Infant formula

Abstract

To provide nutritional compositions, such as infant formulas, that can be used to prevent or reduce the development of allergies in infants.SOLUTION: A baby formula for use in preventing or reducing the incidence of allergic sensitization in babies, wherein the baby formula comprises 0. 8-2. 5g / L 2 ' - fucosyllactose (2 ' FL) and / or 0. 05-0. 2g / L lacto-N-neotetraose (LNnT).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an infant formula for use in preventing or reducing the incidence of allergic sensitization in infants. In particular, the present invention relates to an infant formula comprising 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT). [Background technology]

[0002] The prevalence of allergic diseases such as atopic dermatitis, food allergies, and asthma is increasing worldwide. For example, 300 million people worldwide suffer from asthma, and 11 to 26 million people in the European Union have food allergies (Martins, T.B. et al. (2014) J Allergy Clin Immunol 133:589-91).

[0003] There is increasing evidence that the composition of infant gut microbiota is involved in the immune differentiation pathways and allergy development in infant hosts (Quante M. et al. (2012) BMC Public Health 12:1021). As a result, environmental factors such as diet, pollution, urban living, cleanliness, and birthing method have been linked to immune system development and the onset of allergic diseases (Seppo, AE et al. (2017) J Allergy Clin Immunol 139:708-11 e5; Azad, MB et al. (2018) J Nutr 148:1733-42).

[0004] Breast milk is an immunologically active secretion and contains many components that may regulate immune system development and, in turn, the development of allergic diseases. The influence of human milk oligosaccharides (HMOs), the third most abundant component in breast milk, on the development of allergic diseases has attracted particular interest. HMOs are structurally diverse lactose-based complex glycans containing both short- and long-chain oligosaccharides. The number of HMOs in human breast milk (more than 200 HMOs have been identified) and structural diversity have not been observed in other mammalian milks. The composition of HMOs is influenced by both environmental and genetic factors and varies greatly among maternal populations. The amount of HMOs synthesized in the mammary gland in breast milk varies from approximately 20.9 g / L in colostrum to 12.9 g / L in mature milk.

[0005] There is some in vitro evidence suggesting that HMOs can modulate allergic responses, and certain HMOs (e.g., 2'-fucosyllactose, 2'FL) have been shown to reduce allergic responses in animal models of food allergy. Furthermore, relevant studies have identified concentrations of several HMOs in breast milk that correlate with infant milk or food allergies. However, food-grade synthetic HMOs were unavailable until recently, making it impossible to test HMOs on infants in intervention studies.

[0006] Furthermore, uncertainty remains regarding the identity of specific HMOs that may be beneficial in modulating allergies, and the concentrations of HMOs that may provide beneficial effects.

[0007] Human breast milk and breastfeeding are considered the optimal form of nutrition for healthy infants in the first few months of life. However, there is a need for nutritional sources that can be used in addition to breast milk. Furthermore, breastfeeding is not always possible for infants, and breast milk cannot meet the needs of more vulnerable infants, such as premature infants, so there is a need for breast milk substitutes. Nutritional compositions, such as infant formulas, that meet the nutritional requirements of infants can be used as a substitute for or complement to human breast milk. However, the composition of infant formulas needs to be carefully controlled to meet the nutritional requirements, provide an acceptable taste, and further support the infant's development, especially when targeting allergic infants or infants at risk of allergies.

[0008] Thus, there remains a great need for nutritional compositions, such as infant formulas, that can be used to prevent or reduce the development of allergies in infants, and in particular infant formulas that are effective in preventing or reducing allergic sensitization in infants.

[0009] [Summary of the Invention] The inventors have surprisingly found that the HMOs 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) are most effective in preventing allergic sensitization at intermediate doses. The inventors have found that levels lower or higher than the optimal intermediate dose may be less beneficial.

[0010] The present inventors analyzed HMO concentrations in breast milk from a cohort of mothers and identified two HMOs, 2'FL and LNnT, as being associated with skin sensitization or skin rash. Additionally, by analyzing the distribution of HMO concentrations in clinically diagnosed populations, the present inventors found a nonlinear distribution suggesting a beneficial effect of intermediate doses. The present inventors then conducted animal model studies and confirmed that HMOs indeed exhibit a more beneficial effect at intermediate doses than at low or high doses in preventing allergic sensitization.

[0011] Thus, in one aspect, the present invention provides an infant formula for use in preventing or reducing the incidence of allergic sensitization in an infant, the infant formula comprising 0.8 to 2.5 g / L 2'-fucosyllactose (2'FL) and / or 0.05 to 0.2 g / L lacto-N-neotetraose (LNnT).

[0012] In another aspect, the invention provides a method for preventing or reducing the incidence of allergic sensitization in an infant, the method comprising administering to the infant an infant formula comprising 0.8 to 2.5 g / L of 2'-fucosyllactose (2'FL) and / or 0.05 to 0.2 g / L of lacto-N-neotetraose (LNnT).

[0013] In some embodiments, the infant formula contains 0.8-1.5 g / L, 0.8-1.4 g / L, 0.8-1.3 g / L, 0.8-1.2 g / L, 0.8-1.1 g / L, 0.9-1.1 g / L, or about 1 g / L of 2'FL. In some embodiments, the infant formula contains 0.8-1.2 g / L of 2'FL. In some embodiments, the infant formula contains 0.9-1.1 g / L of 2'FL.

[0014] In some embodiments, the infant formula comprises 1-1.5 g / L, 1-1.4 g / L, 1-1.3 g / L, 1-1.2 g / L, 1-1.1 g / L, or about 1 g / L of 2'FL. In some embodiments, the infant formula comprises 1-1.1 g / L of 2'FL.

[0015] In a preferred embodiment, the infant formula contains about 1 g / L of 2'FL.

[0016] In some embodiments, the infant formula contains 1.5-2.5 g / L, 1.5-2.4 g / L, 1.5-2.3 g / L, 1.5-2.2 g / L, 1.5-2.1 g / L, 1.5-2 g / L, 1.6-2 g / L, 1.7-1.9 g / L, or about 1.8 g / L of 2'FL. In some embodiments, the infant formula contains 1.5-2 g / L of 2'FL. In some embodiments, the infant formula contains 1.7-1.9 g / L of 2'FL.

[0017] In a preferred embodiment, the infant formula contains about 1.8 g / L of 2'FL.

[0018] In some embodiments, the infant formula includes 0.05-0.15 g / L, 0.06-0.14 g / L, 0.07-0.13 g / L, 0.08-0.12 g / L, 0.09-0.11 g / L, or about 0.1 g / L of LNnT. In some embodiments, the infant formula includes 0.08-0.12 g / L of LNnT. In some embodiments, the infant formula includes 0.09-0.11 g / L of LNnT.

[0019] In a preferred embodiment, the infant formula contains about 0.1 g / L of LNnT.

[0020] In a preferred embodiment, the infant formula comprises 2'FL and LNnT.

