
Essential implementation: one, preparation () antigen bacteria of associating antigen vaccine is selected good strains in the field for seed and is selected local popular virulent strain, compares the contrast strain with the international standard virulent strain
Drawn together with bracket.
1, the classification of bacterial strain and antigen: (1) dental caries sick relevant bacterial strain and antigen: 1. streptococcus mutans: (S.mutans Ingbritt) surface protein and glucosyltransferase be streptococcus sobrinus 2.: (S.sobriuns 6715) surface protein and glucosyltransferase
Culture of bacteria, biochemical method separates purification surface protein and glucosyltransferase.③： ( A.viscosus ATCC 19246 ) ( 2 ) ： ①： ( Fusobacterium nucleatum ATCC 25286 ) ②： ( Porphyromonas gingivalis ATCC 33277 ) ③： ( Actinomyces viscosus ATCC 19246 ) ④： ( Actinobacillus actinomycetemitans Y4 ( 3 ) ： ①： ( Helicobacter pylori ATCC 11639 ) ②： ( Fusobacterium nucleatum ATCC 25286 ) ③： ( Porphyromonas gingivalis ATCC 33277 ) ( 4 ) ： ① ( Hp ) ： ( Helicobacter pylori NCTC 11637 ) ②： ( Helicobacter pyloti NCTC 11639 ) ③：Helicobacter pylori* ④： ( 5 ) ： ①Staphylococcus pneumoniae* ②Staphylococcus epidermidis* ③ ( Staphylococcus aures ATCC 14458 ) ( 6 ) ： ① ( Propionibacterium acne NCTC 65102 ) ②Staphylococcus epidermidis* ③ ( Staphylococcus aures ATCC 14458 )
* be that local popular clinical classification is identified bacterial strain, microorganism teaching and research room of Shanghai Second Emdical University
(2) Bacteria Culture and vaccine preparation
(Tryptic Soy Broth TSB) is used for bacterium activation and increase bacterium to adopt trypsase digestion soya broth.(Tryptic Soy Agar TSA) is used for the pure branch of bacterium to the TSA culture medium.Anaerobic culture medium is used for obligate anaerobes and cultivates.The biochemical pipe of trace has sweet mellow wine, sorbierite, gossypose, aesculin, and arginine etc. are used for Bacteria Identification.The bacterium of preservation is inoculated in corresponding medium culture recovery activation, and anaerobism is cultivated 18 hours (37 ℃ of 90%N2 5%CO2) and 2-3 days (obligate anaerobe).The single colonies typical streak inoculation of picking is in the TSA flat board respectively, and anaerobism was cultivated after 48 hours, makes further Physiology and biochemistry and identifies.After biochemical identification, be inoculated in corresponding agar plate.The single bacterium colony of picking agar surface is suspended in the respective liquid culture medium that (1 1mm bacterium colony/ml) cultivate makes bacterial concentration be 10 after cultivating the corresponding time
5-6CFU/ml.Centrifugal (3000g * 30 ').Collect bacterium, it is inferior to give a baby a bath on the third day after its birth with the sodium phosphate buffer of the 0.01M pH6.0 that contains 0.15M NaCl.It is 3 * 10 that the bacterium of collecting is mixed with concentration with sterile water for injection
8The bacterium liquid of individual bacterium/ml.15 pounds made the thalline inactivation in 15 minutes, and-20 ℃ standby.Other method is that bacterium uses the sodium phosphate buffer of the 0.01M pH6.0 that contains 0.15M NaCl to give a baby a bath on the third day after its birth inferior behind 0.5% formalin-inactivated.Being mixed with concentration with sterile water for injection is 3 * 10
8The bacterium liquid of individual bacterium/ml, deposit after the equal portions packing-20 ℃ standby.
Epidemic strain separates to be identified: all make genotype identification with special primer with PCR method.
Hp separates: get people's cancer of the stomach mucous membrane and be coated with ware, roguing on the blood agar plate that contains polymyxins 2500u/L, vancomycin 10mg/L, 5mg/L.
Staphylococcus separates with streptococcus pneumonia: patient's impetigo fester is coated with ware, roguing on the plain agar plate.After morphology is identified, catalase reaction difference staphylococcus and streptococcus, clotting of plasma enzyme positive is selected golden Portugal bacterium, and the positive positive of ovobiocin is selected MRSE.Streptococcus pneumonia is selected in the Optochin sensitization test.
