Compositions and methods for preventing food allergies

Abstract

The present disclosure provides compositions and methods useful for treating and / or preventing food allergies in subjects in need thereof. The compositions include allergenic proteins, at least some of which are naturally folded and some of which are partially unfolded. The partially unfolded allergenic proteins include one or more T cell epitopes and are substantially free of IgE epitopes and / or B cell epitopes. Food compositions that include a mixture of naturally folded and T cell epitope-containing proteins can optimally balance reduced immunogenicity with full antigenic properties. Furthermore, by preserving foods in as natural a form as possible, they induce an overall tolerance training effect.

Description

[Technical field]

[0001]

[0001] This application is directed generally to compositions and methods for altering an allergic response to a food protein in a subject in need thereof. [Background technology]

[0002]

[0002] Food allergies and other types of food hypersensitivity occur when the body's immune system reacts to certain proteins in food. Food allergic reactions vary in severity from mild symptoms including hives and lip swelling to severe, life-threatening symptoms, often called anaphylaxis, which can include fatal breathing problems and shock. The causes of food allergies are poorly understood. Although promising prevention and treatment strategies are being evaluated, food allergies cannot currently be cured.

[0003]

[0003] Therefore, there is a need for new classes of dietary proteins with improved safety profiles. Summary of the Invention [Means for solving the problem]

[0004]

[0004] Provided herein are compositions and methods for treating and / or preventing food allergies in a subject in need thereof, the compositions comprising an allergenic protein, at least a portion of which is natively folded and a portion of which is partially unfolded.

[0005]

[0005] In some embodiments, the present disclosure provides a food composition comprising an allergenic protein, wherein 1%-99% of the allergenic protein in the composition is partially unfolded, the partially unfolded allergenic protein comprises one or more T cell epitopes and is substantially free of IgE epitopes and / or B cell epitopes, and at least 1% of the allergenic protein in the composition is natively folded. In some embodiments, 1%-25%, 25%-50%, 50%-75%, or 75%-99% of the allergenic protein is partially unfolded.

[0006] In some embodiments, the allergenic protein is from peanut, egg, milk, wheat, almond, cashew, pistachio, hazelnut, walnut, pecan, Brazil nut, soybean, oat, pea, cod, salmon, shrimp, or sesame. In some embodiments, the allergenic protein is from peanut and is Ara h 1, Ara h 2, Ara h 3, Ara h 6, or Ara h 9. In some embodiments, the allergenic protein is from egg and is Gal d 1, Gal d 2, Gal d 3, Gal d 4, or Gal d 5. In some embodiments, the allergenic protein is from milk and is Bos d 5 or Bos d 11. In some embodiments, the allergenic protein is from walnut and is Jug r1 or Jug r3. In some embodiments, the allergenic protein is from pistachio and is Pis v 1, Pis v 2, Pis v 3, or Pis V 4. In some embodiments, the allergenic protein is from cashew and is AnaO3. In some embodiments, the allergenic protein is from hazelnut and is Cor a 9, Cor a 14, Cor a 1, or Cor a 8. In some embodiments, the allergenic protein is from pecan and is Car i 1. In some embodiments, the allergenic protein is from almond and is Pru du 1, 2, 3, 4, 5, 6, or 8. In some embodiments, the allergenic protein is from Brazil nut and is Ber e1 or e2. In some embodiments, the allergenic protein is from wheat and is Tri a 19 or Tri a 14. In some embodiments, the allergenic protein is from soybean and is Gly m Bd 60K, Gly m Bd 30K, or Gly m Bd 28K. In some embodiments, the allergenic protein is from pea and is Pis s 1 or Pis s 2.In some embodiments, the allergenic protein is from cod and is Gad m 1, Gad m 2, or Gad m 3. In some embodiments, the allergenic protein is from salmon and is Sal s 1, Sal s 3, Sal s 2, or Onc k 5. In some embodiments, the allergenic protein is from shrimp and is Met e 1, Pan s 1, or Hom a 1. In some embodiments, the allergenic protein is from oat and is avenin.

[0007] In some embodiments, the food composition comprises one or more fats, fibers, and / or vitamins. In some embodiments, the food composition comprises butyrate. In some embodiments, the food composition comprises one or more non-allergenic proteins. In some embodiments, the food composition comprises at least one probiotic, such as bacteria of the genus Lactobacillus or Bifidobacterium. In some embodiments, the food composition comprises a prebiotic, such as an oligosaccharide.

[0008] In some embodiments, the food composition is a powder, a puree, a paste, or a beverage. In some embodiments, the food composition is a puff, a cracker, or a biscuit.

[0009] Also provided is a method for partially unfolding allergenic proteins in a food composition, comprising the steps of treating the food composition using i) heat treatment, ii) instantaneous controlled pressure drop, iii) pepsination, iv) hydrolysis, v) acid or base treatment, and / or vi) fermentation, wherein the treatment reduces the amount of natively folded allergenic proteins in the food composition by at least 50%, and the partially unfolded allergenic proteins comprise one or more T cell epitopes and are substantially free of IgE epitopes and / or B cell epitopes. In some embodiments, the treatment reduces the amount of natively folded allergenic proteins in the food composition by at least 75%, at least 90%, at least 95%, or at least 99%. In some embodiments, 1% to 99% of the allergenic proteins in the food composition are partially unfolded after treatment.

[0010] In some embodiments, the food composition is freeze-dried. In some embodiments, the food composition is a wet composition that is mixed with starch prior to processing.

[0011] In some embodiments, the heat treatment includes boiling or treatment with superheated steam. In some embodiments, the boiling or steam treatment is for about 1-5 minutes, about 5-10 minutes, about 10-20 minutes, about 20-30 minutes, or more than 30 minutes. In some embodiments, the heat treatment includes baking or toasting. In some embodiments, the baking or toasting is for about 1-5 minutes, about 5-10 minutes, about 10-20 minutes, about 20-30 minutes, or more than 30 minutes.

[0011]

[0012] Also provided is a method of treating and / or preventing food allergies in a subject in need thereof, comprising administering to the subject a food composition of the present disclosure. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is 18 months of age or younger. In some embodiments, the subject is 12 months of age or younger. In some embodiments, the subject is 6 months of age or younger. In some embodiments, the food composition is administered orally.

[0012]

[0013] Also provided are kits comprising a container comprising a food composition described herein. In some embodiments, the container is a sachet, bag, or tube. In some embodiments, the container is a spoon. In some embodiments, the container comprises a single dose of the food composition. In some embodiments, the container comprises a multiple dose of the food composition. In some embodiments, the container further comprises a second food composition comprising a second allergenic protein. In some embodiments, the kit comprises a second container. In some embodiments, the second container comprises a second allergenic protein.

[0013]

[0014] In some embodiments, the kit comprises a first container and a second container, and the amount of the partially denatured allergenic protein in the first container is different from the amount of the partially denatured allergenic protein in the second container. In some embodiments, the kit comprises a first container and a second container, and the amount of the partially denatured allergenic protein in the first container is the same as the amount of the partially denatured allergenic protein in the second container. In some embodiments, the kit comprises a first container, a second container, a third container, and a fourth container, and the amount of the partially denatured allergenic protein is different in each container. In some embodiments, the kit comprises a first container, a second container, a third container, and a fourth container, and the amount of the partially denatured allergenic protein is the same in each container.

