RU2819186C2 - Nutritional composition containing 2'-fucosyllactose (2'fl) for improving gastrointestinal barrier - Google Patents

Abstract

FIELD: food products.

SUBSTANCE: invention relates to nutritional compositions for an infant, a toddler or children aged 3 to 8 years. Disclosed is use of a nutritional composition containing 2'-fucosyllactose (2'FL), for improving the gastrointestinal barrier in an infant, which is a child under age of 12 months, in a toddler which is a child at age of 1 to less than 3 years, or in child of 3 years to less than 8 years. Said improvement of the gastrointestinal barrier represents an improved barrier protection. 2'FL is present in amount of 0.25–1.9 g/l or 0.15–1.5 g/100 g of the composition in terms of dry substance weight, preferably in amount of 0.75–1.65 g/l or 0.19–1.5 g/100 g of the composition in terms of dry substance weight.

EFFECT: invention makes it possible to improve protection of the gastrointestinal barrier in an infant, a toddler or children aged 3 to 8 years.

13 cl, 4 dwg, 2 tbl, 6 ex

Description

Scope of the invention

The present invention relates to nutritional compositions containing 2'-fucosyllactose (2'FL) for use in improving the gastrointestinal tract 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 to improve the gastrointestinal barrier in infants and young children through nutritional intervention, for example, through a balanced diet or nutritional supplements.

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 and infections in formula-fed infants than in breast-fed infants.

Certain specific groups of infants and young children have a particular need for compositions that can provide 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 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 components such as human milk oligosaccharides on improving the gastrointestinal barrier have previously been investigated.

2'FL, listed among other oligosaccharides, is described as beneficial for the gastrointestinal barrier in WO2013/032674 (gastrointestinal barrier function), WO2017/144062 (maintaining or restoring gastrointestinal barrier permeability through microbiota), WO2017/ 71716 (treatment of mucosal barrier impairment), WO2017/046711 (treatment of mucosal barrier impairment), WO2016/91265 (reducing intestinal permeability), WO2016/66174 (maintaining mucosal integrity).

Therefore, an improved nutritional composition would be advantageous, 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) for use in improving the gastrointestinal tract barrier. The effect is confirmed by the results of an in vitro study on a model of intestinal epithelial cells. As disclosed in the Examples section, 2'-fucosyllactose (2'FL) was found to be superior to other OGMs tested in improving the gastrointestinal barrier, followed by an OGM mixture containing 2'FL.

Additional experiments identified the mechanisms involved in the improvement of the gastrointestinal barrier due to 2'FL. On the one hand, the present inventors have demonstrated that improvement of the intestinal barrier is achieved by at least down-regulating the TNF-α receptor on epithelial cells, which, in particular, leads to improved protection of the gastrointestinal barrier. This mucosal protective mechanism is associated with improved function, structure, repair and maturation of the gastrointestinal tract barrier and, in particular, with improved function and structure of the gastrointestinal tract barrier.

On the other hand, additional experiments showed that 2'FL was also effective by increasing the production of kynurenic acid by the microbiota of the infant or young child consuming the composition and by subsequent activation of G protein-coupled receptor 35 (GPR35) by kynurenic acid. In fact, kynurenic acid was found to be able to trigger GPR35 receptor activation according to Wang, et al. 2006. Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35. J Biol Chem. 281:22021–22028. GPR35 is involved in increased enterocyte migration, which is an important mechanism following mucosal injury to rapidly restore epithelial integrity to the denuded villi surface area. Tsukahara et al found that activation of GPR35 promotes mucosal repair in the colon (Pharmacological Research 123 (2017) 27–39). This mechanism of mucosal repair is associated with improved function, structure, protection and maturation of the gastrointestinal barrier and, in particular, with improvement of function, structure and protection of 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 containing 2'-fucosyllactose (2'FL) for use in improving the gastrointestinal tract barrier in an infant (child under 12 months of age) or young child (1 year to less than 3 years); or to a nutritional composition in the form of an infant formula for children from 1 to 3 years, containing 2'-fucosyllactose (2'FL), for use in improving the gastrointestinal tract barrier in a child (aged 3 years to less than 8 years). In a specific embodiment, this effect is achieved by increasing the production of kynurenic acid by the microbiota of an infant or young child, respectively a child aged 3 years to less than 8 years, consuming the nutritional composition.

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 more effective barrier repair.

