Infant or young child formula

Abstract

The present invention relates to a nutritional composition for infants and young children containing the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2FL) and lacto-N-neotetraose (LNnT) to modulate the maturation of the gut microbiome. The formula may be an extensively hydrolyzed formula (eHF) or an amino acid-based infant formula (AAF) and may be used for infants with cow's milk protein allergies.

Description

[Technical Field]

[0001] The present invention relates to nutritional compositions for infants and young children and their health benefits in infants. In particular, the present invention relates to an infant or young child formula containing the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2FL) and lacto-N-neotetraose (LNnT) to modulate the maturation of the gut microbiome. The formula may be an extensively hydrolyzed formula (eHF) or an amino acid-based infant formula (AAF) and may be used for infants with cow's milk protein allergies.

[0002] [Background technology] Cow's milk proteins (CMPs) are a major cause of food allergies in infants, affecting 2-3% of children worldwide. Most children with CMP allergies (CMPA) have two or more symptoms: 50-70% have skin symptoms; 50-60% have gastrointestinal symptoms; and 20-30% have respiratory tract symptoms. 10% of children may develop severe, life-threatening symptoms. (Nutten, 2018. EMJ Allergy Immunol, 3(1), pp. 50-59)

[0003] Human breast milk and breastfeeding are considered the optimal form of nutrition for healthy infants in the first few months of life. Breast milk remains the gold standard for feeding infants with CMPA. The European Society for Pediatric Gastroenterology and Hepatology (ESPGHAN) recommends that the complete elimination of cow's milk from the mother's diet is the best treatment for CMPA in breastfed infants (Koletzko, S., et al., 2012. Journal of Pediatric Gastroenterology and Nutrition, 55(2), pp. 221-229).

[0004] When breastfeeding is not possible, specialized infant formulas are recommended. ESPGHAN recommends the use of extensively hydrolyzed protein-based formulas (eHF) for non-breastfed infants with CMPA, which have been proven effective for infants with CMPA. For infants with severe or life-threatening conditions, amino acid-based infant formulas (AAF) may be the first choice (Koletzko, S., et al., 2012. Journal of Pediatric Gastroenterology and Nutrition, 55(2), pp. 221-229).

[0005] There is increasing evidence that the infant gut microbial composition is involved in the immune differentiation pathways and allergy development of the infant host (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).

[0006] Infancy, particularly the first week, three months, six months or twelve months of age, is a critical period for the establishment of a balanced gut microbiota.

[0007] It is known that modulation of the gut microbiota during infancy can have a significant impact on future physical health: for example, the gut microbiome can influence the development of a strong immune system later in life, as well as normal growth and development, and even the development of obesity later in life.

[0008] However, the gut microbiome and its evolution during infant development are a delicate balance between the presence and prevalence (abundance) of many populations of gut bacteria. Some gut bacteria are classified as "generally positive" with respect to their impact on the infant's overall health, while others are classified as "generally negative" (or pathogenic). Certain species of "generally positive" bacteria, such as bifidobacteria, may be present in lower abundance in infants fed conventional infant formula compared to breastfed infants. Similarly, some bacterial populations are considered pathogenic and should be maintained at a low prevalence in the gut microbiota.

[0009] Infants fed infant formula may not benefit from the healthy, balanced gut microbiome seen in infants exclusively or primarily breastfed. The development of a healthy microbiome during the first few years of life is complex and susceptible to disruption by environmental factors. Many taxa of microorganisms coexist in sequentially defined proportions within the highly complex microenvironment of the intestine / gut. When defining the microbiome of an infant or young child, both quantitative and qualitative dimensions should be considered. Furthermore, fluctuations in the gut microbiome over time add additional complexity. The delicate balance of all bacterial families, genera, species, and strains present in each location of the gastrointestinal tract, as well as their fluctuations over time, all contribute to the "gastrointestinal health" of infants and young children.

[0010] Recent studies have observed a steady increase in microbiota age in breastfed infants compared with formula-fed infants who receive little or no breast milk. These early changes in the gut microbiota associated with formula-fed infants are inversely correlated with the infant's immunological and biological maturation during the first few months of life (Stewart CJ et al., Nature 2018;562:583-8; Ho NT et al., Nature Communications 2018;9:4169).

[0011] An adequate and healthy intestinal microbiota is a key factor in the development of the infant's mucosal immune system. It is known that, among other ingredients, non-digestible carbohydrates (prebiotics) can particularly influence the promotion of certain microbiota.

[0012] Human breast milk is an immunologically active fluid and is rich in structurally diverse oligosaccharides collectively known as human milk oligosaccharides (HMOs), which may support immune function through several putative mechanisms. These include a prebiotic effect that contributes to the development and maintenance of a healthy gut microbiome, a key factor in the development of the mucosal immune system (Bode et al. Glycobiology 2012;22(9):1147-1162). HMOs may also function as soluble decoy receptors in the intestine, protecting newborns from enteric pathogens (Newburg et al., Human milk glycans protects infants against enteric pathogens. Annual Review of Nutrition 2005;25:37-58), as well as directly interacting with intestinal epithelial cells, resulting in changes that can interfere with host-microbe interactions (Bode et al., 2012).

[0013] WO 2009060073 to Nestec SA relates to the use of oligosaccharides such as lacto-N-tetraose or lacto-N-neotetraose to promote the development in the first few weeks of an infant's life of a beneficial gut microbiota comparable to that found in breastfed infants, in particular a gut microbiota dominated by significant populations of Bifidobacterium and Lactobacillus species, to the exclusion of other populations such as Bacteroides, Clostridia and Streptococci.

[0014] However, there are currently no solutions available to delay the premature shift to the adult microbiome in formula-fed infants.

[0015] Thus, there remains a great need for nutritional compositions such as infant and young child formulas that can be used to prevent or reduce premature maturation of the gut microbiota, particularly infant and young child formulas that are effective in modulating the maturation of the gut microbiome in infants with cow's milk allergy.

[0016] A need exists to provide such health benefits to these infants or young children in a manner that does not induce side effects, is easy to deliver, and is well tolerated by parents or health care professionals.

[0017] [Summary of the Invention] The present inventors have surprisingly found that supplementation of infant formula with the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) can be advantageously used to prevent or reduce this premature shift to an adult-type gut microbiome in infants who are not or only partially breastfed.