[0014]

[0015] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, examples, and accompanying figures.

[0016] Various objects and advantages of the present disclosure as well as a more complete understanding will become apparent and will be more readily understood by reference to the following detailed description and the appended claims, when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0015] [Figure 1]

[0017] FIG. 1 is a schematic showing the modification of a whole food composition (left of arrow) to produce a composition comprising a mixture of natively folded and partially unfolded proteins. [Figure 2A]

[0018] Figures 2A-2B show the levels of natively folded Gal d 1, 2 (Fig. 2A) and Bos d 5, 11 (Fig. 2B) after toasting for 5 or 10 minutes. [Figure 2B]

[0018] Figures 2A-2B show the levels of natively folded Gal d 1, 2 (Figure 2A) and Bos d 5, 11 (Figure 2B) after toasting for 5 or 10 minutes. [Figure 3A]

[0019] 3A-3B show the levels of natively folded Gal d 1, 2 (FIG. 3A) and Bos d 5, 11 (FIG. 3B) after boiling for 20, 30, or 30 min. [Figure 3B] 3A-3B show the levels of natively folded Gal d 1, 2 (FIG. 3A) and Bos d 5, 11 (FIG. 3B) after boiling for 20, 30, or 30 min. [Figure 4]

[0020] FIG. 4 shows the levels of natively folded Gal d 1, 2 after baking for 10, 20 or 30 min. [Figure 5A]

[0021] Figures 5A-5B show the levels of natively folded Gal d 1, 2 (Figure 5A) and Bos d 5, 11 (Figure 5B) after boiling for 10 min followed by toasting for 5 or 10 min. [Figure 5B]

[0021] Figures 5A-5B show the levels of natively folded Gal d 1, 2 (Figure 5A) and Bos d 5, 11 (Figure 5B) after boiling for 10 min followed by toasting for 5 or 10 min. [Figure 6]

[0022] FIG. 1 presents the upper and lower "x" choice points for the range of the percentage of weak bonds cleaved relative to all weak bonds in a given allergenic protein. [Figure 7]

[0023] FIG. 1 provides the upper and lower "y" choice points for the range of the percent amount by weight of a given allergenic protein relative to the total weight of all proteins in a food composition. [Figure 8]

[0024] FIG. 13. Effect of boiling time on peanut protein structure at acidic pH (initial pH 3.04) by circular dichroism. [Figure 9]

[0025] FIG. 13. Effect of boiling time on peanut protein structure at neutral pH (initial pH 7) by circular dichroism. [Figure 10]

[0026] FIG. 13. Effect of boiling time on peanut protein structure at basic pH (initial pH 8.02) by circular dichroism. [Figure 11]

[0027] FIG. 2: Effect of treatment on the allergenicity of protein Ara h1 (acidic=pH approx. 3, basic=pH approx. 8). [Figure 12]

[0028] FIG. 1 shows the effect of treatment on the allergenicity of protein Ara h3 (acidic=pH approx. 3, basic=pH approx. 8). [Figure 13]

[0029] FIG. 1 shows the effect of treatment on the allergenicity of protein Ara h6 (acidic=pH approx. 3, basic=pH approx. 8). [Figure 14]

[0030] FIG. 13. Effect of boiling time on almond protein structure at acidic pH (initial pH 3.04) by circular dichroism. [Figure 15]

[0031] FIG. 13. Effect of boiling time on almond protein structure at neutral pH (initial pH 7) by circular dichroism. [Figure 16]

[0032] FIG. 13. Effect of boiling time on almond protein structure at basic pH (initial pH 8.02) by circular dichroism. [Figure 17]

[0033] FIG. 1 shows the effect of treatment on the allergenicity of protein Pru du 6 (acidic=pH approx. 3, basic=pH approx. 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016]

[0034] The present disclosure provides compositions and methods useful for treating and / or preventing food allergies in subjects in need thereof. The compositions include allergenic proteins, at least some of which are naturally folded and some of which are partially unfolded. Food compositions containing a mixture of naturally folded and partially unfolded proteins, as shown in FIG. 1, can optimally balance reduced immunogenicity with full antigenic properties. Furthermore, by preserving foods in as natural a form as possible, they induce an overall tolerance training effect.

[0017]

[0035] The use of a composition comprising a mixture of partially unfolded and naturally folded allergenic proteins offers distinct advantages compared to methods for preventing and / or treating food allergies known in the art. The only current method for preventing food allergies, also known as "early allergen introduction", uses a composition comprising only naturally folded (i.e. wild-type) allergenic proteins, which elicits a large immunogenic response. Such compositions cause high IgE activation. The use of isolated hypoallergenic proteins known in the art, which have very low levels of IgE activation, is less desirable for preventing and / or treating food allergies, because these proteins do not provide the context in which humans naturally encounter the proteins (i.e. fat, fiber, nutrients). T cell epitope-containing peptides lack IgE and IgG epitopes and do not provide the natural context. In contrast, the compositions provided herein comprise partially unfolded allergenic proteins that contain one or more T cell epitopes and IgG epitopes, and are substantially free of IgE and / or B cell epitopes. Without wishing to be bound by any theory, it is believed that the fact that these proteins are substantially devoid of IgE and / or B cell epitopes, but retain T cell and IgG epitopes, and retain the context of the natural protein, helps induce its immune tolerance, prophylactically and / or therapeutically. Thus, the disclosed compositions have a reduced immunogenic profile compared to known food supplements in the art, and have an antigenic or tolerogenic profile that more safely and effectively prevents and / or treats food allergies.

[0018]

[0036] The present invention is described more fully below with reference to exemplary, non-limiting embodiments and the accompanying figures. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth below. Rather, these embodiments are provided so that this disclosure will be thorough, and will convey the scope of the invention to those skilled in the art.

[0019] definition

[0037] Unless otherwise defined in this disclosure, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature used in connection with, and techniques of, food science, chemistry, molecular biology, cell biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described in this disclosure are those well known and commonly used in the art.

[0020]

[0038] As used in this disclosure, the following terms have the meanings ascribed to them unless specified otherwise.

[0039] The articles "a," "an," and "the" are used in this disclosure to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021]

[0040] The use of the alternative (eg, "or") should be understood to mean either one, both, and any combination thereof of the alternatives.

[0041] The term "and / or" should be understood to mean either or both of the alternatives.

[0022]

[0042] As used in this disclosure, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In some embodiments, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0023]

[0043] As used in this disclosure, the term "isolated" refers to material that is substantially or essentially free from components that normally accompany it in its natural state. In some embodiments, the terms "obtained" or "derived" are used interchangeably with isolated.

[0024]

[0044] As used herein, a "subject," "individual," or "patient" includes any animal that can consume or use the compositions contemplated herein. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals (e.g., horses, cows, sheep, pigs), and domestic animals or pets (e.g., cats or dogs). In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is a pediatric human. In some embodiments, the pediatric subject is 18 months of age or younger. In some embodiments, the pediatric subject is 12 months of age or younger. In some embodiments, the pediatric subject is 6 months of age or younger.