Brief description of graphic materials

Fig. 1. The effectiveness of OGM in reducing inflammation-induced epithelial barrier dysfunction. Cocultures are treated with OGM and then induce epithelial barrier dysfunction through cytokine-mediated inflammation. All groups, except the negative control (intact barrier), are susceptible to inflammatory provocation. Epithelial barrier integrity is calculated by analyzing the movement of FITC-labeled dextran (FD4) from the apical compartment to the basolateral compartment compared to a negative control (arbitrarily set to 1) and a positive control (arbitrarily set to 0) following an inflammatory challenge. Error bars reflect the minimum standard deviation.

Fig. 2. Resistance to inflammation-induced disruption of intestinal epithelial barrier function by downregulating TNF-α receptor expression. Cocultures are treated with 2'FL and then induce epithelial barrier dysfunction through cytokine-mediated inflammation. The graph shows the expression of TNF-α receptor (rTNF1) mRNA relative to the negative control. All groups, except the negative control (intact barrier), are susceptible to inflammatory provocation. Error bars reflect SD.

In fig. Figure 3 shows the relative excess of kynurenic acid in the supernatant of fermented infant stool in the presence of lactose (lactose), 2'FL, 2'FL + LNT or 2'FL, DFL, LNT, LNnT, 3'SL and 6'SL (6 OGM) , demonstrating increased kynurenic acid levels in the presence of any combination of 2′FL-containing OGMs compared to lactose. * p<0.05 compared to lactose, ** p<0.01 (example 5)

In fig. Figure 4 shows the dose-dependent effect of GPR35 activation in arrestin mode on equimolar 6'SL + LNT with or without 200 μM KYNA and with 100–200 μM KYNA, demonstrating an additive effect at concentrations from 940 μM to 2500 μM. This experiment was performed three times with comparable results (Example 6).

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 dissolution in breast milk, infant formula, infant formula, or breast milk fortified with other nutrients, or may otherwise 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 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.

The term "increasing kynurenic acid production" means increasing, enhancing, and/or stimulating the production of these types of molecules (and/or physiologically acceptable salts or ions thereof) as a result of the action of the microbiota of an infant and/or young child consuming nutritional compositions in accordance with the present invention . In one embodiment, an increase in kynurenic acid production occurs in the intestines of an infant and/or young child consuming the nutritional compositions of the present invention. In one embodiment, the term “increased production of kynurenic acid” means an increase in the production of these types of molecules (and/or physiologically acceptable salts or ions thereof) as a result of the microbiota of an infant and/or young child consuming nutritional compositions in accordance with the present invention. In another embodiment, the term “increasing kynurenic acid production” means stimulating the production of these types of molecules (and/or physiologically acceptable salts or ions thereof) as a result of the microbiota of an infant and/or young child consuming the nutritional compositions of the present invention.

“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 the 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, for example milk, water, etc., for example dissolved infant formula, or infant formula/after-level formula, or infant formula for children from 1 up to 3 years, or a cereal product for baby food, 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'-fucosyllactose (2'FL) for use in improving the gastrointestinal tract barrier. As disclosed in the Examples section, 2'-fucosyllactose (2'FL) was found to be superior to other OGMs tested in improving the gastrointestinal barrier, followed by an OGM mixture containing 2'FL. Thus, one aspect of the invention relates to a nutritional composition containing 2'-fucosyllactose (2'FL) for use in improving the gastrointestinal tract barrier in an infant (child under 12 months of age) or young child (1 year to less than 3 years);

or

nutritional composition in the form of an infant formula for children from 1 to 3 years old, containing 2'-fucosyllactose (2'FL), for use in improving the gastrointestinal tract barrier in a child (aged from 3 years to less than 8 years).

In an embodiment, said gastrointestinal barrier improvement is improved barrier maturation, improved barrier structure, improved barrier function, improved barrier protection, and/or more effective 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 an improvement in 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, allergens and/or toxic compounds from the intestine into the body through the intestinal barrier. As described in Example 3, 2'FL was found to be superior to other OGM test subjects in improving gastrointestinal barrier protection.

Protecting the gastrointestinal tract barrier leads to improvement in the structure and function of the gastrointestinal tract barrier, since the absence of adequate protection of the gastrointestinal tract barrier loses its optimal structure and function, namely under inflammatory provocation.

Thus, in an embodiment, said use is intended to improve gastrointestinal barrier strength, gastrointestinal barrier integrity, tight junction structure, and intestinal epithelial lining integrity.

In another embodiment, the use is intended to improve the stability of the gastrointestinal tract barrier and to reduce the permeability of the gastrointestinal tract barrier. Preferably, said use is intended to reduce the migration of pathogens from the intestine across the intestinal barrier, for example, reducing the migration of synanthropic bacteria from the intestine across the intestinal barrier, reducing the migration of allergens from the intestine across the intestinal barrier, reducing the migration of toxic compounds from the intestine across the intestinal barrier, and reducing susceptibility to disease.

