Nutritional composition comprising GOS and HMO

Abstract

The present invention relates to a combination of a mixture of galactooligosaccharides (GOS) and human milk oligosaccharides (HMO), or a nutritional composition comprising such a combination. The combination or composition is useful for the treatment and / or prevention of stress and / or mood disorders in infants, toddlers and / or children. The mixture of HMO consists of or consists essentially of 2 '-fucosyllactose (2' FL) and difucosyllactose (DFL) or lactose-N-tetrasaccharide (LNT) and 6 '-sialyllactose (6' SL). The invention also relates to the use of a combination of a mixture of galactooligosaccharides (GOS) and human milk oligosaccharides (HMO) or a composition comprising such a combination for the treatment and / or prevention of stress and / or mood disorders in infants, toddlers and / or children, wherein the mixture of HMO consists of or consists essentially of 2 '-fucosyllactose (2' FL) and difucosyllactose (DFL) or lactose-N-tetrasaccharide (LNT) and 6 '-sialyllactose (6' SL).

Description

Technical Field

[0001] The present invention relates to a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) or a composition comprising the combination, particularly for the treatment and / or prevention of stress and / or mood disorders in infants, toddlers and / or children, wherein the mixture of HMOs consists of or essentially consists of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL). The present invention also relates to the use of a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) or a composition comprising the combination, wherein the mixture of HMOs consists of or essentially consists of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL) for the treatment and / or prevention of stress and / or mood disorders in infants, toddlers and / or children. Background Art

[0002] Infants and children are often exposed to psychological stress, such as changes in social situations (e.g., changes in care providers or schools, new family members, medical conditions or diseases). When stress is extreme, prolonged exposure may lead to counter-adaptive physiological and behavioral responses, such as anxiety or distress, increased sensitivity to painful stimuli, depression, and impaired neuronal development or neuronal function. Due to potential side effects on neuronal development, drug interventions may not be suitable for infants and children. Nutritional solutions (i.e., non-drug solutions) are needed to treat or prevent stress.

[0003] Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter. It is present in the central nervous system (CNS), but is also found in large quantities in foods such as crustaceans, plants, and is produced naturally by certain bacteria. Therefore, GABA is naturally present in the intestine.

[0004] Several intestinal flora have been shown to synthesize GABA (Wiley et al., 2021, Mod. Trends Psychiatry, 32: 74-79). There is clinical and preclinical evidence for the role of exogenous GABA in relieving stress and promoting feelings of relaxation (Abdou et al., 2006, BioFactors, 26: 201-208). In preclinical studies, GABA-producing probiotics have been shown to relieve intestinal discomfort triggered by stress (Pokusaeva et al., 2016, Neurogastroenterol. Motil., 29: e12904).

[0005] No nutritional solution has been demonstrated or is useful for naturally increasing GABA synthesised by the microbiota of an infant, toddler and / or child exposed to the nutritional solution, particularly in the treatment and / or prevention of stress and / or mood disorders, for example in maintaining or restoring the functionality of bidirectional transport pathways in the gut-brain axis to healthy levels.

[0006] Therefore, there is a need to provide nutritional solutions that can treat and / or prevent stress and / or mood disorders, for example by maintaining or restoring the functionality of bidirectional transport pathways in the gut-brain axis to healthy levels. Summary of the invention

[0007] The inventors surprisingly found that novel combinations of mixtures of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) can be advantageously used to treat and / or prevent stress and / or mood disorders because the combination shows that the intestinal microbiota of infants, toddlers and / or children increases the biosynthesis of gamma-aminobutyric acid (GABA). The inventors have found that these specific combinations are particularly effective in increasing the biosynthesis of gamma-aminobutyric acid (GABA) by the intestinal microbiota of infants, toddlers and / or children. Therefore, the present invention provides a method for increasing the production of metabolites (i.e., GABA) that have known benefits for stress relief and relaxation. The mixture of HMO consists of or consists essentially of 2'-FL and DFL or LNT and 6'-SL. The effect of the combination of a mixture of GOS and HMO on the biosynthesis of GABA by the microbiota was previously unknown. The combination is particularly effective in restoring the sensitivity of the bidirectional transmission pathway in the gut-brain axis to normal levels.

[0008] In the experiments based on the present invention, the combination of GOS with 2'-FL and DFL or LNT and 6'-SL resulted in an increase in the biosynthesis of GABA by the microbiota. Therefore, the experiments show the role played by the combination in promoting the biosynthesis of GABA.

[0009] Thus, in a first aspect, the present invention provides a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or essentially consists of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0010] In another aspect, the present invention provides a nutritional composition comprising a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO), wherein the mixture of HMO consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL). The nutritional composition is particularly useful for infant formula, first stage infant formula, follow-on or second stage infant formula, growing-up milk, baby food, infant cereal composition, fortifier suitable for infants, toddlers and / or children or supplement suitable for infants, toddlers and / or children.

[0011] In another aspect, the present invention provides a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) for use in treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, wherein the mixture of HMOs consists or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL). Suitably, the combination treats stress and / or mood disorders by increasing gamma-aminobutyric acid (GABA) produced by the intestinal microbiota of infants, toddlers and / or children.

[0012] In another aspect, the present invention provides a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) for maintaining or restoring to healthy levels the functionality of a bidirectional transport pathway in the gut-brain axis in infants, toddlers and / or children, wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0013] In another aspect, the present invention provides a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs) for use in increasing gamma-aminobutyric acid (GABA) production by the intestinal microbiota of an infant, toddler and / or child, wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0014] In another aspect, the present invention provides a nutritional composition for treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, the nutritional composition comprising a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL). Suitably, the composition treats stress and / or mood disorders by increasing gamma-aminobutyric acid (GABA) produced by the intestinal microbiota of infants, toddlers and / or children.

[0015] On the other hand, the present invention provides a nutritional composition for maintaining or restoring to healthy levels the functionality of a bidirectional transport pathway in the gut-brain axis of an infant, toddler and / or child, the nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0016] In another aspect, the present invention provides a nutritional composition for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child, the nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or essentially consists of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0017] In a further aspect, the present invention provides a method for treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, the method comprising administering a combination according to the invention or a nutritional composition according to the invention to the infant, toddler and / or child.

[0018] In another aspect, the present invention provides a method for maintaining or restoring the functionality of a bidirectional transport pathway in the gut-brain axis of an infant, toddler and / or child to a healthy level, the method comprising administering a combination according to the invention or a nutritional composition according to the invention to the infant, toddler and / or child.

[0019] In a further aspect, the present invention provides a method for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child, the method comprising administering a combination according to the invention or a nutritional composition according to the invention to the infant, toddler and / or child.

[0020] In another aspect, the present invention provides the use of a combination according to the invention or a nutritional composition according to the invention for treating and / or preventing stress and / or mood disorders in infants, young children and / or children. Suitably, the combination treats stress and / or mood disorders by increasing gamma-aminobutyric acid (GABA) produced by the intestinal microbiota of infants, young children and / or children.

[0021] In another aspect, the present invention provides the use of a combination according to the invention or a nutritional composition according to the invention for maintaining or restoring to a healthy level the functionality of a bidirectional transport pathway in the gut-brain axis in an infant, toddler and / or child.

[0022] In another aspect, the present invention provides the use of a combination according to the invention or a nutritional composition according to the invention for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Relative abundance of GABA expressed as peak area after 24 hours of fermentation of digested milk matrix supplemented with GOS, 2'FL / DFL, LNT / 6'SL, GOS+2'FL / DFL or GOS+LNT / 6'SL by toddler's microbiota. Points represent individual values, lines are at the median, boxes are 25 / 75 percentiles, and whiskers are at the minimum / maximum range. Different letters represent significant differences.

[0024] Figure 2 : Relative abundance of GABA expressed as the logarithm to base 2 of the ratio between the GABA value after 24 hours of fermentation of a digested milk matrix supplemented with GOS, 2'FL / DFL, LNT / 6'SL, GOS+2'FL / DFL or GOS+LNT / 6'SL by the microbiota of a toddler and the GABA value obtained after fermentation of a separate digested milk matrix. Points represent individual values, lines are in the median, boxes are 25 / 75 percentiles, and whiskers are in the minimum / maximum range. Different letters represent significant differences. DETAILED DESCRIPTION

[0025] definition

[0026] The term "infant" refers to a child under the age of 12 months. The expression "toddler" refers to a child between the age of one year and less than three years old, also called a toddler. The expression "child" refers to a child between the age of three and seven years old. Preferably, the expression "child" refers to a child between the age of three and five years old.

[0027] A "caesarean-born baby, infant, or child" is a baby, infant, or child who was delivered by caesarean section. This means the baby, infant, or child was not delivered vaginally.

[0028] A "vaginally delivered infant, infant, or child" is one that was delivered vaginally rather than by caesarean section.

[0029] "Premature infant" or "preterm infant" refers to an infant, toddler or child who is born prematurely. Typically, it refers to an infant, toddler or child who is born before 37 weeks of gestation.

[0030] "Low birth weight infant" is a newborn baby who weighs less than 2500g (5.5 pounds) due to premature birth or fetal growth restriction. It therefore covers:

[0031] -Infants, toddlers, or children weighing 1500 g to 2500 g at birth (often referred to as "low birth weight" or LBW)

[0032] -Infants, toddlers, or children who weigh 1000 g to 1500 g at birth (called "very low birth weight" or VLBW)

[0033] - Infants, toddlers or children born with a birth weight of less than 1000g (called "extremely low birth weight" or ELBW).

[0034] "Small for gestational age (SGA)" refers to an infant whose birth weight is below the 10th percentile for infants of the same gestational age. Thus, it encompasses an infant, toddler or child whose birth weight is below the 10th percentile for infants of the same gestational age.

[0035] The expression "nutritional composition" refers to a composition that supplies nutrients to an individual. Such nutritional compositions are usually administered orally or parenterally, and they usually include a lipid or fat source and a protein source. A carbohydrate source may also be included. In one embodiment, the nutritional composition of the present invention is a synthetic nutritional composition.

