EP4739149A1

EP4739149A1 - Combination for dietary management of gut health

Info

Publication number: EP4739149A1
Application number: EP24737098.4A
Authority: EP
Inventors: Sophie Hélène NUTTEN; Ralf HEINE; Laurent Favre
Original assignee: Societe des Produits Nestle SA; Nestle SA
Current assignee: Societe des Produits Nestle SA; Nestle SA
Priority date: 2023-07-06
Filing date: 2024-06-28
Publication date: 2026-05-13
Also published as: AU2024288910A1; WO2025008277A1; CN121419685A; JP2026522085A

Abstract

The present invention provides a combination comprising lactose and human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL), lacto-/V-neotetraose (LNnT), 3- fucosyllactose (3'-FL), at least one of 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL), and optionally lacto-N-tetraose (LNT) for use in the dietary management of dysbiosis of the gut microbiota in a subject or for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject.

Description

COMBINATION FOR DIETARY MANAGEMENT OF GUT HEALTH

FIELD OF THE INVENTION

The present invention relates to compositions, particularly a nutritional composition, and methods for the dietary management of gut health; for example in an infant.

BACKGROUND TO THE INVENTION

Mother's milk is recommended for all infants. However, in some cases breast feeding is inadequate or unsuccessful for medical reasons or the mother chooses not to breast feed. Nutritional compositions such as infant formulae have been developed for these situations.

Nutritional compositions for infants and young children are often sold as powders to be reconstituted with water or in some instances as ready to drink or concentrated liquid compositions. Those compositions are intended to cover most or all of the nutritional needs of the infants or young children.

Infant formula manufacturers have therefore made many attempts to induce nutritional health effects close to or similar to the benefits of human breast milk. However, many studies have shown that infant formula do not induce the identical effects on the body compared to human breast milk. For example, infants fed infant formula and infants fed human-breast milk (HBM) can exhibit a different intestinal (gut) microbiota.

HMOs (human milk oligosaccharides) have become the subject of much interest in recent years due to their roles in numerous biological processes occurring in the human organism. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al, Milk Oligosaccharides, Nova Biomedical Books, New York, 2011 , ISBN: 978-1-61122-831-1).

Evidence is accumulating that the resident community of microbes, called the microbiota, in the human digestive tract plays a major role in health and disease. When the composition of the intestinal microbiota is thrown off balance, the host can suffer consequences. Recent research has implicated intestinal microbiota imbalances in individual disorders as diverse as cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism.

Infancy, especially the first weeks, 3 months, 6 months or 12 months of life is a critical period for the establishment of a balanced gut microbiota.

It is known that the modulation of the gut microbiota during infancy can prospectively have a significant influence in the future health status of the body. For example, the gut microbiome can have an influence on the development of a strong immune system later in life, as well as normal growth, and even on the development of obesity or allergy later in life.

Thus, there is a demand for new nutritional interventions for the dietary management of gut health, for example in an infant, in particular where the infant is formula-fed.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that a combination of lactose and specific combinations of human milk oligosaccharides (HMO) synergistically promote beneficial effects for infant gut health, for example by reducing levels of branched chain fatty acids (BCFA). BCFA that are indicative of proteolytic fermentation by the gut microbiota and are associated with the formation of metabolites such as phenol and indole that exert detrimental health effects. Compositions that reduce and/or limit BCFA production may thus have beneficial effects on infant gut health.

In one aspect, the present invention provides a combination comprising lactose and human milk oligosaccharides (HMOs) 2'-fucosyllactose (2’-FL), lacto-ZV-neotetraose (LNnT), 3’-fucosyllactose (3'-FL), at least one of 3’-sialyllactose (3’-SL) or 6’-sialyllactose (6’-SL), and optionally lacto-ZV-tetraose (LNT) for use in the dietary management of dysbiosis of the gut microbiota in a subject. In a related aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3'-FL, LNT, 3’-SL, and 6’-SL for use in the dietary management of dysbiosis of the gut microbiota in a subject.

In another aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, at least one of 3’-SL or 6’-SL, and optionally LNT for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject. In a related aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject.

In a further aspect, the invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, at least one of 3’-SL or 6’-SL, and optional LNT for use in the dietary management of at least one pathogen, preferably a pathogenic bacteria, in a subject. In a related aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’- SL, 6’-SL, and LNT for use in in the dietary management of at least one pathogen, preferably a pathogenic bacteria, in a subject.

In another aspect, the invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, at least one of 3’-SL or 6’-SL, and optionally LNT for use in the reduction of BCFA in a subject. In a related aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT for use in the reduction of BCFA in a subject.

DESCRIPTION OF DRAWINGS

Figure 1 - The impact on branched chain fatty acids (BCFA) for Cow’s Milk Protein Allergy (CMPA) infants (n=12) was tested using the SIFR® technology platform, for 4 HMO blends (M2, M4.1 , M4.2, M6), lactose (L) and combinations thereof (M2+L, M4.1+L, M4.2+L, M6+L) as compared to a no substrate control (NSC). Samples were collected after 6h, 24h and 48h of simulated colonic incubations. Statistical differences between the 4 HMO blends and the NSC are indicated with * (0.01 < p < 0.05), ** (0.001 < p < 0.01) or *** (p < 0.001), while differences between lactose and combinations with the HMO blends and the respective conditions without lactose are indicated with $/$$/$$$. Differences among the HMO blends are indicated with §/§§/§§§ (M4.1/M4.2/M6(+L) versus M2(+L)), #/##/### (M4.2/M6(+L) versus M4.1(+L), &/&&/&&& (M6(+L) versus M4.2(+L)).

Figure 2 - Principal component analysis (PCA) summarizing the impact on microbial community composition of microbial communities derived from CMPA infants (n=12), as tested via the SIFR® technology platform, for 4 HMO blends (M2, M4.1 , M4.2, M6), lactose (L) and combinations thereof (M2+L, M4.1+L, M4.2+L, M6+L) as compared to a no substrate control (NSC). The PCA was calculated based on the (centred) average abundance across the 12 CMPA infants of the 100 most abundant operational taxonomic units (OTUs) as quantified via 16S rRNA sequencing combined with flow cytometry (cells/mL), both at the start (Oh) and at 24h after initiation of the colonic incubations.

Figure 3 - Principal component analysis (PCA) summarizing the impact on the composition of microbial communities derived from CMPA infants (n = 12), as tested via the SIFR® technology platform, for 4 HMO blends (M2, M4.1 , M4.2, M6), lactose (L) and combinations thereof (M2+L, M4.1 +L, M4.2+L, M6+L), as compared to a no substrate control (NSC). The PCA was calculated based on the (centred) average abundances of the significantly affected OTUs (FDR = 0.20) by any of the treatments as quantified via 16S rRNA gene sequencing combined with flow cytometry (cells/mL), at 24h after initiation of the colonic incubations.

DETAILED DESCRIPTION

Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field. All publications mentioned in the specification are herein incorporated by reference.

As used in this specification, the words “comprises”, “comprising”, and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean “including, but not limited to“. The terms “comprises”, “comprising”, and similar words also include the term “consisting of“.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Numeric ranges are inclusive of the numbers defining the range and all percentages disclosed herein are on a w/w basis, unless stated otherwise. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

Combination

In one aspect, the present invention provides a combination of lactose and human milk oligosaccharides (HMOs) 2’-FL, LNnT, 3’-FL, LNT, and at least one of 3’-SL or 6’-SL for use in the dietary management of gut health in a subject. In a related aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3'-FL, LNT, 3’-SL, and 6’- SL for use in the dietary management of dysbiosis of the gut microbiota in a subject.

The combination may be administered to a subject in the form of a composition (e.g. as a composition comprising a lactose mixture and a HMO mixture).

The combination may be administered by any suitable route and in any suitable form. Suitably, the combination is administered by oral and/or enteral administration. In preferred embodiments, the combination is administered by oral administration. The combination may be administered separately, simultaneously or sequentially. In preferred embodiments, the combination is administered simultaneously.

Lactose

Lactose is a disaccharide made up of glucose and galactose. The glucose can be in either the a-pyranose form or the p-pyranose form. Lactose is the main carbohydrate source in breastmilk.

The combination, preferably the nutritional composition, of the present invention may comprise a lactose-containing carbohydrate component, wherein the lactose represents from about 68% to about 97%, optionally from about 70% to about 90%, further optionally from about 72% to about 80%, of the carbohydrate calories. Preferably the nutritional composition is an infant formula as described herein, and any lactose-containing carbohydrate known for use in infant formulas, or otherwise effective for such use, can be used as a lactose-containing carbohydrate component.

In particular embodiments, lactose is from about 49% to about 100% of the non-HMO carbohydrate in the combination, preferably the nutritional composition. In particular embodiments, lactose is about 100% of the non-HMO carbohydrate in the combination, preferably the nutritional composition.

Lactose is present in the nutritional composition in some particular amounts. In an embodiment of the invention, lactose is present in the nutritional composition in an amount up to about 53 g/L or up to about 110g/L of the composition as consumed. In a particular embodiment, lactose is present in an amount of about 37 g/L of the composition as consumed. In a particular embodiment, lactose is present in the nutritional composition in an amount of about 18-110 g/L of the composition as consumed, such as about 24-98 g/L, 49-98 g/L or 58-88 g/L or 65-80 g/L of the composition as consumed. In another particular embodiment, lactose is present in the nutritional composition in an amount of about 73 g/L of the composition as consumed.

In an embodiment of the invention, lactose is present in the nutritional composition in an amount of up to about 34 g per 100 g of composition based on the dry weight of the composition or up to about 67 g per 100g of composition based on the dry weight of the composition. Lactose may be in an amount of about 22-34 g per 100g of the composition based on the dry weight of the composition or about 27 g per 100 g of the composition based on the dry weight of the composition. In an embodiment of the invention, lactose may be in the nutritional composition in an amount of about 44-67 g per 100g of the composition based on the dry weight of the composition. Lactose may be in an amount of about 27 g per 100 g of the composition based on the dry weight of the composition. Lactose may be in an amount of about 55 g per 100 g of the composition based on the dry weight of the composition.

Lactose for use in the present invention is not particularly limited with the exception that it must be suitable for including in a composition, preferably nutritional composition, for administration to a human of any age. Lactose for use in the present invention may be synthesised by any suitable means known in the art, by, e.g. enzymatic, biotechnological, and/or chemical processes including chemical synthesis, fermentation and/or production by suitable bacteria or yeast, or derived or otherwise obtained from cow’s milk, which generally contains about 5% lactose. The lactose can be added in purified or partially purified form, or added as a fractional ingredient of cow’s milk or processed cow’s milk products. Some of the lactose can also be provided as inherent excipients in other added ingredients, e.g., excipient lactose in many protein sources.

The lactose-containing carbohydrate may also contain non-lactose carbohydrates (e.g. a minority of non-lactose carbohydrates), non-limiting sources of which include hydrolyzed or intact, naturally and/or chemically modified, starches sourced from corn, tapioca, rice or potato, in waxy or non-waxy forms. Other non-limiting examples of suitable carbohydrate sources include hydrolyzed corn starch, maltodextrin, glucose polymers, sucrose, corn syrup, corn syrup solids, glucose, fructose, high fructose corn syrup, and combinations thereof.

Suitable doses of lactose are described in e.g. Scientific Opinion on the essential composition of infant and follow-on formulae. EFSA Journal 2014; 12(7):3760, 106 pp. doi:10.2903/j.efsa.2014.3760.

Human Milk Oligosaccharides (HMOs)

HMOs have become the subject of much interest in recent years due to their roles in numerous biological processes occurring in the human organism. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al, Milk Oligosaccharides, Nova Biomedical Books, New York, 2011 , ISBN: 978-1-61122-831-1).

Many different kinds of HMOs are found in human milk. Each oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and/or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk. Almost all HMOs have a lactose moiety at their reducing end while sialic acid and/or fucose (when present) occupy terminal positions at the non-reducing ends. HMOs can be acidic (e.g. charged sialic acid containing oligosaccharides) or neutral (e.g. fucosylated oligosaccharides). In some embodiments, HMOs in the combination comprise, consist essentially of, or preferably consist of 2’-FL, LNnT, 3’-FL, at least one of 3’-SL or 6’-SL, and optionally LNT. Thus, the combination may comprise 2’-FL, LNnT, 3’-FL, and 3’-SL and/or 6’-SL. The combination may comprise 2’-FL, LNnT, 3’-FL, LNT, and 3’-SL and/or 6’-SL. The combination may comprise 2'-FL, LNnT, 3’-FL and 3’-SL. The combination may comprise 2’-FL, LNnT, 3’-FL and 6’-SL. The combination may comprise 2’-FL, LNnT, 3’-FL, 3’-SL and 6’-SL. The combination may comprise 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT. Suitably, the nutritional composition may comprise no other HMO aside from 2’-FL, LNnT, 3’-FL and at least one of 3’-SL or 6’-SL.

In a further embodiment, the HMOs in the composition may comprise, consist essentially of, or consist of 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL and LNT. Thus, the nutritional composition may comprise no other HMO aside from 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL and LNT.

The HMOs may be obtained by any suitable method. Suitable methods for synthesising HMOs will be well known to those of skill in the art. For example, processes have been developed for producing HMOs by microbial fermentation, enzymatic processes, chemical syntheses, or combinations of these technologies (Zeuner et al., 2019. Molecules, 24(11), p.2033).

The 2’-FL may be produced by biotechnological means using specific fucosyltransferases and/or fucosidases either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Alternatively, 2’-FL may be produced by chemical synthesis from lactose and free fucose.

2’-FL may be present in the nutritional composition in some particular amounts. In an embodiment of the invention, 2’-FL is present in the nutritional composition in an amount of up to about 1 .5 g/L of the composition as consumed. 2’-FL may be in the nutritional composition in an amount of 0.2-1 .5 g/L of the composition as consumed, such as 0.25-1.5 g/L or 0.5-1 .5 g/L or 0.8-1.2 g/L of the composition as consumed. In another particular embodiment, 2’-FL is present in the nutritional composition in an amount of about 1.2 g/L of the composition as consumed. In another particular embodiment, 2’-FL is present in the nutritional composition in an amount of about 1.0 g/L of the composition as consumed.