In a specific embodiment, the above-mentioned improvements are achieved by down-regulating the TNF-α receptor on epithelial cells. In a preferred embodiment, the nutritional composition is for use in improving gastrointestinal barrier protection by down-regulating the TNF-α receptor on epithelial cells. In a more preferred embodiment, the nutritional composition is for use in preventing barrier dysfunction, preventing barrier permeability, protecting tight junction structures, and/or protecting the integrity of the intestinal epithelial lining by down-regulating the TNF-α receptor on epithelial cells.

In another specific embodiment of the invention, the above improvements are achieved by increasing the production of kynurenic acid by the microbiota of the infant or young child consuming the nutritional composition. In a preferred embodiment, the nutritional composition is for use in improving gastrointestinal barrier repair by increasing the production of kynurenic acid by the microbiota of the infant or young child consuming the nutritional composition. In a more preferred embodiment, the nutritional composition is for use in restoring the integrity of the gastrointestinal barrier, for example, in restoring a compromised barrier, reducing permeability following an inflammatory challenge to the gastrointestinal mucosa, and/or in restoring the mucosa by increasing kynurenic acid production. microbiota of an infant or young child consuming the nutritional composition.

In another specific embodiment of the invention, the above improvements are achieved by increasing the production of kynurenic acid by the microbiota of a child aged three years to less than eight years consuming the nutritional composition. In a preferred embodiment, the nutritional composition in the form of a formula for children 1 to 3 years of age is intended for use in improving gastrointestinal barrier repair by increasing the production of kynurenic acid by the microbiota of the child consuming the nutritional composition. In a more preferred embodiment, the nutritional composition in the form of an infant formula for children from 1 to 3 years of age is intended for use in restoring the integrity of the gastrointestinal tract barrier, for example, in restoring a damaged barrier, in reducing permeability after inflammatory provocation of the gastrointestinal mucosa and /or when restoring the mucous membrane by increasing the production of kynurenic acid by the microbiota of a child consuming a nutritional composition in the form of a milk formula for children from 1 to 3 years old.

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, said 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 some embodiments, 2'FL may be present in an amount of 0.005-5 g/L of the composition, such as 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 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 the 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 another specific embodiment, 2'FL is present in amounts of 5-500 g/L, 10-400 g/L, 40-300 g/L, 60-200 g/L, 80-180 g/L, 100-150 g /l or 110-130 g/l of the composition. In a specific embodiment, the OGM mixture is present in an amount of 120 g/L. Such amounts are particularly sufficient if the nutritional composition is in the form of a supplement or fortifier.

If the supplement or fortifier is in powder form, 2'FL is preferably provided in the nutritional composition of the present invention in an amount that is 0.05-5 g, 0.1-4.5 g, 0.15-4 g, 0 .2–3.5 g, 0.25–3, 0.3–2.5, 0.35–2, 0.4–1.5 g, 0.45–1 g, 0.5–0, 75 g, for example 0.6 g, per serving.

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

In one embodiment of the present invention, 2'FL is combined with 6-SL and LNT. Without being limited by any theory, in such an embodiment, 2'-FL is responsible for activating the GPR35 receptor through increased production of kynurenic acid by the microbiota of the infant or young child. In such an embodiment, 6SL and LNT are also considered to be responsible for the activation of GPR35, as described in international patent application WO 2019/121929 (same applicant).

In one embodiment, LNT is present in a nutritional composition or formula for children 1 to 3 years of age in an amount of 0.005 to 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 a nutritional composition or formula for children 1 to 3 years of age in an amount of 0.004-2.3 g/100 g of composition based on dry matter weight, LNT may be present in an amount of 0.008-1.2 g/100 g of composition , for example 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.

LNT 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 another embodiment of the invention, the nutritional composition or formula for children from 1 to 3 years of age 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.

A nutritional composition or milk formula for children aged 1 to 3 years in accordance with 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 mass.

6'SL can be isolated from a natural source such as animal milk by chromatography or filtration methods. In an alternative embodiment, it can be produced by biotechnological means using specific sialyltransferases or sialidases, 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 6'SL can be initiated by acceptor substrates, starting from any degree of polymerization (DP) from DP = 1 onwards. In an alternative embodiment, 6'SL can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid).

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, this ratio is 2:1 or about 2:1, preferably this ratio is 1:1 or about 1:1.

In a specific aspect of the invention, a nutritional composition or formula for children from 1 to 3 years of age contains 6'SL and LNT, wherein:

- 6’SL is present in an amount of 0.005–5 g/l of composition and/or in an amount of 0.004–3.8 g/100 g of composition based on dry matter weight; and/or

- LNT is present in an amount of 0.005–3 g/l of composition and/or in an amount of 0.004–2.3 g/100 g of composition based on dry matter weight.

