RU2808968C2 - Nutrient composition containing combination of breast milk oligosaccharides to improve gastrointestinal tract barrier - Google Patents

Abstract

FIELD: nutritional compositions.

SUBSTANCE: invention relates to nutritional compositions for infants or young children. The nutritional composition contains 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL) and is used to improve the gastrointestinal barrier in the infant, child under 12 months of age, and young child from 3 years to less than 8 years.

EFFECT: said improvement of the gastrointestinal tract barrier represents an improved barrier structure and/or improved barrier protection.

9 cl, 3 dwg, 1 tbl, 2 ex

Description

Scope of the invention

The present invention relates to nutritional compositions containing 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL), for use in improving the gastrointestinal barrier. In particular, the present invention relates to improving the gastrointestinal tract barrier in an infant, young child or children aged 3 to 8 years.

Prerequisites for creating the invention

Mother's milk is recommended for all babies. However, in some cases, breastfeeding is insufficient or ineffective for medical reasons, or the mother decides not to breastfeed. Infant formulas have been developed for these situations. Fortifiers have also been developed to fortify mother's milk or infant formula with certain ingredients. In such cases, it would be even more preferable to provide a means of improving the gastrointestinal barrier in infants and young children through nutritional intervention, such as nutritional supplementation or nutritional supplementation.

During postnatal development, the newborn's intestine undergoes a maturation process that culminates in the formation of a functional barrier to macromolecules and pathogenic bacteria. This phenomenon is called intestinal closure and appears to be dependent on diet. In this context, various studies of infants (JPGN, 1995, 21: 383-6) and animal models (Pediatr Res, 1990, 28: 31-7) indicate that barrier maturation occurs more rapidly in infants with breastfeeding than with formula feeding . This may explain the higher prevalence of allergies, inflammation and infection in formula-fed infants than in breast-fed infants.

Certain specific groups of infants and young children are particularly in need of compositions capable of providing health benefits, such as improving intestinal barrier function. Such infants and young children include, for example, premature infants, low birth weight infants, and/or infants or young children with growth restriction. Indeed, the intestinal barrier is more permeable and more susceptible to damage, and its structure and function in these infants is less mature than in a healthy full-term infant. This, in turn, can lead to other problems such as infection, inflammation or allergies. For such infants, it is particularly preferable to supplement pharmacological treatment with nutritional compositions capable of improving intestinal barrier function.

The effects of dietary ingredients, such as human milk oligosaccharides, on improving the gastrointestinal barrier have been previously investigated.

A variety of human milk oligosaccharides have been described as beneficial to the gastrointestinal barrier in WO2013/032674 (gastrointestinal barrier function), WO2017/144062 (maintaining or restoring gastrointestinal barrier permeability through microbiota), WO2017/71716 (treatment of impairment mucosal barrier function), WO2017/046711 (treatment of mucosal barrier dysfunction), WO2016/91265 (reducing intestinal permeability), WO2016/66174 (maintaining mucosal integrity).

However, an improved nutritional composition, in particular a more effective and/or reliable nutritional composition for preterm infants, would be advantageous.

It would be useful to further optimize the effect of nutritional compositions on the gastrointestinal barrier in all infants and children.

There is a clear need to develop acceptable methods for improving the gastrointestinal barrier in infants and young children.

There is also a need to provide such health benefits in a manner that is particularly acceptable to young subjects (infants and young children), which does not involve classical pharmaceutical intervention, since these infants or young children are particularly vulnerable.

There is a need to provide these health benefits to these infants or young children in a manner that does not cause side effects and/or that can be easily provided and acceptable to parents or healthcare professionals.

There is also a need to provide such beneficial effects in a manner that keeps the cost of such provision reasonable and affordable to most.

Thus, there is a clear need to develop alternative methods other than classical pharmaceutical interventions such as the use of pharmaceutical drugs, at least due to the associated risk of side effects.

Summary of the invention

The present invention relates to nutritional compositions containing 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL), for use in improving the gastrointestinal barrier. The effect is confirmed by the results of an in vitro study on a model of intestinal epithelial cells. As described in the examples section, a mixture of 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL was found to be particularly effective in improving the gastrointestinal barrier.

Thus, it is an object of the present invention to provide nutritional compositions that improve the gastrointestinal tract barrier, eg, improve barrier maturation, improve barrier protection, improve barrier structure, improve barrier function, and/or improve barrier repair. All such aspects of the gastrointestinal barrier are interrelated. Proper maturation of the gastrointestinal tract results in proper gastrointestinal structure and function, which in turn is maintained by proper barrier protection and restored by proper barrier repair. Thus, any of these aspects, considered together or separately, contribute to a sustained reduction in susceptibility to disease.

Thus, one aspect of the invention relates to a nutritional composition comprising 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL, for use in improving the gastrointestinal tract barrier in an infant (child under 12 months of age) or young child (from 1 year to less than 3 years); or to a nutritional composition in the form of a milk formula for children from 1 to 3 years old, containing 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL, for use in improving the gastrointestinal tract barrier in a child (aged from 3 years to less than 8 years).

In a preferred embodiment of the invention, said gastrointestinal barrier improvement is improved barrier protection, improved barrier structure and improved barrier function, preferably improved barrier protection and/or improved barrier repair.

Brief description of graphic materials

In fig. Figure 1 shows the effectiveness of OGM6 in providing preventive protection of the epithelial barrier. Co-cultures are treated with OGM and ion permeability is measured prior to the induction of cytokine-mediated inflammation. Ion permeability is calculated by analyzing the change in transepithelial electrical resistance (TEER) over time relative to baseline (time 0 before addition of OGM6) before inflammatory challenge. Error bars represent SD.

In fig. Figure 2 shows the effectiveness of OGM6 in providing preventive protection of the epithelial barrier. Co-cultures are treated with an equal concentration of OGM (60 mg/ml) and ion permeability is measured before induction of cytokine-mediated inflammation. Ion permeability is calculated by analyzing the change in transepithelial electrical resistance (TEER) over time relative to baseline (time 0 before the addition of OGM) before the inflammatory challenge. Error bars represent SD.

In fig. Figure 3 shows the effectiveness of OGM6 in reducing the severity of symptoms of epithelial barrier dysfunction caused by inflammation. Co-cultures are treated with OGM6 and then use cytokine-mediated inflammation to induce epithelial barrier dysfunction. All groups, except the negative control (intact barrier), were provoked by an inflammatory process. The graph depicts the translocation of FITC-labeled dextran (FD4) from the apical to the basolateral compartment after inflammatory challenge compared to controls. Error bars represent SD.

The present invention will be described in more detail below.

Detailed Description of the Invention

Definitions

Before describing the present invention in more detail, the following terms and conventions will be defined.

The term "infant" means a child under 12 months of age (<12 months). The expression "young child" means a child aged one to less than three years (≥1 year to <3 years), also called a toddler. The expression “child” means a child aged three to less than eight years ((≥3 years to <8 years).

“Infant or young child born by cesarean section” means an infant or young child born by cesarean section. This means that the baby or young child was not born through vaginal delivery.

“Infant or young child born by vaginal delivery” means an infant or young child who was born by vaginal delivery rather than by cesarean section.

"Premature" or "premature" means an infant or young child who is born prematurely. Essentially, the term refers to an infant or young child born before the 37th week of pregnancy.

“Low birth weight infant” means a newborn weighing less than 2500 g (5.5 lb) due to either preterm birth or fetal growth restriction. Thus, this term includes:

- an infant or young child who has/had a birth weight between 1500 and 2500 g (commonly referred to as “low birth weight” or LBP);

- an infant or young child who has/had a birth weight between 1000 and 1500 g (commonly referred to as “very low birth weight” or VLBW);

- an infant or young child who has/had a body weight of less than 1000 g at birth (commonly referred to as “extremely low birth weight” or ELBW).

“Infant born small for gestational age (SGA)” means an infant whose birth weight is less than the 10th percentile for infants of the same gestational age.

The expression “nutritional composition” means a composition that is fed to a subject. The nutritional composition is typically for oral administration and typically includes a lipid or fat source and a protein source.

In a specific embodiment, the composition of the present invention is a hypoallergenic nutritional composition. The expression “hypoallergenic nutritional composition” means a nutritional composition that has a low likelihood of causing allergic reactions.

