JP2927076B2 - New protein food ingredients - 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 antigenic and low immunogenic protein food material obtained by the action of transglutaminase.

[0002]

2. Description of the Related Art In recent years, the number of patients with food allergies has increased remarkably. Particularly in infants and children, the problem is significant because it makes it impossible to take nutrition for healthy growth. In fact, the number of patients with food allergies, which are said to account for 10 to 30% of allergic diseases in infants, has increased,
In addition, allergies to plant foods such as soybeans and grains as well as animal foods such as milk, chicken eggs, fish, and meat have recently been reported frequently. Although it is very important for infants to eat good protein foods for their growth, for now,
Low allergenic protein foods satisfying these allergy patients have not been developed. It should be noted that an allergenic protein is a protein having antigenicity (antigenicity) and immunogenicity (immunogenicity) that cause allergy.

[0003] Conventionally, as a method for removing allergens (eg, β-lactoglobulin in milk) that cause allergies, fractionation using a precipitant, chromatography using chemical bonding, selective decomposition using a protease, heat, etc. Denaturation treatment and the like have been reported. As an example, both the casein component and whey component, which are proteins in milk, have allergen activity, and physically removing them can reduce the intake of milk protein, a valuable source of nutrition. You can deny it and consider it virtually impossible. Furthermore, whey protein, which is a major allergen, particularly β-lactoglobulin, is heat-resistant only by heat denaturation, and its reducing effect is not sufficient. In addition, treatment with a protease has problems such as generation of bitter peptides and changes in physical properties of milk proteins. These problems are the same in protein foods having other allergens. Therefore, as can be seen from the above example, reduction of allergenicity is still insufficient, and flavor defects such as bitterness, liquefaction is difficult due to a significant decrease in solubility, and high production cost And other issues.

[0004] In view of the above problems, the present inventors have reported that allergen-reducing protein foods are allergen-reduced using a transglutaminase reaction (Japanese Patent Application Laid-Open No. 3-27253). However, the antigenic properties of allergenic proteins in this way (antigenic)
Although the protein has been successfully eliminated, there is concern that the modified protein may have new immunogenicity (immunogenicity). Therefore,
At present, there is a long-awaited need to provide a protein food material having low antigenicity and low immunogenicity. In addition, as a reminder,
Immunogenic (immunogenic) means having the property of inducing antibody production in the humoral immune system. Antigen (antigenic) means that it has the property of performing an antigen-antibody reaction (precipitation reaction, agglutination reaction, complement activation reaction, phagocytosis enhancement reaction, allergic reaction, etc.) by binding to the produced antibody. Means

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a protein food material which is low in antigenicity and has no new immunogenicity in order to prevent allergy to protein foods.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a glutamine residue or a lysine residue is present in and / or in the vicinity of an antigenic determinant (epitope) of an allergenic protein. Exists,
The inventors of the present invention have come up with the idea of utilizing both transglutaminase and collagen / elastin, which are endogenous in vivo, because of their low antigenicity, and completed the present invention. That is, the present invention provides (1) the action of transglutaminase on (2) the allergenic protein and / or its degraded peptide.
It is a novel protein food material having a low antigenicity and a low immunogenicity obtained by binding an endogenous protein and / or its degradation peptide. The transglutaminase referred to in the present invention is an enzyme that catalyzes an acyl transfer reaction between a γ-carboxamide group of a glutamine residue in a protein and a peptide chain and a primary amine (Fo).
lk et at. , Advances in Enz
ymology, 38, 109-191 (1973) and Folk et al. , Advances in
Protein Chemistry, 31, 1-13
3 (1977)). This reaction also recognizes the ε-amino group of the lysine residue in the protein as a primary amine,
Create crosslinks within and between protein molecules.

On the other hand, when transglutaminase is allowed to act by blocking the ε-amino group of a lysine residue in a protein, amino acids, amino acid esters and peptides which are primary amines can be incorporated. If no primary amine is present in the reaction system, the deamidation reaction proceeds, and glutamine residues can be converted to glutamic acid residues.

[0008] Therefore, if this transglutaminase is utilized, an endogenous protein such as collagen or elastin and / or its degraded peptide can be selectively introduced into the glutamine residue in the epitope of the allergenic protein, and the epitope portion can be introduced. May be able to block the binding to the antibody, and also to introduce an in vivo protein such as collagen and elastin and / or a degradation peptide thereof into glutamine residues other than the epitope of the allergenic protein. However, these steric hindrances may possibly prevent the binding to the antibody. Furthermore, a change in the overall conformation of the allergenic protein due to the interaction with the introduced collagen and elastin can be expected, and it is considered that the protein may not be recognized by the antibody. In addition, since the protein to be introduced and / or its degraded peptide is a protein such as collagen or elastin, which is present in a large amount in the living body, or a peptide thereof, it is unlikely that a newly obtained protein will exhibit immunogenicity.

