RU2782199C2 - Nutritional compositions with 2fl and lnnt for use in prevention and/or treatment of non-rotavirus diarrhea by impact on intestinal microbiota dysbiosis - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to the pharmaceutical industry, namely to the use of a nutritional composition, which contains fucosylated oligosaccharide and N-acetylated oligosaccharide, in the prevention or treatment of non-rotavirus diarrhea caused by E. coli (ETEC, EPEC, and/or EAEC), Salmonella, Shigella, Aeromonas, and/or Campylobacter in infants or young children by prevention or treatment of intestinal microbiota dysbiosis, which occurs before, during, and/or after non-rotavirus diarrhea in the specified infants or young children, where a young child is a child from one year old to three year old, wherein oligosaccharides are contained in the composition in certain amount.

EFFECT: above-described allows for effective prevention and treatment of non-rotavirus diarrhea in children.

16 cl, 9 dwg, 5 tbl, 4 ex

Description

The technical field to which the invention belongs

The present invention relates to nutritional compositions for infants or young children and their impact on health. Such compositions contain specific oligosaccharides and are effective in the prevention and/or treatment of non-rotavirus diarrhea in infants or young children. In particular, it relates to infant formula containing human milk oligosaccharides (HMOs), which bring the composition of the overall intestinal microbiota closer to normal.

State of the art

Mother's milk is recommended for all babies. However, in some cases, breastfeeding is insufficient or ineffective for medical reasons, or the mother refuses to breastfeed. For these cases, nutritional compositions such as infant formulas have been developed.

Nutritional compositions for infants and young children are often commercially available as powders for dissolution in water, or in some cases as ready-to-drink beverage or concentrated liquid compositions. These compositions are intended to meet most or all of the nutritional needs of an infant or young child.

However, it is known that human breast milk is the undisputed "gold standard" in infant nutrition. Consequently, infant formula manufacturers have tried many times to provide nutritional health benefits close to or similar to the beneficial effects of breast milk. However, many studies have shown that infant formula does not provide an identical effect on the body to that provided by breast milk. For example, formula-fed infants and breast-fed infants (HBM) may have different gut microbiota.

Early childhood, especially the first weeks, 3 months, 6 months or 12 months of life, are critical periods for establishing the balance of the intestinal microbiota.

It is known that the modulation of the intestinal microbiota in infancy can have a great impact on the future health of the body. For example, gut flora can influence the development of a strong immune system later in life, normal growth, and even the development of obesity later in life.

However, the gut microbiota and its development during infant development is a delicate balance between the presence and prevalence (number) of many gut bacterial populations. Some gut bacteria are categorized as "generally beneficial" while others are "generally harmful" (or pathogenic) because of their impact on the overall health of the infant.

Certain types of "generally beneficial" bacteria, such as bifidobacteria, may be underrepresented in conventional infant formula-fed infants compared to breastfed infants. Likewise, some populations of bacteria are considered pathogenic, and it is necessary to keep their prevalence low in the gut microbiota.

Indeed, a formula-fed infant may not benefit from the natural, well-balanced gut flora (gut microbiota) of exclusively or predominantly breastfed infants. This natural microbiota seen in breastfed infants is indeed well controlled over time (development over time) and complex. Many taxa of microorganisms coexist in the highly complex microenvironment of the gut, and all of them are in consistently formed proportions. When describing the microbiota of infants or young children, quantitative and qualitative parameters should be taken into account. In addition, an additional complication is that the gut microbiota changes over time.

Healthy gut flora is an indicator of infant health, and altered gut microbiota can be an indicator of (and/or cause of) abnormal health conditions such as diarrhoea, nutritional deficiencies, colic, sleep disturbance, and/or impaired growth and development.

Diarrhea, also called diarrhoea, is a condition in which loose or loose stools are passed at least three times a day. Diarrhea often lasts for several days and can lead to dehydration due to fluid loss. Signs of dehydration often begin with loss of normal skin elasticity and personality changes. With an increase in the severity of the condition, they can progress to a decrease in urination, pale skin, frequent heartbeat, and inhibition of reactions. Diarrhea is also characterized by a dysbiosis of the overall intestinal microbiota that precedes and continues after the onset(s) of diarrhea. Diarrhea is one of the leading causes of child mortality and is one of the four major factors in reducing life expectancy in sub-Saharan Africa and southern Asia. The mortality rate among children under 5 years of age due to diarrhea worldwide is estimated at approximately 15%. The most common cause is intestinal infections caused by viruses, bacteria, or parasites; that is, in conditions known as gastroenteritis. Escherichia coli is the main cause of diarrhea after rotavirus in children in developing countries. Surviving children are also at risk. Frequent cases of diarrhea before the age of 2 years are associated with a subsequent decrease in height by an average of 3.6 cm, rapid heartbeat after exercise, a decrease in IQ by 10 points, and about a year delay in starting school. Thus, it is important to reduce the incidence of diarrhea as well as improve recovery and recovery from it.

This type of intestinal infection usually resolves on its own, but non-specific therapy may bring relief to some patients, and specific therapy may shorten the duration of the disease and stop fecal excretion from the body. In the treatment of patients with diarrhea and dehydration, the main avenues of therapy are fluid and electrolyte administration, dietary regulation (and refeeding is important to compensate for nutritional deficiencies), non-specific antidiarrheal therapy (to relieve symptoms), and specific antimicrobial therapy (usually only in case of dysentery).

However, such existing solutions are not well suited for infants and young children due to their young age and weakness, in particular the use of antidiarrheal compounds is not approved for young children under 2 years of age.

Antibiotics are also unsuitable for infants and young children due to their age and the fact that E. coli is resistant to many antibiotics (Jiang ZD, et al. Prevalence of enteric pathogens among international travelers with acquired diarrhea in Kenya (Mombasa) , India (Goa), or Jamaica (Montego Bay), J. Infect. Dis. 2002; 185 (4): 497–502).

Therefore, alternative therapies have been developed.

Phage therapy has been investigated against E. coli diarrhea (Brüssow H. http://www.ncbi.nlm.nih.gov/pubmed/16000704). Russian pharmacies sell phage mixtures against many bacterial infections, including E. coli diarrhea, but their effectiveness has not been documented in detailed scientific reports. In pilot clinical trials in children infected with mostly enterotoxigenic E. coli (ETEC), neither T4-like phages nor a commercial mixture of coli-phages from Russia had any effect on quantitative criteria for diarrhea. Peak excretion of ETEC with feces was short-lived, with a low titer, and only half of the E. coli isolates from feces were sensitive to the phage.

In particular, studies are underway on the use of probiotics. For example, the ST11 strain of Lactobacillus paracasei improves the outcome of non-rotavirus diarrhea in children in Bangladesh, but has no effect on rotavirus diarrhea, see A. Sarker, Pediatrics 2005. Probiotics do not represent the best solution to treat or prevent diarrhea completely, so how they can only increase certain microbiota taxa. Indeed, probiotics are considered viable microbial preparations that promote the health of the individual by preserving the natural microflora in the gut. It is believed that they attach to the intestinal mucosa, colonize the intestinal tract and in the same way prevent harmful microorganisms from attaching to it. The most important condition for their action is that they must reach the intestinal mucosa in proper viable form and must not be destroyed in the upper gastrointestinal tract, in particular by the low pH prevailing in the stomach. Another difficulty is that the intestinal microbiota is very diverse and complex, and bacteria enter into various interactions with each other.

Other ingredients such as non-digestible carbohydrates (prebiotics) were also investigated. Human milk oligosaccharides (HMO) have specifically been reported to reduce the duration of rotavirus diarrhea in pigs fed this formula (see Li et al. ISME J 2014; 2015 study at the University of Illinois).

These substances can act as soluble decoy receptors in the gut to protect the newborn from enteric pathogens (Newburg et al, Human milk glycans protect infants against enteric pathogens. Annual Review of Nutrition 2005;25:37–58), and they can also directly interact with intestinal epithelial cells, causing changes that may interfere with host-microorganism interactions (Bode et al, 2012).

EP1531832 from Glykos mentions the property of HMOs to act as natural receptors that bind pathogens. In particular, there is mentioned an oligosaccharide-containing substance or receptor that binds diarrheagenic Escherichia coli. Examples of HMOs include 2FL and LNnT.

Similarly, Abbott's WO9956754 describes 2FL compositions that prevent E. coli or V. cholerae from attaching to a host cell receptor.

However, since such substances essentially act as decoy receptors, they are highly specific for the type of pathogen. These decisions also do not take into account microbiota dysbiosis that occurs before, during and/or after the onset(s) of diarrhea.

Prebiotics are also known to affect the stimulation of certain intestinal microorganisms. For example, it has been shown that certain galactooligosaccharides (GOS) and/or certain fructooligosaccharides (FOS) can activate the growth and spread of bifidobacteria in the gut, especially in infants.

Abbott's WO9843495 discloses a nutritional formulation containing an effective amount of lacto-N-neotetraose to stimulate the growth and/or metabolic activity of Bifidobacterium infantis bacteria.

Publication WO2009060073 from Nestec SA refers to the use of an oligosaccharide such as lacto-N-tetraose or lacto-N-neotetraose to promote the development in the first few weeks of an infant's life of a beneficial intestinal microbiota comparable to that found in breastfed infants, especially the intestinal microbiota, dominated by significant populations of Bifidobacterium and Lactobacillus, with the exception of other populations such as Bacteroides, Clostridia and Streptococci.

WO2012158517 describes the use of purified HMOs such as 2'-FL, 3-FL or LDFT to stimulate the growth of bacteria in the gastrointestinal tract of a mammalian subject, including bifidobacteria.

However, most of these studies offer solutions that do not address the treatment or prevention of non-rotavirus diarrhea conditions and all associated symptoms. They can act on the microbiota only by modulating certain taxa of the microbiota, for example, leading to an increase in the number of bifidobacteria or to a decrease in the number of Clostridia.

However, there are currently no solutions to treat or prevent non-rotavirus diarrhea by bringing the total gut microbiota (i.e. total/whole/aggregate/complete microbiota) closer to normal.

No existing solutions seem to take into account the function of the gut microbiota either.

Therefore, for infants or young children, there is a need to reduce the risk of non-rotavirus diarrhea and/or improve cure by shortening the duration of non-rotavirus diarrhea and/or ameliorating the symptoms and consequences of non-rotavirus diarrhea, especially by acting on dysbiosis of the general intestinal microbiota. occurring before, during and/or after manifestations of non-rotavirus diarrhea in said infants or young children.

There is a need to compensate for the abnormal overall intestinal microbiota seen in infants or young children in non-rotavirus diarrhoea. There is a need to restore such a common gut microbiota in terms of both its composition and function.

There is a need to enhance the optimal balance in the overall intestinal microbiota of infants, especially by suppressing or suppressing the growth of pathogenic bacteria, during the first weeks of life when such a balance is established.

There is a need for nutritional compositions for infants or young children that provide them with an overall intestinal microbiota and metabolic profile more similar to those obtained in normal situations (ie, in a healthy state without diarrhea) and/or for breastfed infants.

There is a need to provide these infants or young children with the best nutrition that promotes the development of an overall intestinal microbiota that approximates that in the absence of non-rotavirus diarrhea and/or approximates that of breastfed infants, and that development is short-lived (i.e. . during the period of introduction of nutrition) and / or long-term (ie, after the period of introduction of nutrition).

There is a need for nutritional compositions for infants or young children that provide a microbiota composition that prevents the development of non-rotavirus diarrhea and/or promotes rapid recovery from non-rotavirus diarrhea.

There is a need to provide such 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 that is acceptable to parents or healthcare practitioners.

Disclosure of invention

The present invention relates to a nutritional composition containing at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, for use in the prevention and/or treatment of non-rotavirus diarrhea in infants or young children by acting on dysbiosis of the general intestinal microbiota, that occurs before, during and/or after non-rotavirus diarrhea events in said infants or young children.

With this nutritional composition, the overall intestinal microbiota of said infants or young children will approximate those of infants or young children in the absence of non-rotavirus diarrhoea.

It will also approximate the total intestinal microbiota of infants or young children exclusively breastfed compared to the total intestinal microbiota of infants or young children fed predominantly or exclusively on a traditional nutritional composition that does not contain these oligosaccharides.