[0036] In a specific embodiment, the combination or composition of the present invention is a "synthetic combination" or a "synthetic nutritional composition". The expression "synthetic combination or synthetic nutritional composition" refers to a mixture obtained by chemical and / or biological methods, the chemical properties of which may be the same as the mixture naturally occurring in mammalian milk (i.e., the synthetic combination or synthetic composition is not breast milk).

[0037] As used herein, the expression "infant formula" refers to a food intended specifically for the supply of nutrition to infants during the first few months of life and which itself meets the various nutritional needs of such people (in accordance with Article 2(c) of Directive 91 / 321 / EEC 2006 / 141 / EC of the European Commission of 22 December 2006 on infant formula and follow-on formula). It also refers to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and Special Infants (including foods for special medical purposes). The expression "infant formula" covers both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0038] "Second infant formula" or "follow-on formula" is given from the 6th month onwards. Infant formula constitutes the main liquid element in the gradually diversified diet of such persons.

[0039] The expression "baby food" refers to a food material intended specifically for the nutrition of infants or young children during the first year of life.

[0040] The expression "infant cereal composition" refers to a foodstuff which is intended specifically for the nutrition of an infant or young child during the first year of life.

[0041] The expression "growing-up milk" (or GUM) refers to a milk-based beverage, usually with added vitamins and minerals, which is intended for infants or children.

[0042] The term "fortifier" refers to a liquid or solid nutritional composition suitable for fortifying or admixing human milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients. Thus, the fortifier of the present invention may be administered after being dissolved in human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients, or it may be administered as a separate composition. When administered as a separate composition, the milk fortifier of the present invention may also be identified as a "supplement." In one embodiment, the milk fortifier of the present invention is a supplement.

[0043] The expression "weaning" refers to the period during which breast milk is gradually replaced by other foods in the diet of an infant or young child.

[0044] The expressions "age in days / weeks / months / age" and "days / weeks / months / years since birth" are used interchangeably.

[0045] "Breast milk" is understood to mean mother's breast milk or colostrum.

[0046] "Oligosaccharides" are carbohydrate polymers containing small amounts (usually three to ten parts) of common sugars (monosaccharides).

[0047] The term "galacto-oligosaccharide" refers to a class of non-digestible fibers with prebiotic activity. GOS are formed by enzymatic conversion of lactose. GOS typically comprise a chain of galactose units produced by successive transgalactosylation reactions, with a terminal glucose unit, although a terminal galactose unit may alternatively be present. The degree of polymerization of GOS typically ranges from 2 to 8 monomer units. Such GOS contain β-(1→2), β-(1→3), β-(1→4) or β-(1→6) linked galactose moieties, and may have a degree of polymerization of 3 to 8 galactose units. Thus, the term GOS preferably refers to an oligosaccharide comprising at least three galactose units, more preferably an oligosaccharide comprising at least four galactose units, preferably with a degree of polymerization of 3 to 8 galactose units.

[0048] The term "HMO" refers to human milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, which indicates that they perform important functions that may not be directly related to their caloric value. It has been specifically pointed out in the art that these carbohydrates play a key role in the early development of infants and young children (such as the maturation of the immune system). Many different types of HMO are found in human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N-acetylglucosamine with a variety of bonds between these molecules, so human milk contains a large number of different types of oligosaccharides, and more than 130 such structures have been identified to date. Almost all oligosaccharides have a lactose molecule at the reducing end, and the terminal position of the non-reducing end is occupied by sialic acid and / or fucose (if any). HMOs can be acidic (e.g., oligosaccharides containing charged sialic acid) or neutral (e.g., fucosylated oligosaccharides).

[0049] "Fucosylated oligosaccharides" are oligosaccharides with fucose residues. Such oligosaccharides are neutral. Some examples are 2'FL (2'-fucosyllactose), 3-FL (3-fucosyllactose), difucosyllactose (DFL), lactose difucotetraose (LDFT), lactose-N-fucopentaose (e.g., lactose-N-fucopentaose I, lactose-N-fucopentaose II, lactose-N-fucopentaose III, lactose-N-fucopentaose V), lactose-N-fucohexaose, lactose-N-difucohexaose I, fucosyllactose-N-hexaose, fucosyllactose-N-neohexaose, difucosyllactose-N-hexaose I, difucosyllactose-N-neohexaose II, and any combination thereof.

[0050] The expression "N-acetylated oligosaccharide" encompasses both "N-acetyl-lactosamine" and "oligosaccharides containing N-acetyl-lactosamine". Such oligosaccharides are neutral oligosaccharides with N-acetyl-lactosamine residues. Suitable examples are LNT (lactose-N-tetraose), p-lactose-N-neohexose (p-LNnH), LNnT (lactose-N-neotetraose) and any combination thereof. Other examples are lactose-N-hexaose, lactose-N-neohexose, p-lactose-N-hexaose, p-lactose-N-neohexose, lactose-N-octose, lactose-N-neooctose, iso-lactose-N-octose, p-lactose-N-octose and lactose-N-decasaccharide.

[0051] "HMO precursors" are key compounds that interfere with the production of HMOs, such as sialic acid and / or fucose.

[0052] "Sialyl oligosaccharides" are oligosaccharides containing charged sialic acid, ie oligosaccharides having sialic acid residues. Such oligosaccharides are acidic. Some examples are 3-SL (3' sialyllactose) and 6'SL (6' sialyllactose).

[0053] The combination or nutritional composition of the present invention may be in solid form (e.g., powder) or liquid form. The amount of various ingredients (e.g., oligosaccharides) may be expressed as g / 100 g of composition based on dry weight when the composition is in solid form (e.g., powder), or as a concentration of g / L composition when the composition refers to a liquid form (the latter also encompasses liquid compositions obtained after reconstitution of the powder in a liquid (such as milk, water, ...), such as reconstituted infant formula or larger / second stage infant formula or growing milk or infant cereal products or any other formulation designed to provide nutrition to infants).

[0054] The term "prebiotic" refers to non-digestible carbohydrates that have beneficial effects on the host by selectively stimulating the growth and / or activity of healthy bacteria, such as bifidobacteria in the human colon (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995; 125: 1401-12).

[0055] The term "probiotic" refers to a microbial cell preparation or microbial cell component that has a beneficial effect on the health or well-being of the host. (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999: 10107-10). Microbial cells are generally bacteria or yeast.

[0056] The term "cfu" is to be understood as colony forming units.

[0057] "Gamma-aminobutyric acid" or "GABA" is generally known as an inhibitory neurotransmitter in the central nervous system, and its function of inhibiting neuronal activity is involved, for example, in synchronizing neuronal networks in the hippocampus.

[0058] "Treatment" means addressing a medical condition or disease with the intent to improve or stabilize the outcome of the individual being treated or to address the underlying nutritional needs. Thus, treatment includes dietary or nutritional management of a medical condition or disease by addressing the nutritional needs of the individual being treated. Treatment includes elimination, alleviation, or amelioration of symptoms associated with a medical condition or disease.

[0059] "Prevention" refers to reducing the risk of onset or recurrence of a medical condition or disease. Thus, both primary and secondary prevention are contemplated. "Primary prevention" refers to preventing a medical condition or disease prior to onset, and "secondary prevention" refers to preventing recurrence of a medical condition or disease after the first onset. Thus, prevention includes dietary or nutritional prevention of a medical condition or disease by addressing the nutritional needs of the individual being treated. Prevention includes eliminating or minimizing the risk of developing a medical condition or disease and reducing the risk of developing symptoms associated with the medical condition or disease.

[0060] The term "stress" refers to a state of emotional or mental tension. Stress can be caused by adverse or demanding circumstances.

[0061] The term "stress resilience" refers to the ability to successfully adapt when faced with stress. As a result, individuals can avoid stress-related symptoms.

[0062] The term "mood" refers to positive or negative emotional states of varying intensity that change in response to life circumstances (Polak et al., (2015) Nutrition for brain health and cognitive performance, 95-122). Like emotions, moods are affective states or "feelings" and have different aspects, such as alertness / fatigue, anxiety, and stress. Moods can be distinguished from emotions in terms of duration and the ability to describe triggers. Moods are usually undifferentiated, change slowly, can last from days to weeks, and are "objectless" because people may not know the cause or source of their moods (JA Russell, (2003, Psychological review, 110: 145-172). Emotions have a clear focus (i.e., self-evident causes) and are short-lived, i.e., seconds to minutes. Moods and emotions are closely linked, and usually bad moods are accompanied by negative emotions, and good moods are accompanied by positive emotions.

[0063] The term "mood disorder" refers to a class of diseases that describe significant and severe changes in mood. Mood disorders include: anxiety disorders, depression, bipolar disorder, and SAD (seasonal affective disorder). In some embodiments, the mood disorder is an anxiety disorder or depression. In some embodiments, the mood disorder is anxiety. In some embodiments, the mood disorder is depression.

[0064] The term "anxiety" refers to feelings of uneasiness such as worry, nervousness, or fear, which may be mild or severe, especially about something that is about to happen or may happen in the future.

[0065] The term "anxiety disorder" refers to conditions characterized by intense, excessive, and persistent worry and fear about everyday situations. These symptoms can interfere with daily activities, such as school and relationships. There are several types of anxiety disorders, including generalized anxiety disorder, separation anxiety disorder, panic disorder, social anxiety disorder, and various phobia-related disorders.

[0066] The term "depression" (also called major depressive disorder) refers to a condition characterized by persistent low mood (feeling sad, irritable, empty, or having lost pleasure / interest). These symptoms can interfere with daily activities, such as school and relationships.

[0067] The term "bipolar disorder" refers to a condition that causes extreme mood swings, including high moods (mania) and low moods (depression). Mania is characterized by euphoria, hyperactivity, an inflated ego, and unrealistic optimism.

