Combinations for dietary management of gastrointestinal health

Abstract

The present invention provides a combination of lactose and at least one of the human milk oligosaccharides (HMOs) 2'-fucosyl lactose (2'-FL), lacto- / V-neotetraose (LNnT), 3-fucosyl lactose (3'-FL), 3'-sialyl lactose (3'-SL), or 6'-sialyl lactose (6'-SL), and optionally lacto-N-tetraose (LNT), for use in dietary management of intestinal microbiota dysbiosis in a subject, or for use in dietary management of diseases or disorders related to the metabolism and / or accumulation of BCFAs in a subject.

Description

[Technical Field]

[0001] The present invention relates, for example, to compositions, particularly nutritional compositions, and methods for dietary management of gastrointestinal health in infants. [Background technology]

[0002] Breast milk is recommended for all infants. However, in some cases, breastfeeding may be insufficient or unsuccessful for medical reasons, or mothers may choose not to breastfeed. Nutritional compositions such as infant formula have been developed for these situations.

[0003] Nutritional compositions for infants and toddlers are often sold as powders that are reconstituted with water, or, in some cases, as ready-to-drink or concentrated liquid compositions. These compositions are intended to cover most or all of the nutritional requirements of infants or toddlers.

[0004] Therefore, manufacturers of infant formula have made many attempts to induce nutritional and health benefits similar to or close to those of human breast milk. However, many studies have shown that infant formula does not induce the same effects on the body as human breast milk. For example, infants fed infant formula and infants fed human breast milk (HBM) may exhibit different gut (gastrointestinal) microbiota.

[0005] Human milk oligosaccharides (HMOs) have recently attracted considerable attention due to their roles in numerous biological processes occurring within the human body. 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).

[0006] There is growing evidence that the commensal community of microorganisms in the human digestive tract, known as the gut microbiome, plays a major role in health and disease. When the composition of the gut microbiome becomes unbalanced, the host may suffer consequences. Recent studies suggest that imbalances in the gut microbiome are present in a variety of individual disorders, including cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism.

[0007] Infancy, especially the first week, three months, six months, or twelve months of age, is a crucial period for establishing a balanced gut microbiome.

[0008] It is known that regulating the gut microbiome in infancy can have a significant impact on future health. For example, the gut microbiome can influence the development of a strong immune system later in life, as well as normal growth, and even the development of obesity or allergies later in life.

[0009] Therefore, there is a need for new nutritional interventions for dietary management of intestinal health, for example, in infants, especially those who are being fed formula. [Overview of the project]

[0010] The inventors have surprisingly found that certain combinations of lactose and human milk oligosaccharides (HMOs) synergistically promote beneficial effects on infant intestinal health, for example, by reducing levels of branched-chain fatty acids (BCFAs). BCFAs are associated with the formation of metabolites such as phenols and indoles, which are involved in proteolytic fermentation by the gut microbiota and have harmful effects on health. Therefore, compositions that reduce and / or limit BCFA production may have beneficial effects on infant intestinal health.

[0011] In one aspect, the present invention provides a combination for use in dietary management of dysbiosis of the gut microbiota in a subject, comprising lactose, and at least one of 2'-fucosyllactose (2'-FL), lacto-N-neotetraose (LNnT), 3'-fucosyllactose (3'-FL), 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL), which are human milk oligosaccharides (HMOs), and optionally lacto-N-tetraose (LNT). In a related aspect, the present invention provides a combination for use in dietary management of dysbiosis of the gut microbiota in a subject, comprising lactose, and 2'-FL, LNnT, 3'-FL, LNT, 3'-SL, and 6'-SL, which are HMOs.

[0012] In another aspect, the present invention provides a combination for use in dietary management of a disease or disorder associated with the metabolism and / or accumulation of BCFA in a subject, comprising lactose, and at least one of 2'-FL, LNnT, 3'-FL, 3'-SL or 6'-SL, which are HMOs, and optionally LNT. In a related aspect, the present invention provides a combination for use in dietary management of a disease or disorder associated with the metabolism and / or accumulation of BCFA in a subject, comprising lactose, and 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT, which are HMOs.

