KR20040005540A - The method for production of specific yolk antibody containing anti-helicobacter pylori IgY,anti-helicobacter pylori enzyme(urease, proteases, lipase, phosph olipase)IgY,anti-E.coli(EHEC),anti-salmonella enteritidis IgY,anti-staphylococcus aureus IgY. - Google Patents

Abstract

PURPOSE: A method for producing specific egg yolk antibody to substances causing enteritis and food poisoning is provided, thereby easily and simultaneously producing various antibodies. CONSTITUTION: A method for producing specific egg yolk antibody to substances causing enteritis and food poisoning comprises the steps of: emulsifying enteritis-causing antigens in an immune adjuvant; injecting the mixture to a laying hen at regular intervals; and obtaining the specific egg yolk antibody from eggs of the immunized hen.

Description

The method for production of specific yolk antibody containing anti-helicobacter pylori IgY, anti-helicobacter pylori enzyme (urease, proteases, lipase, phosph olipase) IgY, anti- E. coli (EHEC), anti-salmonella enteritidis IgY, anti-staphylococcus aureus IgY.}

An object of the present invention is to produce a complex specific yolk antibody for the cause of enteritis and food poisoning, and an object of the present invention is to efficiently isolate a specific complex yolk antibody from an effective specific yolk antibody production method and a complex specific yolk.

The present invention is characterized by providing a method for producing a complex specific yolk antibody against the cause of enteritis and food poisoning, diarrhea.

Another invention is characterized by providing an immunoadjuvant and an immune cycle for maintaining and increasing titer for the production of an effective yolk antibody.

Another invention is characterized by providing a technology for producing a complex water-soluble specific yolk antibody against a causative agent of enteritis and food poisoning using sugar and ionized water.

Helicobacter pylori infection is the most important cause of the development of gastroduodenal ulcers, and treatment of Helicobacter pylori can be expected to cure gastroduodenal ulcers. Helicobacter pylori infection has been epidemiologically proven to be the primary cause of gastric cancer, and the World Health Organization has declared Helicobacter pylori infection as a 'group 1 carcinogen', a potent gastric cancer trigger. In addition, the relationship with Helicobacter pylori has been suggested in gastric lymphoma. In Japan and Korea where gastrointestinal diseases such as stomach cancer are common, the prevalence is relatively high, affecting 70-80% of the adult population.

A simple listing of the causes of Helicobacter pylori is as follows.

First is mobility. Helicobacter pylori exhibits strong motility in the gastric mucosa because it possesses motility even at a viscosity 20 times higher than the viscosity of E. coli. Among the isolates of Helicobacter pylori isolated, the motility strains and the weak strains were inoculated into different sterile piglets, and the infection rate was found to be 17-40%, whereas the motility strains showed 100% infection rate. Indicated. Second, there are various enzymes of Helicobacter pylori. The most important microbiological characteristic of Helicobacter pylori as urease is its strong ability to produce urease. The production capacity is more than 100 times compared to the production capacity of the proteus species. About 6% of cell proteins are urease. Urease is an enzyme that breaks down urea into ammonia and carbon dioxide. Therefore, it is possible to effectively decompose urea present at low concentrations in plasma exudates or tissue fluids of the gastric mucosa. The ammonia thus produced neutralizes the attack by hydrochloric acid in the gastric lumen by alkalineizing the environment around the bacteria. Other proteases, lipases, phospholipases and the like weaken the strength of the gastric mucosa, thereby increasing the hydrophilicity of the mucosa and increasing the attack from acids or pepsin. Third, another protein of toxic substances is vacuolating cytotoxin, a protein that causes vacuoles to form in the cytoplasm. This protein is also known to be toxic like the cytotoxin associate protein.