[0068] By "promoting relaxation" is meant enhancing feelings of relaxation, such as increasing feelings of calm.

[0069] "Increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child" means enhancing the biosynthesis of GABA by the intestinal microbiota of an infant, toddler or child administered the combination or nutritional composition of the present invention, compared to the biosynthesis of GABA by the intestinal microbiota of an infant, toddler or child not administered the combination or nutritional composition of the present invention.

[0070] "Maintaining the functionality of the bidirectional transmission pathways in the gut-brain axis at healthy levels" means maintaining the bidirectional transmission pathways in the gut-brain axis in the infant, toddler or child at normal levels, ie, those levels of a healthy infant, toddler or child.

[0071] "Restoring the functionality of the bidirectional transmission pathway in the gut-brain axis to healthy levels" means changing the bidirectional transmission pathway in the gut-brain axis in an infant, toddler or child suffering from a medical condition, disease or dysregulation of the transmission pathway in the gut-brain axis to normal levels, i.e., to those levels of a healthy infant, toddler or child.

[0072] The term "effective amount" preferably refers to an amount of a combination or composition that provides a sufficient amount of the combination of a mixture of GOS and HMOs to produce the desired therapeutic or prophylactic result in an individual. An effective amount can be administered to an individual in one or more doses to achieve the desired therapeutic or prophylactic result.

[0073] Unless otherwise indicated, all percentages are by weight.

[0074] In addition, in the context of the present invention, the term "comprising" or "including" does not exclude other possible elements. The composition of the present invention (including the multiple embodiments described herein) may comprise, consist of or consist essentially of the following elements: the basic elements and necessary limitations of the present invention described herein, as well as any additional or optional ingredients, components or limitations described herein or as required. "Essentially consisting of..." of a given element means that the content of the element is 95% to 100% by weight, preferably 97% to 100% by weight, more preferably 99% to 100% by weight, and most preferably 99.5% to 100% by weight, such as 99.9% to 100% by weight.

[0075] It should be understood that various changes and modifications made to the presently preferred embodiments described herein will be apparent to those skilled in the art. These changes and modifications may be made without departing from the spirit and scope of the present invention and without diminishing the attendant advantages. Therefore, such changes and modifications are intended to be covered by the appended claims.

[0076] use

[0077] In one aspect, the present invention provides a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) for use in treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0078] The combination may be in the form of a nutritional composition.

[0079] Therefore, in another aspect, the present invention provides a nutritional composition for treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, the nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0080] There is clinical and preclinical evidence for the role of exogenous GABA in relieving stress and promoting feelings of relaxation (Abdou et al., 2006, BioFactors, 26: 201-208). In preclinical studies, GABA-producing probiotics have been shown to relieve intestinal discomfort triggered by stress (Pokusaeva et al., 2016, Neurogastroenterol. Motil., 29: e12904). Hepsomali et al. reviewed the effects of oral GABA administration on stress. (Hepsomali et al., (2020), Front. Neurosci., 14: 923). GABA has been shown to reduce human salivary cortisol (Kanehira T et al., (2011), J Nutr Sci Vitaminol (Tokyo), 57: 9-15). However, no nutritional solution has previously been shown to naturally increase GABA synthesized by the microbiota of infants and / or toddlers and / or children exposed to the nutritional solution.

[0081] Treatment includes elimination, alleviation or amelioration of symptoms associated with a medical condition or disease.

[0082] In some embodiments, treatment improves stress tolerance in infants, toddlers, and / or children.

[0083] Symptoms of stress that can be eliminated, reduced or improved include anxiety, pain, depression, increased sensitivity to painful stimuli, and impaired neuronal development or neuronal function. Suitably, treatment can reduce the amount of cortisol in saliva, which is a measure of stress (Jessop DS, Turner-Cobb JM, (2008), Stress, 11: 1-14; Bi et al., (2020), J Anim Physiol Anim Nutr (Berl)., 104: 590-596; Kanehira T et al., (2011), J Nutr Sci Vitaminol (Tokyo), 57: 9-15).

[0084] In some embodiments, the mood disorder is an anxiety disorder or depression.

[0085] Symptoms of anxiety disorders that may be eliminated, reduced, or improved include finding it difficult to concentrate, trouble sleeping, irritability, crying, feeling nervous, fidgeting, or loss of appetite. Appropriately, treatment may reduce scores on the anxiety subscale of the Children's Behaviour Checklist (CB CL), also known as the Achenbach System of Empirically Based Assessment (ASEBA), a tool used to assess children's problem behaviors and abilities (Achenbach 1991; Achenbach and Rescorla 2000).

[0086] Symptoms of depression that may be eliminated, reduced, or improved include finding it difficult to concentrate, trouble sleeping, irritability, tearfulness, anxiety, restlessness, loss of appetite, or feeling sad, heartbroken, empty, or hopeless.

[0087] Suitably, the combination or composition according to the invention treats stress and / or mood disorders by promoting relaxation in infants, toddlers and / or children.

[0088] Suitably, the combination or composition according to the invention treats stress and / or mood disorders by increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of infants, toddlers and / or children. Many species of Lactobacillus and Bifidobacterium, including Bifidobacterium adolescentis, are able to synthesize and secrete the potent inhibitory neurotransmitter gamma-aminobutyric acid (GABA) which is known to be involved in reducing stress, anxiety and depression.

[0089] Thus, in another aspect, the present invention provides a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs) for use in increasing gamma-aminobutyric acid (GABA) production by the intestinal microbiota of an infant, toddler and / or child, wherein the mixture of HMOs consists of or essentially consists of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0090] In another aspect, the present invention provides a nutritional composition for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child, the nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or essentially consists of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0091] Suitably, the biosynthesis of GABA carried out by the intestinal microbiota of an infant, a toddler and / or a child increases from 1.1 times to 20 times, such as increased by 1.2 times to 11 times, preferably 4 times to 11 times. Suitably, the biosynthesis of GABA carried out by the intestinal microbiota of an infant, a toddler and / or a child increases by at least 1.1 times, such as 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times or 20 times.

[0092] Suitably the logarithm to base 2 of the ratio of the GABA value of a combination or composition according to the invention to the GABA value of a corresponding milk control increases from 0.2 to 5, suitably from 0.25 to 3.8.

[0093] The amount of GABA secreted by the intestinal microbiota of infants, young children and / or children (i.e., GABA biosynthesis) can be measured by methods known in the art. For example, the method disclosed herein can be used (see Example 2). Suitably, the amount of GABA in fresh stool samples from infants, young children or children fed with the combination or nutritional composition of the present invention can be compared with samples from infants, young children or children not exposed to the combination or nutritional composition of the present invention (Altaib et al., (2021), Microorganisms, 9: 378).

[0094] The gut-brain axis consists of bidirectional communication between the central nervous system (CNS) and the enteric nervous system (ENS), linking the brain’s emotional and cognitive centers to peripheral gut function. The autonomic nervous system, the hypothalamic-pituitary-adrenal (HPA) axis, and nerves within the gastrointestinal (GI) tract all connect the gut and brain, allowing the brain to influence gut activity, including that of functional immune effector cells; and the gut to influence mood, cognitive abilities, and mental health. Recent advances have described the importance of the gut microbiota in influencing these interactions. This interaction between the microbiota and the gut-brain axis appears to be bidirectional, involving signaling from the gut microbiota to the brain and from the brain to the gut microbiota through neural, endocrine, immune, and humoral connections (e.g., through the vagus nerve). In clinical practice, evidence for microbiota-GBA interactions comes from the association of dysbiosis with CNS disorders (i.e., autism, anxious-depressive behaviors) and functional gastrointestinal disorders.

[0095] Clinical, epidemiological, and immunological evidence suggests that the gut microbiota influences the gut-brain axis (i.e., mental state, mood regulation, neuromuscular function, and regulation of HPA). For example, several mood disorders (such as anxiety disorders, depression, and autism spectrum disorders) now have well-established links to functional GI disturbances, and GI diseases (e.g., irritable bowel syndrome) often involve psychological comorbidities associated with alterations in the gut microbiome.

[0096] Without being bound by theory, by altering the microbiota to increase the biosynthesis of GABA, the combination according to the invention may be able to affect neurotransmission in the paraventricular nucleus of the hypothalamus, the central nucleus of the amygdala, and the bed nucleus of the stria terminalis, among other effects. All three areas are involved in the processing of emotions related to anxiety and mood.

[0097] Thus, in some embodiments, the combination or nutritional composition according to the invention maintains or restores to healthy levels the functionality of the bidirectional transport pathway in the gut-brain axis of an infant, toddler and / or child.

[0098] In another aspect, the present invention provides a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMO) for maintaining or restoring to healthy levels the functionality of a bidirectional transport pathway in the gut-brain axis in infants, toddlers and / or children, wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0099] On the other hand, the present invention provides a nutritional composition for maintaining or restoring to healthy levels the functionality of a bidirectional transport pathway in the gut-brain axis of an infant, toddler and / or child, the nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

[0100] In a further aspect, the present invention provides a method for treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, the method comprising administering a combination according to the invention or a nutritional composition according to the invention to the infant, toddler and / or child.

[0101] In another aspect, the present invention provides a method for maintaining or restoring the functionality of a bidirectional transport pathway in the gut-brain axis of an infant, toddler and / or child to a healthy level, the method comprising administering a combination according to the invention or a nutritional composition according to the invention to the infant, toddler and / or child.

[0102] In a further aspect, the present invention provides a method for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child, the method comprising administering a combination according to the invention or a nutritional composition according to the invention to the infant, toddler and / or child.

[0103] In some embodiments, the method for increasing gamma-aminobutyric acid (GABA) production by the gut microbiota is non-therapeutic.

[0104] In some embodiments of the methods and uses of the invention, the combination or composition according to the invention is administered to an infant, toddler and / or child in an effective amount.