[0021] In some embodiments, the infant formula comprises about 1.5-2 g / L 2'FL and about 0.08-0.12 g / L LNnT. In some embodiments, the infant formula comprises about 1.7-1.9 g / L 2'FL and about 0.09-0.11 g / L LNnT. In some embodiments, the infant formula comprises about 1.8 g / L 2'FL and about 0.1 g / L LNnT.

[0022] In some embodiments, the infant formula is an extensively hydrolyzed infant formula (eHF). In some embodiments, the infant formula is an amino acid-based infant formula (AAF).

[0023] In a preferred embodiment, the infant formula comprises protein, carbohydrates, and lipids.

[0024] In some embodiments, the infant formula comprises: (a) 1.8 to 3.2 g of protein per 100 kcal; (b) 9 to 14 grams of carbohydrates per 100 kcal, and / or (c) 4.0 to 6.0 g of fat per 100 kcal Includes:

[0025] In some embodiments, the infant formula contains about 2.4 g or less of protein per 100 kcal.

[0026] In some embodiments, the infant formula contains 1.8-2.4 g of protein per 100 kcal, 2.1-2.3 g of protein per 100 kcal, or 2.15-2.25 g of protein per 100 kcal. In a preferred embodiment, the infant formula contains about 2.2 g of protein per 100 kcal.

[0027] In some embodiments, no more than about 30% by weight of the lipids are medium chain triglycerides (MCTs).

[0028] In some embodiments, about 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, or 1% by weight or less of the lipids in the infant formula are medium chain triglycerides (MCTs).

[0029] In some embodiments, the infant formula does not contain added MCTs.

[0030] In another aspect, the present invention provides an infant formula comprising 0.05-0.2 g / L, 0.05-0.15 g / L, 0.05-0.1 g / L, or about 0.1 g / L of LNnT, preferably the infant formula comprises about 0.1 g / L of LNnT. [Brief explanation of the drawings]

[0031] [Figure 1] Human milk oligosaccharide (HMO) concentrations in breast milk from mothers of non-allergic infants (N=40) and medically diagnosed allergic / atopic infants (N=29). Milk samples were collected at 3 months. Box plots show the median (horizontal line) within boxes representing the 25th and 75th percentiles, whiskers represent the 10th and 90th percentiles, and circles represent the upper and lower 10%. No statistical differences (Q or adjusted p>0.05) were found between breast milk HMOs in the non-allergic and allergic groups (nonparametric Mann-Whitney-Wilcoxon test). [Figure 2] Quartile analysis of (A) 2'-fucosyllactose (2'FL) concentrations (mg / L) and (B) lacto-N-neotetraose (LNnT) concentrations (μg / mL) in human breast milk shows differences in distribution among population subgroups. Green percentages (highlighted with ovals) indicate statistically significant positive results, while red percentages (highlighted with squares) indicate negative results. [Figure 3] The optimal medium dose of 1% of a 2:1 mixture of 2'FL and LNnT (HMO) in preventing allergic sensitization is demonstrated by the reduction in specific IgG concentrations observed in a mouse model of skin sensitization. DETAILED DESCRIPTION OF THE INVENTION

[0032] 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."

[0033] Infant Formula The term "infant formula" may refer to a food for specific nutritional uses for infants during the first year of life, which formula alone meets the nutritional requirements of humans falling within this category (as defined in Commission Regulation (EU) 2016 / 127 of 25 September 2015).

[0034] In some embodiments, the infant formula is an extensively hydrolyzed infant formula (eHF).

[0035] In some embodiments, the infant formula is a 100% whey-based partially hydrolyzed formula (pHF).

[0036] In some embodiments, the infant formula is an amino acid-based infant formula (AAF).

[0037] In some embodiments, the formula is, for example, an HMO-free infant formula or a supplement that can be mixed into food.

[0038] The term "extensively hydrolyzed infant formula" or "eHF" can refer to an infant formula containing extensively hydrolyzed proteins. eHF can be a hypoallergenic infant formula that provides complete nutrition for infants who cannot digest intact cow's milk protein (CMP) or who have an intolerance or allergy to CMP.

[0039] The term "amino acid-based infant formula" or "AAF" may refer to an infant formula containing only free amino acids as a source of protein. The AAF may contain no detectable peptides. The AAF may be a hypoallergenic infant formula that provides complete nutrition for infants with food protein allergies and / or intolerances. For example, the AAF may be a hypoallergenic infant formula that provides complete nutrition for infants who cannot digest unprocessed CMP or who have an intolerance or allergy to CMP and who may have very serious or life-threatening symptoms and / or sensitivities to multiple foods.

[0040] A "hypoallergenic" composition is one that is unlikely to cause an allergic reaction. Preferably, the infant formula of the present invention is tolerated by more than 90% of infants with cow's milk protein allergy (CMPA). This is in line with the guidance provided by the American Academy of Pediatrics (Committee on Nutrition (2000) Pediatrics 106(2):346-349). Preferably, the infant formula of the present invention may not contain peptides that are recognized by CMP-specific IgE, such as IgE in subjects with CMPA.

[0041] The pHF composition is a hydrolyzed composition to reduce exposure to intact milk allergens. Preferably, the infant formula of the present invention is targeted to the general infant population for the prevention of allergic diseases.

[0042] Infants can be fed infant formula alone, or infant formula can be used as a supplement to human breast milk.

[0043] The infant formula of the present invention may be in powder or liquid form.

[0044] The liquid may be, for example, a concentrated liquid infant formula or a ready-to-feed infant formula. The infant formula may be in the form of a reconstituted infant formula (i.e., a liquid infant formula reconstituted from a powder form). The concentrated liquid infant formula may preferably be diluted, for example, by adding water, to a liquid composition suitable for feeding to an infant.

[0045] In some embodiments, the infant formula is in powder form, which can be reconstituted, for example by adding water, into a liquid composition suitable for feeding to an infant.

[0046] The infant formula, when formulated as directed, may have an energy density of about 60-72 kcal per 100 mL. Preferably, the infant formula, when formulated as directed, may have an energy density of about 60-70 kcal per 100 mL.

[0047] Human milk oligosaccharides The infant formula of the present invention contains at least one of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT).

[0048] Many types of HMOs are found in human breast milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine, with a wide variety of linkages between them, resulting in a great diversity of oligosaccharides in human breast milk. Nearly all HMOs have a lactose moiety at the reducing end, with sialic acid and / or fucose (if present) occupying the terminal positions at the non-reducing end. HMOs can be acidic (e.g., charged sialic acid-containing oligosaccharides) or neutral (e.g., fucosylated oligosaccharides).

[0049] The infant formula of the present invention comprises 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT).

[0050] In some embodiments, the infant formula comprises 2'FL, hi some embodiments, no other types of fucosylated oligosaccharides are present other than 2'FL, i.e., the infant formula of the present invention comprises only 2'FL as a fucosylated oligosaccharide.