[0025]

[0045] As used herein, the term "allergenic protein" refers to a protein capable of eliciting an allergic response in a subject susceptible thereto. An allergic response is a vigorous immune reaction in which the immune system fights off a threat otherwise perceived as harmless to the body. An allergic response may be characterized by the production of antibodies, including IgE antibodies, against the allergenic protein. Symptoms of an allergic reaction may include allergic rhinitis (e.g., sneezing and itchy, runny, or stuffy nose), conjunctivitis (e.g., itchy, red, and / or watering eyes), wheezing, chest tightness, shortness of breath, and / or coughing, hives and / or rash, swelling of the lips, tongue, eyes, or face, stomach pain, vomiting, and / or diarrhea, dry, red, and / or cracked skin.

[0026]

[0046] As used herein, "natively folded" refers to a protein in an effective, functional, properly folded and / or structured form. A naturally folded allergenic protein contains one or more IgE and / or B cell epitopes, which can elicit an immune response in a subject. In contrast, the term "partially unfolded" refers to a protein whose tertiary and / or secondary structure has been altered compared to the naturally folded state. A partially unfolded protein may have a reduced number of IgE and / or B cell epitopes compared to its naturally folded counterpart. In some embodiments, a partially unfolded protein may be free, or substantially or completely free, of IgE and / or B cell epitopes.

[0027]

[0047] As used herein, the term "IgE epitope" refers to an epitope present on an allergenic protein that is recognized by IgE antibodies produced by a subject. In some embodiments, the IgE epitope is a discontinuous epitope, e.g., a conformational epitope.

[0028]

[0048] As used herein, the term "B cell epitope" refers to an epitope present on an allergenic protein that is recognized by B cells produced by a subject. In some embodiments, the B cell epitope is a discontinuous epitope, e.g., a conformational epitope.

[0029] Allergenic Proteins

[0049] Allergenic proteins are found in many different types of food, such as nuts and dairy products. For example, in some embodiments, the allergenic proteins are derived from natural sources. Natural sources can include one or more of peanuts, eggs, milk, wheat, almonds, cashews, pistachios, hazelnuts, walnuts, pecans, Brazil nuts, soybeans, oats, peas, cod, salmon, shrimp, or sesame. A non-limiting list of allergenic proteins is provided in Table 1.

[0030] [Table 1]

[0031]

[0050] As described herein, an allergenic protein may be in a naturally folded state. A naturally folded allergenic protein may contain one or more IgE and / or B cell epitopes and may be capable of eliciting an allergic response in a susceptible subject. In some embodiments, a naturally folded allergenic protein contains one or more T cell epitopes. In some embodiments, a naturally folded allergenic protein contains one or more IgG antibody epitopes.

[0032]

[0051] In some embodiments, the allergenic protein may be in a partially unfolded state. A partially unfolded protein may contain a reduced number of IgE and / or B cell epitopes compared to the same protein in a naturally folded state. For example, partially unfolding a protein may reduce the number of IgE and / or B cell epitopes in the protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to the naturally folded state. In some embodiments, partially unfolding a protein may eliminate the number of IgE and / or B cell epitopes in the protein compared to the naturally folded state. In some embodiments, partially unfolding a protein may eliminate the number of B cell epitopes in the protein compared to the naturally folded state. In some embodiments, partial unfolding of a protein can reduce, but not eliminate, the number of T cell epitopes in the protein compared to the native folded state. In some embodiments, partial unfolding of a protein can not substantially reduce the number of T cell epitopes in the protein compared to the native folded state. In some embodiments, partial unfolding of a protein can reduce, but not eliminate, the number of IgG antibody epitopes in the protein compared to the native folded state. In some embodiments, partial unfolding of a protein can not substantially reduce the number of IgG antibody epitopes in the protein compared to the native folded state.

[0033]

[0052] In some embodiments, any of (a) the percent unfolding, (b) the percent change in conformational structure, (c) the percent change in T cell epitopes, (d) the percent change in IgE epitopes and / or B cell epitopes in the protein, or (e) the percent reduction or freedom of IgE epitopes and / or B cell epitopes in the protein as compared to the native structural state is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 102%, 104%, 105%, 106 %, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66 %, 67%, 68%, 69%, 70, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0034]

[0053] Figure 6 provides details for some embodiments regarding the selection of ranges that define the degree of permanently cleaved weak bonds relative to all weak bonds in a given allergenic protein (e.g., an allergenic protein selected from the right-most column of Table 1) relative to the natively folded state of that allergenic protein. In particular, Figure 6 provides "x" selection points that establish upper and lower limits of the range of percent of cleaved weak bonds (correlated to the degree of unfolding or reduction in number of epitopes of a given protein) relative to all weak bonds of a given allergenic protein in the natively folded state.

[0035] Methods for partially unfolding proteins

[0054] Also provided herein are methods for partially unfolding a protein, e.g., an allergenic protein, and methods for producing a food composition comprising the partially unfolded protein. In certain embodiments, the portion (e.g., the unfolded portion) of the allergenic protein comprises one or more T cell epitopes and is substantially free of IgE epitopes and / or B cell epitopes.

[0036]

[0055] The methods described herein allow for partial unfolding of proteins to a known and controllable extent. In some embodiments, the methods described herein allow for partial unfolding of proteins such that 1% to 99% of the allergenic protein is unfolded. For example, using the methods described herein, about 1% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 99% of the allergenic protein is unfolded.

[0037]

[0056] Exemplary methods for partially unfolding allergenic proteins include, but are not limited to, heat treatment, instantaneous controlled pressure drop, pepsin treatment, hydrolysis, acid treatment, or fermentation. Other methods known to those skilled in the art for partially unfolding proteins may also be used. Treatment with one or more of these methods can reduce the amount of natively folded allergenic proteins, reduce or eliminate IgE and / or B cell epitopes, and / or substantially maintain one or more T cell epitopes on the food composition.

[0038]

[0057] As used herein, "heat treatment" may include any method of applying heat to a natively folded protein without chemically altering (i.e., burning) the protein. Exemplary heat treatment methods may include, for example, moist heat methods (e.g., boiling, or steam) or dry heat methods (e.g., baking or toasting). During heat treatment, the temperature may be constant or may vary over time. In some embodiments, the heat treatment includes boiling or steam treatment. The boiling or steam treatment may be for about 1-5 minutes, about 5-10 minutes, about 10-20 minutes, about 20-30 minutes, or more than 30 minutes. In some embodiments, the heat treatment includes baking or toasting. The baking or toasting may be for about 1-5 minutes, about 5-10 minutes, about 10-20 minutes, about 20-30 minutes, or more than 30 minutes.

[0039]

[0058] As used herein, "instantaneous controlled pressure drop" refers to a method that includes a short heating step (e.g., heating for about 10-60 seconds) that includes saturated steam or pressurized air injection under high pressure applied to a natively folded protein that is first placed under vacuum. Following the first heating step, a sudden drop in pressure (e.g., about 0.5 MPa / sec) toward a vacuum (e.g., about 3-5 kPa) over a short period of time (e.g., about 10-60 milliseconds) creates significant mechanical stress, allowing the product to expand. The pressure drop also allows for rapid cooling, which prevents thermal degradation of sensitive compounds in the composition, e.g., compounds that contribute to one or more sensory properties, such as taste.

