JPH06277056A - Complex of yolk protein and enzymatic decomposition product of galactomannan - Google Patents

Abstract

PURPOSE:To obtain the subject complex having emulsifiability, frothing property, oil-inclusion property and protease digestion resistance and useful for foods, cosmetics, medicines, etc., by storing a powdery mixture of yolk protein with an enzymatic decomposition product of galactomannan under high temperature and high humidity condition. CONSTITUTION:A powdery mixture is prepared by adding ethanol to yolk powder, stirring the mixture for 30min at room temperature, separating insoluble component by filtering under pressure, drying the insoluble component at 40 deg.C under reduced pressure to obtain yolk protein such as defatted yolk powder, adding an aqueous solution containing enzymatic decomposition product of galactomannan as a solute, homogenizing with a homogenizer and drying by spray-drying. The objective complex of yolk protein and enzymatic decomposition product of galactomannan having improved emulsifiability, frothing property, oil-inclusion property, resistance to protease digestion, bondability to bivalent metallic ion, etc., and useful as a high-added value material in the field of foods, cosmetics and medicinal materials can be produced by storing the above mixed powder for 2-21 days in a hot and humid environment at 50-90 deg.C and 50-90% relative humidity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chicken egg yolk protein-galactomannan enzymatic degradation product complex used in the fields of food, cosmetics and pharmaceuticals.

[0002]

2. Description of the Related Art Egg yolk is an emulsion consisting of 50% water, 30% lipid and 20% protein, and its emulsifying property is mainly used as a food material for mayonnaise, dressing, custard cream, pound cake, etc. There is.
In recent years, excessive intake of animal lipids and cholesterol has been regarded as a problem, and attempts have been made to remove the lipid components of egg yolk. However, there is a problem that the excellent emulsifiability of egg yolk decreases with the removal of lipid components, and its development has not progressed.

Egg yolk contains many useful lipids such as phosphatidylcholine and phosphatidylethanolamine. Usually, egg yolk lipids are extracted with organic solvents such as ethanol, hexane, isopropyl alcohol, dimethyl ether, etc., and used as cosmetics and pharmaceutical materials. In this case, the egg yolk protein remains as a residue, but as described above, the functionality of the egg yolk is remarkably lost with the extraction of the lipid, so that the yolk protein has a low utility value and is not effectively used. The current situation.

58% of egg yolk proteins are bound to lipids to form lipoproteins. With the removal of egg yolk lipids,
These become apoproteins, mostly insoluble proteins. 12% of egg yolk protein is a phosphorus-containing protein called phosvitin, which has a chelating effect for divalent metal ions. Therefore, the intake of phosvitin is
It is said that it binds to divalent metal ions such as iron in the intestinal tract and inhibits its absorption in the body. 30% of egg yolk protein is a yolk water-soluble protein called ribetin, and three types of α-, β- and γ-ribetin are known, and the abundance ratio of each is reported to be 2: 3: 5. (J. Biol. C
hem., 179, 349, 1949). It is also immunologically proved that α-ribetin is the same as serum albumin of parent chickens, β-ribetin is the same as serum α ₂ -glocoprotein and γ-ribetin is the same as serum γ-globulin. (Can.
J. Biochem. Physiol., 36, 153, 1958).

In recent years, a method for separating and purifying a parent chicken-derived antibody (γ-ribetin) from egg yolk proteins on an industrial scale has been reported. (Agric. Biol. Chem., 54, 25
31-2535, 1990). An antibody obtained from chicken egg yolk is called an egg yolk antibody, and application development utilizing its specific antigen-recognition ability is progressing.

[0006] That is, if a parent chicken is immunized with an infectious disease pathogen as an antigen, a large amount of specific antibodies that neutralize the infectivity of the pathogen can be obtained from hen egg yolk. Many attempts have been reported to prevent infection by oral administration of the yolk antibody thus obtained to animals (oral passive immunization). The orally administered egg yolk antibody against the pathogen has the effect of specifically binding to the corresponding pathogen at the site of infection in the intestinal tract and neutralizing the infectivity of the pathogen.