An advantage of the nutritional composition of the present invention is that it affects the composition and/or function of the entire gut microbiota, such as the relative taxonomic richness (or quantity), diversity, activity and/or functionality of said microbiota.

In a particularly preferred embodiment, the nutritional composition contains 2'-fucosyllactose (2-FL) and lacto-N-neotetraose (LNnT). In a particularly advantageous embodiment, it contains 2'-fucosyllactose (2-FL) in an amount of 0.8-1.5 g/l of nutritional composition and LNnT in an amount of 0.5-0.8 g/l of nutritional composition.

Brief description of the figures

In FIG. 1 shows the dynamics of titers of population of bacteria, E. coli and enterotoxigenic E. coli in serial stool samples of infants and young children in a hospital during an episode of acute diarrhea.

Fig. 1A. Median titer with interquartile range for the population of bacteria in feces, determined by real-time qPCR with universal primers to bacterial 16S rDNA; expressed as log10 cfu/g stool equivalents from healthy local control infants and young children (H) and patients with diarrhea on the indicated day after hospitalization. Only H is statistically significantly different from other time points (Dunn's tests for multiple comparisons).

Fig. 1b. Median number of viable E. coli, with an interquartile range, measured on McConkey agar as log10 cfu/g fresh stool (ordinate) on a given day after admission of patients with diarrhea (no statistically significant difference).

Fig. 1C and 1D. Distribution of titers and median titers for heat-resistant (st-ETEC, C) and heat-labile (lt-ETEC, D) endotoxin-bearing bacteria in the feces of patients with diarrhea hospitalized with microbiologically confirmed ETEC infection. Real-time PCR titers refer to the indicated days after hospitalization and are compared with healthy control infants and young children (H). Titers are presented as log10 median titers in ETEC CFU equivalents.

Figure 2 refers to the analysis of fecal microbiota by sequencing the 16S rRNA gene in serial stool samples from infants and young children hospitalized with acute bacterial diarrhea.

Fig. 2A. Profiles of bacterial community structure in stool samples from 20 healthy control infants and young children (H) and 56 patients with diarrhea on a given day after hospitalization (D01–D21). The panel shows the relative abundance in percentage of bacterial genera identified by the legend on the right side.

Fig. 2B, 2C and 2D. Median percent abundance of a given genus of bacteria (Bifidobacterium; Streptococcus; Escherichia; respectively) on a given day of diarrhea compared to healthy control infants and young children (H).

In FIG. 3 shows an overall profile of the total mean microbiota at the level of birth compared between a breastfed reference group (BF), a control formula fed group (control), and a study group fed with BT formula as assessed by 16S rRNA gene profiling.

In FIG. 4 shows the relative composition of the three major taxa in three groups—BF, control, and study group—which demonstrates statistically significant differences between study and control groups as measured by 16S rRNA gene profiling: Bifidobacterium (Fig. 4A), Escherichia (Fig. 4B), and Peptostreptococacceae uncl (Fig. 4C). The median with interquartile ranges is shown. A statistically significant difference is indicated by *, p < 0.05; **, p < 0.01; ***, p < 0.001. BF is the reference group for breastfeeding.

In FIG. Figure 5 shows the value of the alpha diversity of the total microbiota in three groups - BF, control and study group - calculated with the parameter PD_whole_tree (phylogenetic diversity throughout the phylogenetic tree) based on 16S rRNA gene profiling. A statistically significant difference is indicated by *, p < 0.05; **, p < 0.01.

In FIG. 6 is a redundancy analysis based on genus-level data measured by 16S rRNA gene profiling and showing a statistically significant separation of the three groups p < 0.001.

In FIG. 7 is a table showing a list of identified genes encoding known pathogenic factors measured by metagenomes. C/T, C/B and T/B mean statistically significant differences between the control and study groups, the control group and the breastfeeding group, and the study group and the breastfeeding group, respectively. The statistical significance of differences between groups was assessed by fitting with a negative binomial regression model, taking into account, if necessary, extra null data. For each group, the number of babies with certain genes is also shown.

In FIG. 8 is a table showing a list of identified genes encoding known antibiotic resistance genes measured by metagenomes. C/T, C/B and T/B mean statistically significant differences between the control and study groups, the control group and the breastfeeding group, and the study group and the breastfeeding group, respectively. The statistical significance of differences between groups was assessed by fitting with a negative binomial regression model, taking into account, if necessary, extra null data. For each group, the number of babies with certain genes is also shown.

In FIG. 9 shows the relative concentrations of important metabolites obtained from stool analysis by ¹ H NMR spectroscopy related to the metabolism of amino acids and other organic acids: phenylalanine (Fig. 9A), tyrosine (Fig. 9B), lactate (Fig. 9C), and isoleucine ( Fig. 9D). * means statistically significant difference according to the Kruskal-Wallis test (p < 0.05). BF is the breastfed reference group.

Implementation 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 expression "toddler" means a child between the ages of one and three (also called a toddler).

"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 end of the 37th week of pregnancy.

Small for gestational age or SGA refers to an infant or young child who is undersized for their gestational age at birth, most commonly defined as body weight below the 10th percentile for that gestational age. In some embodiments, the SGA condition may be associated with intrauterine growth retardation (IUGR), which refers to a condition in which a fetus cannot reach its potential size.

The expression "low birth weight" should be understood as any body weight less than 2500 g at birth.

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 nutritional composition of the present invention is a hypoallergenic nutritional composition. The term "hypoallergenic nutritional composition" means a nutritional composition that is unlikely to cause allergic reactions.

In a specific embodiment, the nutritional 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 mixture of natural origin present in the milk of mammals (ie, the synthetic composition is not breast milk).

The expression "infant formula" as used herein refers to a food product specifically designed for feeding infants during the first months of life, which by itself meets the nutritional needs of this age group (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 the "Special Foods for Infants" standard (including the article "Foods for Special Medical Purposes"). The expression "infant formula" covers both "initial formula" and "advance-level formula" or "pre-fed formula". In some embodiments, the infant formula is a preterm infant formula.

“Follow-up formula” or “pre-fed formula” is given starting at 6 months. It constitutes the main liquid element in the gradually increasing variety of diets for this category of individuals.

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

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

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.

The expressions "fucosylated oligosaccharides containing a 2'-fucosyl epitope" and "2-fucosylated oligosaccharides" include fucosylated oligosaccharides with a certain shape homology, since they contain a 2'-fucosyl epitope, therefore, a certain homology of function can be expected.

The expression "N-acetylated (s) oligosaccharide (s)" means both "N-acetyllactosamine" and "oligosaccharide (s) containing (s) 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 present in solid form (eg powder) or in 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 obtained from a powder after being dissolved in a liquid, e.g. milk, water, etc., e.g. or any other formula intended for baby food). They can also be expressed in g/100 kcal.

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 X days/weeks/months/years", "X days/weeks/months/years of age", and "X days/weeks/months/years from birth" may be used interchangeably.

By "mother's milk" is meant the mother's breast milk or colostrum. HBM stands for human breast milk.

The expressions “infants/young children who are exclusively breastfed with human breast milk”, “infants or young children who are exclusively breastfed”, “infants or young children who are exclusively breastfed”, and “infants/young children who are breastfeeding" can be used interchangeably. They mean infants or young children receiving the vast majority (i.e. at least 90%, or at least 95%, or at least 99%) or all (100%) of the nutrients and/or energy from human breast milk.

The expression "infants or young children fed exclusively on nutritional compositions" refers to infants or young children receiving the vast majority (i.e., at least 90%, or at least 95%, or at least 99%), or all (100%) nutrients and/or energy from artificial nutritional compositions such as infant formula, formula milk for infants or formula milk for children 1 to 3 years of age.

The expression "infants or young children fed predominantly on nutritional compositions" refers to infants or young children receiving nutrients and/or energy primarily from artificial nutritional compositions such as infant formula, complementary milk formula or infant formula for infants. 1 to 3 years. "Mainly" means at least 50% (or at least 60%, or at least 75%) of such nutrients and/or energy, such as 50% to 90%, or 60% to 80%.

The expression "stimulating and/or inducing" in infants or young children in relation to a certain general gut microbiota means the development, increase, establishment, emergence and/or displacement of a particular general microbiota in said infants or young children.

The expression "traditional nutritional composition" means standard artificial nutritional compositions such as infant formula, formula milk for infants or infant formula for children from 1 to 3 years of age, which are already on the market. The expression "traditional nutritional composition without said oligosaccharides" refers to a standard nutritional composition that does not contain "at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide".

The terms "microbial", "microflora" and "microbiota" may be used interchangeably.

The expressions "gut microbiota", "gut microbiota" and "gut microbiota" can be used interchangeably.

The terms "total", "whole", "whole", "complete" and "total" can be used interchangeably, especially in the expression "total microbiota". The terms "total microbiota in the gut/gut" or "total gut microbiota" refer to the entire (or complete, whole, total) microbiota in the gut. It includes:

- the composition of the total microbiota, i.e. the relative taxonomic richness (quantity) and/or diversity of the overall gut microbiota, in other words, the "quantitative" and/or "qualitative" aspects of the microbiota; and/or

- the function of the general microbiota, i.e. activity and/or functionality of the overall gut microbiota, especially metabolic activity/functionality. It can be estimated by measuring the relative abundance of predicted genes in the metagenome or by quantitative profiling of major metabolites, including amino acids, basic organic acids (lactate, succinate, citrate, etc.) and/or carbohydrates.

A suitable and healthy intestinal microbiota is a key factor for the development of the immune system in the infant's mucosa.

The term "dysbiosis" refers to a microbiological imbalance within the body. The terms "dysbiosis of the (total) gut microbiota" or "dysbiosis of the (total) gut microbiota" refer to a (general) imbalance of microorganisms in the gut.

The expression "acting on dysbiosis of the general intestinal microbiota" covers "prevention and/or treatment of dysbiosis of the general intestinal microbiota", i.e.:

- prevention of dysbiosis of the general intestinal microbiota: for example, prevention of manifestations of dysbiosis of the general intestinal microbiota (before, during, after episodes of diarrhea, or at all periods, preferably during episodes);

- treatment of dysbiosis of the general intestinal microbiota: for example, reduction, or reduction, or limitation of dysbiosis of the general intestinal microbiota, which is manifested in the case of diarrhea (either before, during, after episodes of diarrhea, or at all periods, preferably during episodes); and/or

- both actions.

The expressions "diarrhea", "diarrheal" and "episodes of diarrhea" can be used interchangeably. The expression "non-rotavirus diarrhea" may refer to diarrhea caused by Escherichia coli, also called E. coli (e.g., enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and/or enteroaggregative E. coli (EAEC)) , Salmonella, Shigella, Aeromonas and/or Campylobacter.

In a preferred embodiment, the expression refers to diarrhea caused by E. coli.

The terms "oppression" and "reduction" can be used interchangeably.

By "preventing" or "preventing" is meant avoiding and/or reducing the likelihood (ie, reducing the frequency) of the occurrence of a physical condition, situation, or its consequences.

By "treat" or "treatment" is meant a reduction in the duration and/or severity of a physical condition, situation or its consequences.

Prevention and/or treatment of the physical condition, situation, or consequences thereof may occur during treatment (i.e., during the administration of the composition of the present invention, either immediately after the start of administration, or some time after the start, for example, several days or weeks after the start introductions). But it may also include the prevention and/or treatment of said disorders later in life. The term "later in life" includes the effect after the cessation of the intervention or treatment. The "later life" effect can last from 1 week to several months, such as 2 to 4 weeks, 2 to 6 weeks, 2 to 8 weeks, 1 to 6 months, or 2 to 12 months.

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). Microorganism cells are essentially bacteria or yeast.

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

Unless otherwise indicated, all percentages are wt%.

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, in essence, 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.

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

Therefore, the first object of the present invention is a nutritional composition containing at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, for use in the prevention and/or treatment of non-rotavirus diarrhea in infants or young children by acting on general intestinal dysbiosis microbiota that occurs before, during and/or after non-rotavirus diarrhea in said infants or young children.

As illustrated in Example 2, the present inventors proved that the overall gut microbiota in infants with non-rotavirus diarrhea was modified (i.e., dysbiosis of the overall gut microbiota was present) during and after episodes of non-rotavirus diarrhea. In particular, in non-rotavirus diarrhea in infants, there is a strong increase in Streptococcus, a slight increase in Escherichia, and a strong decrease in Bifidobacteria.

