RU2781995C2 - Composition for use in reduction in nociception in infants and young chidren - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to compositions for infants and young children. A composition containing 6'-sialillactose (6'SL) and lacto-N-tetraose (LNT) is used for reduction in nociception in an infant or a young child. At the same time, the composition, in particular, is useful for reduction in stomach pain, for example pain related to infantile colic, irritable bowel syndrome (IBS), and/or recurrent abdominal pain (RAP), and, consequently, also contributes to reduction in crying periods and improvement of sleep quality and life quality in general in infants and young children.

EFFECT: invention allows for reduction in pain perception in infants and young children.

15 cl, 6 dwg, 1 tbl, 4 ex

Description

Scope of the invention

The present invention relates to compositions containing 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT) for use in reducing nociception in an infant or young child. The compositions are particularly useful in reducing abdominal pain, such as pain associated with intestinal discomfort and/or intestinal disorders, and therefore also help reduce crying periods and improve sleep quality and overall quality of life in infants and young children.

Prerequisites for the creation of the invention

Infants, including newborns, experience pain in the same way as adults, as noted, for example, in Goksan et al.; fMRI reveals neural activity overlap between adult and infant pain; eLife 2015;4:e06356. Other studies even suggest that infants may perceive pain more acutely than adults and older children.

Infants and young children may experience different types of pain, from acute pain to habitual pain, which can, for example, be caused by inflammation that can become chronic. In infants and young children, as in adults, pain is associated with reactions such as increased heart rate, faster and shallower breathing leading to reduced oxygen saturation, and increased blood pressure. Such responses, induced by prolonged or repeated exposure to pain, have been found to have a negative effect on the development of infants and young children and may lead, for example, to abnormalities in the development of the pain system such as hyperalgesia, or to dysregulation of stress-related hormones, such as increased cortisol production. See, for example, Ziraldo, Breanne, Infant Pain Management (2010); Senior Honors Theses; 198, which may be obtained from Liberty University, Virginia, USA, and is available as of the date of application at http://digitalcommons.liberty.edu/honors/198.

Infants and young children are particularly susceptible to certain sources of pain, such as abdominal pain. Causes of abdominal pain include abdominal discomfort and abdominal disorders. One example of a common cause of discomfort is, for example, colic. The cause of colic is not well understood, but appears to be related to intestinal hypersensitivity to pain, so intestinal distension caused by the normal passage of gases or feces leads to pain in colic infants, provoking prolonged periods of crying, poor sleep, and reduced quality of life as for the baby and for the parents. Abdominal pain can also result from other causes of discomfort, such as stool patterns, exposure to new foods, bloating, and cramps. Disorders such as inflammatory bowel disease (IBD), diarrhea such as infectious diarrhea, necrotizing enterocolitis (NEC), and functional abdominal pain disorders also occur. Inflammatory bowel disease (IBD) is a serious chronic and destructive disorder of the gastrointestinal tract. It includes Crohn's disease (CD) and ulcerative colitis (UC). Functional abdominal pain disorders and non-destructive gastrointestinal disorders and include irritable bowel syndrome (IBS), abdominal migraine and functional abdominal pain not otherwise specified (FAB-NOS). An additional detailed definition of functional abdominal pain disorders is provided by Hyams et al.; Childhood Functional Gastrointestinal Disorders: Child/Adolescent; Gastroenterology 150 (2016):1456–1468.

Thus, reduction of nociception is desirable for infants and young children. However, the side effects associated with pain medications can be even more problematic in infants and young children than in adults. Thus, it is of particular interest to identify such agents for reducing nociception in infants and young children that are non-pharmacological and associated with low risk to the infant or young child. Of particular advantage would be the identification of ingredients specifically selected for human infants and/or young children and capable of reducing nociception.

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 breast milk or infant formula with certain ingredients. In such cases, it would be even more preferable to provide means to reduce nociception and thereby reduce the incidence of pain in infants and young children through nutritional intervention.

The prior art has already investigated the effect of food ingredients such as human breast milk oligosaccharides on pain. US2016/0243139 discloses the use of synthetic compositions containing one or more human milk mono- or oligosaccharides for the treatment of visceral pain. A wide variety of human milk mono- and oligosaccharides can be used within the meaning of this document, with 2'-FL, 3'FL, DFL, LNnT, 3'-SL, 6'-SL or LNFP-1 being preferred and most preferred being mixture of 2'-FL and LNnT or LNT.

WO2016/139329 relates to compositions for use in improving stool consistency or frequency in infants or young children, such effects being associated with the prevention and/or treatment of colic and/or intestinal discomfort. To achieve this beneficial effect, a nutritional composition containing at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide is used. Fucosylated oligosaccharide and N-acetylated oligosaccharide may be selected from a wide range. The most preferred fucosylated oligosaccharide is 2-FL and the most preferred N-acetylated oligosaccharide is LNnT.

It would be useful to further optimize the impact of nutritional compositions on reducing nociception in all infants and children.

Certain specific groups of infants and young children are in particular need of compositions capable of reducing the perception of pain. Such infants and young children are, for example, premature infants, a low birth weight infant, and/or stunted infants or young children. Indeed, such infants often experience adverse medical conditions and require significantly more frequent medical attention than term infants and normally developing infants. Many of these medical interventions are unfortunately painful for the infant or young child, who thus faces recurring and sometimes severe pain. For such infants, it is particularly preferred to supplement pharmacological pain relief with nutritional compositions capable of reducing nociception.

There is a clear need to develop suitable methods for reducing nociception in infants and young children.

