Drinkable infant compositions

Abstract

To provide a drinkable infant composition comprising a food allergen that can be administered to an infant.SOLUTION: A drinkable infant composition comprising two or more food allergens, wherein one of the food allergens is milk protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a drinking composition for infants containing a food allergen. The present invention also relates to a method for producing a drinking composition for infants and its use for reducing sensitivity to food allergens. [Background technology]

[0002] In Europe and North America, food allergies are estimated to affect nearly 5% of adults and 8% of children (Sicherer, SH and Sampson, HA, 2014. Journal of Allergy and Clinical Immunology, 133(2), pp. 291-307). A population-based study in Australia found that the prevalence of challenge-proven food allergies exceeded 10%, the highest prevalence in the world (Osborne, NJ, et al., 2011. Journal of Allergy and Clinical Immunology, 127(3), pp. 668-676). Overall, the prevalence of food allergies and anaphylaxis has been consistently increasing, with the greatest increase in food allergies or atopic eczema in infants (Koplin, JJ, et al., 2015. Current opinion in allergy and clinical immunology, 15(5), pp. 409-416). Therefore, effective allergy prevention, especially for infants, has become a global public health priority (Ring, J., 2012. Allergy, 67(2), pp. 141-143).

[0003] Nutritional intervention plays a central role in the prevention and treatment of food allergies. Clinical approaches have changed significantly in recent years, with strong interest in the early introduction of complementary foods during infancy (Du Toit, G., et al., 2016. Allergology International, 65(4), pp. 370-377). For example, the Nutrition Committee of the European Society for Paediatric Gastroenterology (ESPGHAN) now recommends that allergenic foods may be introduced when initiating complementary feeding any time after 4 months of age (Fewtrell, M., et al., 2017. Journal of Pediatric Gastroenterology and Nutrition, 64(1), pp. 119-132). For example, it is recommended that infants at high risk for peanut allergy (those with severe eczema, egg allergy, or both) should be introduced to peanuts between 4 and 11 months of age. Therefore, many scientists accept the principle of early introduction of food allergens.

[0004] Introducing food allergens at an early age, for example before 4 months of age, presents challenges related to formality and safety.

[0005] For example, common food allergens, such as eggs, peanuts, nuts, fish, crustaceans, shellfish, and sesame, are typically presented in solid food form, which is unsuitable for infants from a safety standpoint. However, early-life infants are unable to eat solid foods. The Eating Allergy (EAT) study reported that six common food allergens were introduced in the form of solid foods along with breastfeeding to infants aged 3 months and older (Perkin, MR, et al., 2016. Journal of Allergy and Clinical Immunology, 137(5), pp. 1477-1486). Although this study demonstrated a protective effect per protocol, the trial failed overall in the intention-to-treat analysis due in part to the allergen format and due to the lack of compliance in most participants with the study regimen. Therefore, it would be advantageous to administer food allergens in a liquid form.

[0006] The liquid format meets the safety requirements valid for infant formula. The main concerns are microbiological quality and contaminants. Heat denaturation during pasteurization or sterilization of infant drinking compositions can affect the immunogenicity of allergens.

[0007] Therefore, there is a need for a commercially available infant drinking composition containing food allergens that can be administered to young infants. [Brief explanation of the drawings]

[0008] [Figure 1] Diagram showing whey protein denaturation - Rowland. [Figure 2] FIG. 1 shows humanized RBL degranulation assay for residual β-Lg allergenicity. [Figure 3] A diagram showing a reduced sample.

[0009] According to the present invention, there is provided a drinkable composition for infants comprising two or more food allergens, one of the food allergens being a milk protein.

[0010] The infant drinking composition can be ingested in a liquid form. Thus, in some preferred embodiments, the composition is in a powder form, preferably one that can be reconstituted with water. In other preferred embodiments, the composition is in the form of a ready-to-drink.

[0011] Gentle pasteurization and / or sterilization can be used to provide infant drinking compositions containing food allergens. By reducing the holding temperature and / or holding time used during the heat treatment, thermal degradation of the allergens can be reduced or minimized.

[0012] In one embodiment, the infant drinking composition is sterilized. In some embodiments, the infant drinking composition has undergone sterilization at a temperature of 61.9°C to 65°C, preferably 62°C to 64°C, and preferably the sterilization is carried out for at least 30 minutes or at least 35 minutes.

[0013] In some embodiments, the infant drink composition is sterilized. Sterilization may be by indirect or direct ultra-high temperature heating. In some embodiments, the infant drink composition has undergone indirect ultra-high temperature heating, preferably at a temperature of 125°C to 135°C, or 130°C to 134°C, or 131°C to 133°C, for example, about 132°C. Sterilization may be carried out for at least 30 seconds, or at least 60 seconds, for example, 30 to 80 seconds, or 60 to 70 seconds.

[0014] In some embodiments, the infant drinking composition has undergone direct UHT heating, preferably at a temperature of 136°C to 140°C for about 15 to 25 seconds, e.g., about 20 seconds, or at a temperature of 140°C to 144°C for about 5 to 15 seconds, e.g., 10 seconds. In another embodiment, the direct UHT heating may be at a temperature of 150°C to 154°C for about 2 to 4 seconds.

[0015] In some embodiments, the sterilization may be ultra-short-term sterilization (USS). In some embodiments, the infant drinking composition has undergone ultra-short-term sterilization (USS) heat treatment at a temperature of 155°C to 170°C for less than 1 second.

[0016] In some embodiments, the mild heat treatment can be carried out at a temperature of 72°C to 90°C, for example, 72°C to 80°C, for 10 to 30 seconds, or at 80°C to 89°C for 2 to 20 seconds, for example, 80°C to 84°C for 4 to 20 seconds, or at 85°C to 89°C for 1 to 10 seconds.

[0017] In some embodiments, the infant drinking formula composition, or the milk protein-containing component of the infant formula composition, may be subjected to a microfiltration step followed by a mild heat treatment.

[0018] The composition may contain, for example, 0.01 to 0.03 g / mL of the food allergen. In some embodiments, the total amount of the food allergen in the composition is 0.5 to 5 grams per serving, and preferably, the serving size is 15 to 250 mL.

[0019] The food allergen may be any known food allergen. Preferably, the food allergen is selected from the group consisting of milk, egg, cereal (wheat, rye, barley, oat) proteins, soybeans, peanuts, nuts (almonds, hazelnuts, walnuts, cashews, pecans, Brazil nuts, pine nuts, pistachios, macadamia nuts, etc.), fish, crustaceans, shellfish, celery and celery root, mustard, and sesame. In some embodiments, the composition contains three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the food allergens. In one embodiment, the infant drinking composition contains milk protein and egg protein.

[0020] The composition may be for use with infants aged 0 to 12 months, 6 weeks to 12 months, 0 to 6 months, 6 weeks to 6 months, 0 to 4 months, or 6 weeks to 4 months. In one embodiment, the composition is for use with infants aged from about 1 month to about 8 months, e.g., from about 1 month to about 7 months, or from about 1 month to about 6 months. In one embodiment, the composition is for use with infants aged from about 1 month to about 4 months, or from about 1 month to about 3 months.