Some of the patents developed as conventional Helicobacter pylori prophylaxis are as follows. First, in 1992, Taiyo Kagakusa immunized Helicobacter Pyro into the laying hens and immunized them to produce a special antibody. Antibody bound to toxin, pure CB antigen was used (1993). On the other hand, natural extracts have been used, and polyphenols, green tea extracts, have been reported to have an effect on Helicobacter pylori (Taiyo Kugakusa 1993). In 1995, Yasquilt Onshasa's Sashimoto and others extracted polysaccharides containing fucoidan from red algae and brown algae. On the other hand, the development of antibiotics for Helicobacter pylori was developed in 1995 by the treatment of Moestomycine, such as Hestmann's Hedmann, with cimeetidine, tetracycline, etc. Pharmaceutical has developed an antimicrobial thiazole derivative against Helicobacter pylori, an H ₂ receptor antagonist. Actin BioPharma has developed a lectin for the treatment of gastrointestinal ulcers and gastritis caused by Helicobacter Pyro using the binding of pathogens and lectins in the digestive system.

As described above, many new methods such as antibiotics and natural substances have been tried for the prevention and treatment of Helicobacter pylori, but have not reached a definite result. In addition, in the case of using yolk antibodies, complex antibodies other than a single yolk antibody are very incomplete.

Recently, many cases of food poisoning occur every year in the world, and most of them are bacterial food poisoning. The cases of food poisoning reported in the US by the causative organism and the outbreak patient, salmonella accounted for 25% of food poisoning and 18.6% of all patients. In the case of food poisoning in Japan, 70% of cases of bacterial poisoning, 74.3% of patients, 26.0% of Salmonella, 20.0% of Vibrio parahaemolyticus, but 27.1% of Salmonella and E. coli (Escherichia coli-pathogenic) accounts for 21.5%, almost half of Salmonella and E. coli. Escherichia coli is a gram-negative bacillus belonging to the Enterobacteriaceae, and when it is infected through fecal contamination, it causes various diseases such as diarrhea, urinary tract infection, peritonitis, and neonatal sepsis.Especially, Escherichia coli is an important indicator of fecal contamination and a potential indicator of Salmonella. Has been. Escherichia coli forms about 1% of intestinal microorganisms, and pathogenic E. coli with diarrhea includes enteropathogenic Escherichia coli (EPEC), enterotoxin-producing Escherichia coli (ETEC), intestinal hemorrhagic E. coli (EHEC), and enteric tissue invasive Escherichia coli (EIEC). It is reported to be widely distributed in the surrounding environment such as humans, livestock and sewage. Therefore, the management of these food poisoning bacteria is very important in the food industry. Increasing living standards and increasing knowledge of appropriate sanitation and hygiene concepts have lowered the incidence of foodborne illness, but still cannot fully control many types of illness.

In recent years, the number of cases has decreased due to the expansion of food hygiene concept, but the number of patients per incident has increased due to mass production, mass sale of food, and the increase in eating out opportunities. In addition, new strains resistant to the use of chemical additives or antibiotics have appeared, and due to the side effects of the drugs used, many problems have arisen in safety and consumer perception, and thus a different approach has been found. There have been many researches on blocking harmful microorganisms in foods, but the method using egg antibodies is not well known. The production of specific antibodies from chicken eggs can be produced more than other animals, which can have many advantages in terms of economics, hygiene and nutrition when applying relatively large amounts of antibodies to food.

The main technical field of the present invention is for yolk antibodies, which is a form of passive immunization using the immune system of birds. In the case of sub-avian oviparous animals, immune antibodies obtained by active chickens from mother chickens are transferred to and accumulated in egg yolk. In this way immunity is transmitted to the offspring. The largest proportion of antibodies in egg yolk takes a form similar to IgG, an antibody in the blood, commonly referred to as IgY. It has a molecular weight of about 200-220kDa, which is larger than mammalian age (IgG) and has advantages over mammalian antibodies, so its range of application is gradually widening. Maternal antibody implementation through egg yolk has been studied since the 1930s, and research on antibody implementation against diseases has been in progress since the 1960s. As a result of research and development using egg yolk antibodies in Korea, Kim Jung-woo (1998) has applied for a patent for the diagnostic method and diagnostic kit for enterotoxin E. coli (Korean Patent Publication No. 1999-0066438). In addition, the National Veterinary Research and Quarantine Service (1998) has applied for a patent (Korean Patent Laid-Open Publication No. 1999-0079333) for oral immunization using egg yolk antibodies for the prevention and treatment of swine pandemic diarrhea. Lee Seung-bae (1996) has applied for the preparation of yolk antibodies for caries prevention (Korean Patent Publication No. 1997-073607). This is a method for producing a yolk antibody for preventing tooth decay by immuno-treating the streptococcus mutans, the causative agent of caries. Development of egg yolk antibodies against propionibacterium acnes has been carried out in Japan. Developed by solar chemistry and Kanebo Co., Ltd., the acne-causing agents, propionibacterium acnes, Staphylococcus epidermidis, and propionibacterium avidum It was developed to be used as an immunogen.