There is also a need to provide such health benefits in a manner that is particularly suitable for younger subjects (infants and young children), the method need not include classical pharmaceutical intervention as these infants or young children are particularly vulnerable.

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

There is also a need to provide such benefits in a manner that will keep the cost of such provision reasonable and affordable to the majority.

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

Statement of the Invention

The present inventors have found that a composition containing 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT) can advantageously be used to reduce nociception in an infant or young child. Without being bound by any theory, these oligosaccharides are believed to act synergistically to achieve this beneficial effect via activation of the G protein coupled receptor 35 (GPR35). It has been found that 6'SL and LNT are able to trigger the activation of the GPR35 receptor alone and synergistically trigger the activation of such a receptor when used in combination. On the contrary, contrary to what would be expected from the prior art, another human milk oligosaccharide (such as 3'-sialyllactose (3-SL), lacto-N-neotetraose (LNnT), 2'-fucosyllactose (2 -FL) and difucosyllactose) does not activate the GPR35 receptor.

The GPR35 receptor, which is highly expressed, for example in the gastrointestinal tract, has been identified in numerous studies as being involved in the reduction of nociception, and its activation has been described as inducing analgesia (see, e.g., Resta et al.; Kynurenic acid and zaprinast induce analgesia by modulating HCN channels through GPR35 activation; Neuropharmacology 108 (2016), 136–143; Cosi et al.; G-protein coupled 35 (GPR35) activation and inflammatory pain: Studies on the antinociceptive effect of kynurenic acid and zaprinast, Neuropharmacology 60 (2011), 1227–1231; and Zhao et al.; Targetting of the Orphan Receptor GPR35 by Pamoic Acid: A Potent Activator of Extracellular Signal-Regulated Kinase and β-Arrestin2 with Antinociceptive Activity; Mol Pharmacol 78(2010):560 –568). Based on such studies, compounds capable of activating GPR35 receptors have been considered for use as analgesic drugs, and screening for GPR35 activators has been proposed as a way to identify new analgesic drugs. Thus, activation of the GPR35 receptor by 6'SL and LNT provides a solid basis for the use of compositions containing such human milk oligosaccharides to achieve the beneficial effect of reducing nociception. The synergistic activating effect of 6'SL and LNT on GPR35 also provides a solid basis for the excellent nociceptive effect of this human milk oligosaccharide when used in combination.

Accordingly, the present invention thus provides a nutritional composition comprising 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT) for use in a method for reducing nociception in an infant or young child.

The present invention also provides a 1 to 3 year old infant formula containing 6'-sialyllactose (6'SL) and lacto-N tetraose (LNT) for use in a method for reducing nociception in an infant.

Nutritional composition, respectively milk formula for children from 1 to 3 years, in particular, is effective for use in the method

- reduction of pain in the abdomen;

- reduction of pain associated with discomfort in the gastrointestinal tract and/or disorders of the gastrointestinal tract;

- reduction of periods of crying;

- improving the quality of sleep; and

- improving the quality of life

in an infant or young child, respectively, in a child.

The invention also relates to the use of a nutritional composition containing 6'-sialyllactose (6'SL) and lacto-N tetraose (LNT) in a method

- reduction of periods of crying;

- improving the quality of sleep; and

- improving the quality of life

in an infant or young child; and

nutritional composition in the form of milk formula for children from 1 to 3 years, containing 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT), in the method

- reduction of periods of crying;

- improving the quality of sleep; and

- improving the quality of life

The child has.

Brief description of graphic materials

Fig. 1 is the in vitro GPR35 response to LNT in the cellular assay from Example 2. The EC50 value is 4.5 mM.

Fig. 2 is the in vitro GPR35 response to 6'SL in the cellular assay from Example 2. The EC50 value is 6.5 mM.

Fig. 3 is the in vitro GPR35 response to a mixture of human milk oligosaccharides (HMO) consisting of a mixture of 6'SL and LNT in the cell assay from Example 2. The EC50 value is 2.2 mM.

Fig. 4 - analysis of 6'SL (A) and LNT (B) by high performance anion exchange chromatography with pulsed amperometric detection (HPAOC-IAD) before and after treatment with neuraminidase (6'SL) or β-galactosidase (LNT), showing complete cleavage of both HMOs

Fig. 5 shows the in vitro GPR35 response to a mixture of HMOs consisting of a mixture of 6'SL and LNT in the cellular assay from Example 2 (bars shaded with horizontal lines) compared to a mixture of cleaved 6'SL and LNT (bars hatched with vertical lines). The graph shows that upon cleavage of 6'SL and LNT, the activity of activating the receptor is lost.

Fig. 6 - amplitude of contraction measured as a function of colorectal dilatation in non-sensitized mice (AIN93; dots), in sensitized mice (was:AIN93; squares), and in sensitized mice injected with 6'SL and LNT (was:LNT+6'SL ; rhombuses).

Detailed description of the invention

In this document, the following terms have the following definitions.

The term "infant" means a child under the age of 12 months. The term "young child" means a child between the ages of one and less than three years (also called a toddler). The expression "child" means between the ages of three and seven years.

"Infant or young child born by caesarean section" means an infant or young child born by caesarean section. This means that the baby or young child was not born through a vaginal birth.

"Infant or young child born by vaginal delivery" means an infant or young child who was born by vaginal delivery and not by caesarean section.

"Premature" or "premature" means an infant or young child who is born prematurely. Essentially, this 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 delivery or limited fetal growth. 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 LBW);

- 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 birth weight of less than 1000 g (called "extremely low birth weight" or LUBR).

"Infant born small for gestational age (SGA)" means a baby with birth weight less than the 10th percentile for babies of the same gestational age.

