3-fucosyllactose and butyrate for food allergy

Abstract

The present invention relates to nutritional compositions comprising digestible carbohydrates, lipids, proteins, 3-fucosyl lactose (3-FL) and a butyrate source, which are suitable for providing nutrition to an infant or young child suffering from or at risk of developing a food allergy, preferably cow milk protein or egg allergy, wherein the protein is or consists essentially of hydrolysed protein and / or free amino acids, and wherein the butyrate is provided in the form of dietary butyrate and / or by non-digestible saccharides selected from non-digestible polysaccharides, non-digestible oligosaccharides or mixtures thereof capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate, and uses thereof.

Description

Technical Field

[0001] This invention relates to compositions comprising at least 3-fucosyllactose (3-FL) and butyrate, and the use of these compositions in the prevention of food allergies and the induction of immune tolerance in infants. Background Technology

[0002] Human milk is the preferred food for infants. It provides several bioactive factors beneficial to the relatively immature immune system and gut health of newborns in early life. Breastfed infants have a lower incidence of infections and allergies than formula-fed infants. Many components of human milk, including immunoglobulins (such as sIgA), interleukin (IL)-1, IL-6, IL-8, IL-10, interferon-γ (IFN-γ), immune-active cells, transforming growth factor-β (TGF-β), lactoferrin, nucleotides, and human milk oligosaccharides (HMOs), are believed to play a role in protecting against pathogen infection. Furthermore, the gut maturation and microecological development of breastfed infants are considered optimal.

[0003] When breastfeeding is not possible or the mother chooses not to breastfeed, commercially available infant formula suitable for complete nutrition can be purchased. These formulas are typically based on complete milk proteins, particularly whey and casein.

[0004] Food allergens are among the first allergens infants encounter in early life: typically, infants not exclusively breastfed may be exposed to cow's milk proteins. Cow's milk allergy (CMA) is the most prevalent food allergy in early life; approximately 2% to 3% of infants are allergic to cow's milk proteins, and its prevalence has increased over the past few decades [Tsuge et al., "Current insights into atopic march," Children, 2021]. In addition to potentially severe acute clinical manifestations, early-life CMA can have long-term effects, including growth retardation and an increased risk of developing atopic diseases later in life. Therefore, strategies for treating or preventing CMA are crucial.

[0005] Dietary proteins (such as milk and egg proteins) are presented to the immune system via the gastrointestinal tract, and a normal response during the maturation of the immune system will trigger a tolerant immune response to the ingested nutrients. This response is called oral immune tolerance or oral tolerance. Inducing oral immune tolerance is particularly relevant for infants, who are first exposed to dietary proteins after birth and must adapt to this. If oral immune tolerance is not properly established in an infant, food sensitization may occur.

[0006] For example, standard dietary management of CMA in infants and children involves avoiding allergens by eliminating them from the diet. Various formula milk powders have been developed to eliminate milk proteins. For infants with mild to moderate CMA, extensively hydrolyzed formula (eHF) is recommended, while for infants with severe CMA, or those who cannot tolerate eHF or whose CMA symptoms are not relieved by eHF, amino acid-based formula (AAF) is recommended.

[0007] Currently, symptom management for food allergy sufferers consists of actively avoiding allergens. However, there is growing interest in limiting the development of food allergies by preventing anaphylactic sensitization. Anaphylactic sensitization during childhood, especially in early childhood, is crucial and of great interest because it has been shown that the formation of an "anaphylactic phenotype" or "atopic" promotes subsequent sensitization to other allergens. Therefore, childhood allergies may be the first step in causing multiple allergies later in life; this process is often referred to as the "atopic process."

[0008] An infant's immune system is actively developing in the first few years of life, and this immune maturation involves the establishment of type 1 immunity at the expense of type 2 immunity. Taking action—preventing, avoiding, managing, mitigating, or modulating—allergic reactions in such young patients can influence their allergic characteristics not only in the short term but also in the long term. Therefore, allergy prevention is an important aspect when considering the development of early childhood formula concepts.

[0009] Human milk oligosaccharides (HMOs) have become a subject of considerable interest in recent years due to their role in numerous biological processes, including immune-related processes in the human body. Mammalian milk contains at least 200 of these complex oligosaccharides (Kunz C et al., Annu Rev Nutr [Annals of Nutrition] 20: 699-722, 2000).

[0010] Exposure to human milk oligosaccharides (HMOs) via breast milk can promote immune system maturation, either directly or through interactions with intestinal epithelial cells (IECs) and / or through fermentation of HMOs into other bioactive compounds (e.g., short-chain fatty acids, including butyrate) by gut microbiota species [Singh et al., “Recent understanding of human milk oligosaccharides in establishing infant gutmicrobiome and roles in immune system”, Food Res. Int. 2021]. Paparo et al., Allergy. 2021; 76:1398-1415, describe the beneficial effects of butyrate from gut microbiota sources on allergic responses. Corona et al., Children. 2021, 8; 804, provide an overview of the structure-function relationships of various HMOs.

[0011] In early life, human milk oligosaccharides (HMOs) present in breast milk are the primary source of indigestible oligosaccharides (NDOs). A variety of immunomodulatory properties have been attributed to HMOs and SCFAs, including both direct effects on epithelial cells and immune cells and indirect effects via the microbiome [Zuurveld et al., “Immunomodulation by Human Milk Oligosaccharides: The Potential Role in Prevention of Allergic Diseases,” Front. Immunol., 2020], such as inducing mucus production, reducing epithelial barrier permeability, interacting with G protein-coupled receptors (GPRs), inhibiting the NF-κB pathway in histone deacetylases (HDACs) and IECs, and improving regulatory T cell (Treg) formation. Many of these pathways are also actively involved in allergic diseases.

[0012] Functionally, allergies result from a lack of tolerance induction to allergens and an inappropriate type 2 (Th2) immune response to normally harmless proteins. Evidence points to a link between the gut microbiota and the Th2 response, suggesting that the gut microbiota participates in the development of a systemic Th2 response by supporting the induction of immune homeostasis. Therefore, imbalances in the gut microbiota (dysbiosis) (and low levels of bacterial fermentation products, such as SCFAs, including butyrate) may impair the ability to properly establish immune homeostasis and tolerance, thereby increasing the risk of allergies.

[0013] WO 2006 / 115412 A1 relates to a liquid nutritional composition for improving intestinal barrier function and for preventing allergies. The composition comprises lipids having short-chain fatty acyl chains and indigestible, fermentable sugars.

[0014] Zuurveld et al. in Biomolecules [Biomolecules] 2023, 13(2), 263 described how 2'-FL and 3-FL have immunomodulatory properties in neonatal immune maturation by promoting enhanced type I and regulatory mediator secretion in an allergy model.

[0015] WO 2011 / 008086 describes a nutritional composition containing 2'-FL and β-galactooligosaccharides, etc., for use in the treatment of immune disorders. WO 2018 / 210807 A1 describes a nutritional composition containing galactooligosaccharides and HMOs and its use in modifying the gut microbiome. WO 2012 / 158517 A1 further describes 2'-FL, 3-FL, and LDFT for stimulating the growth of bacteria in the human gastrointestinal tract.

[0016] WO 2022 / 253980 A1 describes age-specific infant formula containing 3-FL, 3'-SL, DFL and 2'-FL, while WO 2021116236 A1 describes age-specific nutritional composition systems containing different amounts of 2'-FL, 3-FL, LNT, 6'-SL and 3'-SL for different infant age groups.

[0017] WO 2020 / 245311 A1 relates to a nutritional composition comprising 2'-fucosylated lactose and dietary butyrate, for the purpose of improving gut health, particularly for the treatment or prevention of allergies and for inducing oral tolerance to allergens.

[0018] WO 2022 / 161865 A1 describes a composition comprising Bifidobacterium and a mixture of HMOs consisting of 2'-FL, DFL, LNT, 6'-SL and 3'-SL, for the prevention and / or treatment of allergies by increasing the production of intestinal short-chain fatty acids.

[0019] WO 20201207641 A1 describes a source of butyrate containing a glycerol backbone with one or more butyrate moieties for use in allergic diseases. WO 2018 / 210805 A1 describes preterm infant formula containing butyrate.

[0020] US 2014248415 A1 describes several examples of HMO mixtures comprising both 2'-FL and LNnT in various ratios for a range of health benefits, including immune system maturation and allergy prevention.

[0021] Various approaches have been proposed to reduce the risk of early-life allergies and / or improve the induction of oral tolerance to food allergens in infants and young children; however, there is a continued need to improve the establishment of oral tolerance and to prevent and / or treat food allergies. Summary of the Invention

[0022] The inventors set out to evaluate the effects of the interaction between butyrate and fucosylated human lactose oligosaccharides (2'-fucosylated lactose (2'-FL) and 3-fucosylated lactose (3-FL)) on intestinal epithelial function in a sequential mucosal co-culture model following ovalbumin (OVA)-induced type 2 activation of human intestinal epithelial cells (IECs) and subsequent dendritic cell (DC) maturation and T helper cell function. The inventors found that the combination of the nutrient 3-FL and butyrate has a beneficial effect on immune maturation and prevention of type 2 responses prone to allergies, particularly in the event of food allergen exposure, thus producing a preventative effect against food allergies. It was found that the immune cellular response differed when both 3-FL and butyrate were provided compared to the presence of only one of these components. The mixture of 3-FL and butyrate has been shown to have a beneficial and synergistic effect on the regulation of type 2 immunity by promoting Th1 responses while simultaneously inhibiting Th2 responses, such as IFN secreted by T helper cells. The increased / IL13 ratio is shown. 3-FL, together with butyrate, beneficially promotes immune homeostasis, favoring a type 1 response over a type 2 response, thus deviating from the allergic phenotype. This suggests a beneficial use of 3-fucosyllactose (3-FL) and butyrate sources for providing nutrition to infants or young children who have food allergies, preferably milk protein or egg allergies, or who are at risk of developing or having food allergies, preferably milk protein or egg allergies. Attached Figure Description

[0023] The invention will now be discussed in more detail with reference to the accompanying drawings.

[0024] Figure 1AThe effects of 2'-FL, 3-FL, butyrate, and mixtures thereof on IFN-γ production in naïve T helper cells co-cultured with dendritic cells [DC] sensitized by IECs exposed to OVA are shown.

