JP3071877B2 - Hypoallergenic enzyme-decomposing peptide composition having oral tolerance inducing ability - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

2. Description of the Related Art It is generally 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 reducing the molecular weight of such proteins and peptides to reduce antigenicity. In fact, attempts have been made to reduce allergens by enzymatic degradation of casein and whey protein, which are thought to be the causative agents of milk allergy, and reported the relationship between the molecular weight of the degraded peptide and its antigenicity and immunogenicity. (Takase,
M. et al. , Jpn. Dairy Food S
ic. Otani, H., 33, A5 (1984); e
t al. , Milchwissenschaft, 4
5,217 (1990); Asselm, J. et al. et a
l. , J. et al. Food Sci. , 53, 1208 (19
88)). However, these studies on antigenicity and immunogenicity are all based on the results of in vitro reactivity of enzymatically degraded peptides with antibodies against undegraded protein antigens or the ability to produce in vivo antibodies by parenteral administration. No specific immune response (immunological tolerance or oral tolerance) of the living body during oral administration was considered at all.

[0003] In other words, the conventional peptide composition for degrading enzymatically degraded milk protein has been considered only to reduce the ability to produce an antibody by parenteral administration of a milk protein antigen or the ability to bind the antibody in vitro. It was developed. Examination of active use of 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 allergy Has never been done before. Furthermore, at present, nothing has been done about the development of oral tolerance inducers.

[0004]

The conventional method for reducing allergens in milk proteins has been to enzymatically degrade to low molecular weight peptides in order to eliminate antigenicity. But,
When the molecular weight is extremely reduced, it is expected that a bitter peptide is often produced and the flavor is impaired, and that the ability of the undegraded milk protein to induce oral tolerance in a living body, which is considered to originally have, is lost. In fact, there is an example of an enzymatically degraded peptide of a protein capable of inducing immunological tolerance when administered to a living body in advance (Japanese Patent Application Laid-Open No. 49-13324), but is not known at all for oral administration.

[0005] Therefore, an object of the present invention is to newly develop an enzymatically degraded peptide composition having reduced antigenicity and excellent flavor, and having oral tolerance inducing ability.

[0006]

Means for Solving the Problems As a result of various studies to solve the above problems, the present inventors treated milk-derived proteins with a protease to produce peptides having a molecular weight of 10,000 or less. As a result, it has been found that a low-allergenic enzyme-degrading peptide composition having a low antigenicity and an oral tolerance-inducing ability and a good taste can be obtained, and the present invention has been completed. Hereinafter, the present invention will be described in detail.

[0007] Protein is used as a raw material of the peptide composition according to the present invention. Whey proteins such as β-lactoglobulin and α-lactalbumin, casein and other proteins derived from milk are separated into each component. Use without separation or separation. These proteins are preferably purified, but when the final product is used as a food or a drug for oral administration, it is not always necessary to purify the protein to a pure product.

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

[0009] The term "proteolytic enzyme" broadly refers to an enzyme that hydrolyzes a peptide bond, broadly encompasses proteases, proteinases, endopeptidases, and the like. it can.

[0010] Proteolytic enzymes of plant origin include papain, bromelin, ficin and the like. Enzymes of animal origin include trypsin, chymotrypsin, kallikrein, pancreatin and the like, and in particular, trypsin and α-chymotrypsin. Preferably, a single type or a combination of two or more types can be used. As enzymes of microbial origin, Aspergillus spp. (For example, Aspergillus Dryza)
e IFO30105 etc.), Bacillus subtilis (eg Bacil)
rus subtilis IFO13722, etc.), actinomycetes (eg, Streptomyces griseu)
s IFO13304 etc.) can be used alone or in combination of two or more.

The present invention may be carried out in accordance with a conventional method for hydrolyzing a protein. A protein derived from milk is used as a substrate, and a protease is added thereto. Incubate it.
For example, trypsin, α-chymotrypsin, a protein hydrolase derived from a filamentous fungus of the genus Aspergillus, a protein hydrolase derived from Bacillus subtilis, or a protein hydrolase derived from actinomycete are added to an aqueous milk protein solution, and the pH is 6 to 9, and the temperature is 30 to 40. ℃,
Process for 30 minutes to 24 hours. Next, only a degraded peptide having a molecular weight of 10,000 or less is recovered using a membrane having a molecular sieving effect, such as an ultrafiltration membrane and a UF membrane having a molecular weight of 10,000. If necessary, the obtained peptide may be purified according to a conventional method.

The degraded peptide composition thus obtained is hypoallergenic, while its oral tolerance inducing ability is almost as good as undegraded milk protein, while it is always associated with the degraded peptide. Since the bitter component which has been removed is excellent in flavor and texture, it can be used freely 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 later, it has an excellent oral tolerance inducing ability, so that tablets, granules, capsules, powders, liquids, or other desired agents can be used alone with or without commonly used adjuvants. It can be formulated into a mold and used as a medicament for oral administration for inducing oral tolerance and / or ancillary 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 administered orally, there is no problem in safety at all, and the dose may be appropriately adjusted. . Hereinafter, examples of the present invention will be described.