The expression "nutritional composition" means the composition that feeds the subject. This nutritional composition is usually intended for oral or intravenous use and usually 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 is unlikely to cause allergic reactions.

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

The expression "infant formula" as used herein refers to a food product that is specifically intended to be eaten by infants during the first months of life and that by itself satisfies 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 formula). It also refers to a nutritional composition intended for infants as defined in the Codex Alimentarius (Codex STAN 72-1981) and Infant Specialties (including the entry Food for Special Medical Purpose). The expression "infant formula" covers both "initial formula" and "advance-level formula" or "pre-fed formula".

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

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

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

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

The term "fortifier" refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.

The expression "weaning period" means the period during which the diet of an infant or young child replaces mother's milk with another food.

The expressions "aged days/weeks/months/years" and "days/weeks/months/years after birth" can be used interchangeably.

The expression "decreased nociception" includes one or more of the following:

- reduction of abdominal pain;

- reduction of pain associated with discomfort in the gastrointestinal tract and/or disorders of the gastrointestinal tract;

- reduction of periods of crying;

- improving the quality of sleep; and

- improving the quality of life.

By "mother's milk" is meant the mother's breast milk or colostrum.

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

The term "HMO" refers to the oligosaccharide(s) of breast milk. These carbohydrates are highly resistant to enzymatic hydrolysis, indicating that they may have significant functions not directly related to their calorie content. First of all, 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 HMO 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 bonds between them, resulting in the presence of a huge number of different oligosaccharides in breast milk - at the moment it was more than 130 such structures have been identified. Nearly all of them have a lactose moiety at their reducing end, while sialic acid and/or fucose (if present) end up at the non-reducing ends. HMOs can be acidic (eg, charged sialic acid-containing oligosaccharide) or neutral (eg, fucosylated oligosaccharide).

"Fucosylated oligosaccharide" is an oligosaccharide having a fucose residue. 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, fucosyl lacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyl lacto-N-hexaose I, difucosyl lacto-N-neohexaose II and any combination of them. Although the authors do not wish to be bound by any 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 can prevent and/or limit the action of infection-causing pathogens (of viral or bacterial origin) or of their secreted components (e.g., toxins), primarily by eliminating their binding to natural ligands, and, without regard to any theory , it is believed that this will reduce the risk of infections/inflammations, and in particular the risk of infections and/or inflammations of the lower respiratory tract (LRT)/ear. Additionally, it is believed that fucosylated oligosaccharides stimulate the growth and metabolic activity of specific symbiotic microbes that reduce the inflammatory response and create 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 homology of form, since 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 any theory, it is believed that the 2'-fucosyl epitope of these fucosylated oligosaccharides is particularly characteristic of pathogens (or components secreted by them) that cause LRT and/or ear infections.

The term "N-acetylated oligosaccharide(s)" encompasses both "N-acetyllactosamine" and "oligosaccharide(s) containing N-acetyllactosamine". They are neutral oligosaccharides having an N-acetyllactosamine residue. Suitable examples are LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), and any combination 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-dekaose.

The terms "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".

"HMO precursor" is a key compound that is included in the production of HMO, 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 has an acidic character. 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) or liquid form. The amount of various ingredients (eg, oligosaccharides) may be expressed as g/100 g of the composition on a dry weight basis if presented in solid form, such as 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 that can be made from a powder after being dissolved in a liquid, e.g. milk, water, etc., e.g. up to 3 years, or baby food cereal, or any other formula intended for baby food).

The term "prebiotic" refers to non-digestible carbohydrates that benefit the host by selectively stimulating the growth and/or activity of beneficial 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 organism. (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 yeasts.

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

Unless otherwise indicated, 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 of the embodiments described herein, may contain the 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 or essentially consist of them.

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

Thus, the first object of the present invention is a nutritional composition containing 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT) for use in reducing nociception in an infant or young child.

The second object of the present invention is a milk formula for children from 1 to 3 years, containing 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT), for use in reducing nociception in a child.

In order to achieve such advantages, the nutritional composition, respectively milk formula for children from 1 to 3 years old, of the invention is preferably intended for use in the method

- reduction of abdominal pain in the subject;

- reducing pain associated with gastrointestinal discomfort and gastrointestinal disorders, wherein the gastrointestinal discomfort is preferably caused by colic, bloating and/or cramps, and wherein the gastrointestinal disorder is preferably selected from inflammatory bowel disease (IBD), diarrhea eg infectious diarrhea, necrotizing enterocolitis (NEC) and functional abdominal pain disorders, preferably selected from irritable bowel syndrome (IBS), abdominal migraine and functional abdominal pain without further specification (FAB-NOS);

- reduction of periods of crying in a subject, preferably reduction of periods of crying in a subject experiencing pain, preferably in a subject experiencing abdominal pain, most preferably in a subject experiencing pain associated with gastrointestinal discomfort and/or gastrointestinal disorders, as defined above;

- improving the quality of sleep in a subject, preferably in a subject experiencing pain, more preferably in a subject experiencing abdominal pain, most preferably in a subject experiencing pain associated with gastrointestinal discomfort and/or gastrointestinal disorders as defined above; and

- improving the quality of life of a subject, preferably a subject experiencing pain, more preferably a subject experiencing abdominal pain, most preferably a subject experiencing pain associated with gastrointestinal discomfort and/or gastrointestinal disorders as defined above,

wherein the subject is an infant or young child, respectively a child.

