Human milk oligosaccharides for reducing risk of infection in gastrointestinal tract

Abstract

The present disclosure relates to a composition for reducing the risk of infection in the gastrointestinal tract caused by pathogenic Escherichia coli in a human subject, said composition comprising at least one human milk oligosaccharide wherein the human milk oligosaccharide is 3-fucosyllactose, 3 '-sialyllactose, 6'-sialyllactose and / or a lactose-N-tetrasaccharide.

Description

Technical Field

[0001] The present disclosure relates in part to the use of human milk oligosaccharides for reducing the risk of infection (particularly diarrheal disease) in the gastrointestinal tract caused by Escherichia coli bacteria in human subjects. Further disclosed are combinations of human milk oligosaccharides and specific probiotics. 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'-sialyl lactose (3'-SL and 6'-SI).

[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 intestinal microbiome imbalance 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, Vol67 sup1.2, 183.191).

[0007] When infected with pathogens, the immune system responds through complex mechanisms to eliminate the pathogens while avoiding damage to the host due to excessive immune responses. The secretion of proinflammatory cytokines (such as IFN-γ and TNF-α) plays a vital role in enhancing the immune response to infections (including those caused by E. coli) (Akdis M. et al., 2016 Journal of Allergy and Clinical Immunology, 984-1010; Sanchez-Villamil et al., 2016 Frontiers in Cellular and Infection Microbiology, 6 (OCT), p.219189). For a balanced immune response, the secretion of anti-inflammatory cytokines (such as IL-10 and IL-1 receptor antagonist (IL-1RA)) during bacterial infection is essential for regulating the immune response and preventing excessive inflammation (Raphae et al., 2016 Cytokine, 74 (1), pp.5-17). Therefore, supporting a balanced immune response may be associated with a reduced risk of infection with pathogenic E. coli.

[0008] The intact intestinal barrier plays an important role in effectively defending against gastrointestinal infections. The intestinal barrier is composed of different elements. One element is the single layer of epithelial cells, which forms a physical barrier and plays a vital role in separating luminal microorganisms from the host immune system and circulatory system. Tight junctions between epithelial cells regulate the exchange of substances across the barrier (Bischoff SC et al.; BMC Gastroenterology, 2014, 14, 189). Tight junctions can be disrupted by bacterial pathogens and their virulence factors, resulting in increased permeability of the epithelial layer ( et al., Clinical and Translational Gastroenterology, 2016, 7(10)). Increased permeability and the resulting systemic inflammation are associated with many health problems, including autoimmune diseases (e.g., inflammatory bowel disease, arthritis, allergies), metabolic diseases (e.g., obesity), cancer, and neurological dysfunction (Pilla, R., Suchodolski, JS; Veterinary Clinics of North America: Small Animal Practice, 51, 3, 605-621 and Current Opinion in Clinical Nutrition and Metabolic Care 25(3); 178-185). In addition, it may lead to the systemic spread of pathogenic or opportunistic pathogens (such as pathogenic Escherichia coli).

[0009] 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 contains a variety of strains, ranging from commensals to serious pathogens, depending on the presence of virulence factors (Evans DJ Jr. et al., in: Baron S, Medical Microbiology, 4th edition, Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 25).

[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 affected patients (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 can reduce the risk of colonization and ensuing 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), V01.44, 11, 1-68). Therefore, reducing the colonization of E. coli in the gastrointestinal tract can reduce the risk of diarrheal diseases caused by E. coli.

[0012] Strategies are needed 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), the composition comprising at least one human milk oligosaccharide (HMO), wherein the human milk oligosaccharide is selected from 3-fucosyllactose (3-FL), 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL) and lacto-N-tetraose (LNT).