[0025] Figure 1 (B) illustrates the effect of 2'-FL, 3-FL, butyrate, or a mixture thereof on IL-13 production in naïve T helper cells co-cultured with DCs sensitized by IECs exposed to OVA. In these experiments, the IECs were pre-exposed to 2'-FL, 3-FL, butyrate, or a mixture thereof prior to OVA exposure.

[0026] Figure 2 The ratio of Th1 to Th2 is shown as indicated by the ratio of IFN-γ to IL-13.

[0027] List of preferred embodiments 1. A nutritional composition comprising digestible carbohydrates, lipids, proteins, 3-fucosyllactose (3-FL), and a butyrate source, the nutritional composition being suitable for providing nutrition to human subjects, preferably infants or young children suffering from food allergies, preferably milk protein or egg allergies, or at risk of developing food allergies, preferably milk protein or egg allergies, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed proteins and / or free amino acids, and wherein the butyrate source is provided in the form of dietary butyrate and / or by an indigestible sugar capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate. The indigestible sugars are selected from indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof, and These indigestible sugars are of plant, crustacean, and / or microbial origin.

[0028] 2. The nutritional composition according to Example 1, wherein the nutritional composition is in the form of an instant liquid or in the form of a powder, wherein the powder form is reconstituted with water to become an instant liquid nutritional composition, wherein 3-FL is present in an amount of 10 mg to 1 g 3-FL / 100 ml, more preferably 15 mg to 0.5 g 3-FL, and even more preferably 20 mg to 0.2 g 3-FL / 100 ml of the nutritional composition.

[0029] 3. The nutritional composition according to Example 1 or 2, wherein butyrate is provided in the form of dietary butyrate and / or by indigestible sugars capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate, wherein the nutritional composition comprises 10 mg to 175 mg of dietary butyrate / 100 ml of nutritional composition and / or 1 mg to 1.0 g of indigestible sugars / 100 ml of nutritional composition.

[0030] 4. The nutritional composition according to any one of the foregoing embodiments, wherein the butyrate source comprises dietary butyrate, wherein the dietary butyrate source is selected from the group consisting of: food-grade tricresyl tributyl ester, anhydrous milk fat, butter, and products derived from microbial fermentation.

[0031] 5. The nutritional composition according to any one of the foregoing embodiments, wherein the butyrate source comprises an indigestible oligosaccharide selected from the group consisting of: fructooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucose oligosaccharides, glucose-manniosaccharides, galactomanniosaccharides, mannosaccharides, chitosan oligosaccharides, uronic acid oligosaccharides, and mixtures thereof.

[0032] 6. The nutritional composition according to any one of the foregoing embodiments, wherein the butyrate source comprises an indigestible polysaccharide selected from the group consisting of: low-viscosity pectin, fructooligosaccharides, oat β-glucan, soybean fiber, resistant starch, gum arabic, cellulose, arabinoxylan, xanthan gum, locust bean gum and / or resistant starch.

[0033] 7. The nutritional composition according to any one of the foregoing embodiments, wherein the butyrate source comprises a mixture of indigestible polysaccharides and indigestible oligosaccharides, preferably a mixture of short-chain fructooligosaccharides (scFOS) and long-chain oligosaccharides (lcFOS), more preferably a mixture of scFOS and lcFOS in a weight ratio of 9:1.

[0034] 8. The nutritional composition according to any one of the foregoing embodiments, wherein the butyrate source comprises milk fat and / or fructooligosaccharides, preferably a mixture of short-chain fructooligosaccharides and long-chain fructooligosaccharides.

[0035] 9. The nutritional composition according to any one of the foregoing embodiments, wherein the nutritional composition is infant formula, follow-up formula or toddler milk.

[0036] 10. The nutritional composition according to any one of the preceding claims, wherein the nutritional composition further comprises Bifidobacterium, preferably Bifidobacterium breve.

[0037] 11. The nutritional composition according to any one of the foregoing embodiments, wherein the protein is hydrolyzed whey protein or hydrolyzed rice protein or is substantially composed of hydrolyzed whey protein or hydrolyzed rice protein.

[0038] 12. The nutritional composition according to any one of the foregoing embodiments, wherein the nutritional composition is used in the dietary management of infants or young children suffering from food allergies, preferably egg protein allergies or cow's milk protein allergies, more preferably cow's milk protein allergies, or at risk of food allergies, preferably egg protein allergies or cow's milk protein allergies, more preferably cow's milk protein allergies.

[0039] 13. The nutritional composition according to any one of Examples 1-12, wherein the nutritional composition is used in the treatment or prevention of food allergies, preferably egg protein allergies or cow's milk protein allergies, more preferably cow's milk protein allergies.

[0040] 14. The nutritional composition according to any one of the foregoing embodiments, wherein the nutritional composition is used in the treatment or prevention of food allergies selected from those that stimulate immune function, promote immune maturation, reduce and / or inhibit Th2 response, and improve and / or promote Th1 response, preferably for use in increasing the Th1:Th2 balance.

[0041] 15. Use of lipids, digestible carbohydrates and proteins in the manufacture of a nutritional composition for use in human subjects, preferably infants or young children with allergies or at risk of allergies, preferably human subjects, preferably infants or young children at risk of food allergies, preferably egg or milk protein allergies, or food allergies, preferably egg or milk protein allergies, for dietary management, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed protein and / or free amino acids, and wherein the nutritional composition comprises a combination of 3-FL and butyrate sources.

[0042] 16. A method for treating or preventing food allergies in infants or young children by modulating immune maturation, the method comprising administering to the infant or young child a nutritional composition according to any one of Examples 1 to 11.

[0043] 17. The nutritional composition according to any one of Examples 1 to 11, wherein the butyrate source is not 3-FL.

[0044] 18. The nutritional composition according to any one of Examples 1 to 11, wherein the nutritional composition is a hypoallergenic nutritional composition. Detailed Implementation

[0045] This invention therefore relates to a nutritional composition comprising digestible carbohydrates, lipids, proteins, 3-fucosylated lactose, and butyrate sources, suitable for providing nutrition to human subjects, preferably infants or young children at risk of or already suffering from food allergies, particularly milk protein allergies or egg allergies, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed proteins and / or free amino acids. The nutritional composition according to the invention is a hypoallergenic nutritional composition.

[0046] This invention is particularly advantageous because it provides immune maturation promotion and food allergy prevention, with a lower risk of inducing food allergic reactions when the protein component (which is composed of hydrolyzed protein and / or free amino acids or substantially composed of hydrolyzed protein and / or free amino acids) is used in combination with the immunomodulatory component of 3-FL according to the invention and a separate butyrate source.

[0047] The nutritional composition of the present invention can be in the form of an instant liquid or a powder, which, when reconstituted with water, becomes an instant liquid. This nutritional composition is a synthetic nutritional composition and not human breast milk.

[0048] This invention essentially relates to a nutritional composition for use in human subjects, preferably infants or young children, the nutritional composition comprising a combination of the following: a. 3-Fucose-3-lactose (3-FL), and b. Butyrate, wherein butyrate is provided in the form of dietary butyrate and / or by indigestible sugars selected from indigestible polysaccharides, indigestible oligosaccharides, or mixtures thereof, which are capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate, wherein said indigestible sugars are preferably of plant, crustacean, and / or microbial origin.

[0049] This invention further relates to a nutritional composition comprising digestible carbohydrates, lipids, and proteins, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed proteins and / or free amino acids, and wherein the nutritional composition comprises a combination of 3-FL and a butyrate source, for use in the dietary management of human subjects, preferably infants or young children at risk of or suffering from food allergies, particularly infants or young children with food allergies, especially bovine milk protein and / or egg allergies. In the context of this invention, 3-FL is an immunomodulatory component, not a separate butyrate source. A butyrate source should be understood as a separate source distinct from 3-FL. Use in dietary management includes the prevention of food allergies. In some aspects, use in the dietary management of human subjects, preferably infants or young children at risk of or suffering from allergies, includes the accelerated resolution of said food allergies.

[0050] This can also be described as a method for dietary management of human subjects, preferably infants or young children, who are at risk of or have food allergies, particularly infants or young children with food allergies, especially those with milk protein and / or egg allergies, comprising administering to the human subject, preferably an infant or young child, a nutritional composition comprising digestible carbohydrates, lipids and proteins, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed protein and / or free amino acids, and wherein the nutritional composition comprises a combination of 3-FL and butyrate sources.

[0051] The present invention can also be described as the use of lipids, digestible carbohydrates and proteins in the manufacture of nutritional compositions for use in the dietary management of human subjects, preferably infants or young children, who are at risk of or have food allergies, particularly milk protein and / or egg allergies, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed protein and / or free amino acids, and wherein the nutritional composition contains a combination of 3-FL and butyrate sources.

[0052] The present invention further relates to a nutritional composition comprising digestible carbohydrates, lipids and proteins, wherein the protein is a hydrolyzed protein and / or free amino acids or is substantially composed of hydrolyzed protein and / or free amino acids, and wherein the nutritional composition comprises a combination of 3-FL and butyrate sources, for use in promoting immune maturation and / or preventing the occurrence of food allergies in human subjects, preferably infants or young children, who are at risk of or have food allergies, particularly milk protein or egg allergies.

[0053] Furthermore, the present invention relates to the use of a combination of 3-FL and butyrate sources in a hypoallergenic nutritional composition suitable for human subjects, preferably infants or young children, who are at risk of or have food allergies, particularly to cow's milk protein or eggs, wherein the hypoallergenic nutritional composition comprises digestible carbohydrates, lipids, and proteins, wherein the proteins are hydrolyzed proteins and / or free amino acids or consist substantially of hydrolyzed proteins and / or free amino acids.

[0054] In a preferred embodiment, the protein source consists primarily of free amino acids. In other words, in some aspects, the nutritional composition is preferably an amino acid-based nutritional composition for use in the maturation of the immune system and in the treatment and prevention of food allergies.

[0055] definition In the context of this invention, the term "prevention" means "reducing the risk" or "reducing the severity". The term "preventing a disease" also includes "treating a person who has an increased risk of developing the disease".

[0056] An infant is a child under 12 months of age.

[0057] "Infant formula," "follow-up formula," or "toddler formula" means that it involves an artificially made composition, or in other words, it is a synthetic composition (i.e., a synthetic composition is not breast milk). Therefore, the nutritional composition applied is artificial infant formula, artificial follow-up formula, artificial toddler formula, or synthetic infant formula, synthetic follow-up formula, or synthetic toddler formula.