Propionibacterium acnes separates: whelk patient's acne fester is coated with ware, roguing on the anaerobism agar plate.After morphology was identified, the lipase reaction was selected.
Nuclear Fusobacterium, porphyromonas gingivalis, actinomyces viscosus, actinobacillus actinomycetem comitans bacillus separate: gingivitis patients bacterial plaque, subgingival plaque are coated with ware, and anaerobism is cultivated 18 hours (37 ℃ of 90%N2 5%CO2) or 2-3 days (obligate anaerobe).The single colonies typical streak inoculation of picking is in the TSA flat board respectively, and anaerobism was cultivated after 48 hours, makes further Physiology and biochemistry and identifies.
(3) streptococcus mutans and streptococcus sobrinus surface protein and glucosyl group
Transferase (GTF) purified antigen and subunit vaccine preparation
Select for use the mutans streptococcus S.mutans Ingbritt of international standard bacterial strain mutans streptococcus family, streptococcus sobrinus S.sobrinus6715 to be the operation bacterial strain.Adopt 5% sucrose to replace the trypsase of glucose to digest soya broth (TSB), little aerobic cultivation 18 hours (90%N2,5%CO2,37 ℃), centrifugal (3000g * 30 ').Add ammonium sulfate in the bacterium liquid and reach 60% saturation degree, 4 ℃ are spent the night.Centrifugal (9000g * 40 ', 4 ℃) sediment is the GTF crude extract.Surface protein with 8Mol urea precipitate crude extract.With pH6.04 ℃ of dialysis of 0.01M PBS, changed one time liquid in 12 hours, dialysis is not till have ammonium ion and detect.Polyethylene glycol (molecular weight 22000) is concentrated into 1.5mg pr/ml.Streptococcus mutans and streptococcus sobrinus surface protein and glucosyltransferase (GTF) are further purified, with Sepharose 6B dress post 1.6 * 85cm, crude extract 1.5mg pr/ml, 5ml/ last sample.With 0.01MPBS pH6.0 balance wash-out.8ml/h, the 5ml/ pipe contains the GTF component with the collection of Somagyi method.Dialysis concentrates freeze-drying.
Purity testing: (1) protein gel separation electrophoresis (SDSPAGE) is measured: prepare 12% separation gel, 5% and concentrate glue, 100 milliamperes of electric current electrophoresis of reducing process 3 hours.Examine the blue dyeing of Ma Shi, surface protein, GTF see four protein bands about 190KD, 160KD, 150KD, 50KD respectively.Add protein molecular scale, unpurified mutans streptococcus, streptococcus sobrinus mycoprotein on the vicinity in the sample hole, the latter two are respectively eight and nine major protein bands.
Freund's complete adjuvant: Valelinum Liquidum 8ml, lanolin 4ml, BCG vaccine (100mg/ml) 4ml.At first Valelinum Liquidum and lanolin are mixed through 15 pounds of sterilizations in 20 minutes.Add BCG vaccine down and grind to form emulsion aseptic then, it is fully essential to grind.Incomplete Freund: Valelinum Liquidum 10ml, lanolin 5ml grinds the back sterilization.Prepare 20% tween: get Tween 80 (Tween 80) 1g and add 0.01M PBS 50ml mixing.Preparation bovine serum albumin(BSA) (BSA) (10 mg/mL): get BSA50mg and add the PBS5ml mixing.
Get to freeze and be made into 1mg/ml, 0.15mg/ml, bacteriological filtration with 0.01M PBS pH6.0 in surface protein and GTF 12mg.Add the equivalent Freund's complete adjuvant and mix, making latex is the pulpous state multivalent subunit vaccine.
During inoculation, many thalline of use in conjunction inactivated vaccine and pulpous state multivalent subunit vaccine antigen vaccine prepare antibody milk by the following method.
Two, the preparation of antibody milk
(1) milk cow is selected blue or green middle aged producing cow for use, and requirement is healthy and strong, the output of milk is medium, and the common epidemic prevention already of the system milk of participation.