Example 1 β-protein, one of whey proteins
Lactoglobulin (hereinafter abbreviated as β-LG) was obtained by the method of Otomo et al. (Journal of the Japan Food Industry Association, 35, 755 (1988)).
Collected from milk whey. 500 l of a 0.5% by weight aqueous solution of β-LG was prepared and adjusted to pH 8.0 with 1N sodium hydroxide. Bovine trypsin (Si
gma, T-8003) 2.5 × 10 ⁴ BAEE u
nit / g β-LG was added, and the enzyme reaction was carried out for 1 hour while stirring at pH 8.0 and 37 ° C. After the reaction, β-L
The G enzyme decomposition solution was subjected to a filtration apparatus 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.

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

FIG. 1 and FIGS. 2 to 6 show that there is no undegraded β-LG in the sample and its antigenicity is reduced. In addition, as a result of the sensory test, 0 out of 15 persons complained of bitterness with the β-LG enzyme-decomposing peptide composition.

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

The above experimental results are shown in FIG. 3 shown in FIG. According to this, even in mice administered with the β-LG enzyme-degrading peptide composition, oral tolerance of almost the same strength as in mice administered with undegraded β-LG was induced, and β-LG
It turns out that production of a specific antibody 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. Add 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 enzymatically degraded casein solution was freeze-dried to obtain an enzymatically degraded casein peptide composition. As shown in FIG. 4 of FIG. 8, this casein enzymatically degraded peptide composition was composed of peptides having a molecular weight of 10,000 or less as a result of HPLC gel filtration chromatography.

The above casein enzyme-decomposing composition was used to prepare a solid feed occupying 10% of the total protein, and a mouse (C3H / H) was prepared.
e, 6w, female) (1 group, 5 animals, feed composition based on AIN-76). Four weeks after the start of breeding, each mouse was intraperitoneally immunized with 100 μg of undegraded casein together with Freund's complete adjuvant. Two more weeks later, each mouse was intraperitoneally immunized with 100 μg of undegraded casein together with incomplete Freund's adjuvant. On the 10th day after the second immunization, the serum of each mouse was collected from the tail vein, and the amount of antibody against casein (αs 1 -casein) in the serum was determined by EL.
It was measured by the ISA method. As in Example 2, the positive control was the serum of a mouse bred on a solid feed in which undegraded casein accounted for 10% of the total protein, and the negative control was the serum of a mouse bred on an MF diet.

As a result, as shown in Table 1, oral tolerance of the same level as that of mice to which undegraded casein was administered was induced in mice to which the casein enzyme-degrading peptide composition had been administered, and the production of casein-specific antibodies. Is suppressed.

[0022]

[Table 1]

[0023]

As one of the causes of food allergies observed in infants and infants, it has been pointed out that a part of food proteins is absorbed from the intestinal tract without being digested by digestive enzymes and stimulates the biological immune system. In this regard, since the enzymatically 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 enzymatically degraded peptide composition has almost the same oral tolerance inducing ability as the undegraded milk protein, and thus can sufficiently activate the potential allergic defense mechanism of the living body. Furthermore, the deterioration of the flavor accompanying the enzymatic decomposition of the present enzymatic peptide composition is extremely small.

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

[Brief description of the drawings]

FIG. 1. β-L with commercially available protease (trypsin)
FIG. 4 is an SDS electrophoretogram (sodium dodecyl sulfate polyacrylamide gel electropherogram) after decomposition of G.

FIG. 2. Competitive ELI of reactivity of β-LG enzyme-degrading peptide composition with β-LG monoclonal antibody (21B3)
FIG. 2A is a drawing (FIG. 2A) studied by an SA method (an Elizer method).

FIG. 3 shows a β-LG monoclonal antibody (31A)
FIG. 2B is a drawing showing the reactivity with No. 4) (FIG. 2B).

FIG. 4 shows a β-LG monoclonal antibody (61C
FIG. 2C is a drawing showing the reactivity with 1) (FIG. 2C).

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

FIG. 6 shows a β-LG monoclonal antibody (62A
6 (E) showing the reactivity with (6).

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

FIG. 8: HPLC analysis of casein enzymatically degraded peptide composition
It is the figure which performed (high performance liquid chromatography).

──────────────────────────────────────────────────の Continuing on the front page (51) Int.Cl. ⁷ Identification code FI C12P 21/06 A61K 37/18 (72) Inventor Takeshi Takahashi 2-21-35, Nakatacho, Higashimurayama-shi, Tokyo (72) Inventor Uenogawa Shuichi 400-8 Masuda Nitta, Kasukabe City, Saitama Prefecture (56) References JP-A-2-2319 (JP, A) JP-A-2-182155 (JP, A) JP-A 64-5497 (JP) JP-A-2-234642 (JP, A) JP-A-3-220197 (JP, A) JP-A-3-272694 (JP, A) JP-A-4-320650 (JP, A) 4-248959 (JP, A) JP-A-5-176714 (JP, A) Dairy science and food research, Vol. 39, No. 4, p. A123-129 (1990) Bulletin of the Japanese Society of Animal Husbandry, Vol. 52, No. 1, p. 47-52 (1981) Milchwissenshaft, Vol. 45, no. 4, p. 217-220 (1990) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/17 A61K 38/00 A61P 37/06 A23L 1/305 C07K 14/435 C12P 21/06 JICST file (JOIS )

Claims (1)

(57) [Claims] 1. A low-allergenic enzyme-degrading peptide composition having the ability to induce oral tolerance, which is obtained by treating a protein derived from milk with a protease and having a molecular weight of 10,000 or less, as an active ingredient. Oral tolerance inducer.