[0021] The infant drinking composition may further comprise one or more carriers, preferably the carriers are skim milk powder and / or lactose.

[0022] The infant drinking composition may further comprise probiotics and / or human milk oligosaccharides.

[0023] In one embodiment, at least 20%, preferably at least 30% of the food allergens are unmodified.

[0024] In one aspect, the present invention provides an infant drinking composition as defined herein for use in reducing or preventing allergy to said food allergen in an infant.

[0025] In another aspect, the present invention provides a method for reducing or preventing allergies in infants by administering an effective amount of an infant drink composition as defined herein.

[0026] In another aspect, the present invention provides a method for the manufacture of an infant drinkable composition as defined herein, comprising the steps of: i) blending two or more food allergens to form a mixture, one of the allergens being a milk protein; ii) homogenizing the mixture; iii) sterilizing the mixture; iv) optionally spray drying the mixture; Preferably, a method is provided wherein the sterilization is carried out at a temperature of 61.9°C to 65°C, preferably 62°C to 64°C, and preferably the sterilization is carried out for at least 30 minutes or at least 35 minutes.

[0027] In another aspect, the present invention provides a method for the manufacture of an infant drinkable composition as defined herein, comprising the steps of: i) blending two or more food allergens to form a mixture, one of the allergens being a milk protein; ii) homogenizing the mixture; iii) sterilizing the mixture; iv) optionally spray drying the mixture. The sterilization may be by indirect ultra-high temperature processing as described herein.The sterilization may be by direct ultra-high temperature processing as described herein. DETAILED DESCRIPTION OF THE INVENTION

[0028] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes" or "containing" or "contains" and are inclusive, i.e., open-ended, and do not exclude additional, unrecited components, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0029] Infant drinking composition As used herein, the term "infant drinking composition" means a composition suitable for consumption by an infant. Examples of infant drinking compositions include, but are not limited to, infant nutritional supplements and infant formulas.

[0030] Infants under 4 months of age are usually unable to eat solid food.Therefore, the introduction of food allergens through solid food is impossible, and it is advantageous to ingest food allergens in liquid form.Therefore, in a preferred embodiment, the infant drinking composition can be prepared and / or ingested in liquid form.In some embodiments, the infant drinking composition is intended for use with infants who cannot eat solid food.

[0031] In a preferred embodiment, the infant drinking composition is a nutritional supplement for infants. The nutritional supplement can be provided in addition to breast milk and / or infant formula. Thus, in a preferred embodiment, the infant drinking composition is a nutritional supplement for infants suitable for consumption by infants aged 0 to 12 months, 6 weeks to 12 months, 0 to 6 months, 6 weeks to 6 months, or 4 months to 6 months, preferably 0 to 4 months or 6 weeks to 4 months. In one embodiment, the composition is for use in infants aged from about 1 month to about 8 months, e.g., from about 1 month to about 7 months, or from about 1 month to about 6 months. In one embodiment, the composition is for use in infants aged from about 1 month to about 4 months, or from about 1 month to about 3 months.

[0032] The infant nutritional supplement may further comprise one or more of the following (in addition to those provided by the source of the food allergen): protein; fat (lipids); carbohydrates; and essential vitamins and minerals. The infant nutritional supplement may further comprise sweeteners, flavoring agents, and / or coloring agents.

[0033] In other embodiments, the infant drinking composition does not contain added protein, fat, carbohydrates, and / or essential vitamins and minerals. For example, in some embodiments, the infant drinking composition does not contain any ingredients other than a source of food allergens and, if included, optionally provided by one or more carriers.

[0034] In another embodiment, the infant drinking composition is an infant formula or a follow-on formula. The expression "infant formula" refers to a food product intended for the nutritional intake of infants, particularly those aged 4 to 6 months, which food product itself meets the nutritional requirements of infants at this age. The expression "follow-on formula" refers to a food product intended for the nutritional intake of infants, particularly those aged 4 months and older, which constitutes the main liquid component of the increasingly diverse diet of this population.

[0035] The requirements for infant formulas are known to those skilled in the art. For example, recommendations and requirements are provided by the European Society for Pediatric Gastroenterology and Hepatology (ESPGHAN), e.g., Koletzko, B., et al., 2005. "Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group," Journal of pediatric gastroenterology and nutrition, 41(5), pp. 584-599. Typically, a ready-to-use liquid infant formula (e.g., reconstituted from a powder) provides 60 kcal / 100 mL to 70 kcal / 100 mL. Infant formulas typically contain, per 100 kcal, about 1.8 to 4.5 g of protein; about 3.3 to 6.0 g of fat (lipids); about 300 to 1200 mg of linoleic acid; about 9 to 14 g of carbohydrates selected from the group consisting of lactose, sucrose, glucose, glucose syrup, starch, maltodextrin, and maltose, and combinations thereof; and essential vitamins and minerals.

[0036] In some embodiments, the infant drinking composition further comprises one or more carriers. As used herein, the term "carrier" refers to any useful substance such as an excipient, filler, extender, diluent, colorant, stabilizer, thickener, binder, flavoring agent, etc. Preferably, the one or more carriers include skim milk powder and / or lactose. Most preferably, the carrier is skim milk powder and / or lactose.

[0037] In some embodiments, the infant drinking composition further comprises probiotics and / or human milk oligosaccharides (eg, prebiotics) and / or postbiotics.

[0038] The term "probiotic" refers to microbial cell preparations or components of microbial cells that have a beneficial effect on the health or well-being of the host (Salminen, S. et al. (1999) Trends Food Sci. Technol. 10:107-10).

[0039] Specifically, probiotics can improve intestinal barrier function (Rao, RK (2013) Curr. Nutr. Food Sci. 9:99-107).

[0040] Examples of probiotic microorganisms for use in the compositions of the present invention include yeasts such as Saccharomyces, Debaryomyces, Candida, Pichia and Torulopsis, and bacteria such as Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

[0041] Specific examples of suitable probiotic microorganisms are Saccharomyces cerevisiae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei shirota, Lactobacillus curvatus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus casei ... Lactobacillus falciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus salmonis, Lactobacillus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosis, and Staphylococcus xylosus.

[0042] Exemplary probiotic bacterial strains include Lactobacillus rhamnosus; Lactobacillus rhamnosus LPR (CGMCC1.3724); Bifidobacterium lactis BL818 (CNCM1-3446), sold under the trade name BB12 by Christian Hansen (Denmark), among others, and Bifidobacterium longum BL999 (ATCC BAA-999), sold under the trade name BB536 by Morinaga Milk Industry.

[0043] Prebiotics are typically indigestible, meaning they are not broken down or absorbed in the stomach or small intestine. Thus, they remain intact when they enter the large intestine and are selectively fermented by beneficial bacteria in the large intestine. Examples of prebiotics include certain oligosaccharides, such as fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, or any mixture thereof. In certain embodiments, the prebiotic may be fructooligosaccharides and / or inulin. One example is a combination of 70% short-chain fructooligosaccharides and 30% inulin, which is registered by Neste under the trademark "Prebio1."

