CN101040632B - Milk product with specific immunity of anti-enterobacter sakazakii and the preparing method thereof - Google Patents
Milk product with specific immunity of anti-enterobacter sakazakii and the preparing method thereof Download PDFInfo
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- CN101040632B CN101040632B CN2006100654010A CN200610065401A CN101040632B CN 101040632 B CN101040632 B CN 101040632B CN 2006100654010 A CN2006100654010 A CN 2006100654010A CN 200610065401 A CN200610065401 A CN 200610065401A CN 101040632 B CN101040632 B CN 101040632B
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Abstract
The invention relates to a method for preparing enterobacteria specific immunity milk product which contains enterobacteria specific IgY antibody. The preparation comprises that first cultivating single enterobacteria or various viruses, preparing single or composite antigen, then using the antigen to process high-immunity ejection into egg-laying bird, checking the immunity egg, preparing enterobacteria specific IgY or enterobacteria specific composite IgY, purifying, filtering into the final product, mixing the product with some milk powder, to obtain immunity milk product. The invention can effectively resolve the problem of infant milk powder that polluted by enterobacteria and salmonella.
Description
Technical field
The present invention relates to a kind of Novel immune dairy produce, particularly relate to a kind of immune dairy produce that contains anti-slope fork enterobacteria specific IgY and preparation method thereof.
Background technology
On November 1st, 2002, U.S. food Drug Administration (FDA) detects slope fork enterobacteria (Enterobacter sakazakii) from the babies ' formula milk powder that " Hui Shi " company produces, and recalls from the market the portioned product of the said firm.Subsequently, other national inspection body also successively detects the pathogenic bacteria such as slope fork enterobacteria in the baby milk of large-scale dry milk factories such as " U.S.A praise minister ".
Just caution is human for this: babies ' formula milk powder is not sterile product, may contain pathogenic bacterium, for example slope fork enterobacteria, salmonella.
The World Health Organization (WHO) points out, dies young after two underweight babies of France in 2004 drink the babies ' formula milk powder that suffers germ contamination in hospital, and similarly case also has an example in New Zealand.WHO and the food expert of the United Nations inference, it is to cause infection that babies ' formula milk powder suffers germ contamination, is to cause premature and a sick reason of underweight baby, even may causes serious growth impaired and dead.
Countries in the world suffer the problem day deeply concerned of germ contamination dark to babies ' formula milk powder, in May, 2005,192 member states the WHO conference adopt resolution unanimously last day and point out, there is the serious risk that contains the pathogenic bacteria such as slope fork enterobacteria in milk powder on the market, when appealing that baby for high-risk populations (for example premature, underweight or immunity is poor) prepares formula milk, should step up vigilance.
Because being detected the producer of containing slope fork enterobacteria in the milk powder product all is large pharmaceutical factory, its milk powder product must have high-level gnotobasis in process of production, also certainly passes through strict sterilization process before producing; Still be detected as a result harmful slope fork enterobacteria in its product.This just tells people, and present sterilization measure can not be stopped slope fork enterobacteria.The present inventor thinks the Special Bacteria that this is a kind of vitality extra-heavy, drug resistance is very strong, and present sterilization method is helpless to it; And it can be attached on oneself the dried milk production equipment through sterilize and (has report to point out: once to find the divergent enterobacteria of slope on the equipment of production babies ' formula milk powder, this may be the potentially contaminated source of the milk formula of having sterilized), cause the milk powder manufacturer that it is felt simply helpless.Up to now, the whole world all not yet finds the method for this headachy problem of effective solution; Therefore, the well-known large-scale milk powder in many worlds enterprise in recent years product sales volume significantly comes down, and global milk powder manufacturing enterprise all faces serious threat.
Slope fork enterobacteria is food-borne pathogens, if by the neonate particularly the baby a little less than premature and the immunity suck in the body, very easily suffer from pyemia, bowel necrosis disease and septicemia, under certain conditions, the death rate is up to 80%.In view of this bacterium is very large to infant's harm, it must be removed from baby milk., this special bacteria type kind is changeable, and vitality is very strong again; Present method comprises that Pasteur's high-temperature sterilization all can't eliminate it, can not add chemical sterilization medicine or antibiotic in baby milk again, and general chemistry bactericide and antibiotic do not have much effects to it yet still more.For this present situation, the present invention proposes a kind of innovative approach, prepare a kind of natural safe anti-slope fork enterobacteria specific IgY, it is added in baby milk or the milk powder, this particular milk of infant taking or milk powder, wherein contained anti-slope fork enterobacteria specific IgY will specificity be combined with the pathogenic bacteria such as slope fork enterobacteria of this stubbornness, but it is gone out, thereby it is healthy to avoid the pathogenic bacteria such as slope fork enterobacteria to endanger the infant.Simultaneously, owing to diarrhoeal diseases is first reason of the children's of developing country death, wherein infant rotavirus and Escherichia coli, salmonella are main pathogens; The diarrhoea that particularly rotavirus is caused, the whole world pasts medical help, and according to World Health Organization's statistics, the whole world has 600,000 infants therefore to die young every year.The present invention adopts special technology with slope fork enterobacteria and the compound immune egg-laying bird of a kind of complex antigen of making of salmonella, Escherichia coli and infant rotavirus, the specificity composite IgY for preparing the pathogen such as a kind of anti-slope fork enterobacteria and infant rotavirus, it is added in milk powder or the liquid milk, make a kind of safe milk powder; But the pathogenic bacteria such as residual slope fork enterobacteria and salmonella threaten very large infant rotavirus but can effectively go out simultaneously again to infant's life in the dairy products that both can go out, and the General Promotion infant immunity makes the infant from then on break away from the puzzlement of diarrhoea.Thereby, for global milk powder enterprise provides the means of a kind of effective elimination slope fork enterobacteria, not only can promote the effect of product of these enterprises and quality but also can increase its economic well-being of workers and staff; Simultaneously, protected millions upon millions of infants' health and lives, had a great economic significance and social effect.
Summary of the invention
Defects for existing dairy produce, the object of the invention is to provides a kind of and can effectively get rid of slope fork enterobacteria or effectively get rid of simultaneously slope fork enterobacteria, salmonella, Escherichia coli and baby children rotavirus to the Novel immune dairy produce of infant's invasion and attack for solving the problem of the pathogenic bacteria such as remaining slope fork enterobacteria in the existing dairy produce.
The technical scheme that the present invention solves the problems of the technologies described above is, a kind of milk product with specific immunity of anti-enterobacter sakazakii is provided, and it is characterized in that, contains slope fork enterobacteria specific IgY antibody in this dairy produce.
