JPH055000A - Low-aloergenic emzymolyzed peptide composition with orally tolerant inductivity - Google Patents

Abstract

PURPOSE:To obtain the title composition, little in flavor degradation due to enzymolysis, useful as a food decreased in allergic reaction or orally tolerant inducer, by treating protein of cow milk origin with protein hydrolase to bring its molecular weight to a specified level or lower. CONSTITUTION:beta-lactoglobulin, one of whey proteins, is recovered from cow milk whey through a conventional process and dissolved in water and the resulting solution is adjusted to pH 8.0 with 1 N aqueous sodium hydroxide solution. A protein hydrolase (e.g. bovine trypsin) is then added to the resulting solution to make enzymolysis for one hour while agitating at 37 deg.C, and the resultant solution is put to a filtration unit with a ultrafiltration membrane 10000 in fractionating molecular weight to obtain a fraction containing beta- lactoglobulin partial peptide with a molecular weight of >=10000, which is then lyophilized, thus obtaining the objective peptide composition with a molecular weight of <=10000.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hypoallergenic enzyme-degrading peptide composition obtained by treating milk protein with a protein hydrolase and its use.

[0002]

2. Description of the Related Art Generally, it is known that the antigenicity of a protein or peptide is closely related to its molecular weight. Enzymatic degradation is one of the most effective methods for lowering the antigenicity by lowering the molecular weight of such proteins and peptides. In fact, casein and whey protein, which are considered to be the causative agents of milk allergy, have been attempted to reduce the allergen by enzymatic degradation, and the relationship between the molecular weight of the degraded peptide and the antigenicity and immunogenicity has been reported. (Takase,
M. et al. , Jpn. Dairy Food S
ic. 33, A5 (1984); Otani, H .; e
t al. , Milchwissenschaft, 4
5, 217 (1990); Asselm, J .; et a
l. , J. Food Sci. , 53, 1208 (19
88)). However, all of these studies on antigenicity and immunogenicity were performed based on the in vitro reactivity with the antibody against the undegraded protein antigen of the enzyme-degraded peptide or the in vivo antibody-producing ability by parenteral administration. However, no specific biological immune response (immunity tolerance or oral tolerance) was taken into consideration at the time of oral administration.

That is, the conventional allergen-degrading enzyme-degrading peptide composition of milk protein has only been considered in view of reducing the antibody-producing ability of the milk protein antigen by parenteral administration or the antibody-binding ability in vitro. It was developed. Study to positively utilize the antigen-specific antibody production suppression mechanism (such as induction of antigen-specific suppression T cells) that can be induced in vivo when milk protein is orally administered to the development of foods with reduced allergies Has never been done so far. Moreover, nothing is done at present in the study of the development of an oral tolerance inducer.

[0004]

The conventional methods for reducing allergens of milk proteins have been to enzymatically decompose even low molecular weight peptides in order to eliminate antigenicity. But,
It is expected that when the molecular weight is extremely reduced, bitter peptides are often produced, the flavor is impaired, and in addition, the oral tolerance-inducing ability of the living body, which is supposed to be possessed by undegraded milk protein, disappears. In fact, an enzyme-degraded peptide of a protein that can be administered to a living body in advance to induce immune tolerance is not known at all, although there is an example for parenteral administration (JP-A-49-13324).

Therefore, the problem to be solved by the present invention is to newly develop an enzyme-degrading peptide composition having reduced antigenicity and excellent taste and having oral tolerance inducing ability.

[0006]

[Means for Solving the Problems] In order to solve the above problems, as a result of various studies, the inventors of the present invention treat a milk-derived protein with a proteolytic enzyme to obtain a peptide having a molecular weight of 10,000 or less. As a result, it was found that a low-allergenic enzyme-degrading peptide composition having a low antigenicity and an oral tolerance inducing ability and a good flavor can be obtained, and the present invention has been completed. Hereinafter, the present invention will be described in detail.

