Lactose-n-fucopentasaccharide and bifidobacterium infantis protection from intestinal pathogens

Abstract

The present invention relates to a combination and nutritional composition comprising fucosylated human milk oligosaccharides, i.e., lactose-N-fucopentasaccharide-I (LNFP-I), as the most abundant human milk oligosaccharide in the combination or the nutritional composition, and Bifidobacterium longum subsp. Infantis (Bifidobacterium infantis). The combination and the nutritional composition are useful for treating, preventing and / or inhibiting the growth of intestinal pathogens, in particular in infants.

Description

Technical Field

[0001] This invention relates to combinations and nutritional compositions specifically for infants, toddlers, or children, and their health effects. Such combinations and compositions contain specific oligosaccharides and *Bifidobacterium longum* and effectively protect against intestinal pathogens. Specifically, this invention relates to infant formula containing at least one lactose-N-fucopentose as a human milk oligosaccharide (HMO) and *Bifidobacterium longum* subsp. infantis (*B. infantis*), which provides protection against various intestinal pathogens, namely *Salmonella* and enteropathogenic *Escherichia coli* (EPEC), by inhibiting their growth. Background Technology

[0002] Breastfeeding is recommended for all infants. However, in some cases, due to medical reasons, breastfeeding is insufficient or unsuccessful, or the mother does not choose to breastfeed. Nutritional compositions, such as infant formula, have been developed for these situations.

[0003] Nutritional compositions for infants and young children are typically marketed as water-reconstituted powders or, in some cases, as ready-to-drink or concentrated liquid compositions. These compositions are designed to cover most or all of an infant's or young child's nutritional needs. However, human breast milk is known to represent the ultimate gold standard for infant nutrition. Therefore, infant formula manufacturers have made numerous attempts to induce nutritional and health effects that are close to or similar to the beneficial effects of human breast milk. However, many studies have shown that infant formula does not induce the same effects on the body as human breast milk. For example, infants fed infant formula and infants fed human breast milk (HBM) may exhibit different gut microbiota.

[0004] Specific Bifidobacterium species, such as *Bifidobacterium longum* subsp. *infant* (Bifidobacterium infantis), are among the first to colonize the infant gut, partly due to their ability to metabolize complex human milk oligosaccharides (HMOs), and are proposed to play a key role in infant gut development. Strains belonging to *Bifidobacterium infantis* are particularly well-suited to metabolizing recalcitrant HMOs, which at least partially explains their abundance in the gastrointestinal tract of breastfed infants. They are also proposed to play a key role in early infant immune system maturation and improved intestinal barrier function. It is hypothesized that restoring the early infantile *Bifidobacterium infantis*-dominant microbiota through co-administration of HMOs and selection of *Bifidobacterium infantis* strains is beneficial for infant immune system maturation (Duboux et al., *Microorganisms* 2022, 10(2), 203). It has been reported that carbohydrate utilization patterns may differ among different *Bifidobacterium infantis* strains. Strains belonging to a cluster of strain types (ATCC 15697) have been described as simultaneously and equally well-consuming different types of HMOs, with a preference for neutral and sialylated HMOs. In contrast, the Bi-26 strain, which phylogenetically clusters with LMG11588 (ATCC 17930), shows adaptation to rapid internalization and metabolism, particularly of fucosylated HMOs. Fucosylated HMOs are highly prevalent in human breast milk. 2-Fucosyllactose (2'-FL) is the most abundant fucosylated HMO, accounting for up to 45% of the total HMO content in breast milk, while LNFP-I accounts for a lower proportion (Thum et al. Nutrients. 2021 July; 13(7): 2272).

[0005] Typically, Bifidobacterium infantis has the ability to effectively break down and metabolize various HMO structures, especially fucosylated HMO structures. The main fucosylated HMO present in breast milk is the pentasaccharide lactose-N-fucopentose I (LNFP-I) (Garrido et al., Scientific Reports, Vol. 6, No. 35045 (2016)).

[0006] WO2018 / 024870 A1 relates to infant or young child nutritional compositions comprising at least one fucoidylated oligosaccharide and at least one N-acetylated oligosaccharide, for the prevention and / or treatment of non-rotavirus diarrhea in infants or young children by acting on dysbiosis of the microbiota before and / or after non-rotavirus diarrhea caused by *Escherichia coli* (ETEC, EPEC, and / or EAEC), *Salmonella*, *Shigella*, *Aeromonas*, and / or *Campylobacter*. Preferred fucoidylated oligosaccharide is 2'-fucosylated lactose (2'FL), which has been extensively studied. Without any evidence, examples of fucosylated oligosaccharides include lactose-N-fucopentose (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V), lactose-N-fucohexasaccharide, lactose-N-difucohexasaccharide I, fucosyllacto-N-hexasaccharide, fucosyllacto-N-neohexaccharide, difucosyllacto-N-hexasaccharide I, and difucosyllacto-N-neohexaccharide II. Without any evidence, the addition of probiotics is mentioned in other probiotic products, such as Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co. Ltd. under the trademark BB536, Bifidobacterium breve sold by Danisco under the trademark Bb-03, Bifidobacterium breve sold by Morinaga under the trademark M-16V, and Bifidobacterium infantis sold by Procter & Gamble Co. under the trademark Bifantis.

[0007] WO2021 / 217803 A1 relates to a nutritional composition comprising an HMO (preferably 2'FL) and probiotics including Bifidobacteria for reducing intestinal gas production in infants and young children. Examples of oligosaccharides and probiotics include lactose-N-fucopentoses (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III) and Bifidobacterium infantis. WO2021 / 217803 A1 states that studies have shown that HMOs typically play a very important role in the early growth and development of infants, such as 1) regulating the gut microbiota, especially the proliferation of beneficial bacteria (e.g., Bifidobacteria), 2) indirectly inhibiting the growth of pathogenic bacteria by increasing the competitive advantage of non-pathogenic symbiotic organisms, and also directly acting as anti-adhesion antimicrobial agents to reduce microbial infections.

[0008] WO2021 / 061991 A1 relates to the administration of a mixture of *Bifidobacterium longum* subsp. infantis and concentrated HMOs to adult subjects for several benefits, such as reducing the gut-dominant role of pathogenicity classification. Lactose-N-difucohexose I (LNDFH-I), lactose-N-difucohexose-II (LNDFH-II), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), and lactose-N-fucopentose-III (LNFP-III) are mentioned as potential prebiotics.

[0009] As known from WO2021021746A1, the combination of Bifidobacterium infantis with a milk fat globule membrane complex (MFGM) containing LNT and LNFP provides protection against intestinal pathogens.

[0010] As can be seen from existing technologies, HMOs such as lactose-N-fucopentose (LNFP-I) and lactose-N-fucohexasaccharide (LNDFH-I) have been mentioned, but they have not been extensively studied to date.

[0011] LNFP-I can be isolated from natural sources, such as animal milk, by chromatography or filtration. Suitably, animal milk, as used herein, can be cow's milk, sheep's milk, goat's milk, camel's milk, or buffalo milk. Preferably, the animal milk is cow's milk. Preferably, LNFP-I can be prepared by biotechnological means using specific fucosyltransferases and / or fucosidases, through enzyme-based fermentation techniques (recombinant enzymes or natural enzymes) or microbial fermentation techniques. In the latter case, the microorganisms can express their natural enzymes and substrates, or can be engineered to produce the corresponding substrates and enzymes. Single microbial cultures and / or mixed cultures can be used. Fucosylated oligosaccharides can be formed starting with acceptor substrates initially having any degree of polymerization (DP), beginning with DP = 1. Fucosylated oligosaccharides can also be obtained, for example, from Kyowa, Hakko, Kogyo in Japan, and DSM in the Netherlands, or Elicityl in France. Suitable techniques for generating LNFP-I are known in the art (see, for example, Hu et al., Carbohydr Polym, 2022, 297: 120017 and Derya et al., J Biotechnol., 2020, 318: 31-38). Alternatively, LNFP-I can be generated by chemical synthesis from lactose as the initial acceptor substrate for constructing the LNT backbone and free fucose as the final donor substrate, or by starting with LNTs generated, for example, through biotechnology or chemical synthesis and using fucose, but the methods of generation are hardly scalable.

[0012] These health benefits need to be delivered to these infants or young children in a way that does not cause side effects and / or is not only easy to deliver but also widely accepted by parents or healthcare professionals. Summary of the Invention

[0013] The inventors have surprisingly discovered that HMO blends can be enriched with lactose-N-fucopentose as a fucosylated human milk oligosaccharide, especially LNFP-I.

[0014] The inventors have surprisingly discovered that this combination of LNFP-I-rich HMO blend and Bifidobacterium infantis can be advantageously formulated for therapeutic purposes because the combination (mixture) exhibits remarkable antibacterial properties.

[0015] In other words, the inventors have discovered that these specific combinations are particularly effective in increasing the growth and activity of *Bifidobacterium infantis* in the gastrointestinal tract, especially in infants, toddlers, and / or children, and in inhibiting the growth of intestinal pathogens. Therefore, the present invention provides a pathway to increase the growth and activity of probiotics (i.e., *Bifidobacterium infantis*) with known health benefits. The effects of combinations of *Bifidobacterium infantis* with LNFP-I and optionally other HMOs in the gastrointestinal tract have not been previously reported.

[0016] Therefore, in a first aspect, the present invention provides a combination comprising at least one fucosylated human milk oligosaccharide and at least one Bifidobacterium longum, the combination being used to prevent and / or inhibit the growth of at least one intestinal pathogen in a subject, wherein the at least one fucosylated human milk oligosaccharide comprises lactose-N-fucopentose-I (LNFP-I) as lactose-N-fucopentose, wherein LNFP-I is the most abundant HMO in the combination, and wherein the at least one Bifidobacterium longum is Bifidobacterium longum infantis (Bifidobacterium infantis).

[0017] In a second aspect, the present invention provides a nutritional composition comprising at least one fucosylated human milk oligosaccharide and at least one Bifidobacterium longum, the composition being used to prevent and / or inhibit the growth of at least one intestinal pathogen in a subject, wherein the at least one fucosylated human milk oligosaccharide comprises lactose-N-fucopentose-I (LNFP-I) as lactose-N-fucopentose, wherein LNFP-I is the most abundant HMO in the composition, and wherein the at least one Bifidobacterium longum is Bifidobacterium longum infantis (Bifidobacterium infantis).

[0018] In some embodiments, the nutritional composition contains other HMOs besides those provided in the composition.

[0019] In some embodiments, the nutritional composition does not contain any other HMOs besides the HMOs provided in the composition.

[0020] In some embodiments, the nutritional composition contains Bifidobacterium infantis in an amount of 1 × 10⁻⁶. 3 CFU / g up to 1.5×10 12 CFU / g composition (dry weight).

[0021] In some preferred embodiments, the Bifidobacterium infantis strain is NCC 341 (ATCC 15697) and / or LMG11588 (ATCC 17930).

[0022] The fucosylated HMO consumption patterns exhibited by Bifidobacterium infantis strains NCC 341 (ATCC 15697) and / or LMG 11588 (ATCC 17930) showed that both strains consumed fucosylated HMOs, with LMG 11588 consuming them to a greater extent (Duboux S, Ngom-Bru C, De Bruyn F, Bogicevic B. Phylogenetic, Functional and Safety Features of 1950s B. infantis Strains. Microorganisms. Jan 18, 2022; 10(2):203. doi: 10.3390 / microorganisms10020203.PMID: 35208658; PMCID:PMC8879182).

[0023] In some implementations, the *Bifidobacterium infantis* strain has at least 99% average nucleotide identity (ANI) with *Bifidobacterium longum* subsp. infantis ATCC 15697.

[0024] In some implementations, the *Bifidobacterium infantis* strain has at least 99% average nucleotide identity (ANI) with *Bifidobacterium longum* subsp. infantis ATCC 17930.

[0025] In some embodiments, the total amount of HMO in the nutrient composition ranges from 0.02 g / 100 g to 5 g / 100 g dry weight of the nutrient composition.

[0026] In some embodiments, the nutritional composition is infant formula, stage 1 infant formula, stage 2 or later infant formula, growing milk, infant food, infant cereal composition, (milk) fortifier or supplement.

[0027] In another aspect, the present invention provides a nutritional composition of the present invention for use as a medicine.

[0028] In another aspect, the present invention provides combinations or nutritional compositions of the present invention for enhancing immune responses to bacterial infections such as EPEC and / or Salmonella, for promoting and / or maintaining intestinal health, or for preventing and / or reducing the risk of infection in infants, toddlers or children.

[0029] In another aspect, the present invention provides the use of combinations or nutritional compositions according to the invention for enhancing the growth of Bifidobacterium infantis in the gastrointestinal tract or for increasing the levels of at least one or a series of beneficial metabolites.

[0030] In another aspect, the present invention provides a method for promoting the immunity of a subject in need, the method comprising administering to the subject a combination or nutritional composition comprising Bifidobacterium infantis and LNFP-I.

[0031] In another aspect, the present invention provides a combination comprising Bifidobacterium infantis and LNFP-I or a combination of Bifidobacterium infantis and LNFP-I, wherein LNFP-I is the most abundant HMO in the combination, and in other aspects, the present invention provides uses and methods of using the combination.

[0032] In another aspect, the present invention provides a nutritional composition comprising Bifidobacterium infantis and LNFP-I, wherein LNFP-I is the most abundant HMO in the nutritional composition, and in other aspects, the present invention provides uses and methods of using the nutritional composition.

[0033] In another aspect, the present invention provides a nutritional composition comprising Bifidobacterium infantis LMG 11588 (ATCC 17930) and LNFP-I or composed thereof, wherein LNFP-I is the most abundant HMO in the nutritional composition, and in other aspects, the present invention provides uses and methods of using the nutritional composition.

[0034] In another aspect, the present invention provides a combination of Bifidobacterium infantis strain LMG 11588 (ATCC 17930) and LNFP-I or a combination thereof, wherein LNFP-I is the most abundant HMO in the combination, and in other aspects, the present invention provides uses and methods of using the combination.

[0035] In another aspect, the present invention provides a nutritional composition comprising Bifidobacterium infantis strain LMG 11588 (ATCC 17930) and LNFP-I, wherein LNFP-I is the most abundant HMO in the nutritional composition, and in other aspects, the present invention provides uses and methods of using the nutritional composition.