In a specific aspect of the invention, a nutritional composition or formula for children from 1 to 3 years of age contains 6'SL and LNT, wherein:

- 6’SL is present in an amount of 0.008–2.5 g/l of composition and/or in an amount of 0.006–1.9 g/100 g of composition based on dry matter weight; and/or

- LNT is present in an amount of 0.01–1.5 g/l of composition and/or in an amount of 0.008–1.2 g/100 g of composition based on dry matter weight.

In another specific embodiment, a nutritional composition or formula for children 1 to 3 years of age of the present invention contains 6'SL and LNT, wherein:

- 6’SL is present in an amount of 0.01–1 g/l of composition and/or in an amount of 0.008–0.8 g/100 g of composition based on dry matter weight; and/or

- LNT is present in an amount of 0.04–1.2 g/l of composition and/or in an amount of 0.03–0.9 g/100 g of composition based on dry matter weight.

In another specific embodiment, a nutritional composition or formula for children 1 to 3 years of age of the present invention contains 6'SL and LNT, wherein:

- 6’SL is present in an amount of 0.03–0.7 g/l of composition and/or in a total amount of 0.023–0.5 g/100 g of composition based on dry matter weight; and/or

- LNT is present in an amount of 0.05–1 g/l of composition and/or in an amount of 0.04–0.8 g/100 g of composition based on dry matter weight.

In another specific embodiment, a nutritional composition or formula for children 1 to 3 years of age of the present invention contains 6'SL and LNT, wherein:

- 6’SL is present in an amount of 0.04–0.5 g/l of composition and/or in an amount of 0.031–0.4 g/100 g of composition based on dry matter weight; and/or

- LNT is present in an amount of 0.09–0.8 g/l of composition and/or in an amount of 0.07–0.6 g/100 g of composition based on dry matter weight.

In a specific embodiment, a nutritional composition or formula for children aged 1 to 3 years in accordance with the invention contains 6'SL and LNT, wherein:

- 6’SL is present in an amount of 0.24 or 0.05 g/l of composition and/or in an amount of 0.18 or 0.04 g/100 g of composition based on dry matter weight; and/or

- LNT is present in an amount of 0.5 g/l or 0.1 g/l of the composition and/or in an amount of 0.38 g/100 g of composition or 0.08 g/100 g of composition based on dry matter weight.

In a specific embodiment, 6'SL is contained in a nutritional composition or formula for children 1 to 3 years of age of the present invention in such an amount that the normal intake of the nutritional composition or formula for children 1 to 3 years of age will be provided for an infant or young child , respectively, a child, with a total daily dose of 0.003–6.5 g, preferably 0.005–3.3 g, or 0.006–1.3 g, or 0.02–0.9 g, for example 0.024–0.7 g, per day.

In a specific embodiment, LNT is contained in a nutritional composition or formula for children 1 to 3 years of age of the present invention in such an amount that the normal intake of the nutritional composition or formula for children 1 to 3 years of age will be provided for an infant or young child, respectively child, with a total daily dose of 0.003–3.9 g, preferably 0.006–2 g, or 0.02–1.6 g, or 0.03–1.3 g, for example 0.05–1 g, per day .

In one embodiment, the composition of the invention contains at least one N-acetylated oligosaccharide. The N-acetylated oligosaccharide is preferably LnNT. Without being limited by theory, the inventors have found that LnNT develops the best interactions with intestinal flora and enhances the described effect.

In one embodiment, the N-acetylated oligosaccharide is lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), para-lacto-N-neohexaose (para-LNnH), disialyl lacto-N-tetraose (DSLNT), or any combination thereof, preferably lacto-N-neotetraose (LNnT) and/or lacto-N-tetraose (LNT).

In one embodiment, the N-acetylated oligosaccharide(s) are present in a total amount of 0.025-1.5 g/L of the composition, preferably at least 0.1 g/L or at least 0. 25 g/l, and/or in a total amount of 0.003-0.23 g/100 g, preferably at least 0.015 g/100 g or at least 0.03 g/100 g of the composition based on dry matter weight.

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 of 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.

Mixtures of 2'FL and LNnT are considered particularly advantageous because data indicate that such mixtures provide significant benefit after 48 hours of treatment (and the effect is maintained thereafter), whereas 2'FL without LNnT has a greater effect in the medium term. Thus, in one embodiment, the applications of the invention are intended to rapidly improve the gastrointestinal barrier, for example within 72 hours, preferably within 48 hours or, for example, within 24 hours.