[0007] Heretofore, attempts have been reported to modify a food protein with a polysaccharide by an aminocarbonyl reaction to improve its function. (Agric. Biol. Chem., 54, 3057-3059, 199
0 years etc.). In Japanese Patent Application Laid-Open No. 3-215498, protein derived from animals and plants such as ovalbumin, lysozyme, milk protein, fish meat protein, soybean protein and wheat gluten, and dextran, dextrin, pullulan, xanthan gum, carrageenan, chondroitin sulfate and the like are disclosed. It has been publicly known that a protein-polysaccharide complex having an emulsifying activity and an antibacterial activity can be obtained by binding a branched polysaccharide by an aminocarbonyl reaction. In addition, by adding guar gum enzymatic degradation product to egg white lysozyme,
It is also reported that the antibacterial spectrum of lysozyme could be expanded. (Proceedings of the 1991 Annual Meeting of the Japanese Society of Agricultural Chemistry, P78)

[0008]

The excellent emulsifying power of egg yolk is derived from lipoprotein in which lipid and protein in egg yolk are bound at various ratios. The egg yolk protein after extracting and removing the lipid component from the egg yolk has poor solubility and remarkably loses functions such as emulsifying property. In recent years, with the demand for useful lipids in egg yolk, a large amount of egg yolk protein has been obtained as a defatted egg yolk powder, but its added value is low and it is desired to develop an effective utilization method in industry.

Among egg yolk proteins, egg yolk antibodies derived from parent chickens are attracting attention for their application as antibodies for oral passive immunization. When an egg yolk antibody is used for oral passive immunization, inactivation of antibody activity due to proteolytic enzymes such as pepsin, trypsin, chymotrypsin in the digestive tract becomes a problem. Therefore, in practical application of oral passive immunization, it is important to impart resistance to these proteolytic enzymes to the yolk antibody.

The present invention has been made for the purpose of increasing the value added of egg yolk protein, and the problems to be solved by the invention are solubility, emulsification, foamability, fragrance, oil retention, and protease digestion. It is to develop an egg yolk protein having improved or imparted resistance and functionality such as divalent metal ion binding property.

[0011]

[Means for Solving the Problems] As a result of conducting research on imparting various functionalities to egg yolk proteins, the present inventors have found that
The mixed powder of egg yolk protein and galactomannan enzymatic hydrolyzate was stored under high temperature and high humidity environment, and it was found that the combined complex had various excellent functions, and the present invention was completed. The complex of egg yolk protein and galactomannan enzymatic degradation product was prepared for the first time by the present invention, and its functional improvement effect and functional addition effect were also confirmed for the first time by the present invention.

That is, the present invention can be obtained by storing a mixed powder of egg yolk protein and galactomannan enzymatic decomposition product in a high temperature and high humidity environment. It relates to an egg yolk protein-galactomannan enzymatic degradation product complex.

The egg yolk protein of the present invention means defatted egg yolk powder obtained by removing lipid components from egg yolk, apoprotein obtained by removing lipids from egg yolk lipoprotein, egg yolk water-soluble protein, egg yolk ribetin component, egg yolk antibody, phosvitin, etc. Is a protein contained in.

The galactomannan enzymatic degradation product of the present invention refers to a natural mucilage containing galactomannan as a main component such as guar gum and carob gum whose molecular weight is reduced by β-D-mannase. Regarding the degree of molecular weight reduction,
Usually, those obtained by decomposing sugar chains into an average molecular weight of 100 to 100,000, preferably 10,000 to 40,000 are used.

The mixed powder of the present invention is obtained by dissolving or suspending egg yolk protein in an aqueous solution of enzymatically decomposed galactomannan and pulverizing the solution or suspension by a known method such as freeze drying or spray drying. Say A natural mucilage containing galactomannan as a main component has a high viscosity when dissolved in water, and thus such an operation is impossible. However, when the galactomannan enzymatic degradation product of the present invention is used, its aqueous solution has a very low viscosity,
The mixed powder used in the present invention can be prepared.

In the mixed powder of the present invention, its water content is an important factor in setting the conditions for forming a complex. Normal,
The water content is adjusted to 1-10%. The higher the water content,
The complex formation proceeds faster, and the lower the water content, the slower the complex formation.

In the mixed powder of the present invention, the speed of complex formation is significantly affected by the type of egg yolk protein used. For example, phosvitin of egg yolk proteins and egg yolk antibodies have the property of forming a complex with an enzymatic degradation product of galactomannan very quickly.

The high temperature and high humidity environment of the present invention means a condition that the ambient temperature for storing the mixed powder is 50 to 90 ° C. and the humidity is 50 to 90% as relative humidity. The storage period varies remarkably depending on the type of mixed powder and the water content, but a storage period of 2 to 21 days is usually used for complex formation. It is preferable to examine the high temperature and high humidity conditions and the storage period necessary for forming the complex by conducting a preliminary test or the like for each type of mixed powder.

