KR20030068728A - METHOD FOR PRODUCING IgY AGAINST PANCREATIC AMYLASE AND COMPOSITIONS CONTAINING SAME FOR INHIBITING CARBOHYDRATE UPTAKE - Google Patents

Abstract

PURPOSE: Provided are a method for producing IgY against pancreatic amylase and a composition containing the same for inhibiting carbohydrate uptake. IgY directly inhibits the decomposition of carbohydrate and is thus useful for the improvement of obesity, and the prevention and treatment of diabetes. CONSTITUTION: A method for producing IgY against pancreatic amylase comprises the steps of: preparing IgY from the laying hen immunized by using pancreatic amylase protein as an antigen; and separating IgY from the egg obtained from the immunized laying hen.

Description

METHOD FOR PRODUCING IGY AGAINST PANCREATIC AMYLASE AND COMPOSITIONS CONTAINING SAME FOR INHIBITING CARBOHYDRATE UPTAKE}

The present invention relates to a method for producing yolk antibodies against pancreatic amylase and a composition for inhibiting absorption of dietary carbohydrates using the same, specifically, separating amylase, a carbohydrate degrading enzyme derived from pig pancreas; Producing yolk antibodies by immunizing the laying hens using the pancreatic amylase as an antigen; And separating the yolk antibody from the yolk of the egg laid by the laying hen, the method of producing yolk antibody against pancreatic amylase and the activity of pancreatic amylase which is a carbohydrate degrading enzyme in vivo using the yolk antibody produced by the method The present invention relates to a composition for inhibiting absorption of dietary carbohydrates which inhibits the carbohydrate.

In vivo digestion of ingested foods generally results in some degradation in the stomach, including the initial breakdown of carbohydrates in the oral cavity. However, full-fledged carbohydrate and fat digestion and absorption occur mainly in the area from the duodenum to the ileum, and in general, it is known that resorption of water and salts and absorption of free fatty acids occur mainly in the large intestine. Therefore, the most important part of digestive absorption occurs in the small intestine, where proteolytic enzymes such as trypsinogen, chymotrypsinogen, and carboxy peptidase, lipolytic enzymes such as pancreatic lipase and sphyxin, pancreatic amylase, amylosin Carbohydrate degrading enzymes such as, maltase and various digestive enzymes such as various peptidase, nucleic acid degrading enzyme, alginase, phosphatase, and lactose degrading enzyme which are various glycolytic enzymes in the small intestine Absorbed by the nutrients absorbed through the dark ducts and blood vessels will be moved into the body.

The most important role in carbohydrate and fat absorption in these chorionic cells is the chorionic cell membrane transporter. The chorionic membrane transporter is a membrane transporter (membrane vesicle) present on the surface of the small intestinal chollocytes and the transporter plays a very important role in the transport of monosaccharides by Na cations called sodium dependent glucose cotransporter 1 (SGLT1). That is, the membrane transporter plays a very important role in the active transport of the most representative monosaccharides of the monosaccharides caused by the decomposition of carbohydrates. In addition, the activity of pancreatic amylase and glucoamylase, which are important disaccharide degrading enzymes indispensable for the transport of such monosaccharides, is very high in the chorionic membrane transporter, which is known to increase the absorption of monosaccharides in the body. However, hydrolysis by disaccharide degrading enzymes in the intestinal tract is rarely performed during the upper ileum, which means that the carbohydrate degrading enzymes of the chorionic cell membrane transporter are largely dependent on membrane thinning. Indeed, it can be proved that the absorption of monosaccharides is relatively low compared to the total monosaccharide absorption until it reaches the ileal region where pancreatic amylase activity is found much.

Therefore, considering the dietary patterns of Koreans with the highest intakes of carbohydrates, the main mechanisms are the intestinal cleansing effect using dietary fiber or dietary fiber used for obesity and diabetes, which limit total intake of nutrients, and the inhibition of digestion and absorption of fat. Since the diet product is not suitable, it is required to develop a differentiated method suitable for Koreans who can inhibit the absorption of carbohydrates without inhibiting the absorption of other essential inorganic ions and proteins.

Meanwhile, IgG, an immunoglobulin in the serum of a hen, is passed on to a later generation, so that such an immunoglobulin is also produced in eggs. As a concept of passive immunity, many techniques for producing and using a large amount of antibodies specific to antigens have been studied. Once a hen has 'learned' to produce antibodies to a particular antigen, it is possible to produce antibodies throughout the chicken's life, ie over 10 years, and eggs have 15 ml of egg yolk with an average of 8 mg / ml IgG. It contains. In addition to chickens, other poultry such as turkeys, ducks, geese and the like can be used as a source of eggs.