In a specific embodiment, the composition of the present invention is an “artificial nutritional composition.” The expression “artificial nutritional composition” means a formula produced by chemical and/or biological means that may be chemically identical to the naturally occurring formula found in mammalian milk (i.e., the artificial composition is not breast milk).

The expression "infant formula" as used in this document refers to a food product which is specifically intended for consumption by infants during the first months of life and which in itself meets the nutritional needs of this category of persons (Article 2(c) of European Commission Directive 91/ 321/EEC 2006/141/EC of 22 December 2006 on infant and complementary formulas). It also refers to a nutritional composition intended for use by infants as defined in the Codex Alimentarius (Codex STAN 72-1981) and Infant Specialties (including the article Food for Special Medical Purpose). The expression "infant formula" covers both "infant formula" and "follow-up formula" or "weaning formula".

“Follow-up formula” or “formula for complementary infants” is given starting from the 6th month. It constitutes the main liquid element in the gradually increasing variety of diets for this category of people.

The expression "infant food" means a food product specifically intended for consumption by infants or young children during the first years of life.

The expression “cereal-based infant composition” means a food product specifically intended for consumption by infants or young children during the first years of life.

The expression "formula for children aged 1 to 3 years" (or GUM) refers to a milk-based drink, usually with added vitamins and minerals, that is intended for infants or children.

The term "fortifier" refers to liquid or solid nutritional compositions suitable for fortification or admixture with breast milk, infant formula, infant formula for infants 1 to 3 years of age, or breast milk fortified with other nutrients. Accordingly, the fortifier of the present invention may be administered after being dissolved in human breast milk, infant formula, infant formula, or human breast milk fortified with other nutrients, or otherwise may be administered as a separate composition. When administered as a separate composition, the breast milk fortifier of the present invention may also be defined as a “supplement.” In one embodiment, the milk fortifier of the present invention is an additive.

The expression "weaning period" refers to the period during which mother's milk is replaced by other foods in the diet of an infant or young child.

The expressions “days/weeks/months/years” and “days/weeks/months/years after birth” can be used interchangeably.

The expression "improved gastrointestinal barrier" may include one or more of the following:

- More effective barrier repair, such as (but not limited to) restoration of gastrointestinal barrier integrity, such as repair of a ruptured barrier, decreased permeability after inflammatory challenge of the gastrointestinal mucosa, and mucosal repair.

- Enhanced barrier maturation, such as (but not limited to) barrier maturation and/or development in an infant, preferably a premature infant.

- Improved barrier structure, such as (but not limited to) gastrointestinal barrier strengthening, gastrointestinal barrier integrity, tight junction structure, and intestinal epithelial lining integrity.

- Improved barrier function, such as improving the resistance of the gastrointestinal barrier, reducing the permeability of the gastrointestinal barrier, such as reducing pathogens migrating from the intestine through the intestinal barrier, such as reducing commensal bacteria or bacterial components migrating from the intestine through the intestinal barrier , reducing allergens migrating from the intestine across the intestinal barrier, reducing toxic compounds migrating from the intestine across the intestinal barrier, and reducing susceptibility to disease.

- Enhanced barrier protection, such as (but not limited to) preventing barrier dysfunction, preventing barrier permeability, protecting tight junction structures, protecting the integrity of the intestinal epithelial lining.

In a preferred embodiment, improvement of the gastrointestinal barrier refers to the maturation of the gastrointestinal barrier.

“Mother's milk” refers to the mother's breast milk or colostrum.

An "oligosaccharide" is a saccharide polymer containing a small number (usually three to ten) simple sugars (monosaccharides).

The term "OGM" refers to the oligosaccharide(s) of breast milk. These carbohydrates are highly resistant to enzymatic hydrolysis, indicating that they may exhibit significant functions not directly related to their caloric content. Most notably, they have been shown to play a vital role in the early development of infants and young children, such as the development of the immune system. Many different types of HMOs have been found in breast milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine, forming numerous and varied linkages between them, resulting in the presence of a huge number of different oligosaccharides in breast milk - to date there have been More than 130 such structures have been identified. Almost all of them have a lactose moiety at their reducing end, while sialic acid and/or fucose (if present) occupy terminal positions at the non-reducing ends. OGMs can be acidic (eg, charged sialic acid-containing oligosaccharide) or neutral (eg, fucosylated oligosaccharide).

"Fucosylated oligosaccharide" is an oligosaccharide having a fucose moiety. It has a neutral character. Some examples are 2-FL (2'-fucosyllactose), 3-FL (3-fucosyllactose), difucosyllactose, lacto-N-fucopentaose (e.g. lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II and any combination of them. Although the authors do not wish to be bound by theory, it is believed that the fucosyl epitope of fucosylated oligosaccharides may act as a “trap” on the mucosal surface. Through a competitive effect, it may prevent and/or limit the action of pathogens that cause infections (viral or bacterial origin) or their secreted components (e.g., toxins), primarily by preventing their binding to natural ligands, and, without regard to any theory , it is believed that this will reduce the risk of infection/inflammation, and in particular the risk of infection and/or inflammation of the lower respiratory tract (LRT)/ear. Additionally, it is generally accepted that fucosylated oligosaccharides stimulate the growth and metabolic activity of specific symbiotic microbes, reducing the inflammatory response and creating an unfavorable environment for pathogens, thereby providing colonization resistance.

The expressions "fucosylated oligosaccharides containing a 2'-fucosyl epitope" and "2-fucosylated oligosaccharides" cover fucosylated oligosaccharides with a certain shape homology because they contain a 2'-fucosyl epitope; therefore, a certain homology of function can be expected. Although the authors do not wish to be bound by theory, the 2'-fucosyl epitope of these fucosylated oligosaccharides is believed to be particularly characteristic of pathogens (or their secreted components) that cause UTI and/or ear infections.

The expression "N-acetylated oligosaccharide(s)" covers both "N-acetyllactosamine" and "oligosaccharide(s) containing N-acetyllactosamine." They are neutral oligosaccharides containing an N-acetyllactosamine residue. Suitable examples are LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), and any combinations thereof. Other examples are lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neo-octaose, iso-lacto-N-octaose , para-lacto-N-octaose and lacto-N-decaose.

The expressions “at least one fucosylated oligosaccharide” and “at least one N-acetylated oligosaccharide” mean “at least one type of fucosylated oligosaccharide” and “at least one type of N-acetylated oligosaccharide”.

An "OGM precursor" is a key compound that is included in the production of OGM, such as sialic acid and/or fucose.

A "sialylated oligosaccharide" is an oligosaccharide containing a charged sialic acid, i.e. an oligosaccharide having a sialic acid residue. It is acidic in nature. Some examples are 3-SL (3'-sialyllactose) and 6-SL (6'-sialyllactose).

The nutritional composition of the present invention may be in solid form (eg, powder form) or liquid form. The amount of various ingredients (e.g. oligosaccharides) may be expressed as g/100 g of the composition on a dry matter basis if it is in solid form, such as a powder, or as a concentration in g/L of the composition if it is in liquid form. (the latter case also includes a liquid composition which can be prepared from a powder after being dissolved in a liquid such as milk, water, etc., for example, dissolved infant formula, or infant formula/follow-up formula, or milk formula for children from 1 to 3 years old, or a cereal product for baby food, or a fortifier, or an additive, or any other mixture intended for baby food).

The term “prebiotic” refers to a non-digestible carbohydrate that has a beneficial effect on the host by selectively stimulating the growth and/or activity of health-promoting 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).

The term “probiotic” means preparations of microbial cells or components of microbial cells that have 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. 1999:10 107–10). Microbial cells are essentially bacteria or yeast.

The term "CFU" should be understood as a colony-forming unit.

Unless otherwise stated, all percentages are by weight.

Additionally, in the context of the invention, the terms “comprising” or “comprises” do not exclude other possible elements. The composition of the present invention, including many embodiments described herein, may contain essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components or limitations described herein or otherwise, depending on needs, consist of or essentially consist of them.

Any reference to prior art in this specification should not be construed as an admission that such prior art is widely known or forms part of the general knowledge in the art.

