Oral immune tolerance agent for wheat allergy, and oral composition including same

Abstract

The present invention addresses the problem of providing a means that can prevent or treat wheat allergy, and has a higher efficacy and lower risk than conventional means. The present invention provides an oral immune tolerance agent for wheat allergy, said oral immune tolerance agent including a degradation product of gluten due to a proteolytic enzyme for food, such as a serine protease or a metalloprotease.

Description

Oral immune tolerance inducer for wheat allergy and oral composition containing the same

[0001] The present invention relates to an oral immune tolerance inducer that can be used for the prevention and treatment of wheat allergy, a composition containing the same, and a method for producing the same.

[0002] Wheat is known as a food allergen with a high prevalence in infancy after eggs, milk and tree nuts. However, since it is a food included in staple diets, it is more difficult to avoid than other allergens. As wheat allergens, wheat proteins such as gliadin, glutenin, albumin and globulin are known. Gluten contains these wheat proteins, and mainly gliadin and glutenin are bound in a network structure via water absorption.

[0003] In order to allow wheat allergy patients to ingest wheat protein without inducing allergic symptoms, attempts have conventionally been made to hypoallergenize wheat protein. For example, as in hypoallergenization by deamidation using an organic acid (Patent Document 1), hypoallergenization by bromelain, a proteolytic enzyme for food with high collagenase-like activity (Patent Document 2), hypoallergenization by cellulase and actinase (Patent Document 3), and hypoallergenization by yeast or koji extract (Patent Document 4), methods have been proposed in which wheat protein (gluten) is treated with an acid, an enzyme or the like to decompose the wheat allergen contained therein and achieve hypoallergenization.

[0004] In addition, ω5-gliadin is known as a causative protein that induces exercise-induced anaphylactic shock (WDEIA), and hypoallergenization by producing wheat that does not have the ω5-gliadin gene (1BS-18H) (Non-Patent Document 1) has also been reported.

[0005] On the other hand, regarding food allergies, the dual antigen exposure hypothesis proposed by Lack in 2008, which states that food allergies are induced when food allergens enter the body percutaneously (sensitization), but when the same food allergen is ingested orally, it induces immune tolerance that suppresses the allergic reaction, is widely known. Based on this hypothesis, various food allergens (such as eggs and peanuts) have been tested to see if it is possible to suppress the production of IgE antibodies against food allergens by orally ingesting an appropriate amount of food allergen at the appropriate time (early after weaning).

[0006] For example, Non-Patent Document 2 shows that oral administration of pepsin hydrolysates of gliadin, a wheat protein, to mice for a certain period of time suppresses the production of IgE antibodies, IgG1 antibodies, and Th2 type cytokines, and suppresses anaphylactic shock and allergic reactions in the small intestine when gliadin is administered. In other words, oral immune tolerance to gliadin is induced, reducing its allergenicity. However, Non-Patent Document 2 does not verify the transcutaneous sensitizing ability of gliadin pepsin hydrolysates.

[0007] It is also known that the breakdown products of wheat protein do not necessarily reduce transdermal sensitization ability (in fact, they may even increase transdermal sensitization ability more than wheat protein itself). For example, Non-Patent Literature 3 reports that hydrolyzed gluten produced by acid heat treatment contains a component (Gluparol 19S) in which glutamine contained in glutenin is deamidated and changed to glutamic acid, and that using soap containing this component can cause transdermal and transmucosal sensitization, leading to the development of wheat allergy.

[0008] Japanese Patent Publication No. 2001-218590, Japanese Patent Publication No. Hei 9-172995, Japanese Patent Publication No. 2001-258489, Japanese Patent Publication No. 2005-198582

[0009] Yukinori Yamada et al. Hypoallergenic Wheat Line (1BS-18H) Lacking ω5-Gliadin Induces Oral Tolerance to Wheat Gluten Proteins in a Rat Model of Wheat Allergy. Foods. 2022 Jul 22;11(15):2181. doi: 10.3390 / foods11152181.Xuyang Li et al. Induction of Oral Tolerance by Pepsin-Digested Gliadin Retaining T Cell Reactivity in a Mouse Model of Wheat Allergy. Int Arch Allergy Immunol. 2020;181(6):446-455. doi: 10.1159 / 000506945. Epub 2020 Apr 16.Anne-Sofie Ravn Ballegaard et al. Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins. Mol Nutr Food Res. 2021 Dec;65(23):e2100416. doi: 10.1002 / mnfr.202100416. Epub 2021 Oct 27.

[0010] The present invention aims to provide a more effective and less dangerous method than conventional methods for preventing or treating wheat allergies.

[0011] In this specification, prevention includes primary and secondary prevention. Primary prevention means preventing sensitization in subjects who are not sensitized to wheat (mainly infants and young children), or in other words, suppressing the production of specific IgE antibodies against wheat in subjects who do not produce such antibodies. Secondary prevention means preventing the development of wheat allergy in subjects who are sensitized to wheat but have never developed a wheat allergy (subjects who have never experienced adverse reactions due to wheat exposure; mainly infants and young children). Hereafter, subjects of primary prevention may be referred to as primary prevention subjects, and subjects of secondary prevention may be referred to as secondary prevention subjects. In this specification, treatment means inducing a state of desensitization, a state of sustained unresponsiveness, or a state of acquired tolerance in subjects who have already developed a wheat allergy (who have experienced adverse reactions due to wheat exposure). Hereafter, subjects of treatment may be referred to as treatment subjects.

[0012] Inducing oral immune tolerance is a promising method for preventing or treating wheat allergy. However, when inducing oral immune tolerance, particular attention must be paid to safety. For example, for individuals targeted for secondary prevention and treatment, care must be taken to prevent the onset and induction of wheat allergy symptoms when ingesting food, while for individuals targeted for primary prevention, care must be taken to prevent transcutaneous sensitization. In particular, infants targeted for secondary prevention who have already been sensitized to wheat allergens during the weaning period are at risk of developing allergic symptoms by ingesting wheat protein or substances derived from it. Furthermore, infants targeted for primary prevention who have skin barrier problems such as atopic dermatitis are at risk of transcutaneous sensitization when wheat protein or substances derived from it come into contact with the skin during ingestion, which can trigger the development of wheat allergy in children who were not previously allergic.