[0018] In a controlled, double-blind, randomized, interventional clinical trial, it was surprisingly found that a hypoallergenic infant formula containing the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT) resulted in reduced microbial diversity and reduced gut microbiota age at 12 months of age compared to a corresponding hypoallergenic infant formula without the HMOs.

[0019] Thus, in one aspect, the present invention provides an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) for use in inhibiting or reducing premature maturation of the gut microbiota.

[0020] In one embodiment, the present invention provides an infant or young child formula comprising human milk oligosaccharides (HMO), 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) for use in delaying maturation of the gut microbiota.

[0021] In another aspect, the present invention provides a method for inhibiting or reducing premature maturation of the gut microbiota in an infant in need thereof, comprising administering to the infant an infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT).

[0022] In one embodiment, the present invention provides a method for delaying premature maturation of the gut microbiota in an infant in need thereof, comprising administering to the infant an infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT).

[0023] In one embodiment, the invention provides an infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), for use in inducing a less diverse microbiota at 12 months of age compared to the microbiota at 12 months of age of an infant fed a conventional infant formula that does not contain 2'FL and LNnT.

[0024] In one embodiment, the present invention provides an infant or young child formula comprising the HMO 2'FL and lacto-N-neotetraose (LNnT) for use in inducing a lower microbiota age at 12 months of age compared to infants fed a conventional infant formula that does not contain 2'FL and lacto-N-neotetraose (LNnT).

[0025] Surprisingly, the beneficial effects of the infant or young child formula of the present invention containing the HMOs 2'FL and LNnT on the gut microbiome were observed in 12-month-old infants despite a diversified diet accompanied by a reduced proportion of dietary intake with the formula of the present invention.

[0026] In a preferred embodiment, an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) according to the present invention is consumed by infants up to the age of at least 12 months.

[0027] In a preferred embodiment, an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) according to the present invention is the only or primary infant or young child formula consumed by an infant up to the age of at least 12 months.

[0028] The formula can be used to promote healthy development, promote a healthy immune system, promote healthy intestinal function, and / or prevent microbiome dysbiosis, especially in infants or young children, especially those with cow's milk protein allergies.

[0029] Infant or young child formulas may contain 2'FL and LNnT. In one embodiment, the formula may contain 0.5-3 g / L, 0.8-1.5 g / L, or about 1 g / L of 2'FL, preferably about 1 g / L of 2'FL; and / or the formula may contain 0.2-1 g / L, 0.5-0.8 g / L, or about 0.5 g / L of LNnT, preferably about 0.5 g / L of LNnT. Most preferably, the formula contains about 1 g / L of 2'FL and about 0.5 g / L of LNnT.

[0030] In one embodiment, the infant or young child has a cow's milk protein allergy.

[0031] In one embodiment, the infant or young child formula is a hypoallergenic formula. In one embodiment, the hypoallergenic infant or young child formula is an extensively hydrolyzed formula (eHF) or an amino acid-based formula (AAF). In one embodiment, the infant or young child formula is an eHF. In one embodiment, the infant or young child formula is an AAF. Preferably, the infant formula is an eHF. In an alternative embodiment, the or young child formula is a partially hydrolyzed formula (pHF).

[0032] At least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in the eHF may have a molecular weight of less than about 3000 Da. Preferably, there are no detectable peptides in the eHF greater than or equal to about 3000 Da in size.

[0033] 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 can have a molecular weight of less than about 1500 Da. Preferably, at least about 99% of the peptides in the eHF have a molecular weight of less than about 1500 Da.

[0034] 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 can have a molecular weight of less than about 1200 Da. Preferably, at least 98% by weight of the peptides have a molecular weight of less than about 1200 Da.

[0035] At least about 45 wt.%, at least about 50 wt.%, 45-55 wt.%, or 50-54 wt.% of the peptides in the eHF can be dipeptides and tripeptides. Preferably, about 51-53 wt.%, or more preferably about 52 wt.%, of the peptides in the eHF are dipeptides and tripeptides.

[0036] At least about 45%, at least about 50%, 45-55%, or 50-54% by weight of the peptides in the eHF can have a molecular weight of 240-600 Da. Preferably, about 51-53%, or more preferably about 52% by weight of the peptides in the eHF have a molecular weight of 240-600 Da.

[0037] At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the protein in the eHF can be whey protein. Preferably, the source of protein is whey protein.

[0038] The eHF may contain free amino acids. The free amino acids may 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 free amino acids 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.

[0039] In one embodiment, the infant formula is an eHF containing protein, carbohydrates, and fat, wherein the eHF contains about 2.4 g or less of protein per 100 kcal, or eHF contains about 2.9 g or less of protein per 100 kcal, the infant formula further contains 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT), and about 30% or less by weight of the fat is medium-chain triglycerides (MCT).

[0040] In another embodiment, the infant formula is an AAF containing protein, carbohydrate, and fat, wherein the AAF contains about 2.8 g or less of protein per 100 kcal, preferably about 2.7 g or less of protein per 100 kcal, more preferably about 2.6 g or less of protein per 100 kcal, and further contains 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT), and about 30% or less by weight of the fat is medium-chain triglycerides (MCT). Preferably, the AAF contains 2.5 g or less of protein per 100 kcal.

[0041] In one embodiment, the infant formula contains 1.8-2.4g protein per 100kcal, 2.1-2.3g protein per 100kcal, or 2.15-2.25g protein per 100kcal. Preferably, the infant formula contains about 2.2g protein per 100kcal.

[0042] In one embodiment, about 30% or less, about 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less by weight of the fat in an infant or young child formula can be medium chain triglycerides (MCTs). Preferably, the infant formula does not contain added MCTs.

[0043] In one embodiment, the infant formula may contain 9-14 g of carbohydrates per 100 kcal and / or 4.0-6.0 g of fat per 100 kcal. [Brief explanation of the drawings]

[0044] [Figure 1A] Figure 1A shows a transition model illustrating the development of early to late fecal community type (FCT) clusters over time compared to groups fed the test formula (HMO) (Figure 1A) or the control formula (Figure 1B). [Figure 1B]Figure 1A shows a transition model illustrating the development of early to late fecal community type (FCT) clusters over time compared to groups fed the test formula (HMO) (Figure 1A) or the control formula (Figure 1B). [Figure 2] FIG. 1 shows the differences in gene richness and alpha diversity, as expressed by Shannon diversity, between groups fed test formula (HMO) or control formula in 12-month-old infants. [Figure 3A] FIG. 1 shows the difference in FCT distribution at 12 months of age between groups fed test formula (HMO) or control formula. [Figure 3B] FIG. 1 shows the difference in FCT distribution at 12 months of age between groups fed test formula (HMO) or control formula.

