Mixture of five human milk oligosaccharides for reducing risk of gastrointestinal infections caused by pathogenic escherichia coli

Abstract

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

Description

Technical Field

[0001] The present disclosure relates in part to the use of a mixture of human milk oligosaccharides for reducing the risk of infection in the gastrointestinal tract, particularly diarrheal disease, caused by E. coli bacteria in human subjects. Further disclosed herein are methods, uses, processes, etc. Background Art

[0002] Human milk oligosaccharides (HMOs) are non-digestible carbohydrates found in human milk. Their importance for infant nutrition is reflected in the fact that they are the third most abundant solid component of human milk after lactose and lipids. HMOs can be structurally divided into (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).

[0003] name Chemical structure 2'-FL Fuc(α1,2)Gal(β1,4)Glc 3-FL Gal(β1,4)(Fuc(α1,3))Glc LNT Gal(β1,3)GlcNAc(β1,3)Gal(β1,4)Glc 3'-SL NeuAc(α2,3)Gal(β1,4)Glc 6'-SL NeuAc(α2,6)Gal(β1,4)Glc

[0004] Table 1: Structures of HMOs used in this article

[0005] HMOs in human milk vary widely based on various influences such as genetics, lactation, and geographic location. While most HMO concentrations decrease during lactation, the concentrations of at least two (3'-SL and 3-FL) may increase. The different HMOs may work together in a complementary manner to support the growth and development of the infant.

[0006] It is believed that HMO can reduce the risk of imbalance of the intestinal microbiome caused by harmful bacteria (Weichert, Stefan, et al., Nutrition research 10 (2013): 831-838). In addition, it is believed that HMO can selectively stimulate the growth and metabolic activity of beneficial bifidobacteria to support overall intestinal health (Bode, Lars. Nutrition reviews, 2009, Vol 67 suppl. 2, 183.191). It has been shown that a mixture of HMOs increases the relative abundance of bifidobacteria in the microbiome of formula-fed infants to the abundance of bifidobacteria in the microbiome of human milk-fed infants (Holst A. et al., Nutrients, 2023, 15, 3087).

[0007] Escherichia coli is one of the earliest colonizers and common residents of the infant gut microbiome. It persists because, for example, the expression of pili enables it to attach to colonic epithelial cells (Nowrouzian, F. et al., 2003, Pediatric Research, 54 (1), 8–14). It includes a variety of strains, ranging from commensals to serious pathogens, depending on the presence of virulence factors often 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.), which can therefore be transferred between strains.

[0008] Most E. coli strains are harmless and contained in the intestinal lumen, but may cause infection under favorable circumstances such as disruption of the gastrointestinal barrier (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, LR, 2015, Pediatrics in Review, 36(4), 167–171).

[0009] Certain pathogenic E. coli strains are classified into pathological types associated with unique severe diarrheal diseases in infants, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) (Makvana, S., & Krilov, LR, 2015, Pediatrics in Review, 36 (4), 167-171). In vitro assays have shown that a mixture of human milk oligosaccharides concentrated from human milk has an inhibitory effect on the adhesion of EPEC serotype O119 to epithelial cells (Coppa GV et al., 2006, Pedriatric Research, 59, 3, 2006, 377-382). Mixtures of synthetic human milk oligosaccharides that are not concentrated from human milk have not been studied. Enteropathogenic E. coli (EPEC) of serotype O127 is associated with diarrheal outbreaks in infants under 2 years old (Prabhdeep K., Pradeep KD, 2023, Newborn, Vol 2 Issue 1, 102-113).

[0010] Enterohemorrhagic Escherichia coli (EHEC) is an E. coli pathotype 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 patients affected (Gomes TAT ​​et al., 2016; 47; 3–30). EHEC requires binding to the intestinal epithelium via adhesins to establish infection (McWilliams BD, Torres AG, Microbiol Spectr. 2014; 2(3)). Therefore, reducing this binding may reduce the risk of colonization and subsequent diarrheal disease caused by EHEC and other E. coli pathotypes.

[0011] In general, E. coli is the main cause of infant diarrhea, which is the second most common cause of death in children under five years old worldwide (Johansson, EW et al., 2009, in The United Nations Children's Fund (UNICEF) / World Health Organization (WHO), Vol. 44, 11, 1–68). Therefore, reducing the occurrence and colonization of E. coli in the gastrointestinal tract can reduce the risk of diarrheal diseases caused by E. coli.

[0012] There is a need for strategies that can reduce the risk of diarrheal illness caused by pathogenic E. coli, and in particular EHEC pathotypes. Summary of the invention

[0013] The present disclosure provides compositions, uses, methods, etc. for reducing the risk of infection (such as, for example, diarrheal diseases) in the gastrointestinal tract in human subjects. In particular, the present disclosure relates to a composition for reducing the risk of infection (especially diarrheal diseases) in the gastrointestinal tract in human subjects (especially non-adult subjects), comprising 5 HMOs (2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL) and lactose-N-tetraose (LNT)).

[0014] Although not wishing to be bound by theory, it is believed that a mixture of 5 HMOs (5HMO-mixture consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL) and lacto-N-tetraose (LNT)) can reduce the number of E. coli in the gastrointestinal tract, in particular the number of pathogenic E. coli, and inhibit the binding of pathogenic E. coli to the intestinal mucosal barrier. The binding of pathogenic E. coli to epithelial cells is a step in the infection mechanism. Therefore, by inhibiting this binding step and reducing the total number of E. coli and the number of pathogenic E. coli in the gastrointestinal tract, it is believed that the risk of infection by pathogenic E. coli is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A It is shown that in the presence of galacto-oligosaccharides (GOS) at a dose of 30 mg / ml and 5HMO-mix at different doses from 1 mg / ml to 30 mg / ml, a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O157 has reduced adhesion to monolayers of human colon cancer cells (Caco-2).