In an embodiment of the invention, 2’-FL is present in the nutritional composition in an amount of up to about 1.2 g per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, 2’-FL, may be in the nutritional composition in an amount of 0.38-1.2 g per 100g of the composition based on the dry weight of the composition, such as 0.2-1 ,2g or 0.38-1 ,2g or 0.61-1 ,5g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 2’-FL is present in the nutritional composition in an amount of about 0.9 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 2’-FL is present in the nutritional composition in an amount of about 0.8 g per 100 g of the composition based on the dry weight of the composition.

The 3’-FL may be synthesized by enzymatic, biotechnological, and/or chemical processes. The 3’-FL may be manufactured through fermentation using a genetically modified microorganism. Alternatively, the 3’-FL may be produced as described in WO 2013/139344.

3’-FL may be present in the nutritional composition in some particular amounts. In an embodiment of the invention, 3’-FL is present in the nutritional composition in an amount of up to about 1.1 g/L of the composition as consumed, 3’-FL may be in the nutritional composition in an amount of 0.1 -1.1 g/L of the composition as consumed, such as 0.13-1.1 g/L or 0.25-0.75 g/L or 0.4-0.6 g/L of the composition as consumed. In another particular embodiment, 3’-FL is present in the nutritional composition in an amount of about 0.9 g/L of the composition as consumed. In another particular embodiment, 3’-FL is present in the nutritional composition in an amount of about 0.5 g/L of the composition as consumed.

In an embodiment of the invention, 3’-FL is present in the nutritional composition in an amount of up to about 0.8 g per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, 3’-FL, may be in the nutritional composition in an amount of 0.08-0.8 g per 100g of the composition based on the dry weight of the composition, such as 0.1-0.8g/L or 0.2-0.6g or 0.3-0.5g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 3’-FL is present in the nutritional composition in an amount of about 0.7g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 3’-FL is present in the nutritional composition in an amount of about 0.4 g per 100 g of the composition based on the dry weight of the composition.

The LNnT may be synthesised chemically by enzymatic transfer of saccharide units from donor moieties to acceptor moieties using glycosyltransferases as described, for example, in US Patent No. 5,288,637 and WO 1996/010086. Alternatively, LNnT may be prepared by chemical conversion of Keto-hexoses (e.g. fructose) either free or bound to an oligosaccharide (e.g. lactulose) into N-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharide as described in Wrodnigg, T.M. and Stutz, A.E. (1999) Angew. Chem. Int. Ed. 38: 827-828. N-acetyl-lactosamine produced in this way may then be transferred to lactose as the acceptor moiety. Alternatively, the LNnT may be produced as described in WO 2011/100980 or WO 2013/044928.

LNnT may be present in the nutritional composition in some particular amounts. In an embodiment of the invention, LNnT is present in the nutritional composition in an amount of up to about 0.75 g/L of the composition as consumed, LNnT may be in the nutritional composition in an amount of 0.1-0.75 g/L of the composition as consumed, such as 0.13-0.75 g/L or 0.25-0.75 g/L or 0.4-0.6 g/L of the composition as consumed. In another particular embodiment, LNnT is present in the nutritional composition in an amount of about 0.7 g/L of the composition as consumed. In another particular embodiment, LNnT is present in the nutritional composition in an amount of about 0.5 g/L of the composition as consumed.

In an embodiment of the invention, LNnT is present in the nutritional composition in an amount of up to about 0.6 g per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, LNnT, may be in an amount of 0.08-0.6 g per 100g of the composition based on the dry weight of the composition, such as 0.1-0.6g/L or 0.2-0.6g or 0.3-0.5g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, LNnT is present in the nutritional composition in an amount of about 0.6g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, LNnT is present in the nutritional composition in an amount of about 0.4 g per 100 g of the composition based on the dry weight of the composition.

The 3’-SL may be synthesized by enzymatic, biotechnological, and/or chemical processes. The 3’-SL may be produced as described in WO 2014/153253.

3’-SL may be present in the nutritional composition in some particular amounts. In an embodiment of the invention, 3’-SL is present in the nutritional composition in an amount of up to about 0.24 g/L of the composition as consumed, 3’-SL may be in the nutritional composition in an amount of 0.02-0.24 g/L of the composition as consumed, such as 0.025-0.24 g/L or 0.05- 0.15 g/L or 0.08-0.12 g/L of the composition as consumed. In another particular embodiment, 3’-SL is present in the nutritional composition in an amount of about 0.2 g/L of the composition as consumed. In another particular embodiment, 3’-SL is present in the nutritional composition in an amount of about 0.1 g/L of the composition as consumed.

In an embodiment of the invention, 3’-SL is present in the nutritional composition in an amount of up to about 0.18 g per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, 3’-SL, may be in the nutritional composition in an amount of 0.015-0.18 g per 100g of the composition based on the dry weight of the composition, such as 0.019-0.18 g/L or 0.038-0.11 g or 0.06-0.09 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 3’-SL is present in the nutritional composition in an amount of about 0.15 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 3’-SL is present in the nutritional composition in an amount of about 0.08 g per 100 g of the composition based on the dry weight of the composition.

The 6’-SL may be synthesized by chemical methods including stereoselective 6’-O-sialylation of either 4’,6’-sugar diols or 6’-sugar alcohols using glycosylhalide, thioglycoside or diethylphosphite donor activations. Alternatively, the 6’-SL may be enzymatically produced using glycosyltransferases and sialidases. The 6’-SL may be produced as described in WO 2011/100979.

6’-SL may be present in the nutritional composition in some particular amounts. In an embodiment of the invention, 6’-SL is present in the nutritional composition in an amount of up to about 0.84 g/L of the composition as consumed, 6’-SL may be in the nutritional composition in an amount of 0.04-0.84 g/L of the composition as consumed, such as 0.05-0.84 g/L or 0.1- 0.3 g/L or 0.16-0.24g/L of the composition as consumed. In another particular embodiment, 6’- SL is present in the nutritional composition in an amount of about 0.7 g/L of the composition as consumed. In another particular embodiment, 6’-SL is present in the nutritional composition in an amount of about 0.2 g/L of the composition as consumed.

In an embodiment of the invention, 6’-SL is present in the nutritional composition in an amount of up to about 0.64 g per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, 6’-SL, may be in the nutritional composition in an amount of 0.003-0.64 g per 100g of the composition based on the dry weight of the composition, such as 0.038-0.64 g/L or 0.076-0.23 g or 0.12-0.18 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 6’-SL is present in the nutritional composition in an amount of about 0.53 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, 6’-SL is present in the nutritional composition in an amount of about 0.08 g per 100 g of the composition based on the dry weight of the composition.

The LNT may be synthesized by enzymatic, biotechnological and/or chemical processes. The LNT may be produced as described in WO 2012/155916 or WO 2013/044928. A mixture of LNT and LNnT can be made as described in WO 2013/091660.

LNT may be present in the nutritional composition in some particular amounts. In an embodiment of the invention, LNT is present in the nutritional composition in an amount of up to about 0.96 g/L of the composition as consumed, LNT may be in an amount of 0.06-0.96 g/L of the composition as consumed, such as 0.075-0.96 g/L or 0.15-0.45 g/L or 0.24-0.36 g/L of the composition as consumed. In another particular embodiment, LNT is present in the nutritional composition in an amount of about 0.8 g/L of the composition as consumed. In another particular embodiment, LNT is present in the nutritional composition in an amount of about 0.3 g/L of the composition as consumed.

In an embodiment of the invention, LNT is present in the nutritional composition in an amount of up to about 0.73 g per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, LNT may be in the nutritional composition in an amount of 0.046-0.73 g per 100g of the composition based on the dry weight of the composition, such as 0.057-0.73 g/L or 0.11-0.34 g or 0.18-0.27 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, LNT is present in the nutritional composition in an amount of about 0.6 g per 100 g of the composition based on the dry weight of the composition. In another particular embodiment, LNT is present in the nutritional composition in an amount of about 0.2 g per 100 g of the composition based on the dry weight of the composition.

In some embodiments the combination comprises the HMOs 2’-FL, LNnT, 3’-FL, and 3’-SL. In some embodiments, the HMOs in the combination consist of, or consist essentially of, 2’-FL, LNnT, 3’-FL, and 3’-SL. In a particular embodiment, the combination comprises, consists essentially of the HMOs, or consists of the HMOs 2’-FL (about 0.8-1.2 g/L of the composition as consumed), 3’-FL (about 0.4-0.6 g/L of the composition as consumed), LNnT (about 0.4-0.6 g/L of the composition as consumed), 3’-SL (about 0.08-0.12 g/L of the composition as consumed), and optionally LNT (about 0.24-0.36g/L of the composition as consumed).

In some embodiments the combination comprises the HMOs 2’-FL, LNnT, 3’-FL, and 6’-SL. In some embodiments, the HMOs in the combination consist of, or consist essentially of, 2’-FL, LNnT, 3’-FL, and 6’-SL. In a particular embodiment, the combination comprises, consists essentially of the HMOs, or consists of the HMOs 2’-FL (about 0.8-1.2 g/L of the composition as consumed), 3’-FL (about 0.4-0.6 g/L of the composition as consumed), LNnT (about 0.4-0.6 g/L of the composition as consumed), 6’-SL (about 0.16-0.24 g/L of the composition as consumed), and optionally LNT (about 0.24-0.36g/L of the composition as consumed).

In some embodiments, in the combination, preferably the nutritional composition, comprise with reference to the total amount of HMOs in the combination, preferably nutritional composition: i. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; ii. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; iii. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; and iv. about 2 wt % to about 7 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; and about 4 wt % to about 13 wt % of 6’-SL, preferably about 7 wt % to about 10 wt %, preferably about 9 wt %.

In some embodiments, in the combination, preferably the nutritional composition, comprise with reference to the total amount of HMOs: i. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; ii. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; iii. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; and iv. about 2 wt % to about 7 wt % of 3’-SL; preferably about 4 wt % to about 6 wt %, preferably about 5 wt %; or about 5 wt % to about 14 wt % of 6’-SL, preferably about 7 wt % to about 11 wt %, preferably about 9 wt %.

In some embodiments the invention provides a combination comprising the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL and LNT. In some embodiments, the HMOs in the combination consist of, or consist essentially of, 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL and LNT. In a particular embodiment, the combination comprises, consists essentially of the HMOs, or consists of the HMOs 2’-FL (about 0.8-1 .2 g/L of the composition as consumed), 3’-FL (about 0.4-0.6 g/L of the composition as consumed), LNnT (about 0.4-0.6 g/L of the composition as consumed); 3’-SL (about 0.08-0.12 g/L of the composition as consumed), 6’-SL (about 0.16-0.24 g/L of the composition as consumed), and LNT (about 0.24-0.36g/L of the composition as consumed).

In some embodiments, in the combination, preferably the nutritional composition, comprise with reference to the total amount of HMOs: i. about 19 wt % to about 58 wt % of 2’-FL, preferably about 31 wt % to about 46 wt %, preferably about 38 wt % ; ii. about 10 wt % to about 29 wt % of LNnT, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; iii. about 10 wt % to about 29 wt % 3’-FL, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; and iv. about 2 wt % to about 6 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; v. about 54 wt % to about 12 wt % of 6’-SL, preferably about 6 wt % to about 9 wt %, preferably about 8 wt %; and vi. about 6 wt % to about 17 wt % of LNT, preferably about 9 wt % to about 14 wt %, preferably about 12 wt %.

In a particular embodiment of the present invention, the nutritional composition comprises the 2’-FL and LNnT in a 2’-FL:LNnT weight ratio from 1 :10 to 12:1 , such as from 1 :7 to 10:1 or from 1 :5 to 5:1 or from 2:1 to 5:1 or from 1 :3 to 3:1 , or from 1 :2 to 2:1 , or from 1 :1 to 3:1 , or from 1 :5 to 1 :0.5; for example 2:1 or 10:1. In a particular embodiment of the present invention, the nutritional composition comprises the 2’-FL and LNnT in a 2’-FL:LNnT weight ratio of about 2:1.

The subject may be administered any suitable amounts of HMOs, in any suitable form and via any suitable route of administration (e.g. in any form and via any route described herein).

Suitable doses of human oligosaccharides are described in e.g. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2015. EFSA Journal, 13(11), p.4299; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2019. EFSA Journal, 17(6), p.e05717; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2020. EFSA Journal, 18(5), p.e06097; EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2022. EFSA Journal, 20(5), p.e07331 ; and EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), 2019. EFSA Journal, 17(12), p.e05907.

Suitably, the HMO combination is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.1 g/day, at least about 0. 5 g/day, at least about 1 g/day, or at least about 2 g/day. Suitably, the HMO combination is administered to the subject in an amount of about 10 g/day or less, about 8 g/day or less, or about 5 g/day or less. Suitably, the HMO combination is administered to the subject in an amount of from about 0.05 g/day to about 10 g/day, from about 0.1 g/day to about 10 g/day, from about 0.5 g/day to about 10 g/day, from about 1 g/day to about 8 g/day or from about 2 g/day to about 5 g/day.

Suitably, 2’-FL is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.1 g/day, at least about 0.2 g/day. At least about 0.5 g/day at least about 0.8 g/day, or at least about 1 g/day. Suitably, 2’-FL is administered to the subject in an amount of about 5 g/day or less, about 4 g/day or less, or about 3 g/day or less. Suitably, 2’-FL is administered to the subject in an amount of from about 0.5 g/day to about 5 g/day, from about 0.8 g/day to about 4 g/day or from about 1 g/day to about 3 g/day. Suitably, LNnT is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.2 g/day, at least about 0.3 g/day, or at least about 0.4 g/day. Suitably, LNnT is administered to the subject in an amount of about 2.5 g/day or less, about 2 g/day or less, or about 1 .5 g/day or less. Suitably, LNnT is administered to the subject in an amount of from about 0.2 g/day to about 2.5 g/day, from about 0.3 g/day to about 2 g/day or from about 0.4 g/day to about 1.5 g/day.