[0013] In a further aspect, the present invention provides a combination for use in dietary management of at least one pathogen, preferably a pathogenic bacterium, in a subject, comprising lactose, and at least one of 2'-FL, LNnT, 3'-FL, 3'-SL or 6'-SL, which are HMOs, and optionally LNT. In a related aspect, the present invention provides a combination for use in dietary management of at least one pathogen, preferably a pathogenic bacterium, in a subject, comprising lactose, and 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT, which are HMOs.

[0014] In another aspect, the present invention provides a combination for use in reducing BCFA in a subject, comprising lactose, at least one of 2'-FL, LNnT, 3'-FL, 3'-SL or 6'-SL which are HMOs, and optionally LNT. In a related aspect, the present invention provides a combination for use in reducing BCFA in a subject, comprising lactose, 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT which are HMOs.

Brief Description of the Drawings

[0015] [Figure 1] The effects of four HMO blends (M2, M4.1, M4.2, M6), lactose (L), and their combinations (M2+L, M4.1+L, M4.2+L, M6+L) on branched-chain fatty acids (BCFA) in infants with cow's milk protein allergy (CMPA) (n = 12) were tested using the SIFR® technology platform and compared with a no substrate control (NSC). Samples were collected after 6 hours, 24 hours, and 48 hours of simulated colonic incubation. Statistical differences between the four HMO blends and the NSC are indicated by *(0.01 < p < 0.05), **(0.001 < p < 0.01), or ***(p < 0.001), while differences between the combinations of lactose and HMO blends and each lactose-free condition are indicated by $ / $$ / $$$. Differences between HMO blends are indicated by § / §§ / §§§ (M4.1 / M4.2 / M6(+L) vs M2(+L)), # / ## / (M4.2 / M6(+L) vs M4.1(+L)), & / && / &&& (M6(+L) vs M4.2(+L)). [Figure 2]Principal component analysis (PCA) summarizing the effects of microbial communities from CMPA infants (n=12) on the community composition of four HMO blends (M2, M4.1, M4.2, M6), lactose (L), and combinations thereof (M2+L, M4.1+L, M4.2+L, M6+L) tested via the SIFR® technology platform, compared to a substrate-free control (NSC). PCA was calculated based on the (median) mean abundance of the 100 most abundant operational taxonomic units (OTUs) across the 12 CMPA infants, quantified via 16S rRNA sequencing (cells / mL) combined with flow cytometry, both at the start of colon incubation (0 hours) and 24 hours after the start. [Figure 3] Principal component analysis (PCA) summarizing the effects on the microbial community composition of CMPA infants (n=12) tested via the SIFR® technology platform for four HMO blends (M2, M4.1, M4.2, M6), lactose (L), and combinations thereof (M2+L, M4.1+L, M4.2+L, M6+L) compared to a substrate-free control (NSC). PCA was calculated based on the (central) mean abundance of OTU (FDR=0.20) significantly affected by any of the treatments, quantified via 16S rRNA gene sequencing (cells / mL) combined with flow cytometry 24 hours after the start of colon incubation. [Modes for carrying out the invention]

[0016] Various preferred features and embodiments of the present invention are described herein by non-limiting examples. Those skilled in the art will understand that all features of the present invention disclosed herein can be combined without departing from the scope of the disclosed invention.

[0017] No reference to prior art documents in this specification should be construed as an acknowledgment that such prior art is well known or forms part of a common general understanding in the art. All publications referenced herein are incorporated herein by reference.

[0018] As used herein, the words “comprises,” “comprising,” and similar words should not be interpreted as exclusive or exhaustive. In other words, they mean “including, but not limited to.” The terms “comprises,” “comprising,” and similar words also include the term “consisting of.”

[0019] Unless otherwise specified, the implementation of the present invention will involve the use of prior art that is within the scope of the skills of those skilled in the art. Such art is described in the literature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are commonly understood by those skilled in the art.

[0020] Numerical ranges include the numerical values ​​that define those ranges, and all percentages disclosed herein are weight / weight unless otherwise specified. As used herein, the term “about” means approximately, nearly, roughly, or near. When used with a number or range, the term “about” modifies the value or range by expanding the upper and lower boundaries of the stated number or range. Generally, the terms “about” and “approximately” are used herein to adjust a number or value by more than 10% and less than 10% of the stated value.

