WO2025190896A1

WO2025190896A1 - Mixture of five human milk oligosaccharides for reducing the risk of an infection in the gastrointestinal tract caused by pathogenic e.coli

Info

Publication number: WO2025190896A1
Application number: PCT/EP2025/056528
Authority: WO
Inventors: Erik Juncker BOLL; Stina JENSEN; Sara HESSING; Olivia COLBERG; Anja Wellejus; Gerben HERMES; Andrea Qvortrup HOLST
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2024-03-11
Filing date: 2025-03-11
Publication date: 2025-09-18
Anticipated expiration: 2026-09-11
Also published as: CN120093769A

Abstract

The present disclosure relates to a composition for use in reducing the risk of an infection of the gastrointestinal tract caused by a pathogenic E. coli in a human subject, said composition comprising an effective amount of 2´-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6´-sialyllactose, and lacto-N-tetraose.

Description

MIXTURE OF FIVE HUMAN MILK OLIGOSACCHARIDES FOR REDUCING THE RISK OF AN INFECTION IN THE GASTROINTESTINAL TRACT CAUSED BY PATHOGENIC E.COLI

FIELD

The present disclosure relates, in part, to the use of a mix of human milk oligosaccharides for reducing the risk of infections in the gastrointestinal tract, particularly diarrheal diseases, caused by Escherichia coli bacteria in a human subject. Further disclosed herein are methods, uses, processes, and the like.

BACKGROUND

Human milk oligosaccharides (HMOs) are non-digestible carbohydrates found in human milk. Their importance to infant nutrition is underscored by their position as the third most abundant solid component of human milk, behind lactose and lipids. HMOs can be structurally categorized as (a) fucosylated HMOs such as 2’- and 3-fucosyllactose (2’-FL and 3-FL), (b) neutral non- fucosylated HMOs such as lacto-N-tetraose (LNT) and (c) sialylated HMOs such as 3’- and 6’ sialyllactose (3’-SL and 6’-SL).

Table 1 : Structures of HMOs used herein

HMOs in human milk vary widely based on various influences such as genetics, lactation, and geographic location. While most HMO concentrations decrease over the course of lactation, at least two, 3’-SL and 3-FL, may increase. Different HMOs may work together in complementary ways to support the growth and development of infants.

It has been suggested that HMOs can lower the risk of gut microbiome imbalance due to harmful bacteria (Weichert, Stefan, et al. Nutrition researchi 0 (2013): 831-838). Further, it has been suggested that HMOs can selectively stimulate growth and metabolic activity of helpful bifidobacteria in support of overall gut health (Bode, Lars. Nutrition reviews, 2009, Vol 67 suppl. 2,183.191). It has been shown that mixtures of HMOs increase the relative abundance of bifidobacteria in the microbiome of formula-fed infants towards that in the microbiome of breastfed infants (Holst A. et al., Nutrients, 2023, 15, 3087). E. co!i is among the first colonizers and a common inhabitant of the infant gut microbiome. It persists due to e.g., the expression of fimbriae that enables it to attach to colonic epithelial cells (Nowrouzian, F. et al., 2003, Pediatric Research, 54(1), 8-14). It comprises a wide variety of strains ranging from commensals to severe pathogens depending on the presence of virulence factors that are frequently encoded on genetic elements (Evans DJ Jr. et al. in: Baron S, Medical Microbiology, 4th edition, Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 25 and Kaper et al.; 2004, Nature Reviews Microbiology, 2(2), 123-140.), thus transferable between strains.

Most E. coli strains are harmless and contained within the intestinal lumen but may cause infection given favorable circumstances such as breaches of the gastrointestinal barriers (Kai, A. et al., 2010, Nippon Rinsho. Japanese Journal of Clinical Medicine, 68, 6(1), 203-2075) or through acquired virulence factors (Makvana, S., & Krilov, L. R., 2015, Pediatrics in Review, 36(4), 167-171).

Certain pathogenic E. coli strains are classified into pathotypes that are associated with distinctive severe diarrheal diseases in infants such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) (Makvana, S., & Krilov, L. R., 2015, Pediatrics in Review, 36(4), 167-171). In vitro assays indicate that mixtures of human milk oligosaccharides concentrated from human milk have an inhibitory effect on the adhesion of EPEC serotype 0119 to epithelial cells (Coppa G. V. et al., 2006, Pedriatric Research, 59, 3, 2006, 377 - 382). Mixtures of synthetic human milk oligosaccharides, that were not concentrated from human milk have not been studied. Enteropathogenic E. coli (EPEC) of serotype 0127 is associated with outbreaks of diarrhea in infants younger than 2 years (Prabhdeep K., Pradeep K. D., 2023, Newborn, Vol 2 Issue 1 , 102-113).

Enterohemorrhagic E. coli (EHEC) is a pathotype of E. coli associated with foodborne outbreaks worldwide. Clinical manifestations of EHEC infection range from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome. Infants and children are the main affected patients (Gomes TAT et al, 2016; 47; 3-30). EHEC requires binding to the intestinal epithelium by virtue of adhesins to establish infection (McWilliams BD, Torres AG, Microbiol Spectr. 2014; 2(3)). Thus, reduction of this binding may lower the risk of colonization and ensuing diarrheal disease caused by EHEC and other E. coli pathotypes.