The allergenic protein in the present invention may be of any origin such as animal food, vegetable food, or microbial food. However, most of the food allergies are milk proteins, egg proteins and soy proteins, and these proteins are the main object of the present invention.
Further, a peptide obtained by decomposing these proteins by a microbial treatment such as fermentation, an enzymatic treatment with a protease, and further by a treatment with an acid or an alkali may be used in place of the protein or together with the protein. Further, as the in vivo protein in the present invention, a protein which exists in a large amount in the living body such as collagen, atherocollagen, gelatin, and elastin can be used. Furthermore, these in vivo proteins can be converted into enzymes, acids,
A peptide obtained by hydrolysis using an alkali or the like may be used in place of the in vivo protein or together with the in vivo protein.

The enzyme transglutaminase used in the present invention can be of animal origin, of microbial origin or of plant origin, and its origin is not particularly limited. For example, animal sources include those derived from guinea pig liver (Connellan et al., Jo.
urnal of Biological Chemi
try, 246 (4), 1093-1098 (197)
1)), those widely distributed in mammalian organs and blood (Folk et al., Advancesin E)
nzymology, 38, 109-191 (197)
3) and Folk et al. , Advances
in Protein Chemistry, 31, 1
133 (1977)), and those derived from microorganisms include those produced by bacteria belonging to the genus Streptoverticillium of the actinomycetes (JP-A-64-27471). . Among these transglutaminases, transglutaminase of microbial origin, specifically, actinomycete streptoverticillium origin, is easily and inexpensively available, and acts in the presence or absence of Ca ^2+. desirable.

Under the reaction conditions of the present invention, the concentration of the allergenic protein serving as a substrate slightly varies depending on the type of the protein, but is usually 0.001 μg / ml-10000 m 2.
g / ml, preferably 0.1-100 mg / ml. In addition, the substrate concentration of the endogenous protein to be introduced also slightly varies depending on the type of the protein to be used.
1 μg / ml-90000 mg / ml, preferably 0.1 μg / ml
1-100 mg / ml. Next, the amount of transglutaminase that catalyzes the reaction is not particularly limited,
Usually 0.0001-10000 / g of substrate protein
Unit (U), preferably 0.01-100 U. The reaction temperature or the reaction time is not particularly limited, but is usually 1
-80 ° C, preferably 4-50 ° C, usually 0.01-
The reaction may be performed for 72 hours, preferably for 0.1 to 48 hours. Again, the production conditions of the novel protein food material of the present invention are not limited to the above conditions.

[0012] Incidentally, measurement of activity transglutaminase At a present invention, benzyloxy Cal Bo sulfonyl -L- glutaminyl - conducting a reaction of glycine and hydroxy Shirua Min as a substrate, the presence of trichloroacetic acid generated hydroxamic acid free in to form an iron complex, was carried out by the absorbance at 525nm of the complex is measured to calculate the activity compared with the calibration curve the amount of created separate hydro hexa beam acid. Activity is the enzymatic activity of producing 1μ mole of hydrate b Kisamu acid per minute and 1U (supra JP 64-27471).

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to embodiments.

Example 1 Preparation of Casein-Atelocollagen Complex An aqueous solution obtained by heating and dissolving a mixture of 3% α _s1 -casein (prepared by the Zittle method, molecular weight 23,000) and 6% atelocollagen was added to a transglutaminase derived from a microorganism (specific ratio). (16 U / ml activity) to give a casein substrate of 1 mg or 0.06 U, and kept at 37 ° C. for 24 hours to obtain a casein-atelocollagen complex (sample 1).
I got

[0015] Example 2 Casein - Preparation of gelatin complex alpha _s1 - casein (prepared by Zittle method, molecular weight 23,000) in 3% and mixed aqueous solution of 6% gelatin, transglutaminase microbial origin (specific activity 16U / m
1) was added to give 0.06 U per 1 mg of casein substrate, and the mixture was kept at 37 ° C. for 24 hours to obtain a casein-gelatin complex (sample 2).