Dairy produce provided by the present invention is infant immunization milk or milk powder, and wherein slope fork enterobacteria specific IgY Antibody concentration is 0.01-1 ‰.The slope fork enterobacteria specific IgY antibody that contains in this dairy produce can also be the composite IgY antibody of anti-slope fork enterobacteria, anti-salmonella, Chinese People's Anti-Japanese Military and Political College enterobacteria and anti infantile rotavirus.
Another object of the present invention provides a kind of preparation method of milk product with specific immunity of anti-enterobacter sakazakii, it is characterized in that, comprises following steps:
1) pathogen of obtaining to the residual bacterium that detects in the common dairy produce and in the infected human body is analyzed, screening is also determined modal slope fork enterobacteria as single immunizing antigen, maybe with the slope fork enterobacteria that filters out and salmonella, Escherichia coli and infant rotavirus together as complex immunity antigen;
2) cultivate the above-mentioned slope fork enterobacteria that filters out, or cultivate simultaneously slope fork enterobacteria and adopt conventional method training Yang salmonella, Escherichia coli and infant rotavirus;
3) utilize the above-mentioned slope fork enterobacteria of turning out to prepare single antigen, perhaps adopt slope fork enterobacteria and salmonella, Escherichia coli and baby children rotavirus, preparation contains the complex antigen of slope fork enterobacteria;
4) utilize the described slope fork single antigen of enterobacteria or contain the complex antigen of slope fork enterobacteria, egg-laying bird is carried out high inoculation, and search the immune egg that immune bird produces;
5) get the yolk of described immune egg, prepare corresponding slope fork enterobacteria specific IgY antibody crude extract or composite IgY antibody crude extract;
6) described slope fork enterobacteria specific IgY antibody or composite IgY antibody crude extract are carried out purifying, and carry out filtration treatment with the various bacteriums of filtering and virus, obtain corresponding slope fork enterobacteria specific IgY antibody or composite IgY antibody finished product;
7) get described slope fork enterobacteria specific IgY antibody or composite IgY antibody finished product and mix with liquid milk or milk powder raw material in the ratio that determines in advance, make corresponding anti-slope fork enterobacteria immunity dairy produce.
Among the above-mentioned preparation method, the method for the single slope fork of preparation enterobacteria antigen can be in described step (3): get prior cultured single type kind slope fork enterobacteria 2 * 10
9/ mL or polytypic species slope fork enterobacteria by each 2 * 10
9/ mL mixes and obtains slope fork enterobacteria mixed liquor, and the ratio in 10～1:1～10 adds freund adjuvant again, then inserts in the high-speed homogenization machine, with the homogenize of 8,000～30,000rpm high speed, form water-in-oil emulsion, namely make the single antigen that contains slope fork enterobacteria.
The method of the single slope fork of preparation enterobacteria antigen also can be in the above-mentioned steps (3): get prior cultured single type kind slope fork enterobacteria 2 * 10
9/ mL or polytypic species slope fork enterobacteria by each 2 * 10
9/ mL mixes and obtains slope fork enterobacteria mixed liquor, then insert in the ultrasonic disintegrator in addition Ultrasonic Pulverization, again the fork of the slope after pulverization process enterobacteria thalline mixed liquor is added freund adjuvant in 10～1:1～10 ratios, namely make the single antigen that contains slope fork enterobacteria thalline.
Among the above-mentioned preparation method, the method for preparing slope fork enterobacteria specific IgY antibody crude extract or composite IgY antibody crude extract in described step (5) is: (i) get step 4) in the immune egg of gained clean and clean sterilization, smash with eggbeater immunity egg, remove egg white with the sieving of yolk sieve again, the yolk that stays is stirred, measure the volume of the yolk of gained, add distilled water by 4～6 times of this volume and dilute and mix, adjust between pH to 5.5～6.0 with NaOH solution; (ii) dilution that will adjust the pH value further stirs, and then it is cooled to 2～6 ℃, leaves standstill 12～24 hours; (iii) dilution is inserted in the high-speed centrifuge centrifugation 20 minutes; (iv) supernatant of centrifugation gained is inserted again carry out ultrafiltration and concentrated 10～20 times in the ultrafilter, add sodium alginate in the slurry of the ratio in 0.1～0.3% after concentrated, fully stir; Insert again and carry out centrifugation in the supercentrifuge, get supernatant, remove lipoprotein; And the slurry after the centrifugation inserted in the ultrafilter, through the ultra micro film, carry out filtration sterilization; (v) product after the filtration sterilization is carried out freeze drying with freeze drier, namely make anti-slope fork enterobacteria specific IgY antibody crude extract or specificity composite IgY antibody crude extract.
Among the above-mentioned preparation method, the method of the anti-slope fork of step (7) preparation enterobacteria immunity dairy produce is: the obtained anti-slope fork enterobacteria specific IgY antibody dry powder of the abovementioned steps of learning from else's experience or composite IgY antibody dry powder, add in the milk powder raw material in realizing the ratio that determines, take equivalent to amplify the homogenize method with the fully mixed homogenize of powder stirrer, namely make the phylactic powdered milk that contains anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody.
The method of the anti-slope fork of preparation enterobacteria immunity dairy produce can also be in the above-mentioned steps (7): the obtained slope fork enterobacteria specific IgY antibody liquid of the abovementioned steps of learning from else's experience or composite IgY antibody liquid, mix with liquid milk in realizing the ratio that determines, insert again and fully stir mixed homogenize in the mixer, namely can be made into the immune liquid milk that contains slope fork enterobacteria specific IgY antibody antibody or composite IgY antibody; Also this liquid milk can be carried out spray-drying through freeze drying or under 60～140 ℃ of temperature again, make the phylactic powdered milk that contains anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody.
Because Yolk immunoglobulin (Immunoglobulin of Yolk, be called for short IgY) genus IgG immunoglobulin like protein, can with corresponding antigens generation specific binding, thereby suppress or changed state or the activity of this antigen (for some bacterium or virus), and by opsonic action, promote segmented cell or macrophage engulfing bacterium or virus; It can with viral combination, change the conformation of virus surface, stop viruses adsorption in permissive cell, simultaneously, the virus of formation-IgY immune complex can be by macrophage phagocytic.Studies show that IgY belongs to natural materials, without any flavour of a drug, can not bring out again bacterium and produce drug resistance; Because so being well suited for adding in the baby milk goods as a kind of novel antibacterial material.Simultaneously, the present invention creatively adopts this novel antibodies material of anti-slope fork enterobacteria specific IgY to solve the difficult problem that slope fork enterobacteria pollutes this puzzlement whole world milk products industry of baby milk, also finds originally based on the inventor that IgY is applied in and will bring into play the unexistent advantage of following other antibody here:
1) owing to the difference of evolving, IgY can react with a plurality of antigenic determinants on the antigen, and this has just played a signal amplification; Its sensitivity and antibody activity also comprise the IgG much higher (only needing 50 micrograms/mL concentration that desirable activity specific is just arranged) of ox than general mammal, simultaneously, IgY can not bring out bacterium and produce drug resistance.So just created condition for the drug resistance problem that solves vitality extra-heavy, the diverse obstinate slope fork enterobacteria of hypotype;
2) can not produce Criss-seroreaction or allergic reaction; People's pathogen can not brought in the human body yet.This point is supplied with the very weak infant's use of immunocompetence for add composition as food additive, and particular importance seems.