[0016] Figure 1B It is shown that in the presence of galacto-oligosaccharides (GOS) at a dose of 30 mg / ml and 5HMO-mix at 5 mg / ml and 30 mg / ml, a strain of enteropathogenic Escherichia coli (EPEC) serotype O127 has reduced adhesion to monolayers of human colon cancer cells (Caco-2).

[0017] Figure 2 Shown are the relative abundance of E. coli in the infant in vitro colon model I-TIM-2 and a comparison between donor stool inoculum samples and I-TIM-2 samples fed with SIIEM (without HMO) and SIIEM-HMO (with 5HMO-mix).

[0018] Figure 3 Shown are the relative abundance of E. coli in the infant in vitro colon model I-TIM-2, as well as intra-donor (INF5-7) comparisons between donor stool inoculum samples and I-TIM-2 samples fed with SIIEM (without HMO) and SIIEM-HMO (with 5HMO-mix) in independent experiments with individual donors (INF5-7).

[0019] Figure 4The effect of the 5HMO mixture on the abundance of EPEC in the gut microbiota of healthy infants (n=8) at 44 hours (including 24 hours of introduction of EPEC into the microbiota) is shown. (A) EPEC levels in the NSC and HMO study groups, quantified by qPCR and expressed in copies / mL. Statistical differences between NSC and individual treatments are indicated by *(0.1 <p 调节 <0.2)、**(0.05 <p 调节 <0.1) or ***(p 调节 < 0.05) visualization. (B) Effect of HMO on EPEC levels expressed as log2-fold change vs. NSC, quantified by qPCR. Different symbols represent 8 individual donors. Overall, lower levels of serotype O127 enteropathogenic Escherichia coli (EPEC) were observed in the presence of the 5-HMO mixture (right) compared to the no-substrate control (NSC) without HMO (left) in both the infant ex vivo 20-h fermentation model and the subsequent 24-h E. coli infection model.

[0020] Figure 5 The effects of 5HMO mixture on (A) pH, (B) gas production, and (C) total SCFA at 20 and 44 hours are shown. Statistical differences between NSC and individual treatments are indicated by *(0.1 <p 调节 <0.2)、**(0.05 <p 调节 <0.1) or ***(p 调节 < 0.05). Different symbols represent 8 individual donors. Overall, in the infant ex vivo 20-h fermentation model and the subsequent 24-h E. coli infection model, the metabolic activity of the infant microbiota was modulated in the presence of the 5-HMO mixture as shown by decreased pH, increased gas production, and increased production of short-chain fatty acids compared to non-substrate controls without HMOs. DETAILED DESCRIPTION

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Although any methods and materials comparable or similar to the methods and materials described herein may be used in the practice of the present disclosure, typical methods and materials are described. All methods described herein may be performed in any suitable order, unless otherwise specified herein or otherwise clearly contradicted by context.

[0022] The use of the terms "a" and "an" and "the" and similar indicators in the context of describing the present invention (particularly in the context of the following claims) should be interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. Unless otherwise specified, the terms "comprising", "having", "including" and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, references to numerical ranges herein are intended only to be used as a shorthand method for individually citing each individual value within the range (including both end values), and each individual value is incorporated into this specification as if individually listed herein. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to be used to better illustrate the present invention, and unless otherwise stated, the scope of the present invention is not limited.

[0023] As used herein, the term "and / or" is intended to refer to combinatorial ("and") as well as exclusive ("or") usage, ie, "A and / or B" is intended to mean "A alone, or B alone, or A and B together."

[0024] The terms "effective amount", "effective concentration" or "effective dose" as used herein are defined as the amount, concentration or dosage of a material sufficient to improve the overall health of a subject and impart benefits similar to those shown in the Examples. The actual effective dose in absolute numbers depends on a variety of factors, including the health status of the subject in question and the presence of other ingredients. The "effective amount", "effective concentration" or "effective dose" of a material can be determined by conventional assays known to those skilled in the art.

[0025] As used herein, the term "isolated" refers to the bacterial strain described herein being in a form or environment that does not occur in nature, ie, the strain is at least partially separated from one or more or all of the naturally occurring components with which it is associated in nature.

[0026] As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged when grown or propagated and is derived from a single isolate or pure culture. Probiotics are classified by genus (e.g., Bifidobacterium), species and subspecies (e.g., subspecies lactis animalis), and strain (e.g., DSM 15954 and / or FAO / WHO states that probiotic effects are strain-specific and therefore most probiotic properties of a particular strain cannot be extrapolated to other strains of the same species.

[0027] The term "probiotic" as used herein refers to live or lyophilized microbial cultures, dead microorganisms, microbial fragments, and microbial extracts or supernatants that, when applied to humans or animals, have a beneficial effect on 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).

[0028] Unless otherwise specified, the term "human milk oligosaccharides" or "HMOs" as used herein generally refers to a number of complex carbohydrates found in breast milk that can be in either acidic or neutral form, as well as their precursors. Exemplary non-limiting human milk oligosaccharides include 3'-sialyllactose, 6'-sialyllactose, 3-fucosyllactose, 2'-fucosyllactose, and lacto-N-tetraose.

