KR20020074802A - Method for Preparing Powder of Egg Yolk Having IgY, Powder of Egg Yolk , and Compositions Comprising the Same - Google Patents

Abstract

PURPOSE: A process of preparing egg yolk powder containing antibodies treated with a grapefruit seed extract to maintain the potency of antibodies and to increase preservability and an additive to remove a fishy smell in the yolk and to increase the flavor and preservability thereof is provided. Because the composition containing the egg yolk powder is power, it is convenient to handle and take and can also be encapsulated. CONSTITUTION: The antibodies induced from strains causing various diseases in mammals are injected to laying hens. An egg yolk liquid containing an antibody is separated from the egg produced from the immunized laying hens, added with 250 to 1,000ppm grapefruit extract and 5 to 15% by weight of white sugar and heated at 60 to 70deg.C for 3 to 10min one to three times. The heat treated egg yolk liquid is spray-dried or freeze-dried. The composition comprises 40 to 50% by weight of egg yolk powder, 45 to 55% by weight of lactic acid powder, 0.5 to 1.5% by weight of silica dioxide, 0.5 to 1.5% by weight of magnesium stearate and 0.1 to 2% by weight of a sweetener.

Description

Method for preparing yolk powder containing antibody, yolk powder and yolk powder containing composition prepared from the same {Method for Preparing Powder of Egg Yolk Having IgY, Powder of Egg Yolk, and Compositions Comprising the Same}

The present invention relates to a method for producing an antibody-containing egg yolk powder, a yolk powder prepared therefrom and a yolk powder-containing composition. More specifically, the present invention relates to an antibody-containing egg yolk powder that has been processed to sterilize and dry an antibody-containing immune column obtained by inoculating an antigen into an alga to maximize the titer of the antibody and to facilitate its use, and a composition comprising the same.

Recently, researches on antibody production technology using the egg yolk in which the antibody is introduced after immunization of laying hens by inducing antigen-antibody reactions by injecting antigens prepared from specific cells, mainly in Japan and Canada, have. Japan Daiokaku Co., Ltd. has developed an antibody production method using algae and the use technology of the antibody.Human Rotavirus (HRV), Nippon Nogeikagaku Kaishi Vol.68, No.10, pp. 1457 ~ 1462 (1994), The advantages of the production and use of IgY antibodies against Edwardsiella tarda, Streptococcus mutans are disclosed.

In addition, in Korea, antibodies containing eggs against Helicobacter pylori, a causative agent of gastritis, and streptococcus mutans, a decay bacterium, have been produced in Korea. It has been confirmed that there is a therapeutic effect on the disease.

As described above, algae-derived immune antibodies can be very efficiently used for preventing and treating diseases caused by bacteria, viruses, etc. in animals and humans, and are particularly inexpensive and easily produced than mammalian blood antibodies. It has many advantages. Nevertheless, due to some problems, there are many difficulties in the practical use of immune antibodies derived from algae. Egg immune antibodies (IgY) are unstable at high temperatures (above 70 ° C) and very unstable to strong acids (pH 2-3) in animal and human digestive organs compared to antibodies obtained from mammalian blood (IgG). .

Currently, in order to minimize the loss of the titer of the antibody, in order to not heat the antibody, it is inevitably used in the form of spawning. However, when used in the form of spawning, the titer of the antibody can be preserved, but there is a risk of infection by the lipid of egg yolk, which is additionally taken when the effective amount of antibody required for treatment is taken, and after ingestion of egg yolk The antibody is denatured by strong acidity. Therefore, in order to use the egg immune antibody as an agent for preventing and treating gastrointestinal infection, there is a need for a method of stabilizing the antibody from external factors that reduce the titer of the antibody.

Shimizu et al., In J. of Food Science, 1994, 59 (4), 763-772, added 75 to 80 wt% sucrose or sugar to egg yolk to maintain antibody titers. A method of heat treatment at 15 ° C. for 15 minutes is disclosed. Lin and cunnignham are additives such as 47.7 wt% citric acid, 28.4 wt% alkaline treated soybean oil, 19 wt% skim milk, and the required amount of glucose, sucrose, xanthan gum, or vanilla extract sterilized in egg white Yogurt prepared by inoculating Lactobacillus vulgaris and Streptococcus thermophilus was added (1984), and Korean Patent Application No. 97-574 discloses fermented milk containing IgY antibody.