Collectively, E. coli is the leading cause of infantile diarrhea, which is the second most common cause of death among children under the age of five worldwide (Johansson, E. W. et al., 2009, in The United Nations Children’s Fund (UNICEF)ZWorld Health Organization (WHO), Vol. 44, 11 , 1- 68). Thus, reducing the occurrence and colonization of E. coli in the gastrointestinal tract may lower the risk of diarrheal diseases caused by E. coli.

Strategies are needed that may lower the risk of diarrheal diseases caused by pathogenic E. coli and in particular by the EHEC pathotype. SUMMARY

The present disclosure provides compositions, uses, methods and the like for reducing the risk of an infection in the gastrointestinal tract, as for example a diarrheal disease, in a human subject. In particular, the present disclosure relates to a composition comprising 5 HMOs (2'- Fucosyllactose (2’-FL), 3-Fucosyllactose (3-FL), 3'-sialyllactose (3’-SL), 6'-sialyllactose (6’-SL) and lacto-N-tetraose (LNT)) for lowering the risk of infections in the gastrointestinal tract, in particular diarrheal diseases, in a human subject, in particular in non-adult subjects.

While not wishing to be bound by theory, it is believed that the mixture of 5 HMOs (5HMO-mix consisting of 2'-Fucosyllactose (2’-FL), 3-Fucosyllactose (3-FL), 3'-sialyllactose (3’-SL), 6'- sialyllactose (6’-SL) and lacto-N-tetraose (LNT)) reduces the number of E coli, in particular the number of pathogenic E.coli, in the gastrointestinal tract and inhibits the binding of pathogenic E. coli io the intestinal mucosal barrier. The binding of pathogenic E.coli to epithelial cells is one step in the mechanism of infection. Thus, by inhibiting this binding step and lowering the total number of E. coli and the number of pathogenic E.coli in the gastrointestinal tract, it is believed that the risk of infections with pathogenic E. coli is reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the reduction of adhesion of a strain of enterohemorrhagic E. coli (EHEC) serotype 0157 to monolayers of human colon cancer cells (Caco-2) in the presence of galactooligosaccharides (GOS) at a dosage of 30 mg/ml and 5HMO-mix at different dosages from 1 mg/ml to 30 mg/ml.

Figure 1 B shows the reduction of adhesion of a strain of enteropathogenic E. coli (EPEC) serotype 0127 to monolayers of human colon cancer cells (Caco-2) in the presence of galactooligosaccharides (GOS) at a dosage of 30 mg/ml and 5HMO-mix at 5 mg/mL and 30 mg/mL.

Figure 2 shows the relative abundance of E. coli in an infant in vitro colonic model, l-TIM-2 and the comparisons between samples of the donor fecal inocula and l-TIM-2 samples with the feed SIIEM (without HMOs) and SIIEM-HMO (with the 5HMO-mix).

Figure 3 shows the relative abundance of E. coli in an infant in vitro colonic model, l-TIM-2 and the comparisons within donors (INF5-7) between samples of the donor fecal inocula and l-TIM-2 samples with the feed SIIEM (without HMOs) and SIIEM-HMO (with the 5HMO-mix) in independent experiments with individual donors (INF5-7).

Figure 4 shows the impact of the 5HMO mix on EPEC abundances in the gut microbiota of healthy infants (n = 8) at 44h (including 24h of introducing EPEC into the microbiota. (A) EPEC levels in the NSC and HMO study arms, quantified by qPCR and expressed as copies/mL. Statistical differences between NSC and the individual treatments are visualised via * (0.1 < Padjusted < 0.2), ** (0.05 < padjusted < 0.1) or *** (padjusted < 0.05). (B) The impact of HMO on EPEC levels, expressed as log2Fold change vs NSC, as quantified via qPCR. The different symbols represent the 8 individual donors. Overall, lower levels of Enteropathogenic E. coli (EPEC) of serotype 0127 can be observed in the presence of 5HMOmix (right) compared to a nonsubstrate control (NSC) without HMOs (left) in an infant ex vivo 20hour fermentation model and subsequently a 24 hour E.coli infection model.

Figure 5 shows the impact of 5HMO mix on (A) pH, (B) gas production, (C) total SCFA, at 20h and 44h. Statistical differences between NSC and the individual treatments are visualised via * (0.1 < p adjusted < 0.2), ** (0.05 < P adjusted < 0.1) or *** (p adjusted < 0.05). The different symbols represent the 8 individual donors. Overall, modulation of the metabolic activity of the infant microbiota is indicated by decreased pH, increased gas production, increased production of short-chain fatty acids in the presence of 5HMO mix compared to non-substrate control without HMOs in an infant ex vivo 20hour fermentation model and subsequently a 24 hour E.coli infection model .

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by persons skilled in the art. Although any methods and materials equivalent or similar to those described herein can be used in the practice of the present disclosure, typical methods and materials are described. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range including the two end values, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. As used herein, the term “and/or” is intended to mean the combined (“and”) and the exclusive (“or”) use, i.e. “A and/or B” is intended to mean “A alone, or B alone, or A and B together”.

As used herein the terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of a material sufficient to improve the overall health of the subject and confer benefits similar to the ones demonstrated in the examples. The actual effective dosage in absolute numbers depends on factors including the state of health of the subject in question, and other ingredients present. The "effective amount", "effective concentration", or "effective dosage" of the material may be determined by routine assays known to those skilled in the art.