[0045] [Mode for Carrying Out the Invention] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0046] As used herein, the following terms have the following meanings:

[0047] The term "infant" refers to a child under 12 months of age (<12 months of age).

[0048] The term "young children" refers to children between the ages of 1 and 3 (≥1 to <3 years), also known as toddlers.

[0049] The expression "conventional infant or young child formula" refers to standard synthetic nutritional compositions such as infant formula, follow-on milk or growing-up milk already on the market.

[0050] In this context, the terms "microorganism", "bacterial flora", "microbiome" and "microbiota" can be used interchangeably.

[0051] In this context, the expressions "gut microbiota", "gut microflora" and "gut microbiome" can be used interchangeably.

[0052] An adequate and healthy gut microbiota is a key factor in the development of the infant's mucosal immune system.

[0053] The terms "downregulation" and "reduction" can be used interchangeably.

[0054] The terms "prevent" or "prevention" mean avoiding the occurrence of and / or reducing the incidence (ie, reducing the frequency) of a physical condition, situation, or its consequences.

[0055] The terms "treating" or "treatment" refer to the lessening of the duration and / or severity of a physical condition, a symptom, or its consequences.

[0056] Prevention and / or treatment of a physical condition, state, or consequence thereof can occur during treatment (i.e., during administration of the compositions of the invention, either immediately after initiation of administration or some time later, e.g., days or weeks after initiation). However, they can also encompass prevention and / or treatment in later life. The term "later life" encompasses effects after the intervention or treatment has ceased. Effects in "later life" can last from 1 week to several months, e.g., 2-4 weeks, 2-6 weeks, 2-8 weeks, 1-6 months, or 2-12 months.

[0057] The term "prebiotic" refers to non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and / or activity of healthy bacteria, such as bifidobacteria, in the human colon (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995;125:1401-12).

[0058] The term "probiotic" refers to a preparation of microbial cells or components of microbial cells that have a beneficial effect on the health or quality of life of the host (Salminen S, Ouwehand A, Benno Y, et al., "Probiotics: how should they be defined", Trends Food Sci. Technol. 1999:10 107-10). Microbial cells are broadly defined as bacteria or yeasts.

[0059] The term "cfu" is to be understood as colony forming unit.

[0060] All percentages (%) are by weight unless otherwise specified.

[0061] Additionally, it should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0062] 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."

[0063] As used herein, the term "about" means approximately, in the region of, roughly, or in the vicinity of. When the term "about" is used in conjunction with a numerical value or range, that value or range modifies that value or range by extending the boundaries above and below the stated numerical value(s). In general, the terms "about" and "approximately" are used herein to adjust numerical value(s) above and below the stated value(s) by 10%.

[0064] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any reference cited herein should not be construed as an admission that such reference constitutes prior art to the claims appended hereto.

[0065] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range.

[0066] Formula The term "infant formula" can refer to a food for specific nutritional uses for infants during the first year of life, where the formula alone meets the nutritional requirements of humans falling within this category (as defined in European Commission Regulation (EU) 2016 / 127 of 25 September 2015). It also refers to nutritional compositions intended for infants and as defined in Codex Alimentarius (Codex STAN 72-1981), and special infant foods (including foods for special medical purposes). The expression "infant formula" encompasses both "infant starter formulas" and "follow-up formulas" or "follow-on formulas." In some embodiments, the infant formula is a formula for preterm infants.

[0067] A "follow-up formula" or "follow-on formula" is given from the sixth month onwards and constitutes the main liquid component of the increasingly varied diet of infants in this category.

[0068] The expression "growing-up milk" (or GUM) refers to a milk drink, generally with added vitamins and minerals, targeted at young children or young children.

[0069] In certain embodiments, the nutritional compositions of the present invention are hypoallergenic formulas. The term "hypoallergenic formula" refers to a formula for infants or young children that is unlikely to cause an allergic reaction.

[0070] The infant formula of the present invention is an extensively hydrolyzed infant formula (eHF) or an amino acid-based infant formula (AAF). Preferably, the infant formula is an eHF.

[0071] The term "extensively hydrolyzed infant formula" or "eHF" can refer to an infant formula containing extensively hydrolyzed proteins. An eHF can be a hypoallergenic infant formula that provides complete nutrition for infants who cannot digest intact CMP or who have an intolerance or allergy to CMP.

[0072] 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.

[0073] 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 CMPA. This is in line with the guidance provided by the American Academy of Pediatrics (Committee on Nutrition, 2000. Pediatrics, 106(2), pp. 346-349). Preferably, the infant formula of the present invention does not contain peptides that are recognized by CMP-specific IgE, such as IgE in subjects with CMPA.

[0074] Infants can be fed infant formula alone, or infant formula can be used as a supplement to human breast milk.

[0075] The infant formula of the present invention may be in solid form (eg, powder) or liquid form.

[0076] 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.

[0077] In one embodiment, the infant formula is in powder form, which can be reconstituted, for example by adding water, to a liquid composition suitable for feeding to an infant.

[0078] The amounts of the various ingredients can be expressed on a dry weight basis in g / 100 g of the composition if it is in solid form, e.g., a powder, or as a concentration in g / L of the composition if it refers to a liquid form (the latter also encompasses liquid compositions that can be obtained from a powder after reconstitution in a liquid such as milk, water, e.g., a reconstituted infant formula or follow-on / follow-up formula or an infant cereal product or any other formulation designed for infant nutrition). The amounts of the ingredients can also be expressed in g per 100 kcal.

[0079] 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.

[0080] Human milk oligosaccharides The infant formula of the present invention contains one or more human milk oligosaccharides (HMOs).

[0081] 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 large number of different oligosaccharides in human breast milk, with over 130 such structures identified to date. Nearly all of these oligosaccharides have a lactose residue 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).

[0082] The infant formula of the present invention contains 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT).

[0083] The infant formulas of the present invention may include 2'FL, in some embodiments, no other types of fucosylated oligosaccharides are present other than 2'FL, i.e., the infant formulas of the present invention include only 2'FL as a fucosylated oligosaccharide.

[0084] 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.