Laying chickens deliver all the antibody isotypes found in chickens, namely IgY, IgM and IgA antibodies, to eggs. Egg yolk contains only IgY and IgM and IgA are present only in the egg white portion, and the serum IgY concentration in chickens affects yolk after a single dose of antigen (after about a week). Egg yolk contains between 3 and 25 mg IgY / ml so each egg can provide between 40 and 500 mg IgY by weight.

The advantages of egg yolk antibodies can be obtained in large quantities, and their extraction is very economical because they can be carried out on a large scale without costly investment. In addition, yolk antibodies do not react with mammalian complement, Fc receptor, protein A or protein G, and thus do not cause an immune response, and exhibit great resistance to acids and heat. The FDA approved yolk antibodies as food rather than drugs, and approved them as GRAS (generally accepted as safe) .In this way, using a yolk antibody to produce large quantities of antibodies specific to antigens, US CalaGen Antibodies (IgA) isolated from colostrum by IgA and ImmuCell have been developed for the treatment of diseases caused by intestinal infectious microorganisms, and are currently in clinical trials. Egg yolk antibodies to Streptococcus mutans are used as food additives and feed additives.

Therefore, the present inventors have developed a method for producing yolk antibodies from eggs produced after the pancreatic amylase, an enzyme that degrades macromolecules in the initial digestion of carbohydrates, from the small intestine of pigs and immunized chickens with antigens. The present invention was completed by revealing that the yolk antibody produced by the above method can directly inhibit digestion of carbohydrates in vivo and can be usefully used as a composition for improving and treating obesity and diabetes diseases.

It is an object of the present invention to provide a method for producing yolk antibodies against pancreatic amylase, a carbohydrate degrading enzyme in laying hens.

Another object of the present invention to provide a composition for inhibiting carbohydrate absorption comprising the yolk antibody.

1 is a result of the separation of pancreatic amylase from the membrane protein of the pig small intestine using anion exchange column chromatography according to the present invention,

Figure 2 shows the change in the reaction rate of the pancreatic amylase isolated from Figure 1 with temperature and pH,

FIG. 3 shows the titer of anti-pancreatic amylase antibody in the blood of laying hens according to the number of inoculations when immunized with pancreatic amylase isolated from FIG. 1 ,

Figure 4 shows that the activity of porcine pancreatic amylase is directly inhibited by the yolk antibody prepared according to the present invention,

Figure 5 is the result of measuring the inhibition of the increase in the concentration of sugar in the body by the yolk antibody of the present invention in the water-soluble starch overload test using an animal model rat.

In order to achieve the above object, the present invention provides a method for producing yolk antibodies in laying hens using pancreatic amylase as an antigen.

The present invention also provides a pharmaceutical composition for inhibiting carbohydrate absorption containing the yolk antibody produced by the above method as an active ingredient.

In addition, the present invention provides a food and beverage composition containing the yolk antibody produced by the method as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing yolk antibodies in laying hens using pancreatic amylase as an antigen.

The production method

1) using the pancreatic amylase protein as an antigen to immunize the laying hens and produce yolk antibodies therefrom; And

2) separating the yolk antibody from the yolk of the egg laid by the laying hen.

The above steps will be described in more detail below.

In a preferred embodiment of the present invention step 1), membrane protein is separated from porcine small intestine and the pancreatic amylase is purified using anion exchange column chromatography (see FIG. 1 ). In order to determine the enzymatic activity of purified pancreatic amylase, the optimum reaction temperature and pH of each enzyme activity were examined, and the highest activity was shown at 50 ° C. and neutral pH 5 (see FIG. 2 ).

The pancreatic amylase used as the antigenic protein in the present invention may be isolated from pig small intestine as well as rats, mice, guinea pigs, rabbits or humans. And chemically synthesized ones may be used.

The isolated pancreatic amylase can be used as an antigen to immunize the laying hens to produce egg yolk antibodies therefrom. Although the kind is not specifically limited as a scattering system to be used, For example, it is preferable to use a white leg horn system, a Rhode island type, a high-line brown type, a boas brown type etc. with a high scattering rate.

Antigen proteins are immunized by laying the laying hens by any suitable route, such as subcutaneous, intraperitoneal, intramuscular or intravenous injection or oral administration. Preferred immunization methods are intramuscular injection, and subcutaneous injection into the neck is also possible. Appropriate adjuvant is preferably administered with the antigen to increase immunization, useful adjuvant for this is water-in-oil emulsification adjuvant such as incomplete or complete Freund's adjuvant. Using the appropriate adjuvant is very effective in maintaining high antibody titers in eggs of the immunized laying hens for long periods of time, thus effectively producing the desired antibody-containing material. The dose of antigen may vary depending on the route of administration, generally in the range of 10 to 200 μg / kg body weight or type of antigen and adjuvant, and in a manner in which the immune status can be induced in laying hens without formation of excessive antigenic toxicity.