3) evidence IgY can resist pepsin and enteron aisle trypsase and chymotrypsin to the destruction of its activity, can arrive small intestine by stomach and be absorbed by the body; This just can add milk powder, is effective by oral.
4) made antibody has the uniformity of similar monoclonal antibody; So that end product quality and stable performance.The antibody preparation cost is more much lower than preparation from the mammals such as ox.This just can large scale application provide assurance for the present invention in the milk industry of the whole world, have definite feasible practicality.
5) contain in the egg yolk and enrich lecithin, very useful to infant's brain development, can prepare a kind of IgY antibody that contains lecithin.
Therefore, the inventor is through after a large amount of development test and comparative analysis, but selects the IgY antibody of a kind of novel energy specificity antagonism slope fork enterobacteria of preparation or compound antibody as the desirable material of the pathogen such as the divergent enterobacteria of obstinate slope of going out.
The specific embodiment
One. the preparation of anti-slope fork enterobacteria specific immunity milk powder
1. make anti-slope fork enterobacteria specific IgY or anti-slope fork enterobacteria specificity composite IgY
1.1. the pathogen of obtaining in the residual bacterium that in common baby milk, detects according to inspection and quarantine department in recent years and the infected children's body, screening determines that slope fork enterobacteria and the salmonella of four common type kinds are representative pathogenic bacteria, and alternative is selected Escherichia coli and infant rotavirus as the infantile diarrhea main pathogens.
1.2. cultivate respectively slope fork enterobacteria and salmonella, Escherichia coli and infant rotavirus
1.2.1. slope fork enterobacteria:
Buy four kinds of representational slope fork enterobacterias to U.S. virus center (ATCC), number as follows:
I.No.BAA-894:2001 infects the kind that proposes the human body of slope fork enterobacteria from the U.S. because of edible milk powder;
II.No.29004: colonize in cloaca slope fork enterobacteria (Enterobacter cloacae) [this kind is obtained in clinical children's body by U.S. CDC and cultivated into];
III.No.29544: colonize in slope fork enterobacteria mutation (Enterobacter flavus) [this kind is by proposing to cultivate in clinical children's larynx] in children's body;
IV.No.51329: slope fork enterobacteria type strain.
Then cultivate with following method respectively; Use again SDGU method or gel filtration chromatography method purifying:
The large vegetable soup of culture medium: ATCC#3: the large vegetable soup of nutrition (BD234000)
ATCC#3 agar: nutrient agar (BD213000)
Environment: temperature: 30 ℃
Atmospheric environment: band oxygen
Training Yang step:
[1] opens bottle by the indication of enclosing
[2] with a large vegetable soup of #3 (5-6mL), inhale 0.5-1.0mL or pipette suction 1mL with dropper, drip in the tubule whole grain bacterium ball is soaked work.
[3] with sterile manner isodose is drawn back large vegetable soup.Shake up.
[4] from large vegetable soup, extract several kinds to another large vegetable soup or #3 inclination agar or agar dish.
[5] in 30 ℃, cultivated 24 hours.
The bacterium colony of growing at the #3 dish is: full edge, level and smooth, glittering, and little gallery rise, and with yellow spotting.
1.2.2. salmonella, Escherichia coli, infant rotavirus:
Separate in from infantile diarrhea patient ight soil and obtain salmonella, Escherichia coli and infant rotavirus, entrust China " molecular medicine major test chamber, Jiangxi Province " to adopt conventional method to cultivate, then " the refined minister school of life and health sciences " purifying under Hong Kong Yachen Pharmaceutical Group (Yuandong) Co., Ltd. itself.
1.3. prepare single antigen or complex antigen:
1.3.1. prepare the different single antigen that contains slope fork enterobacteria or slope fork enterobacteria body:
1.3.1.1. first with cultured single type slope fork enterobacteria kind 2 * 10
9/ mL or 2-4 type slope fork enterobacteria kind by each 2 * 10
9/ mL mixes and obtains slope fork enterobacteria mixed liquor; Add freund adjuvant (optimal proportion is 1:1) in the 10-1:1-10 ratio again.Then they are inserted in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, namely makes the single antigen that contains slope fork enterobacteria.
1.3.1.2. tackle the biologically active of the slope fork enterobacteria of polytypic species and resistance in order further to improve prepared IgY, the present invention is first with cultured single type kind liquid 2 * 10
9/ mL or 2-4 type kind slope fork enterobacteria by each 2 * 10
9/ mL mixes, and obtains slope fork enterobacteria mixed liquor, inserts in the ultrasonic disintegrator in addition Ultrasonic Pulverization; Again the thalline mixed liquor after special pulverization process is added freund adjuvant (optimal proportion is 1:1) in the 10-1:1-10 ratio, namely make the single antigen that contains slope fork enterobacteria thalline.
1.3.2. preparation complex antigen:
1.3.2.1. first with cultured No.BAA-894 type slope fork enterobacteria (2 * 10
9/ mL), Escherichia coli (2 * 10
9/ mL) and salmonella (2 * 10
9/ mL) mixing in the 1-10:10-1:10-1 ratio, optimal proportion is 2:0.5:1; Then insert in the ultrasonic disintegrator in addition Ultrasonic Pulverization.
1.3.2.2. the mixture with these three kinds of bacterium mixes in the 10-1:1-10 ratio with the rotavirus liquid that oneself cultivates good and purifying again, optimal proportion is 3:1; With prepared antigen mixed liquor, fully evenly mixed, then, add freund adjuvant in the 10-1:1-10 ratio, optimal proportion is 1:1, inserts in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, namely makes complex antigen.
1.4. prepare immune egg:
1.4.1. adopt single antigen or complex antigen with the said method preparation, bird inlay carried out immunity;
1.4.2. strengthen again injection once every two weeks, planned immunization epidemic disease three times; For the first time immunity is after 20 days, and the hen of searching after the immunity produces immune egg, and coded markings in addition.
1.4.3. above immunization method and frequency of injection can suitably be adjusted and variation according to actual conditions; Also can use above-mentioned same immunological technique, adopt above-mentioned antigen, respectively the duck of laying eggs, the female goose of laying eggs, the different egg-laying birds such as turkey or the ostrich of laying eggs of laying eggs be carried out immunity, obtain corresponding immune egg.