[0085] The infant formulas of the present invention may include LNnT, and in some embodiments, no other N-acetylated oligosaccharides are present other than LNnT, i.e., the infant formulas of the present invention include only LNnT as an N-acetylated oligosaccharide.

[0086] 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. Pat. No. 5,288,637 and WO 96 / 10086. Alternatively, LNnT can be synthesized as described in Wrodnigg, TM; 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.

[0087] In some embodiments, infant formulas of the present invention comprise an oligosaccharide mixture comprising 2'FL and / or LNnT. In preferred embodiments, infant formulas of the present invention comprise an oligosaccharide mixture comprising 2'FL and LNnT. Infant formulas of the present invention may comprise only 2'FL as the fucosylated oligosaccharide and only LNnT as the N-acetylated oligosaccharide.

[0088] 2'FL may be present in infant formulas according to the invention in a total amount of 0.5-3 g per liter of infant formula, e.g., 0.8-1.5 g per liter (when formulated as directed). In some embodiments, the total amount of 2'-fucosyllactose may be 0.85-1.3 g per liter of infant formula, e.g., 0.9-1.25 g, or 0.9-1.1 g, or 1-1.25 g, or 1-1.2 g per liter (when formulated as directed). Preferably, the infant formula (when formulated as directed) contains about 1 g / L of 2'-fucosyllactose.

[0089] LNnT may be present in infant formulas according to the present invention in a total amount of 0.2-1 g, e.g., 0.5-0.8 g, per L of infant formula (when formulated as directed). In some embodiments, the total amount of LNnT may be 0.5-0.75 g, 0.5-0.7 g, or 0.5-0.6 g, per L of infant formula (when formulated as directed). Preferably, the infant formula (when formulated as directed) contains about 0.5 g / L of LNnT.

[0090] All of these different ranges can be combined together.

[0091] Thus, in one embodiment of the present invention, an infant formula (when formulated as directed) comprises 2'FL and LNnT, (i) the total amount of 2'FL is between 0.8 and 1.5 g per liter of infant formula; and / or (ii) The total amount of LNnT is 0.5–0.8 g per liter of infant formula.

[0092] In another embodiment, an infant formula of the invention (when formulated as directed) comprises 2'FL and LNnT, (i) the total amount of 2'FL is between 0.9 and 1.25 g per liter of infant formula; and / or (ii) The total amount of LNnT is 0.5–0.7 g per liter of infant formula.

[0093] In another embodiment, an infant formula of the invention (when formulated as directed) comprises 2'FL and LNnT, (i) the total amount of 2'FL is between 1 and 1.2 g per liter of infant formula; and / or (ii) The total amount of LNnT is 0.5–0.6 g per liter of infant formula.

[0094] In a preferred embodiment, the infant formula of the present invention (when formulated as directed) contains about 1 g / L 2'FL and about 0.5 g / L LNnT.

[0095] Infant formulas of the present invention may contain 0.075-0.5g / 100kcal, 0.1-0.3g / 100kcal, or 0.12-0.25g / 100kcal of 2'FL and about 0.03-0.15g / 100kcal, 0.05-0.12g / 100kcal, or 0.05-0.1g / 100kcal of LNnT. Preferably, infant formulas of the present invention contain about 0.15g / 100kcal of 2'FL and about 0.075g / 100kcal of LNnT.

[0096] The 2'FL and LNnT contained in infant formulas according to the invention are typically present in a 2'FL:LNnT ratio of 2.0:0.54 to 2.0:2.26, for example 2.0:0.76 to 2.0:1.8 or 2.0:0.8 to 2.0:1.4. In particularly advantageous embodiments, this ratio is at or about 2.0:1.

[0097] 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), pp. 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.

[0098] Protein concentration 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 their high metabolic rate.

[0099] The inventors have surprisingly shown that eHF or AAF, which have a lower protein content, can support proper growth and development in allergic infants. Furthermore, the inventors have surprisingly shown that the formulas are safe and tolerable.

[0100] Thus, an eHF of the present invention contains about 2.4 g or less of protein per 100 kcal, or an AAF of the present invention contains about 2.9 g or less of protein per 100 kcal, preferably about 2.8 g or less of protein per 100 kcal. For example, an infant formula of the present invention 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.

[0101] Preferably, the infant formula contains about 1.8 g or more of protein per 100 kcal. For example, the infant formula of the present invention 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).

[0102] The eHF of the present invention may contain 1.8 to 2.4 g of protein per 100 kcal, 1.86 to 2.4 g of protein per 100 kcal, 1.9 to 2.4 g of protein per 100 kcal, 2.0 to 2.4 g of protein per 100 kcal, 2.0 to 2.3 g of protein per 100 kcal, 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein per 100 kcal.

[0103] The AAF of the present invention may contain 1.8 to 2.9 g of protein per 100 kcal, 1.9 to 2.8 g of protein per 100 kcal, 2.0 to 2.7 g of protein per 100 kcal, 2.0 to 2.6 g of protein per 100 kcal, 2.0 to 2.5 g of protein per 100 kcal, 2.0 to 2.4 g of protein per 100 kcal, 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein per 100 kcal.

[0104] Preferably, infant formulas of the present invention comprise 2.0 to 2.4 g of protein per 100 kcal, such as 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein per 100 kcal.

[0105] Preferably, the infant formula contains about 2.2g of protein per 100kcal.

[0106] Protein Source The source of protein can be any source suitable for use in infant formula. Preferably, the protein is cow's milk protein.

[0107] 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.

[0108] 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).

[0109] 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.

[0110] The whey protein may be a mixture of DWP and MSW.

[0111] 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.

[0112] Degree of hydrolysis Hydrolyzed proteins can be rated as "partially hydrolyzed" or "extremely hydrolyzed" depending on the extent to which the hydrolysis reaction is carried out. Currently, there is no consensus on a legal / clinical definition of an extensively hydrolyzed product that complies with the World Allergy Organization (WAO) guidelines for cow's milk allergy (CMA). However, the WAO recognizes that such hydrolyzed formulas have proven useful and are widely used as protein sources for infants with CMA. In the present invention, a partially hydrolyzed protein is one in which 60-70% of the protein / peptide population has a molecular weight of less than 1000 Daltons, whereas an extensively hydrolyzed protein is one in which at least 95% of the protein / peptide population has a molecular weight of less than 1000 Daltons. These definitions are currently used in the industry. Partially hydrolyzed proteins are typically considered hypoallergenic (HA), whereas extensively hydrolyzed proteins are typically considered nonallergenic.