[0040]

[0059] Pepsin treatment refers to the use of pepsin enzyme to partially unfold naturally folded proteins. Pepsin is an endopeptidase that breaks down dietary proteins in the stomach into amino acids that can be easily absorbed in the small intestine. It works by digesting peptide or hydrogen (weak) bonds, which are the main chemical bonds found in proteins that allow complex structures. Such bonds are often IgE binding sites contained in IgE epitopes and / or B cell epitopes. Partial unfolding of proteins using the pepsin treatment technique can be controlled by varying the concentration of the enzyme used and / or the time of treatment.

[0041]

[0060] Hydrolysis refers to a chemical reaction in which a water molecule cleaves one or more peptide bonds in a wild-type folded protein. Typically, this method is used to cleave proteins into peptide chains or amino acids, but can be controlled to reduce the number of IgE binding sites.

[0042]

[0061] Acid or base treatment involves the use of one or more acids or bases to break peptide or hydrogen bonds. As mentioned above, peptide bonds are involved in the secondary or tertiary structure of a protein. Disruption of bonds can eliminate one or more IgE epitopes and / or B cell epitopes in the protein. The acid or base treatment described herein may use weaker or stronger acids or bases to control the degree of protein unfolding. The duration of treatment may also be varied.

[0043]

[0062] Fermentation involves the chemical degradation of protein peptides or hydrogen bonds by bacteria, yeast, or other microorganisms. The fermentation described herein may use one or more organisms in sequence or simultaneously to control protein alterations. Also, the time of fermentation and additional heat or cooling may be varied.

[0044]

[0063] In some embodiments, the method for partially unfolding an allergenic protein comprises treating the isolated protein as described above and then producing a food composition comprising the unfolded protein.

[0045]

[0064] Alternatively, it is envisioned that direct treatment of at least a portion of the food composition to at least partially unfold the allergenic proteins in the portion of the food composition may be used. In such embodiments, suitable techniques typically include treating the food composition with a combination of two or more of the following: heat treatment, instantaneous controlled pressure drop, pepsin treatment, hydrolysis, acid treatment, or fermentation.

[0046]

[0065] The food composition that is subjected to the treatment can be in many different forms. For example, in some embodiments, the food composition is freeze-dried. In some embodiments, the food composition is a dry composition. In some embodiments, the food composition is a wet composition. The wet composition may in some embodiments be mixed with a starch prior to treatment. Exemplary starches that may be used in the present method include, but are not limited to, tapioca starch, cassava flour, corn starch, wheat starch, rice starch, or potato starch.

[0047]

[0066] In some embodiments, the method of partially unfolding an allergenic protein in a food composition comprises treating the food composition using heat treatment, instantaneous controlled pressure drop, pepsin treatment, hydrolysis, acid or base treatment, or fermentation, where the treatment reduces the amount of natively folded allergenic protein in the food composition by at least 50%, and the partially unfolded allergenic protein comprises one or more T cell epitopes and / or IgG epitopes and is substantially free of IgE epitopes and / or B cell epitopes. In some embodiments, the treatment reduces the amount of natively folded allergenic protein in the food composition by at least 75%. In some embodiments, the treatment reduces the amount of natively folded allergenic food protein in the food composition by at least 90%. In some embodiments, the treatment reduces the amount of natively folded allergenic food protein in the food composition by at least 95%. In some embodiments, the treatment reduces the amount of natively folded allergenic food proteins in the food composition by at least 99%. In some embodiments, between 1% and 99%, e.g., between about 1% and about 25%, between about 25% and about 50%, between about 50% and about 75%, or between about 75% and about 99%, of the allergenic proteins in the food composition are partially unfolded after treatment.

[0048]

[0067] In some embodiments, the method of partially unfolding allergenic proteins in a food composition comprises treating the food composition using heat treatment, instantaneous controlled pressure drop, pepsin treatment, hydrolysis, acid treatment, or fermentation, where the treatment further comprises an acidic pH, a neutral pH, or a basic pH. An acidic pH can be less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. A neutral pH can be about 7. A basic pH can be greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, greater than 12, or greater than 13.

[0049] Methods for assessing the degree of unfolding and / or the change or reduction in the number of IgE and / or B cell epitopes in a protein compared to the natively folded state of a given allergenic protein

[0068] It is generally understood that the degree of unfolding is assessed, for example using the techniques described herein, controlled and correlated with the number of epitope differentials (e.g., percentage of IgE epitopes and / or percentage of B cell epitopes relative to the natively folded state) of a given protein. It is generally assumed that there is a direct relationship between the accessibility of an antibody to an antigen and the accessible surface of a protein. Based on this assumption, prediction systems often include as prediction criteria solvent accessibility values ​​calculated from the primary sequences of proteins or their three-dimensional structures.

[0050]

[0069] Techniques for assessing the degree of protein unfolding include solubility analysis, e.g., the more insoluble a protein is in a given solvent, the greater the degree of permanently broken weak bonds and unfolding can be determined. Other techniques, including light scattering, SEM, circular dichroism, and pH shifts, can also be used to quantify conversion of proteins from the native folded state.

[0051]

[0070] In one embodiment, differences in the degree of folding and / or T cell epitopes may be determined by T cell I binding in serum.

[0071] In one embodiment, differences in folding and / or degree of IgE epitopes and / or B cell epitopes may be determined by IgE binding in serum or with monoclonal antibodies.

[0052]

[0072] In one embodiment, differences in folding and / or degree of B cell epitope may be determined by B cell binding in serum.

[0073] The methods used and the units used usually measure the allergen content and biological activity. Examples here are SQ-Unit (Standardised Quality unit), BAU (Biological Allergen Unit), BU (Biological Unit), UM (Unit of Mass), IU (International Unit) and IR (Indicator of Reactivity). Furthermore, guidance to commonly applied acceptable tests to measure the biopotency of allergens can be found, for example, in Note for Guidance on Allergen Product; The European Agency for the Evaluation of Medicinal Product, CPMP_BWP_243_96, London, 1996.

[0053]

[0074] Several laboratory tests are also available to characterize allergens. The most widely used techniques are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), and rocket immunoelectrophoresis (RIE). Quantification of individual allergens can be performed by various quantitative immunoelectrophoresis techniques (QIE), radial immunodiffusion (RIE), or enzyme-linked immunosorbent assay (ELISA). Determination of total allergenic activity is most frequently performed by radio allergosorbent test (RAST), Magic Lite assay (LIA), or related techniques. ELISA-based techniques can also be used.

[0054]

[0075] Techniques for the assessment of protein allergens are detailed in U.S. Publication No. 20110200641, the entire contents of which are incorporated by reference as if fully set forth herein.

[0055] Food Composition

[0076] Provided herein is a food composition for treating and / or preventing food allergies in a subject in need thereof. The composition comprises at least one allergenic protein, at least a portion of which is partially unfolded as detailed above, and optionally one or more additional ingredients, such as fat, fiber, vitamins, minerals, and carbohydrates.

[0056]

[0077] In some embodiments, the food composition comprises an allergenic protein. The amount by weight of the allergenic protein in the food composition is further detailed below. When included in a food composition, 1%-99% of the allergenic protein in the composition is partially unfolded. In some embodiments, 1%-25%, 25%-50%, 50%-75%, or 75%-99% of the allergenic protein is partially unfolded. Other ranges and values ​​for the degree of unfolding are detailed above.