In addition, the conventional technique for the separation of egg yolk antibody is as follows. The specific yolk antibody in yolk is γ-rivetin in the yolk proteins α, β and γ-rivetin (Livetin). The first step for its isolation is the removal of lipoprotein, a lipid component of yolk. Its removal methods include centrifugation (Bade, 1984), organic solvent separation (Paulson, 1980), polyethylene glycol (PEG), sodium dextran sulfate, and xanthan. gum), carrageenan, and the like. The method using centrifugation or carrageenan has high production costs, very low yields, and the use of organic solvents or chemicals is not suitable for edible use, and there are many environmental problems due to the high production of waste. In addition, the pigments in egg yolk were not completely removed when they were separated by these methods. In order to use industrially, research is required to compensate for the shortcomings of the method. Trehalose has a number of excellent functions and gentle sweetness, including the ability to freeze or protect proteins, not coloring or heating reactions by heating. It is also a sugar that is deeply involved in the preservation and regeneration of life.

Therefore, the present invention is effective for the development of yolk antibodies for preventing enteritis and food poisoning, ureases produced by Helicobacter pylori and Helicobacter pylori bacteria, urease, proteases, lipases, phospholipases, E. coli (EHEC), Salmonella enteritidis and Staphylococcus aureus antigens are used to produce eggs with complex antibodies. We have developed an appropriate immune cycle and adjuvant to maintain and raise the titer of yolk antibody. In addition, ionized water and sugar-added ionized water were used to effectively separate the water-soluble yolk antibody. Therefore, the present invention is to provide an effective method for producing gastroenteritis and food poisoning preventive egg and egg yolk antibody method to combine the industrialization thereof.

Therefore, the inventors of the present invention Helicobacter pylori (Helicobacter pylori), urease (Urease), proteases (lipases), phospholipases, E. coli (EHEC), Salmonella enteritidis causes of enteritis and food poisoning (Salmonella enteritidis), Staphylococcus aureus (Staphylococcus aureus) antigens for the development of complex yolk antibodies for the antigen composition and the amount of antigen is established.

In addition, to improve the productivity of complex-specific yolk antibodies, we establish the immune cycle, which is the optimal condition for the egg laying rate and titer of complex-specific yolk antibodies, and adjust the composition of the adjuvant.

In addition, we develop a separation method using ionized water and sugar-added ionized water for effective and stable separation of complex specific yolk antibodies in eggs.

The liver and liver are intended to apply the purified yolk antibody using the separation method of the specific yolk antibody in addition to the egg containing the complex-specific yolk antibody produced by the method in the food industry.

1 is a change in titer of yolk antibody (IgY) upon inoculation of Helicobacter pylori and Escherichia coli, Salmonella enteritidis, Staphylococcus orreas and urease, protease, lipase and phospholipase.

Figure 2 shows the Helicobacter pylori and E. coli and E. coli, Salmonella enteritidis and Staphylococcus oraires and urease, protease, lipase and phospholipase in combination with a combination of a new immunoadjuvant and a comparative test immunoadjuvant It is a change in the titer of Salmonella enteritidis and Staphylococcus Oreas specific yolk antibody (IgY).

Figure 3 shows a combination of urease and protease according to the difference between the neoadjuvant and comparative test immunoadjuvant when inoculating Helicobacter pylori and E. coli, Salmonella enteridis, Staphylococcus orreas and urease, protease, lipase and phospholipase It is a titer change of specific yolk antibody (IgY) of lipase and phospholipase.

Figure 4 shows the helicobacter pylori and E. coli and E. coli, Salmonella enteritidis and Staphylococcus ores and urease, protease and lipase and phospholipase during the inoculation of the feed composition of vitamin A addition and vitamin A addition It is a change in the titer of the specific yolk antibody (IgY) of Salmonella enteritidis and Staphylococcus orares.