(2) livestock farm management (1) has nature epidemic disease source to isolate geographical environment and facility; (2) atmosphere and drinking-water water non-environmental-pollution; (3) feed resource is abundant and pollution-free; (4) cowshed cool in summer and warm in winter; (5) duty is swept and is washed; The biological harmful measure of going out is arranged, and (6) have scientific research facility and place; (7) record is complete.
(3) immunoreagent and inoculation
1, the preparation of surface protein and glucosyltransferase (GTF) milk whey antibody
Get the following application of pulpous state subunit vaccine:
Injection for the first time, every of ox back portion multiple spot injects the 5ml latax.80-200 μ g/ head;
After 15 days, inject the same for the second time;
Inject an injection for the third time later in month, latax 5ml intravenous injection first;
Last injection back one week-ten day blood sampling and milk are surveyed and are tired.
2, the preparation of other various full bacterium milk whey antibody
The deactivation thalline is made into 2 * 10
5Individual bacterium/ml, 2 * 10
6Individual bacterium/ml, 3 * 10
8The bacterium liquid of individual bacterium/ml.The milk cow immunization method is intramuscular injection.
Inject for the first time every every bacterium and inject 0.5ml, every two week back injections for the second time.Injection for the third time after a week.Every the 4th injection in week back.Adopting the milk survey after the per injection tires.This is an inoculation cycle.End after one to two months, once more the booster shot one-period.Blood sampling in one thoughtful ten day of last injection back and milk are surveyed and are tired.Three all after dates detect and confirm that the invalid milk cow of inoculation goes out production line.Later on can the same injection or the lymph node injection, or intravenous injection.
3, injection volume: get in the pulpous state subunit vaccine, every kind of each range of application of antigen is between 50～200 micrograms, and the each range of application of every kind of thalline of vaccine is at 1*10
5～1*10
9Between the CFU/ml.
Each cycle injects the back and obtained original immune milk on the the 7th to the 12 day.
(4) milk whey antibody is collected:
Adopt milk weekly one time, about 500 milliliters, it is anticorrosion to add 2/10000ths Sodium azides.Room temperature was placed after 1-2 hour, put 4 ℃ centrifugal 1000 rev/mins after totally one hour, got the middle level, adding hydrochloric acid acid adjustment basicity is 4.6, precipitation casein, 4 ℃ centrifugal 1000 rev/mins after totally one hour, get whey and add NaOH to transfer PH to reach 7.4 backs aseptic subpackaged, put preserve in-20 ℃ of refrigerators standby.
(5) titration
(1) surface protein and GTF milk whey antibody titer are measured and are adopted immune double diffusion method.Get GTF and be made into 100g/ml, get 2ml with 0.01M PBS pH6.0.Milk whey antibody 1ml, twice dilution 5 times.Prepare 1% agarose with physiological saline, add that 2/10000ths Sodium azides are anticorrosion.Water plate, burrow.Hatched 18 hours for 37 ℃ behind the adding sample, whether perusal has precipitation line.
(2) full bacterium milk whey antibody titer is measured the TA method that adopts.The continuous double dilution of milk whey antibody.Various full bacterial cells are diluted to 3 * 10 with physiological saline
5Individual/ml.Equal-volume whey and bacterium liquid mix, perusal aggegation result.
(3) enzyme linked immunological absorption (ELISA) (sending and receiving) are measured:
Each antigen coated ELISA Plate is measured hole, washes plate, sealing again after the sealing, immunity milk that doubles to dilute and contrast milk point sample incubation, and tagged anti-ox two is anti-, colour developing, ELIASA is surveyed the OD value.
Judge: tire=immunity milk OD/ contrast milk OD; Tire greater than becoming milk for immunity more than 1.6.
Three, felling and transporting of farm immune milk rarely joined, sterilizes, encapsulated
Behind the routine disinfection liquid prewashing nipple, machine squeezes immunity and suckles in jar, adorns cold fortune car behind the filtered through gauze impurity and send to be processed.Through rarely join with non-immunity milk, ultra high temperature short time sterilization routinely behind the homogeneous, envelope or canned.Rare mixing ratio is: 1 portion of immunity milk is joined 10,100,1000,10000 portions of non-immunity milk.
The benefit following points of the multifunction immunity milk that the present invention produced:
1, used bacterial classification adopt international standards strain and local epidemic isolates, special with strong points.