[0014] While not wishing to be bound by theory, it is believed that HMOs inhibit the binding of pathogenic E. coli to the intestinal mucosal barrier and support intestinal barrier integrity. Binding of pathogenic E. coli to epithelial cells is a step in the infection mechanism. Therefore, by inhibiting this binding, it is believed that the risk of infection by pathogenic E. coli is reduced. In addition, it is believed that HMOs can enhance the immune response, which is believed to be beneficial in clearing pathogens such as E. coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is the reduction in adhesion of enterohemorrhagic Escherichia coli (EHEC) serotype O157 to monolayers of human colon cancer cells (Caco-2) in the presence of 3-FL, 3'SL, 6'SL and LNT at doses of 10 mg / ml each. Data are presented as mean + standard deviation (n = 3 independent experiments). Statistical significance was determined by one-way ANOVA compared to the EHEC control group followed by Dunnett's multiple comparison test.

[0016] Figure 2 Lactobacillus rhamnosus Results of transepithelial electrical resistance (TEER) measurements of Caco-2 cell monolayers stimulated with 3-FL, a combination of both, or medium alone, measured in triplicate for 10 hours. (A) Shows the percent change in TEER relative to baseline (y-axis) versus time on the x-axis. (B) Shows the TEER area under the curve (AUC) for 10 hours under the four conditions. Data are mean + SD (n = 3). **** indicates p-value < 0.0001 using one-way ANOVA with multiple comparisons test.

[0017] Figure 3 By using 3-FL, Anti-inflammatory cytokines IL-10 (A) or IL-1RA (B) secreted by human PBMCs co-cultured with or without synbiotic combinations and challenged with LPS of E. coli for 20 h.

[0018] Figure 4 By using 3-FL, IFN-γ (A) and TNF-α (B) secretion from human PBMCs co-cultured with or synbiotic combinations and challenged with E. coli LPS for 20 hours. Significant differences are indicated as follows: * = P < 0.05, ** = P < 0.01, *** = P < 0.005, and **** = P < 0.001. DETAILED DESCRIPTION

[0019] 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.

[0020] The terms "a" and "a" are used in the context of describing the present invention (particularly in the context of the following claims). nn )” and “the” and similar referents should 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 indicated. References to ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value within the range (including both endpoints), 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.

[0021] 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."

[0022] 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.

[0023] As used herein, the term "isolated" refers to the bacterial strain described herein in a form or environment not found in nature, ie, the strain is at least partially free of one or more or all of the naturally occurring components with which it is associated in nature.

[0024] 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 BB- ) classification. FAO / WHO points out that probiotic effects are strain-specific, so most probiotic properties of a particular strain cannot be extrapolated to other strains of the same species.

[0025] 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, produce beneficial effects 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 tem probiotic).

[0026] 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.

[0027] The terms "treatment" or "treat" 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.

[0028] The term "reducing or 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] As used herein, unless otherwise specified, the term "formula" refers to liquid and solid human milk substitutes or replacements suitable for human consumption.

[0034] 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.

[0035] 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.

[0036] The term "enhanced immune response" refers to an increase in the level of the proinflammatory cytokines TNF-α and / or IFN-γ compared to the corresponding cytokine levels observed without the use of the composition of the present invention.

[0037] The term "balanced immune response" refers to stable levels of secreted anti-inflammatory cytokines IL-10 and IL-1RA compared to the corresponding cytokine levels observed in the absence of the composition of the invention.

[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] In the context of two or more nucleotide sequences, the term "[a certain] % sequence identity" refers to the relationship between two polynucleotide sequences determined by sequence comparison (alignment). As used herein, "sequence identity" is determined over the full length of the sequence. "Sequence identity" means that two or more sequences have a given percentage of common nucleotides when compared and aligned. Identity measures the percentage of identical matches of the smaller of two or more sequences relative to a gap alignment (if any) addressed by a specific mathematical model, algorithm or computer program.

[0044] The percent sequence identity of nucleotide sequences can be readily calculated by any method known to those of ordinary skill in the art. In a preferred embodiment, the "percent identity" of two sequences (e.g., polynucleotide or amino acid sequences) is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 as modified by Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms are incorporated in Altschul et al., J. Mol. Biol. 215:403-10, 1990. and If there is a gap between two sequences, you can use Gapped For example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped When you use the corresponding program (such as and ), or the parameters may be appropriately adjusted in a manner understood by a person of ordinary skill in the art.

[0045] Another local alignment technique that can be used is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) J. Mol. Biol. 147: 195-197). A general global alignment technique that can be used is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) J. Mol. Biol. 48: 443-453), which is based on dynamic programming.