[0013] While safety and high efficacy are required when inducing oral immune tolerance, the method described in Non-Patent Literature 2 makes it difficult to safely and broadly prevent and treat wheat allergies. Non-Patent Literature 2 shows that when gliadin pepsin hydrolysates are ingested, they have the ability to induce oral immune tolerance for gliadin and reduce its allergenicity. On the other hand, for food allergies caused by causative allergens other than gliadin, particularly glutenin which is present in gluten in amounts similar to gliadin, oral ingestion of gliadin pepsin hydrolysates alone cannot induce oral immune tolerance because the causative antigens are different. In other words, gliadin hydrolysates alone cannot be a means of broadly preventing and treating wheat allergies that have multiple causative antigens. Furthermore, the safety of transdermal sensitization with gliadin pepsin hydrolysates has not been confirmed.

[0014] Similarly, Non-Patent Literature 1 also states that it is difficult to safely and broadly prevent and treat wheat allergies. Non-Patent Literature 1 claims that wheat without the ω5 gliadin gene (1BS-18H) is a type of wheat that can be safely consumed by wheat allergy patients, including those with WDEIA. It has been shown that oral administration of gluten from common wheat or gluten from 1BS-18H for 5 days before sensitization tends to suppress the production of each specific IgE antibody after sensitization to ω5 gliadin or common gluten compared to the non-administered group, and that a decrease in rectal temperature does not occur when the sensitized antigen is administered after sensitization. In other words, 1BS-18 induces oral immune tolerance to common gluten. However, the allergenicity of wheat proteins other than ω5 gliadin has not been reduced, and it cannot be ruled out that it may induce symptoms in patients sensitized to wheat proteins other than ω5 gliadin. Furthermore, the safety of transdermal sensitization with 1BS-18H gluten has not been confirmed.

[0015] The present inventors have found that the above problems can be solved by using a degradation product (referred to herein as "PDG" (protease-digested gluten product)) obtained by treating general wheat gluten (not gluten lacking specific components, such as the ω5 gliadin gene-deficient wheat gluten described in Non-Patent Literature 1, but gluten containing gliadin and glutenin) with a specific food-grade proteolytic enzyme. Specifically, they have found that oral ingestion of the above degradation product induces oral immune tolerance, suppressing allergic symptoms to gluten, gliadin, or glutenin (such as the production of IgE specific to them), and that even in individuals already sensitized to wheat allergens, oral ingestion of the above degradation product suppresses allergic symptoms (such as a decrease in rectal temperature). Furthermore, they have found that even if areas with impaired skin barrier function (such as areas with skin inflammation) are exposed to the above degradation product, transcutaneous sensitization is unlikely to occur. In other words, the above degradation product can be used as an oral immune tolerance agent that combines high efficacy and low risk, thus completing the present invention. Such superior oral immunotolerance agents that combine these characteristics can only be obtained by using a limited number of preferred food-grade proteolytic enzymes, such as serine proteases and metalloproteases, from among the many types of food-grade proteolytic enzymes available. This is not the case with any food-grade proteolytic enzyme.

[0016] In other words, the present invention provides the following inventions in one aspect: [Item 1] An oral immune tolerance agent for wheat allergy comprising a gluten degradation product (PDG) by a food-grade protease. [Item 2] The oral immune tolerance agent according to Item 1, wherein the food-grade protease comprises a serine protease, a metalloprotease, or both. [Item 3] The oral immune tolerance agent according to Item 2, wherein the serine protease comprises an alkaline protease. [Item 4] The oral immune tolerance agent according to Item 2, wherein the serine protease comprises subtilisin. [Item 5] The oral immune tolerance agent according to Item 2, wherein the metalloprotease comprises basyl leucine. [Item 6] An oral composition for inducing oral immune tolerance, comprising the oral immune tolerance agent according to any one of Items 1 to 5. [Item 7] A method for producing an oral immune tolerance agent for wheat allergy, comprising the step of treating gluten with a food-grade protease. [Item 8] A method for producing an oral composition for inducing oral immune tolerance, comprising the step of incorporating gluten hydrolysate (PDG) by food-grade proteolytic enzymes into a raw material. [Item 9] A method for using gluten hydrolysate (PDG) by food-grade proteolytic enzymes as an ingredient for inducing oral immune tolerance for wheat allergy in an oral composition.

[0017] The inventions described in paragraphs [1] to [9] above can be appropriately converted into inventions of other categories. For example, in other aspects, the present invention provides "PDG used to prevent or treat wheat allergy," "the use of PDG (as an active ingredient) for the prevention or treatment of wheat allergy," "the use of PDG in the manufacture of an oral immune tolerance agent or composition for the prevention or treatment of wheat allergy," and "a method for preventing or treating wheat allergy by oral immune tolerance, comprising administering (oral ingestion of) an effective amount of PDG." Matters described herein in relation to paragraphs [1] to [9] above can also be appropriately converted in accordance with each of the above inventions. Furthermore, the invention-specific features relating to food proteases defined in paragraphs [2] to [5] which are dependent on paragraph [1] above can also be defined as invention-specific features relating to food proteases in each of the inventions described in paragraphs [6] to [9] above, or in inventions converted therefrom (dependent paragraphs are established).

[0018] The oral immune tolerance agent and oral composition containing the same of the present invention can stably induce oral immune tolerance and prevent wheat allergy (primary prevention to prevent sensitization, or secondary prevention to prevent onset) when administered to subjects who are not sensitized to wheat (no specific IgE antibodies are produced) or who are sensitized to wheat but have not developed wheat allergy (adverse reactions have not been triggered by wheat exposure), mainly infants and young children. Furthermore, the oral immune tolerance agent and oral composition containing the same of the present invention are expected to treat wheat allergy (induce a state of desensitization, a state of sustained unresponsiveness, or a state of acquired tolerance) when administered to subjects who have already developed wheat allergy (adverse reactions have been triggered by wheat exposure). The oral immune tolerance agent and oral composition containing the same of the present invention are unlikely to cause transcutaneous sensitization through skin entry, making them extremely useful for achieving an unprecedented level of both efficacy and safety. Furthermore, since the oral immune tolerance agent and oral composition containing the same of the present invention have reduced allergy symptom-inducing potential, it is expected that the risk of symptom induction is low even when ingested orally by infants and young children who have already developed (are sensitized to) wheat allergies, and therefore they can be considered highly safe.