A more detailed description of the invention will be presented below. It should be noted that various aspects, features, examples and embodiments described in this application may be compatible and/or combined with each other.

Nutritional composition

As also stated above. The present invention relates to nutritional compositions containing 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL for use in improving the gastrointestinal tract barrier. As described in the examples section, 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL have been found to be particularly effective in improving the gastrointestinal barrier. Thus, one aspect of the invention relates to a nutritional composition comprising 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL, for use in improving the gastrointestinal tract barrier in an infant (child under 12 months of age) or young child (from 1 year to less than 3 years);

or

to a nutritional composition in the form of a milk formula for children from 1 to 3 years old, containing 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL, for use in improving the gastrointestinal tract barrier in a child (aged 3 years to less than 8 years).

In one embodiment, said gastrointestinal barrier improvement is improved barrier maturation, improved barrier structure, improved barrier function, improved barrier protection, and/or improved barrier repair.

In another embodiment, said gastrointestinal barrier improvement is improved barrier function, improved barrier structure, and/or improved barrier protection.

In yet another embodiment, said gastrointestinal barrier improvement is improved barrier protection.

In yet another embodiment, the use is to improve the prevention of barrier dysfunction, the prevention of barrier permeability, the protection of the tight junction structure, and the protection of the integrity of the epithelial lining of the intestine. In a further embodiment, said prevention of barrier permeability is preventing the migration of pathogens, commensal bacteria, bacterial products, allergens and/or toxic compounds from the intestine into the body through the intestinal barrier. As stated in the Examples, a mixture of 2'FL, DiFL, LNT, LNnT, 3'SL and 6'SL was found to improve, in particular, gastrointestinal barrier protection and gastrointestinal barrier structure.

The protection and structure of the gastrointestinal tract barrier leads to improvement in the function of the gastrointestinal tract barrier, since the gastrointestinal tract barrier, which does not have proper structure and protection, loses its optimal function, namely as a result of inflammatory provocations.

Thus, in one embodiment, said use for improving gastrointestinal barrier protection is more specifically to improve gastrointestinal barrier strength, gastrointestinal barrier integrity, tight junction structure, and intestinal epithelial lining integrity.

In a further preferred embodiment, said use is to increase the resistance of the gastrointestinal tract barrier and to reduce the permeability of the gastrointestinal tract barrier. Preferably, said use is to reduce pathogens migrating from the intestine through the intestinal barrier, for example, reducing commensal bacteria migrating from the intestine through the intestinal barrier, reducing allergens migrating from the intestine through the intestinal barrier, reducing toxic compounds migrating from the intestine through the intestinal barrier, and reducing susceptibility to diseases.

In another embodiment, said use for improving gastrointestinal barrier structure is to increase the strength of the gastrointestinal barrier, increase the integrity of the gastrointestinal barrier, improve the tight junction structure, and improve the integrity of the intestinal epithelial lining.

The above described effects/benefits are preferably achieved in the small intestine.

In yet another embodiment, the nutritional composition is formulated for administration to an infant. In another embodiment, the infant is selected from the group consisting of preterm, small for gestational age and low birth weight infants, preferably the infant is preterm. The nutritional compositions of the invention are considered to be particularly beneficial for these types of infants because the reported beneficial effects are more important for these infants than for “normal” infants because the barrier is less mature and more permeable compared to a healthy full-term infant.

In one embodiment, the nutritional composition contains 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL) in an amount of at least 0.3 g/l, for example at least 0.4 g/l, for example 0.5 g/l. In one embodiment, the nutritional composition contains 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL) in an amount of at least 0.3 g/L, for example at least 0.4 g/L, for example 0.5 g/L of dissolved product if the nutritional composition is to be dissolved in water or any other liquid before use.

In one embodiment, the nutritional composition contains 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL) in an amount of at least 1.5 g/l, for example at least 2.0 g/l, for example 2.5 g/l. In one embodiment, the nutritional composition contains 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL) in an amount of at least 1.5 g/L, for example at least 2.0 g/L, for example 2.5 g/L dissolved product if the nutritional composition is to be dissolved in water or any other liquid before use.

In some embodiments, 2'FL may be present in an amount of 0.005-5 g/L of the composition, for example, 0.01-3 g/L, or 0.02-2 g/L, or 0.1-2.5 g/L l, or 0.15–2 g/l, or 0.25–1.9 g/l, or 0.75–1.65 g/l of the composition. In a specific embodiment, 2'FL is present in an amount of 1 g/L of the composition. In another specific embodiment, 2'FL is present in an amount of 0.25 or 0.26 g/L of the composition. In one embodiment, 2'FL is present in an amount of greater than 0.1 g/L and optionally less than 1 g/L, or greater than 0.2 and less than 0.8 g/L. In one embodiment, 2'FL is present in an amount of at least 0.1 g/L, at least 0.25, at least 0.26, at least 0.5, at least 0.7, at least at least 0.8, at least 1, at least 1.25, at least 1.5 or at least 2 g/l.

2'FL may be present in the nutritional composition in a total amount of 0.004-3.8 g/100 g of composition on a dry matter basis. 2'FL may be present in an amount of 0.008-2.3 g/100 g of the composition, for example 0.015-1.5 g/100 g, or 0.08-1.9 g/100 g, or 0.12-1. 5 g/100 g, or 0.15–1.5 g/100 g, or 0.19–1.5 g/100 g of composition. In a specific embodiment, 2'FL is present in an amount of 0.075 or 0.78 g/100 g of composition. In another specific embodiment, 2'FL is present in an amount of 0.2 g/100 g of composition. In a specific embodiment, 2'FL is present in an amount of at least 0.01 g/100 g, at least 0.02 g/100 g, at least 0.05 g/100 g, at least 0.1 g /100 g, at least 0.2 g/100 g, at least 0.25 g/100 g, at least 0.4 g/100 g, at least 0.5 g/100 g, at least at least 0.75 g/100 g, at least 0.9 g/100 g, at least 1 g/100 g, at least 1.5 g/100 g, at least 2 g/100 g or according at least 3 g/100 g of composition.

In a specific embodiment, 2'FL is contained in the nutritional composition of the present invention in such an amount that a normal intake of the nutritional composition would be provided for an infant or young child, or child, by consuming a total daily dose of 0.003-6.5 g, preferably 0.006- 3.9 g, for example, 0.012–2.6 g, per day. It is believed that a minimum amount of 2'FL is necessary to achieve a measurable, desired effect.

For preterm, low birth weight and small for gestational age infants, the daily dose of 2'FL is preferably 0.05 to 1 g/kg body weight per day, preferably 0.06 to 0.9 g, or 0.07 –0.8 g, or 0.08–0.7 g, or 0.09–0.6 g, or 0.1–0.5 g, or 0.2–0.4 g, most preferably 0. 34 g per kg body weight per day.

In some embodiments, DiFL may be present in an amount of 0.0005-0.5 g/L of the composition, for example, 0.001-0.3 g/L, or 0.002-0.2 g/L, or 0.01-0.25 g/l, or 0.015–0.2 g/l, or 0.025–0.19 g/l, or 0.075–0.165 g/l of the composition. In a specific embodiment, DiFL is present in an amount of 0.1 g/L of the composition. In another specific embodiment, DiFL is present in an amount of 0.025 or 0.026 g/L of the composition. In one embodiment, DiFL is present in an amount of greater than 0.01 g/L and optionally less than 0.1 g/L, or greater than 0.02 and less than 0.08 g/L. In one embodiment, DiFL is present in an amount of at least 0.01 g/L, at least 0.025, at least 0.026, at least 0.05, at least 0.07, at least 0.08, at least 0.1, at least 0.125, at least 0.15 or at least 0.2 g/l.

DiFL may be present in the nutritional composition in a total amount of 0.0004-0.38 g/100 g of composition on a dry matter basis. DiFL may be present in an amount of 0.0008-0.23 g/100 g of the composition, for example 0.0015-0.15 g/100 g, or 0.008-0.19 g/100 g, or 0.012-0.15 g /100 g, or 0.015–0.15 g/100 g, or 0.019–0.15 g/100 g of composition. In a specific embodiment, 2DiFL is present in an amount of 0.0075 or 0.078 g/100 g of composition. In another specific embodiment, DiFL is present in an amount of 0.02 g/100 g of composition. In a specific embodiment, DiFL is present in an amount of at least 0.001 g/100 g, at least 0.002 g/100 g, at least 0.005 g/100 g, at least 0.01 g/100 g, at least 0 .02 g/100 g, at least 0.025 g/100 g, at least 0.04 g/100 g, at least 0.05 g/100 g, at least 0.075 g/100 g, at least 0.09 g/100 g, at least 0.1 g/100 g, at least 0.15 g/100 g, at least 0.2 g/100 g or at least 0.3 g/100 g composition.