[0046] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0047] Numerical ranges used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not.

[0048] 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.

[0049] 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.

[0050] The present disclosure provides a composition comprising at least one human milk oligosaccharide (HMO) selected from 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose and lactose-N-tetraose. While not wishing to be bound by theory, it is believed that a composition comprising at least one of these HMOs can reduce the risk of gastrointestinal infection caused by pathogenic Escherichia coli in human subjects. This is supported by the finding in in vitro experiments that in the presence of these HMOs, the binding of pathogenic Escherichia coli to epithelial cells is inhibited.

[0051] In order to evaluate the effect of the HMO on the binding of pathogenic E. coli to the intestinal epithelium, enterohemorrhagic E. coli (EHEC) strains of pathological type O157 were pre-incubated with or without different doses of HMO. The pre-incubated E. coli were added to the monolayer of human colon adenocarcinoma Caco-2 cells. After a defined incubation time, the number of EHEC adherent to the intestinal cell monolayer was determined. It was found that HMO structure-dependently reduced the binding of EHEC to the cell monolayer ( Figure 1 While not wishing to be bound by theory, it is believed that reducing the binding of EHEC to intestinal epithelial cells may reduce the risk of infection.

[0052] In another in vitro experiment, the effect of substances on the intestinal barrier was studied. There are methods for exploring in vitro intestinal barrier function modeling, for which Caco-2 cell lines are routinely used. Intestinal permeability function is not static, but can be intentionally regulated by exposure to specific stimuli and damage, and can then be evaluated by measuring the transepithelial electrical resistance (TEER) of the cell monolayer. Compared with the unstimulated cell monolayer, the increase in TEER after stimulation indicates that the intestinal barrier integrity is enhanced. On the contrary, relative to unstimulated cells, a decrease in TEER indicates an increase in intestinal permeability, which may increase the risk of systemic spread and disease severity caused by pathogenic bacteria, among other adverse consequences (including systemic inflammation).

[0053] The cytokine profile induced when human PBMCs were pretreated with a composition according to the invention to respond to an E. coli LPS challenge was studied. This allows the study of immune responses against pathogens, which is essential for assessing the effectiveness of pathogen clearance during infection with pathogens. An enhanced and simultaneously balanced immune response is considered to be very important for reducing the risk and / or severity of gastrointestinal infections. The enhancement of anti-pathogen immune responses is associated with an increase in the levels of proinflammatory cytokines such as TNF-α and IFN-γ.

[0054] In certain embodiments, a composition comprising at least one HMO of 3-FL, 3'SL, 6'SL and / or LNT 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 at least one HMO of 3-FL, 3'SL, 6'SL and / or LNT can reduce the risk of diarrheal disease.

[0055] In certain embodiments, compositions comprising at least one HMO of 3-FL, 3'SL, 6'SL and / or LNT 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 of the infection mechanism is attachment to epithelial cells.

[0056] In certain embodiments, compositions comprising at least one HMO of 3-FL, 3'SL, 6'SL, and / or LNT are used to reduce the risk of infection in the gastrointestinal tract by supporting intestinal barrier function.

[0057] In certain embodiments, the composition comprises one, two or three HMOs selected from 3-FL, 3'SL, 6'SL or LNT.

[0058] In certain embodiments, the composition comprises two HMOs selected from 3-FL, 3'SL, 6'SL, or LNT.

[0059] In certain embodiments, the composition comprises an HMO selected from 3-FL, 3'SL, 6'SL, or LNT.

[0060] In certain embodiments, the composition comprises 3'SL and / or 6'SL as the only HMO. In in vitro experiments, these HMOs showed an inhibitory effect on the binding of pathogenic E. coli to epithelial cells.

[0061] In certain embodiments, the composition comprises 3-FL and / or LNT as the only HMO. In in vitro experiments, these HMOs showed an inhibitory effect on the binding of pathogenic E. coli to epithelial cells.

[0062] In certain embodiments, the composition comprises 3-FL as the sole HMO. This HMO has been shown to improve intestinal barrier function in vitro.