[0019] In this invention, by using "specific food-grade proteolytic enzymes" (such as serine proteases and metalloproteases), an oral immune tolerance agent suitable for preventing wheat allergies can be obtained. This is because, for example, the degradation products obtained by treating gluten with "specific food-grade proteolytic enzymes" contain peptides of appropriate molecular weight, or the mixture of various peptides produced contains a mixture with an appropriate molecular weight distribution. In other words, the peptides (mixtures) contained in such degradation products are antigens suitable for inducing immune tolerance in Treg cells (regulatory T cells) (they possess T cell epitopes), while they do not act as antigens that cause B cells to produce IgE antibodies involved in allergic symptoms. Therefore, it is thought that their transcutaneous sensitization ability is reduced. Furthermore, they do not act as antigens against wheat protein-specific IgG antibodies, and therefore, it is thought that they suppress the induction of allergic symptoms.

[0020] In contrast, undigested gluten may act on Treg cells to induce a certain degree of immune tolerance, but it also acts on B cells, which carries the risk of causing transcutaneous sensitization, leading to the production of IgE antibodies or inducing allergic symptoms.

[0021] Furthermore, if a food-grade protease other than the "specific food-grade protease" is used, it may fail to induce immune tolerance or the immune tolerance induction may be unstable because it also degrades the T cell epitopes necessary for Treg cells. Alternatively, if a food-grade protease other than the "specific food-grade protease" is used, the resulting peptide may have a large molecular weight and act on B cells, similar to undegraded gluten, potentially causing transcutaneous sensitization, leading to IgE antibody production or inducing allergic symptoms, thus compelling safety.

[0022] However, the present invention is not unduly limited by such inferences, and even if the effects of the present invention are achieved for other reasons, the present invention is guaranteed by the description herein.

[0023] Figure 1 shows the comparative results of the preventive effect against wheat allergy in Test Example 1 (production of gluten-specific IgE [A], production of gliadin-specific IgE and glutenin-specific IgE [B]). [B] In the double box plot, the left side (dots) represents the production of gliadin-specific IgE, and the right side (shaded lines) represents the production of glutenin-specific IgE. Figure 2 shows the comparative results of the allergy-inducing ability (decreased body temperature due to anaphylactic shock) in Test Example 2. Solid line: solvent, dashed line: protein AY hydrolysate (PDG1), dotted line: orientase 10NL hydrolysate (PDG2), double dotted line: neurase hydrolysate (R-PDG2), dotted line: undegraded gluten, each representing body temperature. Figure 3 shows the comparative results of the transcutaneous sensitization ability (production of gliadin-specific IgE and glutenin-specific IgE) in Test Example 3.

[0024] - Oral Immune Tolerance Agent - The oral immune tolerance agent for the prevention or treatment of wheat allergy of the present invention (which may be referred to herein as "the oral immune tolerance agent of the present invention") comprises gluten degradation products (PDG) by a specific food protease (which may be referred to herein as "specific enzyme").

[0025] The oral immunotolerant agent of the present invention is a substance (active ingredient) that can be used for oral immunotolerance, enabling the prevention or treatment of wheat allergy by oral ingestion. The oral immunotolerant agent of the present invention may be in liquid form dissolved in a suitable solvent such as water or buffer solution, or it may be in powder or other solid form after being prepared in such liquid form and then subjected to processing such as spray drying or freeze drying to form powder. Therefore, the oral immunotolerant agent of the present invention may consist substantially of PDG according to the present invention, or it may be a mixture of PDG with a solvent to make it liquid, or an excipient to make it powder or other solid form.

[0026] (Gluten) The "gluten" used in this invention is a wheat protein fraction containing at least glutenin and gliadin. Gluten can be produced by common methods, such as removing starch from wheat flour, and is available as a product. Since gluten contained in food is often heat-denatured, for the production of the oral immunotolerance agent of this invention, it is preferable that the gluten to be broken down by food-grade protease is also heat-denatured (for example, heated in boiling water).

[0027] (Specific Enzymes) The "specific enzymes" used in this invention refer to various food-grade proteases derived from various microorganisms that are capable of producing the effects of this invention, that is, those for which oral ingestion of gluten treated with such enzymes induces immune tolerance and can prevent or treat wheat allergies.

[0028] Proteases are broadly classified into exopeptidases (sometimes simply called "peptidases"), which act on the peptide bonds at the N-terminus or C-terminus of protein molecules to cleave amino acids one residue at a time, and endopeptidases (sometimes called "proteinases"), which act on peptide bonds in protein molecules in a site-independent manner to cleave them. However, endoproteases are the appropriate term for this specific enzyme.

[0029] Proteases can be classified into serine proteases, metalloproteases (sometimes called "metalloproteases"), aspartic proteases (sometimes called "aspartate proteases"), cysteine ​​proteases (sometimes called "thiolproteases"), etc., based on their catalytic activity for cleaving peptide bonds. Various types of proteases are known within these classifications, and various bacteria are known to produce (or originate from) them. In this invention, an appropriate protease can be selected from among these and used as a specific enzyme, either alone or in combination with others.

[0030] Specific examples of serine proteases include trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1, EC 3.4.21.2), thrombin (EC 3.4.21.5), plasmin (EC 3.4.21.7), elastase (EC 3.4.21.36, EC 3.4.21.70, EC 3.4.21.71), and subtilisin (sometimes called "subtilisin" or "sachilysin") (EC 3.4.21.62). For example, "Protin SD-AY10" (Amano Enzyme Co., Ltd.) and "Orientase 22BF" (HBI Co., Ltd.) are serine proteases containing subtilisin.

[0031] Specific examples of metalloproteases include basilolisin (EC 3.4.24.28) and thermolysin (sometimes called "thermolysin" or "thermolysin") (EC 3.4.24.27). For example, "Orientase 10NL" (HBI Co., Ltd.) is a metalloprotease containing basilolisin, and "Samoase PC10F" (Amano Enzyme Co., Ltd.) is a metalloprotease containing thermolysin.