A protein is used as a raw material of the peptide composition according to the present invention, and whey proteins such as β-lactoglobulin and α-lactalbumin, casein and other milk-derived proteins are separated into each component. Or use without separation. It is convenient to purify these proteins, but when the final product is used as a food or an orally-administered drug, it is not absolutely necessary to purify it to a pure product.

In the present invention, the above-mentioned milk-derived proteins are mixed, if necessary, with two or more kinds and treated with a protein hydrolase to form a low molecular weight peptide having a molecular weight of 20,000 or less, preferably 10,000 or less.

The term "protein hydrolase" broadly refers to an enzyme that hydrolyzes a peptide bond, and broadly includes proteases, proteinases, endopeptidases and the like, and enzymes of plant origin, animal origin, and microbial origin are appropriately used. it can.

Examples of plant-derived protein hydrolases include papain, bromelin and ficin, and examples of animal-derived enzymes include trypsin, chymotrypsin, kallikrein, pancreatin and the like, particularly trypsin and α-chymotrypsin. Preferably, they can be used alone or in combination of two or more. Examples of enzymes of microbial origin include Aspergillus genus bacteria (eg, Aspergillus Dryza).
e IFO30105 etc.), Bacillus subtilis (eg Bacil
lus subtilis IFO13722), actinomycetes (eg Streptomyces griseu)
s IFO13304 etc.) can be used alone or in combination of two or more kinds.

In order to carry out the present invention, it may be carried out according to a conventional method of hydrolyzing a protein, and a protein derived from milk is used as a substrate and a protein hydrolase is added thereto, and a required time is maintained at a predetermined pH and temperature. Just incubate.
For example, trypsin, α-chymotrypsin, proteolytic enzyme derived from Aspergillus filamentous fungus, proteolytic enzyme derived from Bacillus subtilis or proteolytic enzyme derived from actinomycete is added to a milk protein aqueous solution to add pH 6 to 9, temperature of 30 to 40. ℃,
It is processed in the range of 30 minutes to 24 hours. Then, using a membrane having a molecular sieving effect such as an ultrafiltration membrane having a molecular weight of 10,000 or a UF membrane, only the degraded peptide having a molecular weight of 10,000 or less is recovered. Further, if necessary, the obtained peptide may be purified according to a conventional method.

The degraded peptide composition thus obtained has a low allergenicity, while the oral tolerance-inducing ability is almost the same as that of undegraded milk protein. Since the bitterness component is removed and the flavor and texture are excellent, it can be used alone or together with other foods as a new allergy-reducing food material.

Further, the degraded peptide composition according to the present invention comprises
As is clear from the examples described below, the oral tolerance-inducing ability is extremely excellent, and therefore, desired agents such as tablets, granules, capsules, powders, and liquids can be used alone with or without conventional adjuvants. It can be formulated into a mold and used as a pharmaceutical for oral administration for inducing oral tolerance and / or as an adjunct treatment therefor. Since the degraded peptide composition according to the present invention can be originally used as a food, there is no problem in safety, and since it is orally administered, there is no problem in safety, and its dose may be appropriate. .. Examples of the present invention will be described below.

[Example 1] β-, which is one of whey proteins
Lactoglobulin (hereinafter, abbreviated as β-LG) method by Otomo et al. (Journal of Japan Food Industry Society, 35, 755 (1988))
Recovered from milk whey at. 500 l of a 0.5 wt% aqueous solution of β-LG was prepared, and the pH was adjusted to 8.0 with 1N sodium hydroxide. In addition to this, bovine trypsin (Si
gma company, T-8003) 2.5 × 10 ⁴ BAEE u
nit / gβ-LG was added, and the enzyme reaction was carried out for 1 hour with stirring at pH 8.0 and 37 ° C. After the reaction, β-L
The G enzyme-decomposed solution was applied to a filtration device using an ultrafiltration membrane having a molecular weight cutoff of 10,000 to obtain a fraction containing a partial peptide of β-LG having a molecular weight of 10,000 or less.