[0063] In certain embodiments, the composition comprises an effective amount of a probiotic strain. For example, if probiotics are present, the concentration of the probiotics is preferably 0.05 x 10 9 CFU / dose up to 30x10 9 CEU / dose, preferably 0.5x109 CFU / dose up to 25x10 9 CFU / dose. The dose in this article refers to the amount taken in one day.

[0064] In certain embodiments, the composition may include 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 paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus spp. 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, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenellae, Bifidobacterium dentate, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum), Bifidobacterium megaterium, Bifidobacterium pseudosmall chain (Bifidobacterium seudocatenulatum)), or any strain from the genus Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridium, Coprococcus, Dorea, Eubacterium, Faecalibacterium or Roseburia or the family of Rudobacteriaceae, and suitable combinations of the foregoing.

[0065] 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 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.

[0066] In certain embodiments, the composition comprises the probiotic Lactobacillus rhamnosus GG. Preferably, the probiotic is produced and sold by Chr. Hansen A / S. Preferably, the nucleotide sequence of Lactobacillus rhamnosus GG has at least 99.00% identity with SEQ ID NO.1, preferably at least 99.10%, at least 99.20%, at least 99.30%, at least 99.40%, at least 99.50%, at least 99.60%, at least 99.70%, at least 99.80% identity with SEQ ID NO.1, more preferably at least 99.90%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98% identity with SEQ ID NO.1, even more preferably at least 99.99% identity with SEQ ID NO.1, or is identical to SEQ ID NO.1.

[0067] In certain embodiments, the composition comprises the probiotic Lactobacillus rhamnosus GG and the nucleotide sequence of Lactobacillus rhamnosus GG differs from the nucleotide sequence of SEQ ID NO.1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Preferably, the nucleotide sequence of Lactobacillus rhamnosus GG differs from the nucleotide sequence of SEQ ID NO. 1 by no more than 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000, 750, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15 nucleotides.

[0068] The nucleotide sequence of SEQ ID NO. 1 is available in the ncbi database (https: / / www.ncbi.nlm.nih.gov / ) under the reference number GCF_028475085.1 as Lactobacillus rhamnosus GG genome assembly ASM2847508 v1. This database is cited in the version accessible on March 7, 2024.

[0069] Lactobacillus rhamnosus GG It can inhibit pathogenic Escherichia coli (Petrova MI et al., 2016, PLoS ONE, 11(8), e0161337). In vitro experiments showed that It also improves the intestinal barrier function ( Figure 2 ).

[0070] In certain embodiments, the composition comprises Lactobacillus rhamnosus GG and 3-FL, preferably Lactobacillus rhamnosus GG and 3-FL as the only HMO. In vitro experiments have shown that and 3-FL have beneficial effects on intestinal barrier function ( Figure 2 and 3 ).

[0071] In vitro, compared with E. coli LPS challenge, 3-FL and Pretreatment of human PBMC with the combination significantly promoted the secretion of TNF-α and IFN-γ.

[0072] TNF-α is known for stimulating immune cell function and enhancing the phagocytic activity of macrophages and neutrophils, which are essential for clearing bacterial infections such as Escherichia coli. This cytokine not only promotes the recruitment of immune cells to the site of infection, but also enhances its bactericidal function (Trevejo et al., 2001 Proceedings of the National Academy of Sciences of the United States of America, 98 (21), pp. 12162-12167; Jeong et al., 2019 J. Microbiol. Biotechnol., 29 (8), pp. 1248-1254; Jeong et al., 2020 J. Microbiol. Biotechnol., 30 (9), pp. 1395-1403). Similarly, IFN-γ enhances the bactericidal activity of macrophages and has been shown to enhance the expression of major histocompatibility complex (MHC) molecules, which is essential for further promoting adaptive immune responses (Wu et al., 2019 Immunology, 158(4), pp.304-313). The enhancement of these cytokines induced by 3-FL may reduce the risk of E. coli infection.