[0032] Specific examples of aspartic proteases include pepsin (EC 3.4.23.1-3), chymosin (EC 3.4.23.4), and cathepsin D (EC 3.4.23.5). For example, "Orientase AY" (HBI Co., Ltd.) is an aspartic protease.

[0033] Specific examples of cysteine ​​proteases include papain (EC 3.4.22.2), ficin (EC 3.4.22.3) (sometimes called "ficaine"), bromelain (EC 3.4.22.32), and cathepsin B (EC 3.4.22.1).

[0034] Furthermore, proteases can be classified based on the bacteria that produce them, for example, proteases produced by bacteria of the genus Bacillus, proteases produced by bacteria of the genus Aspergillus, and peptidases produced by bacteria of the genus Rhizopus. They can also be classified based on the optimal pH for enzyme action, such as acidic proteases, neutral proteases, and alkaline proteases. These classifications can also be combined to identify specific proteases. For example, "Protin SD-AY10" (Amano Enzyme Co., Ltd.) and "Orientase 22BF" (HBI Co., Ltd.) are alkaline proteases produced by Bacillus licheniformis, "Orientase 10NL" (HBI Co., Ltd.) is a neutral protease produced by Bacillus amyloliquefaciens, and "Samoase PC10F" (Amano Enzyme Co., Ltd.) is a neutral protease produced by Geobacillus stearothermophilus. Furthermore, the acidic peptidase "Neurase F3G" produced by Rhizopus niveus (Amano Enzyme Co., Ltd.), the neutral protease (endopeptidase) and peptidase (exopeptidase) "Protease P 'Amano' 3SD" produced by Aspergillus melleus (Amano Enzyme Co., Ltd.), and the protease and peptidase "Proteax" produced by Aspergillus oryzae (Amano Enzyme Co., Ltd.) also fall under the category of food-grade protein-degrading enzymes.

[0035] In one embodiment of the present invention, the specific enzyme is preferably a serine protease and a metalloprotease, and either one or both may be used in combination. A preferred serine protease is an alkaline protease containing subtilisin, such as "Protin SD-AY10" (Amano Enzyme Co., Ltd.). A preferred metalloprotease is a neutral protease containing basyllosin, such as "Orientase 10NL" (HBI Co., Ltd.).

[0036] (Degradation Products) The degradation products (PDG) of gluten by specific enzymes are presumed to contain a mixture of various peptides produced by enzymatic activity from glutenin, gliadin, and other proteins contained in gluten. The individual peptides (amino acid sequences) and amounts contained in such PDG, in other words, the composition and molecular weight distribution of the peptide mixture, may vary somewhat depending on the type, amount, and titer of the specific enzyme used to obtain the PDG, as well as the conditions of the enzymatic reaction such as pH, temperature, and time. However, such variations are acceptable as long as the effects of the present invention are achieved.

[0037] (Uses) The oral immune tolerance agent of the present invention can be taken alone (for example, as a supplement) or together with other components (for example, in the form of a composition of the present invention as described later). Alternatively, the oral immune tolerance agent of the present invention may be prepared in advance and incorporated into a composition as part of the raw materials for producing a composition of the present invention, particularly as a component for inducing oral immune tolerance.

[0038] However, the uses of the oral immune tolerance agent of the present invention are not limited to those described above, and it can also be used for various purposes related to the prevention or treatment of wheat allergy. For example, an embodiment of the oral immune tolerance agent of the present invention is the use of PDG in vitro for the purpose of analyzing interactions with cells (such as Treg cells, B cells, dendritic cells, and other immune-related cells) or antibodies (such as IgE and IgG) that may be involved in oral immune tolerance of wheat allergy.

[0039] The oral immune tolerance agent of the present invention can be administered to subjects in whom oral immune tolerance is to be induced for the prevention or treatment of wheat allergy. Such subjects may be humans or other mammals (e.g., mice, rats, rabbits, guinea pigs, gerbils, hamsters, ferrets, dogs, miniature pigs, monkeys, cattle, horses, sheep, etc.). When the subject is a human, it is preferable to administer the agent to infants and young children, for example, from around 6 months of age when they begin to eat solid food until around 1 year of age, or up to the age before elementary school when wheat allergies are likely to develop. To prevent wheat allergy, the oral immune tolerance agent of the present invention should be orally administered to subjects who are not sensitized to wheat-derived allergens, such as gliadin and glutenin. To treat wheat allergy, the oral immune tolerance agent of the present invention should be orally administered to subjects who are sensitized to these allergens.

[0040] The recommended daily intake of the oral immune tolerance agent of the present invention, the recommended intake per meal, the number of daily intakes, and the duration (number of days) of intake can be appropriately determined based on the embodiment of the oral immune tolerance agent of the present invention, the age, weight, and sex of the subject to orally administering it, indicators and attributes related to wheat allergy (such as whether the subject is sensitized to wheat-derived allergens or has developed atopic dermatitis), the expected degree of effectiveness in preventing or treating wheat allergy, and the results of non-clinical or clinical tests conducted on humans or non-human mammals.

[0041] Furthermore, when using the oral immune tolerance agent of the present invention for the treatment of wheat allergy, it is possible to implement an embodiment similar to oral immunotherapy in which, for subjects who cannot acquire tolerance early through natural means, the symptom induction threshold is confirmed by prior oral food challenge tests, and then the causative food (wheat allergen) is continuously ingested orally under the guidance of a physician to achieve a state of desensitization or persistent unresponsiveness, ultimately aiming to acquire tolerance. Alternatively, for subjects who are expected to acquire tolerance through natural means, it is possible to implement an embodiment similar to dietary guidance (dietary therapy) in which the safe intake amount is determined by oral food challenge tests, and the subject is instructed to repeatedly ingest the food at home within the limits of that amount, thereby promoting future tolerance acquisition.