[0058] Infant formula refers to an artificially produced nutritional composition designed for use by infants aged from 0 to approximately 4 to 6 months of age, and intended as a substitute for human breast milk. Typically, infant formula is suitable as the sole source of nutrition for infants up to 6 months of age. Such infant formula is also known as starter formula. Preferably, infant formula is a nutritional composition designed for use by infants aged 0 to 6 months.

[0059] Follow-up formula is a nutritional composition intended for infants from approximately 4 to 6 months of age up to 12 months of age, and is designed as a complete or supplemental diet for infants beginning weaning and introducing other complementary foods. Preferably, follow-up formula is a nutritional composition intended for infants aged 6 to 12 months. Infant formula and follow-up formula are subject to stringent regulations, such as EU Regulation Nos. 2016 / 127 and 2016 / 128.

[0060] Toddlers are children aged between one and three years, that is, between 12 and 47 months of age, also known as toddlers.

[0061] Infant formula refers to an artificially produced nutritional composition designed for use in children aged 12 to 48 months, intended as the sole or supplemental food for children.

[0062] "Nutritional composition" means a substance or preparation that satisfies at least a portion of the nutritional requirements of a subject. The nutritional composition according to the invention is preferably selected from infant formula, follow-up formula, and toddler formula. This means that the nutritional composition of the invention is not human milk. Alternatively, the term "formula" means that it relates to an artificially made or, in other words, a synthetic composition. Thus, in one embodiment, the nutritional composition is selected from artificial infant formula, artificial follow-up formula, and artificial toddler formula, or synthetic infant formula, synthetic follow-up formula, and synthetic toddler formula.

[0063] As used in this article, "hypoallergenic" means a composition that is unlikely to cause an allergic reaction or is less likely to do so.

[0064] The term HMO or multiple HMOs refers to one or more human milk oligosaccharides. HMOs are complex carbohydrates found in human breast milk (Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); Chen Adv. Carbohydr. Chem. Biochem. Advances in Carbohydrate Chemistry and Biochemistry 72, 113 (2015)). These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes. Each oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine. HMOs can be classified as neutral or non-acidic HMOs (which may be fucosylated or non-fucosylated) and acidic HMOs having at least one sialic acid residue in their structure. In the context of this invention, lactose is not considered an HMO species. HMOs can be manufactured by means known in the art.

[0065] As used herein, the term "degree of polymerization" (DP) refers to the number of monomer units linked together in a polymer or oligomer.

[0066] As used herein, the term “soluble” in the case of polysaccharides, cellulose, or oligosaccharides means that the substance is at least 50% soluble, as described by L. Prosky et al., J Assoc. Off. Anal. Chem. [Official Journal of the Association for Analytical Chemistry] 71, 1017-1023 (1988).

[0067] As used herein, the term "fermentable" refers to the ability to be broken down (anaerobically) into smaller molecules, particularly short-chain fatty acids and lactates, by microorganisms in the colon. Fermentability can be determined, for example, by the method described in Am. J. Clin. Nutr. [American Journal of Clinical Nutrition] 53, 1418-1424 (1991).

[0068] As used herein, immune maturation refers to the development or bias of the immune system, characterized by an increase in the Th1-Th2 balance towards Th1, which is associated with the healthy adult phenotype and is particularly important in early life immune maturation. The healthy adult phenotype contrasts with the default Th2 phenotype, which is characteristic of atopic conditions, including food allergies, allergic asthma, or certain types of autoimmune diseases. Immune maturation can be assessed by measuring the relative contributions of at least one Th1 biomarker and at least one Th2 biomarker; specifically, the ratio of IFN-gamma (IFN-γ) to IL-13 is used in this paper as a biomarker of Th1-Th2 balance.

[0069] As used herein, the T helper cell 1 response (Th1) refers to the objectively measurable manifestation of the Th1 immunophenotype. Th1 biomarkers include, but are not limited to, certain cytokines, including interferon-γ (IFN-γ) (when secreted by CD4+ T helper cells). Methods for measuring Th1 cytokines are well known in the art and may include, but are not limited to, appropriate cytokine-specific or isotype-specific enzyme-linked immunosorbent assays (ELISA), bioassays, quantitative reverse transcriptase-polymerase chain reactions, etc.

[0070] As used herein, T helper cell 2 response (Th2) refers to the objectively measurable manifestation of the Th2 immunophenotype. Th2 biomarkers include, but are not limited to, certain cytokines, including interleukin-13 (IL-13) (when secreted by CD4+ T helper cells). Methods for measuring Th2 cytokines are well known in the art, and include methods mentioned for measuring Th1 cytokines.

[0071] In this document and its claims, the verb “comprising” and its inflections are used in their non-limiting sense to mean including the items following the word, but not excluding items not specifically mentioned. Furthermore, unless the context explicitly requires the presence of one / a and only one / a type of element, referring to an element by the indefinite article “a / a” (“a” or “an”) does not preclude the possibility of more than one / a type of element. Therefore, the indefinite article “a / a” (“a” or “an”) generally means “at least one / a type”.

[0072] This invention relates to a nutritional composition comprising 3-FL and butyrate sources.

[0073] 3-Fucose-based lactose The nutritional composition of this invention comprises 3-fucosyllactose (3-FL). 3-FL has been found to improve immune function. As used herein, 3-FL has immunomodulatory effects. In the context of this invention, 3-FL is not considered a source of butyrate; although 3-FL can be fermented by the gut microbiome, it produces relatively low levels of butyrate. In some aspects, improved immune function involves reducing the secretion of Th2-type IL-13. Fucosyllactose (FL) is an indigestible oligosaccharide found in human milk. It is not found in cow's milk. It consists of three monosaccharide units linked together (fucose, galactose, and glucose). Lactose is a galactose unit linked to a glucose unit via a β1,4 bond. The fucose unit is linked to the galactose unit of the lactose molecule via an α1,2 bond (2'-fucosyllactose, 2'-FL, Fucα1-2Galβ1-4Glc), or to the glucose unit of lactose via an α-1,3 bond (3-fucosyllactose, 3-FL, Galβ1-4(Fucα1-3)Glc).

[0074] 3-FL (preferably Galβ1-4(Fucα1-3)Glc) is commercially available, for example, from Sigma-Aldrich. Alternatively, it can be isolated from human milk, as described, for example, in Andersson and Donald, 1981, J Chromatogr. [Journal of Chromatography] 211:170-1744, or produced by genetically modified microorganisms, as described, for example, in Huang et al., 2017, Metabolic Eng. [Metabolic Engineering] 41: 23-38.

[0075] Preferably, the nutritional composition according to the invention comprises 10 mg to 1 g 3-FL / 100 ml, more preferably 20 mg to 0.5 g, and even more preferably 20 mg to 0.2 g 3-FL / 100 ml. Based on dry weight, the nutritional composition of the invention preferably comprises 0.075 wt.% to 7.5 wt.% 3-FL, more preferably 0.11 wt.% to 3.75 wt.% 3-FL, and even more preferably 0.15 wt.% to 1.5 wt.% 3-FL. Based on energy, the nutritional composition of the invention preferably comprises 0.015 g to 1.5 g 3-FL / 100 kcal, more preferably 0.023 g to 0.075 g 3-FL / 100 kcal, and even more preferably 0.03 g to 0.3 g 3-FL / 100 kcal. Lower amounts of 3-fucosyllactose are less effective in regulating immune responses, promoting immune maturation, or preventing and / or treating allergies, while excessive amounts will lead to unnecessarily high product costs.

[0076] In one aspect of the invention, the nutritional composition does not contain 2'-FL.

[0077] butyrate The nutritional composition of the present invention contains dietary butyrate or indigestible sugars selected from indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate, and the nutritional composition is suitable for human subjects, preferably infants or young children suffering from allergies, particularly food allergies, or at risk of developing allergies, particularly food allergies. The indigestible sugars selected from NDP, NDO, or mixtures thereof are fermented by the gut microbiota, and thus provide butyrate in the gastrointestinal tract. 3-FL has been found to improve the Th1 / Th2 balance when used with a separate butyrate source. 3-FL alone does not produce high levels of butyrate in the gut. In the context of the present invention, 3-FL is not a butyrate source, and the beneficial effects of 3-FL are obtained when it is provided with a separate butyrate source. In the context of the present invention, 3-FL is also not a butyrate source because fermentation would reduce the availability of the amount of 3-FL. Therefore, in the context of the present invention, the butyrate source can be a direct butyrate source containing butyrate or an indirect source that promotes butyrate production in the gut through fermentation by the gut microbiome.

[0078] Dietary butyrate Based on the weight of the total fatty acyl chains, the nutritional composition preferably contains butyric acid between 0.3 wt.% and 5 wt.%, more preferably between 0.6 wt.% and 5 wt.%, and even more preferably between 1 wt.% and 5 wt.%. In embodiments, the nutritional composition of the present invention comprises tributyric acid glyceride (i.e., a triglyceride having three butyric acid chains attached to the glycerol backbone via ester bonds). In another preferred embodiment, the nutritional composition may comprise synthetic triglycerides, such as those described in EP 3897608 B1, which describes triglycerides containing a single butyrate moiety.

[0079] Preferably, based on the dry weight of the composition, the nutritional composition contains 0.075 wt.% to 1.3 wt.%, preferably between 0.15 wt.% and 1.3 wt.%, and more preferably between 0.25 wt.% and 1.3 wt.%. Alternatively, the nutritional composition contains 0.015 to 0.25 g butyrate / 100 kcal, preferably 0.03 to 0.25 g butyrate / 100 kcal, and more preferably 0.05 to 0.25 g butyrate / 100 kcal. When the nutritional composition is a liquid, the composition preferably contains 0.01 to 0.175 g butyrate / 100 ml, more preferably 0.02 to 0.175 g butyrate / 100 ml, and more preferably 0.035 to 0.175 g butyrate / 100 ml.

[0080] Dietary butyrate can be provided from any suitable source known in the art. Non-limiting sources of dietary butyrate include animal fats and derivatives, such as, but not limited to, anhydrous milk fat, butter, and microbial fermentation derivatives, such as, but not limited to, yogurt and fermented buttermilk. In the context of this invention, other well-known butyrate sources (e.g., milk, butter fat, butter, buttermilk, butter whey, and cream) are less preferred because milk proteins are present in said products. In some embodiments, dietary butyrate is produced synthetically.