In another embodiment, the nutritional composition in accordance with the invention may contain sialylated oligosaccharide(s). It may contain one or more sialylated oligosaccharides. The sialylated oligosaccharide(s) may be selected from the group consisting of 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and any combination thereof. In some embodiments, the composition contains 3'SL and 6'SL. In some specific embodiments, the ratio of 3'-sialyllactose (3'SL) to 6'-sialyllactose (6'SL) may range from 5:1 to 1:10, or from 3:1 to 1:1, or from 1:1 to 1:10. In some specific embodiments, the sialylated oligosaccharide of the composition is 6'-sialyllactose (6'SL).

The sialylated oligosaccharide(s) may 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.

In some specific embodiments of the present invention, the nutritional composition does not contain any sialylated oligosaccharide(s).

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. It may contain one or more types of fucosylated oligosaccharide(s). The fucosylated oligosaccharide(s) may be selected from the list including 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 (for example, fucosyllacto-N- neohexaose I, fucosyllacto-N-neohexaose II), difucosyllacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II, fucosyl-para-lacto-N-hexaose, tri-Fucault-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 consisting of difucosyllactose, 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.

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

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 some embodiments, the fucosylated oligosaccharide(s) (including 2'FL) may be present in a total amount of 0.005-5 g/L of the composition, e.g., 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. In a specific embodiment, the fucosylated oligosaccharide(s) is present in a total amount of 1 g/L of the composition. In another embodiment, the fucosylated oligosaccharide(s) is present in a total amount of 0.25 or 0.26 g/L of the composition. In one embodiment, the fucosylated oligosaccharide(s) is present in a total 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, the fucosylated oligosaccharide(s) is present in a total 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 0.8, at least 1, at least 1.25, at least 1.5 or at least 2 g/l.

The fucosylated oligosaccharide(s) 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. Fucosylated oligosaccharide(s) may be present in a total amount of 0.008-2.3 g/100 g of 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, the fucosylated oligosaccharide(s) is present in a total amount of 0.075 or 0.78 g/100 g of the composition. In another specific embodiment, the fucosylated oligosaccharide(s) is present in a total amount of 0.2 g/100 g of the composition. In a specific embodiment, the fucosylated oligosaccharide(s) is present in a total 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, according to at least 0.5 g/100 g, 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 at least 3 g/100 g of the composition.

In a specific embodiment, the fucosylated 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 young child, or child, at a total daily intake 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 minimal amount of fucosylated oligosaccharide is necessary to achieve a measurable, desired effect.

For preterm, low birth weight and small for gestational age infants, the daily dose of fucosylated oligosaccharides is preferably 0.05 to 1 g/kg body weight per day, preferably 0.06 to 0.9 g, or 0.07 to 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 yet another embodiment, the composition further comprises one or more OGMs selected from the group consisting of LNnT, 2'FL, diFL and 3'SL; for example, contains two or more OGMs selected from the group consisting of LNnT, 2'FL, diFL and 3'S, for example, three or more OGMs selected from the group consisting of LNnT, diFL, LNT, 6'SL and 3'SL , for example, additionally contains all of LNnT, diFL, LNT, 6'SL and 3'SL. As mentioned in Example 3, a mixture of 2'FL, LNnT, 3'SL and DiFL retains the best effect of 2'FL.

In a 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 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 another specific embodiment, the nutritional composition of the present invention contains a mixture of oligosaccharides consisting of 2'FL, 6'SL, LNT, DiFL and 3'SL.

In a specific embodiment of the present invention, the nutritional composition contains 2'FL and does not contain any other oligosaccharide. In a particular embodiment of the present invention, the nutritional composition comprises 2'FL and another oligosaccharide, preferably a human milk oligosaccharide, more preferably lacto-N-neotetraose (LNnT) or LNT (lacto-N-tetraose).

In a specific embodiment of the present invention, the nutritional composition contains 2'FL and LNnT.

In a specific embodiment, the nutritional composition of the present invention contains a mixture of oligosaccharides consisting of 2'FL and LNnT.

It may also be preferable that the composition contains only one OGM. Thus, in one embodiment, HMOs or their precursors other than 2'FL are absent from the nutritional composition or are present in a total amount of less than 0.1% (w/w), preferably, for example, less than 0.01% ( w/w) or more preferably, for example, less than 0.001%. As described in Example 3, the composition containing 2'FL as the only OGM showed the greatest effect.

In a related embodiment, HMOs other than 2'FL and LNnT are absent from the nutritional composition or are present in a total amount of less than 0.1% (w/w), preferably, for example, less than 0.01% (w/w) .) or more preferably, for example, less than 0.001%.

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 a probiotic strain, more preferably from 10e7 to 10e12 CFU, for example from 10e8 to 10e10 CFU, of a 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.