In some embodiments, LNnT may be present in an amount of 0.0005-0.5 g/L of the composition, for example, 0.001-0.3 g/L, or 0.002-0.2 g/L, or 0.01-0.25 g/l, or 0.015–0.2 g/l, or 0.025–0.19 g/l, or 0.075–0.165 g/l of the composition. In a specific embodiment, LNnT is present in an amount of 0.1 g/L of the composition. In another specific embodiment, LNnT is present in an amount of 0.025 or 0.026 g/L of the composition. In one embodiment, LNnT is present in an amount of greater than 0.01 g/L and optionally less than 0.1 g/L, or greater than 0.02 and less than 0.08 g/L. In one embodiment, LNnT is present in an amount of at least 0.01 g/L, at least 0.025, at least 0.026, at least 0.05, at least 0.07, at least 0.08, at least 0.1, at least 0.125, at least 0.15 or at least 0.2 g/l.

LNnT may be present in the nutritional composition in a total amount of 0.0004-0.38 g/100 g of composition based on dry matter weight. LNnT may be present in an amount of 0.0008-0.23 g/100 g of the composition, for example 0.0015-0.15 g/100 g, or 0.008-0.19 g/100 g, or 0.012-0.15 g /100 g, or 0.015–0.15 g/100 g, or 0.019–0.15 g/100 g of composition. In a specific embodiment, LNnT is present in an amount of 0.0075 or 0.078 g/100 g of composition. In another specific embodiment, LNnT is present in an amount of 0.02 g/100 g of composition. In a specific embodiment, LNnT is present in an amount of at least 0.001 g/100 g, at least 0.002 g/100 g, at least 0.005 g/100 g, at least 0.01 g/100 g, at least 0 .02 g/100 g, at least 0.025 g/100 g, at least 0.04 g/100 g, at least 0.05 g/100 g, at least 0.075 g/100 g, at least 0.09 g/100 g, at least 0.1 g/100 g, at least 0.15 g/100 g, at least 0.2 g/100 g or at least 0.3 g/100 g composition.

In some embodiments, 3SL may be present in an amount of 0.0005-0.5 g/L of the composition, for example, 0.001-0.3 g/L, or 0.002-0.2 g/L, or 0.01-0.25 g/l, or 0.015–0.2 g/l, or 0.025–0.19 g/l, or 0.075–0.165 g/l of the composition. In a specific embodiment, 3SL is present in an amount of 0.1 g/L of the composition. In another specific embodiment, 3SL is present in an amount of 0.025 or 0.026 g/L of the composition. In one embodiment, 3SL is present in an amount of greater than 0.01 g/L and optionally less than 0.1 g/L, or greater than 0.02 and less than 0.08 g/L. In one embodiment, 3SL is present in an amount of at least 0.01 g/L, at least 0.025, at least 0.026, at least 0.05, at least 0.07, at least 0.08, at least 0.1, at least 0.125, at least 0.15 or at least 0.2 g/l.

3SL may be present in the nutritional composition in a total amount of 0.0004-0.38 g/100 g of composition on a dry matter basis. 3SL may be present in an amount of 0.0008-0.23 g/100 g of the composition, for example 0.0015-0.15 g/100 g, or 0.008-0.19 g/100 g, or 0.012-0.15 g /100 g, or 0.015–0.15 g/100 g, or 0.019–0.15 g/100 g of composition. In a specific embodiment, 3SL is present in an amount of 0.0075 or 0.078 g/100 g of composition. In another specific embodiment, 3SL is present in an amount of 0.02 g/100 g of composition. In a specific embodiment, 3SL is present in an amount of at least 0.001 g/100 g, at least 0.002 g/100 g, at least 0.005 g/100 g, at least 0.01 g/100 g, at least 0 .02 g/100 g, at least 0.025 g/100 g, at least 0.04 g/100 g, at least 0.05 g/100 g, at least 0.075 g/100 g, at least 0.09 g/100 g, at least 0.1 g/100 g, at least 0.15 g/100 g, at least 0.2 g/100 g or at least 0.3 g/100 g composition.

LNT is present in the nutritional composition in an amount of 0.005-3 g/L of the composition. In some embodiments, LNT may be present in an amount of 0.01-1.5 g/L of the composition, such as 0.04-1.2 g/L, or 0.05-1 g/L, or 0.09-0. 8 g/l composition. In a specific embodiment, LNT is present in an amount of 0.5 g/L of the composition. In another specific embodiment, LNT is present in an amount of 0.1 g/L of the composition.

LNT may be present in the nutritional composition in an amount of 0.004-2.3 g/100 g of composition on a dry matter basis, LNT may be present in an amount of 0.008-1.2 g/100 g of composition, e.g. 0.03-0.9 g/100 g, or 0.04–0.8 g/100 g, or 0.07–0.6 g/100 g of the composition. In a specific embodiment, LNT is present in an amount of 0.38 g/100 g of composition. In another specific embodiment, LNT is present in an amount of 0.08 g/100 g of the composition.

In another embodiment, the nutritional composition may contain 0.005-5 g/L 6'SL, or 0.008-2.5 g/L, or 0.01-1 g/L, or 0.03-0.7 g/L , for example 0.04 or 0.5 g/l.

The nutritional composition of the invention may contain 0.004-3.8 g of 6'SL per 100 g of composition based on dry matter weight, for example 0.006-1.9 g, or 0.008-0.8 g, or 0.023-0.5 g , or 0.031-0.4 g 6'SL per 100 g composition based on dry matter weight, for example, 0.18 g or 0.04 g per 100 g composition based on dry matter weight.

In a specific embodiment, the 6'SL and LNT included in the nutritional composition or formula for children 1 to 3 years of age in accordance with the invention are typically present in a 6'SL:LNT ratio of 3:1 to 1:3, e.g. , from 2:1 to 1:2 or from 2:1 to 1:1. In a particularly advantageous embodiment, the ratio is 2:1 or about 2:1, or the ratio is 1:1 or about 1:1.

In one further embodiment, the nutritional composition of the invention contains 2'FL in an amount of 0.005-5 g/L of the composition, for example, 0.01-3 g/L, or 0.02-2 g/L, or 0.1-2 .5 g/l, or 0.15–2 g/l, or 0.25–1.9 g/l, or 0.75–1.65 g/l composition; DiFL in an amount of 0.0005-0.5 g/l of the composition, for example, 0.001-0.3 g/l, or 0.002-0.2 g/l, or 0.01-0.25 g/l, or 0.015 –0.2 g/l, or 0.025–0.19 g/l, or 0.075–0.165 g/l of the composition;

LNnT in an amount of 0.0005–0.5 g/l of the composition, for example, 0.001–0.3 g/l, or 0.002–0.2 g/l, or 0.01–0.25 g/l, or 0.015 –0.2 g/l, or 0.025–0.19 g/l, or 0.075–0.165 g/l of the composition; 3SL in an amount of 0.0005-0.5 g/l of the composition, for example, 0.001-0.3 g/l, or 0.002-0.2 g/l, or 0.01-0.25 g/l, or 0.015 –0.2 g/l, or 0.025–0.19 g/l, or 0.075–0.165 g/l of the composition; LNT in an amount of 0.005-3 g/l of the composition, for example, in an amount of 0.01-1.5 g/l of the composition, for example, 0.04-1.2 g/l, or 0.05-1 g/l, or 0.09–0.8 g/l of composition; and 6SL in an amount of 0.005–5 g/l, or 0.008–2.5 g/l, or 0.01–1 g/l, or 0.03–0.7 g/l, for example, 0.04 or 0 .5 g/l.

In one embodiment, the composition further comprises one or more additional HMOs or one or more precursors thereof.