As used herein the term "isolated" means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

A bacterial “strain” as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied and that originates from a single isolate or pure culture. Probiotics are classified by their genus (e.g. Bifidobacterium), species and subspecies (e.g. animalis subs p. lactis), and strains (e.g. DSM 15954 and/or BB-12®). FAO/WHO has stated that probiotic effects are strain specific and that most probiotic characteristics of a particular strain cannot therefore be extrapolated to other strains of the same species.

As used herein, the term “probiotic” refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects the host (Hill et al. (2014) Expert Consensus Document, The International Scientific Association for Probiotics and Prebiotics. Consensus statement on the scope and appropriate use of the term probiotic).

The term "human milk oligosaccharide" or "HMO", as used herein, unless otherwise specified, refers generally to a number of complex carbohydrates found in human breast milk that can be in acidic or neutral form, and to precursors thereof. Exemplary non-limiting human milk oligosaccharides include 3'- sialyllactose, 6'-sialyllactose, 3-fucosyllactose, 2'-fucosyllactose, and lacto-N-tetraose.

The terms "treat" or "treating" should not be taken to imply that an individual is treated until total recovery. Accordingly, these terms broadly include amelioration and/or prevention of the onset of the symptoms or severity of a particular condition. The term “lowering the risk of an infection” include prevention of the onset of the symptoms or reduction of the severity of the symptoms. The risk of an infection is reduced or lowered compared to the situation in absence of the claimed composition.

The term "shelf stable" as used herein, unless otherwise specified, refers to a nutritional product that remains commercially stable after being packaged and then stored at 18-24°C for at least 3 months, including from about 6 months to about 24 months, and also including from about 12 months to about 18 months.

The terms "nutritional formulation" or "nutritional composition" as used herein, are used interchangeably and, unless otherwise specified, refer to nutritional liquids, nutritional powders, nutritional supplements, and any other nutritional food product as known in the art. The nutritional powders may be reconstituted to form a nutritional liquid, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human.

The term "nutritional powder" as used herein, unless otherwise specified, refers to nutritional products in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes both spray-dried and dry-mixed dry-blended powders.

The term "newborn" as used herein, unless otherwise specified, refers to a person from birth up to four weeks of age. The term “infant” as used herein, unless otherwise specified, refers to a person 12 months or younger. The term "preterm" as used herein, refers to a baby born prior to 36 weeks of gestation. The term "toddler" as used herein, unless otherwise specified, refers to a person greater than one year of age up to three years of age. The term "child" as used herein, unless otherwise specified, refers to a person greater than three years of age up to twelve years of age.

The term “formula” as used herein, unless otherwise specified, refers to liquid and solid human milk replacements or substitutes that are suitable for consumption by a human.

The term "human milk fortifier" as used herein, unless otherwise specified, refers to liquid and solid nutritional products suitable for mixing with breast milk or formula for consumption by a preterm or term infant.

The terms "susceptible" and "at risk" as used herein, unless otherwise specified, mean having little resistance to a certain condition or disease, including being genetically predisposed, having a family history of, and/or having symptoms of the condition or disease. The terms "modulating" or "modulation" or "modulate" as used herein, unless otherwise specified, refer to the targeted movement of a selected characteristic. The term purity used in this application refers to chemical purity, thus the degree to which a substance is undiluted or unmixed with extraneous material. Hence, the chemical purity is an indicator of the relationship between the at least one HMO and by-products/impurities.

Chemical purity is expressed as a percentage (%) and is calculated using the following formula: Percent purity= 100x (mass of desired compound in sample)/(total mass of sample)

The purity can be determined by any suitable method known to the person skilled in the art. One suitable method is HPLC (high-performance liquid chromatography). In the obtained chromatogram, the ratio of the area underneath the peak(s) representing the amount of HMO(s) to the sum of areas underneath the peaks representing the HMO(s) and all other compounds than said HMO(s) in the chromatogram is calculated.

All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights, as they pertain to listed ingredients, are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified.

Numerical ranges as used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not.

The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any particular preferred embodiment(s) disclosed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The present disclosure provides a composition comprising a mixture of 5 human milk oligosaccharides (HMOs), namely 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'- sialyllactose, and lacto-N-tetraose. This mixture of these 5 HMOs is also referred to as “5HMO- mix”. While not wishing to be bound by theory, is it believed that the composition comprising the 5HMO-mix reduces the risk for a human subject of getting an infection in the gastrointestinal tract caused by a pathogenic E. coli. This is supported by the findings in in vitro experiments that the 5HMO-mix reduces the total abundance of E. coli in the gastrointestinal tract, the reduction of the number of a pathogenic E.coli in an E.coli infection model and that the binding of a pathogenic E. coli to epithelial cells is inhibited in the presence of the 5HMO-mix.

To investigate the influence of the 5HMO-mix on the relative abundance of E. coli , an infant in vitro colonic model in TIM-2 (TNO In vitro model) was set up. The TIM-2 model is a validated, dynamic and computer controlled simulating model (Venema K. et al., The impact of Foods BioActives on Gut Health, 2015, 293-304). Here, the model was set up to simulate the infant colon environment. The bacterial communities from the feces of infants were recreated and the effects of supplementing the 5HMO-mix on microbial composition and activity overtime were studied. The effects were compared to non-supplemented bacterial communities. For this, two media were used, a simulated infant ileal efflux medium (SIIEM), and SIIEM with the 5HMO-mix (2’- Fucosyllactose, 3-Fucosyllactose, Lacto-N-Tetraose, 3’-Sialyllactose and 6’-Sialyllactose) in physiologically relevant ratios and concentrations. It was found that HMO supplementation maintained the relative abundance of HMO-utilizing bacteria, while the relative abundance of E. coli was decreased (Figure 2, Figure 3). While not wishing to be bound by theory, it is believed that the decrease in the relative abundance of E. coli may lower the risk of an infection of the gastrointestinal tract.