All of the above techniques are methods that use egg or egg yolk in liquid form, and are difficult to be applied to various fields, and are difficult to ingest and store, and have the disadvantage that the titer of the antibody may be reduced by the acidity of the stomach. In addition, the fishy smell of the raw material itself and stickiness due to lipids is also a problem in the commercialization.

Prior to the present invention, the present inventors have developed a technique for producing immune eggs containing antibodies by injecting Helicobacter pylori into a laying hen as an antigen (Korean Patent Application No. 2000-11226). In order to increase the product value of the yolk solution obtained through this technology and to expand its field of application, the yolk powder containing antibody is not only sterilized by microorganisms and yeast but also optimized sterilization and drying conditions to maintain the titer of the antibody even during drying. It was to develop an antibody-containing yolk powder composition with improved flavor and preservation together with the coating technology of the antibody to prevent the denaturation or destruction until the antibody is used in the body.

An object of the present invention is to provide a method for producing an antibody-containing yolk powder in a powder state that is easy to consume by processing the yolk containing the antibody.

Another object of the present invention is to provide an egg yolk powder containing an antibody treated with grapefruit seed extract in order to maintain the titer of the antibody and increase its preservation.

It is another object of the present invention to provide a yolk powder containing composition comprising an additive for removing fishy smell of yolk and increasing flavor and preservation.

Another object of the present invention is to provide a method for encapsulating egg yolk powder so that the effective antibody is not denatured or destroyed in the product until the effective antibody is effective.

The above and other objects of the present invention can be achieved by the present invention described below.

1 is a graph showing the change in the specific IgY content contained in egg yolk solution of eggs collected from the egg vaccinated inoculation antigen.

Figure 2 is a graph showing the change in the total IgY content contained in egg yolk solution of eggs collected from the egg laying inoculation antigen.

Figure 3 is a graph showing the change in titer of the antibody according to the repetition of the sterilization process heat treatment of yolk solution at 64.5 ℃ for 5 minutes 50 seconds.

Figure 4 is a graph comparing the titer of the yolk yolk solution, and after adding glucose or white sugar to the yolk solution.

Figure 5 is a graph showing the change in the peroxide value of lipids after the addition of grapefruit seed extract of different concentrations to yolk solution.

Figure 6 is a graph showing the titer of the antibody according to the concentration of grapefruit seed extract added to yolk solution.

7 is a graph comparing the titers of antibodies in egg yolk solution, a concentrate obtained by extracting a water-soluble protein from an egg yolk solution, and a clause powder obtained by freeze-drying the concentrate.

8 is a graph comparing the titer of the total IgY of the spray-dried sample and the lyophilized sample of the antibody-containing yolk solution, Figure 9 is a graph comparing the titer of specific IgY of the sample.

10 is a graph measuring the titer of the antibody according to temperature in order to measure the thermal stability of the antibody in egg yolk powder.

The present invention relates to a method for producing an antibody from algae, separating the yolk solution from the egg containing the antibody, and then spray drying or vacuum drying the yolk powder to process the yolk powder.

Hereinafter, the invention will be described in more detail for each step.

Production of antibodies

(1) Preparation of the antigen

After culturing the cells of the disease-causing strains in mammals, the medium component is removed by centrifugation and the bacteria are inactivated and used as antigen. Examples of strains used in the present invention are Helicobacter pylori (H.pylori), Campylobacter pylori (C.pylori), Staphylococcus aureus (S.aureus), Haemophilus Haemophilus influenzae (H.influenzae), Moraxella catarrhlis (M.catarrhlis), candida albicans (C.albicans), Human Rotavirus (H. Rotavirus), or Streptococos Muteptococcus mutans (S.mutans) and the like.