[0036] In another aspect, the present invention provides a nutritional composition comprising Bifidobacterium infantis strain LMG 11588 (ATCC 17930) and LNFP-I or composed thereof, wherein LNFP-I is the most abundant HMO in the nutritional composition, and in other aspects, the present invention provides uses and methods of using the nutritional composition. Attached Figure Description

[0037] Further features and advantages of the invention are described below in the description of the presently preferred embodiments given with reference to the accompanying drawings, and these features and advantages will be apparent from the description, wherein:

[0038] Figure 1 The image shows the HPAEC chromatographic representation of the HMO blend from Example 1, which is mainly composed of LNFP-I, LNFP-II, LNFP-III, LNDFH-I, and LNT.

[0039] Figure 2 The growth curves of Salmonella typhymurium in the presence of the different used supernatants shown are reported.

[0040] Figure 3 Plate counts of Salmonella typhimurium after incubation in the presence of the different used supernatants shown are reported.

[0041] Figure 4 Plate counts of enteropathogenic Escherichia coli (EPEC) after incubation in the presence of the different used supernatants shown are reported.

[0042] exist Figures 2-4 In this context, "HMO" refers to the HMO blend of Example 1, and "BInf" refers to Bifidobacterium infantis.

[0043] Figure 5 Compared to other HMOs, LNFP-I and *Bifidobacterium longum* subsp. *infantii* LMG 11588 (NCC3039) showed biomass growth. LNFP-I exhibited a unique diauxic shift. Experiments were performed in triplicate, and average values ​​were plotted. Detailed Implementation

[0044] definition

[0045] In the context of this invention, the following terms have the following meanings.

[0046] The term "infant" refers to a child under 12 months of age. The term "toddler" refers to a child between one and three years of age, also known as a toddler. The term "child" refers to a child between three and nine years of age. Preferably, the term "child" refers to a child between three and five years of age.

[0047] The term "nutritional composition" refers to a composition that supplies nutrients to a subject. Such nutritional compositions are typically administered orally or parenterally and generally include a lipid or fat source and a protein source. A carbohydrate source may also be included. In one embodiment, the nutritional composition of the present invention is a synthetic nutritional composition.

[0048] In one specific embodiment, the combination or composition of the present invention is a "synthetic combination" or "synthetic nutritional composition". The expression "synthetic combination or synthetic nutritional composition" refers to a mixture obtained by chemical and / or biological methods, the chemical properties of which may be the same as those of mixtures naturally present in mammalian milk (i.e., the synthetic combination or synthetic composition is not breast milk).

[0049] As used herein, the term "infant formula" refers to a food intended specifically for the nutrition of infants during the first few months of life, and which meets the diverse nutritional needs of such infants (in accordance with Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 concerning infant formula and follow-up formula). It also refers to nutritional compositions intended for use in infants, as defined in the Codex Alimentarius Commission (STAN 72-1981) and in infant specialties (including foods for specific medical purposes). The term "infant formula" encompasses both "Stage 1 infant formula," "Stage 2 infant formula," and "follow-up formula."

[0050] Stage 2 infant formula or follow-up formula is introduced starting from the 6th month. Infant formula constitutes the main liquid component of this group's gradually diversifying diet.

[0051] The term "infant food" refers to food designed specifically to provide nutrition for infants or young children during the first year of their lives.

[0052] The term "infant cereal composition" refers to food designed specifically for supplying nutrition to infants or young children during the first year of life.

[0053] The term "GUM" refers to a milk-based beverage that is typically fortified with vitamins and minerals and is intended for use by toddlers or children.

[0054] The term "fortifier" refers to a liquid or solid nutritional composition suitable for fortifying human milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or mixed with them. Therefore, the fortifier of the present invention can be applied after being dissolved in human breast milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or it can be applied as a stand-alone composition. When applied as a stand-alone composition, the milk fortifier of the present invention can also be identified as a "supplement." In one embodiment, the milk fortifier of the present invention is a supplement.

[0055] The term "weaning period" refers to the period during which breast milk is gradually replaced by other foods in the diet of infants or young children.

[0056] The expressions "age in days / weeks / months / age" and "number of days / weeks / months / years after birth" can be used interchangeably.

[0057] "Breast milk" should be understood as the mother's milk or colostrum.

[0058] "Oligosaccharides" are carbohydrate polymers containing small amounts (usually three to ten parts) of common sugars (monosaccharides).

[0059] The term "nutritional composition" refers to a composition that supplies nutrients to a subject. Such nutritional compositions are typically taken orally and generally contain a carbohydrate source, a lipid source or fat source, and a protein source. In one embodiment, the nutritional composition of the present invention is a synthetic nutritional composition.

[0060] In one specific embodiment, the nutritional composition of the present invention is a "synthetic nutritional composition". The term "synthetic nutritional composition" refers to a mixture obtained by chemical and / or biological means (i.e., the synthetic composition is not breast milk).

[0061] As used herein, the term "infant formula" refers to a food intended specifically for the nutrition of infants during the first few months of life, and which meets the diverse nutritional needs of such infants (in accordance with Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 concerning infant formula and follow-up formula). It also refers to nutritional compositions intended for use in infants, as defined in the Codex Alimentarius Commission (STAN 72-1981) and in infant specialties (including foods for specific medical purposes). The term "infant formula" encompasses both "Stage 1 infant formula," "Stage 2 infant formula," and "follow-up formula."

[0062] Stage 2 infant formula or follow-up formula is introduced starting from the 6th month. Infant formula constitutes the main liquid component of this group's gradually diversifying diet.

[0063] The term "infant food" refers to food designed specifically to provide nutrition for infants or young children during the first year of their lives.

[0064] The term "infant cereal composition" refers to food designed specifically for supplying nutrition to infants or young children during the first year of life.

[0065] The term "GUM" refers to a milk-based beverage that is typically fortified with vitamins and minerals and is intended for use by toddlers or children.

[0066] The term "fortifier" refers to a liquid or solid nutritional composition suitable for fortifying human milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or mixed with them. Therefore, the fortifier of the present invention can be applied after being dissolved in human breast milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or it can be applied as a stand-alone composition. When applied as a stand-alone composition, the milk fortifier of the present invention can also be identified as a "supplement." In one embodiment, the milk fortifier of the present invention is a supplement.

[0067] The term "weaning period" refers to the period during which breast milk is gradually replaced by other foods in the diet of infants or young children.

[0068] The term "HMO" refers to human milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, suggesting that their important functions may not be directly related to their calorific value. It has been specifically noted in the art that these carbohydrates play a crucial role in early infant and toddler development, such as the maturation of the immune system. Many different types of HMOs have been found in human milk. Each individual oligosaccharide is based on a variety of combinations of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine with these molecules, resulting in a large number of diverse oligosaccharides in human milk; more than 130 such structures have been identified to date. Almost all oligosaccharides have a lactose molecule at the reducing end, and the non-reducing end is occupied by sialic acid and / or fucose (if present). HMOs can be acidic (e.g., oligosaccharides containing charged sialic acid) or neutral (e.g., fucoidylated oligosaccharides). "Fucoidylated oligosaccharides" are oligosaccharides containing fucose residues. This oligosaccharide is neutral. Some examples are 2-FL (2'-fucosyllactose), 3-FL (3-fucosyllactose), difucosyllactose, lactose-N-fucopentose (e.g., lactose-N-fucopentose-I, lactose-N-fucopentose-II, lactose-N-fucopentose-III, lactose-N-fucopentose-V), lactose-N-fucohexasose, lactose-N-difucohexasose-I (LNDFH-I), fucosyllactose-N-hexasose, fucosyllactose-N-neohexose, difucosyllactose-N-hexasose-I, difucosyllactose-N-neohexose-II, and any combination thereof.

[0069] The terms “fucosylated oligosaccharides containing α-linked fucosylation epitopes” and “2-fucosylated oligosaccharides” encompass fucosylated oligosaccharides with certain homologous forms. These homologous fucosylated oligosaccharides all contain α-1,2-linked fucosylation epitopes, thus suggesting that they have certain homologous functions.

[0070] The term "N-acetylated oligosaccharide" encompasses both "N-acetyllactosamine" and "oligosaccharides containing N-acetyllactosamine." Such oligosaccharides are neutral oligosaccharides containing N-acetyllactosamine residues. Suitable examples are LNT (lactose-N-tetrasaccharide), para-lactose-N-neohexose (para-LNnH), LNnT (lactose-N-neohexose), and any combination thereof. Other examples include lactose-N-hexose, lactose-N-neohexose, para-lactose-N-hexose, para-lactose-N-neohexose, lactose-N-octasose, lactose-N-neohexose, isolose-N-octasose, para-lactose-N-octasose, and lactose-N-decanose.

[0071] The nutritional compositions of the present invention may be in solid form (e.g., powder) or in liquid form. The amounts of various ingredients (e.g., oligosaccharides) may be expressed in g / 100g composition on a dry weight basis when the composition is in solid form (e.g., powder), or in g / L composition concentration when the composition refers to liquid form (the latter also covers liquid compositions that can be obtained by reconstituted powder in a liquid (such as milk, water, etc.), such as reconstituted infant formula or stage 2 infant formula or growing milk or infant cereal products or any other formulation designed for infant nutrition).

[0072] The term "galacto-oligosaccharides" refers to a class of non-digestible fibers with prebiotic activity. GOS are formed through the enzymatic conversion of lactose. GOS typically consists of chains of galactose units produced by a series of transgalactosylation reactions, with terminal glucose units, although these terminal galactose units may be present alternatively. The degree of polymerization of GOS typically ranges from 2 to 8 monomer units.

[0073] The nutritional compositions of the present invention may be in solid form (e.g., powder) or liquid form. The amounts of various ingredients (e.g., oligosaccharides) may be expressed in g / 100g composition based on dry weight when the composition is in solid form (e.g., powder), or in g / L composition concentration when the composition refers to a liquid form (the latter also covers liquid compositions that can be obtained by reconstituted powder in a liquid (such as milk, water, etc.), such as reconstituted infant formula or stage 2 infant formula or infant cereal products or any other formulation designed to provide nutrition for infants). They may also be expressed in g / 100kcal.

[0074] The term "prebiotic" refers to non-digestible carbohydrates that exert a beneficial effect on the host by selectively stimulating the growth and / or activity of healthy bacteria (such as Bifidobacteria in the human colon) (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995; 125:1401-12).

[0075] The term "probiotics" refers to microbial cell preparations or microbial cell components that have beneficial effects on the health or well-being of a host. (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999: 10 107-10). The microbial cells are generally bacteria or yeast.

[0076] Bifidobacterium longum subsp. infantis (Bifidobacterium infantis) is a Gram-positive, heterofermentative, anaerobic bacterium with the distinctive bifid (i.e., “Y”) shape of Bifidobacteria, a genus of Bifidobacteria found in the human gastrointestinal tract (Nutrients. 2020 June; 12(6): 1581). Bifidobacterium infantis is marketed by Procter & GambIe Co. under the trademark Bifantis. Bifidobacterium infantis can be a type strain (ATCC 15697) or a Bi-26 strain, clustered with LMG 11588 (ATCC 17930).

[0077] Bifidobacterium longum is a bacterium of the genus Bifidobacterium that exists in the human gastrointestinal tract. In 2002, three previously distinct species of Bifidobacterium (Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium suis) were merged with biotype Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium suis into a single species named Bifidobacterium longum (Sakata, S. et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp. 1945-1951).

[0078] Any suitable strain of *Bifidobacterium longum* subsp. infantis can be used in this invention. Such strains are well known to those skilled in the art. Suitable strains include *Bifidobacterium longum* subsp. infantis LMG 11588 (also known as *Bifidobacterium longum* subsp. infantis NCC3039 or *Bifidobacterium longum* subsp. infantis ATCC 17930) and *Bifidobacterium longum* subsp. infantis ATCC 15697 (also known as *Bifidobacterium longum* subsp. infantis NCC 3078), Rosell-33 (sold by Lallemand), and m-63 (sold by Morinaga).

[0079] Bifidobacterium longum subsp. infantis can be a strain that has at least 99% (suitably, at least 99.9%) of the ANI of Bifidobacterium longum subsp. infantis known to the technician.

[0080] Suitably, *Bifidobacterium longum* subsp. *infantitidis* and *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 (also known as *Bifidobacterium longum* subsp. *infantitidis* NCC3039 or *Bifidobacterium longum* subsp. *infantitidis* ATCC 17930) have an ANI of at least 99% (suitably, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%). Preferably, *Bifidobacterium longum* subsp. *infantitidis* and *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 have an ANI of at least 99.9%.

[0081] Bifidobacterium longum subsp. infantis LMG 11588 is publicly available and can be obtained by the Belgian Microbial Collection Center (BCCM) with LMG accession number LMG 11588.

[0082] Bifidobacterium longum infantis subsp. 15697 is publicly available and can be certified by ATCC as ATCC. ® 15697 ™ get.

[0083] The term "cfu" should be understood as colony-forming unit.

[0084] "Treatment" refers to addressing a medical condition or disease with the aim of improving or stabilizing the outcome of treatment for an individual or resolving underlying nutritional needs. Therefore, treatment includes dietary or nutritional management of a medical condition or disease by addressing the nutritional needs of an individual undergoing treatment. Treatment includes the elimination, reduction, or improvement of symptoms associated with the medical condition or disease.

[0085] "Prevention" refers to reducing the risk of developing or recurring medical symptoms or diseases. Therefore, primary prevention and secondary prevention are envisioned.

[0086] Primary prevention refers to preventing a medical condition or disease before its onset, while secondary prevention refers to preventing the recurrence of a medical condition or disease after its initial onset. Therefore, prevention includes dietary or nutritional interventions for a medical condition or disease by addressing the nutritional needs of the individual being treated. Prevention involves eliminating or minimizing the risk of developing a medical condition or disease and reducing the risk of developing symptoms associated with that condition or disease.

[0087] The terms “pathogen” and “pathogenicity” in relation to microorganisms include any such microorganism capable of causing or affecting a disease, symptom, or illness in a host containing that microorganism. Examples of pathogens are intestinal pathogens, such as Salmonella and enteropathogenic Escherichia coli (EPEC).

[0088] The term "effective amount" preferably refers to an amount of combination or composition that provides a sufficient quantity of active agent to produce the desired therapeutic or preventive outcome in a subject. The effective amount may be administered to a subject in one or more doses to achieve the desired therapeutic or preventive outcome.

[0089] The term "SCFA" refers to short-chain fatty acids.

[0090] The statement "increased SCFA production" means that individuals fed the nutritional composition according to the invention have higher levels of systemic and / or colonic SCFAs compared to those fed the standard composition. SCFA production can be measured using techniques known to those skilled in the art, such as gas-liquid chromatography.