If the nutritional composition is a supplement, it may contain 2'-FL and no other additional nutrient other than the excipients necessary to provide a stable nutritional composition.

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'FL, wherein the supplement is in powder form and is offered in a sachet, preferably in a sachet with 1-10 g of 2'FL mixture per sachet, or in the form syrup, preferably a syrup with a total solids concentration of from 5 to 75 g/100 ml (from 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, for example at least 80% of the proteins are unchanged, for example 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, for example, at least 98% of proteins are unchanged. 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 thought 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. % proteins, in particular at least 98% of 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. Composition of infant formula according to example 1

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) 11.2 74.7 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 (OSM) 2FL (g) 0.075 0.5

Example 2

Table 2 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 2. Composition of infant formula according to example 2

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) 11.2 74.7 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 (OSM) 2FL (g) 0.15 1 LNnT (g) 0.075 0.5

Example 3. Direct effect of OGM

Purpose of the study

2'FL, LNT, LNnT, 3'SL, and DiFL were examined for their ability to directly protect and enhance the epithelial barrier before and after inflammatory challenge in an intestinal epithelial cell line coculture model containing Caco-2 and HT29-MTX cells.

Materials and methods

Human Caco-2 and HT29-MTX cell lines were obtained from the American Type Culture Collection (ATCC) and the European Authenticated Cell Culture Collection (ECACC), respectively. Caco-2 and HT29-MTX cells were cultured separately in culture flasks at 37°C in a humidified atmosphere containing 10% CO ² in Dulbecco's modified minimal essential medium (DMEM) supplemented with GlutaMAX (Invitrogen), 1% minimal essential medium , 100 μg/ml streptomycin, 100 IU/ml penicillin, and heat-inactivated fetal bovine serum (FBS; 15% for Caco-2 and 10% for HT29-MTX).

To prepare cocultures for epithelial barrier studies, each cell line was expanded in its respective flask until 90% confluent monolayers were achieved. The cell lines were then trypsinized once with trypsin. A coculture of Caco-2 and HT29-MTX was seeded at a density of 6 x 104/cm2 onto 1.12 cm2 (0.4 µm) Transwell polycarbonate semi-permeable membranes and grown in DMEM supplemented with GlutaMAX, 1% minimal growth medium, 100 µg /ml streptomycin, 100 IU/ml penicillin and 10% heat-inactivated PBS for 21 days.

On the day of the experiment, the medium was replaced with fresh medium at least 4 hours before treatment. Cocultures were first pretreated with 2′FL, LNT, LNnT, 3′SL, DiFL, or a combination of OGM (2′FL, 3′SL, 6′SL, and LNnT) by adding OGM to the apical compartment of the Transwell at a concentration of 30 mg/ml. After 48 hours of pretreatment, epithelial barrier dysfunction was induced by adding TNF-α (2.5 ng/ml) and IFN-γ (10 ng/ml) to the basolateral compartment of the Transwell for an additional 48 hours. At the end of the experiment, the movement of FITC-labeled dextran (4000 Da) from the apical compartment to the basolateral compartment was quantified over a period of 2 hours.

results

As can be seen from the results presented in Fig. 1, 2'FL had the greatest effect on reducing the movement of FITC-labeled dextran (4000 Da) from the apical compartment to the basolateral compartment. This provides evidence that 2'FL is the most successful OGM tested for protecting the gastrointestinal barrier and maintaining proper structure (barrier integrity) and function (reduced permeability, e.g., reducing pathogens, synanthropic bacteria, allergens, and toxic compounds migrating from the intestine through the intestinal barrier and decreased susceptibility to disease) of the gastrointestinal barrier before and after inflammatory provocation. The second largest effect was observed with the combination of 2'FL, 3'SL, 6'SL and LNnT.

Conclusion

The data presented shows that 2'-fucosyllactose (2'FL) is superior to other OGMs tested for improving the gastrointestinal barrier and, in particular, for improving gastrointestinal barrier protection, followed by an OGM mixture containing 2'FL , 3'SL, 6'SL and LNnT.

Example 4 Down-regulation of TNF-α receptor expression by 2'FL

Materials and methods

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 colorectal 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 were separately maintained in flasks. for tissue culture of 75 cm2 (Fischer Scientific) in a humidified atmosphere at 37°C and 10% CO2, saturated with air/water to 95%.