(2) immunization of algae

The antigen prepared as described above was mixed 1: 1 with an adjuvant such as Freund adjuvant and inoculated three times at 1 week intervals of 2 ml each in the chest of the laying hen. It is done at the point where the titer decreases, but it is generally preferred to do it every two months.

(3) Preparation of egg yolk solution containing antibody

After three inoculations, eggs from the laying hens are harvested to separate the yolk solution.

The method for preparing the antibody-containing egg yolk solution is disclosed in more detail in Korean Patent Application No. 2000-11226.

Pretreatment of Egg Yolk Containing Antibody

(1) sterilization treatment

In order to sterilize contaminants such as Salmonella and yeast in egg yolk solution, heat treatment is performed before the yolk solution is processed. The preferred sterilization temperature at which the titer of the antibody is maintained is 60 to 70 ° C. and the time required for sterilization is 3 to 10 minutes. Since the IgY antibody is an immune protein, when the sterilization temperature exceeds 70 ° C, the titer is markedly lowered, and below 60 ° C, no bactericidal effect is exhibited. The sterilization conditions are Escherichia coli group, Salmonella is a complete negative reaction, the condition is less than 1000 cfu / g for the general bacteria, the number of sterilization can be adjusted within the range of 1 to 3 times.

(2) preservative treatment

Algae's fresh eggs are almost aseptic, but the shells are contaminated with germs, molds, soils, washings, and packaging materials by bacteria and mold, and contaminants enter the eggs through moist shells and multiply.

Therefore, in the present invention, the grapefruit seed extract, which is a natural antimicrobial substance, is used to increase shelf life when processing the yolk solution and to use it as a functional material. Since grapefruit seed extract is a natural substance, it is safe for human body, but it is an excellent preservative with antioxidant effect and prevention of microbial contamination.

Grapefruit seed extract of 25ppm or more shows an antimicrobial effect in egg yolk, antioxidant content in the case of 100ppm or more, and the higher the concentration, the higher the antibody titer in egg yolk, grapefruit seed extract is added at a concentration of 100 to 1000ppm Preferably, it is more preferably added at a concentration of 500 to 800 ppm.

A preservative is added to the yolk solution after sterilization to prevent lipid oxidation and protein degeneration of the yolk solution.

(3) addition of sugar

Optionally, 5-15% by weight of glucose or white sugar, preferably white sugar may be added to the yolk solution to maintain the titer of the antibody.

(4) removal of fat-soluble substances

Alternatively, only water-soluble proteins may be extracted to remove fat-soluble substances such as cholesterol in egg yolk. The aqueous protein layer can be recovered by mixing and stirring the yolk solution with water in a volume ratio of 1: 3 to 1:10, eluting the water soluble protein in the yolk solution, and then lyophilizing and removing the pellet layer containing fat.

Dry Powderization of Egg Yolk Containing Antibodies

The yolk solution, which has been sterilized, added with a preservative, is spray-dried or freeze-dried to prepare a powder.

Spray drying is achieved by homogeneously spraying the yolk solution for 1 to 3 seconds at an inlet temperature of 170 to 180 ° C, and drying the chamber at a temperature of 90 to 95 ° C.

Lyophilization takes place at −40 to −60 ° C., 0.03 to 0.04 mBar.

Improved Flavor of Antibody-containing Egg Yolk Powder

The inherent fishy smell of egg yolk is the biggest problem of eating it. The fishy odor is reduced by the powdering of egg yolk, but the excipient is added to remove the remaining fishy odor to improve the palatability and to maintain an effective antibody titer in order to improve palatability. Excipients include lubricants to improve flow, flavoring flavors, salts, and sugars.

Glidants are SiO ₂ , S-Mg, or mixtures thereof, and flavoring agents are banana powder, cocoa powder, vanilla powder, strawberry flavor powder, grape flavor powder, orange flavor powder, coffee flavor powder, cinnamon flavor powder, mugwort powder , Cream flavor powder, or mixtures thereof, sugars are glucose, aspartame, xylitol or mixtures thereof.