[0105] In another aspect, the present invention provides the use of a combination according to the invention or a nutritional composition according to the invention for treating and / or preventing stress and / or mood disorders in infants, young children and / or children. Suitably, the combination treats stress and / or mood disorders by increasing gamma-aminobutyric acid (GABA) produced by the intestinal microbiota of infants, young children and / or children.

[0106] In another aspect, the present invention provides the use of a combination according to the invention or a nutritional composition according to the invention for maintaining or restoring to a healthy level the functionality of a bidirectional transport pathway in the gut-brain axis in an infant, toddler and / or child.

[0107] In another aspect, the present invention provides the use of a combination according to the invention or a nutritional composition according to the invention for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child.

[0108] In some embodiments, the use or method according to the invention is non-therapeutic. Suitably, the use or method is for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child; for promoting relaxation of an infant, toddler and / or child; for promoting calmness of an infant, toddler and / or child; or for improving the mood of an infant, toddler and / or child.

[0109] In some embodiments of the methods and uses of the present invention, the combination or composition according to the present invention is used to treat stress and / or mood disorders.

[0110] In some embodiments of the methods and uses of the present invention, the combination or composition according to the present invention is used to prevent stress and / or mood disorders.

[0111] In some embodiments of the methods and uses of the present invention, the combination or composition according to the present invention is used to treat and / or prevent stress.

[0112] In some embodiments of the methods and uses of the present invention, the combination or composition according to the present invention is used to treat and / or prevent mood disorders.

[0113] Target Group

[0114] In some embodiments of the methods and uses of the invention, the combination or composition is administered to an infant.

[0115] In some embodiments of the methods and uses of the invention, the combination or composition is administered to a young child.

[0116] In some embodiments of the methods and uses of the invention, the combination or composition is administered to a child.

[0117] In one embodiment of the invention, the infant or toddler has an age range of 0 to 36 months.

[0118] In another embodiment, the infant or toddler ranges in age from 0 to 24 months, such as 18 months.

[0119] In yet another embodiment, the infant has an age range of 0 to 12 months, such as 9 or 6 months.

[0120] In some embodiments, the toddler ranges in age from one to three years old, such as two years old.

[0121] In some embodiments, the age range of the child is three to seven years old. In some preferred embodiments, the age range of the child is three to five years old, such as four years old.

[0122] Some specific infants, toddlers and children's colonies are particularly in need of compositions that can treat and / or prevent stress and / or mood disorders. Such infants, toddlers and children are such as premature infants, low birth weight infants and / or infants, toddlers and children of growth retardation. In fact, such individuals usually experience adverse medical conditions, and need significantly more frequent medical interventions than full-term infants and the infants of normal development of experience. Unfortunately, many such medical interventions are painful for infants or toddlers, and therefore face repeated stress and / or mood disorders. For such infants, it is particularly advantageous to supplement medical interventions with the nutritional composition that can treat and / or prevent stress and / or mood disorders.

[0123] The nutritional composition according to the present invention is used in infants, young children and / or children. Infants, young children and / or children may be full-term infants or premature infants. In a specific embodiment, the nutritional composition of the present invention is used for premature infants, infants with low birth weight and / or infants born less than gestational age (SGA). In a specific embodiment, the nutritional composition of the present invention is used for premature infants, low birth weight infants and / or infants born less than gestational age (SGA).

[0124] The nutritional composition of the present invention may also be used for infants, toddlers or children born by caesarean section or vaginally.

[0125] In some embodiments, the composition according to the invention may be used before and / or during the weaning period.The age and duration of administration (or giving or feeding) of the nutritional composition may be determined as required.

[0126] The nutritional composition can be provided, for example, immediately after the infant is born. The composition of the present invention can also be given during the first 1 week of the infant's life, or during the first 2 weeks of life, or during the first 3 weeks of life, or during the first 1 month of life, or during the first 2 months of life, or during the first 3 months of life, or during the first 4 months of life, or during the first 6 months of life, or during the first 8 months of life, or during the first 10 months of life, or during the first 1 year of life, or during the first 2 years of life, or even longer. In some particularly advantageous embodiments of the present invention, the nutritional composition is given (or administered) to the infant within the first 4, 6 or 12 months after the infant is born. In some other embodiments, the nutritional composition of the present invention is administered a few days (e.g., 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days...), or weeks (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks...) or months (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months...) after birth. This may especially refer to the case where the infant is a premature infant, but this is not required.

[0127] In one embodiment, the composition of the present invention is given to an infant or young child as a supplementary composition of breast milk. In some embodiments, an infant or young child receives breast milk during at least the first 2 weeks, the first 1 month, the first 2 months, the first 4 months, or the first 6 months. In one embodiment, the nutritional composition of the present invention is given to an infant or young child after this period of time when breast milk is used to provide nutrition, or is given to an infant or young child together with breast milk during this period of time when breast milk is used to provide nutrition. In another embodiment, during at least a period of time (e.g., after the 1st, 2nd, or 4th month of life), during at least 1 month, 2 months, 4 months, or 6 months, the composition is given to an infant or young child as the only or main nutritional composition.

[0128] combination

[0129] In one embodiment, the mixture of oligosaccharides consists of or consists essentially of 2'FL and DFL.

[0130] In one embodiment of the invention, 2'FL is present in the combination or nutritional composition in an amount of 0.005 g / L to 8 g / L of the combination or composition. In some embodiments, the amount of 2'FL may be 0.01 g / L to 3 g / L, such as 0.04 g / L to 2 g / L or 0.05 g / L to 1.5 g / L or 0.09 g / L to 1.2 g / L. In a specific embodiment, the amount of 2'FL is 1 g / L. In another specific embodiment, the amount of 2'FL is 0.2 g / L.

[0131] 2'FL can be present in the combination or nutritional composition in an amount of 0.004g / 100g to 6.8g / 100g of the combination or composition based on dry weight, 2'FL can be present in an amount of 0.008g / 100g to 2.4g / 100g, such as 0.03g / 100g to 1.6g / 100g or 0.04g / 100g to 1.2g / 100g or 0.07g / 100g to 1.0g / 100g. In a specific embodiment, 2'FL is present in an amount of 0.8g / 100g. In another specific embodiment, 2'FL is present in an amount of 0.16g / 100g.

[0132] In another specific embodiment, the amount of 2'FL is 5g / L to 500g / L, 10g / L to 400g / L, 40g / L to 300g / L, 60g / L to 200g / L, 80g / L to 180g / L, 100g / L to 150g / L, or 110g / L to 130g / L. In a specific embodiment, the amount of 2'-FL is 120g / L. Such amounts are particularly sufficient when the nutritional composition is in the form of a supplement or fortifier.

[0133] When the supplement or fortifier is in powder form, 2'FL is preferably provided in the nutritional composition of the present invention in an amount of 0.05g to 5g, 0.1g to 4.5g, 0.15g to 4g, 0.2g to 3.5g, 0.25g to 3g, 0.3g to 2.5g, 0.35g to 2g, 0.4g to 1.5g, 0.45g to 1g, 0.5g to 0.75g, for example 0.6g per serving.

[0134] In some embodiments, the amount of DFL is 0.0005g / L to 0.5g / L combination or composition, such as 0.001g / L to 0.3g / L combination or composition, or 0.002g / L to 0.2g / L combination or composition, or 0.01g / L to 0.25g / L combination or composition, or 0.015g / L to 0.2g / L combination or composition, or 0.025g / L to 0.19g / L combination or composition, or 0.075g / L to 0.165g / L combination or composition. In a specific embodiment, the amount of DFL is 0.1g / L. In another specific embodiment, the amount of DFL is 0.025g / L or 0.026g / L. In one embodiment, the amount of DFL is greater than 0.01g / L and optionally less than 0.1g / L or greater than 0.02g / L and less than 0.08g / L. In one embodiment, DFL is present in an amount of at least 0.01 g / L, at least 0.025 g / L, at least 0.026 g / L, at least 0.05 g / L, at least 0.07 g / L, at least 0.08 g / L, at least 0.1 g / L, at least 0.125 g / L, at least 0.15 g / L, at least 0.15 g / L, or at least 0.2 g / L.

[0135] In some embodiments, DFL is present in the combination or composition in an amount of 0.0004 g / 100 g to 0.38 g / 100 g of the combination or composition based on dry weight. The amount of DFL may be 0.0008 g / 100 g to 0.23 g / 100 g, such as 0.0015 g / 100 g to 0.15 g / 100 g, or 0.008 g / 100 g to 0.19 g / 100 g, or 0.012 g / 100 g to 0.15 g / 100 g, or 0.015 g / 100 g to 0.15 g / 100 g, or 0.019 g / 100 g to 0.15 g / 100 g. In a specific embodiment, the amount of DFL is 0.0075 g / 100 g or 0.078 g / 100 g. In another specific embodiment, the amount of DFL is 0.02g / 100g. In a specific embodiment, the amount of DFL is at least 0.001g / 100g, at least 0.002g / 100g, at least 0.005g / 100g, at least 0.01g / 100g, at least 0.02g / 100g, at least 0.025g / 100g, at least 0.04g / 100g, at least 0.05g / 100g, at least 0.075g / 100g, at least 0.09g / 100g, at least 0.1g / 100g, at least 0.15g / 100g, at least 0.2g / 100g or at least 0.3g / 100g.

[0136] In a particular embodiment, DFL is provided in the combination or nutritional composition of the present invention in an amount such that normal consumption of the combination or nutritional composition will provide a total daily dose of 0.003g to 3.9g, preferably 0.006g to 3g or 0.05g to 3g or 0.1g to 3g, for example 1.1g to 2.8g per day to an infant, toddler or child consuming it.

[0137] In a particular embodiment, DFL is provided in the combination or nutritional composition of the present invention in an amount such that a serving of the combination or nutritional composition will provide a total dose of 0.003 g to 3.9 g, preferably 0.006 g to 3 g or 0.05 g to 2.5 g or 0.1 g to 2 g, for example 0.02 g to 0.07 g per serving to an infant, toddler or child consuming it.