[0091] The statement "LNFP-I is the most abundant HMO in this combination or nutritional composition" means that LNFP-I is present in an amount higher than any other HMO present in the combination or nutritional composition.

[0092] “Average nucleotide identity (ANI)” is a measure of nucleotide-level genomic similarity between coding regions of two genomes. Average nucleotide identity can be assessed as described here: Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. Oct 2017; 110(10):1281-1286. In an embodiment of the invention, the strain *Bifidobacterium longum* subsp. infant LMG 11588 (also known as ATCC 17930) represents a reference genome to be compared with the microbial genome. Examples of microbial genomes having at least 99.9% ANI with *Bifidobacterium longum* subsp. infant LMG 11588 can be found in PATRIC (https: / / www.patricbrc.org), genome ID 1678.111. In one embodiment of the invention, the *Bifidobacterium longum* subsp. infant strain does not possess potentially transferable antibiotic resistance.

[0093] The ANI (Average Nucleus Indices) of shared genes between two strains is a robust means of comparing the genetic affinity between strains. An ANI value of at least approximately 96% indicates that the strains belong to the same species (Konstantinidis and Tiedje, 2005, Proc Natl Acad Sci USA, 102(7):2567-72; and Goris et al., 2007, Int Syst Evol Microbiol.57(Pt1):81-91), while an ANI value of at least approximately 99% indicates that the bacterial genome belongs to the same strain. The ANI between two bacterial genomes is calculated by pairwise comparisons of all sequences common to any two strains and can be determined, for example, using any of the many publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al., 2017, Antonie van Leeuwenhoek 110:1281-1286); ANI calculator, JSpecies (Richter and Rossello-Mora, 2009, Proc Natl Acad Sci USA 106:19126-19131); and JSpeciesWS (Richter et al., 2016, Bioinformatics 32:929-931). Other methods for determining the ANI of two genomes are known in the art (Konstantinidis, KT and Tiedje, 2005, JM, Proc. Natl. Acad. Sci. USA, 102: 2567-2572; and Varghese et al., 2015, Nucleic Acids Research, 43(14):6761-6771).

[0094] Unless otherwise specified, all percentages are by weight.

[0095] Furthermore, in the context of this invention, the terms "comprising" or "including" do not exclude other possible elements. The compositions of this invention (including the various embodiments described herein) may comprise, consist of, or be substantially composed of the following elements: the essential elements and necessary limitations of the invention as described herein, and any additional or optional ingredients, components, or limitations as described herein or as required.

[0096] According to the present invention, any embodiment, including any preferred, more preferred, most preferred, even more preferred, or exemplary (e.g., having a description such as, or for example) embodiment, can be combined with each other. For example, a preferred embodiment of one feature can be combined with a more preferred embodiment of another feature.

[0097] It should be understood that various changes and modifications to the currently preferred embodiments described herein will be apparent to those skilled in the art. These changes and modifications can be made without departing from the spirit and scope of the invention and without diminishing the accompanying advantages. Therefore, such changes and modifications are intended to be covered by the appended claims.

[0098] Therefore, the first object of the present invention comprises a combination of at least one fucosylated human milk oligosaccharide (HMO) and at least one Bifidobacterium longum, the combination being used to treat, prevent and / or inhibit the growth of at least one intestinal pathogen in a subject, wherein the at least one fucosylated human milk oligosaccharide comprises lactose-N-fucopentose-I (LNFP-I) as lactose-N-fucopentose, wherein LNFP-I is the most abundant HMO in the nutritional composition, and wherein the at least one Bifidobacterium longum is Bifidobacterium longum infantis (Bifidobacterium infantis).

[0099] In some embodiments, the degree of polymerization (DP) of the human milk oligosaccharides in the composition is at least 4, i.e., at least a tetrasaccharide. In some embodiments, each human milk oligosaccharide with a degree of polymerization (DP) less than 4 accounts for less than 2% by weight of the total amount of human milk oligosaccharides in the composition.

[0100] In some embodiments of this combination, lactose-N-fucopentose I (LNFP-I) accounts for at least 5% by weight, preferably at least 20% by weight, more preferably 20% to 100% by weight, more preferably 25% to 95% by weight, such as 50% to 95% by weight, based on dry weight.

[0101] In some embodiments, the combination also includes at least one other lactose-N-fucopentose, preferably lactose-N-fucopentose II and / or lactose-N-fucopentose III as fucosylated human milk oligosaccharides, preferably both.

[0102] In some embodiments, the combination also includes at least one fucoidan as a fucoidylated human milk oligosaccharide.

[0103] In some embodiments, based on dry weight, the total amount of lactose-N-fucopentose accounts for at least 5% by weight of the total amount of human milk oligosaccharides in the combination, preferably at least 20% by weight, preferably 15% to 100% by weight, and more preferably 30% to 95% by weight.

[0104] In some embodiments, the at least one fucoidylated human milk oligosaccharide accounts for at least 5% by weight, preferably at least 10% by weight, more preferably 5% to 90% by weight, more preferably 10% to 80% by weight, such as 10% to 20% by weight, based on dry weight of the total amount of human milk oligosaccharides in the composition.

[0105] In some embodiments of this combination, the at least one fucosylated human milk oligosaccharide contains at least lactose-N-fucohexasaccharide-I (LNDFH-I) as a fucohexasaccharide.

[0106] In some implementations, the at least one enteric pathogen is Salmonella typhimurium.

[0107] In some implementations, the at least one intestinal pathogen is enteropathogenic Escherichia coli (EPEC).

[0108] In some embodiments, the at least one subspecies of Bifidobacterium longum (Bifidobacterium infantis) is strain LMG11588, ATCC 15697 (NCC 341), or a mixture thereof.

[0109] In some embodiments, the combination also includes at least one N-acetylated human milk oligosaccharide, preferably lactose-N-tetrasaccharide (LNT), which is preferably 0.1% to 30% by weight, more preferably 10% to 20% by weight, of the total amount of human milk oligosaccharides in the combination on a dry weight basis.

[0110] In some embodiments, the combination or nutritional composition further comprises at least one N-acetylated human milk oligosaccharide, preferably lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), or a mixture thereof, preferably 1% to 50% by weight, more preferably 5% to 80% by weight, and even more preferably 10% to 60% by weight, based on dry weight. In some embodiments, the nutritional composition is infant formula, stage 1 infant formula, follow-up or stage 2 infant formula, growing milk, baby food, infant cereal composition, (milk) fortifier, or supplement.

[0111] In some embodiments, the combination or nutritional composition does not contain any N-acetylated human milk oligosaccharides, such as lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), or mixtures thereof.

[0112] In some embodiments, the combination or nutritional composition does not contain lactose-N-tetrasaccharide (LNT).

[0113] In some embodiments, the combination or nutritional composition does not contain lactose-N-neotetrasaccharide (LNnT).

[0114] In some embodiments of the nutritional composition, the subjects are infants, toddlers, or children.

[0115] In some embodiments, LNFP-I is present in a total amount of 20 mg / L to 5000 mg / L of the nutritional composition according to the invention after reconstitution in water, or in a total amount of 0.02 g / 100 g to 4 g / 100 g of the nutritional composition or combination according to the invention. Suitably, LNFP-I is present in a total amount of 50 mg / L to 2500 mg / L, for example 60 mg / L to 2000 mg / L, for example 80 mg / L to 1000 mg / L of the nutritional composition or combination according to the invention. Suitably, LNFP-I is present in a total amount of 0.04 g / 100 g to 2 g / 100 g, for example 0.05 g / 100 g to 1.6 g / 100 g, for example 0.07 g / 100 g to 0.8 g / 100 g of the nutritional composition or combination (dry weight).

[0116] In some implementations, LNDFH-I is the next most abundant HMO in the combination after LNFP-I. Therefore, in some implementations, LNDFH-I and LNFP-I are the two most abundant HMOs in the combination.

[0117] In some implementations, LNDFH-I is the next most abundant HMO in this combination after LNFP-I and LNFP-III.

[0118] In some implementations, LNDFH-I is the next most abundant HMO in the HMO blend of this combination, following LNFP-I, LNFP-II, and LNFP-III.

[0119] In some implementations, LNDFH-I is the next most abundant HMO in the HMO blend of this combination, following LNFP-I, LNFP-II, LNT, and LNFP-III.

[0120] In some embodiments, the HMO blend of the combination contains LNFP-I in an amount of 15% to 100% by weight of the total HMO in the blend, preferably 20% to 95% by weight, more preferably 25% to 90% by weight, such as 25% to 30% by weight, such as 27.5% by weight.

[0121] In some embodiments, the HMO blend of the combination contains LNFP-II in an amount of 5% to 50% by weight of the total HMO in the blend, preferably 10% to 35% by weight, more preferably 10% to 20% by weight, such as 10% to 15% by weight, such as 13% by weight.

[0122] In some embodiments, the HMO blend of the combination contains LNFP-III in an amount of 5% to 50% by weight of the total HMO in the blend, preferably 10% to 35% by weight, more preferably 10% to 20% by weight, such as 12% to 18% by weight, such as 16.3% by weight.

[0123] In some embodiments, the HMO blend of the combination contains LNDFH-I in an amount of 5% to 80% by weight of the total HMO in the blend, preferably 10% to 35% by weight, more preferably 10% to 20% by weight, such as 10% to 15% by weight, such as 13.2% by weight.

[0124] In some embodiments, the HMO blend of the combination contains LNT in an amount of 5% to 50% by weight of the total HMO in the blend, preferably 10% to 35% by weight, more preferably 10% to 20% by weight, such as 12% to 16% by weight, such as 14.1% by weight.

[0125] In some embodiments, the HMO blend comprises LNT, LNFP-I, LNFP-II, LNFP-III and LNDFH-I, and if any other HMO is present in the HMO blend, it is present in less than 5% by weight of the total HMO in the blend, preferably 3.5% by weight or less, more preferably 2.0% by weight or less, and even more preferably 1.0% by weight or less.

[0126] In some embodiments, the HMO blend of this combination consists of LNT, LNFP-I, LNFP-II, LNFP-III, and LNDFH-I.

[0127] In one embodiment, the HMO blend of the combination consists of LNFP-I, LNT, LNFP-II, LNFP-III and LNDFH-I.

[0128] In some implementations, the combined HMO consists of LNFP-I.

[0129] In some implementations, the combined HMO blend consists of LNFP-I and LNDFH-I.

[0130] In some implementations, the combined HMO blend consists of LNFP-I, LNFP-II, and LNDFH-I.

[0131] In some implementations, the combined HMO blend consists of LNFP-I, LNFP-III, and LNDFH-I.

[0132] In one embodiment, the HMO blend of the combination consists of LNFP-I, LNFP-II, LNFP-III and LNDFH-I.

[0133] In one embodiment, the combined HMO blend consists of LNFP-I and LNT.

[0134] In one embodiment, the combined HMO blend consists of LNFP-I, LNT, and LNDFH-I.

[0135] In one embodiment, the HMO blend of the combination consists of LNFP-I, LNT, LNFP-II and LNDFH-I.

[0136] In one embodiment, the HMO blend of the combination consists of LNFP-I, LNT, LNFP-III and LNDFH-I.

[0137] In one embodiment, 2'FL and 3 FL constitute less than 5% by weight of the total amount of human milk oligosaccharides in the combination on a dry basis, preferably 3.5% by weight or less, more preferably 2% by weight or less.

[0138] In one embodiment, 2'FL accounts for less than 3.5% by weight of the total amount of human milk oligosaccharides in the composition based on dry matter, preferably 2% by weight or less, and most preferably 1% by weight or less.

[0139] In one embodiment, 3FL accounts for less than 1.5% by weight of the total amount of human milk oligosaccharides in the composition on a dry basis, preferably 0.5% by weight or less, and most preferably up to 0.2% by weight.

[0140] In one embodiment, 2'FL and 3 FL, on a dry basis, constitute less than 5% by weight of the total amount of human milk oligosaccharides in the nutritional composition, preferably 3.5% by weight or less, more preferably 2% by weight or less.

[0141] In one embodiment, 2'FL, based on dry weight, represents less than 3.5% by weight of the total amount of human milk oligosaccharides in the nutritional composition, preferably 2% by weight or less, and most preferably 1% by weight or less.

[0142] In one embodiment, 3FL accounts for less than 1.5% by weight of the total amount of human milk oligosaccharides in the nutritional composition on a dry basis, preferably 0.5% by weight or less, and most preferably up to 0.2% by weight.

[0143] In some embodiments, human milk oligosaccharides are present in the nutritional composition according to the invention in a total amount of 0.5 g / L to 3 g / L (such as 0.8 g / L to 1.5 g / L). In some embodiments, the total amount of human milk oligosaccharides may be 0.85 g / L to 1.3 g / L, such as 0.9 g / L to 1.25 g / L, 0.9 g / L to 1.1 g / L, 1 g / L to 1.25 g / L, or 1 g / L to 1.2 g / L.

[0144] Fucosylated human milk oligosaccharides may be present in the nutritional composition according to the invention in a total amount of 0.5 g / L to 3 g / L (such as 0.8 g / L to 1.5 g / L). In some embodiments, the total amount of fucoidylated human milk oligosaccharides may be 0.85 g / L to 1.3 g / L, such as 0.9 g / L to 1.25 g / L, 0.9 g / L to 1.1 g / L, 1 g / L to 1.25 g / L, or 1 g / L to 1.2 g / L.

[0145] In another aspect, the present invention relates to a nutritional composition comprising an HMO blend as defined in any of the foregoing embodiments and Bifidobacterium infantis.

[0146] In another aspect, the present invention relates to a nutritional composition comprising an HMO blend as defined in any of the foregoing embodiments and Bifidobacterium infantis strain LMG 11588 (ATCC 17930).

[0147] In another aspect, the present invention relates to a blend of HMOs as defined in any of the foregoing embodiments and Bifidobacterium infantis or a combination thereof.

[0148] In another aspect, the present invention relates to a nutritional composition comprising an HMO blend as defined in any of the foregoing embodiments and Bifidobacterium infantis strain LMG 11588 (ATCC 17930) or thereof.

[0149] In another aspect, the present invention relates to a nutritional composition comprising a blend of HMOs as defined in any of the foregoing embodiments and Bifidobacterium infantis or composed thereof.

[0150] In another aspect, the present invention relates to a nutritional composition comprising, or consisting of, an HMO blend as defined in any of the foregoing embodiments and Bifidobacterium infantis strain LMG 11588 (ATCC 17930).