1.5. the preparation of anti-slope fork enterobacteria specific IgY or specificity composite IgY:
1.5.1. at first clean immune egg with circulating water, use again the wipes of alcohol wash disinfection; Smash with the immune egg that eggbeater will be searched, remove egg white with the sieving of yolk sieve, stay yolk, stir, measure the volume of the yolk of gained, doubly add distilled water by the 4-6 of this volume, dilute and mix, adjust between the pH to 5.5-6.0 with NaOH solution.
Further stir 1.5.2. will adjust the dilution of pH value, then it is cooled to 2-6 ℃, left standstill 12 hours-24 hours; Dilution is added in the high-speed centrifuge centrifugation 20 minutes;
1.5.3. separating obtained supernatant added again carries out ultrafiltration and concentrated 10-20 in the ultrafilter doubly, sodium alginate is added in the slurry after concentrated fully stirring by the ratio of 0.1-0.3%; Add again in the supercentrifuge centrifugally, get supernatant, remove lipoprotein; Slurry after the centrifugation is added ultrafilter, cross the ultra micro film, carry out filtration sterilization;
1.5.4. the product after the filtration sterilization is carried out freeze drying with freeze drier, makes respectively anti-slope fork enterobacteria specific IgY or specificity composite IgY crude extract.
1.5.5. use SDS-PAGE electrophoretic determination method, respectively to detecting by the prepared specific IgY of above technique or specificity composite IgY crude extract, recording wherein, pure IgY content is more than 50%.
1.6. the purifying of anti-slope fork enterobacteria specific IgY or specificity composite IgY: above anti-slope fork enterobacteria specific IgY or specificity composite IgY crude extract are dissolved in pH7.0,0.01M PB (phosphate buffer) liquid, successively cross again ion exchange column and gel chromatography column chromatography, namely make anti-slope fork enterobacteria specific IgY or specificity composite IgY finished product.
1.7. to prepared anti-slope fork enterobacteria specific IgY or anti-slope fork enterobacteria specificity composite IgY, carry out respectively activity with reference to conventional " ELISA method " (EUSA) and detect.Testing result shows that anti-slope fork enterobacteria specific IgY all reaches 1:1 to the antibody of slope fork enterobacteria in conjunction with tiring, more than 024.Anti-slope fork enterobacteria specificity composite IgY all reaches 1:1 to the antibody of slope fork enterobacteria, Escherichia coli and salmonella in conjunction with tiring, and more than 024, the antibody of rotavirus is then reached 1:4,096 in conjunction with tiring.The IgY concentration of above test sample book is 1mg/mL.
2. make the immune dairy produce that contains anti-slope fork enterobacteria specific IgY or composite IgY
2.1. dry powder blend method:
Prepared anti-slope fork enterobacteria specific IgY or anti-slope fork enterobacteria specificity composite IgY dry powder are added in the infant immunization milk powder with proper proportion, take equivalent to amplify the homogenize method with the fully mixed homogenize of powder stirrer, namely make and contain anti-slope fork enterobacteria and the antibody of anti-infantile diarrhea pathogen or the safe milk powder of infant immunization of compound antibody.
2.2. liquid mixing method:
After 1.5 prepared anti-slope fork enterobacteria specific IgYs or the filtration sterilization of anti-slope fork enterobacteria specificity composite IgY liquid process, not freeze drying, mix with ordinary fresh milk juice by a certain percentage, insert in the mixer and fully to stir mixed homogenize, namely make and contain anti-slope fork enterobacteria and the antibody of anti-infantile diarrhea pathogen or the infant immunization safe milk of compound antibody.Adopt cool Frozen dry or carry out spray-drying with 60-140 ℃ of temperature and (note: as use spray-drying this infant immunization safe milk, will shorten drying time as far as possible, in order to avoid affect the wherein antibody activity of IgY), namely make the safe milk powder of infant immunization that contains anti-slope fork enterobacteria and anti-infantile diarrhea pathogen antibody or compound antibody.
Two. test example
1. test example 1
Cultivate NO.BAA-894 type slope fork enterobacteria and salmonella 1.1. adopt conventional method.Then process with following two kinds of methods respectively:
1.1.1. with NO.BAA-894 type slope fork enterobacteria (2 * 10
9), salmonella (2 * 10
9) mix the rear freund adjuvant that adds in the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, namely makes the former bacterium antigen of slope fork enterobacteria and salmonella.
1.1.2. with NO.BAA-894 type slope fork enterobacteria (2 * 10
9), salmonella (2 * 10
9) in adding first the ultrasonic disintegrator Ultrasonic Pulverization after the mixing of 1:1 ratio; Again gained thalline mixed liquor is added freund adjuvant with the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, namely makes the somatic antigen of slope fork enterobacteria and salmonella.
1.2. use respectively this two kinds of antigen immune chickens, obtain corresponding immune egg.And chat two kinds of anti-slopes fork enterobacterias and the salmonella specific IgY for preparing in the method before adopting.
1.3. make detectable antigens with NO.BAA-894 type, NO.29004 type, NO.29544 type, NO.51329 type slope fork enterobacteria and salmonella and rotavirus respectively, with conventional " ELISA method " (EUSA) detect prepared two kinds of anti-slopes fork enterobacterias and salmonella specific IgY to these 6 kinds not the antibody combination of synantigen tire.Result such as following table:
(annotate: the concentration in the test sample book is 1mg/mL)
Can find out from above testing result, adopt and of the present inventionly the former bacterium of antigen is added the high-speed homogenization machine after with Ultrasonic Pulverization make the Water-In-Oil somatic antigen, immunity prepared anti-slope fork enterobacteria and salmonella specific IgY behind the bird, the slope fork enterobacteria as other type kind of antigen immune is not also had very high antibody in conjunction with tiring, and namely made IgY can resist the slope fork enterobacteria of multiple-type kind.Namely point out us, " somatic antigen immunity " is more much better than " former bacterium antigen immune " effect.This point has the meaning of particular importance for type kind variation slope fork enterobacteria greatly.Result of the test also shows and since not with rotavirus as antigen immune, prepared two species specificity IgY do not have antibody in conjunction with tiring to rotavirus.
2. test example 2
First with cultured NO.BAA-894 type kind slope fork enterobacteria (2 * 10
9/ mL), Escherichia coli (2 * 10
9/ mL) and salmonella (2 * 10
9/ mL) mix in the 2:1:1 ratio; Then insert in the ultrasonic disintegrator in addition Ultrasonic Pulverization.Mixture with these three kinds of bacterium mixes in the 3:1 ratio with the rotavirus liquid that oneself cultivates good and purifying again; With prepared antigen mixed liquor, fully evenly mixed, then, add freund adjuvant in the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, makes complex antigen.