To evaluate the influence of a composition comprising the 5HMO-mix on the binding of pathogenic E. coli to the intestinal epithelium, a strain of enterohemorrhagic E. coli (EHEC) of serotype 0157 and a strain of enteropathogenic E. coli (EPEC) of serotype 0127 were preincubated or not with the 5HMO-mix at different dosages. The pre-incubated E. coli were added to monolayers of human colon cancer cells (Caco-2). After a defined incubation time, the number of EHEC or EPEC adhering to the intestinal cell monolayers was determined. It was found that the 5HMO-mix dose-dependently reduced binding of EHEC to the cell monolayers (Figure 1 A) and also binding of EPEC to the cell monolayers was reduced (Figure 1 B). While not wishing to be bound by theory, it is believed that the reduction of EHEC and EPEC binding to intestinal epithelial cells, may lower the risk of an infection.

To evaluate the influence of a composition comprising the 5HMO-mix, an EPEC (E.coli 0127) infection was simulated with fecal samples from formula-fed infants in an ex vivo system using the SIFR® technology (Van den Abbeele, P. et al. Bridging preclinical and clinical gut microbiota research using the ex vivo SIFR® technology. Frontiers Microbiol 14, (2023)). This technology provides insights into compositional changes in the gut microbiota. The technology has been validated with clinical data. It was found that the 5HMO-mix significantly lowered EPEC 0127 levels compared to the situation without the 5HMO-mix. While not wishing to be bound by theory, it is believed that the reduction of the EPEC levels observed may lower the risk of an infection with the E.coli pathogen. In certain embodiments, the composition comprising the 5HMO-mix is used to lower the risk of diarrheal diseases. Infections of the gastrointestinal tract often come with diarrhea as a prominent symptom. While not wishing to be bound by theory, it is believed that the application of a composition comprising the 5HMO-mix may lower the risk of diarrheal diseases.

In certain embodiments, the composition comprising the 5HMO-mix is used to reduce the risk of an infection in the gastrointestinal tract by inhibiting the adhesion of E. coli to intestinal epithelial cells. It is known that one step in the mechanism of infection is the attachment to the epithelial cells.

In certain embodiments, the composition comprising the 5HMO-mix is used to reduce the risk of an infection in the gastrointestinal tract caused by the enterohemorrhagic E. coli (EHEC) pathotype, wherein the EHEC preferably has pathotype 0157. In vitro experiments have shown that the 5HMO-mix dose-dependently inhibits the adhesion of this pathotype.

In certain embodiments, the composition comprising the 5HMO-mix is used to reduce the risk of an infection in the gastrointestinal tract caused by the enteropathogenic E. coli (EPEC) pathotype, wherein the EPEC preferably has serotype 0127. Ex vivo experiments have shown that the 5HMO-mix reduces the relative amount of this pathogenic EPEC in the microbiota. While not wishing to be bound by theory, this antipathogenic effect might originate from the strong modulation of metabolite production of the gut microbiome. In ex vivo experiments, the 5-HMO mix stimulated the metabolic activity of the gut microbiota, significantly decreasing pH, and increasing the production of gas as well as of total short chain fatty acid (SCFA) content.

In certain embodiments, the composition is a nutritional composition such as a formula or a dietary supplement.

A preferred composition herein is a nutritional composition such as a formula. The nutritional compositions may be in any product form comprising the ingredients described herein, and which is safe and effective for oral administration. The nutritional compositions may be formulated with optional ingredients such as those described herein.

The nutritional compositions of the present disclosure are preferably formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form that then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof.

The nutritional compositions may be formulated with sufficient kinds and amounts of nutrients to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional product for use in individuals afflicted with specific diseases or conditions or with a targeted nutritional benefit as described below. Specific non-limiting examples of product forms suitable for use as HMO-containing compositions as disclosed herein include, for example, liquid and powdered dietary supplements, liquid and powdered human milk fortifiers, liquid, and powdered formula.

Nutritional liquids include both concentrated and ready-to-feed nutritional liquids. These nutritional liquids are most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the present disclosure.

Nutritional emulsions suitable for use may be aqueous emulsions comprising proteins, fats, and carbohydrates. These emulsions are generally flowable or drinkable liquids at from about 1 °C to about 25°C and are typically in the form of oil- in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.

The nutritional emulsions may be and typically are shelf stable. The nutritional emulsions typically contain up to about 95% by weight of water, including from about 50% to about 95%, also including from about 60% to about 90%, and also including from about 70% to about 85%, of water by weight of the nutritional emulsions. The nutritional emulsions may have a variety of product densities, but most typically have a density greater than about 1 g/mL, including greater than about 1.05 g/mL, including greater than about 1.055 g/mL to about 1.12 g/mL, and also including from about 1.085 g/mL to about 1.10 g/mL. The nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise generally at least 660 kcal/liter, about 675 kcal/liter to about 820 kcal/liter, about 680 kcal/liter to about 800 kcal/liter. In some embodiments, the emulsion may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter. The nutritional emulsion may have a pH ranging from about 3.5 to about 8, from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2. Although the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size is generally at least 1 mL, or even at least 2 mL, or even at least 5 mL, or even at least 10 mL, or even at least 25 mL, including ranges from about 1 mL to about 300 mL, including from about 4 mL to about 250 mL, and including from about 10 mL to about 240 mL.