[0081] In embodiments where dietary butyrate is produced synthetically, the chemical structure of the dietary butyrate can be modified as needed. Furthermore, the synthetically produced dietary butyrate can be purified by any means known in the art to produce a purified dietary butyrate additive, which can be incorporated into the nutritional compositions disclosed herein. Dietary butyrate can be provided in the form of butyrate bound to dairy lipids and / or triglycerides.

[0082] In some embodiments, dietary butyrate may comprise butyrate, such as sodium butyrate, potassium butyrate, calcium butyrate, magnesium butyrate, and combinations thereof. In some embodiments, dietary butyrate comprises a suitable butyrate coated with one or more fats or lipids. In some embodiments where the dietary butyrate comprises a fat-coated butyrate, the nutritional composition may be a dry powder composition incorporating dietary butyrate. Preferably, dietary butyrate is supplied as part of a triglyceride. This is advantageous because butyrate is volatile (and has an odor) when supplied in free or salt form. In triglyceride form, butyrate is released in and after the stomach due to the action of lipases.

[0083] In a preferred embodiment, the weight ratio of 3-FL to dietary butyrate is in the range of 10:1 to 1:10, preferably 5:1 to 1:5, and more preferably 3:1 to 1:3.

[0084] Indigestible sugars In some embodiments, the nutritional composition comprises an indigestible sugar selected from indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof as a source of butyrate. The indigestible sugar selected from indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof as a source of butyrate may optionally further comprise HMOs. The indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof are not digested in the intestine by digestive enzymes present in the human upper digestive tract, but are capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate. The indigestible sugars, together with 3FL, provide the beneficial effects according to the invention.

[0085] Preferred sources of butyrate are indigestible sugars [NDS] capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate. In the context of this invention, NDS used in this composition is defined as NDS or dietary NDS blends capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate. In a preferred aspect, the NDS is an indigestible oligosaccharide.

[0086] Dietary NDS that can act as a substrate to provide butyrate preferably produce acetate, butyrate, propionate and hexanoate, and more preferably, the fiber allows for increased butyrate production in the gastrointestinal tract.

[0087] In a preferred embodiment, fermentation of dietary NDS beneficially leads to the production of fatty acids, including butyrate. Fermentation assays known in the art can be used to evaluate the fermentation characteristics of dietary NDS from a single fiber source or a fiber blend. An exemplary method for evaluating NDS fermentation includes exposing a pool of fecal samples from healthy adults, infants, or young children to NDS under anaerobic conditions at 37°C for at least 24 hours using an NDS fermentation model.

[0088] As used in this article, NDP refers to dietary indigestible polysaccharides with a degree of polymerization greater than 10.

[0089] In preferred embodiments, the NDS is of plant, crustacean, and / or microbial origin, preferably of plant origin. As used herein, “derived from” means the natural source of the NDS. Microbial-derived NDS refers to NDS naturally produced by microorganisms, such as xanthan gum. As used herein, therefore, when the NDS is derived, for example, from or produced in a genetically modified expression vector, “derived from” does not mean the source of production.

[0090] In a preferred embodiment, the NDS that can act as a substrate to provide butyrate does not contain lactose in its molecular structure and / or main chain.

[0091] Exemplary dietary NDS used in accordance with the present invention include indigestible polysaccharides [NDPs], such as hydrolyzed or low-viscosity pectin, fructosaccharides, oat beta-glucan, soybean fiber, resistant starch, gum arabic, cellulose, arabinoxylan, (partially hydrolyzed) guar gum, xanthan gum, locust bean gum, and / or resistant starch. NDPs are fermentable and provide fatty acids, particularly beneficial amounts of butyrate.

[0092] In particular, XG and LBG are NDPs that have been found to undergo microbial fermentation in fermentation models to produce beneficial amounts of butyrate. Another advantage of XG and LBG is that infants and young children with food allergies, who often suffer from gastroesophageal reflux and / or regurgitation, can benefit from thickened nutritional compositions, with the latter NDP also used as a thickener in such compositions.

[0093] Suitable types of NDPs are long-chain fructooligosaccharides (IcFOS) with an average degree of polymerization greater than 10, typically in the range of 10-100, preferably 15-50, and most preferably greater than 20. Preferred types of long-chain fructooligosaccharides are Raftilin HP with an average DP greater than 10.

[0094] If the nutritional composition is based on free amino acids, then the preferred NDO is not milk-derived. Preferably, the indigestible oligosaccharide in such formula is lcFOS. Exemplary fermentable NDOs are inulin or lcFOS.

[0095] In another preferred embodiment, the composition according to the invention comprises a mixture of dietary NDS and dietary butyrate capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate. Preferably, the mixture of dietary NDS and dietary butyrate contains at least 50 wt% dietary NDS; more preferably, the weight ratio of NDS to dietary butyrate in the mixture is in the range of 80:20 to 50:50, and even more preferably 75:25 to 60:40.

[0096] Indigestible oligosaccharides Preferably, the nutritional composition of the present invention comprises NDS, which includes one or more indigestible oligosaccharides (NDOs). In a preferred aspect of the invention, NDS capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate are NDOs. These NDOs may help improve gastrointestinal disorders common in allergic infants and have anti-allergic effects by improving the gut microbiota and intestinal barrier.

[0097] Advantageously and most preferably, the indigestible oligosaccharide is water-soluble (according to the method disclosed below: L. Prosky et al., J. Assoc. Anal. Chem [Journal of the Society for Analytical Chemistry] 71: 1017-1023, 1988) and preferably is an oligosaccharide with a degree of polymerization (DP) of 2 to 10. In a preferred embodiment, the NDO according to the invention with a DP of 2 to 10 further comprises at least an NDO with a DP of 7 or higher.

[0098] Dietary NDS selected from NDPs, NDOs, and optionally additionally containing HMOs are not digested in the intestine by digestive enzymes present in the human upper digestive tract (small intestine and stomach). For example, glucose, fructose, galactose, sucrose, lactose, maltose, and maltodextrin are considered digestible. Oligosaccharide raw materials may contain monosaccharides such as glucose, fructose, fucose, galactose, rhamnose, xylose, glucuronic acid, GalNac, etc., but these are not part of oligosaccharides.

[0099] The NDO according to the invention is fermented by the human gut microbiota. The indigestible oligosaccharide is preferably a prebiotic. The NDO according to the invention is preferably of plant, crustacean, and / or microbial origin, preferably of plant origin. The NDO is preferably selected from the group consisting of: short-chain fructooligosaccharides, indigestible dextrins, polydextrose, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, arabinoxylan oligosaccharides, glucose oligosaccharides, glucan-mannooligosaccharides, galactomannooligosaccharides, mannooligosaccharides, chitosan oligosaccharides, uronic acid oligosaccharides, and mixtures thereof.

[0100] In a preferred embodiment, the indigestible oligosaccharide is not a galactooligosaccharide. Typically, galactooligosaccharides are derived from lactose, which is not a preferred source for use in the treatment and / or prevention of food allergies according to the present invention.

[0101] In one embodiment, the nutritional composition comprises short-chain fructooligosaccharides (scFOS). The short-chain fructooligosaccharides preferably have an average degree of polymerization (DP) in the range of 2-8, preferably 3-7, i.e., short-chain fructooligosaccharides in the context of this invention. The scFOS can be an inulin hydrolysate with an average degree of polymerization in the aforementioned (sub) range; such scFOS products are commercially available, for example, as Raftilose P95 (Orafti) or from Cosucra. Alternatively, scFOS can be enzymatically synthesized from sucrase using fructosyltransferases.

[0102] In embodiments, the nutritional composition may contain a mixture of two or more types of NDS, preferably a mixture of two NDOs or a mixture of NDO and NDP. If the NDS comprises or consists of a mixture of two different sugars, one indigestible sugar may be a short-chain NDO as defined above, and the other indigestible sugar may be a long-chain NDP as defined above. Most preferably, the short-chain oligosaccharides and long-chain polysaccharides are present in a short-chain to long-chain weight ratio within the following range: 1:99-99:1, more preferably 1:1-99:1, more preferably 4:1-97:3, even more preferably 5:1-95:5, even more preferably 7:1-95:5, even more preferably 8:1-10:1, and most preferably about 9:1. Suitable mixtures include mixtures of long-chain fructooligosaccharides and short-chain fructooligosaccharides. Therefore, in one embodiment, the short-chain fructooligosaccharides and the long-chain fructooligosaccharides are present in the following short-chain to long-chain weight ratio range: 1:99-99:1, more preferably 1:1-99:1, more preferably 4:1-97:3, even more preferably 5:1-95:5, even more preferably 7:1-95:5, even more preferably 8:1-10:1, and most preferably about 9:1.

[0103] If the formula is based on free amino acids, then the preferred NDS is not milk-derived. Preferably, the NDS in such formula is a mixture of short-chain fructooligosaccharides and long-chain fructooligosaccharides.

[0104] In particular, mixtures of long-chain and short-chain fructooligosaccharides were found to show favorable butyrate production in fecal fermentation assays. Therefore, another suitable type of oligosaccharide when combined with lcFOS is a short-chain fructooligosaccharide with an average degree of polymerization (DP) of less than 10, preferably up to 8, and more preferably in the range of 2-7.

[0105] Based on the dry weight of the nutritional composition, the nutritional composition preferably contains 0.05 wt% to 20 wt% of the NDS, more preferably 0.5 wt% to 15 wt%, even more preferably 1 wt% to 10 wt%, and most preferably 2 wt% to 10 wt%. When in liquid form, the nutritional composition preferably contains 0.01 to 1.0 g of indigestible sugars, more preferably 0.25 to 0.8 g, even more preferably at least 0.4 g / 100 ml of ready-to-drink nutritional composition, preferably 0.4 to 0.8 g, more preferably 0.6 to 0.8 g / 100 ml of ready-to-drink nutritional composition.

[0106] In certain embodiments, NDS or NDS blends are preferably used. In the context of this invention, an exemplary indigestible carbohydrate blend is a fermentable blend that beneficially provides butyrate in the gut. An exemplary NDS blend is a mixture containing scFOS and lcFOS in a 9:1 ratio.