[0151] In some embodiments, the combination can be prepared by dry mixing, wet mixing, or a combination thereof of an HMO comprising LNFP-I and Bifidobacterium infantis, or of LNFP-I and Bifidobacterium infantis, using standard methods known in the art. A dry powder of Bifidobacterium infantis can be prepared by freeze-drying or spray-drying. The HMO can be purified / prepared by crystallization, spray drying, freeze-drying, or other drying equipment known in the art.

[0152] In some implementations, the infant is provided with a nutritional composition to protect the gut from opportunistic pathogens as the infant's adaptive immune system develops (i.e., to provide colonization resistance).

[0153] In some implementations, the intestinal pathogen is a bacterial infection or intestinal-dominant, including infections or intestinal-dominant infections caused by one or more of the following species, subspecies, or strains: Aeromonas, Bacillus, Blautia, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, and Chlamydia. lamydophila, Citrobacter, Clostridium, Coiynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterobacteriaceae, Enterococcus, Escherichia, Faecalicatena, Francisella Ancisella, Haemophilus, Helicobacter, Hungateha, Klebsiella, Lachnospiraceae, Legionella, Leptospira, Listeria, Morganella, Mycobacterium, Mycoplasma, Neisseria ( Neisseria, Orientia, Plesiomonas, Proteus, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia.Choose one or more of the following: Aeromonas hydrophila, Bacillus cereus, Campylobacter fetus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enterocolitogenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli, Escherichia coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella typhimurium, Salmonella paratyphi, Salmonella typhimurium. *Salmonella typhi*, *Salmonella enterica*, *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Vibrio vulnificus*, *Yersinia enterocolitis*, preferably *Salmonella typhimurium* and / or EPEC.

[0154] In some embodiments, the enteric pathogen is a bacterial infection or a gut-dominant bacterium and / or opportunistic pathogen, including infections caused by drug-resistant bacteria or gut-dominant bacteria. In some embodiments, the drug-resistant bacteria include one or more of the following: antibiotic-resistant bacteria (ARBs), antibiotic-resistant Proteobacteria, carbapenem-resistant Enterobacteriaceae (CREs), extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E), fluoroquinolone-resistant Enterobacteriaceae, extended-spectrum β-lactam-resistant Enterococci (ESBLs), vancomycin-resistant Enterococci (VREs), multidrug-resistant Escherichia coli, or multidrug-resistant Klebsiella pneumoniae.

[0155] In another aspect, the present invention provides a nutritional composition or combination comprising at least one fucosylated human milk oligosaccharide and at least one Bifidobacterium longum for non-therapeutic use in treating, preventing, and / or inhibiting the growth of at least one intestinal pathogen in a subject, wherein the at least one fucosylated human milk oligosaccharide comprises lactose-N-fucopentose I (LNFP-I) as lactose-N-fucopentose, wherein LNFP-I is the most abundant HMO in the nutritional composition or combination, wherein the at least one Bifidobacterium longum is Bifidobacterium longum infantis (Bifidobacterium infantis), preferably LMG 11588, ATCC 15697 (NCC 341) or a mixture thereof, wherein the pathogen is preferably enteropathogenic Escherichia coli (EPEC) and / or Salmonella typhimurium, wherein the nutritional composition is preferably infant formula, stage 1 infant formula, stage 2 or later infant formula, growing milk, infant food, infant cereal composition, (milk) fortifier or supplement, and wherein the subject is preferably an infant, toddler, or child. All embodiments of this nutritional composition or combination are suitable for non-therapeutic use.

[0156] In another aspect, the present invention provides a method for promoting the immunity of a subject in need, the method comprising administering to the subject a combination or nutritional composition according to the present invention.

[0157] In some embodiments, the nutritional composition contains Bifidobacterium infantis in an amount of 1 × 10⁻⁶. 3 CFU / g up to 1.5×10 12 CFU / g composition (dry weight).

[0158] The various health effects of Bifidobacterium infantis as a probiotic have been well documented (see the references cited above).

[0159] Based on dry weight, the nutritional composition according to the present invention may contain 10 per gram of composition. 3 cfu to 10 12 CFU of Bifidobacterium infantis, preferably between 10 7 cfu to 10 12 CFU, such as between 10 8 cfu to 10 10 Bifidobacterium infantis between CFU. Suitable, Bifidobacterium infantis in a quantity of at least about 10 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 The subjects were administered CFU / day. Suitable, Bifidobacterium infantis was given at approximately 10... 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10The subject was given CFU / day or less.

[0160] In one embodiment, *Bifidobacterium infantis* is live. In another embodiment, *Bifidobacterium infantis* is non-replicating or inactivated. In some other embodiments, *Bifidobacterium infantis* can be both live and inactivated.

[0161] The invention will now be described in more detail. It should be noted that the various aspects, features, embodiments, and implementations described in this application are compatible and / or can be combined together.

[0162] The inventors have surprisingly discovered that HMO blends can be enriched with lactose-N-fucopentose, especially LNFP-I.

[0163] The inventors have surprisingly discovered that this combination of LNFP-I-rich HMO blend and Bifidobacterium infantis forms a combination that can be advantageously used for treatment because the combination exhibits remarkable antibacterial properties.

[0164] In some embodiments of this combination, the weight ratio of LNFP-I:LNDFH-I is 90:10 to 30:70 dry weight, preferably 80:20 to 50:20 dry weight, and most preferably 70:30 to 60:40 dry weight. Suitably, the weight ratio of LNFP-I:LNDFH-I is 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, or 30:70 dry weight.

[0165] In some embodiments of this combination, the total LNFP:LNDFH-I weight ratio is 95:5 to 5:95 dry weight, preferably 85:15 to 50:50 dry weight, and most preferably 80:20 to 60:40 dry weight. Suitably, the total LNFP:LNDFH-I weight ratio is 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, or 50:50 dry weight.

[0166] In some embodiments of the combination or the nutritional composition, LNFP-I is present in amounts ranging from 0.02g to 3.8g / 100g, or 0.05g to 3.8g / 100g, or 0.1g to 3.8g / 100g, or 0.1g to 3g / 100g, or 0.1g to 2.5g / 100g, or 0.1g to 2g / 100g, or 0.1g to 1.5g / 100g, or 0.1g to 1g / 100g, or 0.02g to 3g / 100g, or 0.02g to 2.5g / 100g, or 0.02g to 2g / 100g. g / 100g or 0.02g to 1.5g / 100g or 0.02g to 1g / 100g or 0.05g to 3g / 100g or 0.05g to 2.5g / 100g or 0.05g to 2g / 100g or 0.05g to 1.5g / 100g or 0.05g to 1g / 100g or 0.15g to 0.85g / 100g or 0.20g to 0.80g / 100g or 0.30g to 0.70g / 100g or 0.45g to 0.55g / 100g dry weight of combination or nutritional composition.Suitablely, LNFP-I is present in the following amounts: 0.15g / 100g, 0.16g / 100g, 0.17g / 100g, 0.18g / 100g, 0.19g / 100g, 0.20g / 100g, 0.21g / 100g, 0.22g / 100g, 0.23g / 100g, 0.24g / 100g, 0.25g / 100g, 0.26g / 100g, 0.27g / 100g, 0.28g / 100g, 0.29g / 100g, 0.30g / 100g, 0.31g / 100g. g, 0.32g / 100g, 0.33g / 100g, 0.34g / 100g, 0.35g / 100g, 0.36g / 100g, 0.37g / 100g, 0.38g / 100g, 0.39g / 100g, 0.40g / 100g, 0.41g / 100g, 0.42g / 100g, 0.43g / 100g, 0.44g / 100g, 0.45g / 100g, 0.46g / 100g, 0.47g / 100g, 0.48g / 100g, 0.49g / 100g, 0. 50g / 100g, 0.51g / 100g, 0.52g / 100g, 0.53g / 100g, 0.54g / 100g, 0.55g / 100g, 0.56g / 100g, 0.57g / 100g, 0.58g / 100g, 0.59 g / 100g, 0.60g / 100g, 0.61g / 100g, 0.62g / 100g, 0.63g / 100g, 0.64g / 100g, 0.65g / 100g, 0.66g / 100g, 0.67g / 100g, 0.68g / Dry weight of 100g, 0.69g / 100g, 0.70g / 100g, 0.71g / 100g, 0.72g / 100g, 0.73g / 100g, 0.74g / 100g, 0.75g / 100g, 0.76g / 100g, 0.77g / 100g, 0.78g / 100g, 0.70g / 100g, 0.80g / 100g, 0.81g / 100g, 0.82g / 100g, 0.83g / 100g, 0.84g / 100g, or 0.85g / 100g combinations or nutritional compositions.

[0167] In some embodiments of the combination or the nutritional composition, the total amount of LNFP is present in the following ranges: 0.02g to 3.8g / 100g, or 0.05g to 3.8g / 100g, or 0.1g to 3.8g / 100g, or 0.1g to 3g / 100g, or 0.1g to 2.5g / 100g, or 0.1g to 2g / 100g, or 0.1g to 1.5g / 100g, or 0.1g to 1g / 100g, or 0.02g to 3g / 100g, or 0.02g to 2.5g / 100g, or 0.02g to... 2g / 100g or 0.02g to 1.5g / 100g or 0.02g to 1g / 100g or 0.05g to 3g / 100g or 0.05g to 2.5g / 100g or 0.05g to 2g / 100g or 0.05g to 1.5g / 100g or 0.05g to 1g / 100g or 0.15g to 0.85g / 100g or 0.20g to 0.80g / 100g or 0.30g to 0.70g / 100g or 0.45g to 0.55g / 100g dry weight of combination or nutritional composition.Suitablely, the total amount of LNFP is present in the following amounts: 0.15g / 100g, 0.16g / 100g, 0.17g / 100g, 0.18g / 100g, 0.19g / 100g, 0.20g / 100g, 0.21g / 100g, 0.22g / 100g, 0.23g / 100g, 0.24g / 100g, 0.25g / 100g, 0.26g / 100g, 0.27g / 100g, 0.28g / 100g, 0.29g / 100g, 0.30g / 100g, 0.31g / 100g. 0g, 0.32g / 100g, 0.33g / 100g, 0.34g / 100g, 0.35g / 100g, 0.36g / 100g, 0.37g / 100g, 0.38g / 100g, 0.39g / 100g, 0.40g / 100g ,0.41g / 100g,0.42g / 100g,0.43g / 100g,0.44g / 100g,0.45g / 100g,0.46g / 100g,0.47g / 100g,0.48g / 100g,0.49g / 100g,0 .50g / 100g, 0.51g / 100g, 0.52g / 100g, 0.53g / 100g, 0.54g / 100g, 0.55g / 100g, 0.56g / 100g, 0.57g / 100g, 0.58g / 100g, 0.5 9g / 100g, 0.60g / 100g, 0.61g / 100g, 0.62g / 100g, 0.63g / 100g, 0.64g / 100g, 0.65g / 100g, 0.66g / 100g, 0.67g / 100g, 0.68g The following are dry weights of nutrient compositions: 0.69g / 100g, 0.70g / 100g, 0.71g / 100g, 0.72g / 100g, 0.73g / 100g, 0.74g / 100g, 0.75g / 100g, 0.76g / 100g, 0.77g / 100g, 0.78g / 100g, 0.70g / 100g, 0.80g / 100g, 0.81g / 100g, 0.82g / 100g, 0.83g / 100g, 0.84g / 100g, or 0.85g / 100g. Suitablely, the total amount of LNFP is present in amounts less than 0.20 g / 100 g, 0.25 g / 100 g, 0.30 g / 100 g, 0.35 g / 100 g, 0.40 g / 100 g, 0.45 g / 100 g, 0.50 g / 100 g, 0.55 g / 100 g, 0.60 g / 100 g, 0.65 g / 100 g, 0.70 g / 100 g, 0.75 g / 100 g, 0.80 g / 100 g, or 0.85 g / 100 g of the dry weight of the combination or nutrient composition.

[0168] In some embodiments of the combination or the nutritional composition, LNDFHP-I is present in amounts ranging from 0.02g to 3.8g / 100g, or 0.05g to 3.8g / 100g, or 0.1g to 3.8g / 100g, or 0.1g to 3g / 100g, or 0.1g to 2.5g / 100g, or 0.1g to 2g / 100g, or 0.1g to 1.5g / 100g, or 0.1g to 1g / 100g, or 0.02g to 3g / 100g, or 0.02g to 2.5g / 100g, or 0.02g to... 2g / 100g or 0.02g to 1.5g / 100g or 0.02g to 1g / 100g or 0.05g to 3g / 100g or 0.05g to 2.5g / 100g or 0.05g to 2g / 100g or 0.05g to 1.5g / 100g or 0.05g to 1g / 100g or 0.15g to 0.85g / 100g or 0.20g to 0.80g / 100g or 0.30g to 0.70g / 100g or 0.45g to 0.55g / 100g dry weight of combination or nutritional composition.Suitablely, LNDFHP-I is present in the following amounts: 0.15g / 100g, 0.16g / 100g, 0.17g / 100g, 0.18g / 100g, 0.19g / 100g, 0.20g / 100g, 0.21g / 100g, 0.22g / 100g, 0.23g / 100g, 0.24g / 100g, 0.25g / 100g, 0.26g / 100g, 0.27g / 100g, 0.28g / 100g, 0.29g / 100g, 0.30g / 100g, 0.31g / 100g. 00g, 0.32g / 100g, 0.33g / 100g, 0.34g / 100g, 0.35g / 100g, 0.36g / 100g, 0.37g / 100g, 0.38g / 100g, 0.39g / 100g, 0.40g / 100 g, 0.41g / 100g, 0.42g / 100g, 0.43g / 100g, 0.44g / 100g, 0.45g / 100g, 0.46g / 100g, 0.47g / 100g, 0.48g / 100g, 0.49g / 100g, 0 .50g / 100g, 0.51g / 100g, 0.52g / 100g, 0.53g / 100g, 0.54g / 100g, 0.55g / 100g, 0.56g / 100g, 0.57g / 100g, 0.58g / 100g, 0.5 9g / 100g, 0.60g / 100g, 0.61g / 100g, 0.62g / 100g, 0.63g / 100g, 0.64g / 100g, 0.65g / 100g, 0.66g / 100g, 0.67g / 100g, 0.68g The following are dry weights of nutrient compositions: 0.69g / 100g, 0.70g / 100g, 0.71g / 100g, 0.72g / 100g, 0.73g / 100g, 0.74g / 100g, 0.75g / 100g, 0.76g / 100g, 0.77g / 100g, 0.78g / 100g, 0.70g / 100g, 0.80g / 100g, 0.81g / 100g, 0.82g / 100g, 0.83g / 100g, 0.84g / 100g, or 0.85g / 100g.