[0042] The effect of the oral immune tolerance agent of the present invention on preventing or treating wheat allergy can be evaluated by, for example, the following method: (A1) As a treatment group, subjects (e.g., mice) are orally administered the oral immune tolerance agent of the present invention for a certain period of time. As a control group, subjects are orally administered a substance other than the oral immune tolerance agent of the present invention (e.g., water as a negative control, undigested gluten as a positive control). (A2) Gliadin, glutenin, or gluten containing them, which are major allergens contained in wheat, are applied to the inflamed skin of the subjects to induce transdermal sensitization. This step may be performed multiple times (e.g., three times) with a certain period of time in between, as necessary. (A3) Blood is collected from the subjects, and the concentration of antibodies (IgE) specific to gliadin, glutenin, or gluten is measured and compared between the treatment group and the control group. For example, if the antibody concentration in the treatment group tends to be lower (e.g., lower median) compared to the negative control group that received oral water intake, preferably if it is statistically significantly lower, then it can be evaluated that the oral immunotolerance agent of the present invention has been administered orally in advance to suppress the production of IgE antibodies against wheat allergens, that is, that it has an effect of preventing or treating wheat allergy.

[0043] The safety of the oral immune tolerance agent of the present invention can be evaluated from the viewpoint of the risk of causing anaphylactic shock (allergy-inducing ability) and the possibility of percutaneous sensitization (percutaneous sensitizing ability) by the following method, for example: (B1) Similar to (A2) above, wheat allergens such as gliadin and glutenin are applied to the inflamed skin of the subject to induce percutaneous sensitization. (B2) After a certain period has elapsed since sensitization, the subject is administered the oral immune tolerance agent of the present invention intraperitoneally as the treatment group. As a control group, a solvent that does not contain wheat protein (e.g., water) or as a symptom induction group, a substance other than the oral immune tolerance agent of the present invention (e.g., undegraded gluten) is administered intraperitoneally to the subject. (B3) The change in the subject's body temperature (e.g., rectal temperature) before and after administration is measured and compared between the treatment group and the symptom induction group. For example, in the symptom-induced group administered undigested gluten, body temperature decreased and the change was significant, while in the treatment group, similar to the control group where body temperature did not change much, the change in body temperature before and after administration tended to be small. Preferably, if the change is statistically small, then oral ingestion of the oral immune tolerance agent of the present invention does not cause changes (decrease) in body temperature due to anaphylaxis, meaning that the risk due to allergy-inducing ability is low. (C1) As the treatment group, the oral immune tolerance agent of the present invention is applied to the inflamed skin area of ​​the subject for a certain period of time. This step may be performed multiple times with a certain period of time in between as necessary. As the control group, a substance other than the oral immune tolerance agent of the present invention (e.g., water, undigested gluten) is applied to the inflamed skin area of ​​the subject for a certain period of time. (C2) Blood is collected from the subjects, and the concentration of antibodies (IgE) specific to wheat allergens such as gliadin and glutenin is measured and compared between the treatment group and the control group. For example, if the antibody concentration in the treatment group is similar to that of the control group treated with water, preferably without statistically significant difference, then even if the oral immunotolerance agent of the present invention is exposed to the skin, antibodies against wheat allergens such as gliadin and glutenin are unlikely to be produced, meaning that the risk due to transdermal sensitization is low.Alternatively, if the antibody concentration in the treatment group tends to be lower (e.g., the median is lower) than that in the group applied with undegraded gluten, and is preferably statistically significantly lower, it can be evaluated that even when the oral tolerogen of the present invention is exposed to the skin, antibodies against wheat allergens such as gliadin and glutenin are less likely to be produced, that is, the risk due to transcutaneous sensitization ability is low.

[0044] For more specific embodiments of the various evaluation methods described above, reference can be made to the examples of the present specification. Based on the results of such evaluation methods, it is possible to investigate various conditions in the specific enzyme treatment step for producing a preferred embodiment of the present invention, for example, an oral tolerogen, and the method of administering the oral tolerogen to a subject, that is, the amount per dose, the duration of administration, and the like.

[0045] -Method for Producing Oral Tolerogen- The method for producing an oral tolerogen of the present invention comprises at least a step of treating gluten with a specific food-grade proteolytic enzyme (sometimes referred to as a "specific enzyme treatment step" in the present specification).

[0046] (Specific Enzyme Treatment Process) The specific enzyme treatment process can be designed to produce PDG that exhibits the effects of the present invention by using an appropriate amount of the selected specific enzyme according to its type and titer, and by performing the enzymatic treatment at an appropriate temperature, pH, and time according to that specific enzyme. For food-grade proteolytic enzyme products, the titer of the enzyme (protein-degrading power), the range of temperature and pH suitable (optimal or stable) for the enzymatic reaction (change in activity due to temperature and pH), the range of standard reaction time (change in the amount of protein degraded due to reaction time), the range of standard usage amount (mass ratio of enzyme to protein), and other information regarding the enzymatic treatment are made public in documents such as attached instruction manuals and catalogs. Furthermore, for food-grade proteolytic enzymes other than products, the temperature, pH, reaction time, usage amount, etc. suitable for the enzymatic reaction can be determined by conducting tests as appropriate. Those skilled in the art can obtain gluten degradation products exhibiting the effects of the present invention without excessive trial and error by selecting an appropriate food-grade proteolytic enzyme based on the descriptions herein (specific embodiments disclosed by the general description and examples) and publicly known information regarding various food-grade proteolytic enzymes, and by performing a specific enzyme treatment process under appropriate conditions.

[0047] The molecular weight distribution of the peptide mixture can be measured under appropriate conditions using a well-known and conventional method, such as size exclusion chromatography (SEC). If necessary, for example, after obtaining a PDG exhibiting the effects of the present invention by one embodiment of the specific enzyme treatment step, its molecular weight distribution can be measured, and the above-mentioned conditions related to the specific enzyme treatment step can be changed to create another embodiment of the specific enzyme treatment step so that a PDG with a similar molecular weight distribution can be obtained.

[0048] When the product "Protin SD-AY10" (Amano Enzyme Inc.), which is a serine protease (and alkaline protease) containing subtilisin, is used as the specific enzyme, the specific enzyme treatment step can be designed based on the following known information about the product. For more specific embodiments, reference may be made to the examples described later. ・The optimum pH is 10.0 to 11.0, and the enzyme is stable between pH 5.5 and 9.0. ・The optimum temperature is 70°C, and the enzyme is stable at 50°C or lower. ・The specified activity (Folin method, pH 8.0) is 80,000 μ / g or more.