As shown in Example 3, the present inventors have also demonstrated that a composition comprising at least one fucosylated oligosaccharide (2FL) and at least one N-acetylated oligosaccharide (LNnT) can be advantageously used to provide a general intestinal microbiota in infants, close to the microbiota of exclusively breastfed infants compared to the total gut microbiota of infants fed conventional infant formula that does not contain these oligosaccharides. Together, the fecal microbiota and characteristic metabolic signatures show that the addition of 2 individual and highly structure-specific human milk oligosaccharides (HMOs) causes a shift in the estimated total gut microbiota in feces, both in composition and in function, towards the microbiota observed in infants who are on breastfeeding. Without being limited by any particular theory, these oligosaccharides are believed to act synergistically to provide such an effect on the overall gut microbiota. In particular, an increase in the number of Bifidobacterium and a decrease in Escherichia were noted.

Thus, these specific BTs positively affect recovery from non-rotavirus diarrhea and even the incidence of new cases due to their action on the overall intestinal microbiota, which counteracts the dysbiosis observed in non-rotavirus diarrhea.

The nutritional composition of the present invention contains at least one fucosylated oligosaccharide. It may contain one or more types of fucosylated oligosaccharides. The fucosylated oligosaccharide(s) may actually 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, fucosyl lacto-N-neohexaose II), difucosyl lacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosyl lacto-N-neohexaose I, difucosyl lacto-N-neohexaose II, fucosyl-para-lacto-N-hexaose, tri-fuco-para-lacto-N-hexaose I; and any combination thereof.

In some specific embodiments, the fucosylated oligosaccharide is 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 preferred embodiment, the nutritional composition according to 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. the nutritional composition of the present invention contains only 2'-fucosyllactose as the 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 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, 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 by chemical synthesis from lactose and free fucose. In addition, for example, fucosylated oligosaccharides manufactured by Kyowa, Hakko, Kogyo, Japan are commercially available.

The nutritional composition of the present invention also contains at least one N-acetylated oligosaccharide. It may contain one or more types of N-acetylated oligosaccharides. The N-acetylated oligosaccharide(s) may be, for example, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or any combination thereof. 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, the N-acetylated oligosaccharide is LNT. In some other specific embodiments, the N-acetylated oligosaccharide is a mixture of LNT and LNnT. In some specific embodiments, the composition contains both LNT and LNnT in a LNT : LNnT ratio of 5:1 to 1:2, or 2:1 to 1:1, or 2:1.2 to 2:1.6 .

In a preferred embodiment, the nutritional composition according to the invention contains lacto-N-neotetraose (LNnT). In a particular embodiment, no other type of N-acetylated oligosaccharide other than lacto-N-neotetraose (LNnT) is used, i. e. the nutritional composition of the invention contains only lacto-N-neotetraose (LNnT) as the N-acetylated oligosaccharide.

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 Patent No. 5,288,637 and WO 96/10086. In an alternative embodiment, LNT and LNnT can be produced by chemically converting ketohexoses (e.g., fructose), either free or bound to an oligosaccharide (e.g., lactulose), to an 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 the most preferred embodiment of the present invention, the nutritional composition contains 2'-fucosyllactose (2FL) and lacto-N-neotetraose (LNnT).

In another specific embodiment, the nutritional composition of the present invention contains a mixture of oligosaccharides, consisting of 2'-fucosyllactose (2-FL) and lacto-N-neotetraose (LNnT). In other words, the nutritional composition of the invention contains only 2'-fucosyllactose (2-FL) as the fucosylated oligosaccharide and only lacto-N-neotetraose (LNnT) as the N-acetylated oligosaccharide.

The fucosylated oligosaccharide(s) may be present in the nutritional composition according to the present invention in a total amount of 0.5-3 g/l, eg 0.8-1.5 g/l, of the composition. In some embodiments, the fucosylated oligosaccharide(s) may be present in a total amount of 0.85-1.3 g/L of the composition, such as 0.9-1.25 g/L, or 0.9 -1.1 g/l, or 1-1.25 g/l, or 1-1.2 g/l of the composition.

The fucosylated oligosaccharide(s) may be present in the nutritional composition in a total amount of 0.38–2.32 g/100 g, for example 0.62–1.16 g/100 g, of the composition per mass of dry matter. The fucosylated oligosaccharide(s) may be present in a total amount of 0.66–1 g/100 g of the composition, e.g. 0.70–0.97 g/100 g, or 0.70–0.85 g/100 g, or 0.78-0.97 g/100 g or 0.78-0.93 g/100 g of composition.

N-acetylated oligosaccharide(s) may be present in the nutritional composition according to the present invention in a total amount of 0.5-0.8 g/l of the composition.

In some embodiments, N-acetylated(s)
oligosaccharide(s) may be present in a total amount of 0.5-0.75 g/l, or 0.5-0.7 g/l, or 0.5-0.6 g/l of the composition.

The N-acetylated oligosaccharide(s) may be present in the nutritional composition in a total amount of 0.39-0.62 g/100 g of composition based on dry weight, e.g. 0.39-0.58 g/100 g, or 0.39–0.54 g/100 g, or 0.39–0.47 g/100 g.

All of these different ranges can be combined.

Therefore, in one embodiment of the present invention, the nutritional composition comprises at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, wherein:

- fucosylated oligosaccharide(s) are present in a total amount of 0.8–1.5 g/l of the composition and/or in a total amount of 0.62–1.16 g/100 g of the composition, calculated by weight of dry matter; and/or

- N-acetylated oligosaccharide(s) is present in a total amount of 0.5–0.8 g/l of the composition and/or in a total amount of 0.39–0.62 g/100 g of the composition in terms of dry weight.

In another specific embodiment, the nutritional composition of the present invention contains at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, wherein:

- fucosylated oligosaccharide(s) are present in a total amount of 0.9–1.25 g/l of the composition and/or in a total amount of 0.70–0.97 g/100 g of the composition, calculated by weight of dry matter; and/or

- N-acetylated oligosaccharide(s) is present in a total amount of 0.5–0.7 g/l of the composition and/or in a total amount of 0.39–0.54 g/100 g of the composition in terms of dry weight.

In another specific embodiment, the nutritional composition of the present invention contains at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, wherein:

- fucosylated oligosaccharide(s) is (are) present in a total amount of 1–1.2 g/l of the composition and/or in a total amount of 0.78–0.93 g/100 g of the composition, by weight dry matter; and/or

- N-acetylated oligosaccharide(s) is present in a total amount of 0.5–0.6 g/l of the composition and/or in a total amount of 0.39–0.47 g/100 g of the composition in terms of dry weight.

The fucosylated oligosaccharide(s) and the N-acetylated oligosaccharide(s) included in the nutritional composition according to the invention are generally present in the ratio fucosylated oligosaccharide(s): N-acetylated oligosaccharide(s) 2:0.54 to 2:2.26, e.g. 2:0.76 to 2:1.8 or 2:0.8 to 2:1, four. In a particularly advantageous embodiment, this ratio is 2:1, or about 2:1.

The nutritional composition according to the present invention may also contain at least other oligosaccharide(s) (i.e. other than fucosylated oligosaccharide(s) and N-acetylated oligosaccharide(s) ( -s), which are necessarily present in the composition), and/or at least dietary fiber(s), and/or at least their precursor(s). The other oligosaccharide and/or dietary fiber and/or precursor thereof may be selected from the list including galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, sialylated oligosaccharides, sialic acid, fucose, and any of them. combination. They may be present in an amount of 0 to 10% by weight of the composition.

Suitable commercially available products that can be used in addition to the oligosaccharides contained in the mixture of oligosaccharides to obtain nutritional compositions according to the present 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 tradename STA-LITE®.

In one specific embodiment, the nutritional composition according to the invention may contain at least about 0.4 g or at least 0.7 g of oligofructose per 100 kcal of the composition, for example from about 0.4 to about 0.9 g, from about 0.4 to about 0.7 g, about 0.4 to about 0.5 g, about 0.7 to about 0.8 g, or about 0.7 to about 0.9 g of oligofructose per 100 kcal .

In some embodiments, the oligofructose has a polymerization degree of 2 to 10. In some embodiments, at least 80%, 90%, 95%, 99%, or 100% of the oligofructose has a polymerization degree of 2 to 8 (range 2 to 8) .

In a specific embodiment, the nutritional composition according to the invention may contain GOS. "Galactooligosaccharide" is an oligosaccharide that contains two or more uncharged galactose molecules and does not contain an N-acetyl residue. Suitable galactooligosaccharides that may also be added to the nutritional composition of the present invention include Galβ1.3Galβ1.4Glc, Galβ1.6Galβ1.4Glc, Galβ1.3Galβ1.3Galβ1.4Glc, Galβ1.6Galβ1.6Galβ1.4Glc, Galβ1.3Galβ1.6Galβ1.4Glc , Galβ1.6Galβ1.3Galβ1.4Glc, Galβ1.6Galβ1.6Galβ1.6Glc, Galβ1.3Galβ1.3Glc, Galβ1.4Galβ1.4Glc, Galβ1.4Galβ1.4Galβ1.4Glc, and any mixture thereof. Synthetic galactooligosaccharides such as Galβ1.6Galβ1.4Glc, Galβ1.6Galβ1.6Galβ1.6Glc, Galβ1.3Galβ1.4Glc, Galβ1.6Galβ1.6Galβ1.4Glc, Galβ1.6Galβ1.3Galβ1.4Glc, Galβ1.3Galβ1.6Galβ1.4Glc, Galβ1 ,4Galβ1,4Glc, Galβ1,4Galβ1,4Galβ1,4Glc, and mixtures thereof, are commercially available under the trademarks Vivinal® and Elix'or®. Other oligosaccharide suppliers are Dextra Laboratories, Sigma-Aldrich Chemie GmbH and Kyowa Hakko Kogyo Co., Ltd. In an alternative embodiment, specific glycosyltransferases, such as galactosyltransferases, can be used to produce neutral oligosaccharides.

In a specific embodiment, the nutritional composition may also contain at least one cow's milk oligosaccharide. Traditional technologies for fractionating and enriching cow's milk fractions in cow's milk oligosaccharides can be applied (such traditional technologies include column extraction, resin filtration, nanofiltration, special beta-galactosidase enzyme treatment, protein precipitation, crystallization and lactose separation, etc. .). Certain oligosaccharide-enriched cow's milk fractions are commercially available or are described, for example, in EP2526784 A1.

In a specific embodiment, the nutritional composition may also further comprise a mixture of oligosaccharides (BMOS), which contains from 0.1 to 4.0 wt.% N-acetylated (s) oligosaccharide (s), from 92.0 to 98.5 wt.% galactooligosaccharide(s) and from 0.3 to 4.0 wt.% sialylated(s) oligosaccharide(s).

In a particular embodiment, the nutritional composition according to the invention may contain sialylated oligosaccharide(s). It may contain one or more sialylated oligosaccharides.

The sialylated oligosaccharide(s) can be selected from the group consisting of 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), and any combination thereof. In some embodiments of the invention, 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. In an alternative embodiment, they can be produced by biotechnological means using specific sialyltransferases or sialidases, neuraminidase 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 of microorganisms of the same species or mixed cultures. 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 from, for example, Kyowa Hakko Kogyo, Japan.

In specific examples, the composition may contain from 0.05 to 5 g/l of sialylated oligosaccharide(s), or from 0.1 to 4 g/l, or from 0.3 to 2 g/l, or from 0.4 to 1.5 g/l, or 0.4 to 1 g/l, for example 0.5 or 0.9 g/l sialylated oligosaccharide(s). In some specific embodiments, the implementation of the composition may contain from 0.8 to 1.7 g/l sialylated(s) oligosaccharide(s).

The composition according to the invention may contain 0.03-3.88 g of sialylated oligosaccharide(s) per 100 g of the composition based on the weight of dry matter, for example 0.08-3.10 g, or 0.23 -1.55 g or 0.31-1.16 g or 0.31-0.77 g or 0.39-0.7 g or 0.62-1.32 g sialylated oligosaccharide(s) (s) per 100 g of the composition based on the weight of dry matter.

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

In some specific embodiments of the present invention, the nutritional composition does not contain any sialylated oligosaccharides.

