KR20020056612A - The method for production of egg containing anti-Streptococcus mutans IgY, anti-Streptococcus sanguis IgY and anti-Actinomyces israelii IgY individually and egg, yolk, IgY thereof and ice-cream containing complexing specific IgY for anti-Streptococcus mutans, anti-Streptococcus sanguis and anti-Actinomyces israelii - Google Patents

Abstract

PURPOSE: Provided are a method for production of egg containing anti-Streptococcus mutans IgY, anti-Streptococcus sanguis IgY and anti-Actinomyces israelii IgY individually and egg, yolk, IgY thereof and ice-cream containing complexing specific IgY for anti-Streptococcus mutans, anti-Streptococcus sanguis and anti-Actinomyces israelii, thereby preventing oral diseases including tooth decay, periodontal disease, etc. CONSTITUTION: The manufacturing method of egg is characterized by mixing each one of antigens of Streptococcus mutans, Streptococcus sanguis and Actinomyces israelii with an emulsifying agent of aluminium hydroxide; inoculating the each mixture into a hen; mixing the each antigen with ISA25 emulsifying agent then inoculating the each mixture into the hen; and homogenizing each antigen with ISA25 emulsifying agent then inoculating the mixture 9 times into the hen after initiation of egg-laying.

Description

Straptococcus mutans, Straptococcus lanceus, Egg production method containing special immunoprotein against Actinomyces Israeli, Egg and egg yolk produced by the production method, Anti-oral disease special immunoprotein and immunoprotein The method for production of egg containing anti-Streptococcus mutans IgY, anti-Streptococcus sanguis IgY and anti-Actinomyces israelii IgY individually and egg, yolk, IgY approximately and ice-cream containing complexing specific IgY for anti-Streptococcus mutans , anti-Streptococcus sanguis and anti-Actinomyces israelii}

The present invention is to inoculate each chicken by inoculating each of the chickens by inoculating Streptococcus mutans , Streptococcus sanguis , Actinomyces israelii causing oral disease, Produces a special immune protein (IgY) against the bacteria causing oral disease in egg yolk, and provides a technology that uses a combination of special immuno-protein combined with a suitable ratio of each produced special immunoprotein as a food additive.

Another feature of the invention relates to an ice cream containing a complex special immunoprotein.

In connection with the present invention, the conventional Korean technology was invented according to the following principle.

Newborn babies in mammals obtain their mother's immune antibodies through the placenta or breast milk and immunize early infections until their immune function matures.

However, in the case of an animal below the placenta and breast milk, that is, an oviparous animal, the immune antibody obtained by the mother chicken is transferred and accumulated in egg yolk and transmitted to the offspring. Antibodies in egg yolk correspond to antibodies of the mammalian IgG class, but have slightly different protein chemistry and are antibodies derived from egg yolk and are therefore called IgY (Immunoglobulin Yolk) in the field of comparative immunology.

The transfer of immune antibodies from mother chickens to eggs to chicks was discovered in 1893 by Klemberer.

After that, various kinds of antigens (bacteria, viruses, proteins, etc.) were immunized to laying hens until the 1960s, and it was confirmed that the corresponding specific antibodies migrated and accumulated in egg yolk (Markeyson 1965; Fraser et al. 1934, Petterson). Et al. 1962).

Based on these findings, a study was conducted to prevent oral diseases by inoculating the caries-causing bacteria in chickens and extracting the immune proteins produced in egg yolk from eggs.

Lee, Nam-Hyung (1997), in Korea, lays down a diet for raising egg protein and a method for producing eggs containing a large amount of special immunoprotein by inoculating a vaccine of Straptococcus mutans strain as specified by this feed. Korean Patent Application Publication No. 1999-011237). This is an egg production method that ingests feed containing kelp mill, cinnamon, mint or charcoal in the feed to the laying hens, and immunizes the Strain Tococcus mutans to the laying hens to enhance the immunoprotein content. Lee Seung-bae (1996) has applied for a method for preparing egg yolk IgY antibody for caries prevention (Korean Patent Publication No. 1997-073607). This relates to a method for producing egg yolk IgY antibody for preventing caries by immunizing Straptococcus mutans to laying hens.