[0051] 2'FL can be produced by biotechnological means using specific fucosyltransferases and / or fucosidases, either through enzyme-based fermentation techniques (recombinant or natural enzymes) or microbial fermentation techniques. In the latter case, microorganisms can express the natural enzymes and substrates or can be engineered to produce the respective substrates and enzymes. Alternatively, 2'FL can be produced by chemical synthesis from lactose and free fucose.

[0052] In some embodiments, the infant formula includes LNnT, hi some embodiments, no other N-acetylated oligosaccharides are present other than LNnT, i.e., the infant formula of the present invention includes only LNnT as the N-acetylated oligosaccharide.

[0053] LNnT can be chemically synthesized by the enzymatic transfer of a sugar unit from a donor moiety to an acceptor moiety using a glycosyltransferase, as described, for example, in U.S. Patent No. 5,288,637 and WO 1996 / 010086. Alternatively, LNnT can be prepared by chemically converting a free ketohexose or a ketohexose (e.g., fructose) linked to an oligosaccharide (e.g., lactulose) into an N-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharide, as described in Wrodnigg, TM, and Stutz, AE (1999) Angew. Chem. Int. Ed. 38:827-828. The N-acetyl-lactosamine thus produced can then be transferred to lactose as the acceptor moiety.

[0054] In some embodiments, the infant formula comprises an oligosaccharide mixture comprising 2'FL and / or LNnT. In a preferred embodiment, the infant formula comprises an oligosaccharide mixture comprising 2'FL and LNnT. An infant formula of the present invention may, for example, comprise only 2'FL as the fucosylated oligosaccharide and only LNnT as the N-acetylated oligosaccharide.

[0055] 2'FL may be present, for example, in infant formula in a total amount of 0.8 to 2.5 g per liter of infant formula (when formulated as directed).

[0056] In some embodiments, the infant formula contains 0.8-1.5 g / L, 0.8-1.4 g / L, 0.8-1.3 g / L, 0.8-1.2 g / L, 0.8-1.1 g / L, 0.9-1.1 g / L, or about 1 g / L of 2'FL (when formulated as directed). In some embodiments, the infant formula contains 0.8-1.2 g / L of 2'FL (when formulated as directed). In some embodiments, the infant formula contains 0.9-1.1 g / L of 2'FL (when formulated as directed).

[0057] In some embodiments, the infant formula contains 1-1.5 g / L, 1-1.4 g / L, 1-1.3 g / L, 1-1.2 g / L, 1-1.1 g / L, or about 1 g / L of 2'FL (when formulated as directed). In some embodiments, the infant formula contains 1-1.1 g / L of 2'FL (when formulated as directed).

[0058] In a preferred embodiment, the infant formula contains about 1 g / L of 2'FL (when formulated as directed).

[0059] In some embodiments, the infant formula contains 1.5-2.5 g / L, 1.5-2.4 g / L, 1.5-2.3 g / L, 1.5-2.2 g / L, 1.5-2.1 g / L, 1.5-2 g / L, 1.6-2 g / L, 1.7-1.9 g / L, or about 1.8 g / L of 2'FL when formulated as directed. In some embodiments, the infant formula contains 1.5-2 g / L of 2'FL when formulated as directed. In some embodiments, the infant formula contains 1.7-1.9 g / L of 2'FL when formulated as directed.

[0060] In a preferred embodiment, the infant formula contains about 1.8 g / L of 2'FL (when formulated as directed).

[0061] LNnT may be present in infant formula, for example, in a total amount of 0.05 to 0.2 g per liter of infant formula (when formulated as directed).

[0062] In some embodiments, the infant formula contains 0.05-0.15 g / L, 0.06-0.14 g / L, 0.07-0.13 g / L, 0.08-0.12 g / L, 0.09-0.11 g / L, or about 0.1 g / L of LNnT (when formulated as directed). In some embodiments, the infant formula contains 0.08-0.12 g / L of LNnT (when formulated as directed). In some embodiments, the infant formula contains 0.09-0.11 g / L of LNnT (when formulated as directed).

[0063] In a preferred embodiment, the infant formula contains about 0.1 g / L of LNnT (when formulated as directed).

[0064] In a preferred embodiment, the infant formula comprises 2'FL and LNnT.

[0065] In some embodiments, the infant formula contains about 0.8-1.2 g / L of 2'FL and about 0.08-0.12 g / L of LNnT (when formulated as directed).

[0066] In some embodiments, the infant formula contains about 0.9-1.1 g / L of 2'FL and about 0.09-0.11 g / L of LNnT (when formulated as directed).

[0067] In some embodiments, the infant formula (when formulated as directed) contains about 1.8 g / L 2'FL and about 0.1 g / L LNnT.

[0068] In some embodiments, the infant formula contains about 1.5-2 g / L 2'FL and about 0.08-0.12 g / L LNnT (when formulated as directed).

[0069] In some embodiments, the infant formula contains about 1.7-1.9 g / L of 2'FL and about 0.09-0.11 g / L of LNnT (when formulated as directed).

[0070] In some embodiments, an infant formula of the present invention (when formulated as directed) contains about 1.8 g / L 2'FL and about 0.1 g / L LNnT.

[0071] In some embodiments, the infant formula contains 0.12-0.225g / 100kcal, 0.12-0.21g / 100kcal, 0.12-0.195g / 100kcal, 0.12-0.18g / 100kcal, 0.12-0.165g / 100kcal, 0.135-0.165g / 100kcal, or about 0.15g / 100kcal of 2'FL when formulated as directed. In some embodiments, the infant formula contains 0.12-0.18g / 100kcal of 2'FL when formulated as directed. In some embodiments, the infant formula contains 0.135-0.165g / 100kcal of 2'FL when formulated as directed.

[0072] In some embodiments, the infant formula contains 0.15-0.225g / 100kcal, 0.15-0.21g / 100kcal, 0.15-0.195g / 100kcal, 0.15-0.18g / 100kcal, 0.15-0.165g / 100kcal, or about 0.15g / 100kcal of 2'FL (when formulated as directed). In some embodiments, the infant formula contains 0.15-0.165g / 100kcal of 2'FL (when formulated as directed).

[0073] In a preferred embodiment, the infant formula contains about 0.15 g / 100 kcal of 2'FL (when formulated as directed).

[0074] In some embodiments, the infant formula contains 0.225-0.375g / 100kcal, 0.225-0.36g / 100kcal, 0.225-0.345g / 100kcal, 0.225-0.33g / 100kcal, 0.225-0.315g / 100kcal, 0.225-0.3g / 100kcal, 0.24-0.3g / 100kcal, 0.255-0.285g / 100kcal, or about 0.27g / 100kcal of 2'FL when formulated as directed. In some embodiments, the infant formula contains 0.225-2g / 100kcal of 2'FL when formulated as directed. In some embodiments, infant formula contains 0.255-0.285 g / 100 kcal of 2'FL (when formulated as directed).

[0075] In a preferred embodiment, the infant formula contains about 0.27 g / 100 kcal of 2'FL (when formulated as directed).

[0076] LNnT, for example, can be present in infant formula in a total amount of 0.0075 to 0.03 g per 100 kcal of infant formula (when formulated as directed).