[0138] In a particular aspect of the invention, the combination or nutritional composition is an infant formula comprising an HMO mixture consisting of or consisting essentially of 2'-FL and DFL, wherein:

[0139] - 2'-FL in an amount of 1 g / L to 2 g / L composition, preferably 1.3 g / L composition, and / or on a dry weight basis in an amount of 0.8 g / 100 g to 1.5 g / 100 g composition, preferably 1 g / 100 g composition; and / or

[0140] - DFL in an amount of 0.1 g / L to 0.3 g / L composition, preferably 0.2 g / L composition, and / or in an amount of 0.08 g / 100 g to 0.2 g / 100 g composition, preferably 0.2 g / 100 g composition, based on dry weight.

[0141] In another specific aspect of the invention, the combination or nutritional composition is a supplement or fortifier and comprises a HMO mixture consisting of or consisting essentially of 2'-FL and DFL, wherein:

[0142] - 2'-FL in an amount of 100 g / L to 110 g / L composition, preferably 105 g / L composition; and / or

[0143] - The amount of DFL is from 10 g / L to 20 g / L of composition, preferably 15 g / L of composition.

[0144] In particular aspects, 2'-FL and DFL are present in the HMO mixture in a 2'-FL:DFL ratio of 5:1 to 14:1, such as 5:1 to 12:1, 5:1 to 10:1, 6:1 to 10:1, or 8:1 to 10: 1. In particularly advantageous embodiments, the ratio is 9:1 or about 9:1.

[0145] 2'-FL and DFL can be isolated from natural sources (such as animal milk) by chromatography or filtration techniques. Alternatively, HMOs can be prepared by biotechnological means using specific fucosyltransferases and / or fucosidases, by using enzyme-based (recombinant or natural enzyme) fermentation techniques or microbial fermentation techniques. In the latter case, the microorganism can express its natural enzymes and substrates, or can be engineered to produce the corresponding substrates and enzymes. Single microbial cultures and / or mixed cultures can be used. The formation of 2'-FL and DFL can be initiated by acceptor substrates starting from any degree of polymerization (DP) (starting from DP=1). Alternatively, 2'-FL and DFL can be prepared by chemical synthesis from lactose and free fucose. 2'-FL and DFL can also be commercially available, for example, from Glycom A / S in Denmark or Jennewein GmBH in Germany. 2'FL can be synthesized as described, for example, in "Large-scale synthesis of H-antigen oligosaccharides by expressing Helicobacter pylorialpha1,2-fucosyltransferase in metabolically engineered Escherichia colicells" (Drouillard S, Driguez H, Samain E. Angew Chem Int Ed Engl. 2006 Mar 3; 45(11): 1778-80) or obtained from commercial sources.

[0146] In one embodiment of the invention, the combination or nutritional composition comprises a mixture of HMOs consisting of or essentially consisting of 2'FL and DFL. It should be understood that when the expression "a mixture of HMOs consisting of or essentially consisting of 2'FL and DFL" is used in the context of the present invention, such expression does not include the presence of HMOs other than 2'FL and DFL in the mixture. In particular, the mixture does not contain N-acetylated oligosaccharides, sialylated oligosaccharides, fucosylated oligosaccharides other than 2'-FL and DFL, and precursors of HMOs (such as sialic acid or fucose). In some embodiments, the combination or nutritional composition does not contain N-acetylated oligosaccharides, sialylated oligosaccharides, fucosylated oligosaccharides other than 2'-FL and DFL, and precursors of HMOs (such as sialic acid or fucose).

[0147] In one embodiment, the mixture of oligosaccharides consists of or consists essentially of LNT and 6'SL.

[0148] In one embodiment of the invention, LNT is present in the combination or nutritional composition in an amount of 0.005 g / L to 3 g / L of the composition. In some embodiments, the amount of LNT may be 0.01 g / L to 1.5 g / L of the composition, such as 0.04 g / L to 1.2 g / L or 0.05 g / L to 1 g / L or 0.09 g / L to 0.8 g / L of the composition. In a specific embodiment, the amount of LNT is 0.5 g / L of the composition. In another specific embodiment, the amount of LNT is 0.1 g / L of the composition.

[0149] LNT can be present in the combination or nutritional composition in an amount of 0.004g / 100g to 2.3g / 100g composition based on dry weight, LNT can be present in an amount of 0.008g / 100g to 1.2g / 100g composition such as 0.03g / 100g to 0.9g / 100g or 0.04g / 100g to 0.8g / 100g or 0.07g / 100g to 0.6g / 100g composition. In a specific embodiment, LNT is present in an amount of 0.38g / 100g composition. In another specific embodiment, LNT is present in an amount of 0.08g / 100g composition.

[0150] In another embodiment of the invention, the combination or nutritional composition may comprise 0.005 to 5 g / L 6'SL, or 0.008 to 2.5 g / L or 0.01 to 1 g / L or 0.03 to 0.7 g / L, such as 0.04 or 0.5 g / L LNT.

[0151] The nutritional composition according to the invention may comprise 0.004 to 3.8 g LNT per 100 g composition by dry weight, such as 0.006 to 1.9 g or 0.008 to 0.8 g or 0.023 to 0.5 g or 0.031 to 0.4 g LNT per 100 g composition by dry weight, such as 0.18 g or 0.04 g LNT per 100 g composition by dry weight.

[0152] In a particular embodiment, LNT is provided in the nutritional composition of the present invention in an amount such that normal consumption of the nutritional composition will provide a total daily dose of 0.003 g to 3.9 g, preferably 0.006 g to 2 g or 0.02 g to 1.6 g or 0.03 g to 1.3 g, for example 0.05 g to 1 g per day to an infant or toddler (respectively a child) consuming it.

[0153] LNT can be chemically synthesized using an enzymatic transfer method, ie, using a glycosyltransferase to transfer a sugar unit of a donor moiety to an acceptor moiety, as described, for example, in US Pat. No. 5,288,637 and WO 96 / 10086.

[0154] The formation of 6'SL can be initiated by acceptor substrates starting at any degree of polymerization (DP), starting from DP = 1. Alternatively, 6'SL can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid).

[0155] In one embodiment of the invention, 6'SL is present in the combination or nutritional composition in an amount of 0.005 / L to 5g / L of the composition. In some embodiments, the amount of 6'SL may be 0.08g / L to 2.5g / L of the composition, such as 0.01g / L to 1g / L of the composition or 0.03g / L to 0.07g / L of the composition or 0.04g / L to 0.5g / L of the composition. In a specific embodiment, the amount of 6'SL is 0.24g / L of the composition. In another specific embodiment, the amount of 6'SL is 0.05g / L of the composition.

[0156] 6'SL may be present in the combination or nutritional composition in an amount of 0.004g / 100g to 3.8g / 100g of the composition on a dry weight basis, 6'SL may be present in an amount of 0.006g / 100g to 1.9g / 100g of the composition, such as 0.008g / 100g to 0.8g / 100g or 0.23g / 100g to 0.5g / 100g or 0.031g / 100g to 0.4g / 100g of the composition. In a specific embodiment, 6'SL is present in an amount of 0.18g / 100g of the composition. In another specific embodiment, 6'SL is present in an amount of 0.04g / 100g of the composition.

[0157] In a specific aspect, the 6'SL and LNT comprised in the composition or nutritional composition according to the invention are typically present in a ratio of 6'SL:LNT of 3:1 to 1:3, such as 2:1 to 1:2 or 2:1 to 1: 1. In a particularly advantageous embodiment, the ratio is 2:1 or about 2.1, preferably the ratio is 1:1 or about 1:1.

[0158] In a particular aspect of the invention, the combination or nutritional composition comprises 6'SL and LNT, wherein:

[0159] - 6'SL in an amount of 0.005 g / L to 5 g / L of the composition and / or in an amount of 0.004 g / 100 g to 3.8 g / 100 g of the composition on a dry weight basis; and / or

[0160] - LNT in an amount of 0.005 g / L to 3 g / L of the composition and / or in an amount of 0.004 g / 100 g to 2.3 g / 100 g of the composition on a dry weight basis.

[0161] In a particular aspect of the invention, the combination or nutritional composition comprises 6'SL and LNT, wherein:

[0162] - 6'SL in an amount of 0.008 g / L to 2.5 g / L of the composition and / or in an amount of 0.006 g / 100 g to 1.9 g / 100 g of the composition on a dry weight basis; and / or

[0163] - LNT in an amount of 0.01 g / L to 1.5 g / L of the composition and / or in an amount of 0.008 g / 100 g to 1.2 g / 100 g of the composition on a dry weight basis.

[0164] In another embodiment, the combination or nutritional composition of the present invention comprises 6'SL and LNT, wherein:

[0165] - 6'SL in an amount of 0.01 g / L to 1 g / L of the composition and / or in an amount of 0.008 g / 100 g to 0.8 g / 100 g of the composition on a dry weight basis; and / or

[0166] - LNT in an amount of 0.04 to 1.2 g / L of the composition and / or in an amount of 0.03 to 0.9 g / 100 g of the composition on a dry weight basis.

[0167] In another embodiment, the combination or nutritional composition of the present invention comprises 6'SL and LNT, wherein:

[0168] - 6'SL in an amount of 0.03 g / L to 0.7 g / L of the composition and / or in an amount of 0.023 g / 100 g to 0.5 g / 100 g of the composition on a dry weight basis; and / or

[0169] - LNT in an amount of 0.05 g / L to 1 g / L of the composition and / or in an amount of 0.04 g / 100 g to 0.8 g / 100 g of the composition on a dry weight basis.

[0170] In another embodiment, the combination or nutritional composition of the present invention comprises 6'SL and LNT, wherein:

[0171] - 6'SL in an amount of 0.04 g / L to 0.5 g / L of the composition and / or in an amount of 0.031 g / 100 g to 0.4 g / 100 g of the composition on a dry weight basis; and / or

[0172] - LNT in an amount of 0.09 g / L to 0.8 g / L of the composition and / or in an amount of 0.07 g / 100 g to 0.6 g / 100 g of the composition on a dry weight basis.