In general, within a few weeks after the initial immunization (inoculation) the laying hens become sensitive to the antigen. That is, it is immunized against the antigen. Antibodies specific for antigens are produced in the body of a laying hen, and the eggs laid by the laying hens contain specific antibodies. The presence and titer concentrations of specific antibodies to antigens in laying hens and eggs can be confirmed by various immunological tests known in the art. In the present invention, an enzyme-linked immunosorbent assay (ELISA) is used as a preferred embodiment. The antibody activity of egg yolk and serum was measured.

After initial immunization against the antigen, an appropriate dose of one or more additional antigens can be administered to maintain high antibody titers in the laying hens. In each additional antigen administration, appropriate adjuvants may be used with the antigen. The interval between the initial immunization and the administration of the first additional antigen, and the interval between the individual additional antigen administrations, depends on the nature of the antigen and is preferably at least two weeks.

After confirming that the appropriate titers of the specific specific antibodies are present in the eggs laid by the immunized laying hens (see FIG. 3 ), the eggs laid by the laying hens are collected and stored before use as needed. It is convenient to collect several eggs from one or more laying hens immunized against the same antibody and process them together to produce the desired material comprising the antibody.

Step 2) is to separate egg yolk antibodies from egg yolks laid by laying hens. Egg yolks contain high concentrations of fat, so it is necessary to separate them with high efficiency to facilitate the action of egg yolks. Should not be harmful to To this end, extraction using λ-carrageenan and polysaccharides and distilled water or caprylic acid may be used as an extraction material for purifying egg yolk antibodies from eggs recovered from a laying hen. In a preferred embodiment of the present invention, after adding the carrageenan solution to the diluted solution of the egg yolk diluted with distilled water, and centrifugation to precipitate the yolk lipo protein, the supernatant obtained therefrom is salted to separate only the yolk soluble protein. In particular, the carrageenan solution is a gum solution certified as a food additive, and has excellent biostability, antibody recovery rate, and can maintain high titer of recovered antibody.

The isolated and purified yolk antibody effectively inhibited pancreatic amylase activity (see FIG. 4 ), and it was confirmed that the blood glucose increase was reduced by effectively inhibiting the absorption of carbohydrates through the inhibition of the enzyme activity when applied in vivo ( FIG. 5 ) . Reference).

The present invention also provides a pharmaceutical composition for inhibiting carbohydrate absorption containing the yolk antibody produced by the above method as an active ingredient.

The yolk antibody against pancreatic amylase produced in accordance with the present invention directly inhibits the breakdown of carbohydrates in vivo, thereby reducing the amount of increase in blood glucose. It can be usefully used for the prevention and treatment of diseases resulting from, for example, obesity, diabetes, hypertension and the like.

The pharmaceutical formulations can be prepared according to conventional methods using the compositions of the present invention. The formulation may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly Vinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. In humans a typical daily dose of yolk antibody may range from 5 to 30 mg / kg body weight, preferably 10 to 20 mg / kg body weight, and may be administered once or in several doses.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the route of administration chosen, the age, sex, weight of the patient and the severity of the patient's symptoms, and thus the dosage may be The method does not limit the scope of the present invention.

In addition, the present invention provides a food and beverage composition containing the yolk antibody produced by the method as an active ingredient.

Since the food and beverage composition of the present invention inhibits the breakdown of carbohydrates in the body, it is possible to maintain and improve health through inhibition of obesity, blood sugar control, and the like.

The yolk antibody of the present invention may also be incorporated into foods or beverages as additives or food supplements for the purpose of demonstrating the prophylactic and therapeutic effects of diets for improving obesity, or diseases caused by excessive carbohydrate absorption, such as diabetes. . As the food or beverage, meat; Vegetable juices (eg, carrot juice and tomato juice) and fruit juices (eg, orange juice, grape juice, pineapple juice, apple juice and banana juice); Chocolate; Snacks; confectionery; Pizza; Food products made of grain powder such as bread, cakes, crackers, cookies, biscuits, noodle, etc .; Gums; Dairy products such as milk, cheese, yogurt and ice cream; soup; Juicy; Pastes, ketchups and sauces; car; Alcoholic beverages; Carbonated beverages; Vitamin complexes; And various health foods. In this case, the content of the yolk antibody in the food or beverage may be in the range of 0.05 to 0.5% by weight, preferably 0.1 to 0.2% by weight.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Pancreatic Amylase Yolk Antibody