[0073] In addition, maintaining appropriate levels of anti-inflammatory cytokines (such as IL-10 and IL-1RA) during bacterial infection is essential for regulating immune responses and preventing self-injury caused by excessive inflammation (Sun et al., 2015 Mucosal Immunology doi: 10.1038 / mi.2015.49; Akdis et al., 2016 Journal of Allergy and Clinical Immunology, pp. 984-1010). After pretreatment with 3FL, the levels of these cytokines secreted by human PBMCs were maintained, supporting a balanced immune response to E. coli and reducing the risk of tissue damage.

[0074] In certain embodiments, the composition is a nutritional composition, such as a formula or a dietary supplement.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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%, also including about 60% to about 90%, and also including about 70% to about 85% water by weight of the nutritional emulsion. 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, a typical serving size is 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.

[0081] Nutrient solids can be any solid form that can be optionally compressed into tablets, but are generally flowable or substantially flowable granular compositions, or at least in the form of granular compositions. Particularly suitable nutrient solid product forms include spray-dried, agglomerated and / or dry-mixed 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 used 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.

[0082] The present composition can be used for a neonate, infant, toddler or child. The present composition can be used for a neonate. The present composition can be used for an infant. E. coli is a major cause of diarrhea in infants, suggesting that infants may particularly benefit from the present composition.

[0083] The present composition may comprise an effective amount of 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose and / or lacto-N-tetraose.

[0084] The present composition may comprise 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 / or at least 0.01% by weight lacto-N-tetraose.

[0085] The present composition can contain 3-fucosyllactose, 3' sialyllactose, 6'-sialyllactose and / or lacto-N-tetraose in any suitable amount, such as, for example, at least about 0.001 g / dose, at least about 0.01 g / dose, at least about 0.05 g / dose, at least about 0.1 g / dose, at least about 0.5 g / dose. For example, in the case of a dietary supplement, the composition can contain less than about 20 g / dose, less than about 10 g / dose, less than about 5 g / dose, less than about 4 g / dose, less than about 3 g / dose, less than about 2 g / dose, less than about 1.5 g / dose of 3-fucosyllactose, 3' sialyllactose, 6'-sialyllactose and / or lacto-N-tetraose.

[0086] 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 / mL composition of a particular HMO).

[0087] 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).

[0088] 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.

[0089] Preferably, the HMO (particularly 3-FL) used in the present composition has the following particle size characteristics:

[0090] 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%.

[0091] 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%.

[0092] Percent passing #45 mesh (355 μm)—greater than about 95%, greater than about 92%, greater than or equal to about 90%.

[0093] Percent passing #20 mesh (850 μm) - 100%.

[0094] Preferably, when in powder form, the water activity of human milk oligosaccharides is w The water activity is 0.10 to 0.3, preferably 0.10 to 0.25. The water activity can be determined using a known hygrometer; preferably the water activity is determined according to ISO 18787:2017. The water activity ensures the microbial stability of the powder and prevents contamination by unwanted microorganisms. This is especially important for compositions containing probiotic strains.

[0095] 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%.

[0096] The present composition may include probiotics. The composition may be formulated as a combination of probiotic strains and HMOs or as a separate composition. The present composition may include these probiotics in any form suitable for administration to a subject. In a preferred embodiment, the composition may include the bacteria in a dry form, which may be obtained by freeze drying, spray drying, lyophilization, etc.

[0097] If the bacteria are freeze-dried, they are usually mixed with a cryoprotectant before freeze-drying. The term "cryoprotectant" as used herein refers to a substance that can increase the survival rate during freezing and / or drying and improve the storage stability of bacteria. The cryoprotectant used herein preferably comprises a saccharide and / or a sugar alcohol, such as inositol.

[0098] The carbohydrate can be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide, or a mixture of at least two carbohydrates. Useful monosaccharides include, for example, glucose (also referred to as dextrose), fructose, ribose and galactose, and useful disaccharides include, for example, sucrose, trehalose, maltose and lactose. The composition can include one or more monosaccharides or disaccharides, such as one, two or three or even more different carbohydrates.