[0173] In a particular embodiment, the combination or nutritional composition of the present invention comprises 6'SL and LNT, wherein:

[0174] - 6'SL in an amount of 0.24 g / L composition or 0.05 g / L composition and / or on a dry weight basis in an amount of 0.18 g / 100 g composition or 0.04 g / 100 g composition; and / or

[0175] - LNT is present in an amount of 0.5 g / L of the composition or 0.1 g / L of the composition and / or on a dry weight basis in an amount of 0.38 g / 100 g of the composition or 0.08 g / 100 g of the composition.

[0176] In a particular embodiment, 6'SL is provided in the combination or nutritional composition of the invention in an amount such that normal consumption of the nutritional composition or growing-up milk will provide a total daily dose of 0.003g to 6.5g, preferably 0.005g to 3.3g or 0.006g to 1.3g or 0.02g to 0.9g, for example 0.024g to 0.7g per day to the infant, toddler or child consuming it.

[0177] In a particular embodiment, LNT is provided in the nutritional composition or growing-up milk of the present invention in an amount such that normal consumption of the nutritional composition or growing-up milk will provide a total daily dose of 0.003 g to 3.9 g, preferably 0.006 g to 2 g or 0.02 g to 1.6 g or 0.03 g to 1.3 g, for example 0.05 g to 1 g per day to the infant, toddler or child consuming it.

[0178] In one embodiment of the invention, the combination or nutritional composition comprises a mixture of HMOs consisting of or consisting essentially of LNT and 6'SL. It should be understood that when the expression "a mixture of HMOs consisting of or consisting essentially of LNT and 6'SL" is used in the context of the present invention, such expression does not include the presence of HMOs other than LNT and 6'SL in the mixture. In particular, the mixture is free of N-acetylated oligosaccharides, sialylated oligosaccharides, fucosylated oligosaccharides other than LNT and 6'SL, and precursors of HMOs (such as sialic acid or fucose). In some embodiments, the combination or nutritional composition is free of N-acetylated oligosaccharides, sialylated oligosaccharides, fucosylated oligosaccharides other than LNT and 6'SL, and precursors of HMOs (such as sialic acid or fucose).

[0179] In a preferred embodiment of the invention, the HMO mixture may be present in an amount of 0.15 g / L to 15 g / L of the composition, such as 0.5 g / L to 10 g / L or 0.75 g / L to 7.5 g / L or 1 g / L to 5 g / L or 1 g / L to 2 g / L of the composition. In a specific embodiment, the amount of the HMO mixture is 1.5 g / L of the composition. In a specific embodiment, the amount of the HMO mixture is 2 g / L of the composition. Such amounts are particularly sufficient when the nutritional composition is in the form of a complete nutrition, such as an infant formula, or in the case of a growing-up milk.

[0180] In the case where the combination or nutritional composition is in powder form, the HMO mixture may preferably be present in an amount of 0.11 g / 100 g to 11 g / 100 g composition, such as 0.4 g / 100 g to 7.5 g / 100 g or 0.6 g / L to 6 g / L or 0.8 g / 100 g to 3.8 g / 100 g or 0.8 g / 100 g to 1.5 g / 100 g composition, based on dry weight. In a specific embodiment, the amount of the HMO mixture is 1.1 g / 100 g composition, based on dry weight. Such amounts are particularly sufficient when the combination or nutritional composition is in the form of a complete nutrition, such as an infant formula, or in the case of a growing-up milk.

[0181] In another specific embodiment, the amount of the HMO mixture is 5 g / L to 500 g / L, 10 g / L to 400 g / L, 40 g / L to 300 g / L, 60 g / L to 200 g / L, 80 g / L to 180 g / L, 100 g / L to 150 g / L, or 110 g / L to 130 g / L of the composition. In a specific embodiment, the amount of the HMO mixture is 120 g / L. Such amounts are particularly sufficient when the nutritional composition is in the form of a supplement or fortifier.

[0182] When the supplement or fortifier is in powder form, the HMO mixture is preferably provided in the nutritional composition of the present invention in an amount of 0.05g to 5g, 0.1g to 4.5g, 0.15g to 4g, 0.2g to 3.5g, 0.25g to 3g, 0.3g to 2.5g, 0.35g to 2g, 0.4g to 1.5g, 0.45g to 1g, 0.5g to 0.75g, for example 0.6g per serving.

[0183] In a preferred embodiment of the invention the nutritional composition is a synthetic nutritional composition.

[0184] In a preferred embodiment of the invention the HMO mixture of the combination or nutritional composition consists of 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'-SL).

[0185] In a preferred embodiment of the invention, in the combination or in the (synthetic) nutritional composition, the HMOs provided by a HMO mixture consisting of or essentially consisting of 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'-SL) represents 95 to 100 wt.-%, preferably 97 to 100 wt.-%, more preferably 99 to 100 wt.-%, most preferably 99.5 to 100 wt.-%, such as 99.9 to 100 wt.-% of the total amount of HMOs in the combination or the (synthetic) nutritional composition.

[0186] In a preferred embodiment of the present invention the combination or (synthetic) nutritional composition does not comprise any other HMO than the HMO provided by the HMO mixture consisting of 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'-SL).

[0187] In one embodiment, the HMO mixture is provided in the combination or nutritional composition of the invention in an amount such that normal consumption of the nutritional composition will provide a total daily dose of 0.1 g to 10 g, such as 0.2 g to 9 g, 0.3 g to 8 g, 0.4 g to 7 g, 0.5 g to 6 g, 0.6 g to 5 g, 0.8 g to 3 g, 0.9 g to 2 g or 1 g to 1.5 g per day to an infant or toddler (respectively child) consuming it.

[0188] When GOS, 2'FL and DFL are combined, all three oligosaccharides are believed to be responsible for the effects of, for example, treating and / or preventing stress and / or mood disorders.

[0189] When GOS, LNT and 6'SL are combined, all three oligosaccharides are believed to be responsible for the effects of, for example, treating and / or preventing stress and / or mood disorders.

[0190] Mixtures of HMOs as described herein are used in conjunction with GOS.

[0191] In a specific embodiment, GOS are provided in the combination or nutritional composition of the present invention in an amount such that a serving of the combination or nutritional composition will provide a total dose of 0.003 g to 3.9 g to an infant, toddler, or child consuming it, preferably 0.006 g to 3 g or 0.05 g to 2.5 g or 0.1 g to 2 g or 0.2 g to 1.5 g, e.g., 0.36 g to 1.22 g per serving. Suitably, GOS are provided in the combination or nutritional composition of the present invention in an amount such that a serving of the combination or nutritional composition will provide a total dose of 0.001 g, 0.005 g, 0.01 g, 0.005 g, 0.1 g, 0.5 g, 1 g, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, or 4 g per serving, or any range formed therefrom, to an infant, toddler, or child consuming it.

[0192] Other ingredients

[0193] The nutritional composition according to the present invention may also contain other types of oligosaccharides (i.e., in addition to the human milk oligosaccharides mentioned above) and / or their fibers and / or their precursors. Other oligosaccharides and / or their fibers and / or precursors may be selected from a list comprising the following items: HMO, oligofructose (FOS), inulin, oligoxylose (XOS), polydextrose and any combination thereof. Their amount may be between 0% and 10% by weight of the composition. In a specific embodiment, the nutritional composition or growing-up milk may also contain at least one BMO (bovine milk oligosaccharide).

[0194] The additional HMO that may be included in the combination or nutritional composition according to the present invention may be selected from the group consisting of: 3-FL (3-fucosyllactose), lactosyldifucotetraose (LDFT), lactosyl-N-fucopentaose (e.g. lactosyl-N-fucopentaose I, lactosyl-N-fucopentaose II, lactosyl-N-fucopentaose III, lactosyl-N-fucopentaose V), lactosyl-N-fucohexaose, lactosyl-N-difucohexaose I, fucosyllactose-N-hexaose, fucosyllactose-N-neohexose, fucosyllactose-N-neohexose, fucosyllactose-N-hexa ... Sugar, difucosyllactose-N-hexaose I, difucosyllactose-N-neohexose II, p-lactose-N-neohexose (p-LNnH), LNnT (lactose-N-neotetraose), lactose-N-hexaose, lactose-N-neohexose, p-lactose-N-hexaose, p-lactose-N-neohexose, p-lactose-N-neohexose, lactose-N-octaose, lactose-N-neooctaose, iso-lactose-N-octaose, p-lactose-N-octaose, lactose-N-decasose, 3-SL (3' sialyllactose) and any combination thereof.

[0195] In some embodiments, the combination or nutritional composition according to the invention comprises at least one additional HMO. In other embodiments, the combination or nutritional composition according to the invention does not contain any additional HMO.

[0196] The nutritional composition of the present invention may further comprise at least one probiotic (or probiotic strain), such as a probiotic bacterial strain.

[0197] The most commonly used probiotic microorganisms are mainly most bacteria and yeasts of the following genera: Lactobacillus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp.

[0198] In some embodiments, the probiotics are probiotic bacterial strains. In some embodiments, they are specifically bifidobacteria and / or lactobacilli.

[0199] Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103 available from Valio Oy, Finland under the trademark LGG, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM 1-2116, Lactobacillus johnsonii CNCM 1-1225, Streptococcus salivarius DSM 13084 sold under the trade name KI2 by BLIS Technologies Limited, New Zealand, Bifidobacterium lactis CNCM 1-3446 sold specifically under the trademark Bb 12 by Christian Hansen company, Denmark, and Lactobacillus johnsonii CNCM 1-1225 sold under the trade name KI2 by Morinaga Milk Industry Co., Ltd., Japan. Co. Ltd., Japan), Bifidobacterium longum ATCC BAA-999 sold under the trademark BB536 by Danisco, Bifidobacterium breve sold under the trademark Bb-03 by Danisco, Bifidobacterium breve sold under the trademark M-16V by Morinaga, Bifidobacterium infantis sold under the trademark Bifantis by Procter & Gamble Co., and Bifidobacterium breve sold under the trademark R0070 by Institut Rosell-Lallemand in Canada.