With this complex antigen immunity chicken, obtain corresponding immune egg.And the divergent enterobacteria of the anti-slope of chatting the method preparation before adopting and salmonella specificity composite IgY, make detectable antigens with NO.BAA-894 type, NO.29004 type, NO.29544 type, NO.51329 type slope fork enterobacteria and salmonella, Escherichia coli and rotavirus respectively, with conventional " ELISA method " (EUSA) detect prepared anti-slope fork enterobacteria and salmonella specificity composite IgY to these 7 kinds not the antibody combination of synantigen tire.Result such as following table:
(annotate: the concentration in the test sample book is 1mg/mL)
Can find out from above testing result, adopt and of the present invention the former bacterium of bacterial antigens is mixed with rotavirus after with Ultrasonic Pulverization again, then add the high-speed homogenization machine and make the Water-In-Oil complex antigen, immunity prepared anti-slope fork enterobacteria and salmonella specificity composite IgY behind the bird, the slope of 4 kinds of type kinds fork enterobacteria and salmonella, Escherichia coli, rotavirus are had very high antibody in conjunction with tiring, and namely made composite IgY can resist slope fork enterobacteria and salmonella, Escherichia coli and the rotavirus of multiple-type kind.
3. test example 3
With NO.BAA-894 type slope fork enterobacteria (2 * 10
9), salmonella (2 * 10
9) in adding first the ultrasonic disintegrator Ultrasonic Pulverization after the mixing of 1:1 ratio; Again gained thalline mixed liquor is added freund adjuvant with the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the somatic antigen of slope fork enterobacteria and salmonella is made in the homogenize of 000rpm high speed.Use respectively this two kinds of antigen immune chickens, obtain immune egg.And the method for chatting before adopting prepares anti-slope fork enterobacteria and salmonella specific IgY.This anti-slope fork enterobacteria specific IgY liquid through after the filtration sterilization, is mixed with ordinary fresh milk juice by a certain percentage, insert and fully stir mixed homogenize in the mixer; Then carry out spray-drying with 60-140 ℃ of temperature, namely make the safe milk powder of infant immunization that contains anti-slope fork enterobacteria antibody.Dilute the solution that the safe milk powder of this immunity is made 1mg/mL concentration (IgY content) with pure water; Then, make detectable antigens with NO.BAA-894 type, NO.29004 type, NO.29544 type, NO.51329 type slope fork enterobacteria and salmonella respectively, with conventional " ELISA method " (EUSA) detect the prepared immune safe milk power solution that contains anti-slope fork enterobacteria and salmonella specific IgY to these 5 kinds not the antibody combination of synantigen tire.Result such as following table:
(annotate: the concentration in the test sample book is 1mg/mL)
Above testing result shows, adopt the made immune safe milk powder that contains anti-slope fork enterobacteria and salmonella specificity Ig of method of the present invention that slope fork enterobacteria and the salmonella of multiple-type kind are had very high antibody in conjunction with tiring, namely these pathogenic bacteria are had desirable inhibitory action.
4. test example 4
With the NO.BAA-894 type kind slope fork enterobacteria (2 * 10 of cultivating
9/ mL), Escherichia coli (2 * 10
9/ mL) and salmonella (2 * 10
9/ mL) mix in the 2:1:1 ratio; Then insert in the ultrasonic disintegrator in addition Ultrasonic Pulverization.Again that the mixture of these three kinds of bacterium is evenly mixed in the 3:1 ratio with the rotavirus liquid that oneself cultivates good and purifying, add freund adjuvant in the 1:1 ratio again, insert in the high-speed homogenization machine, with 8,000-30, complex antigen is made in the homogenize of 000rpm high speed.
With this complex antigen immunity chicken, obtain corresponding immune egg.And the method for chatting before adopting prepares anti-slope fork enterobacteria and salmonella specificity composite IgY.Prepared anti-slope fork enterobacteria specific IgY or anti-slope fork enterobacteria specificity composite IgY dry powder are added in the infant immunization milk powder with proper proportion, take equivalent to amplify the homogenize method with the fully mixed homogenize of powder stirrer, namely make and contain anti-slope fork enterobacteria and the antibody of anti-infantile diarrhea pathogen or the safe milk powder of infant immunization of compound antibody.Dilute this solution that friendly safe milk powder is made 1mg/mL concentration (IgY content) of exempting from pure water; Then, make detectable antigens with NO.BAA-894 type, NO.29004 type, NO.29544 type, NO.51329 type slope fork enterobacteria and salmonella, Escherichia coli and infant rotavirus respectively, with conventional " ELISA method " (EUSA) detect the prepared immune safe milk power solution that contains anti-slope fork enterobacteria specificity composite IgY to these 7 kinds not the antibody combination of synantigen tire.Result such as following table:
(annotate: the concentration in the test sample book is 1mg/mL)
Above testing result shows, adopt the made immune safe milk powder that contains anti-slope fork enterobacteria specificity composite IgY of method of the present invention that slope fork enterobacteria and salmonella, Escherichia coli and the infant rotavirus of multiple-type kind are had very high antibody in conjunction with tiring, can these pathogen of establishment.
5. antibacterial, the BA of the anti-slope of test example 5--fork enterobacteria specific IgY.
5.1. test material and instrument
5.1.1. anti-slope fork enterobacteria specific IgY 0.1% aqueous solution,
5.1.2. slope fork enterobacteria and salmonella,
5.1.3. nutrient agar,
5.1.4. nutrient broth,
5.1.5.5% sheep blood nutrient broth,
5.1.6. incubator,
5.1.7.721 spectrophotometer,
5.1.8. physiological saline,
5.1.9. suction pipe,
5.1.10. balance.
5.2. test method:
Antibacterial 5.2.1. (MIC) experimental technique:
Get 9 sterile test tube, add bacterium liquid (the slope fork enterobacteria OD600=0.3 of 1mL nutrient broth or 1mL5% cow's serum nutrient broth and the good concentration of 1mL adjusted in every test tube; Salmonella OD600=0.2).Then first test tube adds the IgY aqueous solution of 1mL, and sucking-off 1mL liquid adds the second pipe behind abundant mixing, and fully sucking-off 1mL liquid adds the 3rd pipe behind the mixing ..., doubling dilution successively, thinner ratio is 1:2,1:4,1:8,1:16,1:32.......The tenth pipe is for not adding the aqueous solution of IgY, as positive control.Test tube is placed incubator, 35 ℃ of overnight incubation.Second day is observed test tube nutrient solution clarity, and the finer of high dilution is antibacterial pipe, calculates Mlc (MIC).
5.2.2. sterilization (MBC) experimental technique:
Liquid in the test tube of above-mentioned overnight incubation is shaken up, from test tube, get an oese liquid inoculation and make time culture in blood nutrient agar panel.Blood nutrient agar panel is placed incubator, 35 ℃ of overnight incubation, the second day observed result, the culture dish of sterilization (MBC) is without colony growth.
5.3. result:
5.3.1. antibacterial (MIC) observes: the finer of cultivating after 24 hours high dilution is antibacterial pipe, and the dilution factor of this pipe is antibacterial thinner ratio.