(Step 1) Isolation of Pancreatic Amylase from Porcine Small Intestine

In order to isolate pancreatic amylase involved in digestive enzymes, fresh pig small intestine is added with sodium phosphate saline buffer (hereinafter abbreviated as 'PBS') to separate the liquid in the small intestine. The mixture was homogenized, left to stand by addition of 50 mM calcium chloride and centrifuged to recover only the supernatant. The separated protein solution was adsorbed onto a DEAE-Sepharose column (Amersham Pharmacia Biotech Co., Ltd.) equilibrated with 50 mM Tris-HCl buffer (pH 8) and the same containing 0-1.0 M sodium chloride. Pancreatic amylase was isolated by selectively fractionating proteins with buffer ( FIG. 1 ).

(Step 2) Determination of Enzyme Activity of Isolated Pancreatic Amylase

In order to investigate the activity of the enzyme isolated in step 1, an enzyme reaction was performed by adding water-soluble starch as a substrate. The reaction solution contains a substrate 1%, 50 mM phosphate buffer, enzyme (0.2munit unit), distilled water and the reaction capacity was reacted for 20 minutes at 37 ℃ with 0.1ml. At this time, in order to investigate the optimum reaction temperature and pH of the enzyme, the temperature was reacted at a temperature range of 20 ° C. to 70 ° C., and the pH was reacted at a pH range of 3-9. After the reaction, the enzyme activity was stopped at 100 ° C, and the resulting glucose concentration was quantified using GEL-zyme kit (Shinyang Chemical Co., Ltd.), a GOD-POD colorimetric method using glucose oxidase.

As a result, the highest activity was shown at 50 ° C. and pH 5 ( FIG. 2 ).

(Step 3) Immune of laying hens

Using pancreatic amylase isolated and purified from pig small intestine as the antigen, 20 legs of Boas Brown species laying hens were inoculated three times with Freund's adjuvant, an adjuvant, at a concentration of 100 ㎍ / ㎏. The last four doses were immunized with antigen alone without an adjuvant. Eggs are collected daily and stored at 4 ° C., and blood is collected by the number of immunizations to determine the formation of antibodies to the injected antigen in the inoculated laying hens. ELISA) was performed.

The pancreatic amylase isolated in step 1 as an antigen for enzymatic immunoassay was coated overnight by dispensing 1-100 μg into 96 well microplates, and then treated with 5% skim milk to prevent binding of nonspecific antibodies. It was. The well plate was washed three times with buffer, the antiserum was diluted 3,000 times and reacted as a primary antibody at 37 ° C. for 30 to 60 minutes, and then the unbound antibody was washed with physiological salt-containing phosphate buffer containing 0.02% Tween 20. Washed twice. Here again, alkaline phosphatase-bound anti-chicken IgG (Sigma Aldrich) was diluted 1,000-fold and bound for 60 minutes at room temperature as a secondary antibody. After the reaction was completed, the plate was washed five times with a buffer containing 0.02% Tween 20 and 40 mg of PNPP (p-nitrophenyl phosphate in diethanolamine buffer, Sigma Aldrich), a substrate of alkaline phosphatase, was added for 40 minutes. After the reaction, the absorbance of each well was measured with an ELISA reader at 450 nm. As a result, after the second inoculation, antibodies to maltase began to be generated in the serum of the laying hens and maintained their highest after the third inoculation ( FIG. 3 ).

(Step 4) Isolation of Yolk Antibody

Egg yolk of eggs recovered from the laying hens in which the formation of antibodies to the pancreatic amylase antigen protein was confirmed was separated, and then mixed with distilled water in a volume of 1: 1. The solution of λ-carrageenan (1 mg / ml in D.W., Sigma Aldrich) was added twice and allowed to stand at room temperature for 30 minutes. The mixture was centrifuged (10,000 × g, 15 minutes, 20 ° C.) to take only the supernatant, filtered with a Wattman No. 2 filter paper, and then added 19% sodium sulfate to the filtrate. The brine was performed. This was again centrifuged (10,000 × g, 15 min, 20 ° C.) to recover only the precipitate and then resuspended in PBS, followed by brine and centrifugation (10,000 × g, 15 min) using sodium sulfide. The sample resuspended in PBS was dialyzed with 10 mM PBS, and the dialysate obtained therefrom was lyophilized to obtain an yolk antibody.