The SDS electrophoresis pattern of a sample obtained by freeze-drying this fraction is shown in FIG. 1 shown in FIG. 1, and the antigenicity is shown in FIG.
~ As shown in Figure 2 shown in Figure 6. The antigenicity is Ka
5 types of anti-β-LG monoclonal antibodies (Agric. Biol. Chem., prepared by Minagawa et al.
51, 797 (1987)) and the competitive method EL
The examination was carried out by the ISA method (enzyme immunoassay (2nd edition), p30, medical school). As is clear from the results, β-L
Monoclonal antibodies (21B3, 31A4) showing specificity to 8-26 or 15-26 on the N-terminal side of G reacted slightly with these peptides (Figs. 2A and 2A shown in Figs. 2 and 3). B), it does not react at all with the monoclonal antibodies (61C1, 61B4, 62A6) that recognize the conformation of native β-LG but do not react with the modified β-LG (Fig. 4, 5 and 6). (Fig. 2C, Fig. 2D, Fig. 2E).
-LG proved to be completely degraded.

From FIGS. 1 and 2 to 6, it can be seen that there is no undegraded β-LG in the sample and its antigenicity is reduced. As a result of a sensory test, 0 out of 15 people complained of bitterness to the β-LG enzyme-degrading peptide composition.

[Example 2] β of the sample prepared in Example 1
-A solid feed using the LG enzyme-degraded peptide composition as a protein source was prepared, and mice (C3H / He, 6W, female) were bred (5 mice per group, feed composition based on AIN-76).
Three weeks after the start of breeding, 100 μg of undegraded β-LG was intraperitoneally immunized with Freund's complete adjuvant. After 2 more weeks, 100 μg of undegraded β-LG
Each mouse was intraperitoneally immunized with Freund's incomplete adjuvant. On the 10th day after the secondary immunization, the serum of each mouse was collected from the tail vein, and the amount of antibody to β-LG in the serum was measured by the ELISA method. The positive control for the suppression of antibody production is serum obtained by the same treatment as described above on mice bred with a solid feed containing undegraded β-LG as a protein source, which was the raw material for preparing the sample of Example 1. On the other hand, the negative control is a serum obtained by subjecting a mouse raised on an MF feed (Oriental Yeast Co., Ltd.) containing no milk-derived protein to the same treatment as described above.

The above experimental results are shown in FIG. 3 shown in FIG. According to this, in mice to which the β-LG enzyme-degrading peptide composition was administered, oral tolerance having a strength almost equal to that in mice to which undegraded β-LG was administered was induced, and β-LG
It can be seen that the production of specific antibodies is suppressed.

[Example 3] Casein (Sigma) was dissolved in water to prepare 100 l of a 1.0% by weight aqueous solution.
This was adjusted to pH 8.0 with 1N sodium hydroxide. Bovine trypsin (Sigma, T-8003)
5.0 × 10 ⁴ BAEE unit / g casein was added, and the enzyme reaction was carried out for 24 hours while stirring at pH 8.0 and 37 ° C. After completion of the reaction, the casein enzyme-decomposed solution was freeze-dried to obtain a casein enzyme-decomposed peptide composition. As shown in FIG. 4 shown in FIG. 8, this casein enzyme-degraded peptide composition was found to consist of peptides having a molecular weight of 10,000 or less as a result of HPLC gel filtration chromatography.

The casein enzyme-decomposing composition described above was used to prepare a solid feed containing 10% of the total protein, and the solid feed was prepared using the mouse (C3H / H
e, 6w, female) were bred (1 group, 5 animals, feed composition according to AIN-76). Four weeks after the start of breeding, 100 μg of undegraded casein was intraperitoneally immunized into each mouse together with Freund's complete adjuvant. After a further 2 weeks, 100 μg of undegraded casein was intraperitoneally immunized into each mouse together with Freund's incomplete adjuvant. On the 10th day after the secondary immunization, the serum of each mouse was collected from the tail vein, and the amount of antibody to casein (αs1-casein) in the serum was measured by EL.
It was measured by the ISA method. As in Example 2, the positive control is the serum of a mouse fed on a solid diet in which undegraded casein accounts for 10% of the total protein, and the negative control is the serum of a mouse fed on an MF diet.