In one embodiment, the composition of the invention contains at least one N-acetylated oligosaccharide in addition to LNT and LNnT. In one embodiment, the N-acetylated oligosaccharide is para-lacto-N-neohexaose (para-LNnH), disialyl lacto-N-tetraose (DSLNT), or any combination thereof.

N-acetylated oligosaccharide(s) can be synthesized chemically by enzymatic transfer of saccharide units from donor moieties to acceptor moieties using glycosyltransferases, as described, for example, in US Pat. No. 5,288,637 and WO 96/10086. In an alternative embodiment, LNT and LNnT can be prepared by chemically converting ketohexoses (eg, fructose), either free or bound to an oligosaccharide (eg, lactulose), into N-acetylhexosamine or N-acetylhexosamine-containing oligosaccharide, as described in Wrodnigg, T.M. ; Stutz, A. E. (1999) Angew. Chem. Int. Ed. 38:827–828. The N-acetyllactosamine thus obtained can then be transferred to lactose as an acceptor moiety.

In a particularly advantageous embodiment of the present invention, the N-acetylated oligosaccharide(s) is present in the nutritional composition in certain specific amounts. The term "amount", unless otherwise indicated, refers to the total amount of each of such two components in the nutritional composition. It therefore does not refer to an individual quantity unless it is a single type of such component (in which case both the total and individual quantities will be the same). The same applies to all compounds/ingredients of the invention. As an illustrative example, if only one N-acetylated oligosaccharide (i.e., only one type) is used in a composition (e.g., LNnT), its individual amount (and therefore the total amount of N-acetylated oligosaccharides) will be in the range of 0.75 -1.65 g/l. In the case of several N-acetylated oligosaccharides (i.e. several types), their individual amount will be less (for example, in the case of 2 different types of N-acetylated oligosaccharides, for example LNnT + LNT, each of them can be present in an individual amount of 0.5 g/l ), but the total amount of N-acetylated oligosaccharides will be in the range of 0.75–1.65 g/l.

In a specific embodiment, the N-acetylated oligosaccharide is contained in the nutritional composition of the present invention in such an amount that a normal intake of the nutritional composition would be provided for an infant or toddler, or child, at a total daily intake of 0.003-3.9 g, preferably 0.006- 3.25 g, or 0.03–1.95 g, or 0.03–1.3 g, or 0.03–1 g, for example 0.05–1 g, per day.

For premature, low birth weight and small for gestational age infants, the daily dose is preferably 0.005 to 0.1 g/kg body weight per day, preferably 0.006 to 0.09 g, or 0.007 to 0.08 g, or 0.008-0.07 g, or 0.009-0.06 g, or 0.01-0.05 g, or 0.02-0.04 g, most preferably 0.034 g per kg body weight per day.

In the presence of 2'FL and N-acetylated oligosaccharides, the 2'FL and N-acetylated oligosaccharide(s) contained in the nutritional composition according to the invention are generally present in the ratio of N-acetylated oligosaccharide(s) (s):2'FL from 1:20 to 2:1, preferably from 1:15 to 1:1, most preferably from 1:10 to 1:2. In a particularly advantageous embodiment, this ratio is equal to (or approximately equal to) 1:2, 1:5 or 1:10.

Sialylated 3'SL and 6'SL oligosaccharides can be isolated from a natural source such as animal milk by chromatography or filtration methods. In an alternative embodiment, they can be produced by biotechnological means using specific sialyltransferases or sialyldases, neuraminidases, or by enzyme-based fermentation (recombinant or natural enzymes), by chemical synthesis, or by microbiological fermentation. In the latter case, microorganisms can express their natural enzymes and substrates, or it is possible to create microorganisms that produce the corresponding substrates and enzymes. You can use cultures from microorganisms of the same species or mixed cultures. The formation of sialyloligosaccharides can be initiated by acceptor substrates, starting from any degree of polymerization (DP) from DP = 1 onwards. In an alternative embodiment, sialyllactoses can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid). Sialyl lactoses are also commercially available, for example from Kyowa Hakko Kogyo, Japan.

In another preferred embodiment of the invention, the nutritional composition may comprise 0.005-5 g/L sialylated oligosaccharides, or 0.008-2.5 g/L, or 0.01-1 g/L, or 0.02-0.7 g/L, for example 0.03–0.5 g/l.

The nutritional composition according to the invention may comprise 0.004-3.8 g of sialylated oligosaccharides per 100 g of composition based on dry matter weight, for example 0.006-1.9 g or 0.008-0.8 g or 0.015-0.5 g, for example 0.023–0.4 sialylated oligosaccharides per 100 g of composition based on dry matter weight.

In some specific embodiments of the present invention, the nutritional composition contains sialylated oligosaccharide(s) in an amount of less than 0.1 g/100 g of composition on a dry matter basis.

In a specific embodiment, the sialylated oligosaccharide is contained in the nutritional composition of the present invention in such an amount that a normal intake of the nutritional composition would be achieved for an infant or young child, respectively a child, when consuming a total daily dose of 0.003-6.5 g, preferably 0.005-3 .3 g, or 0.006–1.3 g, or 0.01–0.9 g, for example 0.018–0.65 g, per day.

The nutritional composition of the present invention may also optionally contain at least one oligosaccharide precursor. It may contain one or more oligosaccharide precursors. For example, the precursor to human milk oligosaccharide is sialic acid, fucose, or a mixture thereof. In some specific embodiments, the composition contains sialic acid.

In specific examples, the nutritional composition contains from 0 to 3 g/L of oligosaccharide precursor(s), or from 0 to 2 g/L, or from 0 to 1 g/L, or from 0 to 0.7 g/L, or from 0 to 0.5 g/L, or 0 to 0.3 g/L, or 0 to 0.2 g/L oligosaccharide precursor(s). The composition according to the invention may comprise from 0 to 2.1 g of oligosaccharide precursor(s) per 100 g of composition based on dry matter weight, for example from 0 to 1.5 g, or from 0 to 0.8 g, or from 0 to 0.15 g of oligosaccharide precursor(s) per 100 g of composition based on dry matter weight.

The nutritional composition of the present invention may contain at least one additional fucosylated oligosaccharide in addition to 2'FL and DiFL. It may contain one or more types of fucosylated oligosaccharide(s). The fucosylated oligosaccharide(s) may in fact be selected from the list including 3'-fucosyllactose, lacto-N-fucopentaose (e.g., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N -fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose (e.g. fucosyllacto-N-neohexaose I, fucosyllacto-N- neohexaose II), difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose, difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II, fucosyl-para-lacto-N-hexaose, tri-fuco-para-lacto -N-hexaose I and any combination thereof.

In some specific embodiments, the additional fucosylated oligosaccharide contains a 2'-fucosyl epitope. It may, for example, be selected from the list including lacto-N-fucopentaose, lacto-N-fucohexaose, lacto-N-difucohexaose, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose, difuco -lacto-N-neohexaose, difucosyllacto-N-neohexaose, fucosyl-p-lacto-N-hexaose and any combination thereof, preferably difucosyllactose.

In a particular embodiment, no other type of fucosylated oligosaccharide is used other than 2'-fucosyllactose and difucosyllactose, i.e. the nutritional composition of the invention contains only 2'-fucosyllactose and difucosyllactose as fucosylated oligosaccharides.

The fucosylated oligosaccharide(s) may be isolated from a natural source, such as animal milk, by chromatography or filtration methods. In an alternative embodiment, it(he) can be produced by biotechnological means using specific fucosyltransferases and/or fucosidases, or by using an enzyme-based fermentation method (recombinant or natural enzymes), or a microbiological fermentation method. In the latter case, either microorganisms can express their natural enzymes and substrates, or it is possible to create microorganisms that produce the corresponding substrates and enzymes. Cultures from microorganisms of the same species and/or mixed cultures can be used. The formation of fucosylated oligosaccharide can be initiated by acceptor substrates, starting from any degree of polymerization (DP) from DP = 1 onwards. In an alternative embodiment, fucosylated oligosaccharides can be prepared from lactose and free fucose using chemical synthesis. Fucosylated oligosaccharides are also available, for example from Kyowa, Hakko, Kogyo, Japan.