[0029] The terms "treat" or "treating" should not be construed to imply that an individual is treated until complete recovery. Therefore, these terms broadly include ameliorating and / or preventing the onset or severity of symptoms of a particular condition.

[0030] The term "reducing the risk of infection" includes preventing the onset of symptoms or lessening the severity of symptoms. The risk of infection is reduced or decreased compared to the situation in the absence of the claimed composition.

[0031] Unless otherwise specified, the term "shelf stable" as used herein refers to a nutritional product that remains commercially stable after storage at 18-24°C for at least 3 months (including about 6 months to about 24 months, and also including about 12 months to about 18 months) after packaging.

[0032] As used herein, the terms "nutritional formula" or "nutritional composition" are used interchangeably and, unless otherwise specified, refer to nutritional liquids, nutritional powders, nutritional supplements, and any other nutritional food known in the art. Nutritional powders can be reconstituted to form nutritional liquids, all of which contain one or more of fat, protein, and carbohydrates and are suitable for oral consumption by humans.

[0033] As used herein, unless otherwise specified, the term "nutritional powder" refers to nutritional products in flowable or spoonable form that can be reconstituted with water or other aqueous liquids prior to consumption, and includes spray-dried powders and dry-blended powders.

[0034] Unless otherwise specified, the term "neonate" as used herein refers to a person from birth to 4 weeks of age. Unless otherwise specified, the term "infant" as used herein refers to a person 12 months or younger. The term "premature infant" as used herein refers to an infant born before 36 weeks of gestation. Unless otherwise specified, the term "toddler" as used herein refers to a person older than 1 to 3 years of age. Unless otherwise specified, the term "child" as used herein refers to a person older than 3 to 12 years of age.

[0035] As used herein, unless otherwise specified, the term "formula" refers to liquid and solid human milk substitutes or replacements suitable for human consumption.

[0036] As used herein, unless otherwise specified, the term "human milk fortifier" refers to liquid and solid nutritional products suitable for mixing with breast milk or formula for consumption by preterm or term infants.

[0037] As used herein, unless otherwise indicated, the terms "susceptible" and "at risk" refer to having little resistance to a condition or disease, including genetic susceptibility, family history, and / or symptoms of a condition or disease. As used herein, the terms "modulating" or "modulation" or "modulate" refer to a targeted movement of a selected characteristic, unless otherwise indicated.

[0038] The term purity as used in this application refers to chemical purity and thus to the extent to which a substance is not diluted or mixed with foreign materials. Chemical purity is therefore an indicator of the relationship between at least one HMO and byproducts / impurities.

[0039] Chemical purity is expressed as a percentage (%) and is calculated using the following formula:

[0040] Purity percentage = 100 x (mass of desired compound in sample) / (total mass of sample)

[0041] Purity can be determined by any suitable method known to those skilled in the art. One suitable method is HPLC (High Performance Liquid Chromatography). In the chromatogram obtained, the ratio of the area under the peak representing the amount of HMO to the sum of the areas under the peaks representing the HMO and all other compounds in the chromatogram except the HMO is calculated.

[0042] All percentages, parts and ratios 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.

[0043] Numerical ranges used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not.

[0044] The present invention has been described with reference to various embodiments, aspects, examples, etc. It is not intended that these elements be read independently of each other. Therefore, the present disclosure provides a combination of two or more of the described embodiments, aspects, examples, etc.

[0045] All embodiments described herein are intended to be within the scope of the present disclosure. These and other embodiments of the present invention will become readily apparent to those skilled in the art by reference to the following detailed description of the preferred embodiments described throughout, and the present invention is not limited to any particular preferred embodiment disclosed. Any language in the specification should not be interpreted as indicating that any unclaimed element is necessary to practice the present invention.

[0046] The present disclosure provides a composition comprising a mixture of 5 human milk oligosaccharides (HMOs), namely 2′-fucosyllactose, 3-fucosyllactose, 3′-sialyl lactose, 6′-sialyl lactose and lactose-N-tetraose. The mixture of these 5 HMOs is also referred to as a “5HMO-mixture”. Although not wishing to be bound by theory, it is believed that a composition comprising a 5HMO-mixture can reduce the risk of gastrointestinal infection caused by pathogenic Escherichia coli in human subjects. This is supported by findings in in vitro experiments, namely that the 5HMO-mixture can reduce the total abundance of Escherichia coli in the gastrointestinal tract, reduce the number of pathogenic Escherichia coli in an Escherichia coli infection model, and in the presence of the 5HMO-mixture, the binding of pathogenic Escherichia coli to epithelial cells is inhibited.

[0047] To study the impact of 5HMO-mixture on the relative abundance of Escherichia coli, a TIM-2 infant in vitro colon model (TNO in vitro model) was established. The TIM-2 model is a validated, dynamic and computer-controlled simulation model (Venema K. et al., The impact of Foods Bio-Actives on Gut Health, 2015, 293-304). Here, the model is set to simulate the infant colon environment. The bacterial community from infant feces was reconstructed, and the impact of supplementing 5HMO-mixture on microbial composition and activity over time was studied. The effect was compared with the unsupplemented bacterial community. For this reason, two culture media were used, simulated infant ileal efflux medium (SIIEM) and SIIEM containing 5HMO-mixture (2'-fucosyllactose, 3-fucosyllactose, lactose-N-tetraose, 3'-sialyl lactose and 6'-sialyl lactose) with physiologically relevant ratios and concentrations. It was found that HMO supplementation maintained the relative abundance of HMO-utilizing bacteria, while the relative abundance of Escherichia coli decreased ( Figure 2 , Figure 3 While not wishing to be bound by theory, it is believed that a reduction in the relative abundance of E. coli may reduce the risk of gastrointestinal infection.