[0077] In some embodiments, the infant formula contains 0.0075-0.0225 g / 100 kcal, 0.009-0.021 g / 100 kcal, 0.0105-0.0195 g / 100 kcal, 0.012-0.018 g / 100 kcal, 0.0135-0.0165 g / 100 kcal, or about 0.015 g / 100 kcal of LNnT when formulated as directed. In some embodiments, the infant formula contains 0.012-0.018 g / 100 kcal of LNnT when formulated as directed. In some embodiments, the infant formula contains 0.0135-0.0165 g / 100 kcal of LNnT when formulated as directed.

[0078] In a preferred embodiment, the infant formula contains about 0.015 g / 100 kcal of LNnT (when formulated as directed).

[0079] protein The term "protein" includes peptides and free amino acids. The protein content of an infant formula can be calculated by any method known to those skilled in the art. Preferably, the protein content can be calculated by the nitrogen protein equivalent method, as described, for example, in Maubois, J.L. and Lorient, D. (2016) Dairy Science & Technology 96(1):15-25. Preferably, the protein content is calculated as the nitrogen content x 6.25, as defined in European Commission Regulation (EU) 2016 / 127 of 25 September 2015. The nitrogen content can be determined by any method known to those skilled in the art. For example, the nitrogen content can be measured by the Kjeldahl method.

[0080] Protein concentration The protein content of the infant formula is preferably in the range of 1.8 to 3.2 g of protein per 100 kcal, hi some embodiments, the protein content of the infant formula is in the range of 1.8 to 2.8 g of protein per 100 kcal.

[0081] eHF typically contains 2.6-2.8g of protein per 100kcal, and AAF typically contains 2.8-3.1g of protein per 100kcal, to meet the needs of infants with gastrointestinal pathologies, including severe malabsorption, or who require more protein and calories to compensate for a high metabolic rate.

[0082] Infant formulas such as eHF or AAF with lower protein content may be safe and tolerable while supporting proper growth and development in allergic infants.

[0083] Thus, in some embodiments, an infant formula may contain about 2.4 g or less of protein per 100 kcal. For example, an infant formula may contain about 2.3 g or less of protein per 100 kcal, 2.25 g or less of protein per 100 kcal, or 2.2 g or less of protein per 100 kcal.

[0084] Preferably, the infant formula contains about 1.8 g or more of protein per 100 kcal. For example, the infant formula may contain about 1.86 g or more of protein per 100 kcal, 1.9 g or more of protein per 100 kcal, 2.0 g or more of protein per 100 kcal, or 2.1 g or more of protein per 100 kcal. Preferably, the infant formula contains about 1.86 g or more of protein per 100 kcal, in line with current EU regulations (EFSA NDA Panel (2014) EFSA journal 12(7):3760).

[0085] In some embodiments, infant formula may contain 1.8-2.4g protein per 100 kcal, 1.86-2.4g protein per 100 kcal, 1.9-2.4g protein per 100 kcal, 2.0-2.4g protein per 100 kcal, 2.0-2.3g protein per 100 kcal, 2.1-2.3g protein per 100 kcal, or 2.15-2.25g protein per 100 kcal.

[0086] Preferably, the infant formula contains about 2.2g of protein per 100kcal.

[0087] Protein Source The source of protein can be any source suitable for use in infant formula. Preferably, the protein is cow's milk protein.

[0088] In some embodiments, the infant formula does not contain dairy proteins. In some embodiments, the infant formula does not contain cow's milk proteins. Thus, in some embodiments, 100% of the total protein by weight is non-dairy protein.

[0089] In some embodiments, the infant formula comprises a plant protein. Examples of plant proteins that may optionally be used in the infant formula of the present invention include potato, pea, rice, quinoa, oat, sunflower, or coconut protein, or a combination thereof. Examples of additional non-dairy proteins for use in infant formula include algae protein or leaf protein.

[0090] A highly hydrolyzed / hydrolyzed whey-based formula may be more palatable than a highly hydrolyzed / hydrolyzed casein-based formula, and / or subjects may be sensitized only to casein protein. Thus, preferably, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or about 100% of the protein is whey protein. Preferably, the source of the protein is whey protein.

[0091] The whey protein may be whey derived from cheese production, in particular sweet whey such as that obtained by coagulation of casein with rennet, acid whey obtained by coagulation of casein with acid or by acidification of a ferment, or even mixed whey obtained by coagulation with acid and with rennet. The raw material may be whey that has been desalted by ion exchange and / or by electrodialysis, known as desalted whey protein (DWP).

[0092] The source of whey protein can be sweet whey from which caseinoglycomacropeptide (CGMP) has been completely or partially removed. This whey is called denatured sweet whey (MSW). Removal of CGMP from sweet whey results in a protein material whose threonine and tryptophan content more closely resembles that of human breast milk. The process for removing CGMP from sweet whey is described in EP 880902.

[0093] The whey protein may be a mixture of DWP and MSW.

[0094] In some embodiments, the amount of casein in the infant formula is undetectable, e.g., less than 0.2 mg / kg. The amount of casein can be determined by any method known to one of skill in the art.

[0095] Degree of hydrolysis In eHF, proteins are "extremely hydrolyzed," so that eHF can be tolerated by more than 90% of infants with CMPA.

[0096] The degree of hydrolysis of protein hydrolysates can be characterized by NPN / TN%, which refers to non-protein nitrogen divided by total nitrogen x 100. Non-protein nitrogen refers to amino nitrogen that is free to react with reagents such as trinitrobenzenesulfonic acid (TNBS). NPN / TN% can be determined by any method known to those skilled in the art. For example, NPN / TN% can be measured as described in Adler-Nissen (Adler-Nissen, J. (1979) J. Agric. Food Chem. 27:1256-1262). Preferably, the protein has an NPN / TN% of greater than 90%, greater than 95%, or greater than 98%.

[0097] The degree of hydrolysis can also be determined by the degree of hydrolysis. The "degree of hydrolysis" (DH) is defined as the percentage of broken peptide bonds in a protein hydrolysate and can be determined by any method known to those skilled in the art. Preferably, the degree of hydrolysis is determined by pH stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), or formol titration. (Rutherfurd, SM (2010) Journal of AOAC International 93(5):1515-1522). The degree of hydrolysis (DH) of a protein can be, for example, greater than 90, greater than 95, or greater than 98.

[0098] The degree of hydrolysis can also be determined by peptide molecular weight distribution. Peptide molecular weight distribution can be determined by high-performance size exclusion chromatography (HPSEC / UV), optionally using a UV detector (Johns, P. Wet et al. (2011) Food Chemistry 125(3):1041-1050). For example, peptide molecular weight distribution can be an estimate based on HPSEC peak areas determined at 205 nm, 214 nm, or 220 nm. Preferably, when peptide molecular weight distribution is determined by HPSEC / UV, the "weight percent of peptides" having a certain molecular weight can be estimated by the "peak area fraction as a percentage of the total peak area" having that molecular weight determined at 205 nm, 214 nm, or 220 nm. Preferably, the degree of hydrolysis can be determined by the method described in WO 2016 / 156077. Alternatively, the peptide molecular weight distribution can be determined by any method known to those skilled in the art, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Chauveau, A. et al. (2016) Pediatric Allergy and Immunology 27(5):541-543).