Suitably, 3’-FL is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.1 g/day, at least about 0.15 g/day, or at least about 0.2 g/day. Suitably, 3’-FL is administered to the subject in an amount of about 1 g/day or less, about 0.8 g/day or less, or about 0.6 g/day or less. Suitably, 3’-FL is administered to the subject in an amount of from about 0.1 g/day to about 1 g/day, from about 0.15 g/day to about 0.8 g/day or from about 0.2 g/day to about 0.6 g/day.

Suitably, 3’-SL is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.1 g/day, at least about 0.15 g/day, or at least about 0.2 g/day. Suitably, 3’-SL is administered to the subject in an amount of about 1 g/day or less, about 0.8 g/day or less, or about 0.6 g/day or less. Suitably, 3’-SL is administered to the subject in an amount of from about 0.1 g/day to about 1 g/day, from about 0.15 g/day to about 0.8 g/day or from about 0.2 g/day to about 0.6 g/day.

Suitably, 6’-SL is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.1 g/day, at least about 0.15 g/day, or at least about 0.2 g/day. Suitably, 6’-SL is administered to the subject in an amount of about 1 g/day or less, about 0.8 g/day or less, or about 0.6 g/day or less. Suitably, 6’-SL is administered to the subject in an amount of from about 0.1 g/day to about 1 g/day, from about 0.15 g/day to about 0.8 g/day or from about 0.2 g/day to about 0.6 g/day.

Suitably, LNT is administered to the subject in an amount of at least about 0.05 g/day, at least about 0.2 g/day, at least about 0.3 g/day, or at least about 0.4 g/day. Suitably, LNT is administered to the subject in an amount of about 2.5 g/day or less, about 2 g/day or less, or about 1.5 g/day or less. Suitably, LNT is administered to the subject in an amount of from about 0.2 g/day to about 2.5 g/day, from about 0.3 g/day to about 2 g/day or from about 0.4 g/day to about 1.5 g/day.

Suitably, 2’-FL is administered to the subject in an amount of from about 0.05 g/day to about 5 g/day; LNnT is administered to the subject in an amount of from about 0.05 g/day to about 2.5 g/day; 3’-FL is administered to the subject in an amount of from about 0.05 g/day to about 1 g/day; 3’-SL is administered to the subject in an amount of from about 0.05 g/day to about 1 g/day; 6’-SL is administered to the subject in an amount of from about 0.05 g/day to about 1 g/day; and/or LNT is administered to the subject in an amount of from about 0.05 g/day to about 2.5 g/day.

The term “consisting essentially of” as used herein means that any additional, non-recited members, elements or steps do not materially affect the characteristics of the claimed apparatus, composition, method, etc. Suitably, a composition comprising HMOs which “consist essentially of’ recited HMOs may comprise trace amounts of non-recited HMOs which do not materially affect the characteristics of the composition.

Nutritional composition

The expression “nutritional composition” means a composition which nourishes a subject.

The nutritional composition is usually to be taken orally and it usually includes a lipid or fat source and a protein source.

In a particular embodiment, the nutritional composition is a synthetic nutritional composition. The expression “synthetic nutritional composition” means a mixture obtained by chemical and/or biological means (i.e. the synthetic nutritional composition is not breast milk).

The nutritional composition of the present invention can be in solid form (e.g. powder) or in liquid form. The amount of the various ingredients (e.g. the oligosaccharides) can be expressed in g/100g of composition on a dry weight basis when it is in a solid form, e.g. a powder, or as a concentration in g/L of the composition when it refers to a liquid form (this latter also encompasses liquid composition that may be obtained from a powder after reconstitution in a liquid such as milk, water... , e.g. a reconstituted infant formula or a follow-on/follow-up formula or a growing-up milk or an infant cereal product or any other formulation designed for infant nutrition). Preferably, the nutritional composition in powder form and is reconstituted in water.

In a preferred embodiment, the nutritional composition is for an infant or young child. The infant may be, for example, 0-12 months. The young-child may be, for example, 1-3 years of age. In a particularly preferred embodiment, the nutritional composition is an infant formula or a youngchild formula.

The term “infant formula” may refer to a foodstuff intended for particular nutritional use by infants during the first year of life and satisfying by itself the nutritional requirements of this category of person, as defined in European Commission Regulation (Ell) 2016/127 of 25 September 2015.

The expression “infant formula” encompasses both “starter infant formula” and “follow-up formula” or “follow-on formula”. A “follow-up formula” or “follow-on formula” is given from the 6^th month onwards.

The infant formula of the present invention may be a hypoallergenic infant formula. The infant formula of the present invention may be an extensively hydrolysed infant formula (eHF) or an amino acid-based infant formula (AAF). Alternatively, the infant formula may be a partially hydrolysed infant formula (pHF).

The term “extensively hydrolysed formula” or “eHF” may refer to a formula comprising extensively hydrolysed protein. The eHF may be a hypoallergenic infant formula which provides complete nutrition for infants who cannot digest intact cow’s milk protein (CMP) or who are intolerant or allergic to CMP.

The term “amino acid-based formula” or “AAF” may refer to a formula comprising only free amino acids as a protein source. The AAF may contain no detectable peptides. The AAF may be a hypoallergenic infant formula which provides complete nutrition for infants with food protein allergy and/or food protein intolerance. For example, the AAF may be a hypoallergenic infant formula which provides complete nutrition for infants who cannot digest intact CMP or who are intolerant or allergic to CMP, and who may have extremely severe or life-threatening symptoms and/or sensitisation against multiple foods.

A “hypoallergenic” composition is a composition which is unlikely to cause allergic reactions and wherein the source of protein is provided by extensively hydrolysed protein or by free amino acids. A hypoallergenic infant formula may be tolerated by more than 90% of infants with CMP allergy. This is in line with the guidance provided by the American Academy of Pediatrics (Committee on Nutrition, 2000. Pediatrics, 106(2), pp.346-349).

Infants can be fed solely with the infant formula or the infant formula can be used as a complement of human milk.

The term “young-child formula” may refer to a foodstuff intended to partially satisfy the nutritional requirements of young children ages 1 to 3 years. The expression “young-child formula” encompasses “toddler’s milk”, “growing up milk”, or “formula for young children”. The ESPGHAN Committee on Nutrition has recently reviewed the young-child formula (Hojsak, l.,et al., 2018. Journal of pediatric gastroenterology and nutrition, 66(1), pp.177-185). Suitably, a young-child formula may meet the compositional requirements proposed in Hojsak, I., et al., 2018. Journal of pediatric gastroenterology and nutrition, 66(1), pp.177-185 and/or Suthutvoravut, II., et al., 2015. Annals of Nutrition and Metabolism, 67(2), pp.119-132.

The young-child formula of the present invention may be a hypoallergenic young-child formula. The young-child formula of the present invention may be an extensively hydrolysed young-child formula or an amino acid-based young-child formula. Alternatively, the young-child formula may be a partially hydrolysed young-child formula (pHF).

The infant formula or a young-child formula of the invention may be in the form of a powder or liquid.

The liquid may be, for example, a concentrated liquid formula or a ready-to-feed formula. The formula may be in the form of a reconstituted infant or young-child formula (i.e. a liquid formula that has been reconstituted from a powdered form). The concentrated liquid infant or young child formula is preferably capable of being diluted into a liquid composition suitable for feeding an infant or child, for example by the addition of water.

In some embodiments, the infant or young-child formula is in a powdered form. The powder is capable of being reconstituted into a liquid composition suitable for feeding an infant or child, for example by the addition of water.

The nutritional composition may have an energy density of about 60-72 kcal per 100 mL, when formulated as instructed. Suitably, the nutritional composition may have an energy density of about 60-70 kcal per 100 mL, when formulated as instructed.

The nutritional composition according to the invention can be for example an infant formula, a starter infant formula, a follow-on or follow-up formula, a fortifier such as a human milk fortifier, or a supplement. In some particular embodiments, the composition of the invention is an infant formula, a young-child formula or a supplement. In one preferred embodiment the nutritional composition of the invention is an infant formula.

Within the context of the present invention, the term “fortifier” refers to a composition which comprises one or more nutrients having a nutritional benefit for infants. By the term “milk fortifier”, it is meant any composition used to fortify or supplement either human breast milk, infant formula, growing-up milk or human breast milk fortified with other nutrients. Accordingly, the human milk fortifier of the present invention can be administered after dissolution in human breast milk, infant formula, growing-up milk or human breast milk fortified with other nutrients, or otherwise it can be administered as a stand-alone composition. When administered as a stand-alone composition, the human milk fortifier of the present invention can be also identified as being a “supplement”. In one embodiment, the milk fortifier of the present invention is a supplement. In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (e.g. a human milk fortifier) or a formula fortifier such as an infant formula fortifier or a follow- on/follow-up formula fortifier. In some other embodiments, the nutritional composition of the present invention is a dietary supplement. When the nutritional composition is a supplement, it can be provided in the form of unit doses. The supplement may be in the form of tablets, capsules, pastilles or a liquid for example. The supplement may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface-active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste-masking agents, weighting agents, jellifying agents and gel forming agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients, and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, lignin-sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like.

Further, the supplement may contain an organic or inorganic carrier material suitable for oral or parenteral administration as well as vitamins, minerals trace elements and other micronutrients under the recommendations of Government bodies such as the LISRDA.

Other product formats like beverages and powders (sachet format) can also be chosen. In a further embodiment, the nutritional composition is selected from the group consisting of a beverage product, an amino acid-based beverage, a yogurt product, fermented milk, a fruit juice, a dried powder in sachet format or a cereal bar. These nutritional compositions are well suited for administering plant phenols to, for example, older children and adult humans.

In a further embodiment, the nutritional composition is a food for specific medical purposes such as a health care nutritional composition for oral feeding, and/or a nutritional product for enteral or parental feeding. In the latter case, it will only include ingredients that are suitable for parenteral feeding. Ingredients that are suitable for parental feeding are known to the person skilled in the art. The nutritional composition of the present invention can be in solid (e.g. powder), liquid or gelatinous form.

Protein

The term “protein” includes peptides and free amino acids. The protein content of the nutritional composition may be calculated by any method known to those of skill in the art. Suitably, the protein content may be determined by a nitrogen-to-protein conversion method. For example, as described in Maubois, J.L. and Lorient, D. (2016) Dairy Science & Technology 96(1): 15-25. Preferably the protein content is calculated as nitrogen content x 6.25, as defined in European Commission Regulation (Ell) 2016/127 of 25 September 2015. The nitrogen content may be determined by any method known to those of skill in the art. For example, nitrogen content may be measured by the Kjeldahl method.

The protein content of the nutritional composition of the invention, particularly the infant formula of the invention, is preferably in the range 1.6-3.2 g protein per 100 kcal. In some embodiments, the protein content of the nutritional composition is in the range 1.8-3.0 g protein per 100 kcal.

Infant formulas, such as an eHF or an AAF, with a lower protein content may support appropriate growth and development of allergic infants, as well as being safe and well- tolerated.

Accordingly, in some embodiments, the nutritional composition of the invention, particularly the infant formula of the invention, may comprise up to about 2.5 g protein per 100 kcal. For example, the nutritional composition may comprise about 2.3 g or less protein per 100 kcal, 2.0 g or less protein per 100 kcal, about 1.9 g protein per 100 kcal.

Suitably, the nutritional composition of the invention, particularly the infant formula of the invention, comprises about 1.8 g or more protein per 100 kcal. For example, the nutritional composition may comprise about 1.86 g or more protein per 100 kcal, 1.9 g or more protein per 100 kcal, 2.0 g or more protein per 100 kcal, or 2.1 g or more protein per 100 kcal. In some embodiments, the nutritional composition comprises about 1.86 g or more protein per 100 kcal, in line with present Ell regulations for infant formula (EFSA NDA Panel (2014) EFSA journal 12(7): 3760).

In some embodiments, the nutritional composition of the invention, particularly the infant formula of the invention, may comprise 1.8-2.8 g protein per 100 kcal, 1.86-2.4g protein per 100 kcal, 1.9-2.4 g protein per 100 kcal, 2.0-2.4 g protein per 100 kcal, 2.0-2.3 g protein per 100 kcal, 2.1-2.3 g protein per 100 kcal, or 2.15-2.25 g protein per 100 kcal.

In some embodiments, the nutritional composition of the invention, particularly the infant formula of the invention, may comprise 2.24-3.36 g protein per 100 kcal or 2.5-3.1g protein per 100 kcal.

Protein source

The source of protein may be any source suitable for use in a nutritional composition. The protein source may comprise free amino acids as the only protein source or comprise an animal and/or vegetable derived protein as the only protein source.

In some embodiments, the protein source is comprised or consists of a mix of animal derived protein and vegetable derived protein. In one aspect, the protein source is comprised or consists of a mix of a cow’s milk protein, preferably extensively hydrolysed whey protein, and one or more hydrolysed vegetable proteins, preferably pea protein. In some embodiments, the protein is cow’s milk protein. In some embodiments, the protein is the cow’s milk protein casein, whey, or a combination thereof.

In some embodiments, the nutritional composition does not comprise cow’s milk protein. In some embodiments, the protein source is a hydrolysed vegetable protein such as a rice-based formula or a soy-based formula or a pea-based formula or a combination of at least two of rice, soy and pea.

In some embodiments, the nutritional composition does not comprise dairy protein. Accordingly, in some embodiments, 100% by weight of the total protein is non-dairy protein.

In some embodiments, the combination, preferably nutritional composition, comprises an extensively hydrolysed dairy protein such as a protein derived from goat milk, donkey milk, camel milk, or cow milk. In some embodiments, the combination, preferably nutritional composition, comprises an extensively hydrolysed cow’s milk protein such as extensively hydrolysed casein or extensively hydrolysed whey.

An extensively hydrolysed/hydrolysed whey-based formula may be more palatable than an extensively hydrolysed/hydrolysed casein-based formula and/or the subject may only be sensitised to casein protein. Suitably, therefore, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or about 100% of the protein is whey protein. Preferably, the protein source is whey protein.

The whey protein may be a whey from cheese making, particularly a sweet whey such as that resulting from the coagulation of casein by rennet, an acidic whey from the coagulation of casein by an acid, or the acidifying ferments, or even a mixed whey resulting from coagulation by an acid and by rennet. This starting material may be whey that has been demineralised by ion exchange and/or by electrodialysis and is known as demineralised whey protein (DWP).