Figure 5 shows the urease and protease by the difference between the addition of vitamin A addition group and vitamin A in the composition of the inoculation of Helicobacter pylori and E. coli, Salmonella entericis and Staphylococcus ores and urease and protease and lipase and phospholipase It is a titer change of specific yolk antibody (IgY) of lipase and phospholipase.

Figure 6 is the average titer of the specific yolk antibody (IgY) when inoculating Helicobacter pylori and E. coli, Salmonella enteritidis and Staphylococcus ores and urease, protease, lipase and phospholipase.

Figure 7 shows Helicobacter pylori and E. coli and E. coli, Salmonella enteritidis, Staphylococcus orreas and urease, protease, lipase and phospholipase when inoculated with a combination of a new immunoadjuvant and a comparative test adjuvant The average titer of Salmonella enteritidis and Staphylococcus Oreas specific yolk antibody (IgY).

Figure 8 shows the combination of urease and protease according to the difference between the neoadjuvant and comparative test immunoadjuvant when inoculating Helicobacter pylori, E. coli, Salmonella enteridis, Staphylococcus orreas and urease, protease, lipase and phospholipase It is the average titer of specific yolk antibody (IgY) of lipase and phospholipase.

Figure 9 shows the Helicobacter pylori and E. coli and E. coli, Salmonella enteritidis and Staphylococcus ores and urease, protease, lipase and phospholipase during the composition of the inoculation of vitamin A addition and vitamin A addition in the feed composition It is the average titer of specific yolk antibodies (IgY) of Salmonella enteritidis and Staphylococcus orares.

10 is a combination of urease and protease due to the difference between vitamin A addition and vitamin A addition composition during feed inoculation of Helicobacter pylori, E. coli, Salmonella enteritidis, Staphylococcus orreas and urease, protease, lipase, and phospholipase. It is the average titer of specific yolk antibody (IgY) of lipase and phospholipase.

Figure 11 shows the total specific yolk antibodies due to the difference between the neoadjuvant and comparative test immunoadjuvant at the time of inoculating Helicobacter pylori and E. coli, Salmonella enteritidis, Staphylococcus orreas and urease, protease, lipase and phospholipase IgY) is the average potency.

Figure 12 shows the total specific yolk antibody due to the difference between vitamin A addition and vitamin A addition in the composition of the inoculation of Helicobacter pylori, E. coli, Salmonella entericis, Staphylococcus ores and urease, protease, lipase and phospholipase IgY) is the average potency.

FIG. 13 is a diagram showing a method for separating water-soluble special immunoprotein (IgY) in egg yolk.

FIG. 14 shows the titer of the yolk antibody separated by the normal ionized water and the titer of the yolk antibody separated using the added ionized water.

The present invention for achieving the above-described technical problem is Helicobacter pylori (Helicobacter pylori), urease (Urease), proteases (proteases), lipases (lipases), E. coli (phospholipases), E. coli (EHEC), Salmonella enteritidis (Salmonella enteritidis) and Staphylococcus aureus (Staphylococcus aureus) to the optimal conditions and to share eight complex-specific yolk antibodies in a single egg that can prevent enteritis and food poisoning by using a combination inoculation method for laying hens Characterized in the production method. In addition, the present invention is characterized in that the immunity of the laying hens is carried out at 18 weeks of primary immunity, 20 weeks of secondary, 24 weeks of tertiary, and additional immunity is carried out every two months. In the present invention, the composition of the immunoadjuvant is set to aluminum hydroxide (20%) and RS 25 (10%) at the time of the first immunity, and to the TS 25 (30%) at the time of the second immunity, A 70 (30%) is characterized by. In another aspect, the present invention is characterized by the yolk powder spray-dried complex-specific yolk antibody and yolk produced by the above method. In addition, the present invention is to contain vitamin A in the feed, more specifically, characterized in that any one of 10,000 IU, 20,000 IU, 30,000 IU, 40,000 IU, 50,000 IU, 60,000 IU per 1Kg of feed. In another aspect, the present invention is characterized by egg yolk powder spray-dried with yolk containing the complex specific yolk antibody and vitamin A produced by the above method. In addition, the present invention is characterized by separating specific yolk antibodies using ionized water (pH 3 to 5, pH 9 to 11) and sugar-added ionized water (per trehalose, 1 to 8%). In another aspect, the present invention is characterized by the lyophilized powder of the specific yolk antibody isolated by the above method and its aqueous solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to the drawings of the embodiments or the claims of the present invention.