[0200] The nutritional composition according to the invention may contain 10e3 to 10e12 cfu of the probiotic strain, more preferably between 10e7 and 10e12 cfu, such as between 10e8 and 10e10 cfu of the probiotic strain per g of composition based on dry weight.

[0201] In one embodiment, the probiotics are live. In another embodiment, the probiotics are non-replicating or inactivated. In some other embodiments, live and inactivated probiotics may be present simultaneously. Probiotic components and metabolites may also be added.

[0202] The nutritional composition according to the present invention may be, for example, an infant formula, a first stage infant formula, a larger or second stage infant formula, a growing-up milk, a baby food, an infant cereal composition, a fortifier (such as a human milk fortifier) ​​or a supplement. In some specific embodiments, the composition of the present invention is an infant formula, a fortifier or a supplement intended for the first 4 months or 6 months of age. In a preferred embodiment, the nutritional composition of the present invention is an infant formula.

[0203] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (eg, a human milk fortifier) ​​or a formula fortifier (such as an infant formula fortifier or a follow-on / second infant formula fortifier).

[0204] When the nutritional composition is a supplement, it may be provided in the form of a unit dosage. In such cases, it is particularly useful to define the amount of 2'FL and optionally other oligosaccharides according to the daily dose to be administered to an infant or young child, such as described above.

[0205] When the nutritional composition is a supplement, it may contain 2'-FL and no other additional nutrients necessary to obtain a stable nutritional composition in the top layer of excipients.

[0206] The nutritional composition of the present invention may be in solid (e.g., powder), liquid or gel form. In a particular embodiment, the nutritional composition is a supplement comprising 2'-fucosyllactose (2'-FL), wherein the supplement is in powder form and is provided in sachets, preferably the sachets have 0.1 g / sachet to 20 g / sachet, e.g., 1 g / sachet to 10 g / sachet of 2'-fucosyllactose (2'-FL), or is provided in syrup form, preferably the syrup has a total solid concentration of 5 g / 100 mL to 75 g / 100 mL (5% to 75% (w / v)). When the supplement is in powder form, it may contain a carrier. However, it is preferred that the supplement does not contain a carrier. When the supplement is in syrup form, the HMO is preferably dissolved or suspended in water acidified with citrate.

[0207] The nutritional composition according to the present invention generally contains a protein source. The amount of protein may be 1.6 g / 100 kcal to 3 g / 100 kcal. In some embodiments, particularly when the composition is intended for premature infants, the amount of protein may be between 2.4 g / 100 kcal and 4 g / 100 kcal or more than 3.6 g / 100 kcal. In some other embodiments, the amount of protein may be less than 2.0 g / 100 kcal, for example, 1.8 g / 100 kcal to 2 g / 100 kcal, or less than 1.8 g / 100 kcal.

[0208] Protein sources based on whey, casein and mixtures thereof may be used, as well as soy based protein sources. As far as whey protein is concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof, and may contain alpha-lactalbumin and beta-lactoglobulin in any desired ratio.

[0209] In some advantageous embodiments, the protein source is predominantly whey (ie, greater than 50% of the protein is derived from whey protein, such as 60% or 70%).

[0210] The protein may be intact or hydrolyzed, or a mixture of intact and hydrolyzed proteins. The term "intact" means that the major part of the protein is intact, i.e. the molecular structure is not changed, for example at least 80% of the protein is not changed, such as at least 85% of the protein is not changed, preferably at least 90% of the protein is not changed, even more preferably at least 95% of the protein is not changed, such as at least 98% of the protein is not changed. In a specific embodiment, 100% of the protein is not changed.

[0211] The term "hydrolyzed" means that in the context of the present invention, the protein has been hydrolyzed or broken down into its constituent amino acids. The protein can be fully hydrolyzed or partially hydrolyzed. For example, for infants or young children who are considered to be at risk of developing cow's milk allergy, it may be desirable to provide partially hydrolyzed proteins (the degree of hydrolysis is between 2% and 20%). If hydrolyzed protein is required, a hydrolysis process can be performed as required and as known in the art. For example, a whey protein hydrolyzate can be prepared by enzymatic hydrolysis of a whey fraction in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it is found that the protein undergoes much less lysine blockage during the hydrolysis process. This makes it possible to reduce the degree of lysine blocking from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which greatly improves the nutritional quality of the protein source.

[0212] In one embodiment of the invention, at least 70% of the protein is hydrolyzed, preferably at least 80% of the protein is hydrolyzed, such as at least 85% of the protein is hydrolyzed, even more preferably at least 90% of the protein is hydrolyzed, such as at least 95% of the protein is hydrolyzed, in particular at least 98% of the protein is hydrolyzed. In a specific embodiment, 100% of the protein is hydrolyzed.

[0213] In a particular embodiment, the protein of the nutritional composition is hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be between 8 and 40, or between 20 and 60, or between 20 and 80, or more than 10, 20, 40, 60, 80 or 90.

[0214] Alternatively, the protein component may be replaced with a mixture or synthetic amino acids, for example for premature or low birth weight infants.

[0215] In a particular embodiment, the nutritional composition or growing-up milk according to the invention is a hypoallergenic composition.In another particular embodiment, the composition according to the invention is a hypoallergenic nutritional composition or a growing-up milk.

[0216] The nutritional composition according to the invention typically contains a carbohydrate source. This is particularly preferred in the case where the nutritional composition according to the invention is an infant formula. In this case, any carbohydrate source typically present in infant formulas may be used, such as lactose, sucrose, saccharose, maltodextrin, starch and mixtures thereof, but one of the preferred carbohydrate sources is lactose.

[0217] Nutritional compositions according to the present invention generally contain a lipid source. This is particularly relevant when the nutritional composition of the present invention is an infant formula. In this case, the lipid source may be any lipid or fat suitable for infant formula. Some suitable fat sources include palm oil, structured triglyceride oils, high oleic sunflower oil and high oleic safflower oil, medium chain triglyceride oils. Essential fatty acids linoleic acid and alpha-linolenic acid may also be added, as well as a small amount of oil containing a large amount of preformed arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oil. The ratio of n-6 fatty acids to n-3 fatty acids in the fat source may be from about 5:1 to about 15:1, for example from about 8:1 to about 10:1.

[0218] The nutritional composition of the present invention may also contain all vitamins and minerals that are considered to be necessary for daily diet and in a significant amount of nutrition. The minimum requirements of some vitamins and minerals have been determined. The examples of minerals, vitamins and other nutrients optionally present in the composition of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in salt form. The presence and amount of specific minerals and other vitamins will vary according to the target population.

[0219] If necessary, the nutritional composition of the present invention may contain an emulsifier and a stabilizer such as soybean, lecithin, citric acid monoglyceride, citric acid diglyceride, and the like.

[0220] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, nucleosides, etc.

[0221] The nutritional composition of the present invention may further contain carotenoids. In some embodiments of the present invention, the nutritional composition of the present invention does not contain any carotenoids.

[0222] The nutritional composition according to the invention may be prepared in any suitable manner. The composition will now be described by way of example.

[0223] For example, formula foods such as infant formula foods can be prepared by blending a protein source, a carbohydrate source, and a fat source in appropriate proportions. If used, an emulsifier can be added at this time. Vitamins and minerals can be added at this time, but they are usually added later to avoid thermal degradation. Before blending, any lipophilic vitamins, emulsifiers, etc. can be dissolved in the fat source. Then water (preferably water subjected to reverse osmosis) can be mixed to form a liquid mixture. The water temperature is suitably in the range of about 50°C to about 80°C to help disperse the ingredients. Commercially available liquefiers can be used to form a liquid mixture. In particular, if the final product is in liquid form, fucosylated oligosaccharides and N-acetylated oligosaccharides can be added at this stage. If the final product is a powder, these ingredients can also be added at this stage as needed.

[0224] The liquid mixture is then homogenized, for example in two stages.

[0225] The liquid mixture may then be heat treated to reduce the bacterial load, for example by rapidly heating the liquid mixture to a temperature in the range of about 80°C to about 150°C for a duration between about 5 seconds and about 5 minutes. This may be performed by steam injection, an autoclave, or a heat exchanger (e.g., a plate heat exchanger).

[0226] The liquid mixture is then cooled to between about 60°C and about 85°C, for example by rapid cooling. The liquid mixture is then homogenized again, for example in two stages, with a pressure of between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenized mixture can then be further cooled to add any heat-sensitive components, such as vitamins and minerals. The pH and solids content of the homogenized mixture are conveniently adjusted at this point.

[0227] If the final product is to be a powder, the homogenized mixture is transferred to a suitable drying device, such as a spray dryer or freeze dryer and converted to a powder. The moisture content of the powder should be less than about 5% by weight. Fucosylated oligosaccharides and N-acetylated oligosaccharides may also or alternatively be added at this stage by dry mixing them with the probiotic strain (if used), or by blending them with the probiotic strain in the form of a crystalline syrup and then spray drying or freeze drying the mixture.

[0228] If a liquid composition is preferred, the homogenised mixture may be sterilised and then filled into suitable containers under aseptic conditions or filled into containers first and then retorted.