The source of the whey protein may be sweet whey from which the caseino- glycomacropeptide (CGMP) has been totally or partially removed. This is called modified sweet whey (MSW). Removal of the CGMP from sweet whey results in a protein material with threonine and tryptophan contents that are closer to those of human milk. A process for removing CGMP from sweet whey is described in EP880902.

The whey protein may be a mix of DWP and MSW.

In some embodiments, the amount of casein in the nutritional composition is undetectable, for example less than 0.2 mg/kg. The amount of casein may be determined by any method known to those of skill in the art. Degree of hydrolysis

Hydrolysed proteins may be characterised as “partially hydrolysed” or “extensively hydrolysed” depending on the degree to which the hydrolysis reaction is carried out. Currently there is no agreed legal/clinical definition of Extensively Hydrolyzed Products according to the WAG (World Allergy Organization) guidelines for Cow’s milk protein allergy (CMPA) but there is agreement that according to the WAO that hydrolysed formulas have proven to be a useful and widely used protein source for infants suffering from CMA. In the current invention partially hydrolysed proteins are one in which 60-70% of the protein/peptide population has a molecular weight of less than 1000 Daltons, whereas extensively hydrolysed proteins are one in which at least 95% of the protein/peptide population has a molecular weight of less than 1000 Dalton. See, e.g. BSACI guideline for the diagnosis and management of cow’s milk allergy, Clinical & Experimental Allergy (2014) 44:642-672. extensively hydrolysed proteins are alternatively defined in the industry as one in which at least 95% of the protein/peptide population has a molecular weight of less than 1200 Dalton. See Nutten et al. Allergy (2020) 75:1446-1518. Partially hydrolysed proteins are usually considered as hypoallergenic (HA) whereas extensively hydrolysed proteins are usually considered as non-allergenic.

The hydrolysed proteins of the invention may have an extent of hydrolysis that is characterised by NPN/TN %. Non-Protein Nitrogen over Total Nitrogen is widely used as a measure of soluble peptides created by enzymatic hydrolysis. NPN/TN% means the Non Protein Nitrogen divided by the Total Nitrogen X 100. NPN/TN% may be measured as detailed in Adler-Nissen J-, 1979, J. Agric. Food Chem., 27 (6), 1256-1262. In general, extensively hydrolysed proteins are characterised as having a NPN/TN% of greater than 95%, whereas partially hydrolysed proteins are characterized as having a NPN/TN% in the range 75%-85%. Partially hydrolysed proteins may also be characterised in that 60-70% of their protein/peptide population has a molecular weight of less than 1000 Daltons.

In a preferred embodiment, the protein may have an NPN/TN% greater than 90%, greater than 95% or greater than 98%. In a preferred embodiment where “extensively” hydrolysed proteins are desired the hydrolysed proteins of the invention has a NPN/TN % in the range of greater than 95%. Suitably, the protein may have an NPN/TN% greater than 90%, greater than 95% or greater than 98%. These extensively hydrolysed proteins may also be characterised in that at least 95% of their protein/peptide population has a molecular weight of less than 1000 Daltons.

The extent of hydrolysis may also be determined by the degree of hydrolysis. The “degree of hydrolysis” (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate and may be determined by any method known to those of skill in the art. Suitably the degree of hydrolysis is determined by pH-stat, trinitrobenzenesulfonic acid (TNBS), o- phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), or formol titration methods. (Rutherfurd, S.M. (2010) Journal of AOAC International 93(5): 1515-1522). The degree of hydrolysis (DH) of the protein can, for example, be more than 90, more than 95 or more than 98.

The extent of hydrolysis may also be determined by the peptide molecular mass distribution. The peptide molecular mass distribution may be determined by high performance size exclusion chromatography, optionally with UV detection (HPSEC/UV) (Johns, P.W. et al. (2011) Food chemistry 125(3): 1041-1050). For example, the peptide molecular mass distribution may be a HPSEC peak area-based estimate determined at 205 nm, 214 nm or 220 nm. Suitably when the peptide molecular mass distribution is determined by HPSEC/UV, the “percentage of peptides by weight” that have a certain molecular mass may be estimated by the “fraction of peak area as a percentage of total peak area”, that have the molecular mass, determined at 205 nm, 214 nm or 220 nm. Suitably, the extent of hydrolysis may be determined by the methods described in WO 2016/156077. Alternatively, the peptide molecular mass distribution may be determined by any method known to those of skill in the art, for example by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Chauveau, A. et al. (2016) Pediatric Allergy and Immunology 27(5): 541-543). Theoretically, to bind with cell membrane-bound Immunoglobulin E (IgE), peptides should be greater than about 1500 Da in size (approximately 15 amino acids) and to crosslink IgE molecules and to induce an immune response, they must be greater than about 3000 Da in size (approximately 30 amino acids) (Nutten (2018) EMJ Allergy Immunol 3(1): 50-59).

Suitably, therefore, at least about 95%, at least about 98%, at least about 99% or about 100% of the peptides by weight in the eHF have a molecular mass of less than about 3000 Da. There may, for example, be no detectable peptides about 3000 Da or greater in size in the eHF.

Suitably, therefore, at least about 95%, at least about 98%, at least about 99% or about 100% of the peptides by weight in the eHF have a molecular mass of less than about 1500 Da. Preferably, at least 99% of the peptides by weight have a molecular mass of less than about 1500 Da. There may, for example, be no detectable peptides about 1500 Da or greater in size in the eHF.

Preferably, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the peptides by weight in the eHF have a molecular mass of less than about 1200 Da. More preferably, at least 95% or 98% of the peptides by weight in the eHF have a molecular mass of less than about 1200 Da. Suitably, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the peptides by weight in the eHF have a molecular mass of less than about 1000 Da. Preferably, at least about 95% of the peptides by weight in the eHF have a molecular mass of less than about 1000 Da.

Preferably, the eHF has no detectable peptides about 3000 Da or greater in size; and at least about 95% of the peptides by weight have a molecular mass of less than about 1200 Da.

Having a high proportion of di- and tri-peptides may improve nitrogen (protein) absorption, even in patients with gut impairment. PEPT 1 is a dedicated facilitator transport route for small peptide absorption (e.g. di- and tri-peptides). In the first weeks of life, intestinal PEPT1 is important for nutritional intake, and later for diet transition following weaning.

Thus, at least about 30%, at least about 40%, or at least about 50% of the peptides by weight in the eHF may, for example, be di- and tri-peptides. Preferably, at least about 45%, at least about 50%, 45-55%, or 50-54% of the peptides by weight in the eHF are di- and tri-peptides. More preferably, about 51-53%, or most preferably, about 52% of the peptides by weight in the eHF are di- and tri-peptides. Suitably, at least about 30%, at least about 40%, at least about 50%, or at least 80%, of the peptides by weight in the eHF have a molecular mass of less than 600 Da. Preferably, at least about 45%, at least about 50%, at least 60-80%, or at least 80% of the peptides by weight in the eHF have a less than 600 Da. More preferably, at least about 80of the peptides by weight in the eHF have a molecular mass of between 240 and 600 Da.

The principal recognised cow’s milk allergens are alpha-lactalbumin (aLA), beta-lactoglobulin (bLG), and bovine serum albumin (BSA).

Suitably, therefore, the eHF may have non-detectable aLA content, for example about 0.010 mg/kg aLA or less; the eHF may have non-detectable bLG content, for example about 0.010 mg/kg bLG or less; and/or the eHF may have non-detectable BSA content, for example about 0.010 mg/kg BSA or less. Preferably, the eHF comprises no detectable amounts of aLA, bLG, and BSA. The content of aLA, bLG, and BSA may be determined by any method known to those of skill in the art, for example ELISA.

Method of hydrolysis

Proteins for use in the nutritional composition, preferably the infant formula of the invention, may be hydrolysed by any suitable method known in the art. For example, proteins may be enzymatically hydrolysed, for example using a protease. For example, protein may be hydrolysed using alcalase (e.g. at an enzyme:substrate ratio of about 1-15% by weight and for a duration of about 1-10 hours). The temperature may range from about 40°C to 60°C, for example about 55°C. The reaction time may be, for example, from 1 to 10 hours, and pH values before starting hydrolysis may, for example, fall within the range 6 to 9, preferably 6.5 to 8.5, more preferably 7.0 to 8.0.

Porcine enzymes, in particular porcine pancreatic enzymes may be used in the hydrolysis process. For example, WO1993004593A1 discloses a hydrolysis process using trypsin and chymotrypsin, which includes a two-step hydrolysis reaction with a heat denaturation step in between to ensure that the final hydrolysate is substantially free of intact allergenic proteins. The trypsin and chymotrypsin used in these methods are preparations produced by the extraction of porcine pancreas. WO2016156077A1 discloses a process for preparing a milk protein hydrolysate comprising hydrolysing a milk-based proteinaceous material with a microbial alkaline serine protease in combination with bromelain, a protease from Aspergillus, and a protease from Bacillus.

Free amino acids

The nutritional composition of the invention may comprise free amino acids. In some embodiments free amino acids are the only source of protein.

The levels of free amino acids may be chosen to provide an amino acid profile that is sufficient for infant nutrition, in particular an amino acid profile that satisfies nutritional regulations (e.g. European Commission Directive 2006/141/EC).

Free amino acids may, for example, be incorporated in the eHF of the invention to supplement the amino acids comprised in the peptides.

Example free amino acids for use in the nutritional composition of the invention include histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine and mixtures thereof.

Free amino acids provide a protein equivalent source (i.e. contribute to the nitrogen content). As described above, having a high proportion of di- and tri-peptides may improve nitrogen (protein) absorption, even in patients with gut impairment. Accordingly, having a low proportion of free amino acids may also improve nitrogen (protein) absorption, even in patients with gut impairment.

Suitably, therefore, the free amino acids in the eHF may be present in a concentration of 50% or less, 40% or less, 30% or less, or 25% or less by weight based on the total weight of amino acids. Preferably, the eHF comprises 25% or less by weight of free amino acids based on the total weight of amino acids. More preferably, the free amino acids in the eHF are present in a concentration of 20-25%, 21-23%, or about 22% by weight based on the total weight of amino acids.

The free amino acids content may be determined by any method known of skill in the art. Suitably, the free amino acids content may be obtained by separation of the free amino acids present in an aqueous sample extract by ion exchange chromatography and photometric detection after post-column derivatisation with ninhydrin reagent. Total amino acid content may be obtained by hydrolysis of the test portion in 6 mol/L HCI under nitrogen and separation of individual amino acids by ion-exchange chromatography, as described above.

Carbohydrate

The carbohydrate may be any carbohydrate that is suitable for use in a nutritional composition.

The carbohydrate content of the nutritional composition of the invention, particularly the infant formula of the invention, is preferably in the range 9-14 g carbohydrate per 100 kcal.

In addition to lactose as provided in the combination of the invention, further example carbohydrates for use in the nutritional composition include saccharose, maltodextrin and starch. Suitably, the nutritional composition may comprise mixtures of carbohydrates.

In some embodiments, the carbohydrate content comprises maltodextrin. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60% or at least about 70% by weight of the total carbohydrate content is maltodextrin. In one embodiment, at least about 50% of the total non-HMO carbohydrate content is maltodextrin.

In some embodiments, the carbohydrate comprises lactose and maltodextrin. In some embodiments, at least about 50% of the total non-HMO carbohydrate content is lactose and at least about 50% of the total non-HMO carbohydrate content is maltodextrin.

In some embodiments, about 100 % of the total non-HMO carbohydrate content is lactose.

Fat

The fat content of the nutritional composition of the invention, particularly the infant formula of the invention, is preferably in the range 4.0-6.0 g fat per 100 kcal.

Example fats for use in the nutritional composition of the invention include sunflower oil, low erucic acid rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palm oleic, high oleic sunflower oil and high oleic safflower oil, and microbial fermentation oil containing long chain, polyunsaturated fatty acids.

The fat may also be in the form of fractions derived from these oils, such as palm olein, medium chain triglycerides (MCT) and esters of fatty acids such as arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaeonic acid, linolenic acid, oleic acid, lauric acid, capric acid, caprylic acid, caproic acid, and the like.

Further example fats include structured lipids (i.e. lipids that are modified chemically or enzymatically in order to change their structure). Preferably, the structured lipids are sn2 structured lipids, for example comprising triglycerides having an elevated level of palmitic acid at the sn2 position of the triglyceride. Structured lipids may be added or may be omitted. Oils containing high quantities of preformed arachidonic acid (ARA) and/or docosahexaenoic acid (DHA), such as fish oils or microbial oils, may be added.

Long chain polyunsaturated fatty acids, such as dihomo-y-linolenic acid, arachidonic acid (ARA), eicosapentaenoic acid and docosahexaenoic acid (DHA), may also be added.

Oils containing high quantities of SOFA such as acetate, propionate or butyrate or any other lipidic product derived from microbial fermentation may also be added.

Medium chain triglycerides (MCTs)

A high concentration of MOT may impair early weight gain. MOT is not stored and does not support fat storage. For instance, Borschel et al. have reported that infants fed formula without MOT gained significantly more weight between 1-56 days than infants fed formulas containing 50% of the fat from MCT (Borschel, M. et al. (2018) Nutrients 10(3): 289).

Thus, about 30% or less by weight of the fat may, for example, be medium-chain triglycerides (MCTs) in the nutritional composition of the present invention.

In some embodiments, about 25% or less by weight, 20% or less by weight, 15% or less by weight, 10% or less by weight, 5% or less by weight, 4% or less by weight, 3% or less by weight, 2% or less by weight, 1% or less by weight, 0.5% or less by weight, or 0.1% or less by weight of the fat is medium chain triglycerides (MCTs).

In some embodiments, 0-30% by weight, 0-25% by weight, 0-20% by weight, 0-15% by weight, 0-10% by weight, 0-5% by weight, 0-4% by weight, 0-3% by weight, 0-2% by weight, 0-1% by weight, 0-0.5% by weight, or 0-0.1 % by weight of the fat is medium chain triglycerides (MCTs). In some embodiments, the nutritional composition comprises no added MCTs. Suitably, about 0% by weight of the fat is MCTs and/or the composition comprises no detectable MCTs. Suitably, the nutritional composition comprises no MCTs.