[0048] In order to evaluate the effect of a composition comprising a 5HMO-mixture on the binding of pathogenic E. coli to the intestinal epithelium, strains of enterohemorrhagic E. coli (EHEC) of serotype O157 and strains of enteropathogenic E. coli (EPEC) of serotype O127 were pre-incubated with or without different doses of the 5HMO-mixture. The pre-incubated E. coli were added to a monolayer of human colon cancer cells (Caco-2). After a defined incubation time, the number of EHEC or EPEC adhering to the intestinal cell monolayer was determined. It was found that the 5HMO-mixture dose-dependently reduced the binding of EHEC to the cell monolayer ( Figure 1A ), and the binding of EPEC to the cell monolayer was also reduced ( Figure 1B While not wishing to be bound by theory, it is believed that reducing the binding of EHEC and EPEC to intestinal epithelial cells may reduce the risk of infection.

[0049] To evaluate the effects of compositions containing 5HMO-mixtures, Technology (Van den Abbeele, P. et al., Bridging preclinical and clinical gut microbiota research using the ex vivo technology. Frontiers Microbiol 14, (2023)) simulated EPEC (E. coli O127) infection in an in vitro system using fecal samples from formula-fed infants. The technology provides insight into changes in the composition of the intestinal microbiota. The technology has been validated by clinical data. It was found that the 5HMO-mixture significantly reduced EPEC O127 levels compared to the case without the 5HMO-mixture. While not wishing to be bound by theory, it is believed that the observed reduction in EPEC levels may reduce the risk of infection with E. coli pathogens.

[0050] In certain embodiments, a composition comprising a 5HMO mixture is used to reduce the risk of diarrheal disease. Gastrointestinal infections often present with diarrhea as a prominent symptom. While not wishing to be bound by theory, it is believed that the use of a composition comprising a 5HMO mixture can reduce the risk of diarrheal disease.

[0051] In certain embodiments, compositions comprising a 5HMO-mixture are used to reduce the risk of infection in the gastrointestinal tract by inhibiting the adhesion of E. coli to intestinal epithelial cells. It is well known that one step in the infection mechanism is attachment to epithelial cells.

[0052] In certain embodiments, a composition comprising a 5HMO mixture is used to reduce the risk of infection in the gastrointestinal tract caused by enterohemorrhagic Escherichia coli (EHEC) pathotypes, preferably of pathotype O157. In vitro experiments have demonstrated that the 5HMO mixture dose-dependently inhibits the adhesion of this pathotype.

[0053] In certain embodiments, a composition comprising a 5HMO mixture is used to reduce the risk of gastrointestinal infections caused by enteropathogenic Escherichia coli (EPEC) pathotypes, wherein the EPEC preferably has serotype O127. In vitro experiments have shown that the 5HMO mixture reduces the relative amount of this pathogenic EPEC in the microbiota. Although not wishing to be bound by theory, this antipathogenic effect may result from a strong regulation of the production of metabolites by the intestinal microbiota. In in vitro experiments, the 5-HMO mixture stimulates the metabolic activity of the intestinal microbiota, significantly reduces the pH, and increases the production of gas and the content of total short-chain fatty acids (SCFA).

[0054] In certain embodiments, the composition is a nutritional composition, such as a formula or a dietary supplement.

[0055] The preferred composition herein is a nutritional composition, such as a formula milk powder. The nutritional composition can be in any product form comprising the ingredients described herein, and it is safe and effective for oral administration. Optional ingredients (such as those described herein) can be used to prepare the nutritional composition.

[0056] The nutritional compositions of the present disclosure are preferably formulated in the form of dietary products, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form containing at least one of fat, protein and carbohydrate, and preferably also containing vitamins, minerals or combinations thereof.

[0057] Nutritional compositions can be formulated with sufficient types 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 with a specific disease or condition, or with targeted nutritional benefits as described below. Specific non-limiting examples of product forms suitable for use as the HMO-containing compositions disclosed herein include, for example, liquid and powdered dietary supplements, liquid and powdered human milk fortifiers, liquid and powdered formula milks.

[0058] Nutritional liquids include concentrated nutritional liquids and ready-to-feed nutritional liquids. These nutritional liquids are most commonly formulated as suspensions or emulsions, although other liquid forms are also within the scope of the present disclosure.

[0059] Nutritional emulsions suitable for use may be aqueous emulsions containing proteins, fats and carbohydrates. These emulsions are generally flowable or drinkable liquids at about 1°C to about 25°C and are generally in the form of oil-in-water, water-in-oil or complex aqueous emulsions, although such emulsions are most commonly in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.