Encapsulation of Antibodies

To encapsulate the antibody, 0.1 to 10% by weight of methacrylate unit or polymer solution is added to the yolk solution or the prepared yolk powder.

Encapsulating the antibody as described above maintains the antibody titer stably against external factors such as heat, acidity, etc. during processing.

The invention can be better understood by the following examples, which are intended to illustrate the invention and are not intended to limit the scope of protection defined by the appended claims.

Example

Preparation of the antigen

Helicobacter Philo ( H. pylori) strains were inoculated in Trypticase soy agar (BBL 11043) added with 5% sheep serum (sheep serum) and then incubated for 2 days in a CO ₂ incubator at 37 ℃. The cultured cells were centrifuged to remove the media components, and then the cells were inactivated and used as antigens.

Preparation of Antibody-containing Egg Yolk

The antigen was mixed 1: 1 with Freund's adjuvant oily adjuvant and inoculated three times at 1 ml intervals in 2 weeks intervals in the chest of the laying hen. After the fourth round, the titer of the antibody rapidly dropped (160 days after the first inoculation). Post) inoculation increased the titer of the antibody. Eggs produced in laying hens were collected from three inoculations and egg yolks were separated.

Using the egg yolk solution, the titer stability of the antibody was measured and processed according to the following examples.

Example 1: Content of IgY Antibody in Egg Yolk

The yolk solution prepared by collecting immune eggs at weekly intervals was mixed with a yolk solution: water: lambda-carrageenan in a volume ratio of 1: 1: 2, and then filtered and extracted to prepare a water-soluble protein sample. The total IgY and specific IgY contents in the water soluble protein samples were performed by indirect ELISA.

(1) specific IgY content

The cells were diluted and diluted with buffer so that the absorbance (OD) value was 0.05 at 660 nm. Diluted cells were coated on a microplate and overnight. The microplate was washed with rabbit anti-chick IgG peroxidase conjugate (AB-HRP) diluted to 1 / 10,000. TMB (tetramethylbenzidine) was used as a substrate of HRP, and absorbance was measured at 450 nm using 2N-H ₂ SO ₄ as a reaction stopper. The results are shown in FIG.

Specific IgY 1 unit refers to containing 18% by weight of specific IgY in water-soluble protein.

(2) total IgY content

The microplates were coated by diluting the rabbit anti-cheek IgG antibody, then overnight and washed. The water-soluble protein of diluted egg yolk was added to the microplate, reacted, washed, and added to rabbit anti-cheek IgG antibody-HRP diluted to 1 / 30,000. At this time, TMB was used as a substrate of HRP, and absorbance was measured at 450 nm using 2N-H ₂ SO ₄ as a reaction stopper solution. The results are shown in FIG.

Example 2A-2B: Stability of Antibody Titers

Example 2A: Stability of Antibody Following Heat Treatment

The yolk solution was sterilized twice at 64.5 ° C. for 5 minutes and 50 seconds to observe the change in titer after the first sterilization and the titer after the second sterilization.

As a result, the titer decreased by 4-10% at each sterilization.

Example 2B: Stability with Addition of Sugars

In order to prevent the titer of the antibody from decreasing, 10 wt% of glucose and white sugar were added to the yolk solution, respectively, and the titer of the antibody was measured. The antibody titer graph is shown in FIG. 4 with the yolk solution without sugar as the control group (A), with the addition of glucose as (B) and with the addition of the white sugar as (C). As shown in FIG. 4, in the case of adding (C) the white sugar to the yolk solution, the titer of the antibody was the highest.

Example 3A-3C: Preservative Treatment for Egg Yolk

Example 3A: Antioxidant Effect

In order to confirm the antioxidant effect of lipids according to the preservative treatment, grapefruit seed extract was added to the antibody-containing yolk solution immediately after Harlan at concentrations of 0 ppm, 100 ppm, 250 ppm, 500 ppm, and 750 ppm, and then 30 days at 30 ° C. for 30 days. Peroxide value according to the concentration of is measured and shown in FIG.