[0113] In eHF, proteins are "extremely hydrolyzed," so that eHF can be tolerated by more than 90% of infants with CMPA.

[0114] 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%.

[0115] The degree of hydrolysis can also be determined by the degree of hydrolysis. "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), pp. 1515-1522). The degree of hydrolysis (DH) of a protein can be greater than 90, greater than 95, or greater than 98.

[0116] 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, PW, et al., 2011. Food Chemistry, 125(3), pp. 1041-1050). For example, peptide molecular weight distribution can be an estimate based on HPSEC peak area 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, for example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Chauveau, A., et al., 2016. Pediatric Allergy and Immunology, 27(5), pp. 541-543).

[0117] Theoretically, peptides must be larger than approximately 1500 Da (approximately 15 amino acids) in size to bind to cell membrane-bound IgE, and larger than approximately 3000 Da (approximately 30 amino acids) in size to cross-link IgE molecules and induce an immune response (Nutten, 2018. EMJ Allergy Immunol, 3(1), pp.50-59).

[0118] Thus, preferably, at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in the eHF have a molecular weight of less than about 3000 Da. There may be no detectable peptides in the eHF greater than or equal to about 3000 Da in size.

[0119] Thus, suitably, at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in the 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. There can be no detectable peptides in the eHF greater than or equal to about 1500 Da in size.

[0120] 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.

[0121] 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.

[0122] Preferably, 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] The peptides in the eHF may have a median molecular weight of 300 Da to 370 Da, preferably 320 Da to 360 Da.

[0127] The major recognized allergens in cow's milk are α-lactalbumin (aLA), β-lactoglobulin (bLG), and bovine serum albumin (BSA).

[0128] 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 of the present invention 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.

[0129] 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; the eHF of the present invention contains about 1 g / L 2'-fucosyllactose and about 0.5 g / L lacto-N-neotetraose, and / or about 0.15 g / 100 kcal 2'-fucosyllactose and about 0.075 g / 100 kcal lacto-N-neotetraose; and the eHF does not contain added MCTs.

[0130] 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.

[0131] Porcine enzymes, particularly porcine pancreatic enzymes, may be used in the hydrolysis process. For example, WO 9304593(A1) discloses a hydrolysis process using trypsin and chymotrypsin. This process involves a two-step hydrolysis reaction, with a heat denaturation step between the 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.

[0132] WO2016156077A1 discloses a process for preparing a milk protein hydrolysate, which comprises hydrolyzing a milk-based proteinaceous material with a microbial alkaline serine protease in combination with bromelain, an Aspergillus-derived protease, and a Bacillus-derived protease.

[0133] Free amino acids The infant formula of the present invention may include free amino acids.

[0134] 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).

[0135] For example, free amino acids can be incorporated into the eHF of the present invention to complement the amino acids contained in the peptide.

[0136] In AAF, the protein content of infant formula is provided by free amino acids.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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; the eHF of the present invention contains about 1 g / L 2'-fucosyllactose and about 0.5 g / L lacto-N-neotetraose, and / or about 0.15 g / 100 kcal 2'-fucosyllactose and about 0.075 g / 100 kcal lacto-N-neotetraose; and the eHF does not contain added MCTs.

[0141] 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.

[0142] The carbohydrate can be any carbohydrate suitable for use in infant formula.

[0143] 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.

[0144] In one embodiment, the carbohydrate content comprises maltodextrin, hi one embodiment, 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.

[0145] In one embodiment, the carbohydrate content comprises lactose, hi one embodiment, 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.

[0146] In one embodiment, the carbohydrates include lactose and maltodextrin.

[0147] fat The fat content of the infant formula of the present invention is preferably in the range of 4.0 to 6.0 g of fat per 100 kcal.

[0148] The fat can be any lipid or fat suitable for use in infant formula.

[0149] Examples of fats 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.

[0150] Fats 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.).

[0151] Further examples of fats 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.

[0152] Oils such as fish or microbial oils that are rich in preformed arachidonic acid (ARA) and / or docosahexaenoic acid (DHA) may also be added.

[0153] Long chain polyunsaturated fatty acids such as dihomo-γ-linolenic acid, arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid (DHA) may also be added.

[0154] 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.

[0155] 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 fat (Borschel, M., et al., 2018. Nutrients, 10(3), p. 289).

[0156] Thus, up to about 30% by weight of the fat in the infant formulas of the present invention may be medium chain triglycerides (MCTs).

[0157] 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 fat.

[0158] 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 fat.

[0159] Preferably, the infant formula does not contain added MCTs. Preferably, MCTs are about 0% of the fat by weight and / or the infant formula does not contain detectable MCTs. Preferably, the infant formula does not contain MCTs.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] The infant formulas of the present invention may include one or more carotenoids.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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 alimentarius), Lactobacillus casei subsp. casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbruckii subsp. lactislactis, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidilactici These are: Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosus, and Staphylococcus xylosus.

[0168] The infant formula of the present invention may also contain other substances that may have beneficial effects, such as prebiotics, lactoferrin, dietary fiber, nucleotides, nucleosides, etc.

[0169] Health benefits The infant or young child formula of the present invention, comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), can be advantageously used to inhibit or reduce the aforementioned premature shift to an adult-type gut microbiome in infants who are not or only partially breastfed.

[0170] The hypoallergenic infant formula according to the present invention, comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT), can advantageously be used to induce a reduction in microbial diversity and gut microbiota age at 12 months of age compared to a comparable conventional (commercially available) hypoallergenic infant formula that does not contain said 2'FL and LNnT.

[0171] The infant or young child formula of the present invention has a positive effect on the overall microbiota of the target infant or young child, and the formula inhibits or reduces premature aging of the gut microbiota of infants or young children fed the nutritional composition of the present invention compared to infants or young children fed primarily or exclusively conventional nutritional compositions that do not contain 2'FL and LNnT.

[0172] Surprisingly, the beneficial effects of the infant or young child formula of the present invention containing the HMOs 2'FL and LNnT on the gut microbiome were observed in 12-month-old infants despite dietary diversification that involved a reduction in the proportion of the infant's dietary intake with infant or young child formula.

[0173] An adequate and healthy gut microbiota is a key factor in the development of the infant's mucosal immune system.

[0174] In one aspect, the present invention provides an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) for use in inhibiting or reducing premature maturation of the gut microbiota.