[0057]

[0078] In some embodiments, the food composition comprises a partially unfolded allergenic protein comprising one or more T cell epitopes and IgG epitopes. The presence of T cell epitopes and IgG epitopes in the partially unfolded allergenic protein may promote its tolerance by the immune system of the subject, i.e. the presence of T cell epitopes and / or IgG epitopes may prevent the development of an allergic reaction to the protein.

[0058]

[0079] In some embodiments, the food composition comprises a partially unfolded protein that is substantially free of IgE and / or B cell epitopes. The absence of IgE and / or B cell epitopes in the partially unfolded protein may promote its tolerance by the subject's immune system, i.e. the absence of IgE and / or B cell epitopes may prevent the development of an allergic reaction to the protein.

[0059]

[0080] In some embodiments, one or more portions of the food composition comprise unfolded allergenic protein, and the remainder of the protein comprises naturally folded allergenic protein. Subtracting the amount by weight of partially unfolded or completely unfolded protein present in the food composition from the total amount of protein in the food composition essentially allows for the calculation of the amount of protein remaining in its native state. In some embodiments, at least 1% by weight of the allergenic protein in the composition is natively folded. For example, in some embodiments, 1% to 25%, 25% to 50%, 50% to 75%, or 75% to 99% by weight of the allergenic protein is natively folded.

[0060]

[0081] Without wishing to be bound by any theory, it is believed that a food composition containing both natively folded and partially unfolded forms of the same protein may promote tolerance of the protein by a subject's immune system, an effect that is greater when the subject is exposed to a mixture of the natively folded and partially unfolded forms as opposed to exposure to only the partially unfolded form.

[0061]

[0082] The allergenic proteins of the food composition may be isolated or derived from, for example, peanuts, eggs, milk, wheat, almonds, cashews, pistachios, hazelnuts, walnuts, pecans, Brazil nuts, soybeans, oats, peas, cod, salmon, shrimp, or sesame. In some embodiments, the allergenic protein is any one of the proteins listed in Table 1. In some embodiments, the food composition may include two or more allergenic proteins, for example, three, four, five, six, seven, eight, nine, ten, or more allergenic proteins. In embodiments in which the food composition includes two or more allergenic proteins, each of the allergenic proteins may be selected from those listed in Table 1.

[0062]

[0083] In some embodiments, the food composition comprises an allergenic protein selected from Ara h 1, Ara h 2, Ara h 3, Ara h 6, and Ara h 9. In some embodiments, the food composition comprises an allergenic protein selected from Gal d 1, Gal d 2, Gal d 3, Gal d 4, and Gal d 5. In some embodiments, the food composition comprises an allergenic protein selected from Bos d 5 and Bos d 11. In some embodiments, the food composition comprises an allergenic protein selected from Jug r1 and Jug r3. In some embodiments, the food composition comprises an allergenic protein selected from Pis v 1, Pis v 2, Pis v 3, and Pis V 4. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is AnaO3. In some embodiments, the food composition comprises an allergenic protein selected from Cor a 9, Cor a 14, Cor a 1, and Cor a 8. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is Car i 1. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Pru du 1, Pru du 2, Pru du 3, Pru du 4, Pru du 5, Pru du 6, and Pru du 8. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Ber e1 or e2. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Tri a 19 and Tri a 14. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Pis s 1 and Pis s 2.In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Gad m 1, Gad m 2, and Gad m 3. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Sal s 1, Sal s 3, Sal s 2, or Onc k 5. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is selected from Met e 1, Pan s 1, or Hom a 1. In some embodiments, the food composition comprises an allergenic protein, wherein the allergenic protein is avenin.

[0063]

[0084] FIG. 7 provides, for some embodiments, a selection point "y" that establishes an upper and lower limit of a range of the amount by weight of a given protein in a portion of a food composition, where the given protein has some degree of unfolding "x" (e.g., a range selected from FIG. 6) relative to the total amount by weight of protein in that portion of the food composition.

[0064]

[0085] Thus, using the techniques described herein, designed or "custom" food compositions can be created having one or more proteins that provide varying, precise, and controllable degrees of (1) percent unfolding of a given protein (e.g., typically correlated with a reduction in the number of epitopes of a particular given protein (e.g., a protein selected from the proteins described herein, e.g., those set forth in the right hand column of Table 1)) and (2) percent weight inclusion of that given protein in the food composition.

[0065]

[0086] In one such embodiment, the food composition comprises a first allergenic protein, where a percent range of the total number of weak bonds in the first allergenic protein are permanently cleaved (e.g., unfolded), and the first allergenic protein may optionally maintain or have one or more T cell epitopes. The range of percent of cleaved weak bonds compared to all weak bonds in the first allergenic protein is selected at x in Figure 6. The range of weight percent of the first allergenic protein, by weight, relative to all proteins in the food composition is determined or selected at y in Figure 7, thereby creating a custom food composition.

[0066]

[0087] In another embodiment, the custom food composition comprises a second allergenic protein, where the percentage range of the total number of weak bonds in the second allergenic protein are permanently cleaved (e.g., unfolded), and the second allergenic protein may optionally maintain or have one or more T cell epitopes. The percentage range of cleaved weak bonds compared to all weak bonds in the second allergenic protein is the selected x position in Figure 6. The percentage range of cleaved weak bonds may be the same or different than the range of the first allergenic protein. Similarly, the weight percentage range of the second allergenic protein relative to all proteins in the food composition, including the first allergenic protein, may also be the selected y position in Figure 7, thereby creating a custom food composition.

[0067]

[0088] The food compositions of the invention may comprise a third, fourth, fifth, sixth, seventh or more allergenic proteins. Such proteins may, for example, be selected from the proteins described herein, such as those set out in the right hand column of Table 1.

[0068]

[0089] The first and second allergenic proteins may also be distinguished by the degree of percent unfolding of a given protein (e.g., typically correlated with a reduction in the number of epitopes of a given protein). Thus, in some embodiments, the first and second allergenic proteins may be the same protein type. For example, Cor a 9 may be used for both the first and second allergenic proteins, but the degree of percent unfolding may differ between these same protein types. Alternatively, the first and second allergenic proteins may be different protein types, e.g., Gal d 2 and Cor a 9, each with a particular degree of unfolding.

[0069]

[0090] The first and second allergenic proteins and / or the additional allergenic proteins may be the same in different parts of the custom food composition.

[0091] Similarly, in some embodiments, a protein is partially unfolded using one or more of the above treatments, resulting in a mixture that can then be mixed with a natively folded protein to produce a mixture. For example, a composition containing a natively folded protein can be treated using heat treatment, a momentary controlled pressure drop, pepsin treatment, hydrolysis, or acid or base treatment to result in partial unfolding of the protein. The partially unfolded protein can then be mixed with a natively folded protein to produce a mixture.

[0070]

[0092] In some embodiments, the food composition comprises fat. In some embodiments, the food composition comprises added fat in addition to the fat naturally present therein. The fat may comprise triglycerides and / or cholesterol. In some embodiments, the fat is saturated fat. In some embodiments, the fat is unsaturated fat, such as monounsaturated fat or polyunsaturated fat. In some embodiments, the fat comprises omega-3 fatty acids. In some embodiments, the food composition comprises one or more oils, such as canola oil, avocado oil, sunflower oil, corn oil, olive oil, peanut oil, walnut oil, palm oil, safflower oil, soybean oil, flaxseed oil, sesame oil, or vegetable oil.