The nutritional solids may be in any solid form but are typically in the form of flowable or substantially flowable particulate compositions, or at least particulate compositions, that may optionally be compressed into tablets. Particularly suitable nutritional solid product forms include spray dried, agglomerated and/or dry-blended powder compositions. The compositions can easily be scooped and measured with a spoon or similar other device, and can easily be reconstituted by the intended user with a suitable aqueous liquid, typically water, to form a nutritional composition for immediate oral or enteral use. In this context, "immediate" use generally means within about 48 hours, most typically within about 24 hours, preferably right after reconstitution. The nutritional powders may be reconstituted with water prior to use to a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the powders are reconstituted with water to form compositions comprising generally at least 660 kcal/liter, about 675 kcal/liter to about 820 kcal/liter, about 680 kcal/liter to about 800 kcal/liter. In some embodiments, the reconstituted powder may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the reconstituted powder may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.

The present compositions may be useful in newborns, infants, toddlers, or children. The present compositions may be useful in newborns. The present compositions may be useful in infants. It has been shown that the 5HMO-mix reduces the relative abundance of E. coli in the infant TIM-2 model, thus indicating that the mixture may have a beneficial effect for infants. Also, E. coli is the leading cause of infantile diarrhea, which indicates that infants may particularly benefit from a composition comprising the 5HMO-mix.

The present composition may comprise an effective amount of 2'-fucosyllactose, 3- fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.

The present composition may comprise at least 0.01%, by weight, of 2'-fucosyllactose; at least 0.01%, by weight, 3-fucosyllactose; at least 0.01%, by weight, 3'-sialyllactose; at least 0.01%, by weight, 6'-sialyllactose; and at least 0.01%, by weight, lacto-N-tetraose.

The present compositions may comprise individual HMOs in any suitable amount, such as, for example, at least 0.001 mg/mL, including from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 10 mg/mL, including from about 0.01 mg/mL to about 5 mg/mL (mg of particular HMO per mL of composition).

The present composition may comprise the 5 HMOs in specific ratios, wherein the ratios of 2'- fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose in the composition are: 45% to 60%, by weight, of 2’-fucosyllactose; 8% to 18%, by weight, of 3- fucosyllactose; 2% to 10%, by weight, of 3’-sialyllactose; at least 2% to 10%, by weight, of 6’- sialyllactose; and 20% to 31 %, by weight, of lacto-N-tetraose. Where the composition is a nutritional powder, the concentration of individual HMOs in the nutritional powder is preferably from about 0.001% to about 5%, including from about 0.01% to about 1% (by weight of the nutritional powder). Where the composition is a ready- to-feed nutritional liquid, the concentration of individual HMOs is preferably from about 0.001% to about 0.50%, including from about 0.001% to about 0.15%), including from about 0.01% to about 0.10%, and further including from about 0.01%) to about 0.03% (by weight of the ready-to-feed nutritional liquid). Where the composition is a concentrated nutritional liquid, the concentration of individual HMOs is preferably from about 0.002% to about 0.6%, including from about 0.002% to about 0.3%, including from about 0.02% to about 0.20% (by weight of the concentrated nutritional liquid).

The present composition may be in the form of a powder. Formulating compositions with HMOs can be somewhat problematic. It has been found that a more reproducible and consistent composition can be achieved through controlling the particle size distribution (PSD) of the HMO. While not wishing to be bound by theory, it is believed that having a somewhat narrow PSD improves the flowability of the HMO enabling a more effective mixing with the other ingredients. In addition, it is believed that a PSD within a certain range provides a better solubility profile. Particle size of an HMO may be determined using a standard method, such as using a sieve tower, which separates the powder into the different fractions after a defined time with a predefined amplitude. The sieves used in such a method may be sieves which comply with DIN ISO 3310-1.

It is preferred that the HMOs, in particular 2’FL and 3FL, used in the present compositions have the following particle size characteristics:

Percent through mesh #230 (63 pm) - less than about 20%, less than about 18%, less than about 16%, less than or equal to about 15%.

Percent through mesh #100 (150 pm) - greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.

Percent through mesh #45 (355 pm) - greater than about 95%, greater than about 92%, greater than or equal to about 90%.

Percent through mesh #20 (850 pm) - 100%.

It is preferred that the human milk oligosaccharides when in form of a powder, have a water activity a_w between 0.10 and 0.3, preferably between 0.10 and 0.25. The water activity may be determined using known hygrometers; preferably the water activity is determined according to ISO 18787:2017. The water activity ensures the microbial stability of the powder and prevents contamination with undesired microorganisms.

The present composition preferably comprises HMOs of synthetic origin, such as HMOs produced by microbial fermentation, or alternatively HMOs produced by biocatalysis or chemical synthesis. In particular, microbial fermentation allows production on an industrial scale in high purities, which may be used in nutritional compositions. For the present composition, preferably HMOs are used that have a purity of more than 85%, preferably a purity of more than 90%, more preferably a purity of more than 95%.