2, anti-odontopathy is is especially prevented and treated the dental caries disease, can also prevent and treat periodontitis, has also just reduced pulpitis septicemia and endocarditic possibility occurrence.Milk antibody is directly killed relevant pathogenic bacteria mutans streptococcus, porphyromonas gingivalis, nuclear Fusobacterium, actinomyces viscosus, actinobacillus actinomycetem comitans, actinomyces naeslundii and removing virulence factor, is neutralized a toxin.
3, anti-inflammatory and carcinous gastrointestinal disease be chronic gastritis, gastric ulcer, duodenal ulcer, cancer of the stomach, the relevant helicobacter pylori of gastric lymphoma for example.The milk antibody that the present invention produced can extensively neutralize and effectively remove cause of disease bacterium itself and free toxin, so preventive and therapeutic effect is arranged.Other stomach trouble also there is the effect of prevention and mitigation symptoms.
4, feel pathogenic bacteria for example streptococcus, pneumococcus, MRSE and staphylococcus aureus on the anti-pharyngitis.
5, anti-face or body acne pathogenic bacteria are propionibacterium acnes and free lipase, streptococcus, MRSE and staphylococcus aureus for example.
6, the halitosis that disappears, the halitosis bacterium of the milk antibody capable elimination product sulfide that the present invention produced is helicobacter pylori, nuclear Fusobacterium, porphyromonas gingivalis for example.
Combined vaccine following points advantage of the present invention:
1, multiple bacterial classification deactivation combined matching, booster immunization effect of stimulation mutually;
2, mutans streptococcus surface protein that uses in the combined vaccine and virulence factor subunit vaccine are the mutans streptococcus surface antigen subunit vaccines of human heart common antigen of undoping, and can produce the milk of the no rheumatism factor.Because avoided the whole-bacterial-vaccine utilization to produce the shortcoming of the rheumatism factor that causes the human heart cross reaction;
3, use the golden Portugal bacterium that super antigen is arranged to obtain special anti-pyoderma immunity milk and booster immunization stimulation;
Inocalation method following points advantage of the present invention:
1, it is convenient to implement;
2, easily obtain to stablize the high yield immune milk.
Embodiment one
1, the preparation vaccine is got the bacterial classification line and is cultivated, and the single bacterium colony suspendible of picking agar surface is cultivated, and makes bacterial concentration be 10
5-6CFU/ml.Centrifugal collection bacterium, it is inferior to give a baby a bath on the third day after its birth with sodium phosphate buffer.It is 3 * 10 that the bacterium of collecting is mixed with concentration with sterile water for injection
8The bacterium liquid of individual bacterium/ml.15 pounds made the thalline inactivation in 15 minutes, the packing composition, be stored in-20 ℃ standby.Streptococcus mutans and streptococcus sobrinus surface protein and glucosyltransferase (GTF) purifying, wash-out, collection, dialysis, concentrate, after the freeze-drying, purity testing, get freeze-drying surface protein and GTF 12mg and be made into 1mg/ml, bacteriological filtration with 0.01M PBS pH6.0.Add equivalent Freund's complete adjuvant or Freund and mix, making latex is the pulpous state multivalent subunit vaccine.During inoculation, many thalline of use in conjunction inactivated vaccine and pulpous state multivalent subunit vaccine antigen vaccine, preparation antibody milk.
2, the following application of pulpous state subunit vaccine is got in inoculation, injection for the first time, and every of ox back portion multiple spot injects the 5ml latax.95 μ g/ heads; After 15 days, inject the same for the second time; Inject an injection for the third time later in month, latax 5ml intravenous injection first; Last injection back one week-ten day blood sampling and milk are surveyed and are tired.
The vaccine intramuscular injection.Inject for the first time every every bacterium and inject 0.5ml, altogether 4.5ml.After two weeks, inject every for the second time and inject 4.5ml.After a week, inject every for the third time and inject 4.5ml.Inject 4.5ml every every of the 4th injection in week back.Every the 5th injection in a week back 4.5ml/ only.Every the 6th injection in a week back 4.5ml/ only.The seven, eight injection is the same, adopts milk after the per injection in continuous 4 days and surveys and tire.