<Example 2> Confirmation of inhibition of enzyme activity of egg yolk antibody

In order to confirm whether the yolk antibody isolated and purified from Example 1 could actually inhibit the activity of pancreatic amylase, the enzyme activity of pancreatic amylase was measured in the same manner as in Step 2 of Example 1, respectively. During the enzymatic reaction, yolk antibody was added at a concentration of 1 mg / ml to determine the degree of activity inhibition. In order to compare the activity of yolk antibodies against pancreatic amylase, a control group without an enzyme, no antibody, normal yolk antibody addition group, and yolk antibody immunization to pancreatic amylase were used.

As a result, as shown in Figure 4 , it was confirmed that about 40% of the activity of the pancreatic amylase is inhibited by the yolk antibody prepared according to the present invention.

Example 3 Inhibitory Activity of Carbohydrates in Vivo

Whether the yolk antibody prepared according to the present invention exhibits the absorption inhibitory activity of carbohydrates in vivo was investigated using SD rats. 10 weeks old SD rats (Korean experimental animals) weighing 400 g were divided into 2 groups of 10 rats in each group and fasted for 12 hours, and then the control group was administered with yolk protein of general egg, and the yolk antibody prepared according to the present invention. Was administered at 1 mg or 5 mg / kg body weight. At the time of administration, the injection solution in which the antibody was dispersed in phosphate buffer containing physiological salts was administered orally using horn. After 10 minutes of yolk antibody administration, 200 mg / kg of water-soluble starch was orally administered, and blood glucose was quantified by time. Blood glucose levels were quantified using GEL-zyme kit (Shinyang Chemical), a GOD-POD colorimetric method using glucose oxidase.

As a result, in the control group administered with the protein of common eggs, the blood glucose level increased with time, and increased 17 mg / dl at 30 minutes and then decreased slowly. In the experimental group in which 1 mg or 5 mg of yolk antibody was administered per 1 kg of body weight, the blood glucose level increased by 5 mg / dl at 15 minutes, respectively, and then decreased, showing a blood glucose uptake inhibitory effect of 60% or more with respect to the control group ( FIG. ).

From this, it was confirmed that the yolk antibody prepared according to the present invention effectively inhibits the absorption of glucose in vivo in a concentration-dependent manner.

Preparation Example 1 Preparation of Pharmaceutical Formulation

Capsules were prepared by mixing the following ingredients to fill hard gelatine capsules:

Volume (mg / capsule)

Active Ingredients (An yolk Antibody of Example 1) 20

Dry starch 160

Magnesium Stearate 20

Total 200 mg

Preparation Example 2 Preparation of Functional Food Containing Egg Yolk Antibody

Functional foods comprising the yolk antibody of the present invention were prepared as follows.

(1) Manufacture of dairy products

Egg yolk antibody was added to the milk in an amount of 5% by weight and milk was used to prepare various dairy products such as butter and ice cream.

However, yolk antibodies were added to the coagulated milk protein in cheese production and to the coagulated milk protein obtained after fermentation in yogurt production.

(2) Preparation of Mixed Drinks

1 g of egg yolk antibody was added to 1,000 ml of tomato, carrot, apple or grape juice to obtain a mixed beverage for health promotion.

As described above, the yolk antibody to pancreatic amylase produced from the laying hens can be safely and mass-produced using the laying hens, which is not only economical but also carbohydrates without inhibiting absorption of other essential inorganic ions and proteins. By effectively inhibiting only absorption, it can be usefully used as a composition for improving and treating obesity and diabetes diseases.

Claims (8)

1) using the pancreatic amylase protein as an antigen to immunize the laying hens and produce yolk antibodies therefrom; And 2) A method for producing yolk antibodies against pancreatic amylase, comprising the step of separating the yolk antibodies from the yolk of the eggs laid by the laying hens. The method of claim 1, wherein the pancreatic amylase protein is isolated from porcine small intestine, rat, mouse, guinea pig, rabbit or human. The method of claim 1, wherein the pancreatic amylase protein is expressed by a recombinant microorganism or chemically synthesized. 2. The method of claim 1, wherein in step 1 the laying hens are immunized by one or more injections at an antigen dose of 10 to 200 μg / kg body weight. A pharmaceutical composition for inhibiting carbohydrate absorption, comprising an egg yolk antibody produced by the method of any one of claims 1 to 4 as an active ingredient. The pharmaceutical composition according to claim 5, which is used for the prevention and treatment of obesity or diabetes related diseases. A food and beverage composition comprising the yolk antibody produced by the method of any one of claims 1 to 4 as an active ingredient. 8. A food and beverage composition according to claim 7, which is used for preventing or improving obesity.