In another specific embodiment, the nutritional composition of the present invention contains a mixture of oligosaccharides consisting of 2'FL, 6'SL, LNT, DiFL, LNnT and 3'SL. In other words, it does not contain any additional oligosaccharides from breast milk.

The nutritional composition or formula for children aged 1 to 3 years in accordance with the present invention may also contain other types of oligosaccharide(s) (i.e. different from the above breast milk oligosaccharides) and/or at least fiber(s), and/or at least its precursor(s). The other oligosaccharide and/or fiber and/or precursor thereof may be selected from the list including galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, and any combination thereof. They may be present in an amount from 0 to 10% by weight of the composition. In a specific embodiment, the nutritional composition or formula for children 1 to 3 years of age may also include at least one bovine milk oligosaccharide (BMO).

Suitable commercially available products that can be used to prepare nutritional compositions or formula for children 1 to 3 years of age in accordance with the invention include combinations of FOS with inulin, such as the product sold by BENEO under the trade name Orafti, or polydextrose , marketed by Tate & Lyle under the trademark STA-LITE®.

A nutritional composition or formula for children 1 to 3 years of age in accordance with the present invention may also optionally include at least one oligosaccharide precursor. It may contain one or more oligosaccharide precursors. For example, the precursor to human milk oligosaccharide is sialic acid, fucose, or a mixture thereof. In some specific embodiments, the composition contains sialic acid.

In specific examples, the nutritional composition or formula for children 1 to 3 years of age contains from 0 to 3 g/L of oligosaccharide precursor(s), or from 0 to 2 g/L, or from 0 to 1 g/L, or from 0 to 0.7 g/L, or 0 to 0.5 g/L, or 0 to 0.3 g/L, or 0 to 0.2 g/L of oligosaccharide precursor(s). The composition according to the invention may comprise from 0 to 2.1 g of oligosaccharide precursor(s) per 100 g of composition based on dry matter weight, for example from 0 to 1.5 g, or from 0 to 0.8 g, or from 0 to 0.15 g of oligosaccharide precursor(s) per 100 g of composition based on dry matter weight.

The nutritional composition or formula for children 1 to 3 years of age of the present invention may further include at least one probiotic (or probiotic strain), such as a probiotic bacterial strain.

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

In some specific embodiments, the probiotic is a probiotic bacterial strain. In some specific embodiments, it is, in particular, strains of Bifidobacteria and/or Lactobacilli.

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

A nutritional composition or formula for children 1 to 3 years of age in accordance with the invention may include from 10e3 to 10e12 CFU of probiotic strain, more preferably from 10e7 to 10e12 CFU, for example from 10e8 to 10e10 CFU, of probiotic strain per gram of composition based on weight dry matter.

In one embodiment, the probiotics are viable. In another embodiment, the probiotics are non-propagating or inactivated. In some other embodiments, both viable probiotics and inactivated probiotics can be used. You can also add probiotic components and metabolites.

In one embodiment, the nutritional composition of the invention is a complete nutritional composition (satisfying all or most of the nutritional needs of a subject). In another embodiment, the nutritional composition is a supplement or fortifier intended, for example, to supplement human breast milk or to supplement infant formula or infant formula/follow-up formula.

In some specific embodiments, the composition of the invention is an infant formula, fortifier, or supplement that may be intended for the first 4, 6, or 12 months of life. In a preferred embodiment, the nutritional composition of the invention is an infant formula. Indeed, it is believed that the nutritional intervention according to the invention may be most effective when targeted at an early stage of life (eg, the first 1, 4, 6, 12 months of age).

The nutritional composition according to the invention may, for example, be an infant formula, an infant formula, a weaning or follow-up formula, an infant formula for children aged 1 to 3 years, an infant food, a cereal-based infant composition, a fortifier, such as a human breast milk fortifier or supplement. In some specific embodiments, the composition of the invention is an infant formula, fortifier, or supplement that may be intended for the first 4 or 6 months of life. In a preferred embodiment, the nutritional composition of the invention is an infant formula.

In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier may be a human milk fortifier (eg, a human breast milk fortifier) or a formula fortifier, such as an infant formula fortifier or a baby formula/follow-up formula fortifier.

If the nutritional composition is a supplement, it may be provided in unit dose form. In such cases, it is particularly useful to determine the amount of 2'FL and optionally other oligosaccharides in relation to the daily dose to be administered to the infant or young child, as described above.

The nutritional composition of the present invention may be in solid (eg, powder form), liquid or gelatinized form.

In a specific embodiment, the nutritional composition is a supplement in powder form and is provided in a sachet, tablet, capsule, lozenge, or liquid form, such as a liquid intended to be dropped into breast milk, or into the nutritional composition, or directly into mouth of a baby or young child.

In another embodiment, the additive may further comprise a carrier, protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surfactants , solubilizing agents (oils, fats, waxes, lecithins, etc.), adsorbents, carriers, fillers, auxiliary compounds, dispersing agents, wetting agents, processing aids (solvents), anti-caking agents, taste masking agents, weighting agents, gelling agents agents and gelling agents. The additive may also contain conventional pharmaceutical additives and excipients, excipients and diluents, including, without limitation, water, gelatin of any origin, vegetable gums, lignin sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers , emulsifying agents, buffers, lubricants, dyes, wetting agents, fillers, etc. If the additive is in powder form, it may include a carrier. However, it is preferable that there is no carrier in the additive. If the additive is in the form of a syrup, the OGM is preferably dissolved or suspended in water acidified with citrate.

Additionally, the supplement may contain vitamins, trace minerals and other micronutrients as recommended by government agencies, such as the US Recommended Dietary Allowances (RDA).

The nutritional composition of the present invention may be in solid (eg, powder form), liquid or gelatinized form. In a specific embodiment, the nutritional composition is a supplement containing 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3 'SL) and 6'-sialyllactose (6'SL), wherein the additive is in powder form and offered in a sachet, preferably a sachet with from 1 to 10 g of a mixture of 2'-fucosyllactose (2'FL), difucosyllactose (DiFL) , lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL) in a sachet, or in the form of a syrup, preferably syrup with a total solids concentration of 5 to 75 g/100 ml (5 to 75% (w/v)). If the additive is in powder form, it may include a carrier. However, it is preferable that there is no carrier in the additive. If the additive is in the form of a syrup, the OGM is preferably dissolved or suspended in water acidified with citrate.

A nutritional composition or formula for children aged 1 to 3 years in accordance with the invention typically contains a source of protein. Protein can be contained in amounts from 1.6 to 3 g per 100 kcal. In some embodiments, particularly when the composition is intended for premature infants, the amount of protein may be from 2.4 to 4 g/100 kcal or greater than 3.6 g/100 kcal. In some other embodiments, the amount of protein may be less than 2.0 g per 100 kcal, such as 1.8 to 2 g/100 kcal, or the protein may be contained in an amount less than 1.8 g per 100 kcal.

The type of protein is not considered critical to the present invention, provided that the minimum requirements for essential amino acid content are met and satisfactory growth is achieved. Thus, protein sources based on whey, casein and mixtures thereof, as well as soy-based protein sources can be used. With respect to whey proteins, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions.

In some advantageous embodiments, the protein source is predominantly whey (ie, more than 50% of the proteins are derived from whey proteins, e.g., 60% or 70%).

The proteins may be native or hydrolyzed, or they may be a mixture of native and hydrolyzed proteins. The term “native” means that the main part of the proteins is native, i.e. their molecular structure is unchanged, e.g. at least 80% of the proteins are unchanged, e.g. at least 85% of the proteins are unchanged, preferably at least 90% of the proteins are unchanged, even more preferably at least 95% of the proteins are unchanged, e.g. at least 98% of proteins are changed. In a particular embodiment, 100% of the proteins are unchanged.

The term "hydrolyzed" in the context of the present invention means a protein that has been hydrolyzed or broken down into its constituent amino acids. Proteins can be fully or partially hydrolyzed. It may be desirable to administer partially hydrolyzed proteins (degree of hydrolysis 2 to 20%), for example, to infants or young children believed to be at risk of developing cow's milk allergy. If it is necessary to use hydrolyzed proteins, the hydrolysis process can be carried out as desired and in a manner known in the art. For example, whey protein hydrolysates can be produced by enzymatic hydrolysis of a whey fraction in one or more steps. If the whey fraction used as the starting material is essentially free of lactose, it has been found that during hydrolysis the blocking of protein lysine occurs to a much lesser extent. This allows the degree of lysine blocking to be reduced from about 15 wt.% total lysine to less than about 10 wt.% lysine; for example up to about 7 wt.% lysine, which significantly improves the nutritional quality of the protein source.