[0169] In some embodiments of the combination or the nutritional composition, LNDFHP-I is present in amounts ranging from 0.02g to 3.8g / 100g, or 0.05g to 3.8g / 100g, or 0.1g to 3.8g / 100g, or 0.1g to 3g / 100g, or 0.1g to 2.5g / 100g, or 0.1g to 2g / 100g, or 0.1g to 1.5g / 100g, or 0.1g to 1g / 100g, or 0.02g to 3g / 100g, or 0.02g to 2.5g / 100g, or 0.02g to... 2g / 100g or 0.02g to 1.5g / 100g or 0.02g to 1g / 100g or 0.05g to 3g / 100g or 0.05g to 2.5g / 100g or 0.05g to 2g / 100g or 0.05g to 1.5g / 100g or 0.05g to 1g / 100g or 0.15g to 0.85g / 100g or 0.20g to 0.80g / 100g or 0.30g to 0.70g / 100g or 0.45g to 0.55g / 100g dry weight of combination or nutritional composition.Suitablely, LNDFHP-I is present in the following amounts: 0.15g / 100g, 0.16g / 100g, 0.17g / 100g, 0.18g / 100g, 0.19g / 100g, 0.20g / 100g, 0.21g / 100g, 0.22g / 100g, 0.23g / 100g, 0.24g / 100g, 0.25g / 100g, 0.26g / 100g, 0.27g / 100g, 0.28g / 100g, 0.29g / 100g, 0.30g / 100g, 0.31g / 100g. 00g, 0.32g / 100g, 0.33g / 100g, 0.34g / 100g, 0.35g / 100g, 0.36g / 100g, 0.37g / 100g, 0.38g / 100g, 0.39g / 100g, 0.40g / 100 g, 0.41g / 100g, 0.42g / 100g, 0.43g / 100g, 0.44g / 100g, 0.45g / 100g, 0.46g / 100g, 0.47g / 100g, 0.48g / 100g, 0.49g / 100g, 0 .50g / 100g, 0.51g / 100g, 0.52g / 100g, 0.53g / 100g, 0.54g / 100g, 0.55g / 100g, 0.56g / 100g, 0.57g / 100g, 0.58g / 100g, 0.5 9g / 100g, 0.60g / 100g, 0.61g / 100g, 0.62g / 100g, 0.63g / 100g, 0.64g / 100g, 0.65g / 100g, 0.66g / 100g, 0.67g / 100g, 0.68g The following are dry weights of nutrient compositions: 0.69g / 100g, 0.70g / 100g, 0.71g / 100g, 0.72g / 100g, 0.73g / 100g, 0.74g / 100g, 0.75g / 100g, 0.76g / 100g, 0.77g / 100g, 0.78g / 100g, 0.70g / 100g, 0.80g / 100g, 0.81g / 100g, 0.82g / 100g, 0.83g / 100g, 0.84g / 100g, or 0.85g / 100g.

[0170] In some embodiments of the combination or the nutritional composition, N-acetylated human milk oligosaccharides are present in amounts ranging from 0.02 g to 3.8 g / 100 g, or 0.05 g to 3.8 g / 100 g, or 0.1 g to 3.8 g / 100 g, or 0.1 g to 3 g / 100 g, or 0.1 g to 2.5 g / 100 g, or 0.1 g to 2 g / 100 g, or 0.1 g to 1.5 g / 100 g, or 0.1 g to 1 g / 100 g, or 0.02 g to 3 g / 100 g, or 0.02 g to 2.5 g / 100 g, or 0.02 g. Up to 2g / 100g or 0.02g to 1.5g / 100g or 0.02g to 1g / 100g or 0.05g to 3g / 100g or 0.05g to 2.5g / 100g or 0.05g to 2g / 100g or 0.05g to 1.5g / 100g or 0.05g to 1g / 100g or 0.15g to 0.85g / 100g or 0.20g to 0.80g / 100g or 0.30g to 0.70g / 100g or 0.45g to 0.55g / 100g dry weight of combination or nutritional composition.Suitablely, N-acetylated human milk oligosaccharides are present in the following amounts: 0.15 g / 100 g, 0.16 g / 100 g, 0.17 g / 100 g, 0.18 g / 100 g, 0.19 g / 100 g, 0.20 g / 100 g, 0.21 g / 100 g, 0.22 g / 100 g, 0.23 g / 100 g, 0.24 g / 100 g, 0.25 g / 100 g, 0.26 g / 100 g, 0.27 g / 100 g, 0.28 g / 100 g, 0.29 g / 100 g, 0.30 g / 100 g, 0.31 g / 100g, 0.32g / 100g, 0.33g / 100g, 0.34g / 100g, 0.35g / 100g, 0.36g / 100g, 0.37g / 100g, 0.38g / 100g, 0.39g / 100g, 0.40g / 10 0g, 0.41g / 100g, 0.42g / 100g, 0.43g / 100g, 0.44g / 100g, 0.45g / 100g, 0.46g / 100g, 0.47g / 100g, 0.48g / 100g, 0.49g / 100g, 0.50g / 100g, 0.51g / 100g, 0.52g / 100g, 0.53g / 100g, 0.54g / 100g, 0.55g / 100g, 0.56g / 100g, 0.57g / 100g, 0.58g / 100g, 0. 59g / 100g, 0.60g / 100g, 0.61g / 100g, 0.62g / 100g, 0.63g / 100g, 0.64g / 100g, 0.65g / 100g, 0.66g / 100g, 0.67g / 100g, 0.68g The following are dry weights of nutrient compositions: 0.69g / 100g, 0.70g / 100g, 0.71g / 100g, 0.72g / 100g, 0.73g / 100g, 0.74g / 100g, 0.75g / 100g, 0.76g / 100g, 0.77g / 100g, 0.78g / 100g, 0.70g / 100g, 0.80g / 100g, 0.81g / 100g, 0.82g / 100g, 0.83g / 100g, 0.84g / 100g, or 0.85g / 100g.

[0171] In some embodiments of the combination or the nutritional composition, the N-acetylated human milk oligosaccharide comprises or is composed of lactose-N-tetrasaccharide (LNT) and / or LNnT, preferably LNT.

[0172] Suitable, this nutritional composition can be provided as a serving of 31g total dry weight.

[0173] Suitable of the nutritional composition, it may be intended to be applied twice daily. Therefore, the nutritional composition may be formulated to provide two servings per day.

[0174] According to the present invention, the nutritional composition contains Bifidobacterium infantis as a probiotic.

[0175] In some implementations, the probiotics in the nutritional composition consist of Bifidobacterium infantis.

[0176] In some embodiments, the composition comprises at least one probiotic in an amount of 1×10⁻⁶. 3 cfu / g up to 1.5×10 12 CFU / g composition (dry weight).

[0177] Based on dry weight, the nutritional composition according to the present invention may contain 10 per gram of composition. 3 cfu to 10 12 At least one probiotic of CFU, more preferably between 10 7 cfu to 10 12 CFU, such as between 10 8 cfu to 10 10 At least one probiotic between CFU. Suitably, the at least one probiotic is in a concentration of at least about 10. 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 The subject was administered CFU / day. Suitably, the at least one probiotic was administered at approximately 10... 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10 The subject was given CFU / day or less.

[0178] In one embodiment, the at least one probiotic is live. In another embodiment, the at least one probiotic is non-replicating or inactivated. In some other embodiments, both live and inactivated probiotics may be present simultaneously. Probiotic components and metabolites may also be added.

[0179] Uses to enhance the growth and / or metabolic activity of Bifidobacterium infantis.

[0180] As described above, it has been surprisingly found that the combinations and compositions of the present invention enhance the growth of Bifidobacterium infantis.

[0181] In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention for enhancing the growth and / or metabolic activity of infantile Bifidobacteria in the gastrointestinal tract of infants, toddlers or children.

[0182] In another aspect, the present invention provides a method for enhancing the growth and / or metabolic activity of infantile Bifidobacteria in the gastrointestinal tract of infants, toddlers, or children, the method comprising administering the combination or nutritional composition of the present invention to the infant, toddler, or child.

[0183] In another aspect, the present invention provides the use of at least one probiotic for enhancing the growth and / or metabolic activity of infantile Bifidobacteria in the gastrointestinal tract of infants, toddlers or children, wherein the at least one probiotic is as described elsewhere herein.

[0184] The combination or nutritional composition of the present invention can prevent or treat infections or diseases by enhancing the growth and / or metabolic activity of Bifidobacteria in the gastrointestinal tract of the subject.

[0185] The combination or nutritional composition of the present invention can prevent or treat infections or diseases by increasing the levels of SCFAs and / or other metabolites in the gastrointestinal tract of a subject, as well as the systemic levels of SCFAs and / or other metabolites. Increased systemic levels of SCFAs and / or other metabolites in the subject facilitate the action of the combination or nutritional composition of the present invention outside the gastrointestinal tract (e.g., in the lungs).

[0186] The combination or nutritional compositions of the present invention can be used to treat or prevent conditions associated with a decrease in the number and / or metabolic activity of Bifidobacteria in the gut (see, for example, Rivière, A. et al., 2016. Frontiers in microbiology, 7, p. 979).

[0187] In one aspect, the present invention provides a combination or nutritional composition for treating and / or preventing conditions associated with a decrease in the number and / or metabolic activity of Bifidobacteria in the gut. In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention in the manufacture of a medicament for treating and / or preventing conditions associated with a decrease in the number and / or metabolic activity of Bifidobacteria in the gut. In yet another aspect, the present invention provides a method for treating and / or preventing conditions associated with a decrease in the number and / or metabolic activity of Bifidobacteria in the gut of a subject, the method comprising administering the composition or nutritional composition to the subject.

[0188] Appropriately, conditions associated with a decrease in the number and / or metabolic activity of Bifidobacteria in the subject's gut may be selected from: gastrointestinal diseases, obesity, allergic diseases, and degenerative autism.

[0189] Therefore, in another aspect, the present invention provides combinations or nutritional compositions for enhancing the immune response of infants, toddlers, or children to infection or vaccination. In some embodiments, an enhanced antibody response to infection or vaccination is achieved in infants, toddlers, or children.

[0190] In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention in the manufacture of a medicament for enhancing the immune response of infants, young children or children to infection or vaccination.

[0191] In another aspect, the present invention provides a method for enhancing the immune response of an infant, toddler, or child to an infection or vaccination, the method comprising administering the combination or nutritional composition of the present invention to the infant, toddler, or child.

[0192] In some implementations, antibody responses to infection or vaccination are enhanced in infants, toddlers, or children.

[0193] In some implementations, the immune response to infection is enhanced.

[0194] In some implementation schemes, the immune response to vaccination is enhanced.

[0195] In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention in the manufacture of a medicament for preventing infection in infants, young children or children and / or reducing the risk of infection.

[0196] In another aspect, the present invention provides a method for preventing infection in infants, toddlers or children and / or reducing the risk of infection, the method comprising administering the combination or nutritional composition of the present invention to the infant, toddler or child.

[0197] As used herein, the term “preventing infection and / or reducing the risk of infection” includes preventing infection and / or reducing the risk of infection, delaying or preventing the onset of infection symptoms and / or reducing the number or severity of infection symptoms.

[0198] Infections can be caused by respiratory infections (such as rhinovirus infection, respiratory syncytial virus infection, influenza virus infection) or gastrointestinal infections (including candidiasis, healthcare-associated infections and healthcare-associated diarrhea, antibiotic-associated infections and antibiotic-associated diarrhea).

[0199] In some embodiments, the combination or nutritional composition of the present invention may be administered once daily. In other embodiments, the composition of the present invention is administered in multiple doses, such as two doses daily (also referred to as a “unit dose”). When the nutritional composition is provided in the form of unit doses (or “servings”), it is particularly useful to define the amount of oligosaccharides and probiotics according to the daily dose to be administered to an infant or toddler or child.

[0200] In some implementations, the total amount and range of HMO administered to infants, toddlers, or children is 10 mg / day to 3800 mg / day (suitably, 50 mg / day to 800 mg / day or 100 mg / day to 600 mg / day). 3CFU / Tianzhi 10 12 CFU / day of Bifidobacterium infantis. In one implementation, an HMO total ranging from 200 mg / day to 600 mg / day is administered to infants, toddlers, or children, with a range of 10... 4 CFU / Tianzhi 10 9 Bifidobacterium infantis CFU / day.

[0201] In some implementation methods, HMOs are administered to infants, toddlers, or children in doses ranging from 50 mg / day to 950 mg / day. 3 CFU / Tianzhi 10 12 A dose of Bifidobacterium infantis ranging from CFU / day. In some implementations, HMO and 10... 5 CFU / Tianzhi 10 9 CFU / day of Bifidobacterium infantis.

[0202] In some preferred embodiments, HMOs in doses ranging from 150 mg / day to 450 mg / day are administered to infants, toddlers, or children. 6 CFU / Tianzhi 10 8 CFU / day of Bifidobacterium infantis.

[0203] In some implementations, infants, toddlers, or children are given approximately 50 mg / day (suitably, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, or 950 mg / day) of HMO and approximately 10 3 CFU / day (if appropriate, 10) 4 CFU / day, 10 5 CFU / day, 10 6 CFU / day, 10 7 CFU / day, 10 8 CFU / day, 10 9 CFU / day, 10 10 CFU / day, 10 11 CFU / day, 10 12 Bifidobacterium infantis (CFU / day). In one implementation, approximately 150 mg / day of HMO and approximately 10 mg / day of [unclear] are administered to an infant, toddler, or child. 7CFU / day of Bifidobacterium infantis. In one implementation, approximately 300 mg / day of HMO and approximately 10 cfu / day of Bifidobacterium infantis are administered to an infant, toddler, or child. 9 CFU / day of Bifidobacterium infantis. In one implementation, approximately 450 mg / day of HMO and approximately 10 cfu / day of Bifidobacterium infantis are administered to an infant, toddler, or child. 7 CFU / day of Bifidobacterium infantis. In one implementation, approximately 50 mg / day of HMO and approximately 10 mg / day of [unclear] are administered to an infant, toddler, or child. 7 CFU / day of Bifidobacterium infantis. In one implementation method, approximately 100 mg / day of HMO and approximately 10 mg / day of [unclear text - possibly a specific ingredient or dosage] are administered to the infant, toddler, or child. 6 In one implementation, approximately 150 mg / day of HMO and approximately 50 mg / day of Bifidobacterium infantis are administered to an infant, toddler, or child.