To confirm the formation of the complex, the mixed powder during storage is sampled over time, and known methods such as gel filtration chromatography and SDS-polyacrylamide gel electrophoresis are used to facilitate complexation of the protein. You can check the state of body formation.

[0020]

The present invention will be described in more detail with reference to the following examples and test examples, but the present invention is not limited to these examples. Example 1 Defatted egg yolk-galactomannan enzymatic degradation product complex 10 kg of ethanol was added to 1 kg of egg yolk powder at room temperature 30
After stirring for 1 minute, pressure filtration was performed to separate insoluble matter. To the insoluble matter, add 10 kg of ethanol again, perform the same operation,
The insoluble matter obtained was dried under reduced pressure at 40 ° C and defatted egg yolk powder 430 g
Got Aqueous solution 10 in which 645 g of galactomannan enzymatic degradation product [trade name: Sunfiber, Taiyo Kagaku Co., Ltd.] was dissolved
220 g of defatted egg yolk powder was added to 1 liter and homogenized with a homogenizer, and then 830 g of mixed powder was obtained by a hot air drying (spray dry) method. The mixed powder was stored at 90 ° C for 12 days to obtain an egg yolk protein-galactomannan enzymatic degradation product complex.

Example 2 Phosvitin-galactomannan enzymatic degradation product complex Phosvitin was purchased from Wako Pharmaceutical Co., Ltd. 1 g of phosvitin and 4 g of a galactomannan enzymatic decomposition product [trade name: Sunfiber, Taiyo Kagaku Co., Ltd.] were dissolved in 50 ml of deionized water and freeze-dried to recover a mixed powder. The mixed powder, 60 ℃, relative humidity 78.9% (saturated KB
(Solution r) for 5 days to obtain 4.8 g of phosvitin-galactomannan enzymatic degradation product complex.

Example 3 Egg yolk antibody-galactomannan enzyme degradation product complex Anti-human rotavirus yolk antibody (hereinafter referred to as IgY) was purified from egg yolk of laying hens hyperimmunized with human rotavirus Wa strain (Agric. Biol. Chem., 54 (10), 2531-253
5, 1990). 1 g of anti-human rotavirus IgY and 4 g of a galactomannan enzyme decomposition product [trade name: Sunfiber: Taiyo Kagaku Co., Ltd.] were dissolved in deionized water, and a mixed powder was collected by a freeze-drying method. The mixed powder was stored under the conditions of 70 ° C. and 72% relative humidity (saturated saline) for 2 days to obtain 4.9 g of egg yolk antibody-galactomannan enzyme degradation product complex.

Test Example 1. Confirmation of complex formation <SDS-polyacrylamide gel electrophoresis> Example 1
SDS-polyacrylamide gel electrophoresis was carried out by the method of Lemley (Nature, 227, 680, 1970) using the complex of (1) and the mixed powder as samples. Concentrated gel used 3% polyacrylamide concentration and separation gel used 7.5% polyacrylamide concentration. Apply 25 μg of each sample as protein amount and apply electricity at 30 mA for 2 hours, then use Coomassie Brilliant Blue R250 as usual. Protein staining was performed. As a result, in the mixed powder of Example 1, many stained bands were confirmed in the separation gel, while
The complex was recognized as a dark stained band at the boundary between the concentrated gel and the separation gel. From this, it was confirmed that the complex of Example 1 was a defatted egg yolk-galactomannan enzymatic degradation product complex.

Test Example 2. Confirmation of Complex Formation <Gel Filtration Analysis> The complexes obtained in Examples 2 and 3 and the respective mixed powders before complex formation were dissolved in a gel filtration development buffer (5 mg / ml). 50μl of that Supero
It was applied to a se 12 HR10 / 30 column (manufactured by Pharmacia) and subjected to gel filtration analysis. Gel filtration development buffer is 50 mM Na phosphate, 0.15 M NaCl buffer pH
8.0 was used. The flow rate was 0.5 ml / min, and the absorbance of the column effluent at 280 nm was monitored. The gel filtration patterns of the complex of Example 2 (stored for 5 days) and the mixed powder before complex formation (stored for 0 days) are shown in FIG. In addition, the gel filtration patterns of the complex of Example 3 (stored for 2 days) and the mixed powder before complex formation (stored for 0 days) are shown in FIG. The formation of the complex was confirmed by the shift of the gel filtration peak toward the polymer near the column void volume.