Example 3 Preparation of Casein-Gelatin Peptide Complex A microbial transglutaminase (specific activity 1) was added to a mixed aqueous solution of 3% α _s1 -casein (prepared by the Zittle method, molecular weight 23,000) and 6% of gelatin peptide.
6 U / ml) to give 0.06 U per mg of casein substrate, and kept at 37 ° C. for 24 hours.
A gelatin peptide complex (sample 3) was obtained.

Example 4 Evaluation of antibody binding ability of casein-collagen complex 1 The antibody binding ability of 3 samples of _αs1 -casein and collagen complex prepared in the above three examples was evaluated by ELISA. For comparison, the antibody binding ability of untreated _αs1 -casein was examined. The results are shown in FIG. As shown in FIG. 1, the binding ability (absorbance value on the vertical axis) of the untreated α _s1 -casein to the antibody was high even when the concentration (horizontal axis) was varied, whereas the binding ability of the complex with collagens was high. The binding was reduced at high concentrations. Among these three samples, it was found that the binding ability of the complex with gelatin was significantly reduced.

Example 5 Evaluation of Antibody Binding Ability of Casein-Collagen Complex 2 (Comparison with Commercial Milk) Commercially low-allergenic milk has been reduced in molecular weight by proteolytic enzyme treatment to reduce allergen. ,
There is a disadvantage that the flavor is bad. Thus, the antibody binding ability of the α _s1 -casein-gelatin complex prepared according to the present invention and found to have the lowest antibody binding ability in Example 4 was compared with that of commercially available milk for healthy infants. Was examined by ELISA. For comparison, unprocessed α
The antibody binding ability of _s1 -casein was examined. The results are shown in FIG. As can be seen from the results, the antibody binding ability (absorbance on the horizontal axis) of untreated _αs1 -casein was high. Considering that commercially available milk for healthy infants contains a large amount of β-casein and whey protein in addition to α _s1 -casein, the antibody binding ability of this was also measured by varying the concentration (vertical axis). Interpreted as higher than the result. On the other hand, the antibody binding ability of the α _s1 -casein-gelatin complex was almost completely eliminated, and it was found that those prepared in the present invention had reduced antigen. The α _s1 -casein-gelatin complex was excellent in flavor without bitterness.

Example 6 Evaluation of Immunogenicity Confirmation that the α _s1 -casein-gelatin complex prepared in the present invention is low immunogenic was administered several times to rat veins, and α _{s1 -casein} was _added to the serum. Although it has been confirmed that antibodies to the gelatin complex are not produced,
No new antibodies are produced and it is expected that they have low immunogenicity.

[0020]

According to the present invention, the novel protein food material obtained by the action of transglutaminase is a very useful material not only having low antigenicity but also being characterized by low immunogenicity. In addition, since it has no bitterness and no problem in flavor, it can be expected that it can be widely used as a food material. Further, the process of the present invention can solve the problem of cost increase due to a combination of complicated processes as compared with the conventional method. Therefore, the low immunogenic and low antigenic edible material according to the present invention according to the present invention can be particularly expected as a food for allergic patients.

[Brief description of the drawings]

FIG. 1. ELISA using an anti-α _s1 -casein antibody to confirm that the novel protein food (α _s1 -casein-gelatin complex) prepared in the present invention has no antigenicity
5 shows the results obtained by examining the antigen-antibody reaction.

FIG. 2 shows the results obtained by examining that the antigenicity of the novel protein food (α _s1 -casein-gelatin complex) prepared according to the present invention was lower than that of commercially available milk.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 31/00 637 A61K 31/00 637B 38/17 C12P 21/02 B C12P 21/02 21/06 21/06 A61K 37 / 12 (56) References JP-A-4-91750 (JP, A) JP-A-3-27253 (JP, A) JP-A-3-160957 (JP, A) JP-A-2-171160 (JP, A) (58) Fields investigated (Int.Cl. ⁶ , DB name) A23J 3/34 A23J 3/04 501-3/10 A23L 1/305 A61K 31/00 637 A61K 38/17 C12P 21/02 C12P 21 / 06 JICST file (JOIS) JAFIC file (JOIS)

Claims (2)

(57) [Claims] Claims: 1. An anti-allergenic protein and / or its degrading peptide, which has low antigenicity obtained by binding a (2) endogenous protein and / or its degrading peptide by the action of transglutaminase, and A novel protein food material with low immunogenicity. 2. The novel protein food material according to claim 1, wherein the endogenous protein is collagen, atelocollagen, gelatin and elastin.