[0099] Theoretically, peptides must be larger than approximately 1500 Da (approximately 15 amino acids) to bind to cell membrane-bound IgE, and larger than approximately 3000 Da (approximately 30 amino acids) to cross-link IgE molecules and induce an immune response (Nutten (2018) EMJ Allergy Immunol 3(1):50-59).

[0100] Thus, preferably, at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in eHF have a molecular weight of less than about 3000 Da. For example, there may be no detectable peptides in eHF greater than or equal to about 3000 Da in size.

[0101] Thus, suitably, at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in eHF have a molecular weight of less than about 1500 Da. Preferably, at least 99% by weight of the peptides have a molecular weight of less than about 1500 Da. For example, there may be no detectable peptides in eHF greater than or equal to about 1500 Da in size.

[0102] Preferably, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% by weight of the peptides in the eHF have a molecular weight of less than about 1200 Da. More preferably, at least 95% or 98% by weight of the peptides in the eHF have a molecular weight of less than about 1200 Da.

[0103] Suitably, at least about 80%, at least about 85%, at least about 90%, or at least about 95% by weight of the peptides in the eHF have a molecular weight of less than about 1000 Da. Preferably, at least about 95% by weight of the peptides in the eHF have a molecular weight of less than about 1000 Da.

[0104] Preferably, the eHF has no detectable peptides greater than about 3000 Da in size, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da.

[0105] A high proportion of dipeptides and tripeptides may improve nitrogen (protein) absorption in patients with intestinal dysfunction. PEPT1 is a dedicated transport pathway that facilitates the absorption of small peptides (e.g., dipeptides and tripeptides). In the first few weeks of life, intestinal PEPT1 is important for nutrient uptake and later for food transition after weaning.

[0106] Thus, at least about 30%, at least about 40%, or at least about 50% by weight of the peptides in eHF can be dipeptides and tripeptides. Preferably, at least about 45%, at least about 50%, 45-55%, or 50-54% by weight of the peptides in eHF are dipeptides and tripeptides. More preferably, about 51-53%, or more preferably about 52% by weight of the peptides in eHF are dipeptides and tripeptides.

[0107] Suitably, at least about 30%, at least about 40%, or at least about 50% by weight of the peptides in the eHF have a molecular weight of 240-600 Da. Preferably, at least about 45%, at least about 50%, 45-55%, or 50-54% by weight of the peptides in the eHF have a molecular weight of 240-600 Da. More preferably, about 51-53%, or most preferably about 52% by weight of the peptides in the eHF have a molecular weight of 240-600 Da.

[0108] The peptides in the eHF may have a median molecular weight of, for example, 300 Da to 370 Da, preferably 320 Da to 360 Da.

[0109] The major recognized allergens in cow's milk are α-lactalbumin (aLA), β-lactoglobulin (bLG), and bovine serum albumin (BSA).

[0110] Therefore, preferably, eHF can have an undetectable aLA content, for example, about 0.010 mg / kg or less of aLA, eHF can have an undetectable bLG content, for example, about 0.010 mg / kg or less of bLG, and / or eHF can have an undetectable BSA content, for example, about 0.010 mg / kg or less of BSA. Preferably, eHF does not contain detectable amounts of aLA, bLG, and BSA. The contents of aLA, bLG, and BSA can be determined by any method known to those skilled in the art, for example, ELISA.

[0111] In preferred embodiments, the eHF of the present invention has no detectable peptides greater than about 3000 Da in size, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da; optionally, at least about 45%, at least about 50%, or 45-55% by weight of the peptides have a molecular weight of 240-600 Da and / or are dipeptides or tripeptides; and the eHF does not contain added MCTs.

[0112] Hydrolysis method Proteins for use in the infant formulas of the present invention can be hydrolyzed by any suitable method known in the art. For example, proteins can be enzymatically hydrolyzed using, for example, a protease. For example, proteins can be hydrolyzed using Alcalase (e.g., at an enzyme:substrate ratio of about 1-15% by weight for a duration of about 1-10 hours). The temperature can be in the range of about 40°C to 60°C, e.g., about 55°C. The reaction time can be, for example, 1-10 hours, and the pH value before starting the hydrolysis can be, for example, in the range of 6-9, preferably 6.5-8.5, more preferably 7.0-8.0.

[0113] Porcine enzymes, particularly porcine pancreatic enzymes, may be used in the hydrolysis process. For example, WO 1993004593(A1) discloses a hydrolysis process using trypsin and chymotrypsin. This process involves a two-step hydrolysis reaction, with a heat denaturation step between the two steps to ensure that the final hydrolysate is substantially free of intact allergenic proteins. The trypsin and chymotrypsin used in these methods are preparations made from porcine pancreatic extracts.

[0114] WO2016156077A1 discloses a process for preparing a milk protein hydrolysate, which comprises hydrolyzing a milk-based proteinaceous material with a combination of a microbial alkaline serine protease, bromelain, an Aspergillus-derived protease, and a Bacillus-derived protease.

[0115] Free amino acids The infant formula of the present invention may include free amino acids.

[0116] The concentration of free amino acids may be selected to provide an amino acid profile sufficient for infant nutrition, in particular an amino acid profile that meets nutritional regulations (eg, European Commission Directive 2006 / 141 / EC).

[0117] Free amino acids can be incorporated into the eHF of the present invention to supplement the amino acids contained in the peptide.

[0118] Examples of free amino acids for use in the infant formulas of the present invention include histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine, and mixtures thereof.

[0119] Free amino acids provide an equivalent source of protein (i.e., contribute to the nitrogen content). As mentioned above, a high proportion of dipeptides and tripeptides can improve nitrogen (protein) absorption, even in patients with intestinal dysfunction. Therefore, a low proportion of free amino acids can improve nitrogen (protein) absorption, even in patients with intestinal dysfunction.

[0120] Therefore, the free amino acids in the eHF can be present at a concentration of 50% by weight or less, 40% by weight or less, 30% by weight or less, or 25% by weight or less, based on the total weight of the amino acids. Preferably, the eHF contains 25% by weight or less of free amino acids, based on the total weight of the amino acids. More preferably, the free amino acids in the eHF are present at a concentration of 20-25% by weight, 21-23% by weight, or about 22% by weight, based on the total weight of the amino acids.

[0121] The free amino acid content can be determined by any method known to those skilled in the art. Preferably, the free amino acid content can be obtained by separating the free amino groups present in the aqueous sample extract by ion exchange chromatography and optical detection after post-column derivatization with ninhydrin reagent. The total amino acid content can be obtained by hydrolysis of the test portion in 6 mol / L HCl under nitrogen and separating the individual amino acids by ion exchange chromatography as described above.