[0175] In one embodiment, the present invention provides an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) for use in delaying the maturation of the gut microbiota.

[0176] In one embodiment, the present invention provides an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) for use in delaying the maturation of the gut microbiota into an adult-type gut microbiota.

[0177] In another aspect, the present invention provides a method for inhibiting or reducing premature maturation of the gut microbiota in an infant in need thereof, comprising administering to the infant an infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT).

[0178] In one embodiment, the present invention provides a method for delaying premature maturation of the gut microbiota in an infant in need thereof, comprising administering to the infant an infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT).

[0179] In one embodiment, the invention provides an infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), for use in inducing a less diverse microbiota at 12 months of age compared to the microbiota at 12 months of age of an infant fed a conventional infant formula that does not contain 2'FL and LNnT.

[0180] In one embodiment, the present invention provides an infant or young child formula comprising human milk oligosaccharides (HMOs) 2'FL and lacto-N-neotetraose (LNnT) for use in inducing a lower microbiota age at 12 months of age compared to infants fed a conventional infant formula that does not contain 2'FL and lacto-N-neotetraose (LNnT).

[0181] Surprisingly, the beneficial effects of the infant or young child formula of the present invention containing the HMOs 2'FL and LNnT on the gut microbiome were observed in 12-month-old infants despite a diversified diet accompanied by a reduced proportion of dietary intake with the formula of the present invention.

[0182] In a preferred embodiment, an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) according to the present invention is consumed by infants up to the age of at least 12 months.

[0183] In a preferred embodiment, an infant or young child formula comprising the human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) according to the present invention is the only or primary infant or young child formula consumed by an infant up to the age of at least 12 months.

[0184] In one embodiment, inhibiting or reducing premature maturation of the gut microbiota means inducing a lower microbial diversity at 12 months of age compared to infants fed a conventional infant formula that does not contain 2'FL and LNnT.

[0185] In one embodiment, inhibiting or reducing premature maturation of the gut microbiota means inducing a lower microbiota age at 12 months of age compared to infants fed a conventional infant formula that does not contain 2'FL and LNnT.

[0186] In one embodiment, a lower microbiota age at 12 months of age means having a gut microbiome that is enriched in early fecal community type (FCT) clusters compared to infants fed an infant formula that does not contain 2'FL and LNnT.

[0187] In one embodiment, a lower microbiota age at 12 months of age means having a gut microbiome with reduced late fecal community type (FCT) clusters compared to infants fed an infant formula that does not contain 2'FL and LNnT.

[0188] "Early" FCTs are FCT clusters generally associated with young breastfed infants, e.g., infants up to 6 months of age. In the context of the studies described in the Examples below, early FCTs correspond to FCT clusters GC1, GC2, and GC3.

[0189] "Late" FCTs are FCT clusters commonly associated with older infants, children, and adults. In the context of the studies described in the Examples below, early FCTs correspond to FCT cluster GC5.

[0190] The infant or young child formula of the present invention can be used to promote healthy development, promote a healthy immune system (e.g., prevent or reduce the incidence of infections), promote healthy gut function, and / or prevent microbiome dysbiosis, especially in infants or young children, especially those with cow's milk protein allergies.

[0191] Manufacturing method The infant formulas of the present invention can be prepared in any suitable manner.

[0192] For example, the infant formula can be prepared by blending together a hydrolyzed protein source, a carbohydrate source, and a fat source 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. Any lipophilic vitamins and emulsifiers, etc., can be dissolved in the fat 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] [Example] The present invention will now be further described by way of examples, which are meant to be provided to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way.

[0197] Example 1 - Exemplary Highly Hydrolyzed Infant Formula The following is an exemplary extensively hydrolyzed infant formula according to the present invention. The eHF of the present invention preferably contains nutritionally significant amounts of all nutrients, vitamins, and minerals considered essential in the daily diet. Minimum requirements have been established for certain nutrients, vitamins, and minerals.

[0198] [Table 1]

[0199] Example 2 - Effect of extensively hydrolyzed infant formula supplemented with 2'FL and LNnT on gut microbiome development in infants with cow's milk protein allergy Study design The effect of extensively hydrolyzed infant formula (eHF) supplemented with 2'FL and LNnT on the fecal microbiome in infants with cow's milk protein allergy (CMPA) was investigated in a controlled, double-blind, randomized, multicenter interventional clinical trial with two parallel formula-fed groups.

[0200] Infants aged 0-6 months with CMPA were randomized from enrollment to 12 months of age to receive either lactose-containing commercial eHF (Althera) with or without 2'FL and LNnT, or a parallel eHF (Althera 2.2) with reduced protein levels and two human milk oligosaccharides (HMOs) 2'FL and LNnT (test formula). Commercial eHF is currently approved as a food for specified medical purposes (Regulation (EU) 2016 / 128).

[0201] The study population consisted of full-term infants with physician-diagnosed CMPA according to standard clinical practice and who met at least two of the inclusion criteria. A total of 130 infants were required to complete 4 months of study formula intake.

[0202] The selection criteria were as follows: 1. Full-term infants (37 weeks ≤ gestational age ≤ 42 weeks); 2.2500g≦birth weight≦4500g; 3. Written informed consent has been obtained from the infant's guardian (or parents, if required by national regulations) or legal representative (LAR); 4. Infants up to 6 months of age; 5. Those who are formula-fed exclusively at the time of enrollment or mothers of breastfed CMPA infants who voluntarily chose to feed formula exclusively before enrollment: and 6. Infants diagnosed with CMPA by a physician according to standard clinical practice (and who have not been treated with extensively hydrolyzed milk or amino acid infant formula) who have the presence of at least two of the following symptoms: crying, vomiting, liquid stools or constipation, atopic skin lesions, urticaria, or respiratory symptoms. For a diagnosis based on either a positive IgE blood test, skin prick test, patch test, or food challenge, only one of the above symptoms must be present.

[0203] The exclusion criteria were as follows: 1. Congenital diseases or birth defects that may affect development. 2. Chronic malabsorption documented not to be due to CMPA. 3. Significant prenatal and / or postnatal serious illness other than CMPA prior to enrollment (as determined by the investigator's medical judgment). 4. Parents who are minors. 5. Infants whose parents or caregivers cannot be expected to comply with study procedures. 6.Currently participating in another clinical trial or have participated in one since birth.