[0060] Nutritional emulsions can be and are typically shelf stable. Nutritional emulsions typically contain up to 95% water by weight, including about 50% to about 95% by weight of the nutritional emulsion, also including about 60% to about 90%, and also including about 70% to about 85% water. Nutritional emulsions can have a variety of product densities, but the most common densities are greater than about 1g / mL, including greater than about 1.05g / mL, including greater than about 1.055g / mL to about 1.12g / mL, and also including about 1.085g / mL to about 1.10g / mL. Nutritional emulsions can have a caloric density tailored to the nutritional needs of the end user, although in most cases, the emulsion typically contains at least 660 kcal / L, about 675 kcal / L to about 820 kcal / L, about 680 kcal / L to about 800 kcal / L. In some embodiments, the emulsion can have a caloric density of about 50-100 kcal / liter to about 660 kcal / liter, including about 150 kcal / liter to about 500 kcal / liter. In some specific embodiments, the emulsion can have a caloric density of 25, or 50, or 75, or 100 kcal / liter. The nutritional emulsion can have a pH of about 3.5 to about 8, about 4.5 to about 7.5, including about 5.5 to about 7.3, including about 6.2 to about 7.2. Although the serving size of the nutritional emulsion can vary depending on many variables, common serving sizes are typically 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 about 1 mL to about 300 mL, including about 4 mL to about 250 mL, and including about 10 mL to about 240 mL.

[0061] Nutrient solids can be any solid form, but are generally flowable or substantially flowable granular compositions, or at least in the form of granular compositions, which can be optionally compressed into tablets. Particularly suitable nutrient solid product forms include spray-dried, agglomerated and / or dry blended powder compositions. The composition can be easily scooped out and measured with a spoon or other similar devices, and can be easily reconstructed by the intended user with a suitable aqueous liquid (usually water) to form a nutrient composition that can be immediately taken orally or enterally. In this context, "immediately" use generally refers to within about 48 hours, most typically within about 24 hours, preferably immediately after reconstruction. Nutritional powder can be reconstructed with water before use to a caloric density tailored to the nutritional needs of the end user, although in most cases, powder is reconstructed with water to form a nutrient composition that generally comprises 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 about 50-100 kcal / liter to about 660 kcal / liter, including 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.

[0062] The present composition can be used for neonates, infants, young children or children. The present composition can be used for neonates. The present composition can be used for infants. It has been demonstrated that the 5HMO-mixture reduces the relative abundance of E. coli in the infant TIM-2 model, thereby indicating that the mixture may have a beneficial effect on infants. In addition, E. coli is a major cause of diarrhea in infants, indicating that infants may particularly benefit from a composition comprising a 5HMO-mixture.

[0063] The present composition may comprise effective amounts of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialylactose, 6'-sialylactose and lacto-N-tetraose.

[0064] The present composition may comprise at least 0.01% by weight of 2′-fucosyllactose; at least 0.01% by weight of 3-fucosyllactose; at least 0.01% by weight of 3′-sialyl lactose; at least 0.01% by weight of 6′-sialyl lactose; and at least 0.01% by weight of lacto-N-tetraose.

[0065] The present compositions can include any suitable amount of an individual HMO, such as, for example, at least 0.001 mg / mL, including about 0.001 mg / mL to about 20 mg / mL, including about 0.01 mg / mL to about 10 mg / mL, including about 0.01 mg / mL to about 5 mg / mL (mg specific HMO / mL composition).

[0066] The present composition may comprise five HMOs in a specific ratio, wherein the ratio of 2′-fucosyllactose, 3-fucosyllactose, 3′-sialyl lactose, 6′-sialyl lactose and lacto-N-tetraose in the composition is 45% to 60% by weight of 2′-fucosyllactose; 8% to 18% by weight of 3-fucosyllactose; 2% to 10% by weight of 3′-sialyl lactose; at least 2% to 10% by weight of 6′-sialyl lactose; and 20% to 31% by weight of lacto-N-tetraose.

[0067] If the composition is a nutritional powder, the concentration of the individual HMO 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). If the composition is a ready-to-feed nutritional liquid, the concentration of the individual HMO is 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). If the composition is a concentrated nutritional liquid, the concentration of the individual HMO 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).

[0068] The present composition may be in the form of a powder. Formulating a composition using an HMO may be problematic. It has been found that a more reproducible and consistent composition may be achieved by controlling the particle size distribution (PSD) of the HMO. While not wishing to be bound by theory, it is believed that having a narrower PSD may improve the flowability of the HMO, thereby enabling more efficient mixing with other ingredients. Furthermore, it is believed that a PSD within a certain range provides a better solubility profile. The particle size of the HMO may be determined using standard methods, such as using a sieving tower which separates the powder into different fractions after a specified time with a predefined amplitude. The sieve used in such a method may be a sieve conforming to DIN ISO 3310-1.

[0069] Preferably, the HMO (particularly 2'FL and 3FL) used in the present composition has the following particle size characteristics:

[0070] Percent passing #230 mesh (63 μm)—less than about 20%, less than about 18%, less than about 16%, less than or equal to about 15%.

[0071] Percent passing #100 mesh (150 μm)—greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.

[0072] Percent passing #45 mesh (355 μm)—greater than about 95%, greater than about 92%, greater than or equal to about 90%.

[0073] Percent passing #20 mesh (850 μm) - 100%.

[0074] Preferably, when in powder form, the water activity of human milk oligosaccharides is w The water activity is preferably determined according to ISO 18787:2017. The water activity ensures the microbiological stability of the powder and prevents contamination by unwanted microorganisms.

[0075] The present composition preferably comprises HMO of synthetic origin, such as HMO produced by microbial fermentation, or HMO produced by biocatalysis or chemical synthesis. In particular, microbial fermentation allows production on an industrial scale with high purity, which can be used in nutritional compositions. For the present composition, preferably, the HMO used has a purity of greater than 85%, preferably a purity of greater than 90%, more preferably a purity of greater than 95%.