For the purpose of the present invention, pain reduction is achieved by reducing the perception of pain. The compositions of the present invention affect the mechanism of nociception and, therefore, are able to reduce the perception of pain, regardless of the origin of such pain. Thus, they are effective in reducing, for example, the perception of pain arising in connection with the performance of a medical or surgical event or in connection with any injury, disease, disorder and/or discomfort. The mechanism for reducing pain perception is different from the reduction in pain that can be achieved by treating the injury, disease, disorder, or discomfort that causes the pain. Thus, it is intended that the composition of the present invention is not intended to treat the injury, disease, disorder or discomfort that causes pain, but only to facilitate the perception of pain by the subject.

In a particularly advantageous embodiment of the present invention, 6'SL and LNT are present in a nutritional composition or formula for children 1 to 3 years of age in certain specific amounts.

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

LNT may be present in the nutritional composition or milk formula for children from 1 to 3 years in the amount of 0.004-2.3 g/100 g of the composition, calculated on the dry weight, LNT may be present in the amount of 0.008-1.2 g/100 g of the 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, the LNT is present in an amount of 0.38 g/100 g of the composition. In another specific embodiment, the LNT is present in an amount of 0.08 g/100 g of the composition.

LNTs 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 preferred embodiment of the invention, a nutritional composition or milk formula for children from 1 to 3 years old may include 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, such as 0.04 or 0.5 g/l.

A nutritional composition or milk formula for children from 1 to 3 years of age according to the invention may contain 0.004-3.8 g of 6'SL per 100 g of the composition based on the dry 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 of the composition, calculated on the dry weight, for example, 0.18 g or 0.04 g per 100 g of the composition, calculated on mass of dry matter.

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

In a specific aspect, 6'SL and LNT included in a nutritional composition or milk formula for children from 1 to 3 years in accordance with the invention, as a rule, are present in a ratio of 6'SL : LNT from 3 : 1 to 1 : 3, for example from 2:1 to 1:2 or 2:1 to 1:1. In a particularly advantageous embodiment, this ratio is 2:1 or about 2:1, preferably such a ratio is 1:1 or about 1:1.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

- 6'SL is present in an amount of 0.24 or 0.05 g/l of the composition and/or in an amount of 0.18 or 0.04 g/100 g of the composition, based on the weight of dry matter; 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 the composition or 0.08 g/100 g of the composition, based on the weight of dry matter.

In a specific embodiment, 6'SL is contained in the 1 to 3 year old nutritional composition or formula of the present invention in such an amount that a normal intake of the 1 to 3 year old nutritional composition or formula would be provided for an infant or toddler. , respectively, of 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 0.02-0.9 g, for example 0.024-0.7 g, per day.

In a specific embodiment, LNT is contained in the 1 to 3 year old infant formula or formula of the present invention in such an amount that a normal intake of the 1 to 3 year old infant formula or formula will be provided for an infant or toddler, respectively. child, when consuming 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 specific embodiments of the present invention, the nutritional composition, respectively milk formula for children from 1 to 3 years old, of the present invention may contain additional breast milk oligosaccharides. Even if such human milk oligosaccharides are not effective in activating GPR35 receptors, they can be added for other health benefits.

Thus, in a specific embodiment, the nutritional composition or milk formula for children from 1 to 3 years old contains at least one fucosylated oligosaccharide. It may contain one or more types of fucosylated(s) oligosaccharide(s). The fucosylated oligosaccharide(s) may in fact be selected from a list including 2'-fucosyllactose, 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 (e.g. fucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II), difucosyl lacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosylacto-N-neohexaose I, difucosyl lacto-N-neohexaose II, fucosyl-para-lacto-N-hexaose, three -fuco-para-lacto-N-hexaose I and any combination thereof.

In some specific embodiments, the fucosylated oligosaccharide comprises a 2'-fucosyl epitope. It may, for example, be selected from a list including 2'-fucosyllactose, 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-para-lacto-N-hexaose, and any combination thereof.

In a specific embodiment, the nutritional composition, respectively milk formula for children from 1 to 3 years, in accordance with the invention contains 2'-fucosyllactose (or 2FL, or 2'FL, or 2-FL, or 2'-FL). In a particular embodiment, no other type of fucosylated oligosaccharide other than 2'-fucosyllactose is used, i. e. nutritional composition or milk formula for children from 1 to 3 years 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(them) can be produced by biotechnological means using specific fucosyltransferases and/or fucosidases, either through the use of an enzyme-based fermentation method (recombinant or natural enzymes) or a microbiological fermentation method. In the latter case, either the microorganisms can express their natural enzymes and substrates, or it is possible to create microorganisms that produce the corresponding substrates and enzymes. Single microorganism cultures and/or mixed cultures can be used. Fucosylated oligosaccharide formation can be initiated by acceptor substrates starting at any degree of polymerization (DP) from DP=1 onwards. In an alternative embodiment, fucosylated oligosaccharides can be obtained from lactose and free fucose using chemical synthesis. Fucosylated oligosaccharides are also available from, for example, Kyowa, Hakko, Kogyo, Japan.

In another specific aspect of the invention, the nutritional composition or milk formula for children from 1 to 3 years old may contain at least one N-acetylated oligosaccharide in addition to LNT. It may contain one or more types of N-acetylated oligosaccharides. In some specific embodiments, the N-acetylated oligosaccharide is lacto-N-neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH), or any combination thereof. In some specific embodiments, the N-acetylated oligosaccharide is LNnT. In some specific embodiments, where LNnT is present, the nutritional composition or milk formula for infants 1 to 3 years of age contains both LNT and LNnT in a LNT : LNnT ratio of 5:1 to 1:2, or 2:1 to 1 : 1, or from 2: 1.2 to 2: 1.6.