The bacteria that cause oral diseases in humans are the bacteria that produce glucanase in human dental plaques, such as Actinomycetaceae, Streptococci, and Haemophili. , Gram-negative anaerobes, which are largely divided into species, Streptococcus sanguis , Streptococcus minitor , Actinomyces naeslundii , Actinomyces viscosus , Bacterionema matruchotii , Bacteroides species , Rothia dentocariosa, and the like .

The most common cause of tooth decay is the straptococcus mutans, which is a glucose transferase (glucosyl transferase) produced by the bacterium itself by binding the surface protein of the straptococcus mutans to the tooth surface in the presence of sugar. G Tase) plays an important role in causing tooth decay. Dental caries is initiated by G Tase fermenting various sugars provided from meals to produce insoluble and sticky glucan.

Straptococcus mutans is composed of Cell Associated Glucosyl Transferase (CA G Tase), which synthesizes insoluble glucan, and Cell-Free Glucosyl Transferase (CF G Tase), which synthesizes soluble glucan. Kind action causes the caries to bind to the tooth surface.

However, oral diseases have a problem in that it is necessary to prevent a number of strains that cause oral diseases, not one bacterium, so that a complex special immunoprotein developed by extracting and mixing immunoproteins from eggs containing each specific antibody.

In order to solve the above problems, an object of the present invention is to conventionally produce immunoprotein by antigenizing with only one Straptococcus mutans strain, but in the present invention, Straptococcus mutans (or Streptococcus mutans ) causing oral diseases. , Vaccines of Streptococcus sanguis and Actinomyces israelii strains were injected into different chickens to provide a method for producing specific immunoproteins (IgY) for these three strains. .

It is still another object of the present invention to prepare a complex special immunoprotein prepared by combining a certain proportion of special immunoproteins extracted from eggs or egg yolk produced in the egg production method containing each special immunoprotein, and also the complex special immunity. To provide an ice cream containing protein.

1 is a specific immunoprotein (IgY) titer change for Streptococcus mutans , Streptococcus sanguis , Actinomyces israelii .

FIG. 2 shows total immunoprotein (IgY) titers for Streptococcus mutans , Streptococcus sanguis , Actinomyces israelii .

FIG. 3 is a specific immunoprotein (IgY) titer for Streptococcus mutans , Streptococcus sanguis , Actinomyces israelii .

In order to achieve the above technical problem, the present invention is to antigenize Streptococcus mutans ( Streptococcus mutans ), Streptococcus sanguis , Actinomyces israelii causing oral disease Inoculate each chicken to produce a special immunoprotein (IgY) against bacteria causing oral disease in egg yolks of laying hens, and to provide a complex special immunoprotein containing a specific ratio of each produced special immunoprotein. There is a characteristic.

Another invention is characterized by providing eggs, egg yolk, and complex special immunoproteins containing anti-oral disease special immunoproteins (IgY) produced by the production method.

Another invention relates to an ice cream containing an anti-oral disease complex special immune protein extracted from the egg or egg yolk.

In more detail, the composition of the present invention is first inoculated to chickens by mixing each of the Straptococcus mutans, the Straptococcus lance, and the Actinomyces israeli antigen with an emulsifier sodium hydroxide, and the emulsifier (ISA25) and After the second inoculation by mixing, homogenization is performed to inoculate a total of nine times after the start of spawning, and the inoculation interval is immunized with a dose of about 10 ⁸ / ml for about 2 weeks to immunize special yolk proteins for three antigens in egg yolk. It relates to an egg production method characterized in that it contains.

In more detail, the egg yolk obtained by separating the egg yolk membrane from the egg containing the special immunoprotein obtained for the antigen administered to the chicken using a predetermined container is extracted, and distilled water is added to the extracted egg yolk and stirred. After a certain period of time in a low temperature room, the ratios of the specific immunoproteins to the water-soluble special immunoproteins Straptococcus mutans, Straptococcus lanceus, and Actinomyces israeli antigen contained in the supernatant centrifuged were determined. It is characterized by mix | blending with 1: 1: or 1: 2: 2.

Another aspect of the present invention is to prepare an ice cream containing the anti-oral disease complex immune protein formulated in any one of the specific ratio of the above-mentioned special immuno-protein, anti-oral disease complex immune protein egg yolk powder 5.0% or less, anti-oral disease complex immunity It relates to an ice cream added with less than 0.6% protein IgY extract powder.

Hereinafter, the configuration of the present invention will be described in more detail based on examples.

However, the scope of the present invention is not limited to the claims of the present invention by the embodiment.