[0204] In some implementations, infants, toddlers, or children do not respond to treatment with Bifidobacterium infantis and LNFP-I.

[0205] The combination or nutritional composition of the present invention can be administered by any suitable method known to those skilled in the art. For example, the combination or nutritional composition of the present invention can be administered orally and / or enterally. In some embodiments, the combination or nutritional composition of the present invention is administered orally.

[0206] Methods to promote and / or maintain gastrointestinal health

[0207] The use of probiotics (including Bifidobacterium strains) to maintain healthy gut function in preventive medicine is well documented (Tojo, R. et al., 2014, World journal of gastroenterology: WJG, 20(41), p. 15163). Furthermore, the use of SCFAs to maintain healthy gut function in preventive medicine is also well documented. The combination or composition of the present invention is expected to have the same effect, enhancing the growth of Bifidobacteria and / or increasing the level of SCFAs in the gastrointestinal tract of the subject.

[0208] In another aspect, the present invention provides combinations or nutritional compositions of the present invention for promoting and / or maintaining gastrointestinal health in infants, toddlers or children.

[0209] In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention in the manufacture of a medicament for promoting and / or maintaining the gastrointestinal health of infants, toddlers or children.

[0210] In another aspect, the present invention provides a method for promoting and / or maintaining the gastrointestinal health of an infant, toddler, or child, the method comprising administering the combination or nutritional composition of the present invention to the infant, toddler, or child.

[0211] In one aspect, the present invention provides the use of the combination or nutritional composition of the present invention for promoting and / or maintaining gastrointestinal health. The combination or nutritional composition of the present invention can promote and / or maintain gastrointestinal health by enhancing the growth of Bifidobacteria in the gastrointestinal tract of a subject, or by increasing SCFA levels in the gastrointestinal tract of a subject.

[0212] Methods for treating and / or preventing gastrointestinal diseases

[0213] In some implementations, promoting and / or maintaining gastrointestinal health includes treating and / or preventing gastrointestinal diseases.

[0214] In one aspect, the present invention provides the use of the combination or nutritional composition of the present invention for treating and / or preventing gastrointestinal diseases. In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention for manufacturing a medicament for treating and / or preventing gastrointestinal diseases. In yet another aspect, the present invention provides a method of treating and / or preventing a gastrointestinal disease in a subject, the method comprising administering the combination or nutritional composition of the present invention to the subject.

[0215] As used herein, “gastrointestinal disease” (also known as “GI disease” or “GI morbidity”) can refer to a disease involving the gastrointestinal tract, which includes the esophagus, stomach, small intestine, large intestine, and rectum. In some implementations, gastrointestinal disease is a stomach ailment or an intestinal ailment.

[0216] In some implementations, gastrointestinal diseases are referred to as stomach diseases. “Stomach disease” can refer to any disease that affects the stomach.

[0217] In some implementations, gastrointestinal diseases are referred to as enteropathy. "Enteropathy" can refer to diseases affecting the small intestine (including the duodenum, jejunum, and ileum) or the large intestine (including the cecum, colon, and rectum).

[0218] In some implementations, gastrointestinal diseases are selected from: healthcare-associated diarrhea, antibiotic-associated diarrhea, Helicobacter pylori infection, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), lactose intolerance, infectious diarrhea, and necrotizing enterocolitis.

[0219] Antibiotic-associated diarrhea

[0220] A common complication of antibiotic use is the development of gastrointestinal disorders. These complications range from mild diarrhea to pseudomembranous colitis. Antibiotic-associated diarrhea typically occurs in 5%–35% of patients taking antibiotics and varies depending on the specific type of antibiotic, the host’s health, and exposure to the pathogen. The pathogenesis of antibiotic-associated diarrhea can be mediated by disruption of the normal microbiota, leading to pathogen overgrowth or metabolic imbalance (McFarland, LV, 2008, Future Microbiology, 3(5), p. 563).

[0221] Probiotic blends containing several strains of Bifidobacterium (especially Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) have shown the ability to reduce the incidence of antibiotic-associated diarrhea (Selinger, CP et al., 2013, Journal of Hospital Infection, 84(2), pp. 159-165).

[0222] Helicobacter pylori infection

[0223] Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects the epithelial layer of the stomach. It is a major cause of chronic gastritis and a leading pathogen of gastric cancer and peptic ulcer disease. Recent global systematic reviews estimate that more than half of the world's population is infected with Helicobacter pylori (Hooi, JK et al., 2017, Gastroenterology, 153(2), pp. 420-429).

[0224] It has been reported that after 10 days of administration of a probiotic mixture containing several strains of Bifidobacterium, particularly Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum, the Helicobacter pylori eradication rate in adults was 32.5% (Boltin, D., 2016, Best Practice & Research Clinicalgastroenterology, 30(1), pp. 99–109). Furthermore, this probiotic mixture has been shown to accelerate the healing of gastric ulcers (Dharmani, P. et al., 2013, PLoS One, 8(3), p. e58671).

[0225] Inflammatory bowel disease

[0226] Inflammatory bowel disease (IBD) is a class of inflammatory conditions of the colon and small intestine. Exemplary IBDs include Crohn's disease (CD), ulcerative colitis (UC), and pouchitis. It has been proposed that dysbiosis (i.e., abnormal microbiome composition) and reduced complexity of the gut microbiome ecosystem are common features in patients with IBD (see Manichanh, C. et al., 2012, Nature reviews Gastroenterology & hepatology, 9(10), pp. 599–608).

[0227] In IBD, probiotic blends containing several strains of Bifidobacterium (especially Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) have been shown to reduce UC symptoms in adults (Tursi, A. et al., 2010, The American Journal of Gastroenterology, 105(10), p. 2218) and alleviate symptoms in children (Miele, E. et al., 2009, American Journal of Gastroenterology, 104(2), pp. 437-443).

[0228] In some implementations, IBD refers to Crohn's disease, ulcerative colitis, or pouchitis.

[0229] Irritable bowel syndrome

[0230] Irritable bowel syndrome (IBS) is a bowel dysfunction characterized by chronic and recurrent abdominal pain and altered bowel habits (Chey, WD et al., 2015, Jama, 313(9), pp. 949-958). Increasing evidence suggests that dysbiosis is a hallmark of IBS (Rodiño-Janeiro, BK et al., 2018, Advances in therapy, 35(3), pp. 289-310).

[0231] Administering a probiotic blend containing several strains of Bifidobacterium (especially Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum) for up to 6 weeks resulted in reduced IBS symptoms and improved quality of life in children (Guandalini, S. et al., 2010, Journal of pediatric gastroenterology and nutrition, 51(1), pp. 24-30).

[0232] Other gastrointestinal diseases

[0233] Lactose intolerance is a common condition caused by a reduced ability to digest lactose. The use of Bifidobacterium has been used to improve the symptoms of lactose intolerance (Hidalgo-Cantabrana, C. et al., 2017, Microbiology spectrum, 5(3), pp. 5–3).

[0234] Infectious diarrhea (also known as gastroenteritis) is an inflammation of the gastrointestinal tract caused by infection. Gastroenteritis is usually caused by viruses (such as rotavirus, norovirus, adenovirus, astrovirus, and coronavirus), however, bacteria (such as Campylobacter jeuni, Escherichia coli, Salmonella, Shigella, Clostridium difficile, and Staphylococcus aureus), parasites (such as Giardia lamblia), and fungi can also cause gastroenteritis. There is evidence that live Bifidobacterium lactis has some protective effect against acute diarrhea in healthy children (Chouraqui, JP et al., 2004, Journal of pediatric gastroenterology and nutrition, 38(3), pp. 288-292).

[0235] Necrotizing enterocolitis (NEC) is an intestinal disease affecting preterm infants. It has been shown that probiotic supplementation with Bifidobacterium can reduce the incidence and severity of NEC in the preterm neonatal population (Bin-Nun, A. et al., 2005, The Journal of pediatrics, 147(2), pp. 192-196).

[0236] In one aspect, the present invention provides a combination or nutritional composition for treating and / or preventing lactose intolerance, infectious diarrhea, or necrotizing enterocolitis. In another aspect, the present invention provides the use of the combination or nutritional composition of the present invention in the manufacture of a medicament for treating and / or preventing lactose intolerance, infectious diarrhea, or necrotizing enterocolitis. In yet another aspect, the present invention provides a method for treating and / or preventing lactose intolerance, infectious diarrhea, or necrotizing enterocolitis in a subject, the method comprising administering the combination or nutritional composition of the present invention to the subject.

[0237] Subjects

[0238] In some embodiments of the method and use of the present invention, the combination or the nutritional composition is applied to an infant.

[0239] In some embodiments of the method and use of the present invention, the combination or the nutritional composition is applied to young children.

[0240] In some embodiments of the method and use of the present invention, the combination or nutritional composition is applied to children.

[0241] In yet another implementation, the infant's age ranges from 0 to 12 months, such as 9 or 6 months.

[0242] In some implementation schemes, the age range for children is one to three years old, such as two years old.

[0243] In some implementations, the children's age range is three to nine years old, for example, three to seven years old. In some preferred implementations, the children's age range is three to five years old, for example, four years old.

[0244] In a preferred embodiment, the subjects are infants, toddlers, or children.

[0245] Certain groups of infants, toddlers, and children particularly need the combination or nutritional compositions of the present invention. Such infants, toddlers, and children include, for example, premature infants, low birth weight infants, and / or infants, toddlers, and children with growth retardation. In fact, these subjects often experience adverse medical conditions and require significantly more frequent medical interventions than full-term infants and infants experiencing normal development.

[0246] The nutritional compositions according to the present invention are used in infants, toddlers, and / or children. Infants, toddlers, and / or children may be full-term or preterm. In one specific embodiment, the nutritional compositions of the present invention are used for preterm infants, toddlers, and / or children with low birth weight and / or small for gestational age (SGA). In one specific embodiment, the nutritional compositions of the present invention are used for preterm infants, low birth weight infants, and / or small for gestational age infants (SGA).

[0247] The combination or nutritional composition of the present invention can also be used for infants, toddlers or children born by cesarean section or vaginal delivery.

[0248] In some embodiments, the combination or nutritional composition according to the invention may be used before and / or during weaning. The age and duration of administration (or administration or feeding) of the combination or nutritional composition may be determined as needed.

[0249] The combination or nutritional composition may be given, for example, immediately after the infant's birth. The compositions of the present invention may also be given during the first week of the infant's life, or during the first two weeks of life, or during the first three weeks of life, or during the first month of life, or during the first two months of life, or during the first three months of life, or during the first four months of life, or during the first six months of life, or during the first eight months of life, or during the first ten months of life, or during the first year of life, or during the first two years of life, or even longer. In some particularly advantageous embodiments of the invention, the nutritional composition is given (or applied) to the infant within the first four, six, or twelve months after birth. In some other embodiments, the combination or nutritional composition of the present invention is given a few days (e.g., 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days…), a few weeks (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks…), or a few months (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months…) after birth. This may specifically refer to cases where the infant is premature, but it is not necessary.

[0250] In one embodiment, the nutritional composition of the present invention is given to an infant or young child as a supplementary composition to breast milk. In some embodiments, the infant or young child receives breast milk for at least the first 2 weeks, the first 1 month, the first 2 months, the first 4 months, or the first 6 months. In one embodiment, the nutritional composition of the present invention is given to the infant or young child after this period of breastfeeding, or given to the infant or young child together with breast milk during this period of breastfeeding. In another embodiment, the composition is given to the infant or young child as the sole or primary nutritional composition for at least a period of time (e.g., after the first, second, or fourth month of life), for at least 1 month, 2 months, 4 months, or 6 months.

[0251] Subjects may have low abundance and / or activity of Bifidobacteria in their gastrointestinal tract or may be at risk of low abundance and / or activity of Bifidobacteria. The abundance of Bifidobacteria in the gastrointestinal tract can be determined by any method known to the technician (e.g., any method described in Tang, Q. et al., 2020. Frontiers in cellular and infection microbiology, 10, p. 151). Activity can be assessed, for example, by gas chromatography / or liquid chromatography combined with mass spectrometry or another suitable detector, by measuring typical Bifidobacterial metabolites such as acetate.

[0252] Gastrointestinal samples can be or can be obtained from fecal samples, endoscopic samples (e.g., biopsy samples, luminal brush samples, laser-captured microanatomical samples), aspirated intestinal fluid samples, surgical samples, or through in vivo models or smart capsules. Suitablely, gastrointestinal samples can be or can be obtained from fecal samples. Fecal samples are naturally collected, non-invasive, and can be repeatedly sampled.

[0253] The abundance of Bifidobacteria can be determined from a sample by any suitable method. For example, the abundance of Bifidobacteria can be obtained from a sample by or can be obtained by sequencing methods (e.g., next-generation sequencing (NGS) methods), PCR-based methods, semi-quantitative detection methods, cyclic temperature capillary electrophoresis, immunological methods, cell-based methods, or any combination thereof.

[0254] Subjects may have a condition associated with a reduced number of Bifidobacteria in the gut or may be at risk of developing such a condition. Such conditions are described by Rivière, A. et al., 2016, Frontiers in Microbiology, 7, p. 979, and may include gastrointestinal diseases, obesity, allergies, and degenerative autism.

[0255] Subjects may have gastrointestinal diseases or be at risk of developing gastrointestinal diseases. In some embodiments, subjects may have antibiotic-associated diarrhea or be at risk of developing antibiotic-associated diarrhea. In some embodiments, subjects may have Helicobacter pylori infection or be at risk of developing this infection. In some embodiments, subjects may have IBD or be at risk of developing IBD. In some embodiments, subjects may have IBS or be at risk of developing IBS. In some embodiments, subjects may have lactose intolerance, infectious diarrhea, or necrotizing enterocolitis or be at risk of developing these conditions.

[0256] Other ingredients

[0257] The nutritional compositions according to the invention may further comprise non-HMO oligosaccharides and / or their fiber and / or precursors, optionally selected from the following list: galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, and any combination thereof. Their amounts may be between 0% and 10% by weight of the composition.

[0258] The nutritional composition of the present invention may also contain at least one other probiotic (or probiotic strain), such as at least one other probiotic strain.

[0259] The most commonly used probiotics are mainly bacteria and yeasts belonging to the following genera: Lactobacillus spp., Lacticaseibacillus spp., Limosilactobacillus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp., and Saccharomyces spp.