[0049] When the product "Orientase 10NL" (HBI Inc.), which is a metalloprotease (and neutral protease) containing bacillolysin, is used as the specific enzyme, the specific enzyme treatment step can be designed based on the following known information about the product. For more specific embodiments, reference may be made to the examples described later. ・The pH is usually 6 to 8. ・The temperature is usually 50 to 60°C. ・The reaction time is usually 0.5 to 5 hours. ・The specified activity is 100,000 units / g. ・The enzyme is usually added in an amount of 0.25 to 1% relative to the protein. ・Since the present enzyme is stabilized by calcium ions, it is effective to add approximately 0.03% of calcium chloride to the treatment solution.

[0050] The specific enzyme treatment step can be carried out, for example, by the following procedure: (1) Add an appropriate amount of gluten to an appropriate solvent (e.g., water) to suspend the gluten, and heat the suspension (e.g., boil it) if necessary. (2) Add an acid (e.g., HCl) or an alkali (e.g., NaOH) to the above suspension, adjust the pH to an appropriate value according to the specific enzyme, then adjust (heat) the temperature to an appropriate value according to the specific enzyme, hold for an appropriate time, and allow the gluten to react with the specific enzyme. (3) Adjust (heat, for example, boil) the above suspension to a temperature suitable for inactivating the specific enzyme, and hold at that temperature for an appropriate time.

[0051] (Optional steps) The method for producing the oral immunotolerant of the present invention may include steps other than the specific enzyme treatment step, if necessary. Examples of such optional steps include a spray-drying step and a freeze-drying step for powdering PDG. The conditions for the spray-drying step, freeze-drying step, etc., can be appropriately adjusted to obtain PDG with the desired properties.

[0052] -Composition- The composition of the present invention is for the prevention or treatment of wheat allergy, to be taken orally for oral immune tolerance, in other words, when taken orally, it exerts an effect for the prevention or treatment of wheat allergy by oral immune tolerance, and comprises the oral immune tolerance agent of the present invention, namely, at least a breakdown product (PDG) of gluten by a food protease (specific enzyme).

[0053] In one embodiment of the present invention, the composition of the present invention is a food composition or can be considered a food composition. For example, foods, beverages, food and beverage additives, supplements, or, in the case of animals other than humans, feed, feed additives, etc., are included in the food composition. Such a food composition contains at least one food ingredient, food additive, or other component depending on each form other than the oral immune tolerance agent of the present invention. When the oral immune tolerance agent of the present invention is incorporated into a food composition, for example, when PDG is powdered, additives such as colorants, flavorings, and sweeteners may be added as needed to prepare it as a powdered oral immune tolerance agent.

[0054] The types and embodiments of the food composition are not particularly limited and can be the same as those of general foods. Specific examples of such food compositions include soft drinks (juices, coffee drinks, tea drinks, mineral water, sports drinks, etc.), milk drinks, soy milk drinks, fermented milk (yogurt), lactic acid bacteria drinks, cocoa, drinks prepared from instant powders, jelly, pudding, confectionery (biscuits, cookies, chocolate, etc.), bread, cereals, prepared foods, and seasonings (mayonnaise, dressings, etc.). Furthermore, it is preferable that the food composition be in the form of baby food suitable for consumption by infants. For example, it is preferable to manufacture the various foods exemplified above as wet type (liquid or semi-solid foods such as retort foods or bottled foods) or dry type (powdered, granular, flakey, or solid foods that are eaten after adding water or hot water to return to their original form).

[0055] The amount of the oral immune tolerance agent (PDG) of the present invention in the food composition is not particularly limited, as long as it is within a range that prevents or treats wheat allergy by ingesting the food composition. Such an amount can be determined by considering the amount of PDG (oral immune tolerance agent as an active ingredient) per serving or per day, the weight of the food composition to be ingested, and also taking into account the form and manufacturing method of the food composition as appropriate. The amount of PDG per serving or per day in relation to the food composition is as described above in relation to the oral immune tolerance agent of the present invention.

[0056] In one embodiment of the present invention, the composition of the present invention is or can be considered as an orally ingested composition. The orally ingested composition may be a product that can or can display a functional claim relating to the prevention or treatment of wheat allergy, in accordance with the legal system of the country in which the present invention is implemented, for example, a product called a health functional food, or it may be a product that cannot or does not display a functional claim, for example, an ordinary food. Alternatively, the orally ingested composition may be considered a food, a pharmaceutical, or equivalent to a pharmaceutical, in accordance with the legal system of the country in which the present invention is implemented.

[0057] With respect to the compositions of the present invention (food compositions, orally ingested compositions, etc.), if it is permissible to display the uses, effects, and functionality related to the treatment or prevention of wheat allergy, such displays may be made directly or indirectly. Direct displays include, for example, inscriptions on tangible objects such as the product itself, packaging, containers, labels, and tags. Indirect displays include, for example, advertising and promotional activities through places or means such as websites, stores, exhibitions, signs, bulletin boards, newspapers, magazines, television, radio, mail, and email. Furthermore, as long as the embodiments actually produce effects that contribute to the prevention or treatment of wheat allergy, products that do not explicitly claim such uses, effects, or functionality can be considered to fall under the composition of the present invention.

[0058] -Method for Producing the Composition- The method for producing the composition of the present invention includes a step of incorporating an oral immune tolerance agent, i.e., a gluten degradation product (PDG) by a food-grade protease (specific enzyme) into the raw materials of the composition. Such a production method can be similar to conventional methods for producing compositions, or an embodiment in which the steps and conditions are appropriately adjusted as necessary, depending on the embodiment of the composition (e.g., a food composition), except for the use of PDG as one of the raw materials. The oral immune tolerance agent (PDG) can be prepared in advance by the method described above herein and stored until it is used for the production of the composition.

[0059] [Example 1] Preparation of gluten hydrolysate (PDG1) using "Protin AY" In Example 1, "Protin SD-AY10" (Amano Enzyme Co., Ltd.), a serine protease (and alkaline protease) containing subtilisin, was used as the food-grade protease (abbreviated as "Protin AY"). Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Sodium hydroxide was added to the obtained heated gluten suspension to adjust the pH to 9.0. 0.1 wt% of Protin AY was added to the obtained gluten suspension and held at 50°C for 180 minutes. The obtained gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The PDG1 powder obtained in this way using Example 1 (Protin AY) was suspended in a solvent and used in the test examples described below.