[0122] In a preferred embodiment, the eHF of the present invention has no detectable peptides greater than about 3000 Da in size, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da; optionally, at least about 45%, at least about 50%, or 45-55% by weight of the peptides have a molecular weight of 240-600 Da, and / or are dipeptides or tripeptides, and / or are 20-25%, 21-23%, or about 22% by weight based on the total weight of amino acids; and the eHF does not contain added MCTs.

[0123] carbohydrates The carbohydrate content of the infant formula of the present invention is preferably in the range of 9-14 g of carbohydrate per 100 kcal.

[0124] The carbohydrate can be any carbohydrate suitable for use in infant formula.

[0125] Examples of carbohydrates for use in the infant formulas of the present invention include lactose, sucrose, maltodextrin, and starch. Mixtures of carbohydrates may also be used.

[0126] In some embodiments, the carbohydrate content comprises maltodextrin, hi some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% by weight of the total carbohydrate content is maltodextrin.

[0127] In some embodiments, the carbohydrate content comprises lactose, hi some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% by weight of the total carbohydrate content is lactose.

[0128] In some embodiments, the carbohydrates include lactose and maltodextrin.

[0129] lipids The lipid content of the infant formula of the present invention is preferably in the range of 4.0 to 6.0 g lipid per 100 kcal.

[0130] The lipid can be any lipid or fat suitable for use in infant formula.

[0131] Examples of lipids for use in the infant formulas of the present invention include sunflower oil, low erucic acid rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palm olein, high oleic sunflower oil and high oleic safflower oil, and microbial fermentation oils containing long chain polyunsaturated fatty acids.

[0132] Lipids can also be in the form of fractions derived from these oils, such as palm olein, medium chain triglycerides (MCTs), and esters of fatty acids (e.g., arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linoleic acid, oleic acid, lauric acid, capric acid, caprylic acid, caproic acid, etc.).

[0133] Further examples of lipids include structured lipids (i.e., lipids that have been chemically or enzymatically modified to alter their structure). Preferably, the structured lipids are sn2 structured lipids, such as triglycerides that have an increased proportion of palmitic acid at the sn2 position of the triglyceride. Structured lipids may or may not be included.

[0134] Oils such as fish or microbial oils that are rich in preformed arachidonic acid (ARA) and / or docosahexaenoic acid (DHA) may also be added.

[0135] Long chain polyunsaturated fatty acids such as dihomo-γ-linolenic acid, arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid (DHA) may also be added.

[0136] The infant formula may contain 2-20 mg ARA per 100 kcal, 5-15 mg ARA per 100 kcal, or about 10 mg ARA per 100 kcal and / or 2-20 mg DHA per 100 kcal, 5-15 mg DHA per 100 kcal, or about 10 mg DHA per 100 kcal. Preferably, the infant formula contains about 10 mg ARA per 100 kcal and about 10 mg DHA per 100 kcal.

[0137] Medium Chain Triglycerides (MCTs) High concentrations of MCTs may impair early weight gain. MCTs are not stored and do not support fat accumulation. For example, Borschel et al. reported that infants fed a formula containing no MCTs gained significantly more weight between days 1 and 56 than infants fed a formula containing 50% MCT-derived lipids (Borschel, M. et al. (2018) Nutrients 10(3):289).

[0138] Thus, in the infant formulas of the present invention, up to about 30% by weight of the lipids can be, for example, medium chain triglycerides (MCTs).

[0139] In some embodiments, medium chain triglycerides (MCTs) are about 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0.1% or less by weight of the lipids.

[0140] In some embodiments, medium chain triglycerides (MCTs) are 0-30%, 0-25%, 0-20%, 0-15%, 0-10%, 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 0-0.5%, or 0-0.1% by weight of the lipids.

[0141] Preferably, the infant formula does not contain added MCTs. Preferably, MCTs are about 0% of the lipids by weight and / or the infant formula does not contain detectable MCTs. Preferably, the infant formula does not contain MCTs.

[0142] In a preferred embodiment, the eHF of the present invention has no detectable peptides greater than about 3000 Da in size, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da; 45-55% by weight of the peptides have a molecular weight of 240-600 Da; free amino acids are present at a concentration of 20-25% by weight based on the total weight of amino acids; and the eHF does not contain added MCTs.

[0143] Further ingredients The infant formulas of the present invention also preferably contain nutritionally significant amounts of all vitamins and minerals considered essential in the daily diet, with minimum requirements established for certain vitamins and minerals.

[0144] Examples of vitamins, minerals, and other nutrients for use in the infant formulas of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are typically added in their salt form.

[0145] The infant formulas of the present invention may include one or more carotenoids.

[0146] The infant formula of the present invention may also contain at least one probiotic. The term "probiotic" refers to a preparation of microbial cells or components of microbial cells that have a beneficial effect on the health or well-being of the host. Specifically, probiotics can improve intestinal barrier function.

[0147] Preferred probiotics are generally safe, L(+) lactic acid producing cultures, and have an acceptable shelf life for products that are required to remain stable and effective for up to 24 months.

[0148] Examples of probiotic microorganisms for use in the infant formulas of the present invention include yeasts such as Saccharomyces, Debaromyces, Candida, Pichia, and Torulopsis, as well as species of the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, and the like. Examples of bacteria that may be present include bacteria of the genus Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, and LactoBacillus.

[0149] 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.

[0150] The infant formulas of the present invention may also contain other substances that may have beneficial effects, such as prebiotics, lactoferrin, dietary fiber, nucleotides, nucleosides, short chain fatty acids such as butyrate, and / or postbiotics.

[0151] Reducing and preventing the occurrence of allergic sensitization The term "allergy" refers to a hypersensitivity of the immune system to a substance (allergen) that is normally tolerated. An allergy can be a doctor-diagnosed allergy. Examples of allergic diseases include atopic dermatitis, eczema, food allergies, asthma, and rhinitis.

[0152] Immunization is part of the normal immune response and occurs in healthy individuals when the immune system detects a substance as a threat, which can result in, for example, B cells producing antibodies that bind to the substance.

[0153] The term "allergic sensitization" refers to the sensitization of the immune system to substances (known as allergens, e.g., substances in food or pollen) that are normally tolerated and typically harmless in the absence of an allergic reaction.

[0154] Without wishing to be bound by theory, when an allergen enters the body, it can be captured by antigen-presenting cells and presented to other cells of the immune system, particularly T cells. After interaction between T cells and B cells, the B cells can produce allergen-specific antibodies (IgE). Then, when released into the blood, the IgE antibodies can bind to other immune cells, such as mast cells and basophils. Sensitized individuals can then develop an allergic reaction upon re-exposure to the allergen.

[0155] Thus, allergic sensitization can refer to the priming of the immune system to recognize an allergen. Individuals sensitized in this way can develop an allergic reaction upon re-exposure to the allergen.