As shown in Figure 5, the yolk solution without the grapefruit seed extract was rapidly oxidized, and when treated at a concentration of 100ppm, 250ppm showed some antioxidant effect. On the other hand, treatment with grapefruit seed extract of 500ppm or more showed a sustained and distinct antioxidant effect.

Example 3B: Antibacterial Effect

In order to confirm the antimicrobial effect of the preservative treatment, the grapefruit seed extract was treated in antibody-containing yolk solution at concentrations of 10, 25, 50, and 100 ppm and stored at 5 ° C for 5 days to confirm the degree of microbial contamination against Salmonella and E. coli. It was. The addition of more than 25ppm showed an antimicrobial effect against both microorganisms.

Example 3C: Titer Stability of Antibodies

In order to confirm the titer stability of the antibody according to the preservative treatment, the grapefruit seed extract was treated in the antibody-containing yolk solution at concentrations of 0ppm, 250ppm, 500ppm and 1000ppm, respectively, and the titers of the antibodies were shown in FIG. 6.

The higher the concentration of the grapefruit seed extract, the higher the titer of the antibody, and thus, the grapefruit seed extract is believed to contribute to the titer stability of the antibody.

According to Examples 3A-3C it can be seen that when the grapefruit seed extract is added to the yolk solution at a concentration of 500 ppm or more, it contributes to the antioxidant, antibacterial, and antibody titer stabilization.

Example 4: Determination of titer according to concentration of antibody-containing egg yolk

The yolk solution and water were mixed and stirred in a volume ratio of 1: 5 to elute the water-soluble protein in the yolk solution, and then lyophilized at -20 ° C for 24 hours. The frozen eluate was thawed at 4 ° C. to separate the layer containing the water-soluble protein and the layer containing the pellet containing a large amount of fat, and then the water-soluble protein layer was recovered to obtain an aqueous solution (coarse) from which lipids and solids were removed. The article was concentrated to 1/8 volume using a vacuum concentrator to prepare a concentrate.

The concentrate was lyophilized at −50 ° C., 0.036 mBar to give a high concentration of the powder.

The antibody titers of egg yolk solution and the concentrate and lyophilized powder were measured and shown in FIG. 7. As shown in FIG. 7, the titer of the antibody increased as the concentration step progressed, and the titer of the antibody after lyophilization was 9 times higher than that of the yolk solution.

Example 5: Cholesterol Removal of Antibody-Containing Eggs

Water soluble protein was extracted as in Example 4. Pellets containing fat and cholesterol in egg yolk diluted in water were removed and concentrated and lyophilized by taking only the upper layer containing only water-soluble protein. The lyophilized powder was dissolved in water at room temperature, and then filtered through a filter paper. No filter material was observed.

Preparation of Antibody-containing Egg Yolk Powder

The antibody-containing yolk solution was fixed at an inlet temperature of 180 ° C. for 1 to 3 seconds, and the chamber temperature was 90 ° C. to homogeneously spray dry to prepare a yolk powder.

Separately, yolk powder was prepared by lyophilization at −50 ° C. and 0.036 mBar.

The yolk powder prepared above was compared by measuring the titer, water solubility, and mixing degree of the antibody by the following method.

Example 6: Comparison of Antibody Titer in Egg Yolk Powder

(Total IgY) and (Specific IgY) titers of the above-mentioned spray-dried powder and lyophilized powder antibodies were measured and shown in FIGS. 8 and 9, respectively. As shown in FIGS. 8 to 9, the antibody titer of the lyophilized powder was slightly superior, but there was no significant difference from that of the spray dried powder.

Example 7: Water Soluble of Egg Yolk Powder

Egg yolk portion of the egg is rich in fat-soluble substances such as lipid components and pigments are not well dissolved in water. In order to confirm the solubility in water due to the powdering of egg yolk, 180 g of yolk powder was dissolved in 20 g, 30 g, 60 g, and 90 g of water as follows.