The composition according to the present invention may also optionally contain 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 composition contains 0 to 3 g/l of oligosaccharide precursor(s), or 0 to 2 g/l, or 0 to 1 g/l, or 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).

The composition according to the invention may contain from 0 to 2.1 g of oligosaccharide precursor(s) per 100 g of the composition based on dry weight, for example from 0 to 1.5 g, or from 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 of the present invention may further comprise 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 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 available 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.

In one specific embodiment, the probiotic is Bifidobacterium lactis, for example Bifidobacterium lactis CNCM 1-3446.

The nutritional composition according to the invention may contain 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, based on dry weight.

In one embodiment, the probiotics are viable. In another embodiment, the probiotics are non-replicating or inactivated. In some other embodiments, both viable probiotics and inactivated probiotics can be used.

The nutritional composition of the invention may further comprise at least one phage (bacteriophage) or a mixture of phages, preferably directed against pathogenic microorganisms Streptococci, Haemophilus, Moraxella and Staphylococci.

The nutritional composition according to the invention may, for example, be an infant formula, initial infant formula, formula for feeding infants or follow-up formula, formula for preterm infants, infant formula, cereal-based infant formula, fortifier such as breast milk fortifier, or an additive. 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.

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

The nutritional composition according to the invention essentially 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 may be from 2.4 to 4 g/100 kcal, or more than 3.6 g/100 kcal. In some other embodiments, the amount of protein may be less than 2.0 g per 100 kcal, such as 1.8 to 2.1 g/100 kcal, or 1.8-2 g/100 kcal, or 1.9-2. 1 g of protein per 100 kcal, or the amount may be less than 1.8 g per 100 kcal, such as 1.4–1.8 g/100 kcal or 1.5–1.7 g/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 regard 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. "Alpha-lactalbumin" refers to a high quality, easily digestible whey protein that makes up 20–25% of the total human breast milk protein (HBM) and is the main protein present in HBM. The structure of alpha-lactalbumin consists of 123 amino acids and 4 disulfide bridges, and the molecular weight of the protein is 14.2 kilodaltons. Due to its high content of essential amino acids, especially tryptophan, alpha-lactalbumin is ideal for low protein infant formulas. In one embodiment, the nutritional composition of the present invention contains alpha-lactalbumin in an amount of from about 0.2 to about 0.4 g/100 kcal of the nutritional composition, or in an amount of at least 1.7 g/L, or at least 2, 0 g/l, or at least 2.3 g/l, or at least 2.6 g/l of the nutritional composition.

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 can be fully (ie, deeply) or partially hydrolysed. It may be desirable to administer partially hydrolyzed 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 hydrolysed.

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 a specific embodiment, the nutritional composition according to the invention is a hypoallergenic composition. In another specific embodiment, the composition according to the invention is a hypoallergenic nutritional composition.

The nutritional composition according to the present invention essentially contains a carbohydrate source. 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 according to the present invention essentially contains a source of lipids. This is especially important if the nutritional composition of the 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, high oleic sunflower oil, and high oleic safflower 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, such as from about 8:1 to about 10:1.

In one embodiment, the nutritional composition of the present invention contains high sn-2-palmitate triglycerides, preferably triglycerides having more than 33% palmitic acids at the sn-2 position.

In one embodiment, the nutritional composition of the present invention contains about 5 or 6 g per 100 kcal of fat, and, for example, at least about 7.5 wt.% of this fat, for example, about 7.5-12.0%, consists of palmitic acids in the sn-2 position.

In one embodiment of the invention, the composition contains at least 7.5%, preferably 8%, more preferably at least 9.6% fat, which is sn-2 palmitate, for example about 7.8-11.8%, about 8 0-11.5 wt.%, about 8.5-11.0% or about 9.0-10.0 wt.% of the fat is palmitic acid at the sn-2 position of the triglyceride.

In some embodiments, palmitic acid makes up about 15% to about 25%, such as about 15 to about 20% by weight, of the total fatty acid content of the mixture, and at least about 30%, such as about 35 to about 43% of the total palmitic acid is in the sn-2 position.

Commercially available is BetapolTM B-55 from Lipid Nutrition, which is a mixture of triglycerides derived from vegetable oil in which at least 54% of palmitic acid is in the sn-2 position of the glycerol molecule. In one embodiment, the fat content of the nutritional composition of the invention is about 40-50% by weight BetapolTM B-55, for example from about 43% to about 45% by weight. It will be appreciated by those skilled in the art that the percentage of high sn-2-palmitate fat used and the total amount of sn-2-palmitate in the blend may vary, and that another high sn-2-palmitate oil can be used without departing from the gist and scope of the invention.

The nutritional composition of the invention may also contain all the vitamins and minerals that are 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 of the present invention may contain emulsifiers and stabilizers such as soy, lecithin, mono- and diglyceryl esters of citric acid, and the like.

The nutritional composition 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 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 according to 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, etc. may be dissolved in the fat source prior to mixing. Water, preferably reverse osmosis purified water, may then be mixed in to form a liquid mixture. The temperature of the water conveniently ranges between about 50°C and 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 necessary.

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 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, for convenience, adjust the pH and solids content of the homogenized mixture.

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 powdered. The moisture content of the powder should be less than about 5% by weight. Fucosylated oligosaccharide(s) and N-acetylated oligosaccharide(s) can also be added at this stage or in an alternative embodiment by dry mixing or by mixing them in the form of a crystalline syrup along with the probiotic ( -them) strain(s) (if used), and the mixture is subjected to spray drying or freeze drying.

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 conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, 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 used for infants or young children. It is especially designed for infants under 6 months of age.

The nutritional composition can be administered (or fed or fed with) at age and for a period of time depending on need and whether it is being used for prophylactic or therapeutic purposes.

In some embodiments, the infants or young children are 0-36 months of age, such as 0-12 months or 0-6 months. It is expected that newborn infants (0-4 weeks or 0-8 weeks of age) may benefit from the composition of the invention as their intestinal tract may be more weakened.

In some specific embodiments, the nutritional composition may be an infant formula and may be specifically designed for infants between 0 and 12 months of age who are predominantly fed with infant formula.

In some specific embodiments, the implementation of the nutritional composition is intended for feeding infants or young children from 6-12 months or from 12-36 months. During these periods, infants or young children are at particular risk as the protection of mother's milk decreases and exposure to pathogens increases.

In some embodiments, the implementation of the nutritional composition can, 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 present invention, the nutritional composition is given (or administered) to an infant during the first 4 or 6 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...) or several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10 ...), or a few months (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ...) after birth.

In some preferred embodiments, the nutritional composition is given immediately after the onset of non-rotavirus diarrhea in an infant or young child with non-rotavirus diarrhea.

Depending on the needs, the nutritional composition of the present invention can be given for several days (1, 2, 3, 4, 5, 6, etc.), or for several weeks (1, 2, 3, 4, 5, 6 , 7, 8 or even more), or for several months (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even more).

In some embodiments, the nutritional composition is given until the symptoms of non-rotavirus diarrhea in an infant or young child with non-rotavirus diarrhea have resolved, or up to several days, or weeks, or months after said disappearance.

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.

In one embodiment, the composition of the invention is given to an infant or young child as a composition in addition to mother's milk. In some embodiments, the infant or young child receives breast milk for at least the first 2 weeks, or 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, the composition is given to an infant or young child as the sole or primary nutritional composition for at least one period of time, such as after the 1st, 2nd, or 4th month of life, for at least 1, 2, 4th or 6th 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 breast milk, or to supplement infant formula or complementary formula/follow-up formula.

Infants or young children may be born at term or prematurely. In a specific embodiment, the nutritional composition of the invention is used for infants or young children who were born prematurely. In a specific embodiment, the nutritional composition of the invention is for use in preterm infants.

In one embodiment, the nutritional composition of the present invention may also be used in infants or young children who were born small for their gestational age or had a low birth weight.

Infants or young children with low birth weight may or may not be premature, and similarly, infants or young children who are small for their gestational age may or may not be premature.

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

All infants and young children can benefit from the invention, since they are all or may be at some age susceptible to imbalance of the intestinal microbiota.

In some advantageous embodiments of the invention, the nutritional composition is for use in infants or young children who already have a weakened or imbalanced microbiota or microbiota dysbiosis, such as premature infants, infants born by caesarean section, infants born small for their gestational age or with low birth weight, hospitalized infants/young children, infants/young children who have received or are receiving antibiotic treatment, and/or infants/young children suffering from or have suffered from an intestinal infection and/or intestinal inflammation.

In embodiments of the invention, the target for therapy with the composition of the present invention are infants born prematurely or delivered by caesarean section, or born small for their gestational age, or having a low birth weight, or showing an imbalance or abnormal intestinal microbiota, suffering from or have suffered from an intestinal infection and/or intestinal inflammation, especially if infants are 0-6 months of age. Without being limited by any particular theory, it is believed that the composition of the present invention is even more beneficial to infants, especially if infants have an imbalance in the intestinal microbiota (or are at risk for such). In such infants, of particular interest is the acquisition of a gut microbiota that is similar to the gut microbiota of breastfed (preferably exclusively breastfed) infants. Indeed, this provides them with a normal amount of elements necessary for health, which can be beneficial, especially for such weakened babies.

The nutritional composition of the present invention will, of course, be even more beneficial to infants with possible intestinal microbiota disorders or to weak infants/young children (such as premature infants and/or infants delivered by caesarean section).

In addition, the composition of the invention is expected to benefit even more infants/young children who experience intestinal disorders (such as diarrhea, infections or colic), especially after birth, for example during the first 4 weeks after birth.

In a particularly advantageous embodiment, the nutritional composition is administered to infants or young children with non-rotavirus diarrhea, such as E. coli diarrhea, or who are at high risk for non-rotavirus diarrhea (for example, living in high-risk areas such as Africa). sub-Saharan and southern Asia). The nutritional composition of the present invention will allow infants or young children to:

- receive a total gut microbiota that is close to normal (or a normal environment), for example, get a better balanced microbiota, for example, a total gut microbiota that is close to theirs in the absence of non-rotavirus diarrhea. This is especially true if infants or young children have already developed non-rotavirus diarrhoea.

- to obtain a total intestinal microbiota that is close to that of infants or young children fed exclusively with breast milk, compared with the total intestinal microbiota of infants or young children fed predominantly or exclusively with a traditional nutritional composition that does not contain these oligosaccharides. This may apply when infants or young children already have non-rotavirus diarrhea, or when they are at risk of developing non-rotavirus diarrhea.

The composition of the present invention has a positive effect on the overall microbiota of infants or young children: it stimulates and/or induces in the intestines of infants or young children fed with a nutritional composition of the present invention, the overall intestinal microbiota, close to normal or close to the microbiota of infants or young children of age fed exclusively on human breast milk compared to the total gut microbiota of infants or young children fed predominantly or exclusively on a conventional nutritional composition free of these oligosaccharides (i.e. free of at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide).

The main and unexpected health benefit of the nutritional composition of the present invention is that the composition globally modulates the overall microbiota of infants fed with the nutritional composition of the invention, making it similar to the microbiota of breastfed infants. Not only do some specific taxa of the microbiota change, but the nutritional composition in particular induces a shift in the composition of the overall microbiota towards one that is induced by breastfeeding. In addition, experiments show similarities with the same aspects in breastfed infants in both gut microbiota composition (i.e. relative taxonomic richness and/or diversity of the overall microbiota) and gut microbiota function (i.e. e. its activity and/or functionality, eg resulting metabolites). Thus, in the case of the composition of the intestinal microbiota, the induced/stimulated microbiota is specific in 2 aspects:

"quantitative": the intestinal flora includes more beneficial bacteria and fewer unhelpful or harmful bacteria;

"qualitative": the diversity of bacterial taxa more closely resembles the microbiota of breastfed infants.

The diversity of the overall microbiota can be alpha diversity (details are shown in the examples section) and can be illustrated, for example, as measured by the Phate Phylogenetic Diversity Index (PD_whole_tree).

The health effect provided by the nutritional composition of the invention can be measured in infants or young children aged 0-36 months, optionally 0-12 months. It can be observed after several days or weeks of application of the composition - for example, after 4 weeks, or 6 weeks, or 8 weeks of application. However, observation of this stimulated/induced total microbiota may take 4, 6, or 8 weeks before it can be seen. The effect can be measured, for example, in the feces of infants/young children.