[0204] The test and control formulas are shown below. [Table 2]

[0205] In the test and control formulas, Over 99% by weight of the peptides had a molecular weight below 3000 Da. Over 95% by weight of the peptides had a molecular weight below 1200 Da. Approximately 52% by weight of the peptides were dipeptides and tripeptides (peptides with molecular weights between 600 and 240 Da). Approximately 22.4% by weight of the total amino acids were free amino acids in the test and control formulas. There was no detectable beta-lactoglobulin (i.e., the beta-lactoglobulin content was less than 0.01 mg / kg). There was no detectable casein (i.e., the casein content was less than 0.2 mg / kg).

[0206] Both formulas were in powder form and were prepared for oral administration by the infant in amounts appropriate for their weight, age, and appetite, according to the instructions printed on the product label on the can.

[0207] Infants received the study formula for a minimum of 4 months after baseline (main study period) and for as long as the infant needed as medically indicated (up to 12 months of age).

[0208] The daily intake required by the infants varied according to their age, weight, and appetite. The products were given to the infants ad libitum, but parents or caregivers followed the guidelines printed on the label and / or received advice from study personnel regarding the appropriate daily intake.

[0209] Infants will have up to seven study visits: baseline (at enrollment), monthly (months +1, +2, +3, +4) from baseline until 4 months after baseline, and 6 months after baseline. One additional final visit is planned when the infant reaches 12 months of age.

[0210] Randomization was 1:1 per study formula group and was performed by minimization in Medidata Balance. Stratification was by age at enrollment (0-60 days, 61-120 days, >120 days), sex, and mode of delivery (vaginal or cesarean section). Twins enrolled were randomized to the same formula.

[0211] fecal microbiome Fecal samples were collected from 132 infants (per-protocol set) at baseline (V0), 1 month (V1) and 3 months (V3) from baseline, and at 12 months of age (V6).

[0212] Microbiome composition was profiled by metagenomic sequencing using a metagenomics species (MGS) approach [Nielsen, HB et al., Nature Biotechnology 2014;32:822]. Each MGS represents a known or novel clade at the species or subspecies level. Samples with similar microbiome composition were clustered into five fecal community types (FCTs) and tracked within a transition model to analyze their development over time [Stewart CJ et al., Nature 2018;22(562):583-8].

[0213] Microbial richness and diversity (Shannon index) were compared between groups at each time point. Permutation ANOVA based on Bray-Curtis dissimilarity was used to compare the effects of HMO on the microbiome. Differences in taxonomic composition were assessed by enrichment analysis at the genus, family, and phylum levels.

[0214] Test results The microbiome transition showed a distinctive temporal development from "early" to "late" FCT (Figure 3) and from lower to higher α-diversity. Microbiome development was strongly influenced by age. No significant differences were detected between study groups in V1 and V3, in part due to the wide age range at each time point. At 12 months of age (V6), HMO-treated infants had lower α-diversity (Mann-Whitney U, MWU, MWU, p[richness]<0.003, p[diversity]<0.006) and were enriched for early FCT (two-tailed MWU, p=0.014).

[0215] Figure 1A shows the difference in FCT distribution between the test and control groups stratified by visit. FCT was coded as an ordered factor (GC1 = 1, GC2 = 2, etc.) and the groups were compared pairwise using the MWU test. The figure shows that the test group, the "HMO group" (eHF with 2'FL and LNnT), had a higher prevalence of "early" FCT clusters, especially GC1, GC2, and GC3, compared with the control group, and a lower prevalence of "late" FCT clusters, especially GC5, compared with the control group. This Figure 1B shows a taxonomic overview of the taxa in each FCT cluster at the phylum level, showing the average abundance within each FCT (GC1–GC5) of the most abundant phyla.

[0216] The characteristics of the FCT clusters obtained at the genus level can be summarized as follows:

[0217] GC1 (typical for newborns): Lowest alpha diversity Small amounts of Bacteroidetes (phylum) Abundance of Proteobacteria (phylum) and Enterobacteriaceae (family) Low butyrate production

[0218] GC2 (typical for infants aged 1-8 months): Moderate alpha diversity Abundant Actinobacteria (phylum) and Bifidobacterium (genus) Small amounts of Bacteroidetes (phylum)

[0219] GC3 (typical for infants 3 months to 1 year old): Moderate alpha diversity Abundant Firmicutes (phylum), Lachnospira (family), and Lachnoclostridium (genus)

[0220] GC4 (typical for infants >6 months of age) Moderate alpha diversity Numerous Firmicutes (phylum) Moderate amounts of Bifidobacterium (genus)

[0221] GC5 (typical for infants >9 months of age) Highest alpha diversity Abundant Firmicutes (phylum) and Facaribacterium (genus) Abundant Bacteroidetes (family) Higher butyrate production

[0222] Figure 2 shows the gene richness (Figure 2A) and Shannon diversity index (Figure 2B) of the groups (i.e., how diverse the bacterial population is in each sample). The figure shows that at 12 months of age, infants in the test group "HMO group" (eHF supplemented with 2'FL and LNnT) had lower alpha diversity than the control group.

[0223] Figure 3 shows a transition model illustrating the development of "early" to "late" FCT clusters over time. The transition model shows the progression of samples through each FCT using all 481 samples. Node size represents the proportion of infants in a given cluster per age group (column), and line width represents the proportion of transitions per age group (column). Figure 3 shows the test group "HMO group" (eHF supplemented with 2'FL and LNnT). Figure 3A shows a slower development of "early" to "late" FCT clusters over time compared to the control group. Figure 3B corresponds to a lower microbiota age in the test group "HMO group" compared to the control group.

[0224] From these results, we can conclude that the gut microbiome evolved with age in both study groups, reflecting changes in diet and environmental exposures. Feeding HMO-supplemented EHF was associated with lower microbial diversity and reduced gut microbiota age at 12 months of age. Thus, the addition of 2'FL and LNnT to eHF infant formula was observed to delay the earlier shift toward an adult-type gut microbiome in infants who received no or only some breast milk.

[0225] This observed persistence of infants in an earlier stage of microbiome development, i.e., a younger "microbiome age," can be associated with a reduced overall infection rate in the first year of life (previously published results: Vandenplas Y et al., Oral poster presentation #1885 at EAACI Digital Congress, June 2020), with the greatest reduction observed in the frequency of upper respiratory tract infections.