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, LNnT can be obtained by chemically converting ketohexoses (eg, fructose), either free or associated with an oligosaccharide (eg, lactulose), to N-acetylhexosamine or an 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 nutritional composition or milk formula for children from 1 to 3 years of age contains 2'-fucosyllactose (2FL) and/or lacto-N-neotetraose (LNnT).

In a specific embodiment, the nutritional composition or milk formula for children from 1 to 3 years in accordance with the invention may contain other (s) sialylated (s) oligosaccharide (s) in addition to 6'SL, such as 3'-sialyllactose (3-SL).

The sialylated oligosaccharide(s) may be isolated from a natural source such as animal milk by chromatography or filtration. In an alternative embodiment, they can be produced by biotechnological means using specific sialyltransferases or sialidases, neuraminidase, 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. Single-species cultures or mixed cultures can be used. The formation of sialyl oligosaccharides can be initiated by acceptor substrates starting at any degree of polymerization (DP) from DP = 1 onwards. In an alternative embodiment, sialyllactoses can be obtained 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.

The nutritional composition or milk formula for children from 1 to 3 years in accordance with the present invention may also contain other types of oligosaccharide(s) (i.e. different from the above human milk oligosaccharides) and/or at least fiber(s), and/or at least its (their) predecessor(s). The other oligosaccharide and/or fiber and/or precursor thereof may be selected from a list including galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, and any combination thereof. They may be present in an amount of 0 to 10% by weight of the composition. In a specific embodiment, the nutritional composition or infant formula for children from 1 to 3 years 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 from 1 to 3 years in accordance with the invention include combinations of FOS with inulin, such as the product sold by BENEO under the trademark Orafti, or polydextrose sold by Tate & Lyle under the trademark STA-LITE®.

The nutritional composition or milk formula for children from 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 breast milk oligosaccharide precursor is sialic acid, fucose, or a mixture thereof. In some specific embodiments, the implementation of the composition contains sialic acid.

In specific examples, the nutritional composition or milk formula for children from 1 to 3 years old contains from 0 to 3 g/l of the 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 oligosaccharide precursor(s). A composition according to the invention may comprise 0 to 2.1 g of oligosaccharide precursor(s) per 100 g of composition on a dry weight basis, for example 0 to 1.5 g, or 0 to 0.8 g, or 0 to 0.15 g of oligosaccharide precursor(s) per 100 g of composition based on dry weight.

The nutritional composition or milk formula for children from 1 to 3 years 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, which 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 implementation of the probiotic is a probiotic bacterial strain. In some specific embodiments, the implementation is, in particular, strains of Bifidobacteria and/or Lactobacilli.

Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103, from Valio Oy, Finland, under the trademark 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 inter alia by Christian Hansen, Denmark, under the trademark 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 trademark Bb-03; Bifidobacterium breve sold by Morinaga under the trademark M-16V; Bifidobacterium infantis marketed by Procter & Gambie Co. under the Bifantis trademark; and Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trademark R0070.

A nutritional composition or milk formula for children from 1 to 3 years of age according to the invention may comprise from 10e3 to 10e12 cfu of the probiotic strain, more preferably from 10e7 to 10e12 cfu, for example from 10e8 to 10e10 cfu, of the probiotic strain per gram of composition, by 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.

The nutritional composition according to the invention may, for example, be an infant formula, a starter infant formula, a formula for complementary foods or a follow-up formula, an infant formula for children from 1 to 3 years old, an infant formula, a cereal-based infant formula, 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, which may be 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 can be a human milk fortifier (eg human breast milk fortifier) or a formula fortifier such as an infant formula fortifier or formula fortifier for formula fed/follow-up formula.

If the nutritional composition is a supplement, it may be provided in unit dosage form. In such cases, it is particularly useful to determine the amount of N-acetylated oligosaccharides and optionally other oligosaccharides in relation to the daily dose for administration to an infant or young child, as described above.

The nutritional composition of the present invention may be in solid (eg powder), liquid or gelatinized form. In a particular embodiment, the nutritional composition is a supplement containing 6'SL and LNT in a ratio of 1:3 to 3:1, the supplement being in powder form and offered in a sachet, preferably in a 1-10 g sachet. mixtures of 6'SL and LNT in a sachet, or in the form of a syrup, preferably a syrup with a total solids concentration of 5 to 75 g/100 ml (5 to 75% (w/v)). If the additive is in the form of a powder, it may include a carrier. However, it is preferable that the carrier in the additive was absent. If the supplement is in the form of a syrup, the HMO is preferably dissolved or suspended in water acidified with citrate.

The nutritional composition or milk formula for children from 1 to 3 years of age according to the invention usually contains a protein source. Protein can be contained in an amount of 1.6 to 3 g per 100 kcal. In some embodiments, particularly when the composition is for preterm 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 below 2.0 g per 100 kcal, such as 1.8 to 2 g/100 kcal, or the protein may be present in an amount below 1.8 g per 100 kcal.

The type of protein is not considered critical to the present invention, provided that the minimum requirements for the content of essential amino acids are met and satisfactory growth is ensured. Thus, protein sources based on whey, casein and mixtures thereof, as well as protein sources based on soy, 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 preferred embodiments, the protein source is whey-dominated (ie, more than 50% of the proteins are from whey proteins, eg, 60% or 70%).