[0044] In certain embodiments, the prebiotic may be human milk oligosaccharides. Human milk oligosaccharides (HMOs) are the third most abundant solid component of human breast milk overall, after lactose and fat. HMOs typically contain lactose at the reducing end of the carbohydrate core and often contain fucose or sialic acid at the non-reducing end. Approximately 100 milk oligosaccharides have been isolated and characterized. Many infant formulas have been developed for different purposes using HMO components, such as fucosylated oligosaccharides, lacto-N-tetraose, lacto-N-neotetraose, or sialylated oligosaccharides.

[0045] Postbiotics are non-viable bacterial products or metabolic by-products derived from probiotic microorganisms that have biological activity in the host. Exemplary postbiotics include bioactive components produced during fermentation, such as short-chain fatty acids, enzymes, peptides, polysaccharides, cell surface proteins, or vitamins. Postbiotics can support immune function through the intestine.

[0046] Infant drinking composition can be in powder form, and this powder form can be ingested after reconstitution into liquid form.In a preferred embodiment, infant drinking composition is ingested after reconstitution with water.Preferably, composition can be reconstituted with water and provide one serving.

[0047] In another preferred embodiment, the composition is in the form of a ready-to-drink. The ready-to-drink composition can be provided in a bottle. Preferably, the bottle provides a single serving of food allergen.

[0048] As used herein, the term "serving" refers to the amount recommended for consumption by one infant in one supplementation. Those skilled in the art will recognize that the specific serving or dose size will vary depending on various factors, such as age, weight, general health, diet, and time of administration. Thus, in some embodiments, a serving can provide 0.5-5 g, 1-4 g, or 2-3 g of total food allergens. A serving may comprise, for example, 15-250 mL, 35-200 mL, 50-150 mL, 75-125 mL, 10-100 mL, 50-90 mL, 60-80 mL, or about 70 mL of the infant drinking composition. In one embodiment, a serving provides 0.5-5 g of total food allergens in 15-250 mL or 50-150 mL.

[0049] In some embodiments, the infant drink composition contains 0.01-0.1 g / mL, preferably 0.01-0.05 g / mL, or most preferably 0.01-0.03 g / mL of total food allergens.

[0050] Food allergens As used herein, the term "food allergen" refers to a protein or its derivative that triggers an abnormal immune response. Purified food allergens can be named using the systematic nomenclature of the Allergen Nomenclature Sub-Committee of the World Health Organization and the International Federation of Immunological Societies. Allergen names consist of an abbreviation of the scientific name of the source (genus: 3-4 letters; species: 1-2 letters) and an Arabic numeral. For example, Der p 1 is the first allergen reported from the house dust mite Dermatophagoides pteronyssinus. Food allergens are derived from proteins with various biological functions, including proteases, ligand-binding proteins, structural proteins, pathogenesis-related proteins, lipid transfer proteins, profilins, and calcium-binding proteins.

[0051] A list of food allergens is provided on the official website of the WHO / IUIS Allergen Nomenclature Database, http: / / www.allergen.org / index.php. (Radauer, C., et al., 2014. Allergy, 69(4), pp. 413-419 and Pomes, A., et al., 2018. Molecular immunology).

[0052] The present invention provides a drinkable composition for infants containing two or more food allergens derived from different food materials, one of which is milk. Preferably, the one or more other food allergens are selected from the group consisting of egg, cereal protein (wheat, rye, barley, oat), soybean, peanut, nuts (almonds, hazelnuts, walnuts, cashews, pecans, Brazil nuts, pine nuts, pistachios, macadamia nuts, etc.), fish, crustaceans, shellfish, celery and celery root, mustard, and sesame.

[0053] In some embodiments, the infant drinkable composition comprises three or more of the food allergens (i.e., three or more selected from the group consisting of milk protein, egg protein, wheat protein, soy protein, peanut protein, nut protein, fish protein, crustacean protein, shellfish protein, and sesame protein), or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more of the food allergens.

[0054] In one embodiment, the infant drinking composition comprises milk protein and egg protein. In one embodiment, the infant drinking composition comprises milk protein and peanut protein. In one embodiment, the infant drinking composition comprises milk protein and nut protein. In one embodiment, the infant drinking composition comprises milk protein and wheat protein. In one embodiment, the infant drinking composition comprises milk protein and fish protein. In one embodiment, the infant drinking composition comprises milk protein and soy protein. In one embodiment, the infant drinking composition comprises milk protein and shellfish protein. In one embodiment, the infant drinking composition comprises milk protein and shellfish protein. In one embodiment, the infant drinking composition comprises milk protein and sesame protein. In one embodiment, the infant drinking composition comprises milk protein, egg protein and peanut protein. In one embodiment, the infant drinking composition comprises milk protein, egg protein, peanut protein and nut protein. In one embodiment, the infant drinking composition comprises milk protein, egg protein and fish protein. In one embodiment, the infant drinking composition comprises milk protein, egg protein, peanut protein, nut protein and fish protein. In one embodiment, the infant drinking composition comprises egg protein, wheat protein, soy protein, peanut protein, nut protein, fish protein, crustacean protein, shellfish protein and sesame protein. In one embodiment, the infant drinking composition does not contain any additional food allergens other than those mentioned herein.

[0055] In some embodiments, the amount of each food allergen is about the same, for example, one serving of the infant drink composition may contain about 0.01 g to about 1 g, about 0.05 g to about 0.5 g, or about 0.1 g to about 0.5 g of each food allergen.

[0056] Food allergens, such as peanut allergens, may be mixtures of proteins. In some embodiments, the food allergens include one or more, two or more, three or more, four or more, substantially all, or all of the allergenic components of the food product. For example, in some embodiments, the milk proteins include one or more, two or more, three or more, four or more, substantially all, or all of the allergenic proteins derived from milk. Examples of known allergenic proteins for specific food products are known to those skilled in the art.

[0057] Milk protein Cow's milk is the most common allergen in food allergies in infants, affecting 1.4% to 3.8% of infants (Du Toit, G., et al., 2016. Allergology International, 65(4), pp. 370-377.). This allergy can be IgE-mediated, with immediate reactions such as urticaria, angioedema, and / or anaphylaxis, or non-IgE-mediated, often manifesting with skin or gastrointestinal symptoms (Du Toit, et al.).

[0058] Early exposure to cow's milk proteins has been shown to protect against IgE-mediated cow's milk protein allergy (Katz, Y., et al., 2010. Journal of Allergy and Clinical Immunology, 126(1), pp.77-82).

[0059] Milk allergens (including those that elicit an IgE-mediated response) are known in the art and are described, for example, in Wal, JM, 2002. Annals of Allergy, Asthma & Immunology, 89(6), pp. 3-10 and provided by the WHO / IUIS Allergen Nomenclature Database.

[0060] The dairy protein is preferably a cow's milk protein, and in some embodiments the dairy protein comprises one or more proteins selected from the list consisting of α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), bovine serum albumin (Bos d 6), immunoglobulin (Bos d 7), casein (Bos d 8), such as αS1-casein (Bos d 9), αS2-casein (Bos d 10), β-casein (Bos d 11), and κ-casein (Bos d 11).

[0061] Egg protein Egg allergy is the second most common food allergy, with a prevalence of approximately 2.5% (Du Toit, et al.). Early egg introduction has been shown to reduce the prevalence of egg allergy (Perkin, MR, et al., 2016. Journal of Allergy and Clinical Immunology, 137(5), pp.1477-1486.). The Enquiring About Tolerance (EAT) study found that the incidence of egg allergy was 5.4% in the standard introduction group and 3.7% in the early introduction group (a relative reduction of 31%).