[0107] In another embodiment, an exemplary NDS blend providing butyrate comprises at least a) inulin, b) resistant starch, and / or c) soluble soybean polysaccharide. This blend is a mixture of NDO and dietary fiber, which is fermentable and provides fatty acids, particularly beneficially, butyrate. Another exemplary indigestible carbohydrate blend comprises a) FOS, b) inulin, c) soybean fiber, d) resistant starch, e) gum arabic, and / or f) cellulose. This blend is fermentable and provides fatty acids, particularly a beneficial amount of butyrate. Another exemplary indigestible carbohydrate blend comprises a) arabinoxylan, b) oat β-glucan, c) pectin, and / or d) resistant starch.

[0108] HMO In some embodiments, in addition to NDP and / or NDO, the indigestible sugars providing butyrate as a source may optionally further comprise human milk oligosaccharides [HMOs]. HMOs are NDOs that are not digested in the intestine by digestive enzymes present in the human upper digestive tract but can undergo microbial fermentation in the gastrointestinal tract. HMOs refer to those oligosaccharides present in human milk and fall within the conventional definition adopted by anyone in the art. HMOs include, but are not limited to, fucoidylated oligosaccharides (e.g., diFL, 2FL), N-acetylated oligosaccharides (e.g., lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT)), or sialylated oligosaccharides.

[0109] In some embodiments, HMOs such as 2'-FL can be provided, 2'-FL being fermentable and capable of providing or enhancing SCFA production (including butyrate).

[0110] In the context of this invention, a butyrate source is not a standalone butyrate source other than 3FL. 3FL is an HMO that does not provide a significant amount of butyrate after microbial fermentation. To obtain the beneficial immunological effects according to the invention, 3FL must be combined with a standalone butyrate source; said butyrate source may be dietary butyrate and / or an NDS selected from NDP and / or NDO and optionally further comprising one or more HMOs.

[0111] protein components The protein source in the nutritional composition of the present invention is suitable for human subjects, preferably infants or young children at risk of and / or suffering from food allergies, more particularly dietary protein allergies, and even more particularly milk protein or egg allergies. The nutritional composition according to the present invention is a hypoallergenic nutritional composition. Therefore, the protein or protein source is a hydrolyzed protein, preferably a deeply hydrolyzed protein, free amino acids, or a combination thereof.

[0112] This invention advantageously relates to a composition and its use, wherein the protein source preferably provides 7% to 20% of the total calories of the composition, more preferably 8% to 17% of the total calories, and even more preferably 9% to 15% of the total calories. This invention advantageously relates to a composition and its use, wherein the protein source preferably provides 1.0 g to 3.5 g protein / 100 ml, more preferably 1.2 to 3.0 g / 100 ml, and even more preferably 1.4 to 2.5 g / 100 ml of ready-to-drink nutritional composition. This invention advantageously relates to a composition and its use, wherein the protein source preferably provides 1.5 g to 5.2 g protein / 100 kcal, more preferably 1.8 to 4.5 g / 100 kcal, and even more preferably 2.1 to 3.7 g / 100 kcal. Alternatively, in the compositions and uses according to the invention, the amount of protein source is preferably between 10 and 20 wt% of the total dry weight of the composition, preferably between 11 and 18 wt%, and even more preferably between 12 and 16 wt% of the total dry weight of the composition. Preferably, deeply hydrolyzed proteins, free amino acids, or combinations thereof are the sole protein source.

[0113] Hydrolyzed proteins can be derived from milk proteins, such as whey protein or casein. Suitable alternative sources of hydrolyzed proteins include rice protein hydrolysate. Hydrolyzed proteins are typically obtained by hydrolysis with proteolytic enzymes. Extensively hydrolyzed proteins are typically treated with an ultrafiltration step after hydrolysis with proteolytic enzymes to remove potentially allergenic intact proteins and large peptides. Preferably, the hydrolyzed protein is hydrolyzed whey protein. In another preferred embodiment, the protein is extensively hydrolyzed, preferably extensively hydrolyzed whey protein.

[0114] The hydrolyzed protein, preferably extensively hydrolyzed whey protein, has a degree of hydrolysis of 16% to 50%, more preferably 20% to 30%, and even more preferably 20% to 25%. The degree of hydrolysis is defined as the percentage of peptide bonds broken down by enzymatic hydrolysis, where 100% is the total potential peptide bonds present. Too low a degree of hydrolysis will result in undesirable high levels of intact allergenic proteins or allergenic peptides.

[0115] In contrast, partially hydrolyzed proteins typically have a degree of hydrolysis of 5% to 15%, making them suitable for inducing tolerance because the allergenic portion of the protein remains intact to some extent. Partially hydrolyzed milk proteins (such as partially hydrolyzed whey protein) are suitable for infants at risk of developing bovine milk protein allergy, but not for infants or young children who already have bovine milk protein allergy. In some embodiments, partially hydrolyzed plant proteins, such as partially hydrolyzed rice protein, are used. Partially hydrolyzed rice protein is suitable for human subjects with bovine milk protein allergy. Rice protein hydrolysates suitable for infant formula and infant formula containing rice protein hydrolysates are commercially available.

[0116] Peptide size and molecular weight distribution can be determined using conventional methods known to those skilled in the art, such as HPLC or size exclusion chromatography (SEC), particularly high-performance size exclusion chromatography. An example is described below: Saint-Sauveur et al., “Immunomodulating properties of a whey protein isolate, its enzymatic digest and peptide fractions”, Int. Dairy Journal (2008), Vol. 18(3), pp. 260-270. In short, the total surface area of ​​the chromatogram is integrated and divided into mass ranges expressed as a percentage of the total surface area. The mass ranges are calibrated using peptides / proteins with known molecular weights.

[0117] In the context of this invention, the deeply hydrolyzed protein preferably comprises 55% to 85% peptides with a molecular weight less than 1000 Da, 15% to 45% peptides with a molecular weight between 1000 and less than 5000 Da, and 0% to 1% peptides or proteins with a molecular weight of 5000 Da and above, all based on the total protein of the hydrolysate. In a preferred embodiment, the deeply hydrolyzed protein, preferably deeply hydrolyzed whey protein, comprises peptides having the following size distribution based on the total protein of the hydrolysate: 60%-90% <1 kDa, 10%-40% 1 to <5 kDa, 0%-0.5% 5 to <10 kDa, and 0%-0.2% >10 kDa.

[0118] In a preferred embodiment of the invention, the hydrolyzed protein, preferably extensively hydrolyzed whey protein, comprises less than 100 mg of peptides larger than 5 kDa per gram of protein hydrolysate, preferably less than 50 mg / gram of protein hydrolysate, more preferably less than 10 mg / gram of protein hydrolysate. Preferably, the extensively hydrolyzed protein, preferably extensively hydrolyzed whey protein, comprises less than 30 mg of peptides larger than 10 kDa per gram of protein hydrolysate, preferably less than 10 mg / gram of protein hydrolysate, most preferably less than 5 mg / gram of protein hydrolysate.

[0119] Preferably, the hydrolyzed protein, more preferably extensively hydrolyzed whey protein, contains less than 0.8 micrograms, more preferably less than 0.2 micrograms of sensitizing β-lactoglobulin / g protein. In the context of this invention, the expression "sensitizing β-lactoglobulin" refers to intact or immunogenic β-lactoglobulin and does not consider extensively hydrolyzed β-lactoglobulin. Sensitizing β-lactoglobulin can be determined by methods known in the art, such as ELISA.

[0120] For infants with severe allergies where extensively hydrolyzed proteins (such as extensively hydrolyzed whey protein) remain a problem as a protein source, or for infants with multiple food allergies, it is advantageous that the protein source consists primarily of free amino acids. Preferably, the protein source contains all essential amino acids. Optimal amino acid profiles for infant formula are known in the art and are found in amino acid-based infant formulas (such as Neocate). Preferred examples of amino acid compositions are given in Example 4.

[0121] Probiotics Preferably, the nutritional composition comprises bifidobacteria selected from the genus Bifidobacterium (Bifidobacterium spp.) Bifidobacterium ) and / or Lactobacillus spp. Lactobacillus The nutritional composition comprises lactic acid-producing bacteria, particularly Bifidobacterium. Breastfed infants have a high Bifidobacterium content in their gut microbiota. Adding one or more strains belonging to the Bifidobacterium genus to the nutritional composition will further improve the gut microbiota and its activity. More preferably, the nutritional composition contains Bifidobacterium breve (…). Bifidobacterium breve Such strains of *Bifidobacterium breve* are commercially available or can be isolated from the infant's gut microbiota. An example of a commercially available strain is *Bifidobacterium breve* M-16V from Morinaga. The amount of *Bifidobacterium* and / or *Lactobacillus* is preferably 10. 4 Up to 10 11 CFU / gram of nutrient composition dry weight.

[0122] Butyrate is produced by bacteria that can use lactate as a carbon source (such as anaerobic Corynebacterium). Anaerostipes ) species and genus Roselle ( Roseburia Lactate is formed in the gut. As infants grow, the gut microbiota develops, and lactate-utilizing bacteria develop, which metabolize lactate formed by lactate-producing bacteria (e.g., Bifidobacteria) into propionate and, in particular, butyrate. This development of metabolic characteristics is associated with beneficial effects. Infants with eczema are characterized by decreased levels of both lactate isomers and increased levels of propionate and butyrate at 12 weeks of age. However, this pattern reverses at 26 weeks of age: infants with eczema show increased levels of lactate, while levels of propionate and butyrate decrease. Wopereis et al., Journal of Allergy and Clinical Immunology, Vol. 141, No. 4, April 2018, pp. 1334-1342.e5, especially the presence of butyrate is considered to have beneficial effects. Therefore, it is beneficial to add lactate-utilizing bacteria (e.g., Bifidobacteria) in the presence of fiber, because in older infants and young children, the lactate formed by Bifidobacteria is used by lactate-utilizing bacteria to convert it into butyrate.

[0123] Nutritional composition The nutritional composition according to the invention can be used as infant or follow-up formula, toddler formula, nutritional therapy, food for specific medical purposes, or nutritional supplement. The nutritional composition is preferably an oral composition. The nutritional composition is administered orally, or intended to be administered orally, to subjects in need, particularly children and infants, including toddlers, preferably children up to 6 years of age, preferably typically infants or toddlers aged 0-36 months, more preferably infants aged 0-12 months.