Example 1

Inoculation of chickens with Straptococcus mutans, Straptococcus lanceus, Actinomyces Israeli antigen and production of immunoprotein (IgY)

The lyophilized Straptococcus mutans (KCCM11823) and Straptococcus lance were purchased and used at the Yonsei University Korea Microbial Conservation Center, and the Straptococcus mutans and Straptococcus lance were respectively used in the Otaker (Otake: 1991) culture method. Was added by 5% sucrose in Brain-Heart Infusion (BHI) medium at 37 ° C. at 37 ° C. for 24 hours for Straptococcus mutans, and for 48 hours for Straptococcus lance, and enriched by Actino Meises Israeli was enriched in brain-heart infusion medium at 37 ° C., 10% CO ₂ incubator for 3-7 days.

After each of the above-expanded Straptococcus mutans, Straptococcus lanceus, Actinomyces israeli strain were killed in an incubator to which 5% formalin solution was added, 10,000 g of the killed bacteria were added at 5 ° C. Collected by centrifugation for 15 minutes.

The emulsifier hydroxide aluminum when the three strains each of said first dose, and then mixed with an emulsifier (IAS25) since the second, homogenized and inoculated with a total of nine times after the start of spawning, immunization interval is viable count dose as 2 weeks 10 ⁸ Immunization was inoculated to about / ml.

Example 2

1. Total immunoprotein (IgY) content and special immunoprotein (IgY) content after inoculation of Straptococcus mutans, Straptococcus lanceus, Actinomyces Israeli antigen

Eggs collected during the 33 weeks of laying hens were mixed and homogenized with the same treatment eggs on a weekly basis, and then, after adding 100 ml of distilled water to 10 g of egg yolk, the mixture was stirred and left for 4 hours in a low temperature room at 4 ° C. Water-soluble special immunoproteins were isolated by centrifugation at 10,000 × g for 15 minutes.

The data for analyzing the content of isolated special immunoprotein is shown in Table 1.

The specific immunoprotein (IgY) content of complex strains was higher than that of control, and the specific immunoprotein contents of Straptococcus mutans strains were the highest, and the specific immunoproteins of Straptococcus strains and Actinomyces israeli strains. The content of appeared similar.

Special Immune Protein Content in Eggs of Chickens Specified by the Invention Treatment Special immunoprotein content (mg / yolk) Control 0.30 Complex Strap Toe Caucus Mutans 0.60 Strap Toe Caucasus Above 0.40 Actinomais Israeli 0.45

2. Determination of total immunoprotein (IgY) and special immunoprotein (IgY) titers

The total immunoprotein (IgY) and special immunoprotein (IgY) titers are all measured by a non-competitive sandwich ELISA test, and the special immunoprotein (IgY) titers are coatings containing Straptococcus mutans. Buffer (0.02M Tris, 0.15 NaCl, pH 9.0) was prepared, 100 μl was filled in a microplate well, and incubated at 4 ° C. for 12 hours.

After washing three times with 150 µl of washing buffer (0.02 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4), 100 µl of the sample to be analyzed (yolk soluble immunoprotein) was added and reacted at room temperature for 1 hour. After washing three times with 150 μl wash buffer, 100 μl of rabbit anti-chicken IgG Ab-HRB diluted to 10,000: 1 was added and reacted at room temperature for 1 hour, followed by washing three times with 150 μl wash buffer. , 100 μl of substrate solution (0.1% TM BS, 0.05M citric acid-phosphate buffer, pH 5.0, 0.02% H ₂ O ₂ ) was added.

The mixture was reacted at room temperature for 1 hour, and 50 µl of a reaction stopper solution (2N H ₂ SO ₄ ) was added thereto, and the absorbance (OD) was measured at 450 nm.

The change in titer of the specific immunoprotein obtained in egg yolk collected during 33 weeks of laying hens is shown in FIG. 1.

The measured results are as shown in the total immunoprotein titers in FIG. 2 and the specific immunoproteins (IgY) in FIG. 3.

Referring to Figure 2, when describing the total immunoprotein titers, the titers for the Straptococcus mutans strain is measured the highest, and the titers of the Straptococcus above and Actinomyces israeli strain than the Straptococcus mutans strain Low, but similar. The total immunoprotein titer of the control group corresponding to the normal egg was measured close to 170, and the specific immunoprotein titer necessary for the prevention of oral disease was higher than 170.