Dietary Butyrate

The combination or nutritional composition of the invention may further comprise butyrate, in particular dietary butyrate.

Butyrate may be present in the nutritional composition in an amount from about 1 mg/L to about 30 mg/L of the composition as consumed. Butyrate may be present in the nutritional composition in an amount of from about 1.6 mg/L to about 2.4 mg/L of the composition as consumed, or from about 8 mg/L to about 12 mg/L of the composition as consumed, or from about 16 mg/L to about 24 mg/L of the composition as consumed.

Preferably, butyrate is present in the nutritional composition in some particular amounts. In an embodiment of the invention, butyrate is present in the nutritional composition in an amount about 30 mg/L of the composition as consumed. Butyrate may be in the nutritional composition in an amount of about 10-30 mg/L of the composition as consumed, such as about 13-27 mg/L or 16-24 mg/L or 18-22 mg/L of the composition as consumed. In another particular embodiment, butyrate is present in the nutritional composition in an amount of about 20 mg/L of the composition as consumed.

In an embodiment of the invention, butyrate is present in the nutritional composition in an amount of up to about 23 mg per 100g of composition based on the dry weight of the composition. In an embodiment of the invention, butyrate may be in the nutritional composition in an amount of 12-18 mg per 100g of the composition based on the dry weight of the composition. Butyrate may be in an amount of about 15 mg per 100 g of the composition based on the dry weight of the composition.

The dietary butyrate may, for example, be in the forms disclosed in WO2019/228851 or W02020/127642. The corresponding amount of the butyrate source in the nutritional composition is sufficient to provide butyrate in the nutritional composition in an amount up to about 30 mg/L of the composition as consumed.

The butyrate source may be present in the nutritional composition in an amount sufficient to provide butyrate in the nutritional composition in an amount from about 1 mg/L to about 30 mg/L of the composition as consumed. The butyrate source may be present in the nutritional composition in an amount sufficient to provide butyrate in the nutritional composition in an amount from about 1 .6 mg/L to about 2.4 mg/L of the composition as consumed, or from about 8 mg/L to about 12 mg/L of the composition as consumed, or from about 16 mg/L to about 24 mg/L of the composition as consumed.

Preferably, the amount of the butyrate source in the nutritional composition is sufficient to provide butyrate in the nutritional composition in an amount of about 10-30 mg/L of the composition as consumed, such as about 13-27 mg/L or 16-24 mg/L or 18-22 mg/L of the composition as consumed. In another particular embodiment, the amount of the butyrate source in the nutritional composition is sufficient to provide butyrate in the nutritional composition in an amount of about 20 mg/L of the composition as consumed.

Suitably, the combination or nutritional composition may comprise a compound having the formula: or combinations thereof; wherein R¹, R², R³, R⁴, R⁵ and R⁶ are independently, a long chain fatty acid having between 16 and 20 carbons. In one particular embodiment, each of R¹, R², R³, R⁴, R⁵ and R⁶ is oleic acid, palmitic acid, stearic acid or linoleic acid. In one particular embodiment, each of R¹, R², R³, R⁴, R⁵ and R⁶ is palmitic acid.

In one embodiment, each of R¹, R², R³, R⁴, R⁵ and/or R⁶ as defined herein is an unsaturated fatty acid, preferably monounsaturated. In one particular embodiment, R¹, R², R³, R⁴, R⁵ and R⁶ are each a long chain fatty acid having 18 carbons, wherein the long chain fatty acid having 18 carbons is monounsaturated. In one particular embodiment, R¹, R², R³, R⁴, R⁵ and R⁶ are each oleic acid.

In one embodiment, a combination comprising lactose and HMOs as described herein, may comprise a source of butyrate in the form of one of more compounds having the formula or combinations thereof. In one embodiment, the nutritional composition as described herein comprises about 100 mg/L as consumed of at least one of compounds (5), (6), (7), and (8). In one embodiment, the nutritional composition as described herein comprises about 100 mg of at least one of compounds (5), (6), (7), and (8) and is the equivalent of about 20 mg/L dietary butyrate in the composition as consumed.

An alternative source of dietary butyrate may be a triglyceride composed of a mixture of a C4 fatty acid, such as butyric acid (C4:0), and a C8 fatty acid, such as caprylic acid (C8:0). The triglycerides comprising caprylic acid and butyric acid may be prepared by inter-esterification of a mix of triglycerides comprising either butyric acid (C4:0) or caprylic acid (C8:0). Commercially available sources for BBB, Tributyrin (available e.g. by Sigma-Aldrich). Commercially available sources for CCC, Tricaprylin is e.g. Neobee 895 (available e.g. by Stepan Specialty).

Further ingredients

The nutritional composition, particularly an infant formula or young-child formula of the invention, may also contain all vitamins and minerals understood to be essential in the daily diet in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Example vitamins, minerals and other nutrients for use in the nutritional composition of the invention, particularly the infant formula of the invention, include vitamin A, vitamin B1 , vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in their salt form. The nutritional composition may comprise one or more carotenoids.

The nutritional composition may also comprise at least one probiotic. The term “probiotic” refers to microbial cell preparations or components of microbial cells with beneficial effects on the health or well-being of the host. In particular, probiotics may improve gut barrier function.

Examples of probiotic micro-organisms for use in the nutritional composition of the invention include yeasts, such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, and bacteria, such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, and Lactobacillus. Preferably, the combination as described herein, preferably the nutritional composition, comprises one or more bacterium of the genera Bifidobacterium and/or Lactobacillus.

Preferred probiotics are those which as a whole are safe, are L(+) lactic acid producing cultures and have acceptable shelf-life for products that are required to remain stable and effective for up to 24 months., e.g. Bifidobacterium, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

Specific examples of suitable probiotic microorganisms are: Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbruckii subsp. lactis, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosus and Staphylococcus xylosus, Lacticaseibacillus rhamnosus, Lacticaseibacillus paracasei, Limosilactobacillia, Akkermansia muciniphila, Clostridales, and Prevotella.

The nutritional composition of the invention may also contain other substances which may have a beneficial effect such as prebiotics, lactoferrin, fibres, nucleotides, nucleosides and the like.

Method of manufacture

The nutritional composition of the invention may be prepared in any suitable manner. For example, the nutritional composition described herein may be prepared by blending together the protein source, the carbohydrate source and the fat source in appropriate proportions. If used, the further emulsifiers may be included at this point. The vitamins and minerals may be added at this point but vitamins are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved in the fat source prior to blending. Water, preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. Commercially available liquefiers may be used to form the liquid mixture. The liquid mixture may then be homogenised.

The liquid mixture may then be thermally treated to reduce bacterial loads. This may be carried out, for example, by means of steam injection, or using an autoclave or heat exchanger, for example a plate heat exchanger.

The liquid mixture may then be cooled and/or homogenised. The pH and solid content of the homogenised mixture may be adjusted at this point.

The homogenised mixture may then be transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and converted to powder. If a liquid nutritional composition is preferred, the homogenised mixture may be sterilised, then aseptically filled into a suitable container or maybe first filled into a container and then retorted.

The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

Subject

The subject may be any suitable subject. Suitably, the subject may be a mammal. In preferred embodiments, the subject is a human. In other embodiments, the subject is an animal, preferably wherein the animal is a pet. A pet may be an animal selected from dogs, cats, birds, fish, rodents such as mice, rats, and guinea pigs, rabbits, etc. In some embodiments, the pet is a small dog breed.

In some embodiments, the subject is a juvenile, an adolescent, a child. The term “juvenile” may refer to an individual that has not yet reached adulthood. The term “adolescent” may refer to an individual during the period from the onset of puberty to adulthood. The term “child” may refer an individual between the stages of birth and puberty.

Suitably, the subject is about 3 years of age or older. In some embodiments, the subject is about 4 years of age or older or about 5 years of age or older. In some embodiments, the subject is about 10 years of age or younger. In some embodiments, the subject is about 9 years of age or younger, about 8 years of age or younger, about 7 years of age or younger, about 6 years of age or younger, or about 5 years of age or younger.

In some embodiments, the subject is about 3 years to about 10 years of age, about 3 years to about 9 years of age, about 3 years to about 8 years of age, about 3 years to about 7 years of age, about 3 years to about 6 years of age, or about 3 years to about 5 years of age.

The subject may be an infant, young child or child; preferably the subject is an infant or young child. In some embodiments, the subject is an infant or young child having or is suspected of having Cow’s Milk Protein Allergy (CMPA).

Suitably, the infant may be a subject who is less than 12 months old.

Suitably, the young child may be a subject who is from 12 months to 36 months old (1 to 3 years).

Suitably, the child may be a subject who is from 3 years to 7 years old.

The subject may be an infant or young child who has not been breastfed or wherein breastfeeding has been halted.

Suitably, the subject may be suffering from dysbiosis of the gut microbiota.

The subject may have or be suspected of having Cow’s Milk Protein Allergy (CMPA).

CMPA may be defined as a reproducible immune-mediated allergic response to one or more proteins in cow's milk and is one of the most common presentations of food allergy seen in early childhood. It can be classified according to the underlying immune mechanism: Immunoglobulin (Ig)E-mediated food allergy produces immediate symptoms, which may affect multiple organ systems, typically up to 2 hours after cow's milk ingestion. Non-lgE-mediated food allergy reactions usually manifest between 2 and 72 hours after cow's milk ingestion. Mixed IgE and non-lgE allergic reactions are typically delayed. Diagnosis of IgE-mediated allergy is typically based on presentation of symptoms, such as urticaria, angio-oedema, itching, cough, hoarseness, wheeze, or breathlessness after cow's milk ingestion. Diagnosis of non-lgE- mediated allergy should be suspected if there are one or more symptoms such as gastro- oesophageal reflux disease, abdominal discomfort, constipation, diarrhoea, or atopic eczema, particularly if symptoms are treatment-resistant.

A subject with a CMPA may exhibit outgrowth of the CMPA associated with the induction of oral tolerance after administration of a composition as defined herein, or a nutritional composition as defined here. Oral tolerance may be associated with a partial or complete oral desensitisation in a subject with an allergy, particularly a cow’s milk protein allergy. In the context of the invention, induction of oral tolerance is understood to mean that oral tolerance against an allergen such as a cow’s milk protein is induced as compared to oral tolerance before starting administration of a combination or nutritional composition as defined herein. Some embodiments of the combination as defined herein, or nutritional composition as defined herein, is for use in induction of oral tolerance in a subject with an allergy, particularly a cow’s milk protein allergy. In some embodiments of the combination as defined herein, or nutritional composition as defined herein, is for use in partial or complete oral desensitisation in a subject with an allergy, particularly a cow’s milk protein allergy.

Dietary Management

The term “manage,” “managing,” and “management” as used herein, including in the context of “dietary management”, may refer to the prevention or reduction in severity or frequency of one or more symptoms of a disease or condition, including ameliorating one or more existing symptoms of a disease or condition, preventing one or more existing symptoms of disease or condition, preventing one or more underlying causes of a disease or condition, ameliorating one or more underlying cause of a disease or condition, reducing the prevalence of one or more symptoms of a disease or condition, and/or reducing the occurrence of one or more symptoms of a disease or condition. It will be understood to include stabilizing a disease or condition and preventing progression of a disease or condition.

Dysbiosis

In one aspect the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of dysbiosis of the gut microbiota in a subject.

In another aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, at least one of 3’-SL or 6’-SL, and optionally LNT for use in the treatment of dysbiosis of the gut microbiota in a subject. In another aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT for use in the treatment of dysbiosis of the gut microbiota in a subject.

In a further aspect, the present invention provides the use of a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, at least one of 3’-SL or 6’-SL, and optionally LNT in the dietary management of dysbiosis of the gut microbiota in a subject. In another aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT for use in the dietary management of dysbiosis of the gut microbiota in a subject.

In one aspect the present invention provides a method for the dietary management of dysbiosis of the gut microbiota in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

The present invention also provides a method for the treatment of dysbiosis of the gut microbiota in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

Dysbiosis of the gut microbiota is characterized by a disruption to the microbiome resulting in an imbalance in the microbiota, changes in their functional composition and metabolic activities, or a shift in their local distribution. The most typical features of dysbiosis are a decrease in the diversity of the microbiota, a loss of beneficial microbiota, or an overgrowth of harmful microbiota.

Dysbiosis may be assessed and identified using a range of tests and criteria, for example as described in Wei et al. (2021 ; Applied and Environmental Microbiology; 87(11)). Approaches to determine dysbiosis may comprise e.g. 16S rRNA gene profiling or sequencing to apply one or more of the following: large-scale bacterial marker profiling, relevant taxon-based methods, neighbourhood classification, and combined alpha and beta diversity measures.

In infants and young-children with CM PA, dysbiosis may be characterized by lower levels of Bifidobacteriaceae and enrichment of Proteobacteria and other gram negative gut bacteria. Measurements of Bifidobacteriaceae at about 10% of the microbiota in CMPA infants as compared to levels of about 80% in healthy infants.

Suitably, dysbiosis in the context of the present invention may refer to an abundance of HMO utilizing Bifidobacteriaceae in the gut microbiota of less than about 10%, less that about 5%, less than about 2% or less than about 1%.

Suitably, dysbiosis in the context of the present invention may refer to the overgrowth of a pathogen or an overgrowth of Proteobacteria. In some embodiments, dysbiosis may refer to the overgrowth of one or more of Staphylococcaceae, Clostridiaceae, and Enterococcaceae. In some embodiments, dysbiosis in the context of the present invention may refer to relatively low numbers of Bifidobacteriaceae and relatively high numbers of Enterobacteriaceae and/or Clostridiaceae in view of the total number of bacteria in a sample. Overgrowth of a pathogen or a Proteobacteria can be determined by any means known in the art. In one embodiment, the relative concentration of bacteria are determined by 16S rRNA gene sequencing (as is known in the art and described in the examples) and/or shotgun metagenomic sequences (as it is known in the art), preferably the relative concentration of bacteria are determined by 16S rRNA gene sequencing. Both methods are commercially available, see, e.g., WorldWideWeb microbiomeinsights.com.