5.3.2. sterilization (MBC) is observed: blood nutrient agar panel is cultivated after 24 hours and is observed bacterium colony, and the culture dish without bacterial growth of high dilution is the sterilization thinner ratio.
Antibacterial (MIC) table:
Sterilization (MBC) table:
Result of the test shows: but anti-slope fork enterobacteria specific IgY has the very strong effect of going out to slope fork enterobacteria and salmonella.
Three. embodiment
The embodiment 1--standby anti-slope fork of Aqua pure extract legal system enterobacteria specificity chicken IgY
1.1. buy four kinds of representational slope fork enterobacterias to U.S. virus center (ATCC), number as follows:
I.No.BAA-894:2001 infects the kind that proposes the human body of slope fork enterobacteria from the U.S. because of edible milk powder
II.No.29004: colonize in cloaca slope fork enterobacteria (Enterobacter cloacae) [this kind is obtained in clinical children's body by U.S. CDC and cultivated into]
III.No.29544: colonize in slope fork enterobacteria mutation (Enterobacter flavus) [this kind is by proposing to cultivate in clinical children's larynx] in children's body
IV.No.51329: slope fork enterobacteria type strain;
1.2. then respectively with under chat method and cultivate; Use again SDGU method or gel filtration chromatography method purifying, four kinds of representational slope fork enterobacterias that obtain respectively purifying;
Concrete training Yang method is as follows:
The large vegetable soup of culture medium: ATCC#3: the large vegetable soup of nutrition (BD234000)
ATCC#3 agar: nutrient agar (BD213000)
Environment: temperature: 30 ℃
Atmospheric environment: band oxygen
Training Yang step:
[1] opens bottle by the indication of enclosing
[2] with a large vegetable soup of #3 (5-6mL), inhale 0.5-1.0mL or pipette suction 1mL with dropper, drip in the tubule whole grain bacterium ball is soaked work.
[3] with sterile manner isodose is drawn back large vegetable soup.Shake up.
[4] from large vegetable soup, extract several kinds to another large vegetable soup or #3 inclination agar or agar dish.
[5] in 30 ℃, cultivated 24 hours.
The bacterium colony of growing at the #3 dish is: full edge, level and smooth, glittering, and little gallery rise, and with yellow spotting.
1.3. (each 2 * 10 in the 1:1:1:1 ratio with four kinds of slopes fork enterobacterias that above training Yang obtains
9) mix, then with this mixed liquor again with salmonella (2 * 10
9) mix in the 3:1 ratio, add again the ultrasonic disintegrator Ultrasonic Pulverization; At last gained thalline mixed liquor is added freund adjuvant with the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, makes the somatic antigen of slope fork enterobacteria and salmonella;
1.4. with 50 of the bird inlays of the somatic antigen immune health of the multiple slope fork enterobacteria of made and salmonella, every intramuscular injection 1mL every the injection of two weeks once, amounts to three times;
1.5. for the first time injection began to search immune egg in rear the 20th day, to 7th month 7,500 immune eggs examining was washed down with circulating water, 75% ethanol disinfection, dry broken eggshell, remove egg white with the yolk sieving, leave and take yolk and add 5 times of distilled water and stir evenly, transfer pH to 5.5-6.0 with 1.0N HCl, stir, 2-6 ℃ of hold over night is in 10, centrifugal 20 minutes of 000rpm gets supernatant and carries out ultrafiltration concentration 10-20 doubly; Then add 2.0% sodium alginate solution, to final concentration be 0.1%, be stirred to occur muddy; Add again 2.0%CaCI2 liquid, to final concentration be 0.1%, stir, 4 ℃ were spent the night 10 hours; Centrifugal 20 minutes of 8,000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted, UltiporVFTM DV50 removes virus removal except virus filter; Freeze drying; Namely make anti-slope fork enterobacteria specific IgY crude extract 750 grams;
1.6. above IgY crude extract is successively crossed ion exchange resin column and gel chromatography column chromatography, namely makes anti-slope fork enterobacteria specific IgY sterling 150 grams.
The embodiment 2--standby anti-slope fork of Aqua pure extract legal system enterobacteria specificity composite IgY
2.1. buy most representative slope fork enterobacteria, numbering: No.BAA-894 to U.S. virus center (ATCC);
2.2. then adopt following methods training Yang:
The large vegetable soup of culture medium: ATCC#3: the large vegetable soup of nutrition (BD234000)
ATCC#3 agar: nutrient agar (BD213000)
Environment: temperature: 30 ℃
Atmospheric environment: band oxygen
Training Yang step:
[1] opens bottle by the indication of enclosing
[2] with a large vegetable soup of #3 (5-6mL), inhale 0.5-1.0mL or pipette suction 1mL with dropper, drip in the tubule whole grain bacterium ball is soaked work.
[3] with sterile manner isodose is drawn back large vegetable soup.Shake up.
[4] from large vegetable soup, extract several kinds to another large vegetable soup or #3 inclination agar or agar dish.
[5] in 30 ℃, cultivated 24 hours.
The bacterium colony of growing at the #3 dish is: full edge, level and smooth, glittering, and little gallery rise, and with yellow spotting.