3, milk whey is collected titration and is adopted milk weekly one time, and about 500 milliliters, it is anticorrosion to add 2/10000ths Sodium azides.Room temperature was placed after 1-2 hour, put 4 ℃ centrifugal 1000 rev/mins after totally one hour, got the middle level, acid adjustment basicity precipitation casein, 4 ℃ centrifugal 1000 rev/mins after totally one hour, getting whey, to recall to acid-base value aseptic subpackaged, put preserve in-20 ℃ of refrigerators standby.Surface protein and GTF milk whey antibody titer are measured and are adopted immune double diffusion method.Get GTF and be made into 100g/ml, get 2ml with 0.01M PBSpH6.0.Get the antibody 1ml of milk whey, twice dilution 5 times.Prepare 1% agarose with physiological saline, add that 2/10000ths Sodium azides are anticorrosion.Water plate, burrow.Hatched 18 hours for 37 ℃ behind the adding sample, the perusal precipitation line is obvious.Full bacterium milk whey antibody titer is measured the TA method that adopts.The continuous double dilution of milk whey antibody.Various full bacterial cells are diluted to 3 * 10 with physiological saline
5Individual/ml.Equal-volume whey and bacterium liquid mix, and the perusal aggegation is obvious.Indirect method ELISA measures: each antigen coated ELISA Plate is measured hole, washes plate, sealing again after the sealing, immunity milk that doubles to dilute and contrast milk point sample incubation, and tagged anti-ox two is anti-, colour developing, ELIASA is surveyed the OD value.Judge: tire=immunity milk OD value/contrast milk OD value; Tire greater than being the immunity success more than 1.6.The result tires
Streptococcus mutans S.mutans NCTC Ingbritt surface protein antibody titer is 1: 16.Streptococcus sobrinus S.sobriuns ATCC 6715 GTF antibody titers are GTF immunity 1: 8.The rabbit anteserum antibody of various bacteriums is to the specific antigen bacterium result that tires: 1. actinomyces viscosus Actinomyces viscosus ATCC 19246 is 1: 16,1: 8.2. examining Fusobacterium Fusobacterium nucleatum ATCC 25286 is 1: 8.3. actinobacillus actinomycetem comitans bacillus Actinobacillus actinomycetemitans Y4 is 1: 16,1: 4.4. helicobacter pylori Helicobacter pylori ATCC 11639 is 1: 16,1: 8.5. porphyromonas gingivalis Porphyromonas gingivalis ATCC 33277 is 1: 8.6. pneumococcus Staphylococcus pneumoniae is 1: 16.7. MRSE Staphylococcus epidermidis is 1: 8.8. staphylococcus aureus Staphylococcus aures ATCC14458 is 1: 4,1: 8.9. propionibacterium acne Propionibacterium acne NCTC 65102 is 1: 32.
The first-born, give a birth that the first two began to inoculate and each somatic antibody of colostrum is tired higher to March.
3, finished product production is set up immune cattle according to immune programme for children, behind conventional thimerosal prewashing of antibody peak value day nipple, loads onto milking machine, takes out immunity and suckles in jar, behind the filtered through gauze impurity, adorns cold fortune car and send and non-immunity rare joining of milk.By the rare surely mixing ratio of tiring of milk antibody, be decided to be respectively: 1 portion of immunity milk is joined 10,100,1000,10000 portions of non-immunity milk.Last homogeneous, ultra high temperature short time sterilization, canned listing.
Claims (25)
1; a kind of multi-functional diseases prevention immune milk food; it is characterized by the protecting human body antibody that contains the special neutralization of resisting chronic gastritis and gastroduodenal ulcer, cancer of the stomach pathogen helicobacter pylori, also contain multiple protecting human body antibody periodontitis substance, tooth dental caries disease, sphagitis, halitosis, the special neutralization of acne pathogens.
2, according to the described multifunction immunity milk of claim 1, it is characterized in that using combined vaccine that 13 kinds of virulence malignant bacterias and two kinds of virulence malignant bacteria subunit protein vaccines form and inoculate repeatedly and stimulate the milk that produces;
3, according to the described multifunction immunity milk of claim 2, it is characterized in that it being to select special international standard strain with strong points and local epidemic isolates for use, after deactivation, inoculate milk cow through the pathogenic bacteria of specific culture, obtain special pathogenic bacteria antibody milk.