[0071]

[0093] In some embodiments, the food composition includes fiber. In some embodiments, the food composition includes added fiber in addition to the fiber naturally present therein. Fiber can include parts of plant-derived (or other) foods that are not completely broken down by human digestive enzymes. In some embodiments, the fiber is soluble fiber. In some embodiments, the fiber is insoluble fiber. In some embodiments, the fiber is derived from fruits, vegetables, beans (legumes), whole grains, or meat. In some embodiments, the fiber includes beta-glucan (e.g., cellulose, chitin), hemicellulose (e.g., hexose, pentose), lignin, xanthan gum, resistant starch, arabinoxylan, fructan (e.g., inulin), polyuronides (e.g., pectin), alginic acid (e.g., sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propylene glycol alginate, agar, carrageen), raffinose, or polydextrose.

[0072]

[0094] In some embodiments, the food composition includes one or more vitamins. The vitamins can include, for example, vitamin A, vitamin C, vitamin D, vitamin K, or vitamin E. In some embodiments, the food composition includes B vitamins, such as thiamine, riboflavin, niacin, pantothenic acid, biotin, vitamin B6, vitamin B7, vitamin B8, vitamin B9, vitamin B10, vitamin B11, vitamin B12, vitamin B13, vitamin B14, vitamin B15, vitamin B16, vitamin B17, vitamin B18, vitamin B19, vitamin B20, vitamin B21, vitamin B30, vitamin B40, vitamin B50, vitamin B60, vitamin B71, vitamin B82, vitamin B93, vitamin B104, vitamin B105, vitamin B126, vitamin B130, vitamin B140, vitamin B150, vitamin B160, vitamin B170, vitamin B190, vitamin B206, vitamin B210, vitamin B30, vitamin B41, vitamin B51, vitamin B112, vitamin B114, vitamin B116, vitamin B118, vitamin B120, vitamin B220, vitamin B130, vitamin B140, vitamin B150, vitamin B160, vitamin B230, vitamin B170, vitamin B190, vitamin B240, vitamin B150, vitamin B160, vitamin B250, vitamin B170, vitamin B190, vitamin B260, vitamin B30, vitamin B41, vitamin B150, vitamin B160, vitamin B170, vitamin B240, vitamin B190, vitamin B150, vitamin B150, vitamin B250, vitamin B160, vitamin B170, vitamin B180, vitamin B250, vitamin B190, vitamin B260, vitamin B30, vitamin B190, vitamin B206, vitamin B30, vitamin B41, vitamin 12 , or folic acid.

[0073]

[0095] In some embodiments, the food composition comprises one or more minerals, for example, in some embodiments, the food composition comprises calcium, magnesium, potassium, or sodium.

[0074]

[0096] The food composition may comprise one or more fats, fiber, and / or vitamins. In some embodiments, the food composition comprises one or more fats and one or more fibers. In some embodiments, the food composition comprises one or more fats and one or more vitamins. In some embodiments, the food composition comprises one or more fibers and one or more vitamins. In some embodiments, the food composition comprises fat, fiber, and vitamins.

[0075]

[0097] In some embodiments, the food composition comprises butyrate. Butyrate is a short-chain fatty acid that is typically produced by the gut microbiota. Butyrate can increase the production of Treg cells. Without wishing to be bound by any theory, it is believed that the presence of sufficient butyrate in the digestive tract promotes tolerance of one or more allergenic proteins.

[0076]

[0098] In some embodiments, a food composition comprising an allergenic protein may further comprise one or more non-allergenic proteins, for example, in some embodiments, a food composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-allergenic proteins.

[0077]

[0099] In some embodiments, the food composition comprises at least one probiotic. Probiotics include live bacteria and yeasts that promote gastrointestinal health. Exemplary probiotic bacteria include, but are not limited to, species of Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus. In some embodiments, the food composition comprises Lactobacillus. In some embodiments, the food composition comprises Bifidobacterium. In some embodiments, the food composition comprises Lactobacillus and Bifidobacterium.

[0078]

[0100] In some embodiments, the food containing the allergenic protein may also contain a prebiotic. A prebiotic is a compound in a food that induces the growth or activity of beneficial microorganisms, such as bacteria and fungi. A prebiotic can change the composition of organisms in the gut microbiome. Exemplary prebiotics that may be used in the food compositions described herein include oligosaccharides, galactooligosaccharides, fructooligosaccharides, oligosaccharides, inulin, or mixtures thereof. Oligosaccharides may be isolated from plants, such as chicory root, Jerusalem artichoke, onion (including leek and garlic), legumes, wheat, asparagus, and jicama. In some embodiments, the oligosaccharide may be a food additive, such as inulin or oligofructose.

[0079]

[0101] In some embodiments, the food compositions described herein may be nutritional or dietary supplements. The food compositions described herein may be produced in many different forms. For example, the food compositions described herein may be powders, purees, pastes, or beverages (e.g., formulas). In some embodiments, the food compositions are puffs, crackers, or biscuits.

[0080]

[0102] In some embodiments, the food compositions described herein may be storable for at least 3 months, at least 6 months, at least 9 months, at least 12 months, or more. In some embodiments, the food compositions are storable at room temperature. In some embodiments, the food compositions are storable at 4° C. In some embodiments, the food compositions are stable when frozen.

[0081] Methods for Treating and / or Preventing Food Allergies

[0103] The food compositions described herein may be used to treat and / or prevent food allergies. In some embodiments, a method for treating and / or preventing food allergies in a subject in need thereof comprises administering to the subject one or more food compositions described herein. In some embodiments, the food composition is administered once to the subject. In some embodiments, the food composition is administered multiple times to the subject at effective intervals. In some embodiments, the food composition is administered once per day. In some embodiments, the food composition is administered multiple times per day. In some embodiments, the food composition is administered once per week. In some embodiments, the food composition is administered multiple times per week. In some embodiments, the food composition is administered once per month. In some embodiments, the food composition is administered multiple times per month.

[0082]

[0104] In some embodiments, the subject is an adult subject. In some embodiments, the subject is a pediatric subject. For example, the subject can be 18 months of age or younger, 12 months of age or younger, or 6 months of age or younger.

[0083]

[0105] In some embodiments, the food composition is administered orally. The food composition may be eaten alone or may be mixed and / or combined with another food composition. For example, the powdered food compositions described herein may be combined with a "carrier" food to facilitate administration. Suitable carrier foods may include, for example, yogurt, applesauce, soup, oatmeal, etc.

[0084] kit

[0106] Also provided herein is a kit that includes one or more of the food compositions described herein.The kit can include, for example, one or more containers that include the food composition, and optionally packaging and instructions for use.The instructions can, for example, allow consumers to determine how much of the food composition to use based on the desired protein content per serving.The kit typically includes a label that indicates the intended use of the contents of the kit.

[0085]

[0107] In some embodiments, the kit comprises a container comprising a food composition. In some embodiments, the container comprises a single dose of the food composition. In some embodiments, the container comprises a multiple dose of the food composition. In some embodiments, the container further comprises a second food composition comprising a second allergenic protein.