[0124] Therefore, in some embodiments, the nutritional composition is infant formula, follow-up formula, or toddler formula (also known as growing milk or toddler milk), preferably infant formula or follow-up formula, and most preferably infant formula. The terms "infant formula" and "follow-up formula" are well-defined and subject to consistent control by international regulatory bodies. It recommends nutritional values ​​and formulation compositions that require the prepared milk to contain not less than 60 kcal (250 kJ) and not more than 70 kcal (295 kJ) of energy per 100 ml. The EU, FDA, and other regulatory bodies have set nutritional requirements accordingly. This caloric density ensures an optimal ratio between water and caloric expenditure. In the context of this application, the energy provided by the nutrients is calculated using the following Atwater coefficients: 9 kcal / g lipids, 4 kcal / g protein or g digestible carbohydrates, 2 kcal / g dietary fiber, and other components in zero-kcal products.

[0125] Suitablely, the nutrient composition is in powder form, which is preferably reconstituted with water to form a liquid. When the nutrient composition is in liquid form, the preferred daily administration volume range is about 80 to 2500 ml per day, more preferably about 450 to 1000 ml per day.

[0126] The nutritional composition according to the invention comprises lipids, preferably lipids as known in the art suitable for infant nutrition. The lipids in the nutritional composition are preferably provided at a rate of 2.8 to 7.0 g, more preferably 4.0 to 6.0 g / 100 kcal of the nutritional composition. When in liquid form, the nutritional composition preferably contains 1.9 to 4.7 g lipids / 100 ml, more preferably 2.7 to 4.0 g / 100 ml. Based on dry weight, the nutritional composition of the invention preferably contains 12.5 wt% to 40 wt% lipids, more preferably 19 wt% to 30 wt% lipids.

[0127] The nutritional composition according to the invention may further comprise long-chain polyunsaturated fatty acids (LC-PUFAs). LC-PUFAs are fatty acids wherein the acyl chain has a length of 20 to 24 carbon atoms, and wherein the acyl chain contains at least two unsaturated bonds between the carbon atoms in the acyl chain. More preferably, the nutritional composition comprises at least one LC-PUFA selected from the group consisting of eicosapentaenoic acid (EPA, 20:5 n3), docosahexaenoic acid (DHA, 22:6 n3), arachidonic acid (ARA, 20:4 n6), and docosapentaenoic acid (DPA, 22:5 n3). Preferably, the nutritional composition comprises at least DHA and EPA, more preferably DHA, EPA, and ARA. Such LC-PUFAs have a further beneficial effect on reducing the risk of allergies. Based on total fatty acids, the preferred content of LC-PUFAs in the nutritional composition is no more than 15 wt%, preferably no more than 10 wt%, and even more preferably no more than 5 wt%. Preferably, based on total fatty acids, the nutritional composition contains at least 0.2 wt%, preferably at least 0.25 wt%, more preferably at least 0.35 wt%, and even more preferably at least 0.5 wt% LC-PUFA. Based on total fatty acids, the amount of DHA is preferably at least 0.2 wt%, more preferably at least 0.3 wt%, more preferably at least 0.35 wt%, and even more preferably 0.35-0.6 wt%.

[0128] The nutritional composition contains digestible carbohydrates. Typically, digestible carbohydrates known in the art suitable for infant nutritional compositions are selected from digestible polysaccharides (e.g., starch, maltodextrin), digestible monosaccharides (e.g., glucose, fructose), and digestible disaccharides (e.g., lactose, sucrose). Lactose and / or maltodextrin are particularly suitable. Preferably, the nutritional composition contains lactose. Lactose is the main digestible carbohydrate in milk and has a relatively low glycemic index. Lactose is undesirable for infants requiring amino acid-based formula because it is derived from milk. In this case, preferably, the nutritional composition contains maltodextrin. Maltodextrin consists of D-glucose units linked in chains of variable length. The glucose units are primarily linked by α(1→4) glycosidic bonds and typically consist of a mixture of chains from 3 to 17 glucose units in length. Maltodextrin is classified according to DE (dextrose equivalent), and if maltodextrin is included in the nutritional composition of the present invention as a digestible carbohydrate, maltodextrin preferably has a DE of 3 to 47, more preferably 18 to 23.

[0129] Minerals, vitamins, trace elements and other micronutrients are present as known in the art to comply with directives concerning infant and follow-up formula milk powder and foods intended for medical purposes for infants.

[0130] application The compositions according to the invention are preferably administered enterally, and more preferably orally. The nutritional compositions of the invention are preferably infant formula, follow-up formula, or toddler formula. Examples of toddler formula are toddler milk, toddler formula, and growing-up milk. More preferably, the nutritional composition is infant formula or follow-up formula. The compositions of the invention can be advantageously used as complete nutrition for infants.

[0131] The nutritional compositions of the present invention are preferably suitable for providing nutrition to human subjects with food allergies and human subjects at risk of developing food allergies. The nutritional compositions of the present invention are specifically intended for use in infants and / or toddlers with food allergies and / or infants and / or toddlers at risk of developing food allergies, more preferably infants. In preferred embodiments, the nutritional compositions of the present invention are particularly intended for use in infants and / or toddlers with food allergies, more preferably infants with food allergies.

[0132] Infant formula, follow-up formula, or toddler formula may be in liquid form, preferably an instant liquid, or in powder form. In one embodiment, the infant formula, follow-up formula, or toddler formula is in powder form, suitable for reconstitution with water to provide instant infant formula, follow-up formula, or toddler formula. It should be understood that when the infant formula, follow-up formula, or toddler formula according to the invention is in powder form, the amount of all components in the formula, including 3-FL and dietary fiber and / or dietary butyrate, is defined as the amount that will be present after reconstitution with water, i.e., the amount is defined in mg / 100 ml of instant formula.

[0133] The nutritional composition according to the invention is intended for use in providing nutrition to infants or young children, preferably infants, and preferably infants up to 12 months of age.

[0134] The infant formula, follow-up formula or toddler formula according to the present invention is used to provide nutrition to infants or toddlers, preferably infants, preferably up to 12 months of age.

[0135] The preferred embodiments described above for infant formula, follow-up formula and toddler formula according to the present invention are also applicable to the infant formula, follow-up formula and toddler formula of the present invention used in use.

[0136] The present invention further relates to compositions comprising 3-FL, dietary NDS, and / or dietary butyrate, or compositions according to the invention, for use as pharmaceutical agents. Preferably, the compositions are for use in improving immune function, for use in immune maturation, and for use in the prevention and / or treatment of allergies, particularly food allergies. Immune maturation includes the establishment of an immune response characterized by an increase in the balance of Th1 and Th2 associated with the healthy adult phenotype towards Th1.

[0137] Preferably, the composition is used for improving immune function, for immune maturation, and preferably for reducing and / or inhibiting Th2 responses and improving and / or promoting Th1 responses. Preferably, the composition is used for providing a Th1:Th2 balance, wherein the balance is shifted towards a Th1 response. In embodiments, the Th1 / Th2 balance comprises the IFN-γ to IL-13 cytokine release ratio.

[0138] In specific embodiments, the composition is used to administer to human subjects, particularly infants, who are at risk of developing allergies, especially bovine milk protein or egg allergies, or who have allergies, especially bovine milk protein or egg allergies. Infants known to be at risk of developing allergies include infants whose parents have or have had atopic disorders (e.g., eczema) and / or allergies, most particularly infants with CMA.

[0139] The effects on the immune system according to the present invention will have an effect on inducing oral tolerance to allergens. In the context of the present invention, it should be understood that preventing food allergies focuses on reducing the risk of developing allergies, while inducing oral tolerance is about training the immune system to develop tolerance to specific allergens.

[0140] The effects on both IL-13 and IFN-γ indicate a surprisingly improved modulation of immune cell responsiveness in the presence of the combination of 3-FL and butyrate. Because the nutritional composition of the present invention has an improved effect on reducing Th2 responses and increasing Th1 responses, it will prevent and / or treat allergies. These effects demonstrate an improved effect on tolerance induction and can therefore modulate and / or promote oral tolerance induction, thereby preventing food allergies or accelerating the resolution of food allergies by modulating type 2 hypersensitivity responses. The combination of 3-FL and butyrate sources can inhibit food allergen-induced type 2 responses while enhancing type 1 immunity.

[0141] The nutritional composition according to the invention is preferably used for providing nutrition to infants or young children, preferably infants, who are at increased risk of developing food allergies or who have food allergies. Subjects with food allergies or at risk of developing food allergies are preferably infants and / or toddlers with food allergies. In embodiments, the subjects have food allergies, particularly to dietary proteins, or are at risk of developing food allergies, more preferably to cow's milk protein or egg allergies. The nutritional composition of the invention is preferably used for the prevention and / or treatment of food allergies, more preferably CMA or egg allergies. The nutritional composition of the invention is preferably used in the dietary management of food allergies and the risk of food allergies, more preferably in inducing tolerance to cow's milk protein and preventing CMA.

[0142] Example The present invention is further illustrated by the following examples.

[0143] Example 1: In vitro assessment of the effects of 3-FL and butyrate on immune function In a sequential mucosal co-culture model, we evaluated the effects of the interaction between butyrate and the most common fucosylated human lactose oligosaccharides (2'-fucosylated lactose (2'-FL) and 3-fucosylated lactose (3-FL)) on intestinal epithelial function after ovalbumin (OVA)-induced type 2 activation of intestinal epithelial cells (IECs) and subsequent dendritic cell (DC) maturation and T cell function.

[0144] The effect of epithelial pre-incubation with 2'-FL or 3-FL and / or butyrate was evaluated using intestinal epithelial cell (IEC) / dendritic cell (DC) and DC / T cell co-cultures, under conditions where IECs were also stimulated with ovalbumin (OVA) to induce epithelial inflammation. This experimental model has recently been used to demonstrate the differential immunomodulatory effects of 2'-FL and 3-FL alone [M. Zuurveld et al., J. Innate Immun. [Journal of Innate Immunology], 2022].

[0145] IEC was modeled using the human colon adenocarcinoma HT-29 cell line (passaged 148-156). Confluent HT29 cells in transfer plates were incubated for 24 h with 2'-FL or 3-FL (0.1% w / v) and / or butyrate (0.5 mM). Subsequently, the medium was refreshed to contain fresh HMO and butyrate, and the cells were exposed to ovalbumin (100 µg / mL) under specified conditions for another 24 h. Substrate-exposed medium was collected for cytokine analysis, and monocyte-derived (moDC) cells were added to the substrate-exposed compartments after washing HT29 cells with fresh medium and allowed to remain there for 48 h. moDC cells were collected and subsequently co-cultured with naïve T cells for 5 days. Supernatants were collected from different cell culture steps, and chemokine and cytokine secretion was analyzed by enzyme-linked immunosorbent assay (ELISA).