[0060] [Example 2] Preparation of gluten hydrolysate (PDG2) using "Orientase 10NL" In Example 2, "Orientase 10NL" (HBI Co., Ltd.), a metalloprotease (and neutral protease) product containing basyl leucine, was used as the food-grade protease. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Sodium hydroxide was added to the resulting heated gluten suspension to adjust the pH to 7.0. 0.1 wt% Orientase 10NL was added to the resulting gluten aqueous solution and held at 50°C for 180 minutes. The resulting gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The PDG2 powder obtained in this way using Example 2 (Orientase 10NL) was suspended in a solvent and used in the test examples described below.

[0061] [Reference Example 1] Preparation of gluten hydrolysate (R-PDG1) using "pepsin" In Reference Example 1, "Pepsibio" (Nippon Biocon Co., Ltd.), a pepsin derived from porcine gastric mucosa (referred to as "pepsin"), was used as the food-grade proteolytic enzyme. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Hydrochloric acid was added to the resulting heated gluten suspension to adjust the pH to 2.0. 0.1 wt% pepsin was added to the resulting gluten suspension and held at 40°C for 180 minutes. The resulting gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The R-PDG1 powder obtained in this way using Reference Example 1 (pepsin) was suspended in a solvent and used in the test examples described below.

[0062] [Reference Example 2] Preparation of gluten hydrolysate (R-PDG2) using "Neurase" In Reference Example 2, the acid peptidase "Neurase F3G" (Amano Enzyme Co., Ltd.) (abbreviated as "Neurase") was used as the food-grade proteolytic enzyme. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Hydrochloric acid was added to the resulting heated gluten suspension to adjust the pH to 3.0. 0.1 wt% Neurase was added to the resulting gluten suspension and held at 40°C for 180 minutes. The resulting gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The R-PDG2 powder obtained in this way using Reference Example 2 (Neurase) was suspended in a solvent and used in the test examples described below.

[0063] [Reference Example 3] Preparation of gluten hydrolysate (R-PDG3) using "Samoase" In Reference Example 3, the neutral protease "Samoase PC10F" (Amano Enzyme Co., Ltd.) (abbreviated as "Samoase") was used as the food-grade protein-degrading enzyme. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Sodium hydroxide was added to the obtained heated gluten suspension to adjust the pH to 8.0. 0.1 wt% samoase was added to the obtained gluten suspension and held at 65°C for 180 minutes. The obtained gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The R-PDG3 powder obtained in this way using Reference Example 3 (Samoase) was suspended in a solvent and used in the test examples described below.

[0064] [Reference Example 4] Preparation of gluten hydrolysate (R-PDG4) using "Orientase AY" In Reference Example 4, the acidic protease "Orientase AY" (HBI Co., Ltd.) was used as the food-grade protein-degrading enzyme. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Hydrochloric acid was added to the resulting heated gluten suspension to adjust the pH to 3.0. 0.1 wt% Orientase AY was added to the resulting gluten suspension and held at 50°C for 180 minutes. The resulting gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The R-PDG4 powder obtained in this way using Reference Example 4 (Orientase AY) was suspended in a solvent and used in the test examples described below.

[0065] [Reference Example 5] Preparation of gluten hydrolysate (R-PDG5) using "Proteax" In Reference Example 5, the protease and peptidase "Proteax" (Amano Enzyme Co., Ltd.) was used as the food-grade protein-degrading enzyme. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Sodium hydroxide was added to the resulting heated gluten suspension to adjust the pH to 8.0. 0.1 wt% of Proteax was added to the resulting gluten suspension and held at 60°C for 180 minutes. The resulting gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The R-PDG5 powder obtained in this way using Reference Example 5 (Proteax) was suspended in a solvent and used in the test examples described below.

[0066] [Reference Example 6] Preparation of gluten hydrolysate (R-PDG6) using "Protease P" In Reference Example 6, the neutral protease and peptidase "Protease P 'Amano' 3SD" (Amano Enzyme Co., Ltd.) (abbreviated as "Protease P") was used as the food-grade protein-degrading enzyme. Gluten powder was added to pure water to prepare a gluten suspension with a concentration of 20 mg / mL. The gluten suspension was heated in boiling water and held for 10 minutes. Sodium hydroxide was added to the obtained heated gluten suspension to adjust the pH to 7.0. 0.1 wt% of Protease P was added to the obtained gluten suspension and held at 40°C for 180 minutes. The obtained gluten hydrolysate suspension was heated and boiled for 15 minutes to inactivate the enzyme, and then freeze-dried to obtain a powder. The R-PDG6 powder obtained in this way using Reference Example 6 (Protease P) was suspended in a solvent and used in the test examples described below.

[0067] [Test Example 1] Comparative test animals (BALB / c strain mice, female, 3-4 weeks old) were orally administered 2 mg each of PDG from Examples 1-2 and Reference Examples 1-6 for 5 days to compare the preventive effect of PDG on wheat allergy (production of gluten sIgE, production of gliadin and glutenin sIgE) using PDG from each example and reference example (days 18-14). One week after the first day of oral administration, the test animals were orally administered 2 mg each of PDG for 5 days again (days 11-7). One week later (day 0), the hair on the backs of the test animals was shaved, depilated with cream, and then the procedure of applying and removing tape was repeated three times to induce skin inflammation. 100 μg each of gliadin and glutenin were applied to the inflamed skin areas of the test animals, wrapped with bandages and left for 3 days, then the bandages were removed and left for 11 days. This cycle was repeated three times (day 0-day 9, day 14-day 23, day 28-day 37). Blood was collected on day 38 and the blood concentration of gluten sIgE antibodies was measured. The control group was administered orally with water or 2 mg of gluten (undigested product) instead of PDG using each enzyme, in the same manner.