[0226] Example 3 - Exemplary Amino Acid-Based Infant Formula The following is an example of an amino acid-based infant formula according to the present invention. The AAF of the present invention preferably contains nutritionally significant amounts of all nutrients, vitamins, and minerals considered essential in the daily diet. Minimum requirements have been established for certain nutrients, vitamins, and minerals.

[0227] [Table 3]

[0228] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the disclosed methods, cells, compositions, and uses of the 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. [Item 1] 1. An infant or young child formula comprising the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), for use in inhibiting or reducing premature maturation of the gut microbiota in an infant or young child, and / or delaying the maturation of said gut microbiota, and / or inducing a less diverse microbiota at 12 months of age compared to the microbiota at 12 months of age of an infant fed a conventional infant formula that does not contain 2'FL and LNnT, and / or inducing a lower gut microbiota age at 12 months of age compared to an infant fed a conventional infant formula that does not contain 2'FL and LNnT. [Item 2] 2. An infant or young child formula for use according to item 1, wherein a lower microbiota age at 12 months of age means having a gut microbiome enriched in early fecal community type (FCT) clusters compared to infants fed an infant formula that does not contain said 2'FL and LNnT. [Item 3] 2. An infant or young child formula for use according to item 1, wherein a lower microbiota age at 12 months of age means that the infant has a gut microbiome with fewer late-type fecal community type (FCT) clusters compared to infants fed an infant formula that does not contain said 2'FL and LNnT. [Item 4] 4. An infant formula for use according to any one of items 1 to 3, wherein the infant formula comprises 0.5 to 3 g / L, 0.8 to 1.5 g / L, or about 1 g / L of 2'FL, preferably about 1 g / L of 2'FL. [Item 5] 5. The infant formula for use according to any one of items 1 to 4, wherein the infant formula comprises 0.2 to 1 g / L, 0.5 to 0.8 g / L, or about 0.5 g / L of LNnT, preferably about 0.5 g / L of LNnT. [Item 6] 6. An infant formula for use according to any one of items 1 to 5, wherein the infant formula comprises about 1 g / L of 2'FL and about 0.5 g / L of LNnT. [Item 7] 7. Infant formula for use according to any one of items 1 to 6, wherein the infant formula is eHF and comprises 1.8 to 2.4 g of protein per 100 kcal, preferably 2.0 to 2.4 g of protein per 100 kcal, preferably 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein per 100 kcal. [Item 8] 7. Infant formula for use according to any one of items 1 to 6, wherein the infant formula is AAF and comprises 1.8 to 2.9 g of protein per 100 kcal, preferably 1.9 to 2.8 g of protein per 100 kcal, preferably 2.0 to 2.7 g of protein per 100 kcal, more preferably 2.0 to 2.6 g of protein per 100 kcal, or 2.0 to 2.4 g of protein per 100 kcal. [Item 9] 9. An infant formula for use according to any one of items 1 to 8, wherein the infant formula comprises about 2.2 g of protein per 100 kcal. [Item 10] 10. The infant formula for use according to any one of items 1 to 9, wherein 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 fat in the infant formula is medium chain triglycerides (MCT). [Item 11] 11. An infant formula for use according to any one of items 1 to 10, wherein the infant formula does not contain added MCTs. [Item 12] 12. Infant formula for use according to any one of items 1 to 11, wherein the infant formula comprises 9 to 14 g of carbohydrates per 100 kcal and / or 4.0 to 6.0 g of fat per 100 kcal. [Item 13] 13. Infant formula for use according to any one of items 1 to 12, wherein the infant has a cow's milk protein allergy. [Item 14] A method for inhibiting or reducing premature maturation of the intestinal microbiota and / or delaying maturation of the intestinal microbiota in an infant in need thereof, comprising administering to the infant a formula for infants or young children comprising human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT). [Item 15] A method for inducing a less diverse microbiota at 12 months of age compared to the microbiota at 12 months of age of an infant fed a conventional infant formula that does not contain 2'FL and LNnT, and / or inducing a lower gut microbiota age at 12 months of age compared to an infant fed a conventional infant formula that does not contain 2'FL and LNnT, comprising administering to the infant an infant or young child formula that contains the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT).

Claims

1. 1. An infant or young child formula that is an extensively hydrolyzed formula (eHF) or an amino acid-based formula (AAF) comprising human milk oligosaccharides (HMO) 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), wherein the infant or young child has a cow's milk protein allergy, for use in inhibiting or reducing premature maturation of the gut microbiota in the infant or young child, and / or delaying the maturation of the gut microbiota, and / or inducing a less diverse microbiota at 12 months of age compared to the microbiota at 12 months of age of infants fed a conventional infant formula that does not contain 2'FL and LNnT, and / or inducing a lower gut microbiota age at 12 months of age compared to infants fed a conventional infant formula that does not contain 2'FL and LNnT.

2. 2. The formula of claim 1, wherein the lower microbiota age at 12 months of age means that the infants fed the infant formula without 2'FL and LNnT have a gut microbiome enriched in early fecal community type (FCT) clusters.

3. 2. The formula of claim 1, wherein a lower microbiota age at 12 months of age means that the infant has a gut microbiome with fewer late fecal community type (FCT) clusters compared to infants fed an infant formula that does not contain 2'FL and LNnT.

4. 4. The formula of any one of claims 1 to 3, wherein the formula comprises 0.5 to 3 g / L of 2'FL.

5. 5. The formula of any one of claims 1 to 4, wherein the formula comprises 0.2 to 1 g / L of LNnT.

6. 6. The formula of any one of claims 1 to 5, wherein the formula comprises about 1 g / L 2'FL and about 0.5 g / L LNnT.

7. 7. The formula of any one of claims 1 to 6, wherein the formula is eHF and the formula contains 1.8 to 2.4 g of protein per 100 kcal.

8. 7. The formula of any one of claims 1 to 6, wherein the formula is AAF and the formula contains 1.8 to 2.9 g of protein per 100 kcal.

9. 9. The formula of any one of claims 1 to 8, wherein the formula contains about 2.2g of protein per 100kcal.

10. 10. The formula of any one of claims 1-9, wherein no more than about 25% by weight of the fat in the formula is medium chain triglycerides (MCT).

11. 11. The formula of any one of claims 1 to 10, wherein the formula does not contain added MCTs.

12. A formula according to any one of claims 1 to 11, wherein the formula comprises 9 to 14 g of carbohydrates per 100 kcal and / or 4.0 to 6.0 g of fat per 100 kcal.

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