The compositions of the present disclosure may optionally include anti-inflammatories such as long-chain polyunsaturated fatty acids (LCPUFAs) and/or antioxidants such as carotenoids. LCPUFAs may be included in the compositions to provide nutritional support and to enhance growth and functional development of the intestinal epithelium and associated immune cell populations. Exemplary LCPUFAs for use in the present compositions include, for example, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), linoleic acid, linolenic acid (alpha linolenic acid) and gamma-linolenic acid derived from oil sources such as plant oils, marine plankton, fungal oils, and fish oils. The present compositions preferably comprise total concentrations of LCPUFA of from about 0.01 mM to about 10 mM and including from about 0.01 mM to about 1 mM. Alternatively, the compositions comprise total concentrations of LCPUFA of from about 0.001 g/L to about 1 g L.

Additionally, antioxidants such as carotenoids, and particularly, combinations of the carotenoids, lutein, lycopene, zeaxanthin and/or beta-carotene may be included in the present compositions.

The compositions of the present disclosure may further comprise other optional components that may modify the physical, chemical, aesthetic or processing characteristics of the composition or to serve as pharmaceutical or additional nutritional components. Non-limiting examples of such optional ingredients include preservatives, emulsifying agents, buffers, pharmaceutical actives, nutrients, colorants, flavors, thickening agents and stabilizers, flowing agents, minerals, emulsifying agents, lubricants, sweetening agents, and the like.

A flowing agent or anti-caking agent may be included in the present compositions to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container. Non-limiting examples include tricalcium phosphate, silicates, and combinations thereof. The concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition.

The compositions of the present disclosure may be prepared by any known or otherwise effective manufacturing technique for preparing the selected product solid or liquid form. Many such techniques are known for any given product form such as nutritional liquids or powders and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein.

Those skilled in the art will appreciate that the administration of compositions disclosed herein can be carried out with dose levels and dosing regimens as required depending on the circumstances and on the condition of the subject. Suitable dosage regimes can be determined based on the teaching of the present application. Dosage regimens may be adjusted to provide the optimal support of the subject. It will be appreciated that the exact amounts and rates of administration will depend on a number of factors such as the age, body weight, general health, sex, and dietary requirements of the subject. Based on the teaching herein those skilled in the art can, by routine trial and experimentation, determine suitable dosage regimes on a case-by-case basis.

The present compositions may comprise at least one probiotic strain, for example, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lactobacillus easel subsp. casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius), any strain belonging to the genus Bifidobacterium (including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dent! urn, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum), or any strain from the genera of Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia or the family Coriobacteriaceae, as well as suitable combinations of the foregoing.

The present compositions may comprise at least one strain of a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241 , Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG-17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, Lactobacillus paracasei subsp. paracasei deposited as CCTCC M204012 and suitable combinations thereof.

The present compositions preferably comprise an effective amount of probiotic. For example, where present it is preferred the probiotic has a concentration ranging from 0.05 x 10⁹ CFU/g to 30 x 10⁹ CFU/g, preferably from 0.5 x 10⁹ CFU/g to 25 x 10⁹ CFU/g. EXAMPLES

Example 1

The 5HMO-Mix containing: 2.99 mg/ml 2’-FL, 0.75 mg/ml 3-FL, 1.5 mg/ml LNT, 0.23 mg/ml, 3’- SL, and 0.28 mg/ml 6’-SL, was produced by Chr. Hansen HMO GmbH, Rheinbreitbach, Germany. Stocks of 5HMO-Mix solutions were dissolved in water.

The human epithelial intestinal cancer cell line Caco-2 (ACC 169, DSMZ, passages 5-20) was maintained in Dulbecco’s Modified Eagle Medium (DMEM) GlutaMAX Supplement (Gibco) supplemented with 1% non-essential amino acids (Merck Life Science), 1% penicillinstreptomycin (10,000 U/mL) (Gibco), and 10% heat-inactivated fetal bovine serum (Gibco) at 37°C in a 5% CO2 atmosphere. The cells were seeded at a density of 8x10⁴ cells/well in 24-well culture plates, and the media was replaced every 3-4 days until cells were ready for use in the experiment after 14 days.

A strain of EHEC 0157 (DSM 17076) was grown agitated overnight in Luria-Bertani (LB) broth at 37°C, then washed twice using Hanks balanced salt solution (HBSS, Gibco), and resuspended and ODeoonm-norrnalized to 0.5 in DMEM. The EHEC strain was then diluted 1 :20 in DMEM to approximately 2x10⁶ colony forming units (CFU)Zml and pre-incubated or not with 5-HMO mix at different dosages (range 1 to 30mg/ml) or GOS (30mg/ml) with agitation at room temperature. After 2hrs of pre-incubation, the cell culture media was removed from the Caco-2 cell monolayers, which were then gently washed twice using HBSS. The EHEC strain suspensions (with and without 5-HMO mix or GOS) were then added the Caco-2 cell monolayers, and after 1 hr of incubation, the media containing non-adhering EHEC was removed. The Caco-2 cell monolayers were gently washed trice in HBSS and following 3 washing steps, 0.1% Triton X-100 in HBSS was added to wells to release the adhering EHEC. The number of EHEC adhering to the intestinal cell monolayers was then quantified by serial plating on tryptic soy broth (plates) and counting of CFU) following overnight incubation at 37°C.

Results

The results showed that 5HMO-mix dose-dependently reduced EHEC binding to the intestinal epithelial cell monolayers (up to 60% reduction) with statistical significance (p<0.01) achieved at 10mg/ml or higher compared to the EHEC control treatment group. GOS (tested at 30mg/ml) failed to significantly reduce EHEC binding (see Figure 1A). Data are expressed as means + standard deviation (n = 3 independent experiments). Statistical significance was determined by One-way ANOVA comparison to the EHEC control group followed by Dunnett’s multiple comparisons test.