[0099] The cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

[0100] The cryoprotectant may also contain peptides, proteins, protein hydrolysates or mixtures thereof. Examples of peptides and proteins to be used are casein, pea, whey, albumin, glutamic acid or gelatin, and any isolates or hydrolysates thereof. Other additives may also be present, for example antioxidants such as sodium ascorbate, sodium citrate, trisodium citrate dihydrate and cysteine ​​hydrochloride. Skimmed milk powder and yeast extract may also be ingredients.

[0101] Compositions disclosed herein can 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 related 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 oils, marine plankton, fungal oils and fish oils. 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.

[0102] Furthermore, the present composition may comprise antioxidants, such as carotenoids, and in particular a combination of carotenoids, lutein, lycopene, zeaxanthin and / or beta-carotene.

[0103] The compositions of the present disclosure may further comprise other optional components that may alter the physical, chemical, aesthetic or processing characteristics of the compositions, or serve as pharmaceutical or additional nutritional components. Non-limiting examples of such optional components include preservatives, emulsifiers, buffers, pharmaceutically active substances, nutrients, colorants, flavorings, thickeners and stabilizers, flow agents, minerals, emulsifiers, lubricants, sweeteners, etc.

[0104] 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 allow the powder entity to 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 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.

[0105] 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.

[0106] It will be appreciated by those skilled in the art that the compositions disclosed herein can be administered with dosage levels and dosage 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.

[0107] Example

[0108] Example 1

[0109] HMO 3-FL, LNT, 3'-SL and 6'-SL were produced by Chr. Hansen HMO GmbH, Rheinbreitbach, Germany. A stock solution of HMO solution was dissolved in water.

[0110] Human epithelial intestinal cancer cell line Caco-2 (ACC169, 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) at 37°C in a 5% CO2 atmosphere. Cells were plated 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.

[0111] 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 a single HMO at a dose of 10 mg / ml at room temperature under 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 HMO) 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 adhered to the intestinal cell monolayer was quantitatively counted by continuous plating and CFU counting on tryptone soy broth (plate).

[0112] result

[0113] The results show that individual HMOs (tested at 10 mg / ml) reduced EHEC binding in a structure-dependent manner, with 3FL, 3'SL, 6'SL and LNT reaching statistical significance (p<0.01), and the greatest effect observed for 3'SL and 6'SL (reduction of approximately 40%). Data are presented as mean + standard deviation (n=3 independent experiments). Statistical significance was determined by one-way ANOVA compared to the EHEC control group, followed by Dunnett's multiple comparison test.

[0114] Example 2

[0115] As assessed by transepithelial electrical resistance (TEER) across Caco-2 cell monolayers in vitro, the combined Lactobacillus rhamnosus (SEQ ID NO. 1) and 3-FL (3-fucosyllactose) introduced higher intestinal barrier tightness.

[0116] Caco-2 cell culture

[0117] The human intestinal epithelial Caco-2 cell line (DSMZ ACC 169, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was cultured in complete DMEM medium containing DMEM GlutaMax supplemented with 20% heat-inactivated fetal bovine serum, 1X MEM non-essential amino acids and 1X Pen-Strep-Amp B at 37°C in 5% CO2. Caco-2 cells were used for passage 7-25. When the cell confluence was about 50%, the medium was removed and the cells were washed twice in phosphate buffered saline (PBS). The cells were trypsinized by adding 1 mL of trypsin and placed in a CO2 incubator at 37°C for 3 minutes. About 10 mL of culture medium was added to the trypsinized cells, counted, and a concentration of 100,000 cells / mL was prepared in complete DMEM. 500 μL of cell suspension was used to inoculate a 12 mm to each apical compartment, after which 1.5 mL of supplemented DMEM was added to the basolateral compartment. The cells were cultured on the chambers for 21 days, with the medium replaced twice a week. After 21 days, the transport well plate was moved to the CellZscope (NanoAnalytics, Germany). 1.65 mL and 0.8 mL of complete medium without antibiotics (Abx) were added to the basolateral and apical compartments, respectively. The CellZscope was placed in a 37°C CO2 incubator (5%) overnight, and TEER was measured every hour using automatic data collection. Overnight measurements of TEER before the start of the experiment can determine the baseline TEER in each well and serve as a quality control for stable resistance.