Use again SDGU method or gel filtration chromatography method purifying.This most representative slope fork enterobacteria that obtains purifying;
2.3. entrust obtain salmonella, Escherichia coli, infant rotavirus " molecular medicine major test chamber, Jiangxi Province " middle separation from infantile diarrhea patient ight soil, then adopt conventional method to cultivate repurity;
2.4. preparation complex antigen:
2.4.1. first with cultured No.BAA-894 slope fork enterobacteria 2 * 10
9/ mL, Escherichia coli (2 * 10
9/ mL) and salmonella (2 * 10
9/ mL) mix in the 2:0.5:1 ratio; Then insert in the ultrasonic disintegrator in addition Ultrasonic Pulverization;
2.4.2. the mixture with these three kinds of bacterium mixes in the 2:1 ratio with the rotavirus liquid that oneself cultivates good and purifying again, and is with prepared antigen mixed liquor, fully evenly mixed; Then, add freund adjuvant in the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, makes complex antigen;
2.5. prepare immune egg:
Adopt prepared complex antigen, bird inlay is carried out immunity; Strengthen again injection once every two weeks, planned immunization epidemic disease three times; For the first time immunity is after 20 days, and the hen of searching after the immunity produces immune egg;
2.6. the preparation of anti-slope fork enterobacteria specificity composite IgY:
2.6.1. at first clean immune egg with circulating water, use again the wipes of alcohol wash disinfection; Smash with the immune egg that eggbeater will be searched, remove egg white with the sieving of yolk sieve, stay yolk, stir, measure the volume of the yolk of gained, doubly add distilled water by the 4-6 of this volume, dilute and mix, adjust between the pH to 5.5-6.0 with NaOH solution;
Further stir 2.6.2. will adjust the dilution of pH value, then it is cooled to 2-6 ℃, left standstill 12 hours-24 hours; Dilution is added in the high-speed centrifuge centrifugation 20 minutes;
2.6.3. separating obtained supernatant added again carries out ultrafiltration and concentrated 10-20 in the ultrafilter doubly, sodium alginate is added in the slurry after concentrated fully stirring by the ratio of 0.1-0.3%; Add again in the supercentrifuge centrifugally, get supernatant, remove lipoprotein; Slurry after the centrifugation is added ultrafilter, cross the ultra micro film, carry out filtration sterilization; Product after the filtration sterilization is carried out freeze drying with freeze drier, then make anti-slope fork enterobacteria specificity composite IgY crude extract;
2.6.4. use SDS-PAGE electrophoretic determination method, prepared anti-slope fork enterobacteria specificity composite IgY crude extract is detected, recording wherein, pure IgY content is more than 50%;
2.6.5. the purifying of anti-slope fork enterobacteria specificity composite IgY: will resist slope fork enterobacteria specificity composite IgY crude extract to be dissolved in pH7.0,0.01M PB (phosphate buffer) liquid, successively cross again ion exchange column and gel chromatography column chromatography, make anti-slope fork enterobacteria specificity composite IgY finished product.To prepared anti-slope fork enterobacteria specificity composite IgY, carry out activity with reference to conventional " ELISA method " (EUSA) and detect.Testing result shows, anti-slope fork enterobacteria specificity composite IgY all reaches 1:1 to the antibody of slope fork enterobacteria, Escherichia coli and salmonella in conjunction with tiring, more than 024, the antibody of rotavirus is then reached 1:4,096 (the IgY concentration of above test sample book is 1mg/mL) in conjunction with tiring.
The embodiment 3--standby anti-slope fork of Aqua pure extract legal system enterobacteria specificity duck IgY
3.1. buy wherein a kind of most representative slope fork enterobacteria to U.S. virus center (ATCC), be numbered: No.BAA-894;
3.2. cultivate with before chatting method; Use again SDGU method or gel filtration chromatography method purifying, the most representative slope fork enterobacteria that obtains purifying;
3.3. will train this most representative slope fork enterobacteria (2 * 10 of Yang gained
9) and salmonella (2 * 10
9) mix in the 2:1 ratio, add again the ultrasonic disintegrator Ultrasonic Pulverization; At last gained thalline mixed liquor is added freund adjuvant with the 1:1 ratio, insert in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed forms water-in-oil emulsion, makes the somatic antigen of slope fork enterobacteria and salmonella;
3.4. with 100 of the laying duckses of the somatic antigen immune health of the slope of made fork enterobacteria and salmonella, every intramuscular injection 1mL every the injection of two weeks once, amounts to three times;
3.5. for the first time injection began to search immune egg in rear the 20th day, to 7th month 15,000 immune eggs examining was washed down with circulating water, 75% ethanol disinfection, dry broken eggshell, remove egg white with the yolk sieving, leave and take yolk and add 5 times of distilled water and stir evenly, transfer pH to 5.5-6.0 with 1.0N HCI, stir, 2-6 ℃ of hold over night is in 10, centrifugal 20 minutes of 000rpm gets supernatant and carries out ultrafiltration concentration 10-20 doubly; Then add 2.0% sodium alginate solution, to final concentration be 0.1%, be stirred to occur muddy; Add again 2.0%CaCI2 liquid, to final concentration be 0.1%, stir, 4 ℃ were spent the night 10 hours; Centrifugal 20 minutes of 8,000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted, UltiporVFTM DV50 removes virus removal except virus filter; Freeze drying; Namely make anti-slope fork enterobacteria specificity duck IgY crude extract 1,500g;
3.6. above IgY crude extract is successively crossed ion exchange resin column and gel chromatography column chromatography, namely makes anti-slope fork enterobacteria specificity duck IgY sterling 300 grams.
The embodiment 4--anti-slope fork of standby 10, the 000g of dry powder blend legal system enterobacteria specific IgY infant immunization milk powder
4.1. prepare the complete high quality common baby milk powder 9 that contains various defatted milks, vitamin, amino acid, Bifidobacterium etc. of nutrition by commonsense method first, 990g, and through pasteurization;
4.2. the anti-slope fork enterobacteria specific IgY dry powder 10g that will make by method that this specification is chatted;
4.3. amplify the homogenize method with equivalent prepared IgY dry powder is added in the high quality common baby milk powder that oneself makes, in the dry powder mixing machine, stir;
4.4. dispatch from the factory in the packing machine packing;
4.5. production environment must meet aseptic condition, and strictly carries out under aseptic, dustless condition.
Embodiment 5--prepares the anti-slope fork of 10,000g enterobacteria specific IgY infant immunization milk powder with liquid mixing method
5.1. prepare the complete high quality common infant milk that contains various defatted milks, vitamin, amino acid, Bifidobacterium etc. of nutrition by commonsense method first, and through pasteurization;
5.2. will by method that this specification is chatted make without cryodesiccated anti-slope fork enterobacteria specific IgY slurry;
5.3.0.1% ratio adds the anti-slope of prepared IgY fork enterobacteria specific IgY slurry in the high quality common infant milk that oneself makes, and inserts mixer and stirs;
Carry out spray-drying (note: will shorten drying time as far as possible, in order to avoid affect the wherein antibody activity of IgY) 5.4. adopt 60-140 ℃ of temperature.Namely make the safe milk powder of infant immunization that contains anti-slope fork enterobacteria specific IgY antibody;
5.5. dispatch from the factory in the packing machine packing;
5.6. production environment must meet aseptic condition, and strictly carries out under aseptic, dustless condition.
Embodiment 6--prepares the anti-slope fork of 10,000g enterobacteria specificity composite IgY infant immunization milk powder with liquid mixing method
6.1. prepare the complete high quality common infant milk that contains various defatted milks, vitamin, amino acid, Bifidobacterium etc. of nutrition by commonsense method first, and through pasteurization;
6.2. will by preceding method make without cryodesiccated anti-slope fork enterobacteria specificity composite IgY slurry;
6.3. the ratio with 0.1% adds the anti-slope of prepared IgY fork enterobacteria specificity composite IgY slurry in the high quality common infant liquid milk that oneself makes, and inserts mixer and stirs;
Carry out spray-drying (note: will shorten drying time as far as possible, in order to avoid affect the wherein antibody activity of IgY) 6.4. adopt 60-140 ℃ of temperature.Namely make the safe milk powder of infant immunization that contains anti-slope fork enterobacteria specificity composite IgY antibody;
6.5. dispatch from the factory in the packing machine packing;
6.6. production environment must meet aseptic condition, and strictly carries out under aseptic, dustless condition.