Suitably, dysbiosis in the context of the present invention may be associated with proteolytic fermentation by the gut microbiota. Proteolytic fermentation may be associated with the formation of metabolites such as phenol and indole that exert detrimental health effects.

Dysbiosis of the gut microbiota may be associated with the pathogenesis of both intestinal and extra-intestinal disorders. Intestinal disorders include, for example, inflammatory bowel disease, irritable bowel syndrome (IBS), and coeliac disease, while extra-intestinal disorders include allergy, asthma, metabolic syndrome, cardiovascular disease, and obesity.

Suitably, the intestinal disorder may be one or more of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and coeliac disease.

Suitably, extra-intestinal disorder is one or more of allergy, asthma, atopic dermatitis, metabolic syndrome, cardiovascular disease, obesity, and type 2 diabetes.

Branched chain fatty acids (BCFA)

In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject. In another aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject.

In another aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the treatment of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject. In another aspect, the present invention provides a combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT for use in the treatment of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject.

In one aspect the present invention provides a method for the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject. In a further aspect, the present invention provides a method for the treatment of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

In another aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the reduction of BCFA in a subject.

In one aspect the present invention provides a method for the reduction of BCFA in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of malnutrition in a subject.

In another aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the treatment of malnutrition in a subject.

In one aspect the present invention provides a method for the dietary management of malnutrition in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

In another aspect, the present invention provides a method for the treatment of malnutrition in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

Suitably, the BCFA may comprise isobutyrate, isovalerate, isocaproate and/or 2-methylbutyrate.

Suitably, the BCFA may comprise isobutyrate, isovalerate, and/or isocaproate.

Suitably, the combination reduces levels of BCFA compared to an infant who has not been administered the combination as defined herein.

Suitably, the combination reduces levels of BCFA compared to an infant who has been administered a nutritional composition comprising lactose but not one or more HMOs of the present combination.

BCFA produced by the microbiota include isovaleric, isobutyric, isocaproate and 2-methylbutyric acids, which are produced by the fermentation of the branched-chain amino acids (BCAA) leucine, valine, and isoleucine, respectively. BCFA are indicative of proteolytic fermentation by the gut microbiota and are associated with the formation of metabolites such as phenol and indole. In addition, isovaleric acid can inhibit Na+, K+-ATP-ase, a key enzyme responsible to maintain the basal potential membrane necessary for a normal neurotransmission, providing insights into the participation of BCFA in the gut-brain axis communication.

Suitably, the disease or disorder associated with metabolism of BCFA may be one or more of malnutrition, Rett syndrome, colorectal cancer, anorexia nervosa, and depression.

An increase of BCFA has been evidenced in several pathologies. High levels of fecal BCFA associated with microbiota alterations have been reported in stunted and malnourished children (Li et a! , 2019; Front Microbiol. 2019;10:2635 and Surono et al.; 2021 ; PLoS One. 2021 ;16(1):e0245399) as well as in anorexia nervosa (Mack et al , 2016; Sci Rep.; 6:26752 ). Fecal valeric, isobutyric, and isovaleric acids were found higher in colorectal cancer than in healthy individuals (Wang et al.; 2017; Exp Ther Med. 2017;13(6):2848-54 and Niccolai et al.-, 2019; World J Gastroenterol. 25(36): 5543-58), and it may be differentiating between colorectal and adenomatous polyps patients from healthy controls by the fingerprint of fecal SCFA (Niccolai et al.-, supra.). Higher abundance of isovaleric and isobutyric acids was found in hypercholesterolemic patients, with isobutyric acids also positively correlated with lipid parameters indicative of unfavorable profiles (Granado-Serrano et a!:, 2019; Sci Rep. 2019;9(1):1772). Intestinal dysbiosis and higher concentrations of fecal propionate and isobutyric acids have been reported in Non-alcoholic fatty liver disease (Da Silva et al.; 2018; Sci Rep. 2018;8:1466 ), whereas isovaleric acid has been indicated, together with propionic and butyric acids, as possible gut microbiota predictors of obesity in African American men (Barengolts et al.; 2019; Microorganisms;7(9):320). Rett syndrome is an infrequent X-linked neurodevelopmental disorder that affects female, in which changes in microbiota composition may account for several of the symptoms associated with this pathology. Significantly higher levels of BCFA are associated with Rett syndrome, together with increased microbial genes encoding for propionate, butyrate, and amino acid metabolism (Borghi et al:, 2017; Int J Mol Sci. 2017;18(2):344). High stool levels of isovaleric acid have been found correlated in adults with human depression and high cortisol levels (Szczesniak et a/; Nutr Neurosci. 2016;19(7):279- 83).

Dietary management of colonisation/infection with enteric pathogens

In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of at least one pathogen in a subject. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of at least one pathogen in a subject, wherein the pathogen is an enteric pathogen that is one or more of a bacterial, viral or protozoal pathogen. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of at least one pathogen in a subject, wherein the pathogen is an enteric pathogen that is a viral pathogen, wherein the viral pathogen is a norovirus and/or a rotavirus.

In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of a pathogenic bacteria in a subject. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of a pathogenic bacteria in a subject, wherein the pathogenic bacteria is one or more C. difficile, C. perfringens, Campylobacter or Escherichia coli. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of C. difficile and/or C. perfringens. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of C. difficile and/or C. perfringens, wherein the dietary management decreases the abundance of C. difficile and/or C. perfringens.

In one aspect the present invention provides a method for the dietary management of at least one pathogen in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

Suitably, the pathogen may be associated with dysbiosis in the subject.

For example, the present invention may be directed to the dietary management of C. difficile and/or C. perfringens.

In one aspect the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of at least one pathogen in a subject, wherein the subject is an infant, young child or child who has not been breastfed or wherein breast-feeding has been halted. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of C. difficile and/or C. perfringens, wherein the subject is an infant, young child or child who has not been breastfed or wherein breast-feeding has been halted. In one aspect, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of C. difficile and/or C. perfringens, wherein the dietary management reduces the abundance of C. difficile and/or C. perfringens, and wherein the subject is an infant, young child or child who has not been breastfed or wherein breast-feeding has been halted.

A reduction in the abundance of C. difficile and/or C. perfringens may be determined by analysis of the total gut microbiota.

In one embodiment, a reduction in the abundance of C. difficile and/or C. perfringens may be determined by resolution or reduction of one or more symptoms associated with an overgrowth of C. difficile and/or C. perfringens. In one embodiment, resolution or reduction of at least one of diarrhoea and fever.

In one aspect the present invention provides a method for the dietary management of at least one pathogen in a subject, the method comprising administering a combination as defined herein, preferably in the form of a nutritional composition, to the subject, wherein the subject is an infant, young child or child who has not been breastfed or wherein breast-feeding has been halted.

Proteolytic Fermentation

Besides BCFA, proteolytic fermentation gives rise to the production of compounds such as ammonia, phenols, amines, hydrogen sulfide, and p-cresol, which may be harmful to host health. Because of this, high rates of colonic proteolytic fermentation accompanied by low levels of saccharolytic fermentation are considered detrimental for host health.

Molecules associated with proteolytic fermentation can cause cell damage (e.g. toxic for colonocytes) on the intestinal environment (Aguirre et al., 2016; Res. Microbiol. 167, 114-125) and can contribute to inflammatory conditions (Yao et al.’, 2016; Aliment Pharmacol Ther, 2016. 43(2): p. 181-96, and Popkov et af, 2022; Int J Mol Sci, 23(1)) and have detrimental health effects (Nowak et al. 2006; Anaerobe 12, 80-84). For example, the uremic toxins 4-cresol and 2- or 3-hydroxyphenylacetic acid formed via bacterial tyrosine and phenylalanine fermentation can have negative mucosal and systemic effects (Evenepoel et al. 2009; Kidney Int Suppl, 2009(114): p. S12-9.; and Zheng et al. 2021 ; Front Neurosci, 2021. 15: p. 738220). A reduction in fecal toxic metabolites may therefore contribute to maintaining systemic immune balance.

In one aspect the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use reduction of proteolytic fermentation by the gut microbiota in a subject. In one aspect the present invention provides a method for the reduction of proteolytic fermentation by the gut microbiota in a subject, the method comprising a combination as defined herein, preferably in the form of a nutritional composition, to the subject.

EXAMPLES

The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

Example 1 - Combinations of HMO blends and lactose reduce branched chain fatty acid production in gut microbiota in an ex vivo colonic fermentation model

The combination of 4 HMO blends (M2, M4.1 , M4.2 and M6) with lactose (L) significantly decreased BCFA production in an ex vivo colonic fermentation model (see Figure 1). This effect was not observed for the individual test products.

Figure 1 , bottom panel shows that at 48 hours, combinations of HMO and lactose induce lower levels of BCFA than lactose alone or HMO alone. Combinations of HMOs with lactose, particularly, in the M.4.1+L, M4.2+L and M6+L combinations, significantly decreased BCFA production in a more than additive effect.

Further evidence that the effect of the combination of lactose and HMOs not just an additive effect and that the effect of the combination of HMO blends and lactose are different than the addition of the effect of lactose alone and the effect of HMO alone are provided by Figure 2. This principal component analysis (PCA) summarising the impact on microbial community composition of microbial communities derived from the infants shows that addition of lactose to the HMO blends results in a different pattern as compared to HMO blends without lactose (see PCA1).

Figure 3 that the combination of lactose with HMO blends increased beneficial bacteria (specifically Bifidobacterium species (B. breve, longum, bifidum) which are key for the development of healthy microbiome in infants, and decreased Clostridium difficile, which is responsible for diarrhea) and Ruminococcus gnavus (associated with IBD and inflammation). Despite interpersonal differences among CM PA infants, consistent effects on microbial metabolite production and microbial composition were observed for the HMO and lactose blends. It is thus considered that the beneficial effect is potentiated by using the combination of lactose and HMO instead of HMO alone or with lower level of lactose. Materials & Methods

Four different blends of 2’-FL, 3’-FL, LNnT, 3’-SL, LNT, 6’-SL (M2, M4.1 , M4.2 and M6), lactose (L) and combinations thereof (M2+L, M4.1+L, M4.2+L and M6+L) were compared to a no substrate control (NSC) at the doses indicated in Table 1. Table 1 : Test Products and Doses

The dose of lactose was based on an estimated intake of 70 g lactose per day and assuming an in vivo absorption along the small intestine of 96%. Table 2 - HMO blends

Faecal donor selection criteria

Twelve infants with suspected CM PA fulfilling the following criteria were included in the study: 1-7 months of age; fed an extensively hydrolyzed or amino acid-based formula (as per medical recommendation) or breastfed; gestational age at birth >36 weeks; no antibiotics for 30 days prior to study participation; no previous necrotizing enterocolitis or gut surgery; and no consumption of probiotics or prebiotics for 14 days prior to study participation (L reuteri (DSM 17938), L. rhamnosus LGG (ATCC 53103) and Bifidobacterium animalis subsp. lactis BB- 12 were allowed as commonly used in this population and not known to be consumed by 2’-FL, 3’-FL, 3’-SL, 6’SL and LNnT (See, e.g., Thongaram et al. Journal of Dairy Science (2017) 100:7825-7833). One infant was partially breastfed (infant 2), and one exclusively breastfed (infant 12). Fecal samples were collected by parents/caregivers after having provided informed consent for participation in the study. In total, 10 male and 2 female donor samples were assessed. The age of the test subjects ranged from 2-7 months and was on average 4.3 months (4.2 months for male; 4.8 months for female infants). The study was approved by the Ethics Committee of the University Hospital Ghent (reference number BC-09977).

SIFR® study design

Ex vivo SIFR® experiments were performed as described in Van den Abbeele et al Front Microbiol (2023) 14:1131662. Individual bioreactors were processed in a bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor containing 5mL of a blend of nutritional medium (M0018, Cryptobiotix, Ghent, Belgium), infant-fecal inoculum and the test compounds (i.e. no test product (no substrate control (NSC)), HMO, L or HMO+L), was sealed individually, and then made anaerobic. After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37°C for 48h (MaxQ 6000, Thermo Scientific, Merelbeke, Belgium).

Four study arms were tested for each infant: (i) no test product (C), (ii) HMO, (iii) L, and (iv) HMO+L. Upon gas pressure measurement in the headspace, samples were collected at Oh, 6h, 24h and 48h for key fermentation parameters, untargeted metabolite profiling, and/or microbial composition. The control samples were run in technical triplicate and coefficients of variation for fundamental fermentation parameters pH, gas production, and the three main SCFAs (acetate, propionate and butyrate) below 3% were considered acceptable as previously established for the SIFR® technology (Van den Abbeele et al Front Microbiol (2023) 14:1131662).

Microbiota taxonomic analysis: quantitative 16S rRNA gene profiling

DNA was extracted via the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to manufacturer’s instructions. Subsequently, library preparation and sequencing were performed on an Illumina MiSeq platform with v3 chemistry. 16S rRNA gene V3-V4 hypervariable regions were amplified using primers 341 F (50 -COT ACG GGN GGC WGC AG- 30) and 785Rmod (50 -GAC TAG HVG GGT ATC TAA KCC-30). Pre-processing and OTU (operational taxonomic unit) picking from amplicons was performed with Mothur v1.35.1 (Schloss et al. Appl Environ Microbiol (2009) 75:7537-7541) with representative sequences being annotated with NCBI blast v2.10.0 (Altschul et al. J Mol Biol (1990) 215:403-410). Results were analysed at phylum, family and operational taxonomic unit (OUT) level. To determine total counts, samples were diluted in anaerobic phosphate-buffered saline (PBS), after which cells were stained with SYTO 16 at a final concentration of 1 M and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium). Data was analysed using FlowJo, version 10.8.1. Quantitative insights were obtained by correcting proportions (%; 16S rRNA gene profiling)) with total counts (cells/mL; flow cytometry), resulting in estimated cells/mL of different taxa.