Cell culture model

Both Caco-2 and HT29-MTX cell lines were maintained in Dulbecco's modified minimal essential medium (DMEM; 11965092, Gibco) supplemented with 10% (v/v) heat-inactivated PBS (10270-106, Gibco) and 1% ( v/v) penicillin-streptomycin solution (P4333, Sigma). The growth medium was replaced at least twice a week. Cell lines were subcultured once a week 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 growth medium at 76:24 ratios to simulate colon, and seeded at a density of 6 × 10 cells/cm2 in a semipermeable membrane. made of Transwell™ polycarbonate with a pore size of 0.4 μM and a surface area of 1.12 cm2 (3460, Corning. Caco-2:HT29-MTX culture confluence and integrity were assessed by manually measuring transepithelial electrical resistance every week using a MillicellTM ERS-2 voltmeter Cells were used in experiments 21 days after seeding.

OGM treatments 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 and filtered using a 0.2 μM filter and maintained 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 ratio 0.55:0.7:0.09:0.05:0.18:0.06 or individual OGMs were additionally diluted in the medium for growth and added to the apical compartment of Transwell™ at various final concentrations 48 hours before exposure of the basolateral compartment to the cytokines TNF-α and IFN-γ for an additional 48 hours. Controls were cells not treated with OGM and cells not provoked by the cytokine (negative control), and cells not treated with OGM but provoked by the cytokine (positive control). Additional cells were stimulated with lactose as a control.

mRNA extraction

At the end of the experiment, the growth medium was removed and the cells were washed twice with Dulbecco's phosphate-buffered saline (Sigma). Cells were separated from the Transwell™ membrane by suspending the cells in RLT buffer (Qiagen) and scraping using pipette tips. The two wells were combined and total RNA was extracted using the MiniEasy RNA Extraction Kit (Qiagen) according to the manufacturer's instructions. RNA quality and quantity were measured using a 2100 Bioanalyzer (Agilent). Only RNA with a RIN greater than 9 was used for the experiment. cDNA was prepared using 100 ng of RNA and QuantaBio qScript Supermix cDNA according to the manufacturer's instructions.

qPCR analysis

Quantitative real-time PCR (qPCR) analysis for the human TNF-α receptor 1 gene (rTNF1; NM_001065.3) was performed using Taqman® Assay Preformed Primer (Thermo Fisher Scientific) on an ABI 7300 Real-Time PCR System (Applied Biosystem). The Taqman® pre-assay primer for gapdh (NM_001256799.2) was used as the constitutive gene. All analyzes were performed in triplicate and the difference between the cycle threshold (CT) values obtained for rTNF1 and the constitutive gapdh gene was calculated. The delta-delta-CT equation was used to compare rTNF1 gene expression among fold difference groups.

Statistical analyzes

Statistical differences between treatment types were determined using analysis of variance followed by post hoc multiple comparison analysis. Statistical significance was defined as p ≤ 0.05. Values in the text are means ± SD.

Results and description

TNF-α receptor down-regulating 2'FL on epithelial cells

This example demonstrates one of the mechanisms by which 2'FL exerts its beneficial effects on the gastrointestinal barrier. IFN-γ has been found to disrupt the integrity of the gastrointestinal tract barrier by up-regulating the expression of TNF-α receptors. This, in turn, allows the cytokine TNF-α to bind to its receptor (Wang et al., IFN-γ-induced TNFR2 Upregulation is Required for TNF-dependent Intestinal Epithelial Barrier Dysfunction. Gastroenterology. 2006 Oct; 131(4): 1153 -1163). The combined action of TNF-α and IFN-γ results in a cascade of epithelial cell signaling pathways that ultimately disrupts the integrity of junctional proteins and increased paracellular permeability (Wang et al, 2006). The present example demonstrates that 2'FL effectively inhibits IFN-γ-induced upregulation of TNF-α receptor expression by epithelial cells, thereby attenuating the inflammatory process and preventing profound changes in permeability. Cells treated with 2'FL showed lower expression of TNF-α receptor mRNA compared to positive control cells (Fig. 2), highlighting one of the mechanisms of action of 2'FL in reducing the inflammatory response and subsequent protection of the epithelial barrier.

Example 5

2'FL OGM increases the amount of KYNA (kynurenic acid) produced by the microbiota

Stool fermentation and quantitation of kynurenic acid

Stool samples from ten 3- to 4-month-old infants, seven breastfed (BF) and three formula-fed (FF) infants were used to inoculate carbohydrate-free (CHO-free) YCFA medium. The study design included the addition of various carbohydrates to a CHO-free YCFA medium at a final concentration of 0.5%, lactose (F1 control) or 2'FL alone, a combination of 2'FL and LnNT, or a combination of 2'FL, diFL, 3'SL , 6'SL, LNT and LNnT. Four replicate fermentations (F1–F4) were performed for each subject, and supernatants were collected after 24 h and 48 h. YCFA is a complex medium containing tryptone, yeast extract and various minerals, vitamins and short-chain fatty acids; however, in this case, typical CHO components (glucose, maltose, and cellobiose) were excluded.