Example

I. Development of Complex Yolk Antibody

1. Preparation of Helicobacter pylori antigen

1) Culture of Helicobacter pylori Antigen

Helicobacter pylori was used by the American Type Culture Collection (ATCC). Experimental strains Miller Hilton (Mueller Hinton) medium (Difco Co.) sheep serum (Sheep serum) in a flat plate medium containing 10% and passaged every 2-3 days, 37 ℃, CO ₂ concentration of 10% CO in the environment Incubated in ₂ incubators. The test strain was tested by microscopic observation, Gram staining, urease activity test, oxidase test, and catalase test.

2) Ureaase activity measurement

Ureaase activity was measured using a urease test broth including urea and phenol red. Urea broth and the culture or sample were mixed at a ratio of 4: 1 and reacted for 30 minutes, and then the absorbance at 540 nm was measured to determine the freeace activity.

3) Oxidase Test

A colony was applied to the filter paper and 2 to 3 drops of 1% N, N-dimethyl-p-phenylenediamine oxalate (Junsei, Japan) aqueous solution was dropped, and the colony was found to be positive when the colony became dark purple within 5 to 10 seconds.

4) Catalase test

After spreading the colonies with a cotton swab on the slide glass and dropping 1 drop of 3% hydrogen peroxide solution on the colony was mixed, it was determined to be positive if bubbles were generated.

5) Investigation of Morphology

Visually observe the morphological changes of the bacteria cultured for 3 days and 5 days, and colony colonization of the colonies on a slide glass, followed by gram staining, followed by optical microscopy (× 1,000). It was examined whether it was curved form or coccoid form. Carbon fuchsin was used as a control dye.

6) Preparation of Helicobacter pylori antigen

Cultured Helicobacter pylori strain was recovered by the cell collecter and suspended in saline. The isolated cells were added with sterile physiological saline (PBS, pH 7.2) to 1/2 of the initial amount, and mixed with formaldehyde (formaldehyde) to 0.5% and inactivated at room temperature for 24 hours. Confirmation of inactivation was confirmed that the bacteria do not increase by plating on a solid medium. The inactivated bacteria were centrifuged at 4,000 rpm for 20 minutes to harvest the bacteria, washed three times with sterile physiological saline (pH 7.2) and stored at -70 ° C to use as antigen.

2. Preparation of urease, protease, lipase, phospholipase complex antigen

Helicobacter pylori enriched in anaerobic conditions was centrifuged to separate the cells and the supernatant. The supernatant was added with saturated ammonium sulphate and left at 4 ° C. for 16 hours. The saturated solution was centrifuged to precipitate the precipitate with 1/100 amount of sterile physiological saline solution (PBS, pH7.2), and dialyzed at 4 ° C. for 48 hours. The dialysate was filtered through a 0.45 μm filter and then lyophilized.

3. Preparation of E. Coli (EHEC) Antigens

E. coli (EHEC) strains were inoculated in Tryptic soy agar and incubated for 18 hours in a 37 ° C incubator to colonize one colony in 10 ml of Tryptic soy broth medium. After 2 hours of inoculation, a large amount of Tryptic soy broth medium was inoculated and incubated in a shaking incubator at 37 ° C. for 48 hours. Formaldehyde (formaldehyde) was mixed to 0.5% in the cultured bacteria solution and inactivated at room temperature for 24 hours. Confirmation of inactivation was confirmed that the bacteria do not increase by plating on a solid medium. The inactivated bacteria were centrifuged at 4,000 rpm for 20 minutes to harvest the bacteria, washed three times with sterile physiological saline (pH 7.2) and stored at -70 ° C to use as antigen.