[0156] In some embodiments, allergic sensitization in infants may be characterized by a total IgE concentration greater than 35 kU / L at 6 months of age and / or greater than 53 kU / L at 12 months of age (Martins, TB et al. (2014) J Allergy Clin Immunol 133(2):589-91). In some embodiments, allergic sensitization in adults may be characterized by a total IgE concentration greater than 127 KU / L. One of skill in the art can readily determine the IgE concentration in a sample from a subject using, for example, ImmunoCAP Phadia technology as disclosed in the Examples.

[0157] The infant formula of the present invention may be used to reduce the incidence of allergic sensitization in infants and / or to prevent allergic sensitization in infants.

[0158] As used herein, "reducing the incidence" of allergic sensitization means that the infant formula reduces the likelihood of allergic sensitization.

[0159] As used herein, "preventing" allergic sensitization means that the infant has not yet been sensitized and the infant formula prevents allergic sensitization.

[0160] The term "infant" refers to a child under 12 months of age, for example, a child between 0 and 6 months of age.

[0161] In some embodiments, the infant is at risk of developing one or more allergies, for example, the infant may belong to a family with a history of one or more allergies.

[0162] In one aspect, the invention provides a method of preventing or reducing the incidence of allergic sensitization in an infant, comprising administering to said infant an infant formula of the invention.

[0163] Manufacturing method The infant formulas of the present invention can be prepared in any suitable manner.

[0164] For example, the infant formula can be prepared by blending a protein source, a carbohydrate source, and a lipid source together in appropriate proportions. If an emulsifier is used, it can be added at this point. Vitamins and minerals can also be added at this point, although vitamins are typically added later to avoid thermal degradation. Optional lipophilic vitamins, emulsifiers, etc. can be dissolved in the lipid source before blending. Water, preferably water treated with reverse osmosis, can then be mixed to form a liquid mixture. A commercially available liquefaction device can be used to form the liquid mixture. The liquid mixture can then be homogenized.

[0165] The liquid mixture can then be heat treated to reduce the bacterial content, for example by steam injection or using an autoclave or a heat exchanger, for example a plate heat exchanger.

[0166] The liquid mixture may then be cooled and / or homogenized. The pH and solids content of the homogenized mixture may be adjusted at this point.

[0167] The homogenized mixture can then be transferred to suitable drying equipment, such as a spray dryer or freeze dryer, and converted to a powder. If a liquid infant formula is desired, the homogenized mixture can be sterilized and then aseptically filled into suitable containers, or it can be filled into containers first and then retorted.

[0168] It will be understood that a person skilled in the art can combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0169] Preferred features and embodiments of the present invention will now be described by way of non-limiting examples. [Example]

[0170] Several studies have demonstrated that specific components of breast milk, considered individually, are associated with maternal or child disease states using univariate analysis. However, recent analytical approaches evaluating the relationship of breast milk HMO components alone have not demonstrated an association with allergy. Example 1 identifies the specific doses of 2FL and LNnt required for protection against allergic sensitization. Example 2 confirms the need for specific doses to prevent allergic sensitization in animals.

[0171] Example 1 This study aimed to understand the association between human milk oligosaccharide (HMO) concentrations and allergic sensitization in humans. Although we found no significant association when performing univariate analysis, suggesting that there is no linear association between HMO concentrations and the risk of allergic sensitization, we found that the HMOs 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) prevent allergic sensitization and observed a bell-shaped effect, with optimal protection at intermediate doses, while lower and higher doses may be less effective.

[0172] Materials and Methods Breast milk analysis (German cohort) A cohort of 156 mother-infant pairs was subselected from the Life Child study (Seppo, AE et al. (2017) J Allergy Clin Immunol 139(2):708-11 e5). Sample selection was based on the availability of breast milk samples at 3 months, as well as completion of an allergy questionnaire by the mother at any time during the study and / or completion of an allergy questionnaire for the infant during the first year of life.

[0173] For the purposes of the study, maternal allergic sensitization was defined by a total IgE level greater than 127 KU / L (Martins, TB et al. (2014) J Allergy Clin Immunol 133(2):589-91), and allergy was defined as maternal self-reported asthma, rhinitis, atopic dermatitis, eczema, and / or allergic reactions to specific foods (including vomiting, nausea, diarrhea, exacerbation of eczema or asthma symptoms). For the purposes of this study, infant allergic sensitization was defined as total IgE levels greater than 35 kU / L and 53 kU / L at 6 and 12 months, respectively (Martins, TB et al. (2014) J Allergy Clin Immunol 133(2):589-91), and allergy risk was defined as a positive response at 3, 6 months, or 1 year to the following questions: "Has your child ever been diagnosed with a food allergy by a doctor?", "Has your child ever had eczema / atopic dermatitis?", or "Has your child ever had recurrent itchy rashes lasting more than 15 days?" Maternal and child total IgE were quantified using ImmunoCAP Phadia technology.

[0174] In this cohort, confounding factors, such as allergies and socioeconomic status, were obtained through various questionnaires. Confounders were identified based on available literature related to allergies and breast milk composition and selected based on available data (mode of delivery (delivery), child's sex (sex), child's weight at birth (weight), exclusive breastfeeding at 3 months (breastfeeding), socioeconomic status (ecostatus), and number of siblings (siblings)). This cohort served as an exploratory cohort. Breast milk was expressed, collected, and stored at -80°C from lactating mothers 3 months postpartum (Quante, M. et al. (2012) BMC Public Health 12:1021). One mother-child pair was excluded due to excessive missing data (>50%) for breast milk composition concentrations. This study was designed in accordance with the Declaration of Helsinki and under the supervision of the Ethics Committee of the University of Leipzig (registration number 264-10-19042010). The LIFE Child trial is registered with ClinicalTrials.gov under clinical trial number NCT02550236 (Poulain, T. et al. (2017) European Journal of Epidemiology 32(2):145-58).

[0175] Evaluation of component concentrations Liquid chromatography analysis was performed by centrifuging the samples at 10,000 × g for 5 minutes. Subsequently, the samples were analyzed using an Ultimate 3000-RD UHPLC system (Thermo Fisher Scientific, Waltham, USA) equipped with an RF-200 fluorometer and a two-way, 10-port high-pressure switching valve. The samples were loaded onto an Acquity BEH Glycan and VanGuard BEH Amide column (Waters Corporation, Milford, USA) with a guard column between the injector and the 10-port valve. The column temperature was 55 °C, and the flow rate was 0.5 mL / min. The reliability of the method was verified by a spike reliability assessment in which the calibration curve was verified using standards of the oligosaccharides of interest and a maltotriose control.

[0176] In total, 21 HMOs were identified: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3'FL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), 3'-galactosyllactose (3'GL), 6'-galactosyllactose (6'GL), lacto-N-tetrose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-neofucopentaose (LNnFP), lactodifucotetraose (LDF), lacto-N-tetraose b (LST b), lacto-N-tetraose (LST c), the absolute concentrations of difucosyl lacto-N-hexaose a (DFLNHa), disialyl lacto-N-tetraose (DSLNT), monofucosyl lacto-N-hexaose (MFLNH III), lacto-N-neodifucohexaose (LNnDFH), and lacto-N-difucohexaose (LNDFH 1) were measured.