[0062] Egg allergens are known in the art and are described, for example, in Amo, A., et al., 2010. Journal of agricultural and wood chemistry, 58(12), pp. 7453-7457, and are listed in the WHO / IUIS allergen nomenclature database.

[0063] The egg protein may be a hen egg protein, hi some embodiments, the egg protein comprises one or more proteins selected from the list consisting of ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3), and lysozyme C (Gal d 4). In some embodiments, the egg proteins comprise one or more proteins selected from the list consisting of ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3), lysozyme C (Gal d 4), α-livetin / serum albumin (Gal d 5), egg yolk glycoprotein 42 (YGP42 Gal d 6), myosin light chain 1f (Gal d 7), α-parvalbumin (Gal d 8), β-enolase (Gal d 9), and aldolase (Gal d 10).

[0064] peanut protein Peanut allergy, although less common than milk or egg allergies, can lead to life-threatening anaphylaxis. Results from the Identifying Infants at High Risk for Peanut Allergy (LEAP) study demonstrated that in this cohort of high-risk atopic children, early induction and regular peanut ingestion resulted in a significant reduction (81% relative reduction, intention-to-treat analysis) in the number of children at 60 months with peanut allergy compared with children who avoided peanuts (Du Toit, G., et al., 2013. Journal of Allergy and Clinical Immunology, 131(1), pp. 135-143). The EAT trial also demonstrated a lower prevalence of peanut allergy in the early induction group compared with the standard induction group (0% vs. 2.5%, p=0.003).

[0065] Peanut allergens are known in the art, for example, they are described in Krause, S., et al., 2009. Journal of Allergy and Clinical Immunology, 124(4), pp. 771-778 and are presented in the WHO / IUIS allergen nomenclature database.

[0066] In some embodiments, the peanut seed is Arachis hypogaea. In some embodiments, the peanut protein comprises one or more proteins selected from the list consisting of cupin (vicilin-type, 7S globulin, Ara h 1, and legumin-type, 11S globulin, glycine, Ara h 3), 2S albumin (Ara h 2, 6, 7), profilin (Ara h 5), pathogenesis-related protein, PR-10 (Ara h 8), nonspecific lipid transfer protein type 1 (Ara h 9, Ara h 16, Ara h 17), oleosin (Ara h 10, Ara h 11, Ara h 14, and 15), and defensin (Ara h 12 and 13).

[0067] Wheat protein It has been reported that delaying initial exposure to cereal grains beyond 6 months of age may increase the risk of developing wheat allergy (Poole, JA, et al., 2006. Pediatrics, 117(6), pp.2175-2182.). In a study by Poole et al., the prevalence of wheat allergy reported by parents was 1%, and four of the children surveyed (0.25%) had detectable wheat-specific IgE antibodies. In all four of these children, the first exposure to cereal grains occurred after 6 months of age. Therefore, early introduction of wheat can reduce the risk of developing wheat allergy.

[0068] Wheat allergens are known in the art and are described, for example, in Tatham, AS and Shewry, PR, 2008. Clinical & Experimental Allergy, 38(11), pp. 1712-1726 and presented in the WHO / IUIS allergen nomenclature database.

[0069] In some embodiments, the wheat species is Triticum aestivum. In some embodiments, the wheat protein comprises one or more proteins selected from the list consisting of non-specific lipid transfer protein 1 (Tri a 14), β-amylase (Tri a 17), agglutinin isolectin 1 (Tri a 18), omega-5 gliadin (Tri a 19), gamma gliadin (Tri a 20), thioredoxin (Tri a 25), high molecular weight glutenin (Tri a 26), low molecular weight glutenin GluB3-23 (Tri a 36), alpha purothionin (Tri a 37), mitochondrial ubiquitin lipase activator of NFKB1 (Tri a 41), Tri a 42, and Tri a 43 (hypothetical proteins from cDNA), endosperm transport cell-specific PR60 precursor (Tri a 44), elongation factor 1 (Tri a 45).

[0070] soy protein Approximately 0.4% of infants in the United States are affected by soy allergies (Kattan, JD and Sampson, HA, 2015. The Journal of Allergy and Clinical Immunology: In Practice, 3(6), pp.970-972). Early introduction of soy can reduce the risk of developing soy allergies.

[0071] Soybean allergens are known in the art and are described, for example, in Kattan, JD and Sampson, HA, 2015. Journal of Allergy and Clinical Immunology: In Practice, 3(6), pp. 970-972, and are presented in the WHO / IUIS allergen nomenclature database.

[0072] In some embodiments, the soybean seed is Glycine max. In some embodiments, the soy protein comprises one or more proteins selected from the list consisting of profilin (Gly m 3), pathogenesis-related protein PR-10 (Gly m 4), β-conglycinin (Gly m 5), glycinin (Gly m 6), seed biotinylated protein (Gly m 7), and 2S albumin (Gly m 8).

[0073] Nut and seed protein Early introduction of nuts and seeds can reduce the risk of developing nut allergies (Frazier, AL, et al., 2014. JAMA pediatrics, 168(2), pp.156-162.). For example, Frazier et al. reported that among mothers without nut allergies, children whose mothers consumed a lot of nuts during pregnancy had a lower risk of developing nut allergies. This report supports the hypothesis that early allergen exposure increases tolerance and reduces the risk of food allergies in childhood.

[0074] Nut and seed allergens are known in the art and are described, for example, in Roux, KH, et al., 2003. International archives of allergy and immunology, 131(4), pp. 234-244, and are presented in the WHO / IUIS allergen nomenclature database. In some embodiments, the nut species is selected from one or more of the group consisting of hazelnuts, walnuts, cashews, almonds, pecans, chestnuts, Brazil nuts, pine nuts, macadamia nuts, pistachios, coconuts, nangai nuts, and acorns. In some embodiments, the nut species is hazelnuts, walnuts, cashews, and almonds. In some embodiments, the nut protein comprises one or more proteins selected from the list consisting of lipid transfer protein, profilin, a member of the Bet v1-related family, legumin, vicilin, and albumin. In some embodiments, the nut protein comprises one or more proteins selected from the list consisting of Cor a 1, 2, 8, 9, 11-14 (hazelnut); Jug n 1, 2, 4 (black walnut); Jug r 1-8 (western walnut); Ana o 1-3 (cashew); Pru du 3-6 and Pru du 8 (almond); Car i 1, 2, 4 (pecan); Cas s 5, 8, 9 (chestnut); Ber e 1, 2 (Brazil nut); and Pis v 1-5 (pistachio). In some embodiments, the nut protein comprises one or more proteins selected from the list consisting of Cor a 1, 2, 8, 9, 11-14; Jug n 1, 2, 4; Jug r 1-8; Ana o 1-3; Pru du 3-6.

[0075] Fish protein Early introduction of fish can reduce the risk of developing fish allergy (Kull, I., et al., 2006. Allergy, 61(8), pp. 1009-1015.) For example, Kull et al. reported that regular fish consumption up to the age of one appears to be associated with a reduced risk of allergic disease and sensitization to food allergens and inhalant allergens during the first four years of life.