4. Salmonella enteritidis antigen production

Salmonella enteritidis was inoculated in Tryptic soy agar and incubated for 18 hours in a 37 ° C. incubator. One colony was inoculated in 10 ml of Tryptic soy broth medium. Two hours after the inoculation, a large amount of Tryptic soy broth medium was inoculated and cultured in a shaking incubator at 37 ° C. for 48 hours. Formaldehyde (formaldehyde) was mixed to 0.5% in the cultured bacteria solution and inactivated at room temperature for 24 hours. Confirmation of inactivation was confirmed that the bacteria do not increase by plating on a solid medium. The inactivated bacteria were centrifuged at 4,000 rpm for 20 minutes to harvest the bacteria, washed three times with sterile physiological saline (pH 7.2) and stored at -70 ° C to use as antigen.

5. Preparation of Staphylococcus aureus Antigen

Staphylococcus aureus was inoculated in a Mannitol medium containing 7.5% saline and incubated for 18 hours in a 37 ° C incubator. One colony was inoculated in 10 ml of mannitol salt medium. After 2 hours of inoculation, a large amount of mannitol saline medium was inoculated and incubated in a shaking incubator at 37 ° C. for 48 hours. Formaldehyde (formaldehyde) was mixed to 0.5% in the cultured bacteria solution and inactivated at room temperature for 24 hours. Confirmation of inactivation was confirmed that the bacteria do not increase by plating on a solid medium. The inactivated bacteria were centrifuged at 4,000 rpm for 20 minutes to harvest the bacteria, washed three times with sterile physiological saline (pH 7.2) and stored at -70 ° C to use as antigen.

6. Composition ratio and antigen condition of complex antigen

Helicobacter pylori: Escherichia coli: Salmonella enteritidis: Staphylococcus orares: The ratio of the adjuvant was 3: 1.5: 1.5: 1: 3. Urease, protease, lipase, phospholipase complex antigens were used in powder form. The amount of immunity per laying hen was 0.5 ml. At this time, Helicobacter pylori is adjusted to OD of 0.5 at 660nm to 0.15ml, E. coli and Salmonella enteridis to 0.3 at 660nm to 0.075ml, Staphylococcus orares to 0.3 at 660nm to 0.05ml, and immunoadjuvant 0.15ml It was. And urease (Urease), protease (protease), lipase (lipase), phospholipase complex antigen was emulsified by adding 600㎍.

II. Development of Potency and Synergistic Effect of Complex Yolk Antibody

1. Adjuvant and immune cycle

The complex antigen, as described above, carries out primary immunization to the 18-week-old laying hens, with the adjuvant accounting for 30% of the total immunogen. The composition of the adjuvant was 20% aluminum hydroxide and 10% ISA25. Secondary immunization is carried out 2 weeks after the first immunization, and the composition of the adjuvant is 30% of IS25 (ISA25), the third immunity is performed after 4 weeks of secondary immunity, and the composition of the adjuvant is 70 ( ISA70) 30%. Additional immunization was carried out every two months and the adjuvant was 30% of ISA70.

Comparison of the adjuvant The test group used aluminum hydroxide 30% for the first immunization, and after the second, it was compared with the new adjuvant using 30% ISA25 (ISA25).

2. Measurement of titer of yolk antibody

Anti-Helicobacter pylori, anti-urease, anti-protease, anti-lipase, anti-phospholipase, anti-E. Coli, anti-Salmonella enteritidis, anti-Staphylococcus aureus specific yolk produced by the above method Antibody (IgY) was isolated by the method shown in Example V.

The titer of total yolk antibody (IgY) and the specific yolk antibody (IgY) in the isolated water-soluble protein were performed using ELISA method using anti-antibody reaction.

1) Total yolk antibody (IgY) content

Rabbit anti-chick IgG antibody against diluted chicken IgG was placed in an ELISA assay plate and allowed to stand for 16 hours at 4 ° C for adsorption. After the reaction of the isolated water-soluble protein (antibody) on the plate, washed, and then rabbit rabbit IgG Ab-HRP (rabbit anti-chick IgG Ab-HRP) against IgC of HRP-bound chicken diluted to 1 / 30,000 HRP) is added. Hydrogen peroxide was used here as the substrate of HRP, and TMB was used as a color source. As the reaction stopper, absorbance at 450 nm was measured using ₂ -normal sulfuric acid (2N-H ₂ SO ₄ ).