[0118] Preparation of 3-fucosyllactose (3-FL) in DMEM medium

[0119] 3-Fucosyllactose (3-FL) solution was prepared in complete DMEM medium without Abx.

[0120] Preparation of Lactobacillus rhamnosus (SEQ ID NO.1)

[0121] One day before co-incubation with Caco-2 cells, Lactobacillus rhamnosus was added A frozen stock of (SEQ ID NO. 1) was inoculated in MRS. Dilution lines were generated by transferring 1 mL of the mixed inoculated culture with 9 mL of MRS and repeated 5 times. The strain was incubated anaerobically overnight at 37°C. On the day of co-incubation, the optical density (OD) at 600 nm was measured.600 ) to assess bacterial growth and select cultures representing late exponential phase / early stationary phase. Centrifuge the cultures at 6000 x g for 5 minutes to collect the bacterial pellet. Discard the supernatant and add 35 mL of 37°C warm PBS, wash the bacteria and centrifuge at 6000 x g for 5 minutes. Repeat this washing procedure twice. Harvest the bacterial cells by centrifugation at 6000 x g for 5 minutes and discard the supernatant. Resuspend the bacterial cells in 5 mL of pre-warmed complete DMEM medium without Abx and adjust the OD 600 .

[0122] Use Lactobacillus rhamnosus alone or in combination (SEQ ID NO.1) and 3-FL stimulate Caco-2 cells

[0123] To stimulate Caco-2 cells, pause the CellZscope measurement, remove the CellZscope from the CO2 incubator, and remove the apical medium from each transport well plate. Add bacterial solution (final concentration OD 0.5), 3-FL solution (2% final concentration), combination or medium control (DMEM) to the relevant wells (each in triplicate).

[0124] Place the CellZscope back into the CO2 incubator and resume TEER measurements overnight. Calculate the change in TEER during bacterial stimulation relative to the most recent value recorded immediately before stimulation (baseline measurement set to 100%). Calculate the area under the curve for each well after 10 hours.

[0125] result

[0126] After 10 hours, all test conditions; individual 3-FL alone and the combination of both significantly increased the area under the curve (AUC) TEER measurements. Furthermore, Lactobacillus rhamnosus The combination of 1,2-dihydropyrrolidone and 3-FL had significantly higher AUCTEER measurements.

[0127] Example 3

[0128] Isolation of peripheral blood mononuclear cells (PBMC)

[0129] PBMC isolation was performed in SepMate™ tubes (50 ml) by Ficoll-Plague Plus™ density gradient centrifugation (1200 g, 10 min, RT). Five buffy coats were collected from healthy anonymous donors (Righospitalet, Blodbank, Copenhagen, Denmark) on the day of the experiment. The isolated PBMCs were washed twice in PBMC medium consisting of RPMI medium (Sigma Aldrich), 2-mercaptoethanol (final concentration 50 μM, Sigma Aldrich) and HEPES buffer (final concentration 10 mM, Sigma Aldrich). Subsequently, the PBMCs were isolated using -MC200 TM Count the cells and resuspend in the same medium supplemented with heat-inactivated fetal bovine serum (FBS, final concentration 10%, Gibco). Adjust the PBMC suspension to 2x10 6 cells / ml and seeded in 96-well plates (1x10 5 cells / well) for at least 1 hour (37°C, 5% CO2).

[0130] Bacterial preparation

[0131] Inoculate from frozen stocks from the Chr. Hansen culture collection and grow overnight at 37°C in pH 6.5 MRS (de Man, Rogosa and Sharpe) broth (Difco). A 10-fold dilution series was prepared from the overnight culture and grown overnight under the same conditions. 600 The bacterial cultures were centrifuged at 6000 g for 2 min, washed twice in Hank's balanced salt solution (HBSS), and resuspended in complete DC medium without antibiotics at a stock concentration of 6.7 x 10 7 intact cells / ml.