[0076] Fish allergens are known in the art and are described, for example, in Poulsen, LK, et al., 2001. Allergy, 56, pp. 39-42, and are presented in the WHO / IUIS allergen nomenclature database.

[0077] In some embodiments, the fish species is selected from one or more of the group consisting of cod, herring, trout, tuna, salmon, haddock, chub mackerel, mackerel, eel, sea perch, horse mackerel, sardine, perch, plaice, flounder, plaice, cuttlefish, halibut, hake, megrim, swordfish, anchovy, pike, and carp. In some embodiments, the fish species is selected from one or more of the group consisting of cod, herring, plaice, and mackerel. In some embodiments, the fish species is cod. In some embodiments, the fish protein comprises one or more proteins selected from the list consisting of Gad c 1, Clu h 1, and Ras k 1.

[0078] Crustacean and shellfish proteins The most recent data on prevalence in Asia highlight seafood as a significant sensitizing factor, with a prevalence of up to 40% in children and 33% in adults (Lopata, AL and Lehrer, SB, 2009. Current opinion in allergy and clinical immunology, 9(3), pp. 270-277.). Early introduction of crustaceans or shellfish can reduce the risk of developing shellfish allergy or crustacean allergy (Fleischer, DM, et al., 2013. The Journal of Allergy and Clinical Immunology: In Practice, 1(1), pp. 29-36).

[0079] Crustacean and shellfish allergens are known in the art and are described, for example, in Lopata, AL, et al., 2010. Clinical & Experimental Allergy, 40(6), pp. 850-858, and are presented in the WHO / IUIS allergen nomenclature database.

[0080] In some embodiments, the crustacean species is selected from one or more of the group consisting of crab, lobster, shrimp, and prawn. In some embodiments, the shellfish species is selected from one or more of the group consisting of abalone, snail, whelk, clam, oyster, scallop, mussel, cockle, squid, and octopus. In some embodiments, the crustacean protein comprises one or more proteins selected from the list consisting of tropomyosin (Cha f 1, Cra c 1, Exo e 1, Hom a 1, Lit v 1, Pan s 1, Mac r 1, Mel l 1, Met e 1, Pan b 1, Pen i 1, Pen m 1, Por p 1, Pro c 1), myosin light chain 2 (Hom a 3), and troponin C (Hom a 6). In some embodiments, the shellfish protein comprises one or more proteins selected from the list consisting of Hal m 1 and tropomyosin (Hel as 1, Cra g 1, Sac g 1, Tod p 1).

[0081] Sesame Protein Allergy to sesame, which has only recently been recognized as an allergen in many cases, is estimated to affect 0.10-0.79% of children in studies outside the United States (Sicherer, SH, et al., 2010. Journal of Allergy and Clinical Immunology, 125(6), pp. 1322-1326). Early introduction of sesame can reduce the risk of developing sesame allergy. For example, sesame has been selected as an allergen during the EAT trial (Perkin, MR, et al., 2016. Journal of Allergy and Clinical Immunology, 137(5), pp. 1477-1486).

[0082] Sesame allergens are known in the art and are described, for example, in Beyer, K., et al., 2002. Journal of Allergy and Clinical Immunology, 110(1), pp. 154-159, and are presented in the WHO / IUIS allergen nomenclature database.

[0083] In some embodiments, the sesame seed is Sesamum indicum. In some embodiments, the sesame protein comprises one or more proteins selected from the list consisting of 2S albumin (Ses i 1, 2); 7S vicilin-like globulin (Ses i 3), oleosin (Ses i 4, 5), and 11S globulin (Ses i 6, 7).

[0084] Heat treatment The compositions of the present invention are preferably subjected to mild heat treatment, such as mild pasteurization and / or sterilization. Reducing the holding temperature and / or holding time during heat treatment can reduce the degree of denaturation of allergens.

[0085] "Pasteurization" refers to the partial sterilization of a substance, especially a liquid (such as milk). Standard sterilization conditions are known to those skilled in the art; for example, for sterilization of milk, standard high-temperature, short-time (HTST) sterilization is typically used at a temperature of about 72°C for 15 seconds. The temperature of sterilization is also called the holding temperature, and this temperature remains constant over the holding time.

[0086] In a preferred embodiment, the infant drinking composition has undergone mild sterilization, which reduces thermal damage during sterilization, such as denaturation of allergens. Mild sterilization can be achieved by reducing the holding temperature and / or holding time. Thus, in some embodiments, sterilization is performed at a holding temperature of 72°C to 61.9°C, 70°C to 61.9°C, 68°C to 61.9°C, 66°C to 61.9°C, 65°C to 61.9°C, 64°C to 61.9°C, for example, about 63°C.

[0087] Suitable holding times may be at least 15, 20, 25, 30, or 35 minutes. For example, sterilization can be carried out at 72°C to 61.9°C for 15 to 45 minutes, or at 65°C to 61.9°C for 20 to 40 minutes, preferably at about 63°C for about 35 minutes.

[0088] In some embodiments, the mild heat treatment can be carried out at a temperature of 72°C to 90°C, for example, 72°C to 80°C, for 10 to 30 seconds, or at 80°C to 89°C for 2 to 20 seconds, for example, 80°C to 84°C for 4 to 20 seconds, or at 85°C to 89°C for 1 to 10 seconds.

[0089] In some embodiments, the infant drinking composition or milk protein-containing ingredient is optionally microfiltered before a mild heat treatment, such as mild pasteurization. Suitable microfiltration techniques are known in the art. The use of a microfiltration step can advantageously reduce bacterial load before the pasteurization step, allowing for reduced temperatures and / or shorter holding times. In some embodiments of the present invention, the infant drinking composition has been sterilized by ultra-high temperature (UHT) heat treatment. In preferred embodiments, the sterilization conditions are selected to minimize heat damage (e.g., denaturation of allergens). Such sterilization can be achieved by using a reduced holding temperature and / or a shortened holding time.

[0090] In some embodiments of the present invention, the UHT heat treatment is an indirect heat treatment. In indirect heat treatment, a heat exchanger is used to raise the temperature of the liquid being sterilized to a holding temperature. Indirect UHT heat treatment can be used to minimize heat damage by reducing the holding temperature, as described, for example, in U.S. Pat. No. 4,534,986(A). In some embodiments of indirect UHT heat treatment, the holding temperature is 125°C to 135°C, or 130°C to 134°C, or 131°C to 133°C. In some embodiments of indirect UHT heat treatment, the holding time is at least 30 seconds, or at least 60 seconds, e.g., 30 to 80 seconds, or 60 to 75 seconds. For example, indirect UHT heat treatment is performed at 131°C to 133°C for about 60 to 75 seconds.

[0091] In some embodiments of the present invention, the UHT heat treatment is a direct heat treatment. In direct heat treatment, superheated steam is directly mixed (e.g., injected) into the liquid. Direct heating may be performed for shorter periods of time, which also minimizes thermal damage. In some embodiments, the direct UHT heat treatment is performed at a temperature of 136°C to 140°C for about 15 to 25 seconds, or at a temperature of 140°C to 144°C for about 5 to 10 seconds, or at a temperature of 150°C to 154°C for about 2 to 4 seconds.