In the context of the invention, the health benefit brings the overall intestinal microbiota of infants or young children closer to normal and/or to the microbiota of breastfed infants or young children. The effect is especially visible when compared with infants or young children who do not receive the composition of the present invention.

In some embodiments, the nutritional composition of the present invention will allow to stimulate and/or induce in infants or young children a total gut microbiota that approximates that of those infants or young children in the absence of non-rotavirus diarrhea and/or that approximates that of infants. or young children fed exclusively on human breast milk compared to the total gut microbiota of infants or young children fed predominantly or exclusively on a traditional nutritional composition not containing these oligosaccharides.

Such a beneficial effect may include i) inhibiting, reducing or inhibiting the growth of pathogenic bacteria or reducing the burden of pathogenic bacteria and/or ii) enhancing, increasing or stimulating the growth of beneficial bacteria. In some embodiments, the nutritional composition of the present invention provides for (or includes, or is accompanied by, or is characterized by) an increase in the population of Bifidobacterium and/or suppression of the population of Escherichia and/or Peptostreptococcaceae compared to the total gut microbiota in infants or young children fed predominantly or exclusively conventionally. nutritional composition. This effect can be measured in the feces of said infants or young children, for example at 1, 4, 6 or 8 weeks of age or older and, for example, after 1, 4, 6 or 8 weeks of feeding with said nutritional composition. Fecal microbiota composition can be measured, for example, by 16S rDNA analysis or metagenomic analysis (details are provided in the example section).

By suppressing, reducing and/or inhibiting the growth of pathogenic bacterial populations and/or inducing more beneficial bacteria, quantitatively and qualitatively, the composition of the invention provides a positive effect on health. Such a healthy gut microbiota is ultimately associated with proper nutrient absorption, adequate growth, reduced colic, reduced infection, reduced diarrhea, and improved gut health.

The effect of the invention may be prophylactic (e.g., preventing gut microbiota imbalance, preventing non-rotavirus diarrhea, maintaining a healthy gut microbiota, introducing a healthy gut microbiota) or curative (restoring a healthy gut microbiota when it is disturbed, helping to eliminate or reduce populations of non-rotavirus pathogens). microorganisms in the gut, aid in recovery from non-rotavirus diarrhea, introduction of a healthy microbiota after non-rotavirus diarrhea disorders).

In some embodiments, the nutritional composition of the present invention provides for (or includes, or is accompanied by, or is characterized by) a reduction in pathogen(s) and/or a reduction in disease-causing factor(s) compared to the overall gut microbiota in infants or toddlers. age, fed predominantly or exclusively on a traditional nutritional composition without oligosaccharides. These effects can be measured in the feces of said infants or young children, for example at 1, 4, 6 or 8 weeks of age or older and, for example, after 1, 4, 6 or 8 weeks of feeding with said nutritional composition.

Reducing pathogen(s) means reducing the frequency of occurrence and/or amount (eg load, number) of pathogen(s). It can be measured, for example, by the Luminex PCR method (details are given in the example section). Possible reduction of pathogens such as viruses, bacteria and/or protists. Specific examples of viral pathogens include norovirus (eg norovirus GI/GII) and/or rotavirus (eg rotavirus A). Specific examples of bacterial pathogens include Clostridium difficile (eg Clostridium difficile toxin A/B), Campylobacter, Escherichia coli (eg E. coli O157). Specific examples of pathogenic protozoa include Cryptosporidium.

In specific embodiments, the implementation of the nutritional composition of the present invention causes a decrease in the number of non-rotatirus pathogens.

In one specific example, the nutritional composition provides for the reduction of pathogenic Clostridium difficile bacteria.

In yet another specific example, the nutritional composition causes a reduction in pathogenic E. coli bacteria.

The disease-causing factor(s) may be disease-causing and/or antibiotic resistance genes that can be detected, for example, by metagenomic analysis (details are given in the example section). Specific examples of disease genes are gi:16767513 (yjcB - putative internal membrane protein) from Salmonella enterica LT2, gi:21284341 (cna - collagen adhesin precursor) from Staphylococcus aureus MW2, gi:24528016 (l7045 - L7045) from Escherichia coli 536, gi:15808725 (fecB - FecB) from Shigella flexneri YSH6000 and gi:21282741 (isdA - cell surface protein) from Staphylococcus aureus MW2.

In some embodiments, the nutritional composition of the present invention causes (or contains, or is accompanied by or is characterized by) a decrease in free amino acid production and/or stimulation of lactate production compared to the general gut microbiota in these infants or young children in the absence of non-rotavirus diarrhea, and/or compared to the total gut microbiota in infants or young children fed predominantly or exclusively on a traditional oligosaccharide-free nutritional composition.

Lactate is produced by lactic acid bacteria such as lactobacilli and bifidobacteria, and it can interfere with the growth of other bacteria, including pathogens. Specific examples of free amino acids include phenylalanine, tyrosine, and isoleucine. These effects can be measured in the feces of said infants or young children, for example at 1, 4, 6 or 8 weeks of age or older and, for example, after 1, 4, 6 or 8 weeks of feeding with said nutritional composition. Measurements can be performed using the established metabonomic approach based on proton nuclear magnetic resonance spectrometry ( ¹ H NMR) (Moco et al, 2013, Metabolomics perspectives in Pediatric Research, Pediatr. Res. 73, 570–576).

The health impact on infants or young children can be measured in a variety of ways, as shown in the example below.

In one embodiment, the effect on the overall microbiota is measured by calculating the alpha diversity in each sample and analyzing the distribution (details are provided in the example section).

In one embodiment, the impact on the overall microbiota is measured by calculating beta diversity between groups using a metric that takes into account phylogenetic distances between OTUs (Operational Taxonomic Units), such as using the UniFrac method, and distribution analysis. Alternatively, beta diversity between a control group, a study group, and a breastfeeding group can be assessed using multivariate ordination with hypothesis testing based on randomization procedures (e.g., canonical correspondence analysis (CCA), redundancy analysis (RDA)) or multivariate parametric or non-parametric test (eg Adonis, ANOSIM, multivariate analysis of variance). In one specific example, beta diversity is calculated using RDA.

In one embodiment of the invention, the stimulated and/or induced total gut microbiota in infants and/or young children fed with a nutritional composition of the present invention has a significantly reduced alpha diversity (e.g., reduced by at least 0.10 units, e.g. by at least 0.12 units or at least 0.15 units, e.g. 0.19 units) compared to the total gut microbiota in these infants or young children in the absence of non-rotavirus diarrhoea, and/or compared with the general gut microbiota in infants or young children fed predominantly or exclusively on a conventional nutritional composition free of the oligosaccharides present in the nutritional composition of the invention (e.g., at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide), and therefore , close to that of breastfed infants ania.

The nutritional composition of the present invention may be used for prophylactic and/or therapeutic purposes.

In one specific aspect, the present invention also relates to a nutritional composition for use in promoting healthy growth, for use in promoting a healthy immune system, for use in promoting normal bowel function, and/or for use in the prevention and/or treatment of intestinal microbiota dysbiosis in infants or young children.

The health benefits provided by the composition of the invention may be short, medium and/or long term effects.

The effect may be observed immediately after administration of the composition of the present invention and/or manifest later in life, i.e. after administration of the composition, eg 1 week to several months, eg 2 to 4 weeks, 2 to 6 weeks, 2 to 8 weeks, 1 to 6 months, or 2 to 12 months after said administration.

Other objects

Another object of the present invention is the use of at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide in the preparation of a nutritional composition for the prevention and/or treatment of non-rotavirus diarrhea in infants or young children by affecting the dysbiosis of the general intestinal microbiota, that occurs before, during and/or after non-rotavirus diarrhea in said infants or young children.

Another object of the present invention is a pharmaceutical composition containing at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide for the prevention and/or treatment of non-rotavirus diarrhea in infants or young children by affecting the dysbiosis of the general intestinal microbiota, that occurs before, during and/or after non-rotavirus diarrhea in said infants or young children.

Another object of the present invention relates to a method for the prevention and/or treatment of non-rotavirus diarrhea in infants or young children by affecting the dysbiosis of the general intestinal microbiota that occurs before, during and/or after non-rotavirus diarrhea in these infants or children infants, said method comprising administering to said infant or young child a nutritional composition comprising at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide.

The above embodiments and examples (e.g., relating to types and amounts of oligosaccharide, nutritional composition, administration, target population, etc.) also apply to the various subjects listed (i.e., pharmaceutical compositions, methods, etc.) .

Examples

The following examples demonstrate some specific embodiments of the composition for use in accordance with the present invention. These examples are provided for illustrative purposes only and should not be construed as limiting the present invention, as many variations are possible within the spirit of the 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.

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) 2FL(g) 0.15 one LNnT (d) 0.075 0.5

Table 1. An example of the composition of the nutritional composition (for example, infant formula) in accordance with the present invention

Example 2

Description of the clinical study

120 infants and young children hospitalized with acute diarrhea were included in this prospective single center study. The trial included infants and children aged 6–24 months, boys (to help separate feces from urine), with a history of watery diarrhea for less than 48 hours, from whose parents written informed consent was obtained. Patients with systemic infection, malnutrition (Z score < -3) and significant medical abnormalities were excluded. Patients receiving or requiring antibiotics were also excluded from the trial. Infants and young children must be negative for invasive diarrhoea, Vibrio cholerae by dark field microscopy, and rotavirus by ELISA to be included in the study. Stool samples were tested for ETEC, EPEC and EAEC according to standard procedures at the International Diarrhea Research Centre, Bangladesh (icddr, b) (Svenungsson B, et al. Enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a Swedish clinic for infectious diseases Clin Infect Dis 2000 May 30 (5): 770–8 Vidal M, et al Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections J Clin Microbiol 2005 Oct 43(10): 5362–5).

Age-appropriate feeding was performed early in the form of breastfeeding until satiety or a standardized hospital diet providing approximately 100 kcal/kg body weight per day.

Materials and methods

Complete stool bacterial counts and detection and quantification of E. coli virulent genes were performed by PCR as described in Nadkarni et al, Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. microbiology. 2002 Jan; 148 (Pt 1): 257–66, and in Guion et al, Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR. J.Clin. microbiol. May 2008; 46(5): 1752–7.

Isolation of DNA from feces

Total DNA was isolated using the QIAamp DNA Stool Mini Kit (QIAGEN) according to the manufacturer's instructions except for the addition of a series of mechanical chopping steps (11 × 45 s) using the FastPrep apparatus and Lysing Matrix B tubes (MP Biochemicals) (Junick J, Blaut M. Quantification of human fecal bifidobacterium species by use of quantitative real-time PCR analysis targeting the groEL gene Appl Environ Microbiol 2012 Apr 78 (8): 2613–22 doi: 10.1128/AEM.07749–11. Epub 2012 Feb 3).

Pyrosequencing and analysis of 16S genes

The 16S variable region V1 to V3 was PCR amplified and sequenced on a Roche 454 GS-FLX-Titanium Sequencer as described in Sanchez M, et al. Effect of Lactobacillus rhamnosus CGMCC 1.3724 supplementation on weight loss and maintenance in obese men and women. Br. J. Nutr. 2014 Apr 28; 111(8): 1507–19. The original sequence data was deposited in GenBank Short Read Archive and analyzed using Mothur v.1.33.0 software (Schloss, P. D., et al., 2009, “Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities.” Applied and Environmental Microbiology 75(23): 7537–7541) and QIIME v.1.8 (Caporaso J.G., et al., 2010b, QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335 –336). The background in pyrosequencing results was reduced using the PyroNoise function in the Mothur program (Quince, C., Lanzen, A., Curtis, T.P., Davenport, R.J., Hall, N., Head, I.M. et al., 2009, Accurate determination of microbial diversity from 454 pyrosequencing data. Nat Methods 6: 639–641) according to the standard procedure described in SOP 454 (Schloss et al, 2011, Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl Environ Microbiol 77: 3219–3226). Chimeras were identified using the usearch61 function in QIIME (Edgar et al, 2011, UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27: 2194–2200). The sequences were then trimmed as described in 454 SOP Mothur so that the sequences contained an overlap of the same 16S region. Selection of operating taxonomic units (OTU) de novo with 97% identity was performed using the uclust function (Edgar R.C., 2010, Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460–2461). Taxonomic assignment of representative OTU sequences was performed using the RDP Classifier function at a confidence threshold of 0.6 (Wang Q. et al, 2007, Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261–5267) in the reference database Greengenes v.13.8 (McDonald D. et al, 2012, An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyzes of bacteria and archaea. ISME J 6: 610–618). Diversity analyzes were performed in the QIIME program, and correlations were performed using Calypso at http://bioinfo.qimr.edu.au/calypso. Analysis of the Dirichlet multinomial mixture model (Holmes I. et al., 2012, Dirichlet Multinomial mixtures: Generative models for microbial metagenomics. PLoS One 7:e30126) was performed using the Mothur software.

results

Conducted a detailed microbiological analysis of serial samples of feces from the first 56 patients; 77% were cultured in the clinical laboratory with pathogenic E. coli (ETEC: 23; enteroaggregative (EAEC): 10; enteropathogenic (EPEC): 2; mixed E. coli: 8 patients). PCR of all stool DNA confirmed the diagnosis of E. coli: 28 ETEC (23 elt ⁺ est ⁺ ; 3 elt; 2 est ⁺ ); 5 EPEC (4 eae ⁺ bfp ⁺ ; 1 eae ⁺ ); 7 EAEC (daaD ⁺ ).