In one embodiment, at least 70% of the proteins are hydrolyzed, preferably at least 80% of the proteins are hydrolyzed, e.g. at least 85% of the proteins are hydrolyzed, even more preferably at least 90% of the proteins are hydrolyzed, e.g. at least 95% of the proteins are hydrolyzed , in particular, at least 98% of the proteins are hydrolyzed. In a specific embodiment, 100% of the proteins are hydrolyzed.

In one specific embodiment, the proteins of the nutritional composition are hydrolyzed, fully hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of a protein can be from 8 to 40, or from 20 to 60, or from 20 to 80, or greater than 10, 20, 40, 60, 80 or 90.

In an alternative embodiment, the protein component may be replaced with a formula or artificial amino acid, for example for premature or low birth weight infants.

In a specific embodiment, the nutritional composition or formula for children from 1 to 3 years of age in accordance with the invention is a hypoallergenic composition. In another specific embodiment, the composition in accordance with the invention is a hypoallergenic nutritional composition or formula for children from 1 to 3 years of age.

The nutritional composition or formula for children aged 1 to 3 years in accordance with the present invention typically contains a source of carbohydrates. This is particularly preferred when the nutritional composition of the invention is an infant formula. In this case, any carbohydrate source commonly found in infant formulas can be used, such as lactose, sucrose, sucrose, maltodextrin, starch and mixtures thereof, although one preferred carbohydrate source is lactose.

The nutritional composition or formula for children aged 1 to 3 years in accordance with the present invention typically contains a source of lipids. This is especially important if the nutritional composition of the present invention is an infant formula. In this case, the source of lipids may be any lipid or fat that is suitable for use in infant formulas. Some suitable fat sources include palm oil, structured triglyceride oil, high oleic acid sunflower oil and high oleic acid safflower oil, a medium chain triglyceride oil. You can also add the essential fatty acids linoleic acid and α-linolenic acid, as well as small amounts of oils containing large amounts of arachidonic acid and docosahexaenoic acid in a prepared form, such as fish oils or microbial oils. The ratio of n-6 to n-3 fatty acids in the fat source can range from about 5:1 to about 15:1; for example from about 8:1 to about 10:1.

The nutritional composition or formula for children aged 1 to 3 years of the invention may also contain all the vitamins and minerals considered essential in the daily diet in nutritionally significant quantities. Some vitamins and minerals have minimum requirements. Examples of minerals, vitamins and other nutrients that are optionally present in the composition of the 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 the form of salts. The presence and amounts of specific minerals and other vitamins will vary depending on the intended population.

If necessary, the nutritional composition or infant formula for children 1 to 3 years of age of the invention may contain emulsifiers and stabilizers such as soy, lecithin, mono- and diglyceryl esters of citric acid, and the like.

The nutritional composition or infant formula for children 1 to 3 years of age of the invention may also contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, nucleosides, and the like.

The nutritional composition or infant formula for children 1 to 3 years of age of the invention may also contain carotenoid(s). In some specific embodiments, the nutritional composition of the invention does not contain carotenoids.

The nutritional composition or formula for children aged 1 to 3 years in accordance with the invention can be prepared by any suitable method. Next, the composition will be described using a specific example.

For example, a formula, such as an infant formula, can be prepared by mixing a protein source, a carbohydrate source and a fat source in appropriate proportions. At this stage, you can include emulsifiers if used. Vitamins and minerals can be added at this stage, but are usually added later to prevent thermal decomposition. Any lipophilic vitamins, emulsifiers, etc. may be dissolved in the fat source before mixing. Water, preferably purified by reverse osmosis, can then be added to form a liquid mixture. The water temperature conveniently ranges from about 50°C to about 80°C to facilitate dispersion of the ingredients. Commercially available thinners can be used to form a liquid mixture.

Fucosylated oligosaccharide(s) and N-acetylated oligosaccharide(s) may be added at this stage, especially if the final product is to be in liquid form. If the final product is to be a powder, these components can also be added at this stage if desired.

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

The liquid mixture can then be thermally treated to reduce the bacterial load by rapidly heating the liquid mixture, for example to a temperature in the range of about 80°C to about 150°C for a period of time from about 5 seconds to about 5 minutes. Heating can be accomplished by injection of steam using an autoclave or a heat exchanger such as a plate heat exchanger.

The liquid mixture can then be cooled to a temperature of from about 60°C to about 85°C, for example by flash cooling. The liquid mixture can then be homogenized again, for example in two stages: under a pressure ranging from about 10 MPa to about 30 MPa in the first stage and from about 2 MPa to 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. At this stage, the pH and solids content of the homogenized mixture are adjusted for convenience.

If the final product is to be a powder, the homogenized mixture is transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, and converted into a powder. The moisture content of the powder should be less than about 5 wt.%. At this stage it is also possible or alternatively to add the fucosylated oligosaccharide(s) and N-acetylated oligosaccharide(s) by dry mixing or by mixing them in the form of a crystal syrup together with the probiotic(s). them) strain(s) (if used), and the mixture is spray-dried or freeze-dried.

If a liquid composition is preferred, the homogenized mixture can be sterilized and then aseptically filled into suitable containers, or it can be filled into containers first and then sterilized in an autoclave.

In another embodiment, the composition of the invention may be an additive. The supplement may be presented, for example, in the form of tablets, capsules, lozenges or liquid. The additive may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizing agents (oils, fats, waxes, lecithins, etc.), adsorbents, carriers, fillers, auxiliary compounds, dispersing agents, wetting agents, processing aids (solvents), anti-caking agents, taste-masking agents, weighting agents, gelling agents and gelling agents. The additive may also contain conventional pharmaceutical additives and excipients, excipients and diluents, including, without limitation, water, gelatin of any origin, vegetable gums, lignin sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers , emulsifying agents, buffers, lubricants, dyes, wetting agents, fillers, etc.

Additionally, the supplement may contain an organic or inorganic carrier material suitable for oral or parenteral administration, as well as vitamins, trace minerals and other trace elements in accordance with the recommendations of government authorities, such as the US Recommended Dietary Allowances (RDA).

The nutritional composition according to the invention is intended for use in infants or young children. Infants or young children may be born full term or premature. In a specific embodiment, the nutritional composition of the invention is for use in infants or young children who were born preterm, low birth weight, and/or small for gestational age (SGA). In a specific embodiment, the nutritional composition of the invention is for use in preterm infants, low birth weight infants, and/or small for gestational age (SGA) infants.

The nutritional composition of the present invention can also be used in infants or young children born by cesarean section or by vaginal delivery.

In some embodiments, the composition in accordance with the invention may be intended for use before and/or during the weaning period.

The age at which the nutritional composition is administered (or used) or fed to it, as well as the duration of such administration, depends on the needs.

The nutritional composition may, for example, be given to infants immediately after birth. The composition of the invention may also be given during the first week of life of an infant, or during the first 2 weeks of life, or during the first 3 weeks of life, or during the first 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 year of life, or during the first two years of life, or even longer. In some particularly advantageous embodiments of the invention, the nutritional composition is given (or administered) to an infant during the first 4, 6 or 12 months after the birth of said infant. In some other embodiments, the nutritional composition of the invention is given over a period of several days (e.g., 1, 2, 3, 5, 10, 15, 20, etc.), or several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), or several months (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) after birth. First of all, this may be relevant, but not necessarily, in the case where the baby was born prematurely.

In one embodiment, the composition of the invention is given to an infant or young child as a supplemental composition to breast milk. In some embodiments, the infant or young child receives breast milk for at least the first 2 weeks, the first 1, 2, 4, or 6 months. In one embodiment, the nutritional composition of the invention is given to an infant or young child after or during such a period of breast milk feeding. In another embodiment of the invention, the composition is given to an infant or young child as the sole or primary nutritional composition for at least one period of time, for example after the 1st, 2nd or 4th month of life, for at least 1 , 2, 4 or 6 months.