The total yolk antibody (IgY) titer measurement results are shown in FIG. 11. As shown in FIG. 11, the titer of total specific yolk antibody was increased when the immunoadjuvant was used by the new composition than when aluminum hydroxide and ISA25 were used.

B) Measurement of titer of specific yolk antibody (IgY)

The titer of each specific yolk antibody of eight complex antigens was measured by ELISA method. Helicobacter pylori, Escherichia coli, Salmonella enteritidis, and Staphylococcus orreas antigens were diluted with an absorbent buffer (pH9.0) so that the absorbance (OD) value was 0.05 at 660 nm. Urease, protease, lipase, and phospholipase antigens were diluted to 100 μg / ml using products from Merck and Sigma aldrich. The diluted antigen was adsorbed onto the ELISA reaction plate and left for 16 hours. The filtered water-soluble special immune protein was added and washed for 1 hour at room temperature. The rabbit antibody IgG Ab-HRP (Habbit anti-chick IgG Ab-HRP) against the IgG of HRP-coupled chicken diluted to 1 / 10,000 was washed. ) Was added. Hydrogen peroxide was used here as the substrate of HRP, and TMB was used as a color source. As the reaction stopper, absorbance at 450 nm was measured using ₂ -normal sulfuric acid (2N-H ₂ SO ₄ ).

The results of measuring the content of the specific yolk antibody (IgY) are shown in FIGS. 1 to 5. And the results of the average content of the specific yolk antibody (IgY) is shown in Figures 6-10.

As shown in FIG. 1, each specific yolk antibody against the eight complex antigens was present in the yolk sac. Since the first inoculation in the 18-week-old laying hens and the second inoculation two weeks later, high levels of specific yolk antibodies were detected even in egg yolks of the first egg.

In Figure 2, Helicobacter pylori, Escherichia coli, Salmonella enteritidis. The specific yolk antibody against Staphylococcus orares showed higher titer of the specific yolk antibody when SA25 was used when the new immunoadjuvant was used. In addition, the new immunoadjuvant appeared early in the early stage of antibody formation and had higher antibody titers.

In Figure 3, the specific yolk antibody to urease, protease, lipase, phospholipase showed higher titers of the specific yolk antibody than when using SA25 when using a new adjuvant. In addition, the new immunoadjuvant appeared early in the early stage of antibody formation and had higher antibody titers.

In Figure 4, Helicobacter pylori, Escherichia coli, Salmonella enteritidis. The specific yolk antibody against Staphylococcus aureus showed an early antibody formation period and a higher antibody titer when fed vitamin A than the general diet when using a new immunoadjuvant.

In Figure 5, the complex specific yolk antibody against urease, protease, lipase, phospholipase appeared early in the early antibody-forming period when fed vitamin A than fed a general feed when using a new immune adjuvant and antibody titer Was even higher.

Anti-helicobacter pylori, anti-urease, anti-protease, anti-lipase, anti-force in one egg produced from laying hens inoculated with eight complex antigen emulsions prepared according to the examples of I, II, Eggs containing eight complex-specific yolk antibodies that simultaneously share lipase, anti-E. Coli, anti-Salmonella enteridis, and anti-Staphylococcus orreas specific yolk antibody (IgY) were produced.

III. Egg yolk powder

Anti-Helicobacter pylori, anti-urease, anti-protease, anti-lipase, anti-phospholipase, anti-E. Coli, anti-Salmonella enteritidis, anti-Staphylococcus aureus specific yolk produced by the above method Egg yolk powder was prepared by spray drying the yolk containing both antibodies (IgY).

Ⅳ. Isolation of Water-soluble Special Immune Protein in Egg Yolk by Addition of Trehalose to Deionized Water

Trehalose was added to the ionized water in 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% to make sugar-added ionized water, that is, sugar ionized water. With this sugar ionized water, water-soluble special immunoproteins in egg yolk were isolated as in the method of FIG. The titer of the water-soluble special immune protein was higher than that of the water-soluble special immune protein separated by normal ionized water. This suggests that the breakdown of water-soluble special immune protein in egg yolk proceeded when it was separated by general ionized water. After freezing at -20 ° C. for 3 days for each control and each treatment, thawing in 20 ° C. water was followed by measuring the titer of water-soluble special immune protein for each control and treatment. As a result, the titer was higher in all treatments than the control, and there was almost no difference in titer from 7% to 10%, which was very stable at 7% to 10% concentration. However, 1% to 6% also showed higher titers than the control, and when frozen and thawed after the addition of sugars, the water-soluble special immune protein was protected without destruction and was found to be very effective during long-term preservation.