Test Example 3. Comparison of Emulsifying Properties of Complexes Emulsifying properties were determined by the method of Pearce et al. (J. Agric. Food Chem., 2
6, 716-723, 1978). Examples 1 and 2
The complex and the mixed powder before complex formation (preservation treatment) were dissolved in water (1 mg / ml). Mix 3.0 ml of each solution with 1.0 ml of corn oil, and add 10,000 ml of hiscotron.
Homogenized at rpm for 1 minute to obtain an emulsion. Immediately after emulsification, take 50 μl from the bottom and add 5 ml of 0.1% SDS aqueous solution.
It was diluted with and the absorbance at 500 nm was compared as emulsifying activity. In the complex of Example 1, the emulsifying activity was increased by 1.8 times as compared with the mixed powder before complex formation. The complex of Example 2 had a 2.3-fold increase.

Test Example 4. Comparison of enzyme digestion resistance of the complex Trypsin and chymotrypsin had an enzyme digestion buffer of 50 mM Tris, 10 mM CaCl ₂ buffer (pH 8.0) and pepsin had an enzyme digestion buffer of 0.
07M acetate buffer (pH 4.0) was used. Example 3
The complex and the mixed powder obtained in step 1 were dissolved in an enzyme digestion buffer, and trypsin, chymotrypsin, and pepsin were added to each to perform enzyme digestion. For trypsin and chymotrypsin digestion, substrate: enzyme = 1: 20 pH
After digesting at 8.0 and 37 ° C for 8 hours, 1/10 amount of 40 mM PMSF / isopropyl alcohol was added to the enzyme digestion solution to stop the enzyme reaction. In pepsin digestion, substrate: enzyme = 1: 200, pH 4.0, 37 ° C., 4
After digesting for 1 hour, 1/10 amount of 1% Na to the enzyme digestion solution
_The enzymatic reaction was stopped by adding a ₂ CO ₃ solution and returning the pH to neutral. For each enzyme digestion solution, the virus neutralizing antibody activity of anti-human rotavirus IgY was determined by the method of Ebina et al. (Microbiol. Immunol., Vol.34, 617-629,
(1990) and compared with the neutralizing antibody activity of the control to determine the residual ratio of neutralizing antibody activity of IgY. A pure anti-human rotavirus IgY was used as a control. The results of the residual ratio of neutralizing antibody activity are shown in Table 1.

[0026]

[Table 1]

The anti-human rotavirus IgY-galactomannan enzyme degradation product complex showed a high residual ratio of neutralizing antibody titer against any enzyme digestion. It is considered that the enzymatic degradation product of galactomannan formed a complex with IgY, which made it difficult to be affected by pepsin, trypsin, and chymotrypsin.

[0028]

EFFECTS OF THE INVENTION The complex of the present invention has improved or added functional properties such as emulsification property, foaming property, solubility property, fragrance property, oil retention property, protease digestion resistance, and divalent metal ion binding ability. It can be used as a high value-added material in the food, cosmetics and pharmaceutical fields.
INDUSTRIAL APPLICABILITY The complex of the present invention can be used as a source of a galactomannan enzyme hydrolyzate, and in that case, the intestinal bacterial flora improving effect, serum cholesterol lowering effect, fecal improving effect, etc. of the decomposed product of the present invention It is given to the complex added products. Conventionally, egg yolk protein has a low added value and its use has been limited. For the first time according to the invention,
It has become possible to use egg yolk protein with high added value.

[0029]

[Brief description of drawings]

FIG. 1 is a diagram showing a gel filtration analysis pattern of a mixed powder of a phosvitin-galactomannan enzymatic degradation product and a phosvitin-galactomannan enzymatic degradation product complex of Example 2.

FIG. 2 is a diagram showing a gel filtration analysis pattern of a mixed powder of an egg yolk antibody-galactomannan enzyme hydrolyzate and an egg yolk antibody-galactomannan enzyme hydrolyzate complex of Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yu Fujiki 9-5 Akabori Shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. (72) Inventor Kim Takehisa 9-5 Akahori-shinmachi, Yokkaichi, Mie Prefecture Gaku Co., Ltd.

Claims (4)

[Claims] 1. An egg yolk protein-galactomannan enzymatic degradation product complex obtained by storing a mixed powder of an egg yolk protein and a galactomannan enzymatic degradation product under a high temperature and high humidity environment. 2. The egg yolk protein-galactomannan enzymatic degradation product complex according to claim 1, wherein the egg yolk protein is defatted egg yolk. 3. The egg yolk protein-galactomannan enzymatic degradation product complex according to claim 1, wherein the egg yolk protein is phosvitin. 4. The egg yolk protein-galactomannan enzymatic degradation product complex according to claim 1, wherein the egg yolk protein is an egg yolk antibody.