The proteins may be native or hydrolysed, or they may be a mixture of native and hydrolyzed proteins. The term "native" means that the bulk of the proteins are native, i. their molecular structure is not altered, e.g. at least 80% of the proteins are not altered, e.g. at least 85% of the proteins are not altered, preferably at least 90% of the proteins are not altered, even more preferably at least 95% of the proteins are not altered, e.g. at least 98% of the proteins are changed. In a specific 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 cleaved into its constituent amino acids. Proteins may be fully or partially hydrolysed. It may be desirable to administer partially hydrolysed proteins (degree of hydrolysis between 2 and 20%), for example, to infants or young children who are 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 prepared by enzymatic hydrolysis of a whey fraction in one or more steps. If the whey fraction used as starting material is substantially free of lactose, it has been found that the blocking of protein lysine occurs to a much lesser extent during hydrolysis. This allows the degree of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, up to about 7 wt.% lysine, which significantly improves the nutritional quality of the protein source.

In one embodiment of the invention, at least 70% of the proteins are hydrolyzed, preferably at least 80% of the proteins are hydrolyzed, e.g. at least 85% of the proteins are hydrolyzed, even more preferably at least 90% of the proteins are hydrolyzed, e.g. at least 95% of the proteins are hydrolyzed in particular, at least 98% of the proteins are hydrolysed. 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 the protein can be from 8 to 40, or from 20 to 60, or from 20 to 80, or more than 10, 20, 40, 60, 80, or 90.

In an alternative embodiment, the protein component may be replaced with a mixture or an artificial amino acid, such as for preterm or low birth weight infants.

In a specific embodiment, the nutritional composition or milk formula for children from 1 to 3 years 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 milk formula for children from 1 to 3 years.

The nutritional composition or milk formula for children from 1 to 3 years of age in accordance with the present invention usually contains a source of carbohydrates. This is especially 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 milk formula for children from 1 to 3 years in accordance with the present invention usually 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 lipid source can 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 sunflower oil, and high oleic safflower oil, medium chain triglyceride oil. You can also add 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 finished form, such as fish oils or microbial oils. The ratio of n-6 to n-3 fatty acids in the fat source may be from about 5:1 to about 15:1; for example, from about 8:1 to about 10:1.

The nutritional composition or milk formula for children from 1 to 3 years of the invention may also contain all the vitamins and minerals considered indispensable in the daily diet, in nutritionally significant amounts. 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 may vary depending on the intended population.

If necessary, the nutritional composition or milk formula for children from 1 to 3 years 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 milk formula for children from 1 to 3 years of the invention may also contain other substances that may have a beneficial effect, such as lactoferrin, nucleotides, nucleosides, and the like.

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

The nutritional composition or milk formula for children from 1 to 3 years of age in accordance with the invention can be obtained by any suitable method. Next, the composition will be described on a specific example.

For example, a formula, such as infant formula, can be made by mixing a protein source, a carbohydrate source, and a fat source in appropriate proportions. At this stage, emulsifiers can be included if they are used. Vitamins and minerals can be added at this stage, but are usually added later to prevent thermal decomposition. Any lipophilic vitamins, emulsifiers, and the like may be dissolved in the fat source prior to mixing. Water, preferably purified by reverse osmosis, can then be mixed in to form a liquid mixture. The temperature of the water conveniently ranges from about 50° C. to about 80° C. to facilitate dispersion of the ingredients. Commercially available thinners can be used to form the 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 end 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., over a period of time from about 5 seconds to about 5 minutes. The heating can be carried out by steam injection 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 pressure in the range of 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 point, the pH and solids content of the homogenized mixture is adjusted for convenience.

If the end 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 to powder. The moisture content of the powder should be less than about 5% by weight. Fucosylated oligosaccharide(s) and N-acetylated oligosaccharide(s) can also or alternatively be added at this stage by dry mixing or by mixing them in the form of a crystal syrup together with the probiotic (- them) strain(s) (if used), and the mixture is spray dried or freeze dried.

If a liquid composition is preferred, the homogenized mixture may be sterilized and then aseptically filled into suitable containers, or may be first filled into containers and then sterilized by autoclaving.

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 traditional 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, colorants, wetting agents, fillers, and the like.

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 agencies, such as the recommended intakes (RDA) of the United States.

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

The nutritional composition of the present invention may also be used in infants or young children delivered by caesarean section or by vaginal delivery.

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

The age at which a 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 can also be given during the first week of an infant's life, 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 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 when the baby was born prematurely.

In one embodiment, the composition of the invention is given to an infant or young child as a composition to supplement mother's 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 such a breast milk feeding period, or is given during such a breast milk feeding period. In another embodiment of the invention, the composition is given to an infant or young child as the sole or main nutritional composition for at least one period of time, for example after 1, 2 or 4 months of age, 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 subject's nutritional needs). 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 complementary formula/follower formula.

Examples

The following examples demonstrate some specific embodiments of the composition for use in accordance with the present invention. The examples are for illustrative purposes only and should not be construed as limitations of the present invention.

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 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 fifty Mn (mcg) eight fifty Se (mcg) 2 13 Vitamin A (µg RE) 105 700 Vitamin D (mcg) 1.5 ten Vitamin E (mg TE) 0.8 5.4 Vitamin K1 (mcg) eight 54 Vitamin C (mg) ten 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.15 1.0 Niacin (mg) one 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 fifteen Choline (mg) ten 67 Fe (mg) 1.2 eight I (µg) fifteen 100 Cu (mg) 0.06 0.4 Zn (mg) 0.75 5 Oligosaccharides
(OGM) 6'SL (g) 0.035 0.24 LNT (g) 0.07 0.45

Example 2

Principle of analysis

Six human milk oligosaccharides (LNnT, LNT,
2-FL, di-FL, 6'SL and 3-SL) for their ability to activate the GPR35 receptor in an in vitro assay. Screening was performed on the DiscoverX instrument (Fremont, California, USA) using the PathHunter analytical system with β-arrestin. This assay monitors the activation of GPR35 expressed in cultured cells using complementation of enzyme fragments with β-galactosidase (β-Gal) as a functional reporter. The enzyme is separated into two inactive complementary parts, EA and PK, which are expressed in the cell as fusion proteins. EA is fused to β-arrestin and PK is fused to GPR35. If GPR35 is activated and β-arrestin is recruited to the receptor, PK and EA complementation occurs to restore β-Gal activity, which is measured using chemiluminescent detection reagents.