[0021] combination In one embodiment, the present invention provides a combination of lactose and at least one of the human milk oligosaccharides (HMOs) 2'-FL, LNnT, 3'-FL, LNT, and 3'-SL or 6'-SL for use in dietary management of gastrointestinal health in a subject. In a related embodiment, the present invention provides a combination of lactose and the HMOs 2'-FL, LNnT, 3'-FL, LNT, 3'-SL, and 6'-SL for use in dietary management of intestinal dysbiosis of the gut microbiota in a subject.

[0022] The combination may be administered to the subject in the form of a composition (for example, as a composition containing a lactose mixture and an HMO mixture).

[0023] The combination may be administered by any suitable route and in any suitable form. Preferably, the combination is administered orally and / or enterally. In a preferred embodiment, the combination is administered orally. The combination may be administered separately, simultaneously, or sequentially. In a preferred embodiment, the combination is administered simultaneously.

[0024] Lactose Lactose is a disaccharide composed of glucose and galactose. Glucose can be either α-pyranose or β-pyranose. Lactose is the primary carbohydrate source in breast milk.

[0025] The combination of the present invention, preferably the nutritional composition, may also contain a lactose-containing carbohydrate component, with lactose accounting for about 68% to about 97%, optionally about 70% to about 90%, and further optionally 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 formula, or otherwise effective for such use, can be used as the lactose-containing carbohydrate component.

[0026] In certain embodiments, lactose constitutes about 49% to about 100% of the non-HMO carbohydrates in the combination, preferably in the nutritional composition.

[0027] Lactose is present in the nutritional composition in some specific amounts. In one embodiment, lactose is present in the nutritional composition in an amount of up to about 53 g / L or up to about 110 g / L of the ingested composition. In a particular embodiment, lactose is present in an amount of about 37 g / L of the ingested composition. In a particular embodiment, lactose is present in the nutritional composition in an amount of about 18 g / L to 110 g / L of the ingested composition, for example, about 24 g / L to 98 g / L, 49 g / L to 98 g / L, or 58 to 88 g / L, or 65 g / L to 80 g / L of the ingested composition. In another particular embodiment, lactose is present in the nutritional composition in an amount of about 73 g / L of the ingested composition.

[0028] In one embodiment of the present invention, lactose is present in the nutritional composition in an amount of up to approximately 34 g per 100 g of composition based on the dry weight of the composition, or up to approximately 67 g per 100 g of composition based on the dry weight of the composition. Lactose may be present in an amount of approximately 22 g to 34 g per 100 g of composition based on the dry weight of the composition, or approximately 27 g per 100 g of composition based on the dry weight of the composition. In one embodiment of the present invention, lactose may be present in the nutritional composition in an amount of approximately 44 g to 67 g per 100 g of composition based on the dry weight of the composition. Lactose may be present in an amount of approximately 27 g per 100 g of composition based on the dry weight of the composition. Lactose may be present in an amount of approximately 55 g per 100 g of composition based on the dry weight of the composition.

[0029] The lactose used in this invention is not particularly limited, except that it must be suitable for inclusion in a composition for administration to a human being of any age, preferably a nutritional composition. The lactose used in this invention can be synthesized by any suitable means known in the art, for example, by enzymatic, biotechnological and / or chemical methods including chemical synthesis, fermentation and / or production by suitable bacteria or yeast, or can be derived from milk generally containing about 5% lactose or obtained by other means. Lactose can be added in a purified or partially purified form, or as a fraction of milk or processed milk products. Some lactose can also be provided as an intrinsic excipient in other additives, for example, as excipient lactose in many protein sources.

[0030] Lactose-containing carbohydrates may also contain non-lactose carbohydrates (e.g., a small number of non-lactose carbohydrates), non-limiting sources of which include hydrolyzed or untreated natural and / or chemically modified starches from corn, tapioca, rice, or potatoes, in waxy or unwaxed form. 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.

[0031] The appropriate dosage of lactose is described, for example, in 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.

[0032] Human milk oligosaccharides (HMOs) HMOs have recently attracted considerable attention due to their roles in numerous biological processes occurring within the human body. 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).