[0086]

[0108] In some embodiments, the kit includes a second container. The second container may include a second allergenic protein in some embodiments. In some embodiments, the kit includes a first container and a second container, and the amount of the partially denatured allergenic protein in the first container is different from the amount of the partially denatured allergenic protein in the second container. For example, the amount of the partially denatured allergenic protein in the first container may be greater or less than the amount in the second container. In some embodiments, the kit includes a first container and a second container, and the amount of the partially denatured allergenic protein in the first container is the same as the amount of the partially denatured allergenic protein in the second container.

[0087]

[0109] In some embodiments, the kit comprises a first container, a second container, a third container, and a fourth container, and the amount of partially denatured allergenic protein is different in each container. For example, the amount of partially denatured allergenic protein may be greater in the second container than in the first container, greater in the third container than in the second container, and / or greater in the fourth container than in the third container. In some embodiments, the amount of partially denatured allergenic protein may be less in the fourth container than in the third container, less in the third container than in the second container, and / or less in the second container than in the first container. In some embodiments, the kit comprises a first container, a second container, a third container, and a fourth container, and the amount of partially denatured allergenic protein is the same in each container.

[0088]

[0110] The container containing the food composition can have any size and / or shape. For example, in some embodiments, the container is a sachet, bag, or tube. In some embodiments, the container is a spoon, such as a preloaded spoon.

[0089] Incorporation by Reference

[0111] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not an admission or in any manner intended to suggest that they constitute valid prior art or form part of the common general knowledge in any country in the world, and should not be construed as such. EXAMPLES

[0090]

[0112] The present disclosure is further illustrated by the following examples, which are not to be construed as limiting the scope or spirit of the disclosure to the specific procedures described herein. It should be understood that the examples are provided to illustrate specific embodiments, and no limitation to the scope of the disclosure is intended thereby. It should be further understood that without departing from the spirit of the present disclosure, resort can be made to various other embodiments, modifications, and equivalents thereof that may suggest themselves to those skilled in the art.

[0091] Example 1

[0113] Production of food compositions containing partially unfolded allergenic proteins from milk and eggs The inventors hypothesized that a food containing a mixture of naturally folded proteins and T cell epitope-containing proteins could optimally balance reduced immunogenicity with sufficient antigenic properties. Moreover, by preserving the food in as natural a form as possible, an overall tolerance training effect could be achieved. Therefore, the inventors attempted to prepare milk and egg compositions that contain various levels of partially denatured allergenic proteins, while also retaining some of the allergenic proteins in their naturally folded structure.

[0092]

[0114] The inventors have used heat treatments including toasting, boiling, baking, and combinations thereof to partially denature allergenic proteins. It was hypothesized that the length of heat treatment affects the degree to which the food is denatured. It was further hypothesized that the specific mechanism of heating (e.g., boiling vs. baking) is also important.

[0093]

[0115] Milk and eggs containing known allergenic proteins were subjected to various different types of heat treatments for different times. For example, samples were toasted for about 0, 5, or 10 minutes, boiled for about 0, 10, 20, or 30 minutes, baked for about 0, 10, 20, or 30 minutes, or boiled for 10 minutes and then toasted for 5 or 10 minutes. In this example, the toasting method was performed by heating the spray-dried version of the milk or egg using a heat source placed adjacent to it, the heat source having a temperature of about 167°C. Boiling was performed by heating the moistened milk or egg composition alone or in water, with the temperature maintained at about 100°C. Baking involved mixing the moist form of the milk or egg composition with starch (cassava flour or tapioca starch) in a 1:1 ratio. The mixture was then placed in an oven at a temperature of about 167°C, and the food product was allowed to rise to the required temperature without burning. After heat treatment, the level of reactive allergenic proteins was measured using a standard ELISA.

[0094]

[0116] As shown in Table 1 and FIG. 2B, toasting for 5 min reduced the levels of reactive Bos d 5 and Bos d 11 compared to untreated milk samples. After 10 min of toasting, reactive Bos d 5 and Bos d 11 were not detectable. Boiling the samples for longer times and baking substantially reduced, but did not eliminate, reactive Bos d 5 and Bos d 11 (FIG. 3B). In samples boiled for 10 min and subsequently toasted, reactive Bos d 5 was not detectable (FIG. 5B). However, reactive Bos d 11 was still detectable after 10 min of boiling and 5 min of toasting.

[0095]

[0117] As shown in Table 2, Figures 2A, and 3A, toasting for 5 or 10 min or boiling for 10, 20, or 30 min reduced, but did not eliminate, reactive Gal d 1 and Gal d 2. Baking for 0, 10, 20, or 30 min did not eliminate reactive Gal d 1, but baking for 30 min eliminated reactive Gal d 2 (Figure 4). Detectable levels of reactive Gal d 1 and Gal d 2 were maintained after boiling for 10 min and toasting for 5 or 10 min (Figure 5A).

[0096] [Table 2]

[0097] [Table 3]

[0098]

[0118] Together, these data indicate that the heat treatment methods tested can be used to partially denature allergenic proteins from milk and eggs to a controllable extent. It also shows how different heating methods / treatment times can be used to reduce or eliminate the reactivity of selective proteins compared to other allergenic proteins in the same original composition. For example, in these experiments, the method with the lower heat level (i.e., boiling) appeared to preferentially denature one protein while protecting the other. Notably, the combination of boiling and toasting the moist starting composition eliminated the need to use starch.

[0099] Example 2

[0119] Method for unfolding peanut proteins to produce hypoallergenic compositions Peanut flour was diluted to a 20% slurry using high purity deionized water (18 MΩ) and then heated until reflux or bubbling was observed, which marked the starting boiling time. Boiling proceeded for 60 minutes, with sampling taken at 5, 10, 15, 30, 45, and 60 minute intervals. The slurry was covered with a watch glass and aluminum foil except when sampling. For the low pH condition, a starting pH of 3 was achieved using 0.1 M sulfuric acid, and for the high pH condition, a starting pH of 8 was achieved using 0.03 M sodium hydroxide. Samples were freeze-dried prior to MARIA testing.

[0100]

[0120] The effect of treatment on peanut protein structure was assessed by circular dichroism and the results are shown in Figure 8 for low pH conditions, Figure 9 for neutral conditions and Figure 10 for high pH conditions.

[0101]

[0121] Table 3 and Figures 11, 12, and 13 summarize the effect of pH and boiling duration on protein allergenicity measured with proteins Ara h 1, Ara h 3, and Ara h 6 for peanuts.

[0102] [Table 4]

[0103]

[0122] As observed in Table 3 and Figures 11 and 12, extreme reduction of allergenicity of Ara h 1 and 3 is possible with short boiling times (5 min) and without pH adjustment. Moreover, with acidic pH adjustment (pH 3), the antibody response to Ara h 1 and 3 is less than 0.4 μg / g even without heat treatment. Boiling for only 5 min under acidic conditions is sufficient to reduce Ara h 1 and 3 below the test detection limit. In contrast, as shown in Figure 13, Ara h 6 requires 30 min of boiling without pH adjustment to reduce allergenicity by 50%. Boiling for 60 min under acidic conditions does not further reduce the allergenicity of Ara h 6. Ara h 6 has been found to be protease resistant and heat stable, which can be explained by the six disulfide bonds in its core.