4, according to the described multifunction immunity milk of claim 2, select special international standard strain with strong points and local epidemic isolates for use, the purified multivalent subunit vaccine that does not contain the human heart common antigen that makes of the inoculation same period, the antibody milk that systemic immunity obtains.
5, according to the described multifunction immunity milk of claim 2, be characterized in using a kind of combined immunization method, water-soluble inactivated vaccine of use in conjunction and the liposoluble multivalent subunit vaccine behind the purifying respectively are according to obtaining milk after the injection of following method branch.
Inject time: inject injection for the second time after 15 days for the first time; Injection for the third time after injecting for three weeks first, last injection in back around the injection first, blood sampling in thoughtful ten day of last injection back and milk are surveyed and are tired.It is an inoculation cycle.End after one to two months, once more the booster shot one-period.Three all after dates detect and confirm that the invalid milk cow of inoculation goes out production line.The injection site: get the pulpous state subunit vaccine, first inferior to ox back portion multi-point injection; The vaccine intramuscular injection.Later on can the same injection or the lymph node injection, or intravenous injection.Injection volume: get in the pulpous state subunit vaccine, every kind of each range of application of antigen is between 80～200 micrograms, and the each range of application of every kind of thalline of vaccine is at 1*10
5～1*10
9Between the CFU/ml.Each cycle injects the back and obtained original immune milk on the the 7th to the 12 day.
6, according to the described multifunction immunity milk of claim 5, it is characterized in that the multifunction immunity milk of producing behind the compatibility in proportion in listing milk.
7, according to the described multifunction immunity milk of claim 5, it is characterized in that not diluting and the multifunction immunity milk that goes on the market.
8, according to the described multifunction immunity milk of claim 5, it is characterized in that original multifunction immunity milk and immunity milk for thinner ratio be 1: 0.5 the multifunction immunity milk that proportioning obtained.
9, according to the described multifunction immunity milk of claim 5, it is characterized in that original multifunction immunity milk and immunity milk for thinner ratio be 1: 1 the multifunction immunity milk that proportioning obtained.
10, according to the described multifunction immunity milk of claim 5, it is characterized in that original multifunction immunity milk and immune milk not for thinner ratio be 1: 10 the multifunction immunity milk that proportioning obtained.
11, according to the described multifunction immunity milk of claim 5, it is characterized in that original multifunction immunity milk and immune milk not for thinner ratio be 1: 100 the multifunction immunity milk that proportioning obtained.
12, according to the described multifunction immunity milk of claim 5, it is characterized in that original multifunction immunity milk and immune milk not for thinner ratio be 1: 1000 the multifunction immunity milk that proportioning obtained.
13, according to the described multifunction immunity milk of claim 5, it is characterized in that original multifunction immunity milk and immune milk not for thinner ratio be 1: 10000 the multifunction immunity milk that proportioning obtained.
14, make gargle, the oral liquid of preventing and treating gingivitis, periodontitis according to the described multifunction immunity milk of claim 5.
15, make the oral liquid of preventing and treating halitosis according to the described multifunction immunity milk of claim 5.
16, make the oral liquid of preventing and treating acne according to the described multifunction immunity milk of claim 5.
17, make the liquid of washing one's face of preventing and treating acne according to the described multifunction immunity milk of claim 5.
18, make health bathing and the external use liquid of preventing and treating acne or being used to prevent impetigo, furuncle disease, folliculitis according to the described multifunction immunity milk of claim 5.
19, make the live body body cavity flushing liquid of preventing and treating chronic gastropathy, cancer of the stomach, gingivitis, periodontitis, tooth dental caries disease, sphagitis, halitosis, acne, diarrhoeal diseases according to the described multifunction immunity milk of claim 5.
20, make the oral liquid of preventing and treating campylobacter jejuni property diarrhoeal diseases according to the described multifunction immunity milk of claim 5.
21, make the oral liquid of preventing and treating chronic gastritis, gastric ulcer, duodenal ulcer disease, cancer of the stomach, gastric lymphoma disease according to the described multifunction immunity milk of claim 5.
22, the whey antibody and the blood antibody of exempting to make according to the described multifunction immunity milk of claim 6 epidemic disease method, make an addition to and be used to prevent and treat chronic gastropathy, cancer of the stomach, gingivitis, periodontitis, tooth dental caries disease, sphagitis, halitosis, acne, diarrhoeal diseases in medicine and food, the articles for use, or be used to prevent impetigo, furuncle disease, folliculitis.