Referring to FIG. 3, the titer of the specific immunoprotein was determined to be 1.2, Straptococcus mutans 0.5, and Actinomyces Israeli 0.55. It was rarely measured in the control egg.

In this experiment, the most immunoproteins for the Straptococcus mutans strain were made, while the other were less.

Example 3

Blending process for mixing immunosuppressive protein against extracted Straptococcus mutans, Straptococcus lanceus, Actinomyces israeli antigen

A complex immune protein was prepared in which an immunoprotein against Straptococcus mutans, Straptococcus lanceus, Actinomyces israeli was combined at 1: 1: 1 or 1: 2: 2.

Example 4

Manufacture of complex special immunoprotein-added ice cream

The preparation ratio of ice cream containing 5.0% of anti-oral disease complex immunoprotein yolk powder and 0.6% of anti-oral disease complex immunoprotein extract powder is shown in Table 2.

Special Ice Cream Formulation Example Raw material Compounding ratio A Compounding ratio B Cream (35% fat) 34.0 34.0 Skim condensed milk (30% solids) 25.0 25.0 Skim milk (8% solids) 10.0 11.0 Skim milk powder 2.0 4.0 Per liquid (80% bric) 19.5 20.0 stabilizator 0.6 0.6 Anti-oral disease bacteria yolk powder 5.0 - Anti-oral Disease IgY Extract Powder - 0.6 Spices A reasonable amount A reasonable amount water 3.9 4.8

The present invention is not limited to the technical spirit described in the specific embodiments, diagrams or drawings described above, and those skilled in the art without departing from the gist of the invention claimed in the claims. As long as anyone can carry out various modifications, such changes are within the scope of the claims.

Combined with each specific immunoprotein (IgY) against the Streptococcus mutans , Streptococcus sanguis , Actinomyces israelii antigen produced by the present invention Eggs containing complex special immunoproteins and ice creams containing complex special immunoproteins extracted from these eggs can be ingested to provide excellent efficacy in preventing oral diseases.

In addition, the complex special immunoprotein extracted from egg yolk of the egg produced by the production method according to the present invention is water-soluble, so it is easy to clean the mouth by burning in water, has a high sugar content, such as confectionery or ice cream special immunity By adding protein, expected effects on dental caries, gum disease and the like are expected.

Claims (9)

Streptococcus mutans , Streptococcus sanguis , Actinomyces israelii antigens were mixed with an aluminum hydroxide emulsifier to inoculate each other firstly , and then Each antigen was mixed with the ISA25 emulsifier and inoculated secondly, and the respective antigens were homogenously mixed with the ISA25 emulsifier and inoculated and immunized a total of 9 times after initiation of spawning to produce an anti-oral disease-specific immunoprotein for each antigen. Egg production method characterized by producing each egg containing IgY). According to claim 1, When the inoculation after the start of spawning, each of the homogenized mixed antigen and ISA25 emulsifier is dosed 10 ⁸ / ㎖ bacteria, the inoculation interval is inoculated two weeks inoculation immunization anti-oral disease for each antigen Egg production method characterized by producing each egg containing a special immunoprotein (IgY). An egg containing anti-oral disease special immunoprotein (IgY) produced by the method of claim 1 or 2. Egg yolk powder containing an anti-oral disease special immunoprotein (IgY) prepared by lyophilizing the egg yolk from which the egg yolk film is removed from a predetermined container of the egg produced by the method of claim 1 or 2. Each of the eggs produced by the production method according to claim 1 or 2, distilled water is added to the egg yolk and stirred, and then allowed to stand in a low temperature room for a certain period of time, each anti-oral disease water-soluble special immunoprotein prepared by centrifugation (IgY ). Each of the special immunoproteins (IgY) of claim 5, each of the specific immunoproteins for Streptococcus mutans , Streptococcus sanguis, Actinomyces israelii antigen IgY) Anti-oral disease complex immunoprotein (IgY) which mix | blended in any one which mix | blended 1: 1: or 1: 2: 2. Ice cream containing an anti-oral disease complex immunoprotein (IgY) of claim 6. 8. The ice cream according to claim 7, which contains 0.6% or less of the anti-oral disease complex immunoprotein (IgY). Ice cream containing 5.0% or less of the anti-oral disease complex immunoprotein (IgY) yolk powder of claim 4.