Example 2

The 5HMO-Mix containing: 2.99 mg/ml 2’-FL, 0.75 mg/ml 3-FL, 1.5 mg/ml LNT, 0.23 mg/ml, 3’- SL, and 0.28 mg/ml 6’-SL, was produced by Chr. Hansen HMO GmbH, Rheinbreitbach, Germany. Stocks of 5 H MO- Mix solutions were dissolved in water.

A strain of EPEC 0127 E2348/69 was grown agitated overnight in Luria-Bertani (LB) broth at 37°C, then washed twice using Hanks balanced salt solution (HBSS, Gibco), and resuspended and ODeoonm-norrnalized to 0.5 in DMEM. The EPEC strain was then diluted 1 :20 in DMEM to approximately 2x10⁶ colony forming units (CFU)/ml and pre-incubated or not with 5-HMO mix at different dosages (5mg/mL and 30mg/ml) or GOS (30mg/ml) with agitation at room temperature. After 2hrs of pre-incubation, the cell culture media was removed from the Caco-2 cell monolayers, which were then gently washed twice using HBSS. The EPEC strain suspensions (with and without 5-HMO mix or GOS) were then added to the Caco-2 cell monolayers, and after 1 hr of incubation, the media containing non-adhering EPEC was removed. The Caco-2 cell monolayers were gently washed trice in HBSS and following 3 washing steps, 0.1% Triton X-100 in HBSS was added to wells to release the adhering EPEC. The number of EPEC adhering to the intestinal cell monolayers was then quantified by serial plating on tryptic soy broth (plates) and counting of CFU) following overnight incubation at 37°C.

Results

The results showed that 5HMO-mix dose-dependently reduced EPEC binding to the intestinal epithelial cell monolayers (up to 30% reduction) with statistical significance (p<0.01) achieved at 30mg/ml compared to the EPEC control treatment group. GOS (tested at 30mg/ml) failed to significantly reduce EPEC binding (see Figure 1 B). Data are expressed as means + standard deviation (n = 3 independent wells). Statistical significance was determined by One-way ANOVA comparison to the EPEC control group followed by Dunnett’s multiple comparisons test.

Example 3 Method

Fecal samples from three healthy, Danish, 2-6-month-old infants (INF 5 to 7) were used as inoculation material in three independent experiments using the validated, computer-controlled, dynamic model of the infant colon, l-TIM-2. Each experimental run using feces from one of the infants consisted of four independent compartments operating in parallel. For the general set-up of the TIM-2 model see Venema K. et al., The impact of Foods Bio-Actives on Gut Health, 2015, 293-304. For each experiment two compartments received the simulated infant ileal efflux medium, SIIEM (consisting of (g/L demineralized H2O): 25.5 lactose, 1 .7 casein, 28.9 whey, 0.1 ox-bile, 0.8 CaCI₂-2H₂O, 0.01 FeSO₄-7H₂O, 0.004 hemin, 4.7 K₂HPO₄, 4.5 KCI, 0.75

MgSO₄ 7H₂O, 4.0 mucin, 2.5 yeast extract, 8.4 NaCI, 1.5 NaHCCh, 4.5 peptone, 0.8 cysteine HCI, 10.0 polysorbate 80, and 1.5 ml vitamin solution consisting of (mg/L H₂O): 1 menadion, 2 biotin, 0.5 vitamin B12, 10 pantothenic acid, 5 nicotinamide, 5 para-aminobenzoic acid and 4 thiamine) and two compartments received SIIEM-HMO, which additionally contained a mixture of five of the most abundant HMOs in breastmilk (5HMO-Mix), 2’-Fucosyllactose (2’-FL), 3- Fucosyllactose (3-FL), Lacto-N-Tetraose (LNT), 3’-Sialyllactose (3’-SL) and 6’Sialyllactose (6’- SL) in average physiological concentrations (5.75 g/L) and proportions (52:13:26:4:5).

Samples were taken before inoculation from the fecal mixture that was used to inoculate the system and subsequently from the compartments (simulated lumen) at 24h, 48h, 72h and 96h. DNA was extracted from inoculum and luminal samples. Bacterial composition was determined by shotgun metagenomics. Statistical analyses were performed using R Statistical Software v4.1 .0. The microbiome data were center log-ratio transformed (clr) to account for compositionality.

Pairwise comparisons between samples from the donor feces and from l-TIM-2 with the two different feed types, SIIEM and SIIEM-HMO, were performed using a linear model with false discovery rate (FDR) correction to account for multiple testing. Corrected P-values <0.05 were considered statistically significant.

Results

In the infant in vitro colonic model, I-TIM2, the bacterial communities were recreated and maintained from the feces of infants. This allowed studying the effects of supplementing the 5HMO-mix on the microbial composition and activity overtime in comparison to nonsupplemented bacterial communities. Under supplementation with the 5-HMO mix, the relative abundance of HMO-utilizing bacteria was maintained (data not shown), while the relative abundance of E. coli was decreased (Figure 2, Figure 3).