The total number of bacteria in the feces, determined by qPCR, was stable during hospitalization, but less than in the control (see Fig. 1A). Viable E. coli counts were < 5% of all bacteria in the feces of patients with diarrhea (Fig. 1B) and were only 10 times greater in patients with ETEC than in patients with non-E. coli diarrhea. Heat-resistant (Fig. 1C) and heat-labile (Fig. 1D) toxin gene counts in microbiologically confirmed ETEC infections reached peak titers on day 2 of hospitalization (representing <1% of all stool bacteria) followed by a rapid fall in all three treatment groups. As illustrated in FIG. 1, a slight increase in Escherichia coli was first observed and then a decrease, but E. coli did not dominate the gut microbiota in E. coli-induced diarrhea in children.

In FIG. 2 shows that Streptococcus accounted for 52% of bacteria in the feces during the acute phase of diarrhea compared to 7% for Escherichia (FIGS. 2C and 2D). Non-rotavirus diarrhea caused by E. coli is accompanied, in particular, by a strong increase in Streptococcus, a slight increase in Escherichia, and a strong decrease in Bifidobacteria. During hospitalization, there was an intersecting increase in Bifidobacterium and a decrease in Streptococcus (FIGS. 2B and 2C). At day 4, the composition of the total stool microbiota was similar to that 21 days after hospitalization, but did not recover to that of healthy infants and young children (Fig. 2A).

Interestingly, in infants and young children with earlier resolution of diarrhea (< 4 days), the Bifidobacterium/Streptococcus ratio also showed earlier normalization and the percentage of Escherichia was lower than those with later resolution of diarrhea (> 5 days). ).

In patients with diarrhea, massive multiplication of streptococci in the feces is shown, which in the process of recovery were successively replaced by bifidobacteria.

Example 3

Description of the clinical study

The safety study was carried out at Dipartimento Materno Infantile, Unità Operativa Complessa di Neonatologia e Terapia Intensiva Neonatale, AOUP “Paolo Giaccone”, Palermo, Italy and Kinderartsenpraktijk, Hasselt, Belgium.

This study was a randomized, controlled, two-centre intervention clinical trial with 2 parallel formula feeding groups. The study population for the formula-fed groups consisted of healthy full-term male and female infants aged 0 to 14 days at study entry who were exclusively formula-fed at study entry. Eligible infants were randomly assigned to the two study formulas (control or test) using mode of delivery (vaginal or caesarean section) and gender as stratification factors. In Stage 1, these randomized infants were exclusively fed with either test or control formula from study entry until 4 months of age, in amounts appropriate to their weight, age, and appetite. Parents/caregivers, investigators, study support staff, and clinical project leader were unable to identify study mixtures.

The following infant formulas were used in the study:

• control formula was given to the control group: it was a standard whey-dominated infant formula containing long-chain polyunsaturated fatty acids (LC-PUFA) and no probiotics (66.9 kcal/100 ml diluted formula, 1.889 g protein/100 kcal powder with a ratio of whey : casein 71.6% : 28.4%, detailed composition is shown in table 2);

• the test mixture was given to the study group: it was the control mixture except that part of the lactose was replaced with two 2 HMOs (2FL and LNnT) in the following amounts: 0.5–0.6 g LNnT and 1.0–1.2 g 2 'FL per liter of dissolved mixture (detailed composition is shown in table 2).

As a reference group (breastfeeding group = group BF), stool samples were collected from infants who were exclusively breastfed for at least 3 months at least 3 months of age.

Parameter Control mix, 100 g Test mixture, 100 g Energy value (kcal) 518.7 518.7 Water (g) 2.6 2.6 Fat (g) 27.5 27.5 Fatty acids, saturated (g) eleven eleven Fatty acids, monounsaturated (g) 9.4 9.4 Alpha-linolenic acid C18:3 n-3 (mg) 510 510 Docosahexaenoic acid C22:6 n-3 (DHA) (mg) 61 61 Arachidonic acid C20:4 n-6 (ARA) (mg) 61 61 Linoleic acid C18:2 n-6 (mg) 4270 4270 Fatty acids, polyunsaturated (g) 4.9 4.9 Protein (g) 9.8 9.8 Available carbohydrates (g) 58 58 lactose (g) 56 55 OGM LNnT (d) 0 0.39–0.47 2FL(g) 0 0.77–0.93 Sugar (g) 56 56 Ash (g) 2.1 2.1 Sodium (mg) 162 162 Potassium (mg) 575 575 Chloride (mg) 348 348 Calcium (mg) 317 317 Phosphorus (mg) 178 178 Magnesium (mg) 43 43 Manganese (mcg) 121 121 Selenium (mcg) 13 13 Iron (mg) 4.8 4.8 Copper (mg) 0.37 0.37 Zinc (mg) 5 5 Iodine (mcg) 102 102 Fluoride (mcg) 60 60 Vitamin A (retinol) (mcg RE) 542 542 Vitamin D (calciferol) (mcg D) 7.6 7.6 Vitamin E (tocopherol) (mg TE) 8.4 8.4 Vitamin K (phytoquinone) (mcg) 45 45 Vitamin C (ascorbic acid) (mg) 80 80 Vitamin B1 (thiamine, base) (mg) 0.6 0.6 Vitamin B2 (riboflavin) (mg) 0.67 0.67 Niacin (mg) 4.1 4.1 Vitamin B6 (pyridoxine, base) (mg) 0.34 0.34 Folic acid (mcg) 80.9 80.9 Pantothenic acid (mg) 3.4 3.4 Vitamin B12 (cyanocobalamin) (mcg) 1.8 1.8 Biotin (mcg) fourteen fourteen Choline (mg) 48 48 Inositol (mg) 48 48 Taurine (mg) 33 33 Carnitine, L- (mg) 9.5 9.5 Nucleotides (mg) fifteen fifteen Adenosine 5'-monophosphate (mg) 3.8 3.8 Cytidine 5'-monophosphate (mg) 6 6 Guanosine 5'-monophosphate (mg) 1.2 1.2 Uridine 5'-monophosphate (mg) four four Ca/P (ratio) 1.781 1.781 Linoleic/alpha-linolenic (ratio) 8.373 8.373 Vitamin C/Fe (ratio) 16.667 16.667 Phenylalanine, L- (mg) 488 488 Alanine, L- (mg) 391 391 Arginine, L- (mg) 258 258 Cystine, L- (mg) 247 247 Histidine, L- (mg) 226 226 Isoleucine, L- (mg) 502 502 Leucine, L- (mg) 1045 1045 Methionine, L- (mg) 205 205 Threonine, L- (mg) 460 460 Tryptophan, L- (mg) 200 200 Tyrosine, L- (mg) 399 399 Valine, L- (mg) 542 542

Table 2. Composition of the control mixture and the test mixture

Fecal microbiota was assessed for each group at 3 months of age using different methods, see Table 3.

Number of samples ITT PP PP images,
rDNA 16S Images PP, Luminex PCR PP images, metagenome PP images, metabolites Control 87 75 63 54 65 64 Test 88 71 58 fifty 58 57 On the chest
breastfeeding - 38 33 - 34 32

Table 3 Number of infants in initiating treatment (ITT) and complying (PP) populations and number of stool samples available for protocol compliance or breastfeeding reference population for analysis microbiota by total 16S rDNA sequencing, pathogen-specific Luminex PCR amplification, global metagenomic sequencing, and profiling of metabolites by NMR.

Materials and methods

Collection of stool samples

Parents collected stool samples at home from all study participants 48 hours prior to the 3-month-old visit. To do this, parents were provided with a kit (thermos bag, ice pack, vessels with a spatula, sealed plastic bags, instruction sheet). Parents were asked to take 2 samples, store them at home in a freezer at -20°C, and bring the stool samples in a thermos bag containing a frozen ice pack to the site where the samples were stored frozen at -80°C. Next, the samples were transported to the Nestle Research Center, Switzerland, on dry ice and stored frozen at -80°C until analysis.

Isolation of DNA from feces

Total DNA was isolated using the QIAamp DNA Stool Mini Kit (QIAGEN) according to the manufacturer's instructions except for the addition of a series of mechanical chopping steps (11 × 45 s) using the FastPrep apparatus and Lysing Matrix B tubes (MP Biochemicals) (Junick and Blaut, 2012, Quantification of human fecal bifidobacterium species by use of quantitative real-time PCR analysis targeting the groEL gene, Appl Environ Microbiol 78: 2613–2622).

16S gene amplification and sequencing

Next, PCR amplification of the 16S V3–V4 variable regions was performed using universal primers (Klindworth et al., 2013, Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41: e1 ) and sequencing using Illumina Miseq technology as previously described (Caporaso et al., 2012, Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6: 1621–1624).

16S data analysis

After qualitative filtering of 6,710,039 microbiota-descriptive sequences, 154 protocol-compliant (PP) samples (see Table 3), with an average coverage of 42,739 sequences per sample, were classified into 173 OTUs. Three samples with less than 10,000 sequences were excluded from the 16S rDNA analysis.

Raw sequence data were analyzed using a combination of Mothur software packages (Schloss et al., 2009, Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75: 7537–7541) and QIIME (Caporaso et al., 2010, QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335–336). The paired end sequences were demultiplexed and spliced as described (Kozich et al., 2013 Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79: 5112–5120). The sequences were then split into separate fasta files for each sample using the Mothur commands [deunique.seqs(), degap.seqs() and split.groups()]. Conversion to QIIME format using add_qiime_labels.py and further analysis steps were performed in QIIME. Checking for chimerization and selection of OTUs with 97% identity was performed using the Uchime algorithm (Edgar et al., 2011, UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27: 2194–2200) using pick_open_reference.py. Assignment to taxonomic categories was performed on representative sequences using the RDP Classifier with a confidence threshold of 0.6 (Wang et al., 2007, Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261–5267) . Alignment of representative OTU sequences was performed using the PyNast method (Caporaso et al., 2010, PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26: 266–267), as well as Uclust as a pairwise alignment method. The resulting multiple alignments were then filtered and used to build a phylogenetic tree using the FastTree method (Price et al., 2009, FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26: 1641–1650). After qualitative filtering (Bokulich et al., 2013, Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10: 57–59), alpha diversity analyzes were performed in QIIME, as well as redundancy analysis (RDA) (Kindt R and Coe R., 2005, Tree diversity analysis. A manual and software for common statistical methods for ecological and biodiversity studies. Nairobi: World Agroforestry Center - ICRAF) at the genus level using the Calypso websites at http://bioinfo.qimr .edu.au/calypso.

Metagenomic analysis

The microbial composition of fecal samples from the three groups was determined by high-throughput multiplex sequencing of fecal-isolated DNA using an Illumina HiSeq instrument using 100 bp paired-end reads. (PE 100). DNA libraries were obtained using the Nextera XT protocol. Samples were sequenced on 6 high throughput flow cells.