[0229] In another embodiment, the composition of the present invention can be a supplement. The supplement can be in the form of, for example, a tablet, a capsule, a lozenge or a liquid. The supplement can also contain a protective hydrophilic colloid (such as a gum, a protein, a modified starch), a binder, a film former, an encapsulating agent / material, a wall / shell material, a matrix compound, a coating, an emulsifier, a surfactant, a solubilizer (oils, fats, waxes, lecithins, etc.), an adsorbent, a carrier, a filler, a co-compound, a dispersant, a wetting agent, a processing aid (solvent), a flow agent, a taste masking agent, a weighting agent, a gelling agent and a gel forming agent. The supplement can also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including but not limited to: water, gelatin from any source, vegetable gum, lignin sulfonate, talc, sugars, starch, gum arabic, vegetable oil, polyalkylene glycol, flavoring agent, preservative, stabilizer, emulsifier, buffer, lubricant, colorant, wetting agent, filler, etc.

[0230] Additionally, supplements may contain organic or inorganic carrier materials suitable for oral or parenteral administration, as well as vitamins, minerals trace elements and other micronutrients as recommended by government agencies such as the USRDA.

[0231] Example

[0232] Example 1

[0233] Table 1 below gives an example of the composition of an infant formula according to the invention. The composition is given by way of example only.

[0234] Table 1 : Example 1 Composition of infant formula

[0235]

[0236]

[0237] Example 2

[0238] method

[0239] In vitro digestion and fermentation

[0240] According to the standardized INFOGEST 2.0 method, the milk matrix (standard milk follow-on formula) was subjected to oral, gastric and small intestinal digestion procedures (Brodkorb et al., Nature Protocols, 14: 991–1014 (2019)). In vitro fermentation was then achieved for 24 hours in a reactor containing the following items:

[0241] -10% digested milk matrix

[0242] - 80% fermentation medium containing microbiota derived from fresh stool samples recovered from 3 healthy toddlers aged 3.2 to 3.3 years

[0243] -10% of the test composition consisting of

[0244] ○ Buffer only

[0245] ○ The final concentration of galacto-oligosaccharide was 2 g / L

[0246] ○ A blend of 2'FL and DFL (ratio 9:1) with a final concentration of 2 g / L

[0247] ○ A blend of LNT and 6'SL with a final concentration of 2 g / L (ratio 2:1),

[0248] ○ Combination of (i) GOS and (ii) a blend of 2'FL and DFL (ratio 9:1), both at a final concentration of 2 g / L

[0249] ○ Combination of (i) GOS and (ii) a blend of LNT and 6'SL (ratio 2:1), both at a final concentration of 2 g / L

[0250] Each combination was tested in triplicate. After 24 h, the fermentation medium was collected for metabolomics analysis.

[0251] Metabolomics

[0252] Non-targeted LC-MS / MS was performed using a Thermo Scientific Vanquish LC coupled to a Thermo Q Exactive HF MS. An electrospray ionization interface was used as the ionization source. Analyses were performed in negative and positive ion modes. The ionization profile was determined using the method described by Doneanu et al. (Waters application note (2011) https: / / www.waters.com / webassets / cms / library / docs / 720004042en.pdf ) was performed using a slightly modified version of the protocol described by . Peak areas were extracted using Compound Discoverer 3.1 (Thermo Scientific). In addition to the automated compound extraction by Compound Discoverer 3.1, manual extraction of compounds contained in the in-house library was performed using Skyline 21.1 (MacCoss Lab Software).

[0253] For each blend B, we calculated log2(GABA(B) / GABA(milk)), ie the logarithm to base 2 of the ratio between the GABA value of the blend and the GABA value of milk.

[0254] Statistical analysis

[0255] Mixed linear models with random intercepts were used to assess the effects of the blends on the levels of each metabolite.

[0256] Goodness of fit was assessed using marginal and conditional R2. Marginal R2 (marginal coefficient of determination) represents the variance explained by the fixed effects, while conditional R2 is interpreted as the variance explained by the entire model, including both fixed and random effects. In addition, we examined the standardized residuals for each model to check whether outliers in the data were of potential concern.

[0257] The ratio of the inter-cluster variance to the total variance is called the intraclass correlation coefficient (ICC) and is typically between 0.6 and 0.8. This can also be interpreted as the correlation between observations within the same cluster (donor).

[0258] P values ​​calculated approximately using the Kenward-Roger method were used to determine which blends were significantly associated with the outcome.

[0259] result

[0260] Exposure of the microbiome to digested milk matrix in the presence of GOS, a blend of 2'FL and DFL or a blend of LNT and 6'SL did not trigger any effect on GABA abundance ( Figure 1In contrast, the presence of a blend containing GOS plus HMO (2'FL / DFL or LNT / 6'SL) potently increased GABA synthesis by the microbiota compared to the individual components ( Figure 2 ). The amount of GABA secreted when exposed to GOS+2'FL / DFL was higher than that when exposed to GOS+LNT / 6'SL.

Claims

1. A combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs) for use in the treatment and / or prevention of stress and / or mood disorders in infants, toddlers and / or children, wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

2. A nutritional composition for the treatment and / or prevention of stress and / or mood disorders in infants, toddlers and / or children, the nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'SL).

3. The combination for use according to claim 1 or the nutritional composition for use according to claim 2, wherein the combination or the nutritional composition maintains or restores the functionality of the bidirectional transmission pathway in the gut-brain axis to a healthy level.

4. The combination for use according to claim 1 or 3 or the nutritional composition for use according to claim 2 or 3, wherein the combination or the nutritional composition increases the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of the infant, toddler and / or child and thereby treats stress and / or mood disorders in the infant, toddler and / or child.

5. The combination for use according to any one of claims 1, 3 or 4, or the nutritional composition for use according to any one of claims 2 to 4, wherein the mood disorder is anxiety or depression.

6. The nutritional composition for use according to any one of claims 2 to 5, wherein the GOS are present in an amount of 2.4 g / L or 1.1 g / 100 g to 2.8 g / 100 g of the composition on a dry weight basis.

7. The combination for use according to any one of claims 1 or 3 to 5, or the nutritional composition for use according to any one of claims 2 to 6, wherein the mixture of HMOs consists of or consists essentially of 2'FL and DFL.

8. The combination or nutritional composition for use according to claim 7, wherein the ratio of 2'-FL to DFL is from 6:1 to 10:1 by weight.

9. The combination for use according to any one of claims 1 or 3 to 5, or the nutritional composition for use according to any one of claims 2 to 6, wherein the mixture of HMOs consists or consists essentially of LNT and 6'SL.

10. The combination or nutritional composition for use according to claim 9, wherein the ratio of LNT to 6'-SL by weight is from 2:1 to 1:

1.

11. The nutritional composition for use according to any one of claims 2 to 10, wherein: (i) a) 2'FL is present in an amount from 0.005 g / L to 8 g / L or from 0.004 g / 100 g to 6.8 g / 100 g of the composition on a dry weight basis; and / or b) wherein DFL is present in an amount from 0.1 g / L to 0.3 g / L or from 0.08 g / 100 g to 0.2 g / 100 g of the composition on a dry weight basis; or (ii) a) LNT is present in an amount from 0.005 g / L to 3 g / L or from 0.004 g / 100 g to 2.3 g / 100 g of the composition on a dry weight basis; and / or b) 6'SL is present in an amount of 0.005 g / L to 5 g / L, or 0.004 g / 100 g to 3.8 g / 100 g of the composition on a dry weight basis.

12. The nutritional composition according to any one of claims 2 to 11, wherein the nutritional composition further comprises a protein source, a carbohydrate source and a lipid source.

13. The nutritional composition according to any one of claims 2 to 12, wherein the nutritional composition further comprises at least one probiotic, the amount of the probiotic being 10 3 cfu / g to 10 12 cfu / g of the composition (dry weight).

14. The nutritional composition according to any one of claims 2 to 13, wherein the nutritional composition is an infant formula, a first stage infant formula, a follow-on or second stage infant formula, a growing-up milk, a baby food, an infant cereal composition, a fortifier or a supplement.

15. The combination for use according to any one of claims 1 or 3 to 5 or 7 to 10, or the nutritional composition for use according to any one of claims 2 to 14, wherein the infant is a preterm infant, a low birth weight infant and / or an infant born small for gestational age (SGA).

16. A method for treating and / or preventing stress and / or mood disorders in infants, toddlers and / or children, the method comprising administering to the infant, toddler and / or child an effective amount of the combination as defined in any one of claims 1 or 3 to 5 or 7 to 10 or 15 or an effective amount of the nutritional composition as defined in any one of claims 2 to 15.

17. A method for maintaining or restoring the functionality of bidirectional transport pathways in the gut-brain axis of an infant, toddler and / or child to a healthy level, the method comprising administering to the infant, toddler and / or child an effective amount of the combination as defined in any one of claims 1 or 3 to 5 or 7 to 10 or 15 or an effective amount of the nutritional composition as defined in any one of claims 2 to 15.

18. A method for increasing the production of gamma-aminobutyric acid (GABA) by the intestinal microbiota of an infant, toddler and / or child, the method comprising administering to the infant, toddler and / or child an effective amount of the combination as defined in any one of claims 1 or 3 to 5 or 7 to 10 or 15 or an effective amount of the nutritional composition as defined in any one of claims 2 to 15.

19. The method of claim 18, wherein the method is non-therapeutic.

20. A nutritional composition comprising a combination of a mixture of galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs), wherein the mixture of HMOs consists of or consists essentially of 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL) or lacto-N-tetraose (LNT) and 6'-sialyllactose (6'-SL), wherein the nutritional composition is an infant formula, a first infant formula, a follow-on or second infant formula, a growing-up milk, a baby food, an infant cereal composition, a fortifier suitable for infants, toddlers and / or children, or a supplement suitable for infants, toddlers and / or children.

21. The nutritional composition of claim 20, wherein the mixture of HMOs consists of or consists essentially of 2'-FL and DFL.

22. The nutritional composition of claim 21, wherein the ratio of 2'-FL to DFL is from 6:1 to 10:1 by weight.

23. The nutritional composition of claim 20, wherein the mixture of HMOs consists of or consists essentially of LNT and 6'-SL.

24. A combination or composition according to claim 23, wherein the ratio of LNT to 6'-SL by weight is from 2:1 to 1:1.

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