[0146] Sensitization of DCs with IECs exposed to OVA (OVA as an allergic inflammatory trigger) resulted in a slight increase in IFNγ secretion, which tended to increase further if the IECs were pre-incubated with a combination of 2'-FL or 3-FL and butyrate. Figure 1A Furthermore, IEC-sensitized DCs exposed to OVA produced enhanced IL-13 secretion during T cell co-culture. Figure 1B Furthermore, pre-incubation of OVA-IEC with butyrate alone had no significant effect on downstream OVA-induced T helper cell cytokine secretion. Figure 1B However, pre-incubation with 3-FL prevented OVA-IEC / DC-mediated IL-13 secretion from T cells, and this effect was still observed when 3-FL was combined with butyrate. Figure 1B This is not the case with 2'-FL, where enhanced IL-13 secretion is unaffected by its combination with butyrate. Therefore, under these inflammatory conditions, butyrate primarily affects the immunomodulatory characteristics of 3'-FL, while the role of 2'-FL remains. However, in both cases, incubation with either HMO and butyrate during IEC / DC culture tended to enhance IFN-γ secretion by T cells downstream, but only the combination of 3-FL and butyrate maintained low levels of IL-13 and caused a shift in the Th1 / Th2 response ratio towards a direction favorable for allergy prevention. Figure 2 ).

[0147] When IEC / DC pre-incubation with butyrate was combined with either 2'-FL or 3-FL, our results revealed distinct immunomodulatory effects in an in vitro model of OVA exposure. An overall enhancement of both inflammatory and regulatory cytokine secretion was observed in the presence of 2'-FL, and this overall enhancement persisted when tested in combination with butyrate, but further promoted type 1 immunity. Therefore, although an interaction between butyrate and 2'-FL was hypothesized and observed at the level of IEC / DC interaction in this OVA-induced mucosal immune activation model, the effects observed after combined 2'-FL + butyrate pre-incubation largely correspond to the effects observed after pre-incubation with 2'-FL alone.

[0148] Nevertheless, combined pre-incubation with butyrate and 3-FL significantly enhanced the secretion of IL6 and TGFβ by sensitized DCs, a phenomenon not observed with pre-incubation of butyrate or 3-FL alone in the presence of OVA (data not shown). Furthermore, IFN-γ secretion tended to increase in the 3-FL + butyrate combination (a phenomenon not observed with pre-incubation of butyrate or 3-FL alone), while maintaining very low IL-13 levels.

[0149] Overall, these in vitro studies suggest that the combination of 3-FL and butyrate may suppress OVA-induced type 2 responses by shifting the immune homeostasis relative to type 2 responses toward type 1 responses (IFN-γ / IL-13 ratio). Thus, the combination of 3-FL and butyrate is found to inhibit OVA-induced type 2 responses while enhancing type 1 immunity.

[0150] Example 2: Extensively hydrolyzed whey protein suitable for infants with allergies and those at risk of allergies. 3FL and fiber as a source of dietary butyrate in infant formula A packaged powdered infant formula, after being reconstituted with water according to the instructions on the packaging, contains (13.46 g of powder per 100 ml final volume): -66 kcal -1.6 g protein (extensively hydrolyzed whey protein) -7.1 g of digestible carbohydrates (mainly lactose) -3.4 g fat (vegetable oil, fish oil) -0.4 g prebiotics: scFOS / lcFOS in a 9:1 ratio (source: Raftilose P95 and Raftiline HP) -0.1 g 3-FL (Chr. Hansen) Such as minerals, vitamins, trace elements and other micronutrients that comply with the guidelines.

[0151] Example 3: Extensively hydrolyzed whey protein, 3'-distilled, suitable for infants with allergies and those at risk of allergies. FL and fiber as a source of dietary butyrate in infant formula A packaged powdered infant formula for older infants, after being reconstituted with water according to the instructions on the packaging, contains (14.44 g of powder in 100 ml final volume) per 100 ml: -68 kcal -1.6 g protein (extensively hydrolyzed whey protein) -7.8 g of digestible carbohydrates (mainly lactose), and -3.2 g fat (vegetable oil, fish oil) -0.7 g prebiotics: scFOS / lcFOS in a 9:1 ratio (source: Raftilose P95 and Raftiline HP) -20 mg 3-FL (Chr. Hansen) Such as minerals, vitamins, trace elements and other micronutrients that comply with the guidelines. 。

[0152] Example 4: Suitable for infants with allergies and infants at risk of allergies, containing free amino acids, 3-FL, and dietary fiber. Butyrate-containing infant formula A packaged infant formula, after being reconstituted with water according to the instructions on the packaging, contains (14.7 g of powder per 100 ml final volume): -67 kcal -1.8 g protein equivalents (free amino acids): L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamine, glycine, L-histidine, L-isoleucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-carnitine -7.3 g of digestible carbohydrates (mainly dry glucose syrup) and includes 1.0 g of waxy corn starch.

[0153] -3.4 g fat (approximately 50 wt.% milk fat, the remainder being vegetable oil, fish oil, and microbial oil). Based on total fatty acids, the amounts are: butyric acid 1.48 wt.%, arachidonic acid 0.52 wt.%, eicosapentaenoic acid 0.11 wt.%, and docosahexaenoic acid 0.52 wt.%. -0.7 g prebiotics: scFOS / lcFOS in a 9:1 ratio (source: Raftilose P95 and Raftiline HP) -0.1 g 3-FL (Chr. Hansen) - Such as minerals, vitamins, trace elements and other micronutrients that comply with the Infant Formula Directive.

[0154] Example 5: Infant formula based on rice protein hydrolysate A rice protein hydrolysate infant formula in powder form, packaged with instructions on the packaging to reconstitute it with water and to use it for dietary management of infants aged 0-12 months with milk allergy.

[0155] Approximately 13.5 g of powder was reconstituted with water to make 100 ml of formula milk powder.

[0156] Per 100 g of powder, the composition comprises: -495 kcal -12.4 g protein equivalent, rice protein hydrolysate, L-Trp, L-Tyr, L-Ile -50.6 g of digestible carbohydrates (mainly maltodextrin) -26.0 g lipids (primarily plant lipids and containing microbial oils, based on total fatty acid delivery of 0.51 wt% arachidonic acid and 0.51 wt% docosahexaenoic acid) -4.6 g of an indigestible oligosaccharide mixture, which consists of the following o4.0 scFOS (Source: RaftilineHP) 0.45 g lcFOS (Source: Raftiline HP) 0.45 g 2'-fucosylated lactose (Chr. Hansen) o0.2 g 3-fucosyllactose - Minerals, trace elements, vitamins and other micronutrients comply with international directives and FSMP regulations for infant formula.

[0157] Example 6 - Fermentation characteristics of xanthan gum, locust bean gum, and indigestible oligosaccharides scGOS / lcFOS Stool samples were collected from formula-fed infants (5.5 months of age) and breastfed infants (3 months of age). The infants had no gastrointestinal problems and had not used antibiotics in the past month. Stool samples were pooled, homogenized, aliquoted into smaller aliquots, and mixed with 10% glycerol in an anaerobic chamber. The aliquots were then stored at -80°C.

[0158] Indigestible oligosaccharides were added at a concentration of 100 mg dietary fiber (DP ≥ 2) per 6 ml of fecal suspension, as shown in Table 1. The source of GOS was Vivinal GOS (Friesland Campina, Netherlands), and the source of lcFOS was Raftilin HP (Orafti). Xanthan gum (Keltrol Grindsted 808 MAS-SHClear) was obtained from CP Kelco, and locust bean gum (Grindsted LBG 860) was obtained from Danisco, Valencia, Spain.

[0159] Table 1 Conditions for the fermentation of fecal slurry

[0160] In the experiment, the fecal tank was thawed in a 37°C water bath for 20 minutes. Then, the fecal tank was placed in an anaerobic chamber. The feces and fermentation medium were mixed at a 1:5 ratio in a Falcon tube. At t = 0, a sample of the fecal suspension was taken, and 6 ml of this suspension, along with the target substrate, was added to a sterile Falcon tube and thoroughly mixed. Next, 6 ml of the feces / substrate suspension was placed into a dialysis tube, and air was removed from the gaps. The dialysis tube was placed in a 100 ml Scott bottle containing 100 ml of dialysis medium. The Scott bottle was sealed and incubated at 37°C. Dialysis medium (dialysate) and fecal suspension (lumen) samples were collected at t = 24 and t = 48 hours to determine SCFA, D- and L-lactates, and gas volume.

[0161] The fermentation medium (McBain and MacFarlane) contained 3.0 g / L buffered peptone water, 2.5 g / L yeast extract, 3.0 g / L tryptone, 0.4 g / L L-cysteine-HCl, 0.05 g / L bile salts, 2.6 g / L K₂HPO₄·3H₂O, 0.2 g / L NaHCO₃, 4.5 g / L NaCl, 0.5 g / L MgSO₄·7H₂O, 0.3 g / L CaCl₂·2H₂O, and 0.005 g / L FeSO₄·7H₂O. Each component was added sequentially to 800 ml of water, and the pH was adjusted to 5.5 ± 0.1 with K₂HPO₄ or NaHCO₃, bringing the volume to 1 liter. The medium was sterilized at 121°C for 15 minutes and placed in an anaerobic chamber for at least 16 hours before use.

[0162] The dialysis medium contained 2.6 g / L K₂HPO₄·3H₂O, 0.2 g / L NaHCO₃, 4.5 g / L NaCl, 0.5 g / L MgSO₄·7H₂O, 0.3 g / L CaCl₂·2H₂O, and 0.005 g / L FeSO₄·7H₂O. The pH was adjusted to 5.5 ± 0.1 using K₂HPO₄ or NaHCO₃. The medium was not sterilized due to sediment formation. The medium was placed in an anaerobic chamber for at least 16 hours before use.

[0163] pH was measured by directly immersing the 423 pH electrode (Mettler Toledo, Columbus, Ohio, USA) connected to the Handy-lab pH meter (Schott Glass, Mainz, Germany) into the sample.

[0164] The gas volume is determined using a device that measures both pressure and volume. Before measurement, the bottles are shaken thoroughly.