[0076] Compositions disclosed herein may optionally include anti-inflammatory drugs, such as long-chain polyunsaturated fatty acids (LCPUFA) and / or antioxidants, such as carotenoids. LCPUFA may be included in the composition to provide nutritional support and enhance the growth and functional development of intestinal epithelium and associated immune cell groups. Exemplary LCPUFA for use in the present composition include, for example, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), linoleic acid, linolenic acid (α-linolenic acid) and γ-linolenic acid derived from oil sources such as vegetable oil, marine plankton, fungal oil and fish oil. The present composition preferably includes a total concentration of about 0.01mM to about 10mM and includes about 0.01mM to about 1mM LCPUFA. Alternatively, the present composition includes a total concentration of about 0.001g / L to about 1g / L LCPUFA.

[0077] Furthermore, the present composition may include antioxidants, such as carotenoids, and in particular a combination of carotenoids, lutein, lycopene, zeaxanthin and / or beta-carotene.

[0078] The compositions of the present disclosure may further comprise other optional ingredients that may alter the physical, chemical, aesthetic or processing characteristics of the compositions, or be used as pharmaceuticals or additional nutritional ingredients. Non-limiting examples of such optional ingredients include preservatives, emulsifiers, buffers, pharmaceutically active substances, nutrients, colorants, flavorings, thickeners and stabilizers, flow agents, minerals, emulsifiers, lubricants, sweeteners, and the like.

[0079] Flowing agents or anti-caking agents may be included in the present composition to delay the aggregation or caking of the powder over time and to facilitate the flow of the powder entity 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 depends on the product form, other selected ingredients, desired flow properties, etc., but the most common range is from about 0.1% to about 4%, including about 0.5% to about 2%, by weight of the nutritional composition.

[0080] The compositions of the present disclosure can be prepared by any known or other effective manufacturing techniques for preparing a selected product solid or liquid form. Many such techniques are known for any given product form (such as a nutritional liquid or powder) and can be easily applied to the nutritional compositions described herein by those of ordinary skill in the art.

[0081] It will be appreciated by those skilled in the art that the compositions disclosed herein can be administered with dosage levels and dosing regimens as needed, depending on the situation and the condition of the subject. Suitable dosage regimens can be determined according to the teachings of this application. The dosage regimen can be adjusted to provide optimal support for the subject. It will be appreciated that the exact dosage and rate will depend on many factors, such as the age, weight, general health, sex, and dietary requirements of the subject. Based on the teachings herein, those skilled in the art can determine suitable dosage regimens according to specific circumstances through routine tests and experiments.

[0082] The present composition may comprise at least one probiotic strain, such as 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 casei 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 angularis, Bifidobacterium animalis subsp. lactis lactis), Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentate, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium megaterium, Bifidobacterium pseudocatenulatum), or any strain from the genus Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridium, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia, or the family Rhodococcus, and suitable combinations of the foregoing.

[0083] The present composition may comprise at least one strain of bacteria selected from the following: 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 subsp. 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.

[0084] The present composition preferably comprises an effective amount of probiotics. For example, when present, the concentration of probiotics is preferably 0.05 x 10 9 CFU / g to 30x 10 9 CFU / g, preferably 0.5x 10 9 CFU / g to 25x 10 9 CFU / g.

[0085] Example

[0086] Example 1

[0087] Chr. Hansen HMO GmbH, Rheinbreitbach, Germany, produces a 5HMO mixture 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. A stock solution of the 5HMO mixture solution was dissolved in water.

[0088] Incubate at 37 °C in 5% CO 2 Human epithelial intestinal cancer cell line Caco-2 (ACC 169, DSMZ, passage 5–20) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX supplement (Gibco) supplemented with 1% non-essential amino acids (Merck Life Science), 1% penicillin-streptomycin (10,000 U / mL) (Gibco), and 10% heat-inactivated fetal bovine serum (Gibco) in a 4% CO atmosphere. Cells were cultured at 8 × 10 4 Cells were seeded at a density of 10 cells / well in 24-well culture plates and the medium was changed every 3-4 days until the cells were ready for experiments after 14 days.

[0089] EHEC O157 (DSM 17076) strain was grown overnight in Luria-Bertani (LB) broth at 37°C with agitation, then washed twice with Hanks balanced salt solution (HBSS, Gibco) and resuspended in DMEM to an OD of 600nm Normalized to 0.5. Then, the EHEC strain was diluted 1:20 in DMEM to approximately 2x10 6Colony forming units (CFU) / ml, and pre-cultured with or without 5-HMO mixture or GOS (30mg / ml) of different doses (1 to 30mg / ml) at room temperature with stirring. After 2 hours of pre-culture, the cell culture medium was removed from the Caco-2 cell monolayer, and then gently washed twice with HBSS. Then, the EHEC strain suspension (with and without 5-HMO mixture or GOS) was added to the Caco-2 cell monolayer, and after 1 hour of cultivation, the culture medium containing non-adherent EHEC was removed. The Caco-2 cell monolayer was gently washed three times in HBSS, and after 3 washing steps, 0.1% Triton X-100 in HBSS was added to the cells to release adherent EHEC. Then, after overnight culture at 37°C, the number of EHEC adhering to the intestinal cell monolayer was quantitatively counted by continuous plating and CFU) counting on tryptone soy broth (plate).