Egg yolk powder: H ₂ 0 = 180g: 20g = 9: 1

Egg yolk powder: H ₂ 0 = 180g: 30g = 6: 1

Egg yolk powder: H ₂ 0 = 180g: 60g = 3: 1

Egg yolk powder: H ₂ 0 = 180g: 90g = 2: 1

The mixture of the ratio was homogenized with a stirrer and left at 4 ° C. for 6 hours, and then the dissolution state was visually observed and filtered using a filter paper. As a result, no separation of all four samples occurred and no filtrate was obtained. Powdered egg yolk solution was found to improve the solubility in water.

Example 8: Thermal Stability of Antibodies in Egg Yolk Powder

In order to test the thermal stability of the antibody in egg yolk powder, the yolk powder was dissolved in distilled water to prepare five 5% aqueous solution samples. One of these samples was a control group that was not subjected to warming, and the other four samples were warmed for 5 minutes in a 60 ° C, 70 ° C, 80 ° C, and 90 ° C water bath and cooled to room temperature, respectively. The activity of specific IgY antibodies was measured accordingly.

10 is a graph showing the change in activity of the antibody with temperature. As shown in FIG. 10, the antibody activity was maintained at 80% or higher in the samples treated at 60 ° C. and 70 ° C. compared to the antibody activity (100%) of the control group, whereas the antibody activity was rapidly increased in the samples treated at 80 ° C. or higher. Decreased. Therefore, it was found that the optimum conditions of the heat treatment for sterilization were 60 to 70 ° C.

Example 9A-9H: Flavor Improvement of Egg Yolk Powder

The antibody-containing yolk solution was sterilized at 64.4 ° C. for 5 minutes 20 seconds and then treated with 500 ppm of grapefruit seed extract. Then, the yolk powder was prepared by spray drying in the method of Example 6. In order to improve the flowability of the powder of egg yolk powder, 3 parts by weight of lubricant SiO ₂ (Aerosil 200) and 2 parts by weight of S-Mg were added to 80 parts by weight of the powder, and to remove fishy smell and improve flavor. Various additives were added and stirred as mentioned above. The taste, appearance and flowability of the prepared composition were observed and the results are shown in Table 1 below. At this time, all raw materials used in the whole process was sieving to 35mesh (500㎛) and maintained a dehumidification state. All parts in the following are parts by weight.

Example 9A

80 parts of egg yolk powder, 12 parts of crystal glucose, 3 parts of SiO ₂ (Aerosil 200), 2 parts of S-Mg, 1.5 parts of salt, 1.5 parts of cocoa powder

Example 9B

Egg yolk powder 80 parts, crystal glucose 13.5 parts, SiO ₂ (Aerosil 200) 3 parts, S-Mg 2 parts, salt 1 part, banana powder 0.5 part

Example 9C

80 parts egg yolk powder, 13 parts crystal glucose, 3 parts SiO ₂ (Aerosil 200), 2 parts S-Mg, 0.5 parts salt, 1.5 parts banana powder

Example 9D

80 parts egg yolk powder, 8 parts crystal glucose, 5 parts xylitol, 3 parts SiO ₂ (Aerosil 200), 2 parts S-Mg, 0.5 parts salt, 1.5 parts banana powder

Example 9E

80 parts of egg yolk powder, 7 parts of crystal glucose, 5 parts of xylitol, 3 parts of SiO ₂ (Aerosil 200), 2 parts of S-Mg, 1.5 parts of salt, 1.5 parts of banana powder

Example 9F

80 parts of egg yolk powder, 3 parts of SiO ₂ (Aerosil 200), 2 parts of S-Mg, 12.5 parts of crystal glucose, 0.5 parts of aspartame, 0.5 parts of salt, 1.5 parts of banana powder

Example 9G

80 parts egg yolk powder, 3 parts SiO ₂ (Aerosil 200), 2 parts S-Mg, 12.7 parts lactose, 0.3 parts aspartame, 0.5 parts salt, 1.5 parts banana powder

Example 9H

80 parts of egg yolk powder, 3 parts of SiO ₂ (Aerosil 200), 2 parts of S-Mg, 12.4 parts of glucose, 0.2 parts of aspartame, 0.4 parts of sodium chloride (p), 0.5 parts of salt, 1.5 parts of banana powder