Ways

The GPR35 cell line was propagated from the original frozen cultures and cultured 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 prior to testing. All tests were performed in duplicate.

HMO samples were delivered to DiscoverX as concentrated stock solutions in phosphate buffered saline (PBS). On the day of the test, a 5-fold working solution of the intermediate in PBS was prepared. Five μl of a 5-fold sample was added to the cells and incubated at room temperature for 3–5 hours.

An assay signal was generated by a single addition of 12.5 μl (50% v/v) PathHunter Detection Reagent Mix followed by one hour incubation at room temperature. Microplate readings were recorded after signal acquisition on a PerkinElmer Envision™ chemiluminescent signal detection instrument.

Zaprinast (which is known as a GPR35 activator) was used as a positive control.

Percentage activity was calculated using the following formula:

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

Dose-response plots were generated using iterative 3-fold dilutions of the highest concentration tested (10 mM).

conclusions

LNT activated GPR35 with an EC50 value of 4.5 mM (FIG. 1). 6'SL activated GPR35 with an EC50 value of 6.5 mM (FIG. 2). For 3SL, 2FL, di-FL and LNnT, no GPR35 activation was obtained.

Since LNT and 6'SL were found to be activators, the two HMOs were tested in combination. An equimolar mixture of 6'SL and LNT activated GPR35 with an EC50 value of 2.2 mM (FIG. 3). These results demonstrate synergy between 6'SL and LNT.

A mixture of all six HMOs was also tested in an equimolar ratio with the same results as for the combination of LNT and 6'SL. These results demonstrate that the synergistic effect of LNT and 6'SL is maintained even when mixed with other human milk oligosaccharides.

Example 3

The following tests were carried out by digesting LNT and 6'SL and using the digested material to perform a GPR35 activation assay to validate the test results from example 2.

Cleavage of LNT by β-galactosidase

LNT was digested with β-galactosidase purified from Aspergillus black (Megazyme, ref: E-AGLAN) using the following protocol: in a 1.5 ml tube with external thread and screw cap, 84 mg of LNT were mixed in 900 μl of 100 mM acetate buffer (pH 4.5) and 100 µl of enzyme (400 U). This tube was incubated for 72 hours at 55°C in a ThermoMix shaker with agitation speed of 450 rpm. Enzyme inactivation was carried out by heat treatment for 5 minutes at 100°C. The inactivated tube was stored at -20°C until further analysis.

Cleavage of 6'SL by neuraminidase

6'SL was digested with neuraminidase purified from Clostridium enteritis (Megazyme, ref: E-SIALCP) using the following protocol: in a 1.5 ml tube with external thread and screw cap, 78 mg of 6'SL were mixed in 900 μl of 50 mM phosphate buffer (pH 6.0) and 50 µl of enzyme (50 U). This tube was incubated for 72 hours at 37°C in a ThermoMix shaker with agitation speed of 450 rpm. Enzyme inactivation was carried out by heat treatment for 5 minutes at 100°C. The inactivated tube was stored at -20°C until further analysis.

Split control

The authors confirmed the complete cleavage of LNT and 6'SL by quantifying them using high performance anion exchange chromatography with pulsed amperometric detection (HEAOC-IAD). The results of this analysis are shown in Fig. 4. Peaks clearly confirm complete cleavage of both LNT and 6'SL.

Sample preparation, test samples and results

Samples were diluted in 1x PBS (pH ~ 7) at 5x concentration and sent to DiscoverX. The GPR35 activation assay was performed as described in Example 2. The results are shown in FIG. 5. The response of GPR35 to the cleaved and uncleaved mixture of LNT and 6'SL showed a response with the uncleaved mixture and a background response to the cleaved mixture, thus demonstrating the specificity of the GPR35 response to LNT and 6'SL.

Example 4

The effectiveness of the combination of 6'SL and LNT in reducing nociception has been confirmed in vivo.

Water avoidance stress (WAS) and nutrition interventions

C57Bl.6/J mice were purchased from Janvier SA (Les Genestes-Saint-Iles, France). Upon arrival, they were housed in polypropylene cages in a temperature-controlled room with a 12-hour light cycle for 1 week prior to electrode implantation (see below). Five days after surgery, mice were subjected to WAS daily for 9 days by placing them individually for 1 h on a 3 x 3 cm platform in a 40 x 40 cm pool filled with cold tap water up to 1 cm from the top surface of the platform. For false WAS, animals were placed on a platform in a pool without water for 1 h/day. Animals had free access to water and feed. Special diets were started 3 days before the WAS procedure and continued until the end of the experiment. Three groups of 12 mice were randomized to the following nutritional interventions:

Group 1: control diet (AIN93), false WAS (unsensitized)

Group 2: control diet (was:AIN93), WAS (sensitized)

Group 3: experimental diet (WAS:LNT+6'SL), AIN93 supplemented with 6'SL 0.09% w/v and LNT 0.18% w/v), WAS (sensitized)

Colorectal distension (CRD)

To measure abdominal contractions as an indicator of pain, mice were fitted with 2 nickel-chromium alloy wire electrodes implanted in the external oblique abdominal muscle and one under the abdominal skin. The operation was performed under anesthesia with xylazine/ketamine (both 1.2 mg, subcutaneously). A small balloon (Fogarty, Edwards Laboratories Inc., Santa Anna, USA) was inserted into the rectum and fixed at the base of the tail. The balloon was gradually inflated from 0 to 0.10 ml in 0.02 ml increments. Each stretching step lasted 10 s and alternated with a 5 min recovery period without stretching.