As a result, as shown in Table 1, the mice to which the casein enzyme-degrading peptide composition was administered also induced oral tolerance with the same level of strength as the mice to which undegraded casein was administered, thus producing a casein-specific antibody. It can be seen that is suppressed.

[0022]

[Table 1]

[0023]

As one of the causes of food allergy observed in infants, it has been pointed out that a part of food protein is absorbed from the intestinal tract without being decomposed by digestive enzymes and stimulates the living body immune system. In this regard, since the enzyme-degraded peptide composition of the present invention has a molecular weight of 10,000 or less, the antigenicity of the original undegraded milk protein is reduced. On the other hand, the present enzyme-degraded peptide composition has almost the same oral tolerance-inducing ability as that of undegraded milk protein, and therefore can sufficiently activate the potential allergic defense mechanism of the living body. Further, the deterioration of the flavor due to the enzymatic decomposition of the present enzymatically decomposed peptide composition is extremely small.

Therefore, the peptide composition of the present invention is extremely useful as a new allergen-reducing food material.

[Brief description of drawings]

FIG. 1 β-L by a commercially available protease (trypsin)
It is the SDS electrophoretic diagram (Sodium dodecyl sulfate polyacrylamide gel electrophoretic diagram) after decomposition | disassembly of G.

FIG. 2 shows the competitive ELI of the reactivity of the β-LG enzyme-degrading peptide composition with the β-LG monoclonal antibody (21B3).
It is the drawing (FIG. 2A) examined by the SA method (the eraser method).

FIG. 3 is also a β-LG monoclonal antibody (31A
4 is a drawing showing the reactivity with (4) (FIG. 2B).

FIG. 4 is also a β-LG monoclonal antibody (61C).
It is drawing (FIG. 2C) which showed the reactivity with 1).

FIG. 5 is also a β-LG monoclonal antibody (61B).
FIG. 4D is a drawing showing the reactivity with 4) (FIG. 2D).

FIG. 6 is also a β-LG monoclonal antibody (62A).
6 is a drawing showing the reactivity with 6) (FIG. 2E).

FIG. 7 is a drawing in which the oral tolerance-inducing ability of a β-LG enzyme-degrading peptide composition was investigated from an increase in anti-β-LG antibody titer.

FIG. 8: HPLC analysis of a casein enzyme-degrading peptide composition
It is a figure subjected to (high performance liquid chromatography).

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Takeshi Takahashi 2-21-35 Maita-cho, Higashimurayama-shi, Tokyo (72) Inventor Shuichi Uenogawa 400-8 Masuda-Nitta, Kasukabe-shi, Saitama

Claims (1)

Claims: 1. A low allergenic enzyme-degrading peptide composition having an oral tolerance-inducing ability, characterized in that a protein derived from milk is treated with a protein hydrolase to have a molecular weight of 10,000 or less. object. 2. The hypoallergenic enzyme-degrading peptide composition having the ability to induce oral tolerance according to claim 1, wherein the protein derived from milk is casein or whey protein. 3. The hypoallergenic enzyme-degrading peptide composition having an oral tolerance-inducing ability according to claim 1, wherein the protein hydrolase is an enzyme derived from an animal, a plant and / or a microorganism. 4. The protein hydrolase is trypsin and / or
Or α-chymotrypsin, The hypoallergenic enzyme-degrading peptide composition having the ability to induce oral tolerance according to claim 3. 5. The hypoallergenic enzyme-degrading peptide having an oral tolerance-inducing ability according to claim 3, wherein the proteolytic enzyme is an enzyme derived from Aspergillus filamentous fungus, Bacillus subtilis and / or actinomycete. Composition. 6. An oral tolerance inducer, which comprises the peptide composition according to any one of claims 1 to 5 as an active ingredient.