[0092] In some embodiments, the sterilization is an ultra-short time sterilization (USS) heat treatment at a temperature of 155°C to 170°C for less than 1 second.

[0093] In some embodiments of the present invention, the infant drinking composition is heat sterilized and then aseptically filled.

[0094] In some embodiments of the present invention, at least 20%, preferably at least 30%, of the two or more food allergens in the sterilized nutritional composition according to the present invention are unmodified. A high proportion of unmodified food allergens may be the result of a heat treatment described herein that reduces the holding temperature and / or shortens the holding time.

[0095] In some embodiments of the invention, at least 20%, preferably at least 30% of the milk proteins are native. In some embodiments of the invention, at least 40% of the milk proteins are native. In some embodiments of the invention, at least 50% of the milk proteins are native.

[0096] In some embodiments, at least 30% of each of the two or more food allergens is native, e.g., 30% to 100%, or 30% to 95%, or 30% to 90%, or 40% to 80%, or 50% to 60% of the food allergens are native.

[0097] According to the present invention, a "denatured" protein is one in which the three-dimensional structure of the protein is perturbed or disrupted. Thus, typically in a denatured protein, one or more of the interactions consisting of hydrogen bonds, salt bridges, disulfide bonds, and non-polar hydrophobic interactions are perturbed. A "denatured" protein typically has its primary structure (i.e., peptide bonds) intact.

[0098] The proportion of native food allergens can be determined by any method known to those skilled in the art, such as high pressure liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), bicinchoninic acid assay (BCA), Kjeldahl nitrogen (KN), circular dichroism analysis (CD), native polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), Fourier transform infrared spectroscopy (FTIR), or fluorescence spectroscopy.

[0099] If the nutritional composition contains two or more food allergens, each food allergen can be separated and analyzed individually. The food allergens can also be separated and analyzed together. In some embodiments, all food allergens, on average (mean), are native to the extent specified herein.

[0100] Any method known to those skilled in the art can be used to separate food allergens from nutritional compositions. Examples include HPLC, FPLC, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, free-flow electrophoresis, and affinity chromatography. These are described, for example, in Scopes, RK, 2013. Protein purification: principles and practice. Springer Science & Business Media. HPLC allows separation of compounds based on their interaction with a stationary phase. Common HPLC methods include reversed-phase partition HPLC (RP-HPLC) and size exclusion HPLC.

[0101] In some embodiments of the present invention, the Rowland method is used to determine the percentage of intact food allergens. Preferably, such food allergens are milk allergens.

[0102] In the Rowland method, undenatured whey protein nitrogen (serum protein nitrogen, SPN) is defined as the nitrogen not precipitated by acetic acid and sodium acetate (non-casein nitrogen, NCN) minus non-protein nitrogen (NPN), where SPN = NCN - NPN (Rowland, SJ, 1938. 175. Journal of Dairy Research, 9(1), pp. 30-46, and Rowland, SJ, 1938. 176). NCN and total nitrogen (TN) are determined by the Kjeldahl method. NCN is determined from the filtrate after precipitation of SPN.

[0103] Therefore, in the case of milk allergens, the amount of undenatured milk allergen protein can be expressed as the percentage of serum protein nitrogen (undenatured whey protein nitrogen) "SPN" in total protein: SPN (% of total protein) = ((NCN - NPN) / (TN - NPN)) x 100.

[0104] In some embodiments of the present invention, the percentage of intact food allergens is determined using Kjeldahl nitrogen, which is a known method for determining the degree of protein denaturation (Parris, N. and Baginski, MA, 1991. Journal of Dairy Science, 74(1), pp. 58-64).

[0105] In some embodiments of the invention, at least 20%, preferably at least 30% of the milk proteins are undenatured.

[0106] Preventing food allergies The present invention provides an infant drinking composition as described herein for use in reducing or preventing food allergies in infants, in particular allergies to milk protein, egg protein, wheat protein, soy protein, peanut protein, nut protein, fish protein, crustacean protein, shellfish protein and sesame protein.

[0107] The present invention also provides a method for reducing or preventing food allergies in infants by administering an effective amount of the infant drinking composition described herein.

[0108] In some embodiments, the allergic response is a specific IgE-related immune response and / or a T cell-mediated hypersensitivity response. Thus, in some embodiments, reducing or preventing allergy comprises reducing or preventing a specific IgE-related immune response and / or a T cell-mediated hypersensitivity response.

[0109] The infant drinking composition may contain milk proteins to prevent or reduce allergies to milk. The infant drinking composition may contain egg protein to prevent or reduce allergies to eggs. The infant drinking composition may contain wheat protein to prevent or reduce allergies to wheat. The infant drinking composition may contain soy protein to prevent or reduce allergies to soy. The infant drinking composition may contain peanut protein to prevent or reduce allergies to peanuts. The infant drinking composition may contain nut protein to prevent or reduce allergies to nuts. The infant drinking composition may contain fish protein to prevent or reduce allergies to fish. The infant drinking composition may contain shellfish protein to prevent or reduce allergies to shellfish. The infant drinking composition may contain shellfish protein to prevent or reduce shellfish allergies. The infant drinking composition may contain sesame protein to prevent or reduce allergies to sesame.

[0110] In one embodiment, preventing or reducing food allergies in infants also includes inducing cross-tolerance, which can reduce or prevent the development of allergies to food allergens other than those contained in the infant drinking composition.

[0111] Before administering the composition of the present invention, a step of assessing the risk of the infant developing the food allergy may be carried out. This step may involve administering a small amount of the allergen to the skin of a relative and / or completing a questionnaire for the relative. For example, an infant with a parent or sibling who has a food allergy may be at higher risk of developing a food allergy and may be administered a drinkable composition for infants containing the food allergen to prevent or treat the food allergy.

[0112] process The infant drinking composition according to the present invention can be prepared by any suitable method. For example, the composition can be prepared by blending appropriate amounts of food allergens together, optionally blending with one or more carriers such as an amino acid-based infant formula, and then mixing the dry-blended mixture with water to form a liquid mixture. The liquid mixture is then stirred homogeneously. The temperature is then gradually increased and sterilization is carried out. If the final product is a powder, the liquid mixture is then optionally spray-dried. The composition may be homogenized before or after sterilization.

[0113] In one aspect, the present invention provides a method for producing an infant drinkable composition, comprising the steps of: i) blending two or more food allergens to form a mixture, one of the allergens being a milk protein; ii) homogenizing the mixture; iii) sterilizing the mixture; iv) optionally spray drying the mixture; Preferably, the method provides that the sterilization is a mild sterilization as discussed herein.

[0114] In a preferred embodiment, sterilization is carried out at a temperature of 61.9°C to 65°C, preferably 62°C to 64°C, and preferably the sterilization is carried out for at least 30 minutes or at least 35 minutes. In one embodiment, sterilization is carried out at about 63°C for 30 to 35 minutes.

[0115] In some embodiments, the liquid mixture is homogenized and then pasteurized. In other embodiments, the liquid mixture is pasteurized and then homogenized.