Will be by the prepared safe milk powder of infant immunization of anti-slope fork enterobacteria specificity composite IgY antibody that contains of above embodiment method to infants, not only can suppress the pathogenic bacteria such as slope fork enterobacteria and salmonella, stop the infection of these pathogenic bacteria, especially meaningfully, also can prevent and treat infant rotavirus and Escherichia coli, salmonellal diarrhoea.
Claims (4)
1. the preparation method of an anti-slope fork enterobacteria immunity dairy produce is characterized in that, comprises following steps:
1) pathogen of obtaining to the residual bacterium that detects in the common dairy produce and in the infected human body is analyzed, screening and definite NO.BAA-894 type slope fork enterobacteria be as single immunizing antigen, maybe with the NO.BAA-894 type slope fork enterobacteria that filters out and salmonella, Escherichia coli and infant rotavirus together as complex immunity antigen;
2) cultivate the above-mentioned NO.BAA-894 type slope fork enterobacteria that filters out, or cultivate simultaneously NO.BAA-894 type slope fork enterobacteria, salmonella, Escherichia coli and infant rotavirus;
3) utilize the above-mentioned NO.BAA-894 type slope fork enterobacteria of turning out to prepare single antigen, perhaps adopt NO.BAA-894 type slope fork enterobacteria and salmonella, Escherichia coli and infant rotavirus, preparation contains the complex antigen of NO.BAA-894 type slope fork enterobacteria;
4) utilize the described NO.BAA-894 type slope fork single antigen of enterobacteria or contain the complex antigen of NO.BAA-894 type slope fork enterobacteria, egg-laying bird is carried out high inoculation, and search the immune egg that immune bird produces;
5) get the yolk of described immune egg, prepare corresponding anti-slope fork enterobacteria specific IgY antibody crude extract or composite IgY antibody crude extract;
6) described anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody crude extract are carried out purifying, and carry out filtration treatment with the various bacteriums of filtering and virus, obtain corresponding anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody finished product;
7) get described anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody finished product and mix with liquid milk or milk powder raw material in the ratio of 0.01-1 ‰, make corresponding anti-slope fork enterobacteria immunity dairy produce;
Wherein the method for the single slope fork of preparation enterobacteria antigen is in described step (3): get prior cultured NO.BAA-894 type slope fork enterobacteria 2 * 10
9Then/mL inserts in the ultrasonic disintegrator in addition Ultrasonic Pulverization, again the thalline mixed liquor after pulverization process is added freund adjuvant in 10～1:1～10 ratios, namely makes the single antigen that contains slope fork enterobacteria thalline;
The method for preparing the complex antigen that contains NO.BAA-894 type slope fork enterobacteria in described step (3) is: get prior cultured NO.BAA-894 type slope fork enterobacteria 2 * 10
9/ mL, Escherichia coli 2 * 10
9/ mL and salmonella 2 * 10
9/ mL is in 1～10: 10～1: 10～1 ratio is mixed, then insert and carry out Ultrasonic Pulverization in the ultrasonic disintegrator, the mixture of getting again these three kinds of thalline and the rotavirus liquid of cultivating in advance good and purifying are in 10～1: 1～10 ratio is mixed and is made the antigen mixed liquor, it is fully evenly mixed to mix liquid, then in 10～1: 1～10 ratio adds freund adjuvant, insert again in the high-speed homogenization machine, with 8,000-30, the homogenize of 000rpm high speed, form water-in-oil emulsion, namely make the complex antigen that contains NO.BAA-894 type slope fork enterobacteria;
Described anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody finished product antagonism NO.BAA-894 type, NO.29004 type, NO.29544 type and NO.51329 type slope fork enterobacteria.
2. the preparation method of anti-slope fork enterobacteria immunity dairy produce according to claim 1, it is characterized in that, in described step (7), the obtained anti-slope fork enterobacteria specific IgY antibody dry powder of the abovementioned steps of learning from else's experience or anti-slope fork enterobacteria specificity composite IgY antibody dry powder, add in the milk powder raw material in the ratio that determines in advance, take equivalent to amplify the homogenize method with the fully mixed homogenize of powder stirrer, namely make the phylactic powdered milk that contains anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody.
3. the preparation method of anti-slope fork enterobacteria immunity dairy produce according to claim 1, it is characterized in that, in described step (7), the obtained slope fork enterobacteria specific IgY antibody liquid of the abovementioned steps of learning from else's experience or composite IgY antibody liquid, mix with liquid milk in the ratio that determines in advance, insert again and fully stir mixed homogenize in the mixer, namely can be made into the immune liquid milk that contains slope fork enterobacteria specific IgY antibody or composite IgY antibody; Maybe this liquid milk is carried out spray-drying through freeze drying or under 60～140 ℃ of temperature again, make the phylactic powdered milk that contains anti-slope fork enterobacteria specific IgY antibody or composite IgY antibody.
4. milk product with specific immunity of anti-enterobacter sakazakii, it is characterized in that, it adopts preparation method claimed in claim 1 to prepare, and wherein, contains the anti-slope fork enterobacteria specific IgY antibody that makes as antigen with NO.BAA-894 type slope fork enterobacteria in the described dairy produce; Described anti-slope fork enterobacteria specific IgY antibody antagonism NO.BAA-894 type, NO.29004 type, NO.29544 type and NO.51329 type slope fork enterobacteria.
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| CN1465261A (en) * | 2002-11-13 | 2004-01-07 | 上海光明乳业股份有限公司 | Immune formula milk powder and preparation process thereof |
| CN1481900A (en) * | 2003-06-16 | 2004-03-17 | 雅臣药业集团（远东）有限公司 | Specific IgY and composite IgY for Rheum and its novel formulation |
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| CN1481900A (en) * | 2003-06-16 | 2004-03-17 | 雅臣药业集团（远东）有限公司 | Specific IgY and composite IgY for Rheum and its novel formulation |
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Effective date of registration: 20150513 Address after: 518000 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A (located in Shenzhen Qianhai business secretary Co. Ltd.) Patentee after: Shenzhen Cheng Cheng Biotechnology Co., Ltd. Address before: 518000, a bio incubator building in central high tech Zone, Shenzhen, Guangdong, 1-204, Nanshan District Patentee before: Shenzhen Yachen Biotechnology Technology Co., Ltd. |
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Effective date of registration: 20170821 Address after: Room 9, block C, building, science and Technology Park, 1 Shenzhen Road, Longhua street, Longhua New District, Guangdong, China Patentee after: Shenzhen elegant intelligent biological engineering Co., Ltd. Address before: 518000 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A (located in Shenzhen Qianhai business secretary Co. Ltd.) Patentee before: Shenzhen Cheng Cheng Biotechnology Co., Ltd. |
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