[0132] Stimulation of PBMCs

[0133] PBMCs from 5 healthy donors were used 3-FL stimulation or no treatment. The final concentration is 1x10 6 IC / well, corresponding to an MOI of 1:10 (PBMC: ), the final concentration of 3-FL was 0.5%, and the synbiotic combination was 1x10 6 IC / well plus 0.5% 3-FL, all wells were treated with tetracycline (final concentration of 4 μg / ml, SigmaAldrich). After 3 hours of culture, all wells were treated with lipopolysaccharide (LPS) of Escherichia coli (Catalog No. L2654, Sigma Aldrich) at a final concentration of 500 ng / ml. After a total of 20 hours of culture, the cell supernatant was collected on an AcroPrep filter plate (PallTM, New York, USA), centrifuged (1500 x g, 5 min, 4°C), and stored at -80°C before cytokine measurement.

[0134] Cytokine quantification

[0135] According to the manufacturer's instructions, use Multi-point detection system MESO Scale QuickPlex TM The levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1RA), and interferon-γ (IFN-γ) secreted by PBMC were quantified by ELISA (MSDMaryland, USA). The data are expressed as picograms per milliliter (pg / ml).

[0136] Statistical analysis

[0137] The levels of secreted IFN-γ were compared using the Friedman test and Dunn's multiple comparison test. For the remaining cytokines (TNF-a, IL-1RA, and IL-10), RM one-way ANOVA and Tukey's multiple comparison test were used. An alpha level of 0.05 was considered significant.

[0138] result

[0139] like Figure 3 As shown, compared with the secretion induced by E. coli LPS challenge alone, the secretion induced by 3-FL, or synbiotic combination co-cultured PBMCs did not result in the expression of the anti-inflammatory cytokine IL-10 ( Figure 3 A) and IL-1RA ( Figure 3 B) produced significant differences in secretion.

[0140] In addition, although 3-FL had no significant effect on IFN-γ secretion, Its secretion was significantly promoted, and the synbiotic combination showed a trend of enhancing IFN-γ levels ( Figure 4 A) 3-FL does not cause any significant changes in TNF-α secretion, while significantly increased its levels, and the synbiotic combination resulted in an even higher significant increase ( Figure 4 B).

Claims

1. A composition for reducing the risk of infection in the gastrointestinal tract caused by pathogenic Escherichia coli of the enterohemorrhagic Escherichia coli (EHEC) pathotype in a human subject, the composition comprising at least one human milk oligosaccharide, wherein: The human milk oligosaccharide is 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose and / or lactose-N-tetraose.

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 The EHEC is preferably of serotype O157.

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

6. A composition for use according to any one of the preceding claims, wherein The risk of infection in the gastrointestinal tract is reduced by enhancing the immune response, preferably by increasing the levels of pro-inflammatory cytokines; and preferably further balancing the immune response by maintaining the levels of secreted anti-inflammatory cytokines compared to the levels of said anti-inflammatory cytokines observed when the composition is not used to reduce the risk of infection in the gastrointestinal tract.

7. A composition for use according to any one of the preceding claims, wherein The composition comprises at least 0.5x10 9 CFU / dose of probiotics.

8. The composition for use according to claim 7, wherein The probiotic is Lactobacillus rhamnosus GG.

9. A composition for use according to any one of the preceding claims, wherein The composition comprises an effective amount of 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose and / or lacto-N-tetraose.

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

11. The composition for use according to claim 10, wherein The composition comprises 3-fucosyllactose, and the particle size distribution of the 3-fucosyllactose is less than about 20% through #230 mesh (63 μm), greater than about 65% through #100 mesh (150 μm), greater than about 92% through #45 mesh (355 μm), and 100% through #20 mesh (850 μm) as measured using sieves conforming to DIN ISO 3310-1 standard.

12. The composition for use according to any one of claims 10 or 11, wherein The water activity of human milk oligosaccharides is w Between 0.10 and 0.30, preferably between 0.10 and 0.

25.

13. The composition for use according to claim 7 or 8, wherein The composition comprises the human milk oligosaccharide 3-fucosyllactose, wherein 3-fucosyllactose is preferably the only human milk oligosaccharide.

14. The composition according to any one of claims 1 to 13, wherein The subject is a newborn or an infant.

15. The composition according to any one of claims 1 to 13, wherein The subject is an infant or a child.

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