As evidenced above, hepatobacter pylori, urease, protease, lipase, phospholipase, which are the causative agents of enteritis, and high yolk antibodies with E. coli, Salmonella enteritidis, and Staphylococcus aureus, which are the causative agents of food poisoning, By developing functional eggs, a variety of antibodies can be produced simply and simultaneously. In addition, it is expected to be used in many fields because it can be used as a variety of material additives by using the separation of specific yolk antibodies using sugar-added ionized water, which has an excellent effect on economy and stability compared to other separation methods and the advantage of removing pigments.

Claims (8)

Helicobacter pylori antigens, urease antigens, protease antigens, lipase antigens, phospholipase antigens, E. coli (EHEC) antigens, Salmonella enteritidis, Staphylococcus orreas antigens that cause enteritis, Anti-Helicobacter pylori, anti-urease, anti-protease, anti-lipase, anti-phospholipase, anti-E. Coli, anti-Salmonella enteritidis, anti Method for producing complex specific yolk antibody (IgY) against Staphylococcus. The method of claim 1, The ratio of the immunogen for inoculation for the production of the complex specific yolk antibody is Helicobacter pylori: Escherichia coli: Salmonella enteridis: Staphylococcus orares: The ratio of the adjuvant is 3: 1.5: 1.5: 1: 3, secreted by Helicobacter pylori The protease, lipase, phospholipase is in the form of a powder, the method of the immunity is 0.5ml per laying hens. The method of claim 1, Helicobacter pylori is adjusted to OD of 0.5 at 660nm to 0.15ml, Escherichia coli and Salmonella enteridis to 0.3 at 660nm to 0.075ml, Staphylococcus ureus to 0.3 at 660nm, 0.05ml and 0.15ml for immunoadjuvant. And emulsifying the urease, protease, lipase and phospholipase complex antigen powder secreted by Helicobacter pylori by adding 600 µg. The method according to claim 1 or 2, In the production of complex specific yolk antibodies against the causes of enteritis and food poisoning, the composition of the immunogen in the first immunity is 20% aluminum hydroxide, 10% of ISA25, and 25% of secondary immune (ISA25) 30%, the method after the third immunity to 70% (ISA70) 30%. The method according to claim 1, 2, 4, Method of obtaining a titer and synergistic effect of the complex specific yolk antibody by the immunization cycle of laying hens 18 weeks of age, 2 weeks after the second, 4 weeks after the third. A method of adding vitamin A in a feed, characterized in that any one of 10,000 IU, 20,000 IU, 30,000 IU, 40,000 IU, 50,000 IU, 60,000 IU is added per kg of feed of laying hens. Egg yolk powder prepared by separating the egg yolk and egg yolk from the egg yolk antibody and vitamin A produced by claim 1 to 6 by spray drying the yolk containing the complex yolk antibody. The complex specific egg yolk produced according to claim 1, 2, 3, 4, 5 and 6 is diluted with an appropriate multiple of the yolk solution with alkaline ionized water and acidic ionized water to which sugar is added and the water-soluble specific egg yolk at 5 ° C. In the method of separating the antibody layer and the insoluble protein layer by gravity drop method Water-soluble complex specific yolk antibody and insoluble protein, characterized in that the added content of the sugar in the acidic ionized water and alkaline ionized water is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% Purification method Using any one of pH 11, 9, 10 and acidic ionized water of pH 5, 4, 3 to separate the water-soluble special immune protein layer and the insoluble protein layer of the specific egg yolk antibody solution, Dilution ratio of 4, 5, 6, 7, 8, 9, 10, 11, 12 when diluting egg yolk with acidic ionized water and alkaline ionized water to separate the water-soluble special immune protein layer and the insoluble protein layer of the complex specific yolk antibody solution Using any one of 13, 14, 15, 16, 17, 18, 19, and 20 times