For metabolite analysis, samples were extracted and divided into equal parts for analysis on LC/MS/MS and polar LC platforms. Proprietary software was used to match ions to an internal library of standards to identify metabolites and to quantify metabolites by peak area integration.

results

We assessed the effect on KYNA of adding fermented infant stool samples to the medium in vitro with various combinations of HGM. We found that, compared with lactose, 2'FL or the combination of 2'FL and LNnT significantly increased supernatant KYNA levels by 1.76-fold and 1.61-fold (P<0.01 and P<0.05, respectively) in fermented supernatant (Fig. 3). The 6-OHM mixture tended to increase KYNA levels (mean increase 1.56-fold compared to lactose, p=0.07). There are no significant differences between OGM treatments, indicating that the increase in KYNA levels is mainly due to 2′FL.

Example 6

6'SL+LNT and KYNA have an additive effect on GPR35 activation

Way

A cell line stably expressing GPR35 was propagated from frozen stock solutions according to standard procedures. Cells were seeded in a total volume of 20 μl in 384-well white-walled microplates and incubated at 37°C for the required time before testing. All tests were performed in duplicate. On each test day, five times the working intermediate was prepared in phosphate-buffered saline (PBS) for each concentration tested. 5 μl of quintuple sample was added to the cells and incubated at 37°C or room temperature for 90 minutes. The analytical signal was obtained by adding a single 12.5 or 15 μL (50% v/v) PathHunter Developing Reagent Mix (DiscoverX) followed by incubation for one hour at room temperature. Readings from the microplates were recorded after signal acquisition on a PerkinElmer EnvisionTM instrument for detecting chemiluminescent signals. Compound activity was analyzed using the CBIS data analysis package (ChemInnovation, California). Activity as a percentage was calculated using the following formula:

Activity in % = 100% x (average RLU of the test sample - average RLU of the vehicle control) / (average maximum value for the control ligand - average RLU of the vehicle control).

results

Stimulation of GPR35 with 6'SL and LNT together with 200 μM KYNA showed additive effects at OGM concentrations ranging from 940 μM to 2500 μM.

Claims (15)

1. The use of a nutritional composition containing 2'-fucosyllactose (2'FL) to improve the gastrointestinal tract barrier in an infant, which is a child under 12 months of age, in a young child, which is a child over 1 year to less than 3 years, or in a child aged from 3 years to less than 8 years, wherein said gastrointestinal barrier improvement represents improved barrier protection, and wherein 2'FL is present in an amount of 0.25-1.9 g/l or 0.15-1.5 g/100 g of composition based on dry matter weight, preferably in an amount of 0.75-1.65 g/ l or 0.19-1.5 g/100 g of composition based on dry matter weight. 2. The use of claim 1, wherein said use is intended 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. 3. Use 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. The use of any of the preceding claims, wherein said improvement, said prevention, and/or said protection is achieved by down-regulating the TNF-α receptor on epithelial cells. 5. Use according to any of the preceding claims, wherein said improvement, said prevention and/or said protection is achieved by increasing the production of kynurenic acid by the microbiota of an infant or young child consuming the nutritional composition, respectively a child aged three years to less than eight years consuming nutritional composition in the form of milk formula for children from 1 to 3 years old. 6. Use according to any of the preceding claims, wherein said nutritional composition further comprises one or more HMOs and/or precursors thereof, for example selected from the group consisting of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT) , 6'-sialyllactose (6'SL), difucosyllactose (diFL) and 3'-sialyllactose (3'SL). 7. Use according to any of the preceding claims, wherein said nutritional composition further comprises LNnT. 8. Use according to any of the preceding claims, wherein said nutritional composition further comprises 3'SL, 6'SL and LNnT. 9. Application according to any one of paragraphs. 1-3, wherein OGM or their precursors other than 2'FL are absent or present in said nutritional composition in a total amount of less than 0.1% (w/w), preferably, for example, less than 0.01% (w/w) ./mass.) or more preferably, for example, less than 0.001%. 10. Application according to any one of paragraphs. 6-8, wherein OGMs other than 2'FL and LNnT are absent or present in said nutritional composition in a total amount of less than 0.1% (w/w), preferably, for example, less than 0.01% (w/w). /wt.) or more preferably, for example, less than 0.001%. 11. Use according to any of the preceding paragraphs, wherein said nutritional composition is selected for administration to an infant. 12. Use according to claim 11, 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. 13. Use 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, a cereal-based infant composition base, fortifier or additive and is intended for administration to an infant or young child.