[0033] Many different types of HMOs are found in human breast milk. Each oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine, and has a wide variety of bonds between them, resulting in a very diverse range of oligosaccharides in human breast milk. Almost all HMOs have a lactose moiety at the reducing end, and the non-reducing end is occupied by sialic acid and / or fucose (if present). HMOs can be acidic (e.g., charged sialic acid-containing oligosaccharides) or neutral (e.g., fucosyl oligosaccharides).

[0034] In some embodiments, the HMO in the combination includes, essentially consists of, or preferably consists of, at least one of 2'-FL, LNnT, 3'-FL, 3'-SL, or 6'-SL, and optionally LNT. Thus, the combination may include 2'-FL, LNnT, 3'-FL, and 3'-SL, and / or 6'-SL. The combination may include 2'-FL, LNnT, 3'-FL, LNT, and 3'-SL, and / or 6'-SL. The combination may include 2'-FL, LNnT, 3'-FL, and 3'-SL. The combination may include 2'-FL, LNnT, 3'-FL, and 6'-SL. The combination may include 2'-FL, LNnT, 3'-FL, 3'-SL, and 6'-SL. The combination may include 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT. The nutritional composition may appropriately not contain any other HMOs other than at least one of 2'-FL, LNnT, 3'-FL, and 3'-SL or 6'-SL.

[0035] In further embodiments, the HMOs in the composition may include, essentially consist of, or comprise 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT. Thus, the nutritional composition may not contain any other HMOs besides 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT.

[0036] HMOs can be obtained by any suitable method. Suitable methods for synthesizing HMOs are well known to those skilled in the art. For example, methods for producing HMOs have been developed by microbial fermentation, enzymatic methods, chemical synthesis, or combinations thereof (Zeuner et al., 2019. Molecules, 24(11), p.2033).

[0037] 2'-FL can be produced by biotechnological means using specific fucosyltransferases and / or fucosidases, either through enzyme-based fermentation techniques (recombinant or natural enzymes) or microbial fermentation techniques. In the latter case, the microorganisms can either express the natural enzymes and substrates, or be engineered to produce the respective substrates and enzymes. Alternatively, 2'-FL can be produced by chemosynthesis from lactose and free fucose.

[0038] 2'-FL may be present in the nutritional composition in some specific amounts. In one embodiment of the present invention, 2'-FL may be present in the nutritional composition in an amount of up to about 1.5 g / L of the ingested composition. 2'-FL may be present in the nutritional composition in an amount of 0.2 to 1.5 g / L of the ingested composition, for example, 0.25 g / L to 1.5 g / L, or 0.5 g / L to 1.5 g / L, or 0.8 g / L to 1.2 g / L. In another specific embodiment, 2'-FL may be present in the nutritional composition in an amount of about 1.2 g / L of the ingested composition. In another specific embodiment, 2'-FL may be present in the nutritional composition in an amount of about 1.0 g / L of the ingested composition.

[0039] In one embodiment of the present invention, 2'-FL is present in the nutritional composition in an amount of up to about 1.2 g per 100 g of composition based on the dry weight of the composition. In another embodiment of the present invention, 2'-FL may be present in the nutritional composition in an amount of 0.38 g to 1.2 g per 100 g of composition based on the dry weight of the composition, for example, 0.2 g to 1.2 g, or 0.38 g to 1.2 g, or 0.61 g to 1.5 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, 2'-FL is present in the nutritional composition in an amount of about 0.9 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, 2'-FL is present in the nutritional composition in an amount of about 0.8 g per 100 g of composition based on the dry weight of the composition.

[0040] 3'-FL can be synthesized by enzymatic, biotechnological, and / or chemical methods. 3'-FL can be produced by fermentation using genetically modified microorganisms. Alternatively, 3'-FL can be produced as described in International Publication 2013 / 139344.