[0177] Univariate analysis To test for associations between HMOs and allergic sensitization or allergy, logistic regression and chi-square tests were performed comparing individual HMO concentrations with the incidence of atopic dermatitis. The sum of the 21 HMOs was also included in the statistical analysis and classified as "total HMO."

[0178] For statistical analysis, the following confounding variables were included in the model: sex, mode of delivery, parental allergy, Se and Le status of breast milk, and household pets. The nonparametric Mann-Whitney-Wilcoxon test was used to compare differences in the levels of individual HMOs between allergic and non-allergic infants.

[0179] result Association between HMO and allergies in a German cohort Demographic data of the study population at baseline are shown in Table 1 .

[0180] [Table 1]

[0181] [Table 2]

[0182] A total of 21 HMO concentrations were measured and are shown in Figure 1. When only medically diagnosed and / or sensitized infants were used, no significant associations were found between any HMO concentration and medically diagnosed allergic versus non-allergic infants. Q values ​​or p values ​​corrected for multiple testing were all >0.05.

[0183] Association of HMO concentrations with risk of allergic sensitization and skin rash We next evaluated the possibility that HMOs may be associated with allergic sensitization. Because our cohort analyzed less than 30% of the population for sensitization, we expanded our analysis to include not only infants who tested positive for sensitization but also infants at increased risk for sensitization based on long-term skin rash or eczema. The distribution of 2'FL and LNnT levels in this expanded population revealed a nonlinear distribution, suggesting that only certain doses of 2FL and LNnT may be associated with a reduced risk of allergic sensitization (Figure 2), with higher doses lacking protective effects.

[0184] A population quartile analysis, shown in Figure 2, identified breast milk concentrations of 2'FL and LNnT associated with reduced allergic sensitization. Breast milk concentrations of 2'FL below 2.5 mg / L and LNnT below 0.2 g / L appear to be associated with a particularly beneficial effect.

[0185] Example 2 The purpose of this example was to confirm the bell-shaped preventive effect of human milk oligosaccharides (HMOs) in preventing allergies in mice. Preclinical Assays The animal study protocol was approved by the Service Veterinaire du Canton de Vaud, Switzerland. Briefly, 5-week-old female BALB / c mice were fed a diet supplemented with either 0%, 0.2%, 1%, 5%, or 10% (by weight) of an HMO mixture of 2'FL and LNnT (2:1 by weight) starting from week 0 of the protocol. The backs of the mice were shaved and washed with a 70% isopropanol solution (VWR; Nyon, Switzerland), and transepidermal water loss (TEWL) was measured as described below. At week 3, 100 μL of 2 mg / mL Aspergillus fumigatus (Af) protein extract (Greer Laboratories; Lenoir, NC, USA) (sensitized group (S)) or 100 μL of 0.9% NaCl solution (non-sensitized group (NS); Merck; Zoug, Switzerland) was applied to a 1 × 1 cm patch of sterile gauze (Hartmann; Dermaplast, Chatenois, France) and secured to the skin with a bio-occlusive transparent dressing (Systagenix; Bioclusive, San Antonio, Texas, USA, Switzerland) and Band-Aid (Mefix; Wasquehal, France). TEWL was then measured again. After a 2-week rest period, a second identical patch was applied for another week, and TEWL was then measured again. Mice were then intranasally exposed to Af diluted with 0.9% NaCl, anesthetized with isoflurane, and euthanized after blood collection from the abdominal aorta.

[0186] Quantification of specific IgG1 96-well plates (Nunc Maxisorp; VWR) were coated overnight at 4°C with 50 μg / ml Af protein extract (Greer Laboratories) in carbonate buffer. Plates were then washed with PBS-0.05% Tween (Biorad, Reinach, Switzerland) and blocked with PBS-1% BSA (Sigma) for 1 h at 37°C. Diluted serum was incubated for 2 h at 37°C. After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (Southern Biotech, Bioconcept Allschwil, Switzerland) followed by the HRP substrate tetramethylbenzidine (TMB) (KPL, Socochim, Lausanne, Switzerland) for 2 h at 37°C. The reaction was stopped using 1 N hydrochloric acid (HCl, Merck). The absorbance was measured at 450 nm and the results were expressed as optical density (OD) values.

[0187] statistical analysis Statistical analyses were performed using JMP Pro 14 (SAS Software, Cary, USA). Figures were drawn using GraphPad Prism 6 (GraphPad Software, San Diego, USA). For all tests, associations were considered significant if the p-value was >0.05.

[0188] For animal studies, groups were compared using the exact Wilcoxon nonparametric statistical test. Statistical analysis was performed using the software R2.14.1. Results with a p-value ≤ 0.05 were considered significant. Data are expressed as median ± standard error of the median (SE).

[0189] Beneficial effects of HMOs on allergic sensitization To confirm that 2'FL and LnNT prevent allergic sensitization, we tested the efficacy of the 2'FL:LnNT mixture in an animal model. Animals were sensitized on the skin with Aspergillus fumigatus in a mouse skin sensitization model.

[0190] The observed changes in specific IgG with HMO concentration from these studies confirmed that 2'FL and LNnT were particularly effective in preventing allergic sensitization at a dose of 1% (mouse model of skin sensitization, Figure 3), with lower or higher doses being less effective, as indicated by the bell-shaped curve.

[0191] Consideration We have shown that 2'FL and LNnT prevent allergic sensitization and that a bell-shaped effect is observed, indicating improved protection at intermediate doses.

[0192] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the uses and methods disclosed herein 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.

Claims

1. 1. An infant formula for use in preventing or reducing the incidence of allergic sensitization in infants, said infant formula comprising 0.05 to 0.2 g / L of lacto-N-neotetraose (LNnT).

2. An infant formula as described in claim 1 containing 0.05 to 0.15 g / L or 0.1 g / L of LNnT.

3. An infant formula as described in claim 1 or 2, which is an extensively hydrolyzed infant formula (eHF) or an amino acid-based infant formula (AAF).

4. An infant formula described in any one of claims 1 to 3, further comprising protein, carbohydrates and lipids. (a) 1.8 to 3.2 g of protein per 100 kcal; (b) 9-14 g of carbohydrates per 100 kcal; and / or 5. An infant formula according to any one of claims 1 to 4, comprising (c) 4.0 to 6.0 g of lipid per 100 kcal.

6. An infant formula described in any one of claims 1 to 5, containing 2.4g or less of protein per 100kcal.

7. An infant formula as claimed in any one of claims 1 to 6, comprising 1.8 to 2.4 g, 2.1 to 2.3 g, 2.15 to 2.25 g or 2.2 g of protein per 100 kcal.

8. An infant formula described in any one of claims 1 to 7, wherein not more than 30% by weight of the lipids are medium-chain triglycerides (MCTs).

9. An infant formula described in any one of claims 1 to 8, wherein less than 25% by weight, less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, or less than 1% by weight of the lipids are medium chain triglycerides (MCTs).

10. An infant formula described in any one of claims 1 to 9, which does not contain added MCTs.

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