[0116] In one aspect, the present invention provides a method for producing an infant drinkable composition, comprising the steps of: i) blending two or more food allergens to form a mixture, one of the allergens being a milk protein; ii) homogenizing the mixture; iii) sterilizing the mixture; iv) optionally spray drying the mixture; Preferably, the sterilization is as discussed herein. Preferably, if the infant drinking composition is in powder form, the homogenized liquid mixture is dried, for example spray dried.

[0117] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods, processes, and compositions discussed in this invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to those skilled in the art, are intended to be within the scope of the following claims.

[0118] Preferred features and embodiments of the present invention will now be described by way of non-limiting examples. [Example]

[0119] Example 1. Infant nutritional supplement A drinkable infant nutritional supplement is prepared for clinical trials. In this example, the nutritional supplement contains milk protein and egg white protein in combination with an amino acid-based infant formula (Alfamino Infant Formula (Nestlé)).

[0120] The dietary supplement composition (based on 3.1% moisture by weight in the final product) comprises: Skim milk powder 43.2% by weight, Alfamino (amino acid-based hypoallergenic infant formula) 37.3% by weight, Egg white powder 16.4% by weight.

[0121] The composition is processed by standard processing steps, including homogenization at 120 / 30 bar and mild pasteurization. Optionally, the process can include spray drying to provide a powder composition ready for reconstitution.

[0122] Pasteurization is carried out at a hold temperature of 63°C for a hold time of 2100 seconds to ensure that egg proteins are not significantly damaged. A critical limit (i.e., minimum hold temperature) is set at 61.9°C to ensure safety.

[0123] Example 2. Gentle sterilization The milk protein-containing dietary supplement was subjected to mild pasteurization as defined above, and the degree of denaturation was measured using the Rowland method.

[0124] The degree of denaturation is expressed as the ratio of serum protein nitrogen (undenatured whey protein nitrogen) "SPN" to total protein = ((NCN-NPN) / (TN-NPN)) x 100.

[0125] NCN = non-casein nitrogen; NPN = non-protein nitrogen; TN = total nitrogen. Before heat treatment, the percentage of undenatured milk protein was 45%. After mild pasteurization, the percentage of native protein dropped to 36%, thus leaving a significant amount of native milk protein.

[0126] Example 3. Effect of heat treatment on protein degradation Unprocessed skim milk powder (24% total solids) was subjected to the following steps: (i) hot microfiltration (14 μm filter at 52° C.) and the permeate was heat treated by direct steam injection (DSI) at 83° C. for 6 seconds followed by spray drying to form powder "Prototype A"; (ii) cold microfiltration (14 μm filter at 15° C.) and the permeate was heat treated by direct steam injection (DSI) at 83° C. for 6 seconds followed by spray drying to form powder "Prototype B"; and (iii) mild pasteurization (as specified in Example 1) at 63° C. for 35 minutes followed by spray drying "Prototype C".

[0127] Raw (unprocessed) milk (9-10%, TS) samples 1-16 (Table 1) were subjected to the selected heat treatments as described in Table 1, with or without homogenization prior to the heat treatment. The homogenized milk samples (homogenized at 150 / 30 bar) were spray dried to obtain the final milk powder.

[0128] The Rowland method was used to measure the degree of protein denaturation for Prototype A, Prototype B, and Prototype C, as well as for the heat-treated milk samples (heat treatments shown in Table 1). The degree of denaturation is expressed as the percentage of serum protein nitrogen (undenatured whey protein nitrogen) "SPN" to the total protein, as in Example 2. The denaturation rate is expressed as the percentage of denatured whey protein to the total protein.

[0129] Undenatured whey protein (SPN)% = ((NCN-NPN) / TN) x 100 Whey protein denaturation rate (%) = 100 - [100 x SPN] / [(TN - NPN) x 0.2] [where [(TN-NPN) x 0.2] is the total whey protein nitrogen in milk, assuming a whey:casein weight ratio of 1:4]. The results are shown in Figure 1.

[0130] [Table 1]

[0131] It can be seen from Figure 1 that high temperature long treatment (80°C, 90°C / 20 min) and UHT treatment resulted in the highest degree of whey protein denaturation. The pasteurized milk sample, HTST (high temperature short time) sterilized milk sample and prototypes A to C had similar denaturation profiles.

[0132] Example 4. Immune activity The immunological activity of the milk samples of Example 4 was assessed using a humanized rat basophilic leukemia (RBL) degranulation assay (Bioceros Holding BV).

[0133] RBL cells expressing high-affinity human FcεRI α-chains were sensitized with an oligoclonal pool of anti-BLG-chimeric human IgE antibodies (i.e., mouse IgG heavy and light chain variable regions combined with human IgE heavy and light chain constant regions, as described by Knipping & Simons, PLoS ONE 2014;9(8):e106025) and then exposed to milk samples containing BLG at different concentrations (0 μg / mL, 0.0032 μg / mL, 0.016 μg / mL, 0.08 μg / mL, 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL, and 10,000 μg / mL of protein). Degranulation of RBL cells was assessed by measuring extracellular β-hexosaminidase activity. The results are shown in FIG.

[0134] [Table 2]

[0135] "% Remaining BLG / % Initial BLG" refers to the ratio of the measured residual BLG concentration (in μg / g protein) for each milk sample divided by the initial (theoretical) BLG concentration (in μg / g protein). For the calculation of the initial (theoretical) BLG concentration, a whey:casein weight ratio of 1:4 and BLG = 50% of the total whey protein in the milk were assumed.

[0136] Heat treatment at 80°C for 20 minutes resulted in the highest residual allergenicity compared to other heat treatments, while HTST pasteurization and Prototype C resulted in very low residual allergenicity. Raw milk showed no detectable residual β-Lg allergenicity.

[0137] Example 5. Maintenance of immunogenicity A. SDS-PAGE The infant nutritional supplement "PREMEA" of Example 1 was analyzed by gel electrophoresis (SDS-PAGE). It was stored at 4°C, 25°C, and 37°C for 6 months. Gel electrophoresis was performed using a Novex NuPAGE® system (Thermo Fisher Scientific) according to the manufacturer's protocol. Precast NuPAGE® 4-12% Bis-Tris gels were used for separation in combination with MES SDS running buffer.

[0138] The results are shown in Figure 3. The following protein bands were identified based on the band assignments in Figure 3 (bands 1 to 9). Egg white powder 1. Ovomucin (less than 100 kDa) 2. Conalbumin (77.7 kDa) 3. Ovalbumin (44.5 kDa) 4. Ovomucoid (30-40 kDa) 5. Lysozyme (14.3 kDa) Skim milk powder 6. Serum albumin (66 kDa) 7. Casein (28-35kDa) 8. β-lactoglobulin (18.8 kDa) 9. α-lactalbumin (14.4 kDa)

[0139] Conclusion: Protein bands derived from egg and milk are present in the prototype. These results indicate that the major milk and egg proteins (including the most allergenic ones, such as ovomucoid or β-lactoglobulin) are not degraded by the processing of the final product. The band intensities of the milk and egg allergens in the prototype indicate that their amounts are significant in the prototype.