23, according to the described multifunction immunity milk of claim 2, streptococcus mutans and streptococcus sobrinus surface protein and glucosyltransferase (GTF) purified antigen and subunit vaccine are prepared as follows in the combined vaccine that it is characterized in that using:
Produce GTF and surface protein crude extract from the mutans streptococcus S.mutans Ingbritt of international standard bacterial strain mutans streptococcus family, streptococcus sobrinus S.sobrinus 6715.5% sucrose replaces the trypsase digestion soya broth (TSB) of glucose, little aerobic cultivation.Produce GTF, precipitation reaches 60% saturation degree with ammonium sulfate.Surface protein with 8Mol urea precipitate crude extract.After dialysing, concentrating, cross the post purifying with Sepharose 6B, collection contains the GTF component.Dialysis again concentrates freeze-drying.After surveying purity, get surface protein and GTF 12mg and be made into 1mg/ml, 0.15mg/ml, bacteriological filtration respectively with 0.01M PBS pH6.0.Add the equivalent Freund's complete adjuvant and grind the back sterilization, add 20% tween, bovine serum albumin(BSA) (BSA) mixing.Making latex is the pulpous state multivalent subunit vaccine.
24, according to the described multifunction immunity milk of claim 2, the many thalline combination vaccine in the combined vaccine that it is characterized in that using is produced as follows:
(Tryptic Soy Broth TSB) is used for bacterium activation and increase bacterium to adopt trypsase digestion soya broth.(Tryptic Soy Agar TSA) is used for the pure branch of bacterium to the TSA culture medium.Anaerobic culture medium is used for obligate anaerobes and cultivates.Microbionation is cultivated 18 hours (37 ℃ of 90%N2 5%CO2) and 2-3 days (obligate anaerobe) in corresponding culture medium anaerobism.The single colonies typical streak inoculation of picking is in the TSA flat board respectively, and anaerobism was cultivated after 48 hours, was inoculated in corresponding agar plate after biochemical identification.The single bacterium colony of picking agar surface is suspended in the respective liquid culture medium that (1 1mm bacterium colony/ml) makes bacterial concentration be 10 after cultivating
5-6CFU/ml.Centrifugal (3000g * 30 ').Collect bacterium, it is inferior to give a baby a bath on the third day after its birth with the sodium phosphate buffer of the 0.01M pH6.0 that contains 0.15M NaCl.It is 3 * 10 that the bacterium of collecting is mixed with concentration with sterile water for injection
8The bacterium liquid of individual bacterium/ml.15 pounds made the thalline inactivation in 15 minutes, and-20 ℃ standby.Other method is that bacterium uses the sodium phosphate buffer of the 0.01M pH6.0 that contains 0.15M NaCl to give a baby a bath on the third day after its birth inferior behind 0.5% formalin-inactivated.Be mixed with concentration with sterile water for injection and be respectively 2 * 10
5Individual bacterium/ml, 2 * 10
6Individual bacterium/ml, 3 * 10
8The bacterium liquid of individual bacterium/ml, deposit after the equal portions packing-20 ℃ standby.
25, according to the described multifunction immunity milk of claim 2, contain the Staphylococcus aureus thalline in the combined vaccine that it is characterized in that using, thereby utilize super antigen to stimulate multivalence high concentration antibody milk.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01126326 CN1398531A (en) | 2001-07-24 | 2001-07-24 | Immunological milk with several health functions |
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| CN 01126326 CN1398531A (en) | 2001-07-24 | 2001-07-24 | Immunological milk with several health functions |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100379349C (en) * | 2004-03-10 | 2008-04-09 | 王庭桂 | Method for preparing immune milk anti-pathogenic microoganism in digestive tract |
| CN101072508B (en) * | 2004-10-06 | 2011-04-13 | 农业生物技术有限公司 | Milk production method |
-
2001
- 2001-07-24 CN CN 01126326 patent/CN1398531A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100379349C (en) * | 2004-03-10 | 2008-04-09 | 王庭桂 | Method for preparing immune milk anti-pathogenic microoganism in digestive tract |
| CN101072508B (en) * | 2004-10-06 | 2011-04-13 | 农业生物技术有限公司 | Milk production method |
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