[0090] result

[0091] The results showed that the 5HMO-mixture dose-dependently reduced EHEC binding to intestinal epithelial cell monolayers (up to 60% reduction) compared to the EHEC control treated group, reaching statistical significance (p<0.01) at 10 mg / ml or higher. GOS (tested at 30 mg / ml) failed to significantly reduce EHEC binding (see Figure 1A ). Data are presented as mean + standard deviation (n = 3 independent experiments). Statistical significance was determined by one-way ANOVA compared with the EHEC control group followed by Dunnett's multiple comparison test.

[0092] Example 2

[0093] Chr. Hansen HMO GmbH, Rheinbreitbach, Germany, produces a 5HMO mixture 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. A stock solution of the 5HMO mixture solution was dissolved in water.

[0094] Incubate at 37 °C in 5% CO 2Human epithelial intestinal cancer cell line Caco-2 (ACC 169, DSMZ, passage 5–20) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX supplement (Gibco) supplemented with 1% non-essential amino acids (Merck Life Science), 1% penicillin-streptomycin (10,000 U / mL) (Gibco), and 10% heat-inactivated fetal bovine serum (Gibco) in a 4% CO atmosphere. Cells were cultured at 8 × 10 4 Cells were seeded at a density of 10 cells / well in 24-well culture plates and the medium was changed every 3-4 days until cells were ready for experiments after 14 days.

[0095] EPEC O127 E2348 / 69 strain was grown overnight in Luria-Bertani (LB) broth at 37°C with agitation, then washed twice with Hanks balanced salt solution (HBSS, Gibco) and resuspended in DMEM to an OD of 600nm Normalized to 0.5. Then, the EPEC strain was diluted 1:20 in DMEM to approximately 2x10 6 Colony forming units (CFU) / ml, and pre-cultured with or without 5-HMO mixture or GOS (30 mg / ml) at room temperature with stirring. After 2 hours of pre-culture, the cell culture medium was removed from the Caco-2 cell monolayer, and then gently washed twice with HBSS. Then, the EPEC strain suspension (with and without 5-HMO mixture or GOS) was added to the Caco-2 cell monolayer, and after 1 hour of culture, the culture medium containing non-adherent EPEC was removed. The Caco-2 cell monolayer was gently washed three times in HBSS, and after 3 washing steps, 0.1% Triton X-100 in HBSS was added to the cells to release adherent EPEC. Then, after overnight culture at 37°C, the number of EPEC adhered to the intestinal cell monolayer was quantified by continuous plating and CFU counting on tryptone soy broth (plate).

[0096] result

[0097] Results showed that 5HMO-mixture dose-dependently reduced EPEC binding to intestinal epithelial cell monolayers (up to 30% reduction) compared to EPEC control treated groups, reaching statistical significance at 30 mg / ml (p<0.01). GOS (tested at 30 mg / ml) failed to significantly reduce EPEC binding (see Figure 1B). Data are presented as mean + standard deviation (n = 3 independent wells). Statistical significance was determined by one-way ANOVA compared with the EPEC control group followed by Dunnett's multiple comparison test.

[0098] Example 3

[0099] method

[0100] In three independent experiments using a validated, computer-controlled infant colon dynamic model I-TIM-2, stool samples from three healthy Danish 2-6 month old infants (INF 5 to 7) were used as inoculation materials. Each experiment using stool from one of the infants consisted of four independent compartments operated in parallel. For the general setting 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 simulated infant ileal efflux medium SIIEM (composed of (g / L demineralized H 2 O): 25.5 lactose, 1.7 casein, 28.9 whey, 0.1 ox bile, 0.8 CaCl 2 ·2H 2 O, 0.01FeSO 4 7H 2 O, 0.004 hematin, 4.7K 2 HPO 4 、4.5KCl、0.75MgSO 4 7H 2 O, 4.0 mucin, 2.5 yeast extract, 8.4 NaCl, 1.5 NaHCO 3 , 4.5 peptone, 0.8 cysteine ​​HCl, 10.0 polysorbate 80 and 1.5 ml vitamin solution, the composition of the vitamin solution is (mg / LH 2 O): 1 menaquinone, 2 biotin, 0.5 vitamin B12, 10 pantothenic acid, 5 nicotinamide, 5 p-aminobenzoic acid and 4 thiamine), and two compartments received SIIEM-HMO, which additionally contained a mixture of the five most abundant HMOs in breast milk (5HMO-mix) at average physiological concentrations (5.75 g / L) and ratios (52:13:26:4:5): 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyl lactose (3'-SL) and 6'-sialyl lactose (6'-SL).

[0101] Samples were collected from the fecal mixture used to inoculate the system before inoculation, and then from the compartment (simulated lumen) at 24, 48, 72, and 96 hours. DNA was extracted from the inoculum and lumen samples. Bacterial composition was determined by shotgun metagenomics. Statistical analysis was performed using R statistical software v4.1.0. Microbiome data were centered log-ratio transformed (clr) to account for compositionality.

[0102] Pairwise comparisons were performed between samples from donor feces and samples from I-TIM-2 with two different feed types, SIIEM and SIIEM-HMO, using a linear model with false discovery rate (FDR) correction to account for multiple testing. Corrected P values ​​< 0.05 were considered statistically significant.

[0103] result

[0104] In the infant in vitro colon model I-TIM2, the bacterial community was reconstituted and maintained from infant feces. This allowed the study of the effects of supplementation with the 5HMO-mix on microbial composition and activity over time compared to unsupplemented bacterial communities. In the case of 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 decreased ( Figure 2 , Figure 3 ).