The first reads were cut using the DynamicTrim (v2.1) algorithm from the SolexaQA package (Cox, M.P. et al., BMC Bioinformatics, 2010, SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinformatics 11:485 –490) with a probability cutoff of 0.05. The resulting sliced sequences were then filtered for a length threshold of 25 bp using the LengthSort (v2.1) algorithm from the SolexaQA package. Filtered reads were mapped to the full human hg19 genome using the bowtie v2.2.5 algorithm (Langmead, B. and Salzberg, S., Nature Methods., 2012, Fast gapped-read alignment with Bowtie 2. Nature Methods 9:357–359) to remove human reads. On average, 0.9%, 0.02%, and 3.3% of human reads were detected in the control group, study group, and breastfeeding group, respectively. To improve computational efficiency, the number of reads was further reduced using the unique.seqs function from the mothur v1.35 package (Schloss, P.D., et al., Appl Environ Microbiol, 2009, Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities, Appl Environ Microbiol 75: 7537–7541), which returns only unique sequences found. This stage reduced the number of reads by ~50%. After qualitative filtering, a median number of 76 to 80 million sequences was obtained from 157 samples for a set of "completed protocol" (PP) (see Table 3) with equal coverage in the control, study and reference (BF) group. A median number of 36 to 42 million unique sequences was obtained, also evenly distributed between groups. One sample with a total of 53661 sequences was excluded from the metagenomic analysis. The remaining reads were then used for profiling the composition of microbial populations using the MetaPhlAn v2 package (Segata, N. et al., Nature Methods, 2012, Metagenomic microbial community profiling using unique clade-specific marker genes. Nature Methods 9:811–814).

The presence of genes encoding known pathogens or antibiotic resistance genes was examined using ShortBRED. ShortBRED is an information processing system that takes a set of protein sequences, groups them into families, extracts a set of distinctive strings ("markers"), and then searches for these markers in metagenomic data and determines the presence and number of protein families of interest. Pathogenic factor markers were based on all protein sequences (2447 protein sequences) from the R3 release of the Virulence Factor of Pathogenic Bacteria Database (http://www.mgc.ac.cn/VFs/ — Chen, L.H. et al ., 2012, Toward the genetic diversity and molecular evolution of bacterial virulence factors Nucleic Acids Res 40 (Database issue): D641–D645). Antibiotic resistance gene markers were based on all protein sequences (7828 protein sequences) from version 1.1 of the Antibiotic Resistance Genes Database (http://ardb.cbcb.umd.edu/ - Liu, B. and Pop. , M., NAR, 2009, ARDB-Antibiotic Resistance Genes Database Nucleic Acids Res 37 (Database issue): D443–D447).

The statistical significance of differences between groups was assessed by fitting with a negative binomial regression model, taking into account, if necessary, extra null data.

Determination of pathogens by the Luminex method in stool samples

Fecal samples were subjected to a series of mechanical grinding steps (3 × 60 s) using a FastPrep apparatus and Lysing Matrix B tubes (MP Biochemicals), and simultaneous isolation of DNA and RNA was performed using the QIAamp MinElute Virus Spin Kit (QIAGEN). Nucleic acids were determined using sequence-specific primers from the Gastrointestinal Pathogen Panel (xTAG GPP) using the Luminex 200 system as recommended by the manufacturer (Luminex Molecular Diagnostics, Inc., Toronto, Canada). The analyzed analytes were adenovirus serotypes 40/41, Campylobacter (C. jejuni, C. coli and C. lari), toxins A/B Clostridium difficile, Cryptosporidium (C. parvum and C. hominis), Entamoeba histolytica, Escherichia coli O157, enterotoxigenic LT/ST toxins E. coli (ETEC), Giardia (G. lamblia also known as G. intestinalis and G. duodenalis), norovirus GI/GII, rotavirus A producing Shiga-like toxin E. coli (STEC) stx1/stx2, Vibrio cholera and Yersinia enterocolitica. Salmonella and Shigella results were not considered due to inconsistency in controls.

Analysis of feces for metabolites

In order to gain information beyond the aspect of fecal microbiota composition, the inventors investigated the biochemical composition of feces using a recognized metabonomic approach based on proton nuclear magnetic resonance spectroscopy (1H NMR). 1H NMR metabonomics provides a quantitative profile of key metabolites, including amino acids, basic organic acids (lactate, succinate, citrate, etc.) and carbohydrates, and therefore offers a unique window for tracking intestinal metabolic functions.

Metabolic profiling of fecal samples was adapted from a previously published method (Martin et al., 2014, Impact of breast-feeding and high- and low-protein formula on the metabolism and growth of infants from overweight and obese mothers. Pediatr Res 75, 535– 543. doi: 10.1038/pr. 2013.250). Briefly, 80–100 mg of frozen stool was taken from a stool sample tube, weighed, and freeze-dried. The dried samples were suspended in 1.2 ml of deuterated phosphate buffer solution 0.2 M KH2PO4 containing 0.3 mM sodium azide as an antibacterial agent and 1 mM 3-(trimethylsilyl)-[2,2,3,3-2H4]- Sodium 1-propionate as NMR chemical shift referent. The homogenates were centrifuged at 17,000 xg for 10 minutes and 5,500 µl of the supernatant were transferred into 5 mm NMR tubes. Metabolic 1H NMR profiles were obtained using a Bruker Avance III 600 MHz spectrometer equipped with a 5 mm cryoprobe at 300 K (Bruker, Biospin, Germany) using standard and water-suppressed spin echo pulse sequences and processed using the TOPSPIN software package ( vs. 3.2., Bruker). Data processing and analysis were performed as described previously (Martin et al., 2014). Significant metabolites identified by multivariate data analysis were relatively quantified using signal integration and analyzed using Kruskal-Wallis criteria. Given the exploratory nature of the work, p values were not adjusted for multiple comparisons.

results

Fecal microbiota composition based on 16S rDNA analysis

The average overall profile for the three groups at the birth level showed that although the composition of the total microbiota of the control and study groups showed a similar pattern characteristic of formula feeding, the study group showed a greater tendency towards similarity with the BF group than the control group. See fig. 3, which shows the overall profile for 3 groups.

Statistical analysis revealed several taxa, differently represented in the control and study groups. Indeed, when performing a statistical analysis of differences in microbiota composition at the level of genera (Wilcoxon rank test without correction for multiple comparisons), the control and study groups differed in six taxa (see Table 4). Three taxa had median null values across all groups, and numbers were reported for only a few outliers. The rest are shown in Fig. 4. The significance of these three taxa (Bifidobacterium, Escherichia and Peptostreptococacceae_uncl) as the main distinguishing features between the control and study groups was confirmed by random forest analysis (mean reduction error from 0.013 to 0.006). In particular, there is an increase in the population of Bifidobacteria and the suppression of populations of Escherichia and/or Peptostreptococcaceae compared to the total microbiota of the control group.

Genus p-value q FDR value Test Control BF (referent) Escherichia 0.008207 0.15888 1.616 4.432 2.196 Bifidobacterium 0.010146 0.15888 82.678 74.433 90.684 Coprobacillaceae_g__uncl 0.010592 0.15888 0 0 0 Peptostreptococcaceae_g__uncl 0.0258 0.2608 0.172 0.313 0 dorea 0.033119 0.2608 0 0 0 Megamonas 0.035533 0.2608 0 0 0

Table 4. To assess statistically significant differences between the control and study groups, the Wilcoxon rank test at the birth level, denoted by the p-value, was used. The BF group was not used in statistical tests, but the values are presented as reference. Values for study group, control group and BF group are medians of relative abundance for the indicated genus. The q FDR values are p-values corrected for multiple comparisons with a given false positive rate.

Alpha diversity for each sample was calculated using a score that takes into account the phylogenetic distances between OTUs and their distribution among the three comparison groups, see FIG. 5. Although the diversity in the BF group is significantly lower than the diversity in the groups of both mixtures, the diversity in the study group is significantly reduced (mean reduced by 0.19 units) compared to the control group and, therefore, is closer to the BF group.

The difference in the composition of the total microbiota according to the 16S rDNA data for the three groups was assessed using ordination (see Fig. 6). Statistics based on random permutations in redundancy analysis (RDA) showed that the three groups could be significantly distinguished at the level of birth (p < 0.001). The centroids of the BF group and the control group were clearly different, while the test group was in an intermediate position between the BF group and the control group.

Pathogen loading of stool samples

A portion of the stool sample set was available for specific pathogen load analysis using the Luminex xTAG Gastrointestinal Pathogen Panel. Table 5 shows the number of infants with at least one pathogen detected in feces collected at 3 months of age. The proportion of infants with a detectable viral pathogen was very similar in the test and control groups at 28% and 31.5%, respectively. The most common was norovirus. On the other hand, detectable bacterial pathogens were found in the feces of 14% of formula-fed infants, while in the control group, at least one bacterial pathogen was detected in 26% of fecal samples. However, this difference did not reach statistical significance (disagreement coefficient 0.46, p = 0.15). In these European infants, Clostridium difficile, by toxin A/B, was the most commonly identified pathogen by a wide margin. Eukaryotic pathogens (protozoa) were very rare, in 2% of infants treated with test formula and 5.6% of infants treated with control formula, with Cryptosporidium occurring in feces.

pathogens Test Control n,
% n,
% Disagreement ratio p-value (95% CI) Viral 14/50 17/54 0.846 0.83 28% 31.5% (0.364–1.967) Adenovirus 40/41 4/50 0/54 Norovirus Gl/Gll 10/50 15/54 Rotavirus A 1/50 2/54 Bacterial 7/50 14/54 0.465 0.149 fourteen% 26% (0.170–1.270) C. difficile toxin A/B 7/50 12/54 Campylobacter 0/50 1/54 E. coli O 157 0/50 1/54 ETEC LT/ST 0/50 0/54 STEC stx1/stx2 0/50 0/54 Vibrio cholerae 0/50 0/54 Yersinia enterocolitia 0/50 0/54 Protozoa 1/50 3/54 0.347 0.619 2% 5.6% (0.035–3.45) Cryptosporidium 1/50 3/54 Entamoeba histolytica 0/50 0/54 Giardia 0/50 0/54

Table 5. Number of infants with at least one pathogen present in feces at 3 months of age. The coefficient of disagreement and the p-value of Fisher's two-sided exact probability test were calculated.

Pathogen loading of infant stool samples was also assessed using a metagenomic dataset. In the case of Clostridium difficile, 36% of the infants in the study group and 46% in the control group were carriers according to metagenomic data. A similar pattern was observed for C. difficile toxin A/B when analyzed by Luminex PCR, where detectable levels were observed in 14% of infants in the study group and 22% of infants in the control group.

Selected first functional aspects of the stool microbiota

In addition to studying pathogens, the inventors also made queries on metagenomic data for the presence of genes encoding known pathogenic or antibiotic resistance genes. In FIG. 8 summarizes the results for known disease genes with or without correction for multiple comparisons. In total, the inventors identified 7 genes encoding known pathogenic factors, the level of which was significantly different in the control and study groups. Of these 7 genes, 5 genes clearly differed between the control and test groups, and between the control group and the BF group, but not between the test group and the BF group, indicating that the test group was closer to the BF reference group for these genes. Another 2 different genes were found to be different between all 3 groups.

With regard to genes encoding antibiotic resistance, the inventors identified a total of 8 genes encoding known antibiotic resistance genes, the levels of which were found to be significantly different between the control and study groups, see FIG. 6. Of these 8 genes, 4 genes obviously differed between the control and test groups, and between the control group and the BF group, but not between the test group and the BF group, and this shows that the test group was closer to the BF reference group for these genes . The 4 other genes differed between control and test groups, but did not differ between any of the mixture groups and the BF reference group.

Characteristic metabolic signs in feces

Multivariate analysis of the data revealed significant metabolites distinguishing between the test group and the control group, as well as the breastfed reference group (see Fig. 9). The content in the feces of some amino acids and organic acids was different in the study and control groups, and the differences observed in the study group tended towards the values observed in the feces of breastfed infants. More specifically, phenylalanine and isoleucine levels differed between test and control formula fed infants and differed from those observed in the feces of breastfed infants. Tyrosine levels did not differ between the test and control groups, but differed from the breastfed reference group. On the other hand, lactate levels were higher in test formula-fed infants than in control formula-fed infants, while reference samples from breastfed infants did not achieve statistically significant differences from those of formula-fed infants.