Visceral sensitivity to CRC was assessed by electromyography of the abdominal muscles.

results

The results are shown in FIG. 6. The amplitude of the contraction, which is associated with pain perceived by mice, was significantly increased due to the fact that WAS (was:AIN93, group 2) mice were sensitized compared to non-sensitized mice (AIN93, group 1). The introduction of a mixture of 6'SL and LNT contributed to a decrease in the amplitude of contraction and, thus, perceived pain in sensitized mice (was:LNT+6'SL, group 3). For CRC 0.06 ml or more, the contraction amplitude could not even be statistically distinguished from that observed in non-sensitized mice. This is a very significant achievement, since a CRC of 0.06 ml corresponds to a typical pain threshold, while smaller volumes of dilation are more likely to be associated with discomfort.

Claims (27)

1. Use of a nutritional composition containing 6'-sialyllactose (6'SL) and lacto-N-tetraose (LNT) to reduce nociception in an infant or young child. 2. Use according to claim 1, said use being for the reduction of abdominal pain in an infant or young child. 3. Use according to claim 1 or 2, said use being for the reduction of pain associated with gastrointestinal discomfort and/or gastrointestinal distress. 4. Use according to claim 3 wherein the gastrointestinal discomfort is caused by colic, bloating and/or cramps and/or the gastrointestinal disorder is selected from inflammatory bowel disease (IBD), diarrhea such as infectious diarrhea, necrotizing enterocolitis (NEC) and functional abdominal pain disorders, preferably selected from irritable bowel syndrome (IBS), abdominal migraine and functional abdominal pain without further specification (FAB-NOS). 5. Use according to any one of the preceding claims, wherein 6'SL and LNT are present in a 6'SL:LNT weight ratio of 3:1 to 1:3, for example 2:1 to 1:2 or 2:1 to 1: one. 6. Use according to any one of the preceding claims, wherein a. 6'SL is present in an amount of 0.005-5 g/l of the composition or in an amount of 0.004-3.8 g/100 g of the composition based on the weight of dry matter; and/or b. LNT is present in an amount of 0.005-3 g/l of the composition, or in an amount of 0.004-2.3 g/100 g of the composition, based on the weight of dry matter. 7. Use according to any one of the preceding claims, wherein a. 6'SL is present in an amount of 0.008-2.5 g/l of the composition or in an amount of 0.006-1.9 g/100 g of the composition based on the weight of dry matter; and/or b. LNT is present in an amount of 0.01-1.5 g/l of the composition, or in an amount of 0.008-1.2 g/100 g of the composition, based on the weight of dry matter. 8. Use according to any one of the preceding claims, wherein a. 6'SL is present in an amount of 0.01–1 g/l of the composition, or in an amount of 0.008–0.8 g/100 g of the composition, based on the weight of dry matter; and/or b. LNT is present in an amount of 0.04-1.2 g/l of the composition, or in an amount of 0.03-0.9 g/100 g of the composition, based on the weight of dry matter. 9. Use according to any one of the preceding claims, wherein a. 6'SL is present in an amount of 0.03-0.7 g/l of the composition or in a total amount of 0.023-0.5 g/100 g of the composition based on the weight of dry matter; and/or b. LNT is present in an amount of 0.05-1 g/l of the composition or in an amount of 0.04-0.8 g/100 g of the composition based on the weight of dry matter. 10. Use according to any one of the preceding claims, wherein a. 6'SL is present in an amount of 0.04-0.5 g/l of the composition or in an amount of 0.031-0.4 g/100 g of the composition based on the weight of dry matter; and/or b. LNT is present in an amount of 0.09-0.8 g/l of the composition, or in an amount of 0.07-0.6 g/100 g of the composition, based on the weight of dry matter. 11. Use according to any one of the preceding claims, wherein a. 6'SL is present in an amount of 0.24 or 0.05 g/l of the composition, or in an amount of 0.18 or 0.04 g/100 g of the composition, based on the weight of dry matter; and/or b. LNT is present in an amount of 0.5 g/l or 0.1 g/l of the composition, or in an amount of 0.38 g/100 g of the composition or 0.08 g/100 g of the composition, based on the weight of dry matter. 12. Use according to any one of the preceding claims, wherein said nutritional composition contains at least other(s) oligosaccharide(s), and/or fiber(s), and/or its(their) precursor(s), selected from the list including fucosylated oligosaccharides, N-acetylated oligosaccharides, sialylated oligosaccharides, galactooligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharides (XOS), inulin, polydextrose, sialic acid, fucose, and any combination thereof. 13. The use according to any one of the preceding claims, wherein said composition further comprises at least one probiotic in an amount of 10 ³ to 10 ¹² CFU/g of said composition (dry weight basis). 14. The use according to any one of the preceding claims, wherein said nutritional composition is an infant formula, initial infant formula, infant formula for formula-fed infants or follow-up infant formula, milk formula for infants from 1 to 3 years old, infant formula, cereal-based infant formula base, enricher or additive. 15. Use according to any one of the preceding claims, wherein said nutritional composition is for use in preterm infants, low birth weight infants and/or infants born small for their gestational age (SGA).

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