Example 9A 9B 9C 9D 9E 9F 9G 9H flavor salty salty Good Good salty Sweet Sweet Good Fishy Remaining Improving Remaining Improving Improving Improving Improving Improving Constellation Uniformity Uniformity Uniformity Lump Lump Uniformity Lump Uniformity Flow Good Good Good Bad Bad Good Bad Good

Example 10A-10B: Mixing of yolk powder and lactic acid bacteria powder

After mixing the antibody-containing yolk powder and lactic acid bacteria powder in the following manner, the taste was examined through the sensory test, and the titer of the antibody was measured. All percentages below are by weight.

Example 10A

50% of lactic acid bacteria powder and 47.5% of egg yolk powder were uniformly mixed, and then 1% of silicon dioxide was uniformly mixed firstly in a dehumidifying state, and 1% of magnesium stearate and 0.5% of banana powder were added to mix uniformly. At this time, all raw materials were used by sieving at 35 mesh (500 μm).

Example 10B

50% of lactic acid bacteria powder and 47% of egg yolk powder were uniformly mixed, and then 1% of silicon dioxide was uniformly mixed firstly in a dehumidified state, and 1% of magnesium stearate and 1% of banana powder were added and mixed uniformly. At this time, all raw materials were used by sieving at 35 mesh (500 μm).

Example 10A Example 10B flavor Good Good Fishy Slightly remaining Improving Constellation Uniform powder Uniform powder Flow Good Good

Example 11A-11D: Encapsulation of Antibodies

Example 11A

To 100 g of the antibody-containing egg yolk solution, 5 g of EUDRAGIT L30D-55 (product name: Rohm America Inc., pH 7.0) was added as a methacrylate polymer solution, mixed well, and lyophilized at -50 ° C and 0.036 mBar. The yolk solution which was not treated with the methacrylate polymer solution was used as a control.

Example 11B

25 g of gelatin (manufactured by Fishers Scientific, Ottawa), 5 g of starch (manufactured by Sigma, St.Louis, MO), 5 g of antibody-containing yolk solution were dissolved in 500 g of water, mixed at 40 ° C. for 30 minutes, and then lyophilized as in Example 11A. I was. The lyophilized powder was dissolved in 100 ml of an aqueous 0.5% (w / v) EUDRAGIT L-100 (trade name: Rohm America Inc., pH 8.0) solution as a methacrylate polymer solution. The mixture was left at 4 ° C. overnight and then lyophilized again. The same treatment as above was used as a control except that it was not treated with a methacrylate polymer solution.

Example 11C

5 g of Arabian gum (manufactured by Sigma) was dissolved in 250 ml of boiling water, cooled to room temperature, 5 g of antibody-containing egg yolk solution was dissolved in the solution, and then lyophilized. The lyophilized powder was dissolved in 0.5% (w / v) aqueous solution of EUDRAGIT S-100 (trade name, manufactured by Rohm America Inc., pH 8.0) as a methacrylate polymer solution, and then allowed to stand overnight at 4 ° C. I was. The same treatment as above was used as a control except that it was not treated with a methacrylate polymer solution.

Example 11D

2 g of egg phospholipid (manufactured by Avanti, CA) and 2 g of cholesterol (manufactured by Sigma) were dissolved in 50 ml of chloroform / methanol solution and then distilled to dryness. 2g of IgY (10 mM HEPES buffer 200mL, pH 7.5) was added thereto, and liposomes were generated by treatment with ultrasonic waves (Branson Sonicator, German) for 30 minutes at room temperature. The solution was mixed with an aqueous 0.5% w / v EUDRAGIT RD-100 (trade name, manufactured by Rohm America, Inc., pH 8.0) as a methacrylate polymer solution. The same treatment as above was used as a control except that it was not treated with a methacrylate polymer solution.

Residual activity (%) of the antibodies in each control group and the coated test group was measured and shown in Table 3.