[0104]

[0123] Interestingly, basic conditions prior to boiling increase the allergenicity of all three tested allergens. However, boiling under basic conditions for 30 minutes was the most successful in reducing the allergenicity of Ara h 6, resulting in approximately 5% of the original level. These conditions also reduced the allergenicity of Ara h 1 and 3 to less than 5% of the original level. Boiling peanut flour under basic conditions for 30 minutes or more is the preferred condition for reducing allergenicity.

[0105] Example 3

[0124] Method for unfolding almond proteins to produce hypoallergenic compositions Almond flour was diluted to a 30% slurry using high purity deionized water (18 MΩ) and then heated until reflux or bubbling was observed, which marked the start of the boiling time. Boiling proceeded for 60 minutes, with sampling taken at 5, 10, 15, 30, 45, and 60 minute intervals. The slurry was covered with a watch glass and aluminum foil except when sampling. For the low pH condition, a starting pH of 3 was achieved using 0.1 M sulfuric acid, and for the high pH condition, a starting pH of 8 was achieved using 0.03 M sodium hydroxide. Samples were freeze-dried prior to MARIA testing.

[0106]

[0125] The effect of treatment on almond protein structure was assessed by circular dichroism and the results are shown in Figure 14 for low pH conditions, Figure 15 for neutral conditions and Figure 16 for high pH conditions.

[0107]

[0126] Table 4 and Figure 17 summarize the influence of pH and boiling duration on protein allergenicity measured with protein Pru du 6 for almonds. The allergenicity of almonds was tested using Pru du 6. All boiling conditions reduced allergenicity to an extreme degree. Under neutral conditions, at least 5 minutes of boiling is required. The two replicates of 5 minutes of boiling show a large variability, so a longer time is preferred to ensure sufficient time for heat transfer. As observed with peanuts, changing the pH to pH 3 without heat is sufficient to suppress most of the allergenic response to Pru du 6. Boiling under basic conditions is also effective in reducing the allergenicity of Pru du 6, but the same results are achieved in shorter times under acidic conditions.

[0108]

[0127]

[0109] [Table 5]

Claims

1. 1. A food composition comprising a first portion of an allergenic protein: between 1% and 99% of the allergenic proteins in the composition are partially unfolded, the partially unfolded allergenic proteins comprising one or more T cell epitopes and substantially free of IgE and / or B cell epitopes; A food composition, wherein at least 1% of the allergenic proteins in said composition are natively folded.

2. 2. The food composition of claim 1, wherein 75% to 99% of the allergenic proteins are partially unfolded.

3. 2. The food composition of claim 1, wherein the allergenic protein comprises at least one selected from the following: (a) a peanut allergenic protein selected from Ara h 1, Ara h 2, Ara h 3, Ara h 6, or Ara h 9; (b) a walnut allergenic protein selected from Jug r1 or Jug r3; (c) a pistachio allergenic protein selected from Pis v 1, Pis v 2, Pis v 3, or Pis v 4; (d) a cashew allergenic protein selected from AnaO3; (e) hazelnut allergenic proteins selected from Cor a 9, Cor a 14, Cor a 1, or Cor a 8; (f) a pecan allergenic protein selected from Car i 1; (g) almond allergenic proteins selected from Pru du 1, 2, 3, 4, 5, 6, or 8; (h) a Brazil nut allergenic protein selected from Ber e1 or e2; (i) a wheat allergenic protein selected from Tri a 19 or Tri a 14; (j) a soybean allergenic protein selected from Gly m Bd 60K, Gly m Bd 30K, or Gly m Bd 28K; (k) a pea allergenic protein selected from Pis s 1 or Pis s 2; (l) a cod allergenic protein selected from Gad m 1, Gad m 2, or Gad m 3; (m) a salmon allergenic protein selected from Sal s 1, Sal s 3, Sal s 2, or Onc k 5; (n) a shrimp allergenic protein selected from Met e 1, Pan s 1, or Hom a 1; or (o) an oat allergenic protein selected from avenin;

4. 4. The food composition of claim 3, comprising at least one additive selected from fat, fiber, one or more vitamins, butyrate, one or more non-allergenic proteins, or at least one probiotic, said probiotic optionally being selected from the genus Lactobacillus or Bifidobacterium.

5. A food composition according to any one of claims 1 to 4, comprising a prebiotic.

6. 6. The food composition according to claim 1 which is a powder, puree, paste or beverage.

7. 7. The food composition according to any one of claims 1 to 6, which is a puff, a cracker or a biscuit.

8. 1. A method for partially unfolding an allergenic protein in a food composition, comprising: i) heat treatment, ii) Instantaneous controlled pressure drop; iii) Pepsin treatment; iv) hydrolysis; v) Acid treatment, or vi) Fermentation using a suitable treatment for a given protein selected from said treatment reduces the amount of natively folded allergenic protein in said food composition by at least 50%, The method, wherein the partially unfolded allergenic protein comprises one or more T cell epitopes and is substantially free of IgE epitopes and / or B cell epitopes.

9. 9. The method of claim 8, wherein the treatment reduces the amount of natively folded allergenic protein in the food composition, the amount by weight of unfolded protein being in the range "y" selected from Figure 7, and the remainder of the amount by weight of protein being natively folded allergenic protein.

10. 10. The method of claim 8, wherein said treatment reduces the amount of natively folded allergenic food proteins in said food composition by at least 90%.

11. A method for treating and / or preventing food allergies in a subject in need thereof, comprising administering to said subject a food composition described in any one of claims 1 to 8.

12. 2. The food composition of claim 1, wherein about 1% of the total number of weak bonds in the allergenic protein first portion are permanently cleaved.

13. 2. The food composition of claim 1, wherein about 75% of the total number of weak bonds in the allergenic protein first portion are permanently cleaved.

14. 2. The food composition of claim 1, wherein about 90% of the total number of weak bonds in the allergenic protein first portion are permanently cleaved.

15. A kit comprising a container containing the food composition of any one of claims 1 to 7 or 12 to 14.

16. 16. The kit of claim 15, wherein the container further comprises a second food composition comprising a second allergenic protein.

17. 16. The kit of claim 15, further comprising a second container containing a second allergenic protein.

18. 16. The kit of claim 15, comprising a first container and a second container, wherein the amount of partially denatured allergenic protein in the first container is different from the amount of partially denatured allergenic protein in the second container.

19. 15. The food composition of any of claims 1 to 7 or 12 to 14, further comprising a second portion of a second allergenic protein, which may be the same as or different from the allergenic protein of the first portion, and wherein between 0% and 100% of the total number of weak bonds in the second allergenic protein of the second portion are permanently cleaved.

20. 2. A food composition comprising a first allergenic protein, wherein a percentage range of the total number of weak bonds in said first allergenic protein are permanently cleaved, said first allergenic protein maintains one or more T cell epitopes, and an unfolded portion of said protein is substantially free of IgE epitopes and / or B cell epitopes relative to the native structure of said protein, said percentage range of cleaved weak bonds compared to all said weak bonds in said first allergenic protein is at a selected x position in Figure 1, and a percentage range of the amount by weight of said first allergenic protein relative to all proteins in said food composition by weight is at a selected y position in Figure 2.

21. The method of claim 8 , wherein the heat treatment comprises an acidic pH, a neutral pH, or a basic pH.

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