[0260] In some specific embodiments, the probiotics are probiotic bacterial strains. In some specific embodiments, they are specifically Bifidobacterium and / or Lactobacillus.

[0261] Suitable probiotic strains include: *Lactobacillus rhamnosus* ATCC 53103, *Lactobacillus rhamnosus* CGMCC 1.3724, *Lactobacillus paracasei* CNCM I-2116, *Lactobacillus johnsonii* CNCMI-1225 (from Valio Oy, Finland); *Streptococcus salivarius* DSM 13084 (sold by BLIS Technologies Limited, New Zealand under the name KI2); and *Bifidobacterium lactis* CNCM 1-3446 and *Bifidobacterium longum* CNCM (specially sold by Christian Hansen, Denmark under the trademark Bb 12). I-2618 (Bifidobacterium longum NCC2705), Bifidobacterium breve sold by Danisco under the trademark Bb-03, Bifidobacterium breve sold by Morinaga under the trademark M-16V, Bifidobacterium infantis sold by Procter & GambIe Co. under the trademark Bifantis, and Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trademark R0070.

[0262] Suitable probiotic strains include *Bifidobacterium animalis* subsp. *lactobacter* CNCM 1-3446, deposited on June 7, 2005, at the French National Collection of Microorganism Cultures (CNCM), Pasteur Institute (Institut Pasteur, 25 Rue Du Docteur Roux, F-75724 ParisCedex 15 (France)), or BL818, specially marketed by Christian Hansen of Denmark under the trademark Bb 12, or *Bifidobacterium animalis* subsp. *lactobacter* (also known as DSM-15954), and *Bifidobacterium longum* CNCM. I-2618 (Bifidobacterium longum NCC2705), Bifidobacterium breve sold by Danisco under the trademark Bb-03, Bifidobacterium breve sold by Morinaga under the trademark M-16V, Bifidobacterium breve sold by Morinaga under the trademark B-3, Bifidobacterium breve sold by Yakult under the trademark BBG-01, and Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trademark R0070.

[0263] In some preferred embodiments, the *Bifidobacterium longum* subsp. strain can be selected from... Bifidobacterium longum subspecies strain CNCM I-2169, Bifidobacterium longum subsp. longum strain CNCM I-2171, Bifidobacterium longum subsp. longum strain ATCC 15708, Bifidobacterium longum subsp. longum strain DSM 20097, Bifidobacterium longum subsp. longum strain NCIMB 8809, Bifidobacterium longum subsp. longum strain CNCM I-2618 (NCC 2705), Bifidobacterium longum subsp. longum strain CNCM I-2170, Bifidobacterium longum subsp. longum strain ATCC 15707, or combinations thereof, especially Bifidobacterium longum CNCM I-2618 (NCC 2705).

[0264] Suitablely, *Bifidobacterium longum* subsp. *longum* and *Bifidobacterium longum* subsp. *longum* NCC 2705 (also known as *Bifidobacterium longum* subsp. *longum* CNCM I-2618) have an ANI of at least 99% (suitably, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%). Preferably, *Bifidobacterium longum* subsp. *longum* and *Bifidobacterium longum* subsp. *longum* NCC 2705 have an ANI of at least 99.9%.

[0265] Bifidobacterium longum NCC 2705 was deposited at the Pasteur Institute (Institute Pasteur, 25 Rue Du Docteur Roux, F-75724 Paris Cedex 15 (France)) on January 29, 2001, under the Budapest Convention, with accession number CNCM I-2618.

[0266] Based on dry weight, the nutritional composition according to the present invention may contain 10 per gram of composition. 3 cfu to 10 12 CFU contains at least one other probiotic strain, more preferably between 10 7 cfu to 10 12 CFU, such as between 10 8 cfu to 10 10 Probiotic strains between CFUs.

[0267] In one embodiment, the probiotics are live. In another embodiment, the probiotics are non-replicating or inactivated. In some other embodiments, both live and inactivated probiotics may be present simultaneously. Probiotic components and metabolites may also be added.

[0268] The nutritional compositions according to the invention may be, for example, infant formula, stage 1 infant formula, follow-up or stage 2 infant formula, growing milk, baby food, infant cereal compositions, fortifiers (such as human milk fortifiers), or supplements. In some specific embodiments, the compositions of the invention are infant formula, fortifiers, or supplements intended for infants aged 4 months or 6 months. In a preferred embodiment, the nutritional compositions of the invention are infant formula.

[0269] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier may be a breast milk fortifier (e.g., human milk fortifier) ​​or a formula food fortifier (such as an infant formula fortifier or a follow-up formula / stage 2 infant formula fortifier).

[0270] When a nutritional composition is a supplement, it can be provided in unit doses. In such cases, it is particularly useful to limit the amount of oligosaccharides and probiotics according to the daily dose to be administered to an infant, toddler, or child, as described above.

[0271] The nutritional composition of the present invention may be in solid (e.g., powder), liquid, or gel form. In one particular embodiment, the nutritional composition is a supplement, wherein the supplement is in powder form and provided in sachets, preferably with 0.1 g / sachet to 20 g / sachet, for example, 1 g / sachet to 10 g / sachet, or provided in syrup form, preferably with a total solids concentration of 5 g / 100 mL to 75 g / 100 mL (5% to 75% (w / v)). When the supplement is in powder form, it may contain a carrier. However, it is preferred that the supplement does not contain a carrier. When the supplement is in syrup form, the components are preferably dissolved or suspended in water acidified with citrate.

[0272] The nutritional compositions according to the invention typically contain a protein source. The amount of protein can be from 1.6 g / 100 kcal to 3 g / 100 kcal. In some embodiments, particularly when the composition is intended for preterm infants, the amount of protein can be between 2.4 g / 100 kcal and 4 g / 100 kcal or more than 3.6 g / 100 kcal. In some other embodiments, the amount of protein can be less than 2.0 g / 100 kcal, for example, from 1.8 g / 100 kcal to 2 g / 100 kcal, or less than 1.8 g / 100 kcal.

[0273] Protein sources based on whey, casein, or mixtures thereof, or soy-based protein sources, may be used. Regarding the whey protein of interest, the protein source may be based on acidic whey or sweet whey, or mixtures thereof, and may contain any desired proportions of α-lactalbumin and β-lactoglobulin.

[0274] In some advantageous implementations, the protein source is whey-based (i.e., more than 50% of the protein comes from whey protein, such as 60% or 70%).

[0275] The protein can be whole or hydrolyzed, or a mixture of whole and hydrolyzed proteins. The term "whole" means that the major components of the protein are intact, i.e., the molecular structure is not altered, for example, at least 80% of the protein is unchanged, such as at least 85% of the protein is unchanged, preferably at least 90% of the protein is unchanged, even more preferably at least 95% of the protein is unchanged, such as at least 98% of the protein is unchanged. In one specific embodiment, 100% of the protein is unchanged.

[0276] The term "hydrolyzed" means, in the context of this invention, that a protein has been hydrolyzed or broken down into its constituent amino acids. The protein may be completely or partially hydrolyzed. For example, providing partially hydrolyzed protein (with a degree of hydrolysis between 2% and 20%) may be desirable for infants or young children considered at risk of bovine milk allergies. If a hydrolyzed protein is required, the hydrolysis process can be carried out as needed and as is known in the art. For example, whey protein hydrolysates can be prepared by enzymatic hydrolysis of whey fractions in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it has been found that the protein undergoes far less lysine blockage during the hydrolysis process. This allows the degree of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which significantly improves the nutritional quality of the protein source.

[0277] In one embodiment of the invention, at least 70% of the protein is hydrolyzed, preferably at least 80%, such as at least 85%, even more preferably at least 90%, such as at least 95%, and particularly at least 98%. In one specific embodiment, 100% of the protein is hydrolyzed.

[0278] In one specific embodiment, the protein in the nutritional composition is hydrolyzed, fully hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be between 8 and 40, or between 20 and 60, or between 20 and 80, or greater than 10, 20, 40, 60, 80, or 90.

[0279] Alternatively, the protein component may be replaced with a mixture or synthetic amino acids or peptides, for example, for premature or low birth weight infants.

[0280] In one embodiment, the nutritional composition or growing milk according to the invention is a hypoallergenic composition. In another embodiment, the composition according to the invention is a hypoallergenic nutritional composition or growing milk.

[0281] The nutritional compositions according to the invention typically contain a carbohydrate source. This is particularly preferred when the nutritional compositions of the invention are for infant formula. In this case, any carbohydrate source commonly found in infant formula can be used, such as lactose, sucrose, saccharin, maltodextrin, starch, and mixtures thereof, but one of the preferred carbohydrate sources is lactose.

[0282] The nutritional compositions according to the invention typically contain a lipid source. This is particularly relevant when the nutritional compositions of the invention are for infant formula. In this case, the lipid source can be any lipid or fat suitable for use in infant formula. Some suitable fat sources include palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, and medium-chain triglyceride oil. Essential fatty acids linoleic acid and α-linolenic acid may also be added, as well as small amounts of oils containing high levels of pre-formulated arachidonic acid and docosahexaenoic acid (DHA), such as fish oil or microbial oil. The ratio of n-6 fatty acids to n-3 fatty acids in the fat source can be from about 5:1 to about 15:1, for example from about 8:1 to about 10:1.

[0283] The nutritional compositions of the present invention may also contain all vitamins and minerals considered essential for a daily diet and required in significant amounts. Minimum requirements for certain vitamins and minerals have been determined. Examples of minerals, vitamins, and other nutrients optionally present in the compositions of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are typically added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the target population.

[0284] If necessary, the nutritional compositions of the present invention may contain emulsifiers and stabilizers, such as soybean, lecithin, mono- and di-citrate citrate, etc.

[0285] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, nucleosides, etc.

[0286] The nutritional compositions of the present invention may also contain carotenoids, such as lutein (free lutein, lutein esters, lutein salts). In some embodiments, the weight ratio of lutein (μg) to docosahexaenoic acid (mg) is 1.5:1 to 10:1, preferably 2:1 to 5:1. In some embodiments of the present invention, the nutritional compositions of the present invention do not contain any carotenoids.

[0287] The nutritional compositions according to the invention can be prepared in any suitable manner. The compositions will now be described by way of example.

[0288] For example, formulated foods (such as infant formula) can be prepared by blending protein sources, carbohydrate sources, and fat sources together in appropriate proportions. If used, an emulsifier may be added at this stage. Vitamins and minerals may be added at this stage, but they are usually added later to avoid thermal degradation. Before blending, any lipophilic vitamins, emulsifiers, etc., can be dissolved in the fat source. Water (preferably water subjected to reverse osmosis) can then be mixed in to form a liquid mixture. The water temperature is suitably in the range of about 50°C to about 80°C to aid in the dispersion of the components. Commercially available liquefying agents can be used to form the liquid mixture.

[0289] Especially if the final product is in liquid form, fucoidylated oligosaccharides and optional N-acetylated oligosaccharides can be added at this stage. If the final product is a powder, these components can also be added at this stage as needed.

[0290] Then, the liquid mixture is homogenized in, for example, in two stages.

[0291] The liquid mixture can then be heat-treated to reduce the bacterial load, for example by rapidly heating the liquid mixture to a temperature ranging from about 80°C to about 150°C for a duration between about 5 seconds and about 5 minutes. This can be done by steam injection, autoclaving, or a heat exchanger (e.g., a plate heat exchanger).

[0292] The liquid mixture is then cooled, for example, to between about 60°C and about 85°C by rapid cooling. It is then homogenized again, for example, in two stages, where the pressure in the first stage is between about 10 MPa and about 30 MPa, and the pressure in the second stage is between about 2 MPa and about 10 MPa. The homogenized mixture can then be further cooled to add any heat-sensitive components, such as vitamins and minerals. The pH and solids content of the homogenized mixture can then be conveniently adjusted.

[0293] If the final product is a powder, the homogenized mixture is transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, and converted into a powder. The moisture content of the powder should be less than about 5% by weight. Oligosaccharides may also be added at this stage, or alternatively, by dry mixing them with probiotic strains in the form of crystalline syrup, or by blending them with probiotic strains, followed by spray drying or freeze drying of the mixture.

[0294] If a liquid composition is preferred, the homogenized mixture can be sterilized and then filled into a suitable container under aseptic conditions, or it can be filled into a container first and then distilled.

[0295] In another embodiment, the composition of the present invention may be a supplement. The supplement may be in the form of, for example, tablets, capsules, lozenges, or liquids. The supplement may also contain protective hydrophilic colloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulation agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavor masking agents, weighting agents, gelling agents, and gelling agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients, and diluents, including but not limited to: water, gelatin of any source, plant gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, fillers, etc.

[0296] In addition, supplements may contain organic or inorganic carrier materials suitable for oral or parenteral administration, as well as vitamins, trace minerals, and other micronutrients recommended by government agencies such as the USRDA.

[0297] In one embodiment, the nutritional composition comprises

[0298] -Based on dry weight, and based on the total amount of the nutritional composition, 0.02% to 5%, preferably 0.1% to 1.8% of at least one human milk oligosaccharide.

[0299] -10% to 40%, preferably 20% to 30%, of at least one lipid source, such as palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, and medium-chain triglyceride oil. Essential fatty acids, namely linoleic acid and α-linolenic acid, and small amounts of oils containing high levels of pre-formulated arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oil, can also be added.

[0300] -10 3 cfu to 10 12 CFU, preferably 10 5 cfu to 10 10 CFU of Bifidobacterium longum subsp. infantis (Bifidobacterium infantis), preferably LMG 11588 and / or ATCC 15697;

[0301] -0.1% to 1.5%, preferably 0.5% to 1%, of at least one vitamin and / or mineral, such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine.

[0302] -2% to 20%, preferably 5% to 15%, of at least one protein source.

[0303] -30% to 80%, preferably 45% to 65% of carbohydrate sources, preferably lactose, sucrose, cane sugar, maltodextrin, starch and mixtures thereof, more preferably lactose.

[0304] -Optionally, at least one of emulsifiers and stabilizers, such as soybean, lecithin, monoglyceride citrate and diglyceride citrate, and substances capable of having beneficial effects, such as lactoferrin, nucleotides, nucleosides and carotenoids;

[0305] The at least one human milk oligosaccharide in the nutritional composition is, based on dry weight, a percentage of %.

[0306] (a) 0.015% to 3.8%, preferably 0.08% to 1.2% of lactose-N-fucopentose I (LNFP-I).

[0307] (b) 0% to 3.8%, more preferably 0.08% to 1.5% of at least one fucoidan, preferably lactose-N-fucohexose-I (LNDFH-I).