Residual Activity Measurement Method

Residual activity was measured by digestibility test with pepsin.

Pepsin extracted from swine gastric juice (Sigma) was added at a ratio of 1:50 (w / w) to 25 mg / ml of the encapsulated IgY solution. The mixed solution was left at pH 2.0 and 37 ° C. for 1 hour, followed by ELISA and SDS-PAGE.

result

In Example 11A, IgY encapsulated with methacrylate retained at least 90% of activity, compared to only 4% of IgY of the control group was maintained by pepsin. Example 11B was treated with gelatin and starch in the control group, so reprocessing showed 96% residual activity. Example 11C shows 9% residual activity for the control IgY treated with gum arabic and 86% residual activity when retreated with methacrylate. Example 11D shows 7% residual activity for the control IgY treated with liposomes and more than 90% residual activity when retreated with methacrylate. Therefore, it can be seen that encapsulation with methacrylate polymer can protect against degradation of pepsin.

Residual Activity of Antibody (%) Control Coated capsules Example 11A 4 92 Example 11B 18 96 Example 11C 9 86 Example 11D 7 91

The present invention provides an antibody-containing yolk powder prepared by processing the yolk solution under optimal sterilization and drying conditions and an antibody-containing yolk powder composition having improved flavor and preservation in order to increase the product value of the antibody-containing yolk solution and expand its field of use. In addition, it has the effect of providing a method of coating and stabilizing the antibody and stabilizing the antibody using a grapefruit seed extract as a preservative in order to prevent denaturation or destruction until the antibody is used in the body.

Simple modifications and variations of the present invention can be readily understood by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

Claims (11)

Immunizing the laying hens by injecting an antigen derived from a disease-causing strain into a mammal; Separating the antibody-containing yolk solution from the egg produced in the hens that were immunized; Heat-treating the antibody-containing egg yolk solution 1 to 3 times at 60 to 70 ° C. for 3 to 10 minutes; And Spray drying or lyophilizing the heat treated antibody-containing egg yolk solution; A method for producing an antibody-containing egg yolk powder. The method of claim 1, further comprising adding a 250 ppm to 1000 ppm grapefruit seed extract to the antibody-containing yolk solution after the separation step. The method of claim 1, wherein the method further comprises adding 5 to 15% by weight to the antibody-containing yolk solution after the separation step. The method of claim 1, further comprising eluting the water-soluble protein by mixing 10 parts by weight of the antibody-containing yolk solution with 50 parts by weight of water after the separation step. A sterilized antibody-containing yolk powder prepared by the method of any one of claims 1 to 4, characterized by excellent stability of the antibody and excellent preservation. 40 to 50% by weight of egg yolk powder of claim 5; Lactic acid bacteria powder 45 to 55% by weight; 0.5 to 1.5 wt% silicon dioxide; 0.5 to 1.5 wt% magnesium stearate; Antibody-containing egg yolk powder composition comprising 0.1 to 2% by weight sweetener. 70 to 90 parts by weight of egg yolk powder of claim 5; 1 to 5 parts by weight of SiO ₂ ; S-Mg 1 to 3 parts by weight; 10 to 15 parts by weight of glucose; 0.3 to 1 part by weight of salt; Antibody-containing egg yolk powder composition comprising 1 to 3 parts by weight of sweetener. The egg yolk powder composition of claim 7, further comprising 0.1 to 0.3 parts by weight of aspartame or 3 to 7 parts by weight of xylitol. The method of claim 6 or 7, wherein the sweetener is banana powder, cocoa powder, vanilla powder, strawberry flavor powder, grape flavor powder, orange flavor powder, coffee flavor powder, cinnamon flavor powder, mugwort flavor powder, cream flavor powder, and Egg yolk powder composition, characterized in that selected from the group consisting of a mixture thereof. A method of coating the antibody in egg yolk by treating the yolk powder of claim 5 with 0.1 to 10% by weight of a methacrylate polymer. A method of coating the antibody in egg yolk by treating the antibody-containing yolk solution of claim 1 with 0.1 to 10% by weight of a methacrylate polymer.