In one embodiment, the nutritional composition of the invention is a complete nutritional composition (satisfying all or most of the nutritional needs of a subject). In another embodiment, the nutritional composition is a supplement or fortifier intended, for example, to supplement human breast milk or to supplement infant formula or infant formula/follow-up formula.

All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety.

The present invention will be further described in detail in the following non-limiting examples.

Examples

Example 1

Table 1 below shows an example of the composition of a nutritional composition (eg, infant formula) in accordance with the present invention. This composition is provided for illustrative purposes only.

Table 1. Example of the composition of a nutritional composition (eg, infant formula) in accordance with the present invention

Nutrients per 100 kcal per liter Energy value (kcal) 100 670 Protein (g) 1.83 12.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg) 101 675 Lactose (g) 10.6 70.7 Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (µg) 8 50 Se (µg) 2 13 Vitamin A (µg RE) 105 700 Vitamin D (mcg) 1.5 10 Vitamin E (mg, TEQ) 0.8 5.4 Vitamin K1 (mcg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50 Folic acid (mcg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (mcg) 0.3 2 Biotin (mcg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 I (µg) 15 100 Cu (mg) 0.06 0.4 Zn (mg) 0.75 5 Oligosaccharides
(OGM) 2FL (g) 0.13 0.9 LNnT (g) 0.0075 0.05 LNT (g) 0.04 0.25 DFL (g) 0.013 0.09 3SL (g) 0.014 0.1 6SL (g) 0.022 0.15

Example 2

A mixture of 6 HMOs conferred resistance to inflammation-induced epithelial barrier dysfunction

Cell lines

The human colorectal adenocarcinoma cell line Caco-2 (HTB-37) was obtained from the American Type Culture Collection (ATCC, Manassas, USA) at passage 21 and used in experiments at passages 23 to 33. The human colon adenocarcinoma cell line HT29 ( HTB-38; ATCC), pre-adapted with methotrexate (MTX), was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) at passage 51 and used in experiments at passages 23 to 33. Both cell lines separately were maintained in 75 ^cm2 surface area tissue culture flasks (Fischer Scientific) in a humidified atmosphere at 37°C and 10% CO2, 95% air/water saturated atmosphere.

Cell culture model.

Both Caco-2 and HT29-MTX cell lines were maintained in Dulbecco's minimal essential medium (DMEM; 11965092, Gibco) supplemented with 10% (v/v) heat-inactivated PBS (10270-106, Gibco) and 1% (v/v) /vol.) solution of penicillin and streptomycin (P4333, Sigma). The nutrient medium was replaced at least twice a week. Cell lines were subcultured weekly at preconfluent densities with 0.4% trypsin-EDTA (T3924, Sigma).

For experimental studies, Caco-2 and HT29-MTX cells were stained with trypan blue (T8154, Sigma), counted, resuspended in complete culture medium at 76:24 ratios to simulate colon, and seeded at a density of 6 × 10 ⁴ cells per cm ² in semipermeable Transwell™ polycarbonate membrane with a pore size of 0.4 μM and a surface area of 1.12 cm ² (3460, Corning. Caco-2:HT29-MTX culture confluence and integrity were assessed by manually measuring transepithelial electrical resistance every week using a Millicell™ ERS voltmeter -2 Cells were used in experiments 21 days after seeding.

OGM treatment and a model of inflammation-induced epithelial barrier dysfunction.

OGM, obtained from Glycom (Härsholm, Denmark), was diluted in sterile water at a concentration of 250 mg/ml, filtered using a 0.2 μM filter and kept at −20°C for a maximum of 6 months. On the day of treatment, the cell growth medium was replaced with fresh medium with all supplements, but without phenol red. Mixtures 2'FL:3'SL:6'SL:LNnT:LNT:diFL in the proportion 0.55:0.7:0.09:0.05:0.18:0.06 were additionally diluted in a nutrient medium and added into the apical compartment of Transwell™ at various final concentrations 48 hours before exposure to the cytokines TNF-α and INF-γ in the basolateral compartment for an additional 48 hours. Controls were cells not treated with OGM and not treated with cytokines (negative control), and cells not treated with OGM but provoked with cytokines (positive control).

Assessment of epithelial permeability.

Permeability was assessed using two metrics: transepithelial electrical resistance (TEER) and translocation of FITC-labeled dextran (FD4; 4000 Da, Sigma) from the apical to the basolateral Transwell™ compartment.

TEER was measured dynamically every 5–15 minutes by placing a Transwell™ seeded with Caco-2:HT29-MTX into a cellZscope (Nano Analytics) in a humidified atmosphere at 37°C and 10% CO2, 95% air/water saturated. atmosphere during the entire period of the experiment. Culture medium and treatments were replenished every 48 hours. TEER was measured as Ω per cm ² and percentage changes in TEER were calculated relative to the baseline value (measuring TEER before the start of any treatment).

FD4 translocation was measured by adding filter-sterilized FD4 solution to the Transwell™ apical compartment at a final concentration of 1 mg/ml at the end of the inflammatory challenge. Basolateral samples were collected before and every 30 minutes for 2 hours after apical addition of FD4. FD4 translocation was measured by interpolating the fluorescence intensity of the samples relative to a standard curve and expressed as ng/ml. FD4 translocation was normalized to positive control and negative control cells. The +ve control represents the minimum resistance to FD4 translocation, and the -ve control represents the maximum resistance to FD4 translocation.

results

OGM6 increased TEER before inflammatory challenge in a dose-dependent manner compared with lactose or untreated controls (Fig. 1). In addition, OGM6 demonstrated a significant increase in pre-inflammatory TEER compared to individual OGMs, indicating the effectiveness of the complex mixture compared to individual OGMs (Figure 2). Following inflammatory challenge, OGM6 significantly prevented the increase in FD4 translocation from the apical to basolateral compartment in a dose-dependent manner compared with lactose-treated and untreated control cells (Fig. 3). Overall, this highlights that OGM6 provides health benefits by prophylactically strengthening the epithelial barrier and conferring resistance to inflammation-mediated barrier dysfunction.

Claims (9)

1. Use of a nutritional composition containing 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL), to improve the gastrointestinal barrier in an infant, which is a child under 12 months of age, a young child, which is a child between 1 year of age and less than 3 years of age, or a child between 3 years of age and less than 8 years of age, wherein said gastrointestinal barrier improvement is improved barrier structure and/or improved barrier protection. 2. Use of the nutritional composition of claim 1, wherein said use for improving barrier protection is to improve the prevention of barrier dysfunction, prevent barrier permeability, protect the tight junction structure, and protect the integrity of the intestinal epithelial lining. 3. Use of a nutritional composition according to claim 2, wherein said prevention of barrier permeability is the prevention of migration of pathogens, allergens and/or toxic compounds from the intestine into the body through the intestinal barrier. 4. Use of the nutritional composition of claim 1, wherein said use for improving barrier structure is to increase the strength of the gastrointestinal barrier, increase the integrity of the gastrointestinal barrier, improve the tight junction structure and improve the integrity of the intestinal epithelial lining. 5. Use of a nutritional composition according to any of the preceding paragraphs, which is selected for administration to the infant. 6. Use of the nutritional composition of claim 5, wherein said infant is selected from the group consisting of preterm, small for gestational age and low birth weight infants, preferably the infant is preterm. 7. Use of a nutritional composition according to any of the preceding claims, wherein the nutritional composition contains 2'-fucosyllactose (2'FL), difucosyllactose (DiFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3' -sialyllactose (3'SL) and 6'-sialyllactose (6'SL) in an amount of at least 0.3 g/l, for example at least 0.4 g/l, for example 0.5 g/l. 8. Use of a nutritional composition according to any of the preceding paragraphs, wherein said nutritional composition is an infant formula, an initial infant formula, an infant formula for weaning children or a subsequent level infant formula, a milk formula for children from 1 to 3 years of age, an infant food, an infant composition grain-based, fortifier or supplement and is intended for administration to an infant or young child. 9. Use of a nutritional composition according to claims. 1-4, 7, wherein said nutritional composition is a nutritional composition in the form of a milk formula for children from 1 to 3 years of age and is intended for administration to a child aged from 3 years to less than 8 years.

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