[0041] 3'-FL may be present in the nutritional composition in some specific amounts. In one embodiment of the present invention, 3'-FL may be present in the nutritional composition in an amount of up to about 1.1 g / L of the ingested composition, and 3'-FL may be present in the nutritional composition in an amount of 0.1 g / L to 1.1 g / L of the ingested composition, for example, 0.13 g / L to 1.1 g / L, or 0.25 g / L to 0.75 g / L, or 0.4 g / L to 0.6 g / L. In another specific embodiment, 3'-FL may be present in the nutritional composition in an amount of about 0.9 g / L of the ingested composition. In yet another specific embodiment, 3'-FL may be present in the nutritional composition in an amount of about 0.5 g / L of the ingested composition.

[0042] In one embodiment of the present invention, 3'-FL is present in the nutritional composition in an amount of up to about 0.8 g per 100 g of composition based on the dry weight of the composition. In another embodiment of the present invention, 3'-FL may be present in the nutritional composition in an amount of 0.08 g to 0.8 g per 100 g of composition based on the dry weight of the composition, for example, 0.1 g / L to 0.8 g / L, or 0.2 g to 0.6 g, or 0.3 g to 0.5 g per 100 g of composition based on the dry weight of the composition. In another specific embodiment, 3'-FL is present in the nutritional composition in an amount of about 0.7 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, 3'-FL is present in the nutritional composition in an amount of about 0.4 g per 100 g of composition based on the dry weight of the composition.

[0043] LNnT can be chemically synthesized by the enzymatic transfer of sugar units from a donor moiety to an acceptor moiety using glycosyltransferase, as described, for example, in U.S. Patent No. 5,288,637 and International Publication No. 1996 / 010086. Alternatively, LNnT can be prepared by chemically converting either a free or oligosaccharide-bound ketohexose (e.g., fructose) to N-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharide, as described in Wrodnigg, TM and Stutz, AE (1999) Angew. Chem. Int. Ed. 38:827-828. The N-acetyl-lactosamine thus produced can then be transferred to lactose as the acceptor moiety. Alternatively, LNnT may be produced as described in International Publication No. 2011 / 100980 or International Publication No. 2013 / 044928.

[0044] LNnT may be present in the nutritional composition in some specific amounts. In one embodiment, LNnT is present in the nutritional composition in an amount of up to about 0.75 g / L of the ingested composition, and LNnT may be present in the nutritional composition in an amount of 0.1 g / L to 0.75 g / L of the ingested composition, for example, 0.13 g / L to 0.75 g / L or 0.25 g / L to 0.75 g / L or 0.4 g / L to 0.6 g / L of the ingested composition. In another specific embodiment, LNnT is present in the nutritional composition in an amount of about 0.7 g / L of the ingested composition. In yet another specific embodiment, LNnT is present in the nutritional composition in an amount of about 0.5 g / L of the ingested composition.

[0045] In one embodiment of the present invention, LNnT is present in the nutritional composition in an amount of up to about 0.6 g per 100 g of composition based on the dry weight of the composition. In another embodiment of the present invention, LNnT may be present in an amount of 0.08 g to 0.6 g per 100 g of composition based on the dry weight of the composition, for example, 0.1 g / L to 0.6 g / L, or 0.2 g / L to 0.6 g, or 0.3 to 0.5 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, LNnT is present in the nutritional composition in an amount of about 0.6 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, LNnT is present in the nutritional composition in an amount of about 0.4 g per 100 g of composition based on the dry weight of the composition.

[0046] 3'-SL can be synthesized by enzymatic, biotechnological, and / or chemical methods. 3'-SL can be produced as described in International Publication 2014 / 153253.

[0047] 3'-SL may be present in the nutritional composition in some specific amounts. In one embodiment of the present invention, 3'-SL may be present in the nutritional composition in an amount of up to about 0.24 g / L of the ingested composition, and 3'-SL may be present in the nutritional composition in an amount of 0.02 to 0.24 g / L of the ingested composition, for example, 0.025 g to 0.24 g / L, or 0.05 g to 0.15 g / L, or 0.08 g to 0.12 g / L. In another specific embodiment, 3'-SL may be present in the nutritional composition in an amount of about 0.2 g / L of the ingested composition. In yet another specific embodiment, 3'-SL may be present in the nutritional composition in an amount of about 0.1 g / L of the ingested composition.