[0068] The results are shown in Figures 1[A] and [B]. As can be seen from [A], oral administration of PDG1 from Example 1, PDG2 from Example 2, R-PDG2 from Reference Example 2, and R-PDG6 from Reference Example 6 suppressed gluten sIgE production more than oral administration of water (negative control). On the other hand, oral administration of R-PDG1 from Reference Example 1, R-PDG3 from Reference Example 3, R-PDG4 from Reference Example 4, and R-PDG5 from Reference Example 5 did not suppress gluten sIgE production as much as the negative control. Furthermore, as can be seen from [B], with regard to gliadin sIgE and glutenin sIgE, oral administration of PDG1 from Example 1, PDG2 from Example 2, and R-PDG2 from Reference Example 2 suppressed the production of gliadin sIgE and glutenin sIgE compared to oral administration of water (negative control). On the other hand, oral administration of the PDGs from the other Reference Examples did not suppress the production of these sIgEs to the same extent, indicating that the effect of PDG differed depending on the food-grade proteolytic enzyme used.

[0069] Based on the results of Test Example 1, PDG1 from Example 1, PDG2 from Example 2, and R-PDG2 from Reference Example 2 are expected to have some effect in preventing wheat allergy through oral immune tolerance, and therefore will be used in further tests. In addition, unhydrolyzed gluten (control, conventional technology) will also be used in further tests.

[0070] [Test Example 2] Evaluation of allergy-inducing ability (hyperthermia due to anaphylactic shock) The fur on the backs of test animals (BALB / c strain mice, female, 6 weeks old) was shaved, the fur was removed with cream, and then the procedure of applying and removing tape was performed three times to induce skin inflammation. 100 μg each of gliadin and glutenin were applied to the inflamed skin, and a bandage was wrapped around it and left for 3 days, after which the bandage was removed and left for 11 days. This cycle was repeated three times (day 0 to day 9, day 14 to day 23, day 28 to day 37). Blood was collected on day 38 and the blood concentrations of gliadin sIgE antibody and glutenin sIgE antibody were measured. Seven days later (day 45), the test animals were intraperitoneally administered water or undegraded gluten (control), or PDG1 from Example 1, PDG2 from Example 2, or R-PDG2 from Reference Example 2, each at a dose of 2 mg / kg (intraperitoneal loading test). Body temperature (rectal temperature) was measured at the time of administration and every 10 minutes thereafter for 60 minutes, and the change in body temperature was calculated.

[0071] The results are shown in Figure 2. When the subjects (experimental animals) were already sensitized with gliadin and glutenin, ingestion of undigested gluten or R-PDG2 from Reference Example 2 resulted in a decrease in body temperature due to allergic reaction (anaphylactic shock). However, ingestion of PDG1 from Example 1 or PDG2 from Example 2 resulted in a milder decrease in body temperature and a shorter recovery time, indicating a reduction in allergic inducing ability. These results suggest that even among PDG and undigested gluten, which can suppress the production of IgE specific to gluten, gliadin, or glutenin, there are differences in the degree of reduction in allergic inducing ability, i.e., the degree of safety.

[0072] [Test Example 3] Evaluation of transcutaneous sensitization ability: The fur on the backs of test animals (BALB / c strain mice, female, 6 weeks old) was shaved, the fur was removed with cream, and then the procedure of applying and removing tape was performed three times to induce skin inflammation. Water or undegraded gluten (control), or PDG1 from Example 1, PDG2 from Example 2, or R-PDG2 from Reference Example 2 were applied to the inflamed areas. After wrapping with a bandage and leaving it for 3 days, the bandage was removed and left for 11 days. This cycle was repeated three times (day 0 to day 9, day 14 to day 23, day 28 to day 37). Blood was collected on day 38 and the blood concentrations of gliadin sIgE antibody and glutenin sIgE antibody were measured.

[0073] The results are shown in Figures 3[A] and [B]. When PDG1 from Example 1, PDG2 from Example 2, or R-PDG2 from Reference Example 2 were used for transdermal sensitization, the production of both gliadin sIgE antibody [A] and glutenin sIgE antibody [B] was almost negligible, comparable to that of water. On the other hand, when undigested gluten was used for transdermal sensitization, the production of both gliadin sIgE antibody [A] and glutenin sIgE antibody [B] increased significantly. These results indicate that even among PDGs and undigested gluten products, which can suppress the production of IgE specific to gluten, gliadin, or glutenin, there are differences in the degree of reduction in transdermal sensitization ability, i.e., the degree of safety.

[0074] Based on the results of the above Test Examples 1 to 3, PDG1 in Example 1 and PDG2 in Example 2 are expected to be effective in preventing or treating wheat allergy through oral immune tolerance, and are highly safe with reduced allergenicity and transcutaneous sensitization potential, demonstrating their potential as excellent oral immune tolerance agents. On the other hand, R-PDG2 in Reference Example 2 shows expected effects through oral immune tolerance and reduced transcutaneous sensitization potential, but has high allergenicity and cannot be said to be sufficiently safe as an oral immune tolerance agent. Furthermore, the unhydrolyzed gluten used as a control may show some effect through oral immune tolerance, but retains high allergenicity and transcutaneous sensitization potential, making it highly dangerous as an oral immune tolerance agent. The present invention demonstrates that by using a specific food-grade proteolytic enzyme selected from a large number of candidates, it is possible to manufacture an oral immune tolerance agent that balances usefulness as an oral immune tolerance agent with safety regarding allergenicity and transcutaneous sensitization potential.

Claims

1. An oral immunoreceptor for wheat allergy containing gluten breakdown products by food-grade proteolytic enzymes.

2. The oral immunotolerance agent according to claim 1, wherein the food protease comprises a serine protease, a metalloprotease, or both.

3. The oral immunotolerance agent according to claim 2, comprising an alkaline protease as the serine protease.

4. The oral immunotolerance agent according to claim 2, comprising subtilisin as the serine protease.

5. The oral immunotolerance agent according to claim 2, comprising basilolicin as the metalloprotease.

6. An oral composition for inducing oral immune tolerance, comprising an oral immune tolerance agent according to any one of claims 1 to 5.

7. A method for producing an oral immunoreceptor for wheat allergy, comprising the step of treating gluten with a food-grade proteolytic enzyme.

8. A method for producing an oral composition for inducing oral immune tolerance, comprising the step of incorporating a hydrolyzed product of gluten using a food-grade protease into the raw materials.

9. A method for using a food-grade protein-degrading product of gluten, which is incorporated into an oral composition as an ingredient for inducing oral immune tolerance for wheat allergy.

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