Example 4

Method

Ex vivo, reactor-based, high-throughput SIFR® (Systemic Intestinal Fermentation Research) technology was performed as recently described (Van den Abbeele, P. et al. Bridging preclinical and clinical gut microbiota research using the ex vivo SIFR® technology. Frontiers Microbiol 14, (2023)). Fecal samples were donated from healthy, formula-fed infants aged 2-4 months (n = 8). The identity of the EPEC strain E. coli 0127 E2348/69 was confirmed and pure E. coli 0127 E2348/69 culture was used to make a qPCR standard curve. The study was designed with a study arm supplemented with 5-HMO mix (5g/L) tested against a no-substrate control (NSC), and individual fecal suspensions were initially incubated for 20 hours. Subsequently, EPEC was spiked at 10⁷ cells/ml exponential phase pregrown EPEC, into the microbiota and incubated for an additional 24 hours, resulting in a total incubation time of 44 hours. Samples were collected at Oh, 20h and 44h for analysis of key fermentative parameters, EPEC levels and bacterial composition. Measurements of total short-chain fatty acids (including acetate, propionate, butyrate and valerate) was determined with a GC-FID approach. Further, pH and gas production were measured with standard methods. The bacterial composition was determined using quantitative shotgun sequencing coupled with flow cytometry. Quantification of EPEC levels was obtained with an EPEC-specific qPCR kit (Escherichia coli Typing eae EHEC or EPEC genome (NZYTech, Lissabon, Portugal). Standardised Illumina library preparation resulting in 3M total DNA sequencing was used for taxonomic analysis. Data was log-transformed and absolute phylogenetic data used as input. rCCA was executed using the mixOmics package with the shrinkage method for estimation of penalisation parameters in R (https://www.r-project.org/ Rohart, F., Gautier, B., Singh, A. & Cao, K.-A. L. mixOmics: An R package for ‘omics feature selection and multiple data integration. PLOS Comput Biol 13, e1005752 (2017)). Statistical evaluation of the treatment effects on key fermentative parameters, cell counts across the 8 donors was performed with a paired t-test, thus accounting for the fact that values are compared between samples of a given donor. Statistical differences were visualized via * (0.1 < p < 0.2), ** (0.05 < p < 0.1) or *** (p < 0.05).

Results

The 5-HMO mix significantly lowered EPEC levels by decreasing EPEC for 7 out of 8 infants in the 24-hour infection model from 20h to 44h (p = 0.04; as assessed via qPCR; see Figure 4). This antipathogenic effect could originate from the strong modulation of metabolite production of the gut microbiome. The 5-HMO mix stimulated the metabolic activity of the gut microbiota, significantly decreasing pH, and increasing the production of gas and the total SCFAs at both 20h of pre-growth without EPEC and at 44h after 24 additional hours of introduction of EPEC in the microbiota (see Figure 5).

Claims

1 . A composition for use in reducing the risk of an infection in the gastrointestinal tract caused by a pathogenic E. coli in a human subject, said composition comprising 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.

2. The composition for use according to claim 1 , wherein the infection in the gastrointestinal tract is a diarrheal disease.

3. The composition for use according to any one of the preceding claims, wherein the risk of an infection in the gastrointestinal tract is reduced through the inhibition of adhesion of E. coli to intestinal epithelial cells.

4. The composition for use according to any one of the preceding claims, wherein the risk of an infection in the gastrointestinal tract is reduced through a reduction of the relative abundance of E. coli.

5. The composition for use according to any one of the preceding claims, wherein the pathogenic E. coli is of the enterohemorrhagic E. coli (EHEC) pathotype, wherein the EHEC preferably has serotype 0157.

6. The composition for use according to any one of the preceding claims, wherein the pathogenic E. coli is of the enteropathogenic E. coli (EPEC) pathotype, wherein the EPEC preferably has serotype 0127.

7. The composition for use according to any one of the preceding claims, wherein the composition is a nutritional composition.

8. The composition of any one of claims 1 to 7, wherein the composition comprises an effective amount of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.

9. The composition of any one of claims 1 to 8, wherein the composition comprises at least 0.01 %, by weight, of 2’-fucosyllactose; at least 0.01 %, by weight, 3-fucosyllactose; at least 0.01 %, by weight, 3'-sialyllactose; at least 0.01 %, by weight, 6'-sialyllactose; and at least 0.01 %, by weight, lacto-N-tetraose.

10. The composition of claim 9, wherein the ratios of 2'-fucosyllactose, 3-fucosyllactose, 3'- sialyllactose, 6'-sialyllactose, and lacto-N-tetraose in the composition are: 45%, by weight, to 60%, by weight, of 2’-fucosyllactose; 8%, by weight, to 18%, by weight of 3- fucosyllactose; 2%, by weight, to 10%, by weight, of 3’-sialyllactose; at least 2%, by weight, to 10%, by weight, of 6’-sialyllactose; and 20%, by weight, to 31 %, by weight, of lacto-N- tetraose.

11 . The composition of any one of claims 1 to 10, wherein the composition is a powder.

12. The composition of claim 11 , wherein the 2’-fucosyllactose has a particle size distribution, as measured using sieves complying to DIN ISO 3310-1 , of less than about 20% through mesh #230 (63 pm), greater than about 65% through mesh #100 (150 pm), greater than about 92% through mesh #45 (355 pm), and 100% through mesh #20 (850 pm).

13. The composition of any one of claims 11 or 12, wherein the water activity a_w of the human milk oligosaccharides is between 0,10 and 0,30, preferably between 0,10 and 0,25.

14. The composition of any one of claims 1 to 13, wherein the composition comprises at least 0.5 x 10⁹ CFU/g of a probiotic.

15. The composition of any one of claims 1 to 14, wherein the subject is a newborn oran infant.

16. The composition of any one of claims 1 to 14, wherein the subject is a toddler or a child.