[0105] Example 4

[0106] method

[0107] In vitro, reactor-based, high-throughput The technique was performed as recently described (Van den Abbeele, P. et al., Bridging preclinical and clinical gut microbiota research using the ex vivo technology. Frontiers Microbiol 14, (2023)). Fecal samples were donated by healthy formula-fed infants aged 2-4 months (n=8). The identity of the EPEC strain E. coli O127 E2348 / 69 was confirmed, and a qPCR standard curve was prepared using a pure culture of E. coli O127 E2348 / 69. The study designed a study group that was supplemented with a 5-HMO mixture (5 g / L) and tested against a no substrate control (NSC), and a single fecal suspension was initially cultured for 20 hours. Subsequently, EPEC was incubated at 10 7Exponentially pre-grown EPEC at cells / ml was added to the microbiota and re-cultured for 24 h, with a total culture time of 44 h. Samples were collected at 0 h, 20 h, and 44 h to analyze key fermentation parameters, EPEC levels, and bacterial composition. The content of total short-chain fatty acids (including acetate, propionate, butyrate, and valerate) was determined using the GC-FID method. In addition, pH and gas production were measured using standard methods. Bacterial composition was determined using quantitative shotgun sequencing combined with flow cytometry. EPEC levels were quantified using an EPEC-specific qPCR kit (Escherichia coli typing eae EHEC or EPEC genome (NZYTech, Lisbon, Portugal)). Standardized Illumina library preparation yielded 3M total DNA sequencing for taxonomic analysis. The data were log-transformed and absolute phylogenetic data were used as input. rCCA was performed using the mixOmics package and shrinkage method to evaluate the penalty parameter 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)). Paired t-tests were used to statistically evaluate the treatment effects on key fermentation parameters and cell counts for 8 donors, taking into account the fact that values were compared between samples for a given donor. Statistical differences were visualized by *(0.1 < p < 0.2), **(0.05 < p < 0.1), or ***(p < 0.05).

[0108] Results

[0109] The 5-HMO mixture significantly reduced EPEC levels, decreasing EPEC in 7 out of 8 infants in the 24-h infection model from 20 h to 44 h (p = 0.04; evaluated by qPCR; see Figure 4 ). This anti-pathogen effect may stem from a strong modulation of metabolite production by the gut microbiome. The 5-HMO mixture stimulated the metabolic activity of the gut microbiota, significantly reducing the pH and increasing gas and total SCFA production at 44 h after 20-h pre-growth without EPEC and 24 h after introducing EPEC into the microbiota (see Figure 5 ).

Claims

1. A composition for reducing the risk of infection in the gastrointestinal tract caused by pathogenic Escherichia coli in a human subject, the composition comprising 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose and lactose-N-tetraose, wherein The pathogenic Escherichia coli is an enterohemorrhagic Escherichia coli (EHEC) pathological type or an enteropathogenic Escherichia coli (EPEC) pathological type. Preferably, the subject is a newborn, infant, toddler or child.

2. The composition for use according to claim 1, wherein Such infections in the gastrointestinal tract are diarrheal diseases.

3. A composition for use according to any one of the preceding claims, wherein Reduces the risk of infection in the gastrointestinal tract by inhibiting the adhesion of Escherichia coli to intestinal epithelial cells.

4. A composition for use according to any one of the preceding claims, wherein Reduce the risk of infection in the gastrointestinal tract by reducing the relative abundance of E. coli.

5. A composition for use according to any one of the preceding claims, wherein The pathogenic E. coli of the enterohemorrhagic E. coli (EHEC) pathotype has serotype O157.

6. A composition for use according to any one of the preceding claims, wherein The pathogenic E. coli of the enteropathogenic E. coli (EPEC) pathotype has serotype O127.

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

8. The composition according to any one of claims 1 to 7, wherein The composition comprises effective amounts of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialylactose, 6'-sialylactose and lacto-N-tetraose.

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

10. The composition according to claim 9, wherein The ratios of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose 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'-sialyl lactose; at least 2% by weight to 10% by weight of 6'-sialyl lactose; and 20% by weight to 31% by weight of lacto-N-tetraose.

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

12. The composition according to claim 11, wherein The particle size distribution of the 2'-fucosyllactose, measured using sieves conforming to DIN ISO 3310-1 standard, is less than about 20% passing through #230 mesh (63 μm), greater than about 65% passing through #100 mesh (150 μm), greater than about 92% passing through #45 mesh (355 μm), and 100% passing through #20 mesh (850 μm).

13. The composition according to any one of claims 11 or 12, wherein The water activity of human milk oligosaccharides is w Between 0.10 and 0.30, preferably between 0.10 and 0.

25.

14. The composition according to any one of claims 1 to 13, wherein The composition comprises at least 0.5x10 9 CFU / g of probiotics.

15. Use of a composition for reducing the risk of infection in the gastrointestinal tract caused by pathogenic Escherichia coli in a human subject, wherein: The composition comprises 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose and lactose-N-tetraose, wherein the pathogenic Escherichia coli is an enterohemorrhagic Escherichia coli (EHEC) pathological type or an enteropathogenic Escherichia coli (EPEC) pathological type.

16. The use according to claim 15, wherein The risk of infection in the gastrointestinal tract is reduced by increasing the content of total short-chain fatty acids (SCFAs).

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