[0165] SCFAs (acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid) were quantitatively determined using a Shimadzu-GC2025 gas chromatograph with a flame ionization detector. Hydrogen was used as the mobile phase. 2-Ethylbutyric acid was used as an internal standard to determine the levels of SCFAs. Calibration curves were constructed based on peak areas, and the concentrations in the samples were calculated.

[0166] Lactate was determined using an enzymatic method using an L-lactate assay kit containing D- and L-lactate dehydrogenases (Boehringer Mannheim, Mannheim, Germany). First, the sample was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was then heated at 100°C for 10 min to inactivate all enzymes, followed by centrifugation at 13,000 rpm for 10 min.

[0167] result: Isobutyric acid, valeric acid, and isovaleric acid levels were below the detection limit. In a mixture containing scG / lcF, lactic acid, primarily L-lactic acid, was formed at t = 24 h.

[0168] When xanthan gum or locust bean gum is the only fiber, butyric acid and, especially, propionic acid are present in high amounts. Therefore, the SCFA profile shows a lower percentage of acetic acid and a higher percentage of butyric and propionic acid.

[0169] These effects occurred at t = 24 and 48 h. Table 2 shows SCFA production and gas production after 48 h, which represents the infant's colonic transit time.

[0170] Table 2 Formation of butyrate after 48 hours of fermentation by the infant gut microbiota (in µmol / g fiber)

[0171] Overall, fermentation characteristics showed that mixtures of scFOS with lcFOS, LBG, and especially XG beneficially led to butyrate formation.

[0172] Example 7 - Utilizing the fermentation characteristics of FOS and Bifidobacterium breve in fecal samples from 4-month-old breastfed infants.

[0173] Stool samples were obtained from breastfed infants aged 4 months.

[0174] probiotic strains Freeze-precipitate (count stock solution) of *Bifidobacterium breve* M-16V (Morinaga) containing the pNZ44St plasmid conferring streptomycin resistance. This allows for selective plating of the strain on TOS-propionate agar supplemented with streptomycin. The final dose of *Bifidobacterium breve* per feeding was 1 x 10⁻⁶. 8 CFU.

[0175] Compare the following experimental groups (sterile stock solutions): scFOS / lcFOS: 5 g / L (10% solution) scFOS / lcFOS, wt / wt ratio is 9:1; scFOS / lcFOS 5 g / L scFOS / lcFOS (10% solution) + 1.10 8 cfu / ml Bifidobacterium breve M-16V Growth medium: Colonic microecological culture medium: yeast extract 1 g / L, ammonium sulfate 2 g / L, K2HPO4 2 g / L, NaHCO3 3.2 g / L, NaCl 4.5 g / L, MgSO4·7H2O 0.5 g / L, cysteine ​​HCl 0.5 g / L, CaCl2·2H2O 0.4 g / L, bile salts 25 mg / L, menadione, metal and vitamin solutions, heme (10 mg / L), mucus 2.5 g / L.

[0176] Infant fecal samples were thawed under anaerobic conditions and a fecal suspension of approximately 4% (w / v) was prepared in a colonic microecological medium containing 25 mM acetate and 12 mM lactate, 25 mg / L bile acids (Sigma), 2.5 g / L porcine gastric mucoprotein, 15 mmol / L ammonium sulfate, and 1 g / L tryptone (carbon-free), adjusted to pH 5.5 (to mimic the pH of feces from breastfed infants). The diluted fecal sample was homogenized, allowed to settle for 5 minutes, filtered through a tea sieve to remove large particles, and subsequently filtered through a Millex 100 µm vacuum filter.

[0177] Fill BioLector Pro plates (BOH2 round wells, M2P-labs) with pH electrodes using fecal samples. Seal the plates with ventilated silicone foil with slits. Incubate the plates in BioLector Pro (85% moisture, 37°C, 600 rpm, anaerobic). At T = 0, add 80 μL of sterile experimental composition as a bolus feed. If appropriate, add 1 x 10⁻⁶ ppm. 8 The concentration of CFU was increased by adding thawed M-16V precipitate. For infant samples using continuous pH control, the experiment was started at a set point of 5.5. After 4 hours, the experiment was paused, and fecal slurry from each well was collected and briefly centrifuged. All procedures were performed under sterile anaerobic conditions. The supernatant was frozen for further analysis, while the fecal precipitate was resuspended in fresh medium containing carbohydrates and pipetted back into the original wells of the BOH2 plate. This procedure was repeated for 3 days, with manual feeding twice daily (every 4 hours), followed by automated, continuous, slow carbohydrate feeding (80 µl overnight) via a microfluidic system using a BioLector system.

[0178] The supernatant from the fermented feces was thawed and aliquoted. The SCFAs (acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid) were quantitatively determined by gas chromatography. The ammonia concentration was quantitatively determined by a rapid ammonia kit (Megazyme). After heat inactivation, D-lactic acid and L-lactic acid were quantitatively determined by a D-lactic acid / L-lactic acid kit (R-Biopharm AG).

[0179] result: Butyrate production following exposure to a mixture of scFOS and lcFOS is beneficially further increased upon exposure to Bifidobacterium breve.

[0180] Table 3 The gut microbiota of breastfed infants forms fermentation end products (in mmol / L).

[0181] .

Claims

1. A nutritional composition comprising digestible carbohydrates, lipids, proteins, 3-fucosylated lactose (3-FL), and a butyrate source, the nutritional composition being suitable for providing nutrition to human subjects, preferably infants or young children suffering from food allergies, preferably milk protein or egg allergies, or at risk of developing food allergies, preferably milk protein or egg allergies. The protein is a hydrolyzed protein and / or free amino acids or is essentially composed of hydrolyzed protein and / or free amino acids. The butyrate source is provided in the form of dietary butyrate and / or by indigestible sugars capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate; and These indigestible sugars are selected from indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof, and These indigestible sugars are of plant, crustacean, and / or microbial origin.

2. The nutritional composition according to claim 1, wherein the nutritional composition is in the form of an instant liquid or a powder, the powder being reconstituted with water to become an instant liquid nutritional composition, wherein the 3-FL is present in an amount of 10 mg to 1 g 3-FL / 100 ml, more preferably 15 mg to 0.5 g 3-FL, or even more preferably 20 mg to 0.2 g 3-FL / 100 ml of the nutritional composition.

3. The nutritional composition according to claim 1 or 2, wherein the butyrate is provided in the form of dietary butyrate and / or by an indigestible sugar capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate, wherein the nutritional composition comprises 10 mg to 175 mg of dietary butyrate / 100 ml of nutritional composition and / or 1 mg to 1.0 g of indigestible sugar / 100 ml of nutritional composition.

4. The nutritional composition according to any one of the preceding claims, wherein the butyrate source comprises dietary butyrate, wherein the dietary butyrate source is selected from the group consisting of: food-grade tricresyl tributyl ester, anhydrous milk fat, butter, and products derived from microbial fermentation.

5. The nutritional composition according to any one of the preceding claims, wherein the butyrate source comprises an indigestible oligosaccharide selected from the group consisting of: fructooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucose oligosaccharides, glucose-manniosaccharides, galactomanniosaccharides, mannosaccharides, chitosan oligosaccharides, uronic acid oligosaccharides, and mixtures thereof.

6. The nutritional composition according to any one of the preceding claims, wherein the butyrate source comprises an indigestible polysaccharide selected from the group consisting of: low-viscosity pectin, (partially hydrolyzed) guar gum, fructooligosaccharides, oat beta-glucan, soybean fiber, resistant starch, gum arabic, cellulose, arabinoxylan, xanthan gum, locust bean gum, and / or resistant starch.

7. The nutritional composition according to any one of the preceding claims, wherein the butyrate source comprises a mixture of indigestible polysaccharides and indigestible oligosaccharides, preferably a mixture of short-chain fructooligosaccharides (scFOS) and long-chain oligosaccharides (lcFOS), more preferably a mixture of scFOS and lcFOS in a weight ratio of 9:

1.

8. The nutritional composition according to any one of the preceding claims, wherein the butyrate source comprises milk fat and / or fructooligosaccharides, preferably a mixture of short-chain and long-chain fructooligosaccharides.

9. The nutritional composition according to any one of the preceding claims, wherein the nutritional composition is infant formula, follow-up formula or toddler milk.

10. The nutritional composition according to any one of the preceding claims, wherein the nutritional composition further comprises Bifidobacterium, preferably Bifidobacterium breve.

11. The nutritional composition according to any one of the preceding claims, wherein the protein is hydrolyzed whey protein or hydrolyzed rice protein or is substantially composed of hydrolyzed whey protein or hydrolyzed rice protein.

12. The nutritional composition according to any one of the preceding claims, wherein the nutritional composition is used in the dietary management of infants or young children suffering from food allergies, preferably egg protein allergies or cow's milk protein allergies, more preferably cow's milk protein allergies, or at risk of food allergies, preferably egg protein allergies or cow's milk protein allergies, more preferably cow's milk protein allergies.

13. The nutritional composition according to any one of claims 1-12, wherein the nutritional composition is used in the treatment or prevention of food allergies, preferably egg protein allergies or cow's milk protein allergies, more preferably cow's milk protein allergies.

14. The nutritional composition according to any one of the preceding claims, wherein the nutritional composition is used in the treatment or prevention of food allergies selected from those that stimulate immune function, promote immune maturation, reduce and / or inhibit Th2 responses, and improve and / or promote Th1 responses, preferably for use in increasing the Th1:Th2 balance.

15. Use of lipids, digestible carbohydrates and proteins in the manufacture of a nutritional composition for use in human subjects, preferably infants or young children with allergies or at risk of allergies, preferably human subjects, preferably infants or young children at risk of food allergies, preferably egg or milk protein allergies, for dietary management, wherein the protein is a hydrolyzed protein and / or free amino acids or consists substantially of hydrolyzed protein and / or free amino acids, and wherein the nutritional composition comprises a combination of 3-FL and a butyrate source, wherein the butyrate source is provided in the form of dietary butyrate and / or by an indigestible sugar capable of undergoing microbial fermentation in the gastrointestinal tract to produce butyrate; The indigestible sugars are selected from indigestible polysaccharides [NDP], indigestible oligosaccharides [NDO], or mixtures thereof, and These indigestible sugars are of plant, crustacean, and / or microbial origin.

16. A method for treating or preventing food allergies in infants or young children by modulating immune maturation, the method comprising administering to the infant or young child a nutritional composition according to any one of claims 1 to 12.

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