[0308] (c) 0% to 3%, preferably 0.08% to 1.2% of lactose-N-fucopentose II (LNFP-II).

[0309] (d) 0% to 3.8%, preferably 0.04% to 1.2% of lactose-N-fucopentose III (LNFP-III).

[0310] (e) Optionally 0% to 1.5%, preferably 0.04% to 2.3% of at least one N-acetylated human milk oligosaccharide, preferably lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), or a mixture thereof.

[0311] (f) optionally 0% to 1.9%, preferably 0.4% to 1.2%, more preferably 0.6% to 1.2% of at least one other human milk oligosaccharide other than (a) to (e).

[0312] Example

[0313] Materials and methods

[0314] Example 1

[0315] Analysis of HMO blends rich in lactose-N-fucopentose (LNFP) and lactose-N-fucohexaose-I (LNDFH-I) Leave .

[0316] The HMO blend was prepared as follows: Human milk was defatted by centrifugation. Ethanol was added to the defatted milk to a final concentration of approximately 66% (v / v). After incubation overnight at -20°C, the precipitate was removed by centrifugation and decantation through a paper filter. The solution was freeze-dried and redissolved in MilliQ water. The solution was loaded onto a preparative Bio-Gel P-2 gel (Bio-Rad, Hercules, California, USA) size exclusion column and separated. The fractions were collected and analyzed using an HPAEC (High Performance Anion Exchange Chromatography) system equipped with a CarboPac PA1 column (Thermo Fisher Scientific, Dionex, California, USA) and a pulsed current analyzer detector.

[0317] Fractions containing LNFP, LNDFH-I, and LNT were combined and freeze-dried. Figure 1 The relative abundance of individual HMOs in the obtained blends (referred to as HMO blends) is shown.

[0318] The composition of the obtained HMO blends is shown in Figure 1 middle.

[0319] Example 2

[0320] The combination of the HMO blend according to the invention with Bifidobacterium infantis neutralizes enteropathogenic Escherichia coli and Salmonella. bacteria .

[0321] The HMO blend prepared in Example 1 was tested below in combination with *Bifidobacterium longum* subsp. infantile (Bifidobacterium infantis, ATCC 15697). The combination containing 1% (w / v) HMO blend was grown overnight at 37°C under anaerobic conditions with commercially available glucose-free MRS (DeMan, Rogosa, and Sharpe) broth, starting from a single colony, together with *Bifidobacterium longum* subsp. infantile (ATCC 15697). In parallel, other polysaccharides of the comparative examples were also incubated at 1% (w / v). Each overnight culture was diluted to an initial OD600 of 0.1 in DMEM (Dürbeco modified Igor medium) containing 0.1% glucose as a carbon source. This medium was used without any other carbohydrate supplements or containing an additional 1% (w / v) HMO blend or 1% (w / v) of other polysaccharides. Therefore, the DMEM medium was adjusted under anaerobic conditions at 37°C during the overnight incubation. The conditioned culture medium was then centrifuged, and the supernatant was filtered through a 0.22-micron filter to remove Bifidobacterium infantis. The culture medium was then centrifuged and filtered to obtain prepared used culture medium or other polysaccharides as comparative examples.

[0322] Used culture media were incubated with Salmonella typhimurium and enteropathogenic Escherichia coli at different dilutions. Salmonella growth was monitored over time. For Salmonella and EPEC, cell counts were performed after plate inoculation.

[0323] Comparative example

[0324] The non-inventory-based polysaccharide was used to neutralize enteropathogenic Escherichia coli and Salmonella with Bifidobacterium infantis. .

[0325] Repeat the procedure of Example 2 six times, except that each time the HMO blend prepared in Example 1 is replaced with the following substance.

[0326] Glucose (Glc)

[0327] Lactose (Lac)

[0328] 2' Fucosyl lactose (2'FL)

[0329] Lactose-N-tetrasaccharide (LNT)

[0330] Lactose-N-neotetrasaccharide (LNnT)

[0331] 6' Sialolactose (6'SL).

[0332] result .

[0333] The LNFP-I-rich HMO blends of the present invention exhibit the strongest neutralizing activity against both *Salmonella typhimurium* and *Escherichia coli* compared to glucose (Glc), lactose (Lac), 2'-fucosylated lactose (2'FL), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), or 6'-sialylated lactose (6'SL). In particular, neither LNT nor 2'FL had the same effect on *Salmonella typhimurium* growth as the HMO blends of the present invention; however, LNT and 2'FL are considered suitable substrates for *Bifidobacterium infantis*, and therefore, without being bound by any theory, it can be speculated that an alternative mechanism of action is triggered by the HMO blends of the present invention.

[0334] The best inhibition of pathogen growth was observed with the LNFP-I-rich HMO blend according to the invention (Example 1). However, the combination of Bifidobacterium infantis with LNT alone produced lower inhibition. Therefore, it is quite reasonable that the inhibitory (neutralizing) effect observed with the LNFP-I-rich HMO blend according to the invention is due to HMO LNFP-I, LNFP-II and LNFP-III and LNDFH-I, more particularly the most abundant LNFP-I, rather than due to any N-acetylated human milk oligosaccharides, especially LNT.

[0335] Neutralization of Salmonella typhimurium is illustrated in Figure 2 and Figure 3 middle.

[0336] Examples of neutralization of enteropathogenic Escherichia coli include Figure 4 middle.

[0337] Growth was assessed by monitoring the optical density at 600 nm using a photometer.

[0338] Example 3

[0339] Table 1 below provides examples of the composition of infant formula according to the present invention. This composition is given by way of example only.

[0340] Table 1: Composition of the infant formula in Example 1

[0341]

[0342]

[0343] Example 4

[0344] In vitro growth assay

[0345] Bifidobacterium infantis LMG11588 was grown overnight under anaerobic conditions in a minimal medium (MRSAPI) containing 0.25% glucose. After 16 hours of growth, the overnight culture was inoculated with Bifidobacterium infantis LMG11588 in MRSAPI medium supplemented with 0.25% carbon source at an OD of 0.05, in a total volume of 1200 µL.

[0346] The following conditions were tested:

[0347] - Glucose (positive control)

[0348] - No substrate, with no added sugar (negative control)

[0349] -LNFP-I

[0350] -LNT

[0351] The bacteria were grown at 37°C under anaerobic (CO2) conditions for 48 hours without pH control. The initial conditions were set at pH 5.7. Biomass growth was continuously monitored over time to evaluate whether the selected carbon source could support the growth of *Bifidobacterium infantis* LMG11588. The experiment was performed in triplicate. Figure 5 The average value is depicted in the text.

[0352] Biomass increase was continuously monitored over a 48-hour period as a measure of whether and how different carbon sources support the growth of Bifidobacterium infantis LMG11588.

[0353] No growth (as measured by biomass increase) was observed without the addition of a carbon source (no substrate), while the addition of monosaccharides, such as glucose, or the less complex HMO LNT, induced exponential growth, which began to stagnate after 10 hours. *Bifidobacterium infantis* LMG11588 could utilize LNFP-I for growth and, after the adaptation period, exhibited two phases of growth, indicating that *Bifidobacterium infantis* can utilize LNFP-I as the sole carbon source to ensure biomass increase.

[0354] It should be understood that various changes and modifications to the currently preferred embodiments described herein will be apparent to those skilled in the art. These changes and modifications can be made without departing from the spirit and scope of the invention and without diminishing the accompanying advantages. Therefore, such changes and modifications are intended to be covered by the appended claims, provided they remain within the scope of the independent claims.

[0355]

[0356]

[0357]

[0358]

Claims

1. A combination comprising at least one fucosylated human milk oligosaccharide (HMO) and at least one Bifidobacterium longum, said combination for treating, preventing and / or inhibiting the growth of at least one intestinal pathogen in a subject, wherein said at least one fucosylated human milk oligosaccharide comprises lactose-N-fucopentose-I (LNFP-I) as lactose-N-fucopentose, wherein LNFP-I is the most abundant HMO in said combination, wherein said at least one Bifidobacterium longum is Bifidobacterium longum infantis (B. infantis), wherein said subject is preferably an infant, toddler or child.

2. The combination according to claim 1 for the stated purpose, wherein, based on dry weight, lactose-N-fucopentose I (LNFP-I) accounts for at least 5% by weight, preferably at least 20% by weight, more preferably 20% by weight to 100% by weight, and more preferably 25% by weight to 95% by weight of the total human milk oligosaccharides in the combination.

3. The combination for the stated purpose according to claim 1 or 2, wherein the combination further comprises at least one other lactose-N-fucopentose besides LNFP-I, preferably lactose-N-fucopentose II (LNFP-II) and / or lactose-N-fucopentose III (LNFP-III) as other fucosylated human milk oligosaccharides, preferably both.

4. The combination of any one of the preceding claims for the stated use, wherein the combination further comprises at least one fucoidan as another fucoidylated human milk oligosaccharide.

5. The combination for the use according to any one of the preceding claims, wherein, based on dry weight, the total amount of lactose-N-fucopentose accounts for at least 5% by weight, preferably at least 20% by weight, more preferably 15% to 100% by weight, and more preferably 30% to 95% by weight of the total amount of human milk oligosaccharides in the combination.

6. The combination for the use according to any one of the preceding claims, wherein the at least one fucoidylated human milk oligosaccharide comprises at least 5% by weight, preferably at least 10% by weight, preferably 5% to 90% by weight, and more preferably 10% to 80% by weight of at least one fucoidan as other fucoidylated human milk oligosaccharide, based on a dry weight percentage of the total amount of human milk oligosaccharides in the combination.

7. The combination of any one of the preceding claims for the stated use, wherein the at least one fucosylated human milk oligosaccharide comprises lactose-N-fucohexose-I (LNDFH-I) as a fucohexose or is composed of lactose-N-fucohexose-I (LNDFH-I) as a fucohexose.

8. A combination of any of the preceding claims for the stated use, wherein the at least one intestinal pathogen is Salmonella typhymurium.

9. A combination of any of the preceding claims for the stated use, wherein the at least one intestinal pathogen is enteropathogenic Escherichia coli (E. coli) (EPEC).

10. The combination of any one of the preceding claims for the stated use, wherein the at least one subspecies of Bifidobacterium longum infantis (Bifidobacterium infantis) is LMG 11588, ATCC 15697 (NCC 341) or a mixture thereof.

11. The combination for the use according to any one of the preceding claims, wherein the combination further comprises at least one N-acetylated human milk oligosaccharide, preferably lactose-N-tetrasaccharide (LNT), preferably 0.1% to 30% by weight, more preferably 10% to 20% by weight, based on dry weight of the total amount of human milk oligosaccharides in the combination.

12. A nutritional composition comprising a combination according to any one of the preceding claims for the stated purpose.

13. The nutritional composition according to claim 12, wherein the nutritional composition is infant formula, stage 1 infant formula, stage 2 or later infant formula, growing milk, infant food, infant cereal composition, (milk) fortifier or supplement.

14. A nutritional composition or combination comprising at least one fucosylated human milk oligosaccharide and at least one Bifidobacterium longum for non-therapeutic use in treating, preventing, and / or inhibiting the growth of at least one intestinal pathogen in a subject, wherein the at least one fucosylated human milk oligosaccharide comprises lactose-N-fucopentose I (LNFP-I) as lactose-N-fucopentose, wherein LNFP-I is the most abundant HMO in the nutritional composition or combination, wherein the at least one Bifidobacterium longum is Bifidobacterium longum subsp. infantis (Bifidobacterium infantis), preferably LMG 11588, ATCC 15697 (NCC). 341) or mixtures thereof, wherein the pathogen is preferably enteropathogenic Escherichia coli (EPEC) and / or Salmonella typhimurium, wherein the nutritional composition is preferably infant formula, stage 1 infant formula, stage 2 or 2 infant formula, growing milk, infant food, infant cereal composition, (milk) fortifier or supplement, wherein the subject is preferably an infant, toddler or child, wherein at least one fucoidylated human milk oligosaccharide and the corresponding amount are preferably as defined in any one of claims 2 to 7.

15. A method for promoting immunity in a subject in need, the method comprising administering to the subject a combination according to any one of claims 1 to 11 or a nutritional composition according to claim 12 or 13.

16. A nutritional composition for the stated use according to any one of claims 12 to 14, a non-therapeutic use of the nutritional composition according to claim 14, or the method according to claim 15, wherein the nutritional composition comprises -Based on dry weight, and based on the total amount of the nutritional composition, 0.02% to 5%, preferably 0.1% to 1.8% of at least one human milk oligosaccharide. -10% to 40%, preferably 20% to 30%, of at least one lipid source, such as palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, medium-chain triglyceride oil; essential fatty acids, namely linoleic acid and α-linolenic acid, and small amounts of oils containing large amounts of pre-formulated arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oil, may also be added. -10 3 cfu to 10 12 CFU, preferably 10 5 cfu to 10 10 CFU of Bifidobacterium longum subsp. infantis (Bifidobacterium infantis), preferably LMG 11588 and / or ATCC 15697; -0.1% to 1.5%, preferably 0.5% to 1%, of at least one vitamin and / or mineral, such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. -2% to 20%, preferably 5% to 15%, of at least one protein source. -30% to 80%, preferably 45% to 65% of carbohydrate sources, preferably lactose, sucrose, cane sugar, maltodextrin, starch and mixtures thereof, more preferably lactose. -Optionally, at least one of emulsifiers and stabilizers, such as soybean, lecithin, monoglyceride citrate and diglyceride citrate, and substances capable of having beneficial effects, such as lactoferrin, nucleotides, nucleosides and carotenoids; The at least one human milk oligosaccharide in the nutritional composition is, based on dry weight, a percentage of %. (a) 0.015% to 3.8%, preferably 0.08% to 1.2% of lactose-N-fucopentose I (LNFP-I). (b) 0% to 3.8%, more preferably 0.08% to 1.5% of at least one fucoidan, preferably lactose-N-fucohexose-I (LNDFH-I). (c) 0% to 3%, preferably 0.08% to 1.2% of lactose-N-fucopentose II (LNFP-II). (d) 0% to 3.8%, preferably 0.04% to 1.2% of lactose-N-fucopentose III (LNFP-III). (e) Optionally 0% to 1.5%, preferably 0.04% to 2.3% of at least one N-acetylated human milk oligosaccharide, preferably lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), or a mixture thereof. (f) optionally 0% to 1.9%, preferably 0.4% to 1.2%, more preferably 0.6% to 1.2% of at least one other human milk oligosaccharide other than (a) to (e).

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