BCFA analysis

Branched chain fatty acids (BCFA) (sum of isobutyrate, isovalerate and isocaproate) were determined via a GC-FID (Gas Chromatography With Flame Ionization Detection) approach.

Data analysis

All univariate and multivariate analyses were performed by GraphPad Prism (v9.3.1 ; www.graphpad.com), while Regularized Canonical Correlation Analysis (rCCA) was executed using the mixOmics package with the shrinkage method for estimation of penalization parameters (version 6.16.3) in R (4.1.1 ; WorldWideWeb r-project.org) (Rohart et al. PLOS Computational Biology (2017) 13: e1005752). Significance of supplementation effect effects compared with the C were assessed via repeated measures ANOVA analyses (based on paired testing among the 12 infants), with p-value-correction according to Benjamini-Hochberg (Journal of the Royal Statistical Society: Series B (Methodological) (1995) 57:289-300). For analysis of microbial composition, three measures were taken. First, aforementioned statistical analysis was performed on the log -transformed values. Second, a value of a given taxonomic group below the limit of detection (LOD) was considered equal to the overall LOD as described recently (Van den Abbeele et al Front Microbiol (2023) 14:1131662). Finally, a threshold was set in order to retain the 100 most abundant OTUs in the analysis, to avoid excessive p-value corrections.

EMBODIMENTS

Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras).

1. A combination comprising lactose and human milk oligosaccharides (HMOs) 2'- fucosyllactose (2’-FL), lactoW-neotetraose (LNnT), 3-fucosyllactose (3’-FL), and at least one of 3’-sialyllactose (3’-SL) or 6’-sialyllactose (6’-SL) for use in the dietary management of dysbiosis of the gut microbiota in a subject. 2. The combination for use according to para 1 , comprising the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and lacto-N-tetraose (LNT).

3. The combination for use according to para 1 or para 2 wherein the dysbiosis includes increased production of branched chain fatty acids (BCFA), such as isobutyrate, isovalerate, and/or isocaproate.

4. The combination for use according to any one of paras 1 to 3, wherein the dysbiosis of the gut microbiota is associated with an intestinal disorder and/or an extra-intestinal disorder.

5. The combination for use according to para 4, wherein the intestinal disorder is one or more of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and coeliac disease.

6. The combination for use according to para 5 wherein the IBD is Crohn’s Disease (CD) or ulcerative colitis (UC).

7. The combination for use according to para 4, wherein the extra-intestinal disorder is one or more of allergy, asthma, atopic dermatitis, metabolic syndrome, cardiovascular disease, obesity, and type 2 diabetes.

8. The combination for use according to any one of paras 1 to 7, wherein dysbiosis diagnosed by presence of relatively low numbers of Bifidobacteriaceae and relatively high numbers of Enterobacteriaceae and/or Clostridiaceae in view of the total number of bacteria in a faecal sample of the subject.

9. The combination for use according to any one of paras 1 to 8, wherein the subject has or is suspected of having Cow’s Milk Protein Allergy (CM PA).

10. A combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, and at least one of 3’-SL or 6’-SL for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of BCFA in a subject.

11 . The combination for use according to para 10, comprising the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT.

12. The combination for use according to para 10 or para 11 , wherein the BCFA comprise isobutyrate, isovalerate, and/or isocaproate.

13. The combination for use according to any one of paras 10 to 12, wherein the disease or disorder associated with metabolism of BCFA is increased proteolytic fermentation and/or decreased saccharolytic fermentation. 14. The combination for use according to any one of paras 10 to 12, wherein the disease or disorder associated with metabolism of BCFA is one or more of malnutrition, Rett syndrome, colorectal cancer, anorexia nervosa, and depression.

15. The combination for use according to any one of paras 10 to 12, wherein the disease or disorder associated with metabolism and/or accumulation of BCFA is an accumulation of one or more of isobutyrate, isovalerate, and isocaproate.

16. A combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, and at least one of 3’-SL or 6’-SL for use in the dietary management of

(a) a pathogen in a subject;

(b) a pathogen in a subject, wherein the pathogen is one or more of bacterial, viral or protozoal pathogen;

(c) a pathogen in a subject, wherein the pathogen is one or more viral pathogens, preferably rotavirus or norovirus;

(d) a pathogen in a subject, wherein the pathogen is one or more bacterial pathogens, preferably C. difficile, C. perfringens, Campylobacter or Escherichia coli.

17. The combination for use according to para 16, comprising the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT.

18. The combination for use according to para 16 or para 17, wherein the pathogen is at least one of C. difficile and C. perfingens.

19. The combination for use according to para 18, wherein there is a resolution or reduction in one or more symptoms associated with an overgrowth of C. difficile and/or C. perfringens.

20. The combination for use according to para 19, wherein the symptom is at least one of diarrhoea and fever.

21. The combination for use according to any one of paras 16 to 20, wherein the subject is an infant, young child or child who has not been breastfed or wherein breast-feeding has been halted.

22. The combination for use according to any one of the preceding paras, wherein the combination of lactose and HMOs reduces levels of BCFA in the gut of the subject.

23. The combination for use according to para 22, wherein the BCFA comprise isobutyrate, isovalerate, and/or isocaproate. 24. A combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, and at least one of 3’-SL or 6’-SL for use in the reduction of BCFA in a subject.

25. The combination for use according to para 24, comprising the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT.

26. The combination for use according to para 24 or para 25, wherein BCFA are reduced in the gut microbiota of the subject.

27. The combination for use according to any one of paras 24 to 26, wherein the BCFA comprise isobutyrate, isovalerate, and/or isocaproate.

28. The combination for use according to any one of paras 24 to 27, wherein the reduction of BCFA is associated with reduction of proteolytic fermentation.

29. The combination for use according to any one of the preceding paras, wherein the combination is provided in the form of a nutritional composition.

30. The nutritional composition for use according to para 29, wherein the nutritional composition is: a. an infant formula, a starter infant formula, a follow-on or follow-up infant formula, a growing-up milk, a fortifier or a supplement, b. a beverage product, a yoghurt product, a pudding product, fermented milk, a fruit juice, bar, mousse, snack, chips, meal, or meal replacement, or c. a health care nutritional composition for oral feeding, a nutritional product for enteral feeding or a parenteral feeding product.

31. The nutritional composition for use according to para 29 or para 30, wherein the nutritional composition is an infant formula, a starter infant formula, a follow-on or follow-up infant formula.

32. The nutritional composition for use according to any one of paras 29 to 31 , wherein the nutritional composition is an infant formula.

33. The nutritional composition for use according to any one of paras 30 to 32, wherein the HMOs in the nutritional composition comprise or consist of, with reference to the total amount of HMOs in the nutritional composition: i. the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, and 6’SL comprising or consisting of: a. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; b. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; c. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; d. about 2 wt % to about 7 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; and e. about 4 wt % to about 13 wt % of 6’-SL, preferably about 7 wt % to about 10 wt %, preferably about 9 wt %; ii. the HMOs 2’-FL, LNnT, 3’-FL, and one of 3’-SL or 6’SL comprising or consisting of: a. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; b. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; c. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; and d. about 2 wt % to about 7 wt % of 3’-SL; preferably about 4 wt % to about 6 wt %, preferably about 5 wt %; or about 5 wt % to about 14 wt % of 6’-SL, preferably about 7 wt % to about 11 wt %, preferably about 9 wt %; iii. the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’SL and LNT comprising or consisting of: a. about 19 wt % to about 58 wt % of 2’-FL, preferably about 31 wt % to about 46 wt %, preferably about 38 wt %; b. about 10 wt % to about 29 wt % of LNnT, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; c. about 10 wt % to about 29 wt % 3’-FL, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; and d. about 2 wt % to about 6 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; e. about 54 wt % to about 12 wt % of 6’-SL, preferably about 6 wt % to about 9 wt %, preferably about 8 wt %; and f. about 6 wt % to about 17 wt % of LNT, preferably about 9 wt % to about 14 wt %, preferably about 12 wt %.

34. The nutritional composition for use according to any one of paras 30 to 33, comprising the HMOs 2’-FL (about 0.8-1.2 g/L of the composition as consumed), 3’-FL (about 0.4-0.6 g/L of the composition as consumed), LNnT (about 0.4-0.6 g/L of the composition as consumed); 3’-SL (about 0.08-0.12 g/L of the composition as consumed), 6’-SL (about 0.16-0.24 g/L of the composition as consumed), and LNT (about 0.24-0.36 g/L of the composition as consumed).

35. The nutritional composition for use according to any one of paras 30 to 34, comprising the HMOs 2’-FL (about 1 g/L of the composition as consumed), 3’-FL (about 0.5 g/L of the composition as consumed), LNnT (about 0.5 g/L of the composition as consumed); 3’-SL (about 0.1 g/L of the composition as consumed), 6’-SL (about 0.2 g/L of the composition as consumed), and LNT (about 0.3 g/L of the composition as consumed).

36. The nutritional composition for use according to any one of paras 30 to 35, comprising lactose in an amount of about 23-50 g/L or 29-44 g/L of the composition as consumed.

37. The nutritional composition for use according to any one of paras 30 to 35, comprising lactose in an amount of about 37 g/L of the composition as consumed.

38. The nutritional composition for use according to any one of paras 30 to 35, comprising lactose in an amount of about 58-88 g/L or 65-80 g/L of the composition as consumed.

39 The nutritional composition for use according to any one of paras 30 to 35, comprising lactose in an amount of about 73 g/L of the composition as consumed.

40. The nutritional composition for use according to any one of paras 30 to 39, wherein the nutritional composition is an extensively hydrolysed formula (eHF).

41 . The nutritional composition for use according to para 40, wherein the protein source in the extensively hydrolysed formula (eHF) is an extensively hydrolysed casein, whey, or combination thereof.

42. The nutritional composition for use according to para 40, wherein the protein source in the extensively hydrolysed formula (eHF) is an extensively hydrolysed whey.

43. The nutritional composition for use according to any one of paras 30 to 39, wherein the nutritional composition is an amino acid-based formula (AAF).

44. The nutritional composition for use according to any one of paras 30 to 39, wherein the nutritional composition is a partially hydrolysed infant formula (pHF).

45. The nutritional composition for use according to para 44, wherein the protein source in the partially hydrolysed formula (pHF) is a partially hydrolysed casein, whey, or combination thereof.

46. The nutritional composition for use according to para 40, wherein the protein source in the partially hydrolysed formula (pHF) is a partially hydrolysed whey. 47. The nutritional composition for use according to any one of paras 40 to 42 and 44 to 46, wherein the casein, whey, or combination thereof is from goat milk, donkey milk, camel milk or cow milk.

48. The nutritional composition for use according to any one of paras40 to 42 and 44 to 47 wherein the casein, whey, or combination thereof is from cow milk.

49. The nutritional composition for use according to any one of paras 30 to 39, comprising a vegetable protein.

50. The nutritional composition for use according to any one of paras 30 to 49, wherein the nutritional composition is an infant formula, a starter infant formula, a follow-on or follow-up infant formula and comprises:

(a) 1.6-3.2 g protein per 100 kcal;

(b) 9-14 g carbohydrate per 100 kcal; and/or

(c) 4-6 g fat per 100 kcal.

51 . The combination or nutritional composition for use according to any one of the preceding paras, wherein the combination or nutritional composition further comprising butyrate in an amount from about 1 mg/L to about 30 mg/L of the composition as consumed.

52. The combination or nutritional composition for use according to any one of the preceding paras, wherein the combination or nutritional composition further comprises butyrate in an amount of from about 1.6 mg/L to about 2.4 mg/L of the composition as consumed, or from about 8 mg/L to about 12 mg/L of the composition as consumed, or from about 16 mg/L to about 24 mg/L of the composition as consumed.

53. The combination or nutritional composition for use according to para 51 or para 52, wherein the combination or nutritional composition comprises butyrate in an amount of from about from about 16 mg/L to about 24 mg/L of the composition as consumed or about 20 mg/L of the composition as consumed.

54. The combination or nutritional composition for use according to any one of paras 1 to 50, wherein the combination or nutritional composition further comprises a compound having the formula: or combinations thereof as a source of dietary butyrate; wherein R¹, R², R³, R⁴, R⁵ and R⁶ are independently, a long chain fatty acid having between 16 and 20 carbons.

55. The combination or nutritional composition for use according to para 54, wherein each of R¹, R², R³, R⁴, R⁵ and R⁶ are oleic acid.

56. The combination or nutritional composition for use according to para 55, wherein the combination or nutritional composition comprises the equivalent of from about 1 mg/L to about 30 mg/L dietary butyrate in the composition as consumed.

57. The combination or nutritional composition for use according to para 55 or para 56, wherein the combination or nutritional composition comprises the equivalent of from about 1.6 mg/L to about 2.4 mg/L dietary butyrate in the composition as consumed, or from about 8 mg/L to about 12 mg/L dietary butyrate in the composition as consumed, or from about 16 mg/L to about 24 mg/L dietary butyrate in the composition as consumed.

58. The combination or nutritional composition for use according to any one of paras 55 to 57, wherein the combination or nutritional composition comprises the equivalent of from about 16 mg/L to about 24 mg/L dietary butyrate in the composition as consumed or about 20 mg/L dietary butyrate in the composition as consumed.

59. The combination or nutritional composition for use according to para 55, wherein the combination or nutritional composition comprises the equivalent of about 30 mg/L dietary butyrate in the composition as consumed.

60. The combination or nutritional composition for use according to para 55 or para 59, wherein the combination or nutritional composition comprises the equivalent of about 16-24 mg/L dietary butyrate in the composition as consumed.

61 . The combination or nutritional composition for use according to any one of paras 55, 59 and 60, wherein the combination or nutritional composition comprises the equivalent of about 20 mg/L dietary butyrate in the composition as consumed.

62. The combination or nutritional composition for use according to any one of paras 1 to 50, wherein the combination or nutritional composition further comprises a triglyceride composed of a mixture of butyric acid (C4:0) and caprylic acid (C8:0) as a source of dietary butyrate.

63. The combination or nutritional composition for use according to para 62, wherein the combination or nutritional composition comprises the equivalent of from about 1 mg/L to about 30 mg/L dietary butyrate in the composition as consumed.