[0048] In one embodiment of the present invention, 3'-SL is present in the nutritional composition in an amount of up to about 0.18 g per 100 g of composition based on the dry weight of the composition. In another embodiment of the present invention, 3'-SL may be present in the nutritional composition in an amount of 0.015 g to 0.18 g per 100 g of composition based on the dry weight of the composition, for example, 0.019 g / L to 0.18 g / L, or 0.038 g to 0.11 g, or 0.06 g to 0.09 g per 100 g of composition based on the dry weight of the composition. In another specific embodiment, 3'-SL is present in the nutritional composition in an amount of about 0.15 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, 3'-SL is present in the nutritional composition in an amount of about 0.08 g per 100 g of composition based on the dry weight of the composition.

[0049] 6'-SL can be synthesized by a chemical method involving stereoselective 6'-O-sialylation of either a 4',6'-sugar diol or a 6'-sugar alcohol using donor activation of a glycosyl halide, thioglycoside, or diethyl phosphite. Alternatively, 6'-SL can be produced enzymatically using glycosyltransferases and sialidases. 6'-SL can also be produced as described in International Publication No. 2011 / 100979.

[0050] 6'-SL may be present in the nutritional composition in some specific amounts. In one embodiment of the present invention, 6'-SL may be present in the nutritional composition in an amount of up to about 0.84 g / L of the ingested composition, and 6'-SL may be present in the nutritional composition in an amount of 0.04 g / L to 0.84 g / L of the ingested composition, for example, 0.05 g / L to 0.84 g / L, or 0.1 g / L to 0.3 g / L, or 0.16 g / L to 0.24 g / L. In another specific embodiment, 6'-SL may be present in the nutritional composition in an amount of about 0.7 g / L of the ingested composition. In yet another specific embodiment, 6'-SL may be present in the nutritional composition in an amount of about 0.2 g / L of the ingested composition.

[0051] In one embodiment of the present invention, 6'-SL is present in the nutritional composition in an amount of up to about 0.64 g per 100 g of composition based on the dry weight of the composition. In another embodiment of the present invention, 6'-SL may be present in the nutritional composition in an amount of 0.003 g to 0.64 g per 100 g of composition based on the dry weight of the composition, for example, 0.038 g / L to 0.64 g / L, or 0.076 g to 0.23 g, or 0.12 g to 0.18 g per 100 g of composition based on the dry weight of the composition. In another specific embodiment, 6'-SL is present in the nutritional composition in an amount of about 0.53 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, 6'-SL is present in the nutritional composition in an amount of about 0.08 g per 100 g of composition based on the dry weight of the composition.

[0052] LNT can be synthesized by enzymatic, biotechnological, and / or chemical methods. LNT can be produced as described in International Publication 2012 / 155916 or International Publication 2013 / 044928. A mixture of LNT and LNnT can be prepared as described in International Publication 2013 / 091660.

[0053] LNT may be present in the nutritional composition in some specific amounts. In one embodiment, LNT is present in the nutritional composition in an amount of up to about 0.96 g / L of the ingested composition, and the amount of LNT may be 0.06 g / L to 0.96 g / L of the ingested composition, for example, 0.075 g / L to 0.96 g / L, or 0.15 g / L to 0.45 g / L, or 0.24 g / L to 0.36 g / L. In another specific embodiment, LNT is present in the nutritional composition in an amount of about 0.8 g / L of the ingested composition. In yet another specific embodiment, LNT is present in the nutritional composition in an amount of about 0.3 g / L of the ingested composition.

[0054] In one embodiment of the present invention, LNT is present in the nutritional composition in an amount of up to about 0.73 g per 100 g of composition based on the dry weight of the composition. In another embodiment of the present invention, LNT may be present in the nutritional composition in an amount of 0.046 g to 0.73 g per 100 g of composition based on the dry weight of the composition, for example, 0.057 g to 0.73 g / L, or 0.11 g to 0.34 g, or 0.18 g to 0.27 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, LNT is present in the nutritional composition in an amount of about 0.6 g per 100 g of composition based on the dry weight of the composition. In yet another specific embodiment, LNT is present in the nutritional composition in an amount of about 0.2 g per 100 g of composition based on the dry weight of the composition.

[0055] In some embodiments, the combination includes HMOs 2'-FL, LNnT, 3'-FL, and 