[0140] B. Ovomucoid quantification Egg white contains 23 different glycoproteins. Among these, ovomucoid (Gal d 1) constitutes approximately 11% of the total egg white protein and has been shown to be the major egg allergen (1, 2). To compare the immunogenicity of proteins present in infant nutritional supplements after processing with that of the raw materials used, ovomucoid was quantified as a representative egg allergen.

[0141] method: Ovomucoid was detected and quantified using a commercially available ELISA kit (BioKits Egg Assay Kit; Neogen Corporation, USA, ref. 902072T) according to the manufacturer's instructions. The polyclonal antibody used in this kit specifically detects ovomucoid (Gal d 1), which is significantly more robust for individual epitopes when assaying proteins that exhibit subtle changes such as denaturation, polymorphism, or conformational changes.

[0142] The raw egg white material used to make the prototypes, as well as the prototypes, were stored and analyzed for 6 months at different temperatures (4° C., 25° C., and 37° C.) The amount of ovomucoid measured in the prototypes was then back-calculated from the % egg material present in the prototypes, allowing a direct comparison between the raw material and the final product.

[0143] result: The ovomucoid content in egg white is 205.9±24.7 mg / g protein. Similar ovomucoid contents were determined in prototypes stored at 4°C, 25°C, and 37°C (158.3±43.5 mg / mL; 175.8±42.7 mg / mL, and 222.1±20.9 mg / mL, respectively).

[0144] Conclusion: Quantitative analysis showed that there was little difference in immunogenicity (epitopes recognized by anti-ovomucoid polyclonal antibodies) between raw egg white material and the final processed product stored for 6 months at 4°C, 25°C, and 37°C. These results demonstrate that the ovomucoid in the samples was not degraded during storage.

[0145] Example 6. Quantitation of β-lactoglobulin and ovalbumin Three samples of raw milk (unprocessed milk) and raw egg white mixtures were prepared by mixing three different amounts of milk with one amount of egg white. The protein content was calculated as 100 g / L for egg white protein and 33 g / L for milk protein. The samples were pretreated at 55°C for 5 minutes, followed by mild pasteurization at different conditions: (i) 63°C for 30 minutes; (ii) 70°C for 3 minutes; and (iii) 70°C for 20 minutes. The samples had a pH of 6.7.

[0146] A set of samples of the infant nutritional supplement "PREMEA" and milk / egg white samples (i) to (iii) at pH 6.7 in Example 1 was subjected to ultracentrifugation (10,000 g / 1 hour) to remove aggregated proteins. Native proteins and soluble denatured proteins were quantified by protein separation by gel electrophoresis (SDS-PAGE) as in Example 5, followed by protein quantification in which the staining intensity of the electrophoretic bands was scanned to measure the concentration.

[0147] A second set of samples of the infant nutritional supplement "PREMEA" of Example 1 and milk / egg white samples (i)-(iii) were acidified to pH 4.6 and subsequently centrifuged at 14000g / 10 min to remove precipitates (denatured and aggregated proteins). Quantification of native protein was performed as described above.

[0148] The denaturation degree of β-lactoglobulin and ovalbumin was quantified as follows: Denaturation rate (%) = [1 - (I pH4.6 / I pH6.7 )][where I pH4.6 is the concentration of native protein and I pH6.7 is the concentration of total protein (native protein + soluble denatured protein)]. The results are shown in Tables 3, 4 and 5. Denaturation of milk / egg white proteins

[0149] [Table 3]

[0150] [Table 4]

[0151] Denaturation of milk / egg white proteins

[0152] [Table 5]

Claims

1. A drinking composition for infants comprising two or more food allergens derived from different foods, one of the food allergens being a milk protein.

2. 2. A drinking composition for infants according to claim 1, which is a powder for reconstitution with a liquid, preferably water, before administration.

3. 10. The infant drinking composition according to claim 1, which is in the form of a ready-to-drink.

4. 4. A drinking composition for infants according to any one of claims 1 to 3, wherein the composition has undergone sterilisation at a temperature of 61.9°C to 65°C, preferably 62°C to 64°C, preferably for at least 30 minutes.

5. The infant drinkable composition according to any one of claims 1 to 4, which has been subjected to heat sterilization.

6. 6. The infant drinking composition according to claim 5, wherein the heat sterilization is an indirect ultra-high temperature heat treatment, preferably at a temperature of 125°C to 135°C, or 130°C to 134°C, or 131°C to 133°C, and preferably the sterilization is carried out for at least 30 seconds, or at least 60 seconds; or the heat sterilization is a direct ultra-high temperature heat treatment, preferably at a temperature of 136°C to 140°C for about 15 to 25 seconds, or at a temperature of 140°C to 144°C for about 5 to 10 seconds, or at a temperature of 150°C to 154°C for about 2 to 4 seconds; or the sterilization is an ultra-short time sterilization (USS) heat treatment at a temperature of 155°C to 170°C for less than 1 second.

7. 7. The infant drinkable composition according to claim 1, wherein the food allergen is selected from the group consisting of egg protein, wheat protein, soy protein, peanut protein, nut protein, fish protein, crustacean protein, shellfish protein and sesame protein.

8. 8. The infant drinkable composition of claim 7, comprising three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the food allergens.

9. 9. A drinking composition for infants according to claim 7 or 8, comprising milk protein and egg protein.

10. The infant drinkable composition according to any one of claims 1 to 9, for use on infants aged 0 to 12 months, 6 weeks to 12 months, 0 to 6 months, 6 weeks to 6 months, 0 to 4 months, or 6 weeks to 4 months.

11. 11. The infant drinking composition according to any one of claims 1 to 10, wherein the total amount of the food allergen in the composition is 0.5 to 5 grams per serving, and preferably the serving size is 15 to 250 mL.

12. A drinking composition for infants according to any one of claims 1 to 11, wherein at least 20%, preferably at least 30% of the milk allergens are unmodified.

13. A drinking composition for infants according to any one of claims 1 to 12 for use in reducing or preventing allergies to said food allergens in infants.

14. A method for producing the infant drink composition according to any one of claims 1 to 13, comprising: i) blending two or more food allergens to form a mixture, wherein one of the allergens is a milk protein; ii) homogenizing the mixture; iii) sterilizing the mixture; iv) optionally spray drying the mixture; Preferably, the sterilization is carried out at a temperature of 61.9°C to 65°C, preferably 62°C to 64°C, and preferably said sterilization is carried out for at least 30 minutes or at least 35 minutes.

15. A method for producing the infant drink composition according to any one of claims 1 to 13, comprising: i) blending two or more food allergens to form a mixture, wherein one of the allergens is a milk protein; ii) homogenizing the mixture; iii) sterilizing the mixture; iv) optionally spray drying the mixture; Preferably, the sterilization is an indirect UHT heat treatment at a temperature of 125°C to 135°C, or 130°C to 134°C, or 131°C to 133°C, for 30 to 80 seconds, or 60 to 75 seconds. Alternatively, the sterilization is by direct UHT heat treatment at a temperature of 136°C to 140°C for about 15 to 25 seconds, or at a temperature of 140°C to 144°C for about 5 to 10 seconds, or at a temperature of 150°C to 154°C for about 2 to 4 seconds. Alternatively, the sterilization is an ultra-short time sterilization (USS) heat treatment at a temperature of 155°C to 170°C for less than 1 second.

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