64. The combination or nutritional composition for use according to para 62 or para 63, wherein the combination or nutritional composition comprises the equivalent of from about 1.6 mg/L to about 2.4 mg/L dietary butyrate in the composition as consumed, or from about 8 mg/L to about 12 mg/L dietary butyrate in the composition as consumed, or from about 16 mg/L to about 24 mg/L dietary butyrate in the composition as consumed.

65. The combination or nutritional composition for use according to any one of paras 62 to 64, wherein the combination or nutritional composition comprises the equivalent of from about 16 mg/L to about 24 mg/L dietary butyrate in the composition as consumed or about 20 mg/L dietary butyrate in the the composition as consumed.

66. The combination or nutritional composition for use according to para 62, wherein the combination or nutritional composition comprises the equivalent of about 30 mg/L dietary butyrate in the combination or nutritional composition as consumed.

67. The combination or nutritional composition for use according to para 62 or para 66, wherein the combination or nutritional composition comprises the equivalent of about 16- 24 mg/L dietary butyrate in the combination or nutritional composition as consumed.

68. The combination or nutritional composition for use according to any one of paras 62, 66, and 67, wherein the combination or nutritional composition comprises the equivalent of about 20 mg/L dietary butyrate in the combination or nutritional composition as consumed.

69. The combination for use according to any one of paras 1 to 29 and 50 to 68, wherein the HMOs in the combination comprise or consist of, with reference to the total amount of HMOs in the combination: i. the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, and 6’SL comprising or consisting of: a. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; b. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; c. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; d. about 2 wt % to about 7 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; and e. about 4 wt % to about 13 wt % of 6’-SL, preferably about 7 wt % to about 10 wt %, preferably about 9 wt %; ii. the HMOs 2’-FL, LNnT, 3’-FL, and one of 3’-SL or 6’SL comprising or consisting of: a. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; b. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; c. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; and d. about 2 wt % to about 7 wt % of 3’-SL; preferably about 4 wt % to about 6 wt %, preferably about 5 wt %; or about 5 wt % to about 14 wt % of 6’-SL, preferably about 7 wt % to about 11 wt %, preferably about 9 wt %; iii. the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’SL and LNT comprising or consisting of: a. about 19 wt % to about 58 wt % of 2’-FL, preferably about 31 wt % to about 46 wt %, preferably about 38 wt %; b. about 10 wt % to about 29 wt % of LNnT, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; c. about 10 wt % to about 29 wt % 3’-FL, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; and d. about 2 wt % to about 6 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; e. about 54 wt % to about 12 wt % of 6’-SL, preferably about 6 wt % to about 9 wt %, preferably about 8 wt %; and f. about 6 wt % to about 17 wt % of LNT, preferably about 9 wt % to about 14 wt %, preferably about 12 wt %.

70. The combination for use according to any one of paras 1 to 29 and 50 to 69, comprising the HMOs 2’-FL (about 0.8-1.2 g/L of the combination as consumed), 3’-FL (about 0.4-0.6 g/L of the combination as consumed), LNnT (about 0.4-0.6 g/L of the composition as consumed); 3’-SL (about 0.08-0.12 g/L of the combination as consumed), 6’-SL (about 0.16-0.24 g/L of the combination as consumed), and LNT (about 0.24-0.36 g/L of the combination as consumed).

71 . The combination for use according to any one of paras 1 to 29 and 50 to 70, comprising the HMOs 2’-FL (about 1 g/L of the combination as consumed), 3’-FL (about 0.5 g/L of the combination as consumed), LNnT (about 0.5 g/L of the combination as consumed); 3’-SL (about 0.1 g/L of the composition as consumed), 6’-SL (about 0.2 g/L of the combination as consumed), and LNT (about 0.3 g/L of the combination as consumed).

72. The combination for use according to any one of paras 1 to 29 and 50 to 71 , comprising lactose in an amount of about 23-50 g/L or 29-44 g/L of the combination as consumed.

73. The combination for use according to any one of paras 1 to 19 and 50 to 72, comprising lactose in an amount of about 37 g/L of the combination as consumed.

74. The combination for use according to any one of paras 1 to 29 and 50 to 71 , comprising lactose in an amount of about 58-88 g/L or 65-80 g/L of the combination as consumed.

75 The combination for use according to any one of paras 1 to 29 and 50 to 74, comprising lactose in an amount of about 73 g/L of the combination as consumed.

76. The combination or nutritional composition for use according to any one of the preceding paras wherein the subject is an infant, young child or child; preferably wherein the subject is an infant or young child.

77. The combination or nutritional composition for use according to any one of the preceding paras, wherein the combination or nutritional composition further comprises one or more bacterium of the genera Bifidobacterium and/or Lactobacillus.

78. The combination or nutritional composition defined according to any one of the preceding paras for use in the dietary management of cow’s milk protein allergy (CMPA).

79. The combination or nutritional composition for use according to any one of the preceding paras, wherein the dietary management is the reduction in severity or frequency of one or more symptoms of a disease.

80. A combination or nutritional composition as defined in any one of claims 1 to 77.

Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

Claims

1. A combination comprising lactose and human milk oligosaccharides (HMOs) 2'- fucosyllactose (2’-FL), lacto-/V-neotetraose (LNnT), 3-fucosyllactose (3’-FL), and at least one of 3’-sialyllactose (3’-SL) or 6’-sialyllactose (6’-SL) for use in

(a) the dietary management of dysbiosis of the gut microbiota in a subject;

(b) the dietary management of a disease or disorder associated with metabolism and/or accumulation of branched chain fatty acids (BCFA) in a subject; or

(c) the reduction of BCFA in a subject.

2. The combination for use according to claim 1 , wherein the composition comprises the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and lacto-N-tetraose (LNT).

3. The combination for use according to claim 1 or claim 2, wherein the combination is for use in the dietary management of dysbiosis of the gut microbiota, and wherein the dysbiosis includes increased production of BCFA, such as isobutyrate, isovalerate, and/or isocaproate.

4. The combination for use according to any one of claims 1 to 3, wherein the combination is for use in the dietary management of dysbiosis of the gut microbiota in a subject, and wherein the dysbiosis of the gut microbiota is associated with an intestinal disorder and/or an extra- intestinal disorder; optionally wherein the intestinal disorder is one or more of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and coeliac disease and/or wherein the extra-intestinal disorder is one or more of allergy, asthma, atopic dermatitis, metabolic syndrome, cardiovascular disease, obesity, and type 2 diabetes.

5. The combination for use according to claim 1 or claim 2, wherein the combination is for use in the dietary management of a disease or disorder associated with metabolism and/or accumulation of branched chain fatty acids (BCFA), and

(a) wherein the BCFA comprise isobutyrate, isovalerate, and/or isocaproate; and/or

(b) wherein the disease or disorder associated with metabolism of BCFA is increased proteolytic fermentation and/or decreased saccharolytic fermentation.

6. A combination comprising lactose and HMOs 2’-FL, LNnT, 3’-FL, and at least one of 3’- SL or 6’-SL for use in the dietary management of

(a) a pathogen in a subject;

(b) a pathogen in a subject, wherein the pathogen is one or more of bacterial, viral or protozoal pathogen; (c) a pathogen in a subject, wherein the pathogen is one or more viral pathogens, preferably rotavirus or norovirus; or

(d) a pathogen in a subject, wherein the pathogen is one or more bacterial pathogens, preferably C. difficile, C. perfringens, Campylobacter or Escherichia coir, preferably wherein the subject is an infant, young child or child who has not been breastfed or wherein breast-feeding has been halted.

7. The combination for use according to claim 6, comprising the composition comprises the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’-SL, and LNT.

8. The combination for use according to claim 6 or claim 7, wherein

(a) the pathogen is at least one of C. difficile and C. perfringens’, or

(b) the pathogen is at least one of C. difficile and C. perfringens, and wherein there is a resolution or reduction in one or more symptoms associated with an overgrowth of C. difficile and/or C. perfringens, preferably wherein the symptom is at least one of diarrhoea and fever.

9. The combination for use according to any preceding claim, wherein the combination of lactose and HMOs reduces levels of BCFA in the gut of the subject; optionally wherein the BCFA comprise isobutyrate, isovalerate, and/or isocaproate.

10. The combination for use according to any one of claims 1 to 9, comprising the HMOs 2’- FL (about 0.8-1.2 g/L of the combination as consumed), 3’-FL (about 0.4-0.6 g/L of the combination as consumed), LNnT (about 0.4-0.6 g/L of the combination as consumed); 3’-SL (about 0.08-0.12 g/L of the combination as consumed), 6’-SL (about 0.16-0.24 g/L of the combination as consumed), and LNT (about 0.24-0.36 g/L of the combination as consumed).

11. The combination for use according to any one of the preceding claims, wherein the combination is provided in the form of a nutritional composition and wherein the nutritional composition is an infant formula, a starter infant formula, a follow-on or follow-up infant formula or a growing-up milk, preferably an infant formula.

12. The combination for use according to any one of claims 1 to 10 or the nutritional composition for use according to claim 11 , wherein the HMOs in the combination or nutritional composition comprise or consist of, with reference to the total amount of HMOs in the combination or nutritional composition: i. the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, and 6’SL comprising or consisting of: a. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; b. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; c. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; d. about 2 wt % to about 7 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; and e. about 4 wt % to about 13 wt % of 6’-SL, preferably about 7 wt % to about 10 wt %, preferably about 9 wt %; ii. the HMOs 2’-FL, LNnT, 3’-FL, and one of 3’-SL or 6’SL comprising or consisting of: a. about 22 wt % to about 65 wt % of 2’-FL, preferably about 35 wt % to about 52 wt %, preferably about 43 wt %; b. about 11 wt % to about 33 wt % of LNnT, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; c. about 11 wt % to about 33 wt % 3’-FL, preferably about 17 wt % to about 26 wt %, preferably about 22 wt %; and d. about 2 wt % to about 7 wt % of 3’-SL; preferably about 4 wt % to about 6 wt %, preferably about 5 wt %; or about 5 wt % to about 14 wt % of 6’-SL, preferably about 7 wt % to about 11 wt %, preferably about 9 wt %; iii. the HMOs 2’-FL, LNnT, 3’-FL, 3’-SL, 6’SL and LNT comprising or consisting of: a. about 19 wt % to about 58 wt % of 2’-FL, preferably about 31 wt % to about 46 wt %, preferably about 38 wt %; b. about 10 wt % to about 29 wt % of LNnT, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; c. about 10 wt % to about 29 wt % 3’-FL, preferably about 15 wt % to about 23 wt %, preferably about 19 wt %; d. about 2 wt % to about 6 wt % of 3’-SL; preferably about 3 wt % to about 5 wt %, preferably about 4 wt %; e. about 54 wt % to about 12 wt % of 6’-SL, preferably about 6 wt % to about 9 wt %, preferably about 8 wt %; and f. about 6 wt % to about 17 wt % of LNT, preferably about 9 wt % to about 14 wt %, preferably about 12 wt %.

13. The nutritional composition for use according to claim 12, comprising the HMOs 2’-FL (about 0.8-1 .2 g/L of the composition as consumed), 3’-FL (about 0.4-0.6 g/L of the composition as consumed), LNnT (about 0.4-0.6 g/L of the composition as consumed); 3’-SL (about 0.08- 0.12 g/L of the composition as consumed), 6’-SL (about 0.16-0.24 g/L of the composition as consumed), and LNT (about 0.24-0.36 g/L of the composition as consumed).

14. The combination for use according to any one of claims 1 to 10 or the nutritional composition for use according claim 12 or claim 13, comprising

(a) lactose in an amount of about 23-50 g/L or 29-44 g/L of the combination or nutritional composition as consumed;

(b) lactose in an amount of about 37 g/L of the combination as consumed;

(c) lactose in an amount of about 58-88 g/L or 65-80 g/L of the combination or composition as consumed; or

(d) lactose in an amount of about 73 g/L of the combination or composition as consumed.

15. The combination or nutritional composition for use according to any one of the preceding claims, wherein the combination or nutritional composition further comprises butyrate

(a) in an amount from about 1 mg/L to about 30 mg/L of the combination or nutritional composition as consumed;

(b) in an amount of from about 1.6 mg/L to about 2.4 mg/L of the combination or nutritional composition as consumed, or from about 8 mg/L to about 12 mg/L of the combination or nutritional composition as consumed, or from about 16 mg/L to about 24 mg/L of the combination or nutritional composition as consumed;

(c) in an amount of from about from about 16 mg/L to about 24 mg/L of the composition or nutritional composition as consumed; or

(d) in an amount of about 20 mg/L of the composition or nutritional composition as consumed.

16. The combination for use according to any one of claims 1 to 10, 14 and 15 or the nutritional composition for use according to any one of the claims 12 to 15, wherein the combination or nutritional composition further comprises

(a) a compound having the formula: or combinations thereof as a source of dietary butyrate; wherein each of R¹, R², R³, R⁴, R⁵ and R⁶ are oleic acid; or

(b) a triglyceride composed of a mixture of butyric acid (C4:0) and caprylic acid (C8:0) as a source of dietary butyrate.

17. The combination for use according to claim 16 or the nutritional composition for use according to claim 16, wherein the combination or nutritional composition comprises the equivalent of

(a) from about 1 mg/L to about 30 mg/L of the combination or nutritional composition as consumed;

(b) from about 1.6 mg/L to about 2.4 mg/L of the combination or nutritional composition as consumed, or from about 8 mg/L to about 12 mg/L of the combination or nutritional composition as consumed, or from about 16 mg/L to about 24 mg/L of the combination or nutritional composition as consumed;

(c) about 30 mg/L dietary butyrate in the combination or nutritional composition as consumed;

(d) about 16-24 mg/L dietary butyrate in the combination or nutritional composition as consumed; or

(e) about 20 mg/L dietary butyrate in the combination or nutritional composition as consumed.

18. The combination or nutritional composition for use according to any one of the preceding claims wherein the combination or nutritional composition further comprises one or more bacterium of the genera Bifidobacterium and/or Lactobacillus.

19. The combination or nutritional composition defined according to any one of the preceding claims for use in the dietary management of cow’s milk protein allergy (CM PA).