Conclusion

All of these different analyzes show that the overall microbiota composition of the test group (i.e., infants fed with infant formula according to the invention) tends to be more similar to the BF group (i.e., exclusively breastfed infants) than the control group. group (i.e. infants fed with conventional nutritional composition).

Indeed, this randomized, placebo-controlled, two-center clinical trial showed that fortification of standard infant formula with two specific human milk oligosaccharides 2'FL and LNnT alters the gut microbiota at 3 months of age, as shown by analysis of stool samples. It should be noted that the composition of the total microbiota and functional parameters for infants fed with the test formula not only differed from the control group, but also approached the breastfed reference group. In particular, this intermediate position of the fecal microbiota of the test group between the control group and the breastfed reference group is observed in the alpha diversity plot, in the analysis of redundancy at the genus level, and also when comparing the relative abundance of specific taxa at the genus level. Bacteria from the taxa Bifidobacterium, Escherichia and Peptostreptococcaceae make the main contribution to the observed shift of the test group towards the BF reference group. Such a shift in the composition of the microbiota is additionally confirmed by the observed change in the content of metabolites of intestinal bacteria in the feces, which are formed during the breakdown of milk, which can be seen from the data of 1H-NMR metabonomics. This shows that HMO 2'FL and LNnT in the test mixture affect not only the composition, but also the function of the gut microorganism.

In order to further emphasize the beneficial effect on the health of the infant, the inventors also studied the presence of specific true pathogens and pathogens in general. The bacterial pathogen C. difficile shows apparently lower levels, both by toxin-specific A/B PCR amplification and by metagenomic analysis, in the study group compared to the control group and approaches the lower levels observed in the reference group BF according to metagenomic data, although it does not reach statistical significance. It should be noted that several known pathogenic and antibiotic resistance genes determined by metagenomic analysis in the study group had a different level compared to the control group and were similar to the number observed in the BF reference group. Taken together, and assuming that the BF reference group is standard, these observations indicate that the microbial ecology in the gut of the host in the study group may be less favorable for putative harmful bacteria.

The higher fecal levels of the free amino acids phenylalanine, tyrosine, and isoleucine in the formula groups compared to the BF reference group may either be due to increased proteolytic activity or an excess of amino acids included in the mixture but not absorbed in the upper intestine. This suggests increased processing of food proteins by bacteria.

The present test mixture data show that the addition of HMO 2'FL and LNnT to the mixture generally reduces fecal free amino acids and stimulates lactate production. These changes describe the potential of 2'FL and LNnT to induce changes in gut microbial metabolism towards the metabolite levels seen in the feces of BF infants and hence towards induction of metabolic equivalence with breast milk.

Taken together, the fecal microbiota and characteristic metabolic signatures show that the addition of two individual and highly structurally specific HMOs to the initial infant formula shifts the gut microbiota assessed in feces, both in composition and in function, towards the microbiota observed in the infants of the group. b.f. In general, the test group is located between the infants of the control group and the infants of the BF group. However, according to some specific indicators, the study group is even indistinguishable from the BF reference group.

A nutritional composition containing at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, such as 2FL and LNnT, is very effective in infants or young children in terms of stimulation and/or induction in these infants or young children of the general a gut microbiota that is similar to the total gut microbiota of infants or young children fed exclusively on human breast milk, compared to the total gut microbiota of infants or young children fed predominantly or exclusively on a traditional nutritional composition that does not contain these oligosaccharides.

Therefore, it is also considered particularly effective for use in promoting normal bowel function and/or for use in the prevention and/or treatment of gut microbiota dysbiosis in infants or young children.

Example 4

Description of the clinical study

Goals. To determine the effect of oral administration of HMO on acute diarrhea and on the development of prolonged and persistent diarrhea in pediatric patients hospitalized with acute diarrhea.

Ways. A single-center, double-blind, randomized controlled clinical trial was conducted in non-breastfeeding boys and girls hospitalized in icddr, b, b (Dhaka, Bangladesh) with acute diarrhea. Children were randomized into groups (i) HMO and ORS-zinc (test product), and (ii) drinking water and ORS-zinc (standard of care treatment). Age-matched group (iii) included breastfed children hospitalized with acute diarrhea who continued to breastfeed during hospitalization and served as an open reference group.

Children hospitalized with acute diarrhea and eligible for inclusion criteria were randomly assigned to two groups. Both groups consisted of non-breastfed infants who received the standard care of oral rehydration with a glucose-based solution supplemented with zinc. Group (i) additionally received the test product, which is HMO in acidified drinking water, at a dose of 1.5 g per day for 14 days. The HMOs consisted of 2FL and LNnT, taken in a weight ratio of 2:1. Group (ii) received as a placebo only acidified drinking water, which cannot be distinguished by color and taste from the test product. Comparison between groups (i) and (ii) will allow to evaluate the therapeutic and prophylactic activity of the test product, consisting of two synthetic human milk oligosaccharides. Group (iii) consisted of fully or partially breastfed children hospitalized with acute diarrhea who received only standard care containing glucose-based oral rehydration solution supplemented with zinc. A comparison between groups (i) and (iii) will assess the therapeutic and prophylactic activity of breastfeeding, and a comparison with group (ii) will show whether the two synthetic human milk oligosaccharides in the test product reproduce the therapeutic and prophylactic activity of human milk containing a complex mixture of oligosaccharides and other protective agents.

The children included in the study were observed for diarrhea in the study ward for no more than 5 days. Children who continued to have diarrhea on day 5 were transferred to a long-term care unit (LTW) where they remained until the diarrhea resolved. Children whose diarrhea resolved during their stay in the study ward were discharged from the hospital on the day the diarrhea ended. Each child included in the study remained under observation in the study ward for at least 48 hours. The mother or caregiver of a child discharged from the hospital received instructions, a picture of the Bristol Stool Scale, and a cell phone for daily reporting of stool consistency, recurrence of diarrhea, or adverse events. On days 7 and 14 post-hospital, the mother/guardian and child were invited for a follow-up visit to the clinic for evaluation. A stool sample should be collected within 24 hours before the last visit.

Outcome indicators/parameters. Quantitative parameters of diarrhea (duration of diarrhea, number of stools, frequency of stools, presence of vomiting) and nutritional status to assess the effect of treatment. By analyzing the microbiota in feces, the stimulating effect of HMO on the reproduction of Bifidobacteria and restoration of balance after dysbiosis was evaluated.

Claims (18)

1. Use of an artificial nutritional composition containing at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide in the prevention or treatment of non-rotavirus diarrhea caused by E. coli (ETEC, EPEC and/or EAEC), Salmonella, Shigella , Aeromonas and/or Campylobacter in infants or young children by preventing or treating general intestinal microbiota dysbiosis that occurs before, during and/or after non-rotavirus diarrhea in said infants or young children where the young child is a child from one year to three years, and where fucosylated(s) oligosaccharide(s) is(are) in a total amount of 0.5-3 g/l or in a total amount of 0.38-2.32 g/100 g of the composition based on the weight of dry matter ; and N-acetylated oligosaccharide(s) is (are) in a total amount of 0.05-1 g/l of the composition or in a total amount of 0.03-0.78 g/100 g of the composition based on weight dry matter. 2. Use according to claim 1, wherein the fucosylated oligosaccharide(s) are present in a total amount of 0.8-1.5 g/l of the composition or in a total amount of 0.62-1.16 g /100 g of the composition based on the weight of dry matter. 3. Use according to claim 1 or 2, where the N-acetylated oligosaccharide(s) is (are) in a total amount of 0.5–0.8 g/l of the composition or in a total amount of 0.39– 0.62 g/100 g of composition based on dry weight. 4. Use according to any of the preceding claims, wherein the fucosylated oligosaccharide is selected from the list consisting of 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, 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 I, fucosyllacto-N-neohexaose II, difucosyllacto-N-hexaose I, difucosyllacto- N-neohexaoses I, difucosylacto-N-neohexaoses II, fucosyl-para-lacto-N-hexaoses, and any combination thereof, with preferably the fucosylated oligosaccharide being 2-fucosylactose (2'FL). 5. Use according to any one of the preceding claims, wherein the fucosylated oligosaccharide is a 2'-fucosyl epitope. 6. Use according to any one of the preceding claims, wherein the N-acetylated oligosaccharide is lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or a combination thereof. 7. Use according to any one of the preceding claims, wherein the N-acetylated oligosaccharide is lacto-N-neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH) or a combination thereof, preferably the N-acetylated oligosaccharide is lacto -N-neotetraose (LNnT). 8. Use according to any one of the preceding claims, wherein the composition comprises 2'-fucosyllactose and lacto-N-neotetraose (LNnT) or an oligosaccharide mixture consisting of 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT). 9. Use according to any one of the preceding claims, wherein the composition further comprises at least other(s) oligosaccharide(s) and/or dietary fiber(s) and/or their precursor(s) selected from the list containing galactooligosaccharide (GOS), xylooligosaccharide (FOS), xylooligosaccharide (XOS), inulin, polydextrose, sialylated oligosaccharides, sialic acid, fucose, and any combination thereof. 10. Use according to any one of the preceding claims, wherein the composition further comprises at least one probiotic in an amount of 10 ³ to 10 ¹² CFU/g of said composition by weight of dry matter. 11. The use according to any one of the preceding claims, wherein the composition is an infant formula, initial infant formula, formula for breastfed infants or follow-up formula, preterm infant formula, infant formula, cereal-based infant formula, fortifier or nutritional supplement. 12. Use according to any one of the preceding claims, wherein the composition is intended for infants under 6 months of age. 13. Use according to any one of the preceding claims, wherein the total gut microbiota refers to the composition and/or function of the entire gut microbiota, such as the relative taxonomic richness, diversity, activity and/or functionality of said microbiota. 14. Use according to any one of the preceding claims, wherein stimulation and/or induction in infants or young children of a total gut microbiota that approximates that of those infants or young children in the absence of non-rotavirus diarrhea and/or that approximates that of those infants or young children is provided in infants or young children fed exclusively on human breast milk compared to the total gut microbiota in infants or young children fed predominantly or exclusively on a conventional nutritional composition not containing these oligosaccharides. 15. Use according to claim 14, wherein said stimulation and/or induction involves an increase in Bifidobacterium population and/or suppression of Escherichia and/or Peptostreptococcaceae populations compared to the total gut microbiota in these infants or young children in the absence of non-rotavirus diarrhea and /or compared to the total gut microbiota in infants or young children fed predominantly or exclusively with a conventional nutritional composition not containing the named oligosaccharides, and/or said stimulation and/or induction involves the reduction of pathogens, especially the bacterial pathogen Clostridium difficile, and /or a decrease in disease-causing factor(s) compared to the total gut microbiota in these infants or young children in the absence of non-rotavirus diarrhea and/or compared to the total gut microbiota in predominantly fed infants or young children or and exclusively conventional nutritional composition not containing said oligosaccharides and/or said stimulation and/or induction involves a reduction in free amino acid production and/or stimulation of lactate production compared to the total gut microbiota in these infants or young children in the absence of non-rotavirus diarrhea , and/or compared to the total gut microbiota in infants or young children fed predominantly or exclusively with a conventional nutritional composition not containing said oligosaccharides, and/or said stimulated and/or induced total gut microbiota in infants or young children has alpha -diversity significantly reduced compared to the total gut microbiota in these infants or young children in the absence of non-rotavirus diarrhoea, and/or compared to the total gut microbiota in predominantly or exclusively fed infants or young children a highly conventional nutritional composition not containing these oligosaccharides and/or said stimulated and/or induced total gut microbiota in infants or young children has alpha diversity reduced by at least 0.10 units, such as at least 0.12 units or at least 0.15 units, such as 0.19 units, compared to the total gut microbiota in these infants or young children in the absence of non-rotavirus diarrhea, and/or compared to the total gut microbiota in infants or young children of age fed predominantly or exclusively with a conventional nutritional composition not containing these oligosaccharides and/or said stimulation and/or induction of the overall gut microbiota can be measured in the feces of said infants or young children, e.g. at 1, 4, 6 or 8 weeks of age, or older and, for example, after 1, 4, 6 or 8 weeks of feeding said nutritional composition. 16. Use according to any one of the preceding claims, wherein said nutritional composition is given to infants in the first 1, 2, 4, 8 or 12 weeks of life or during the first 2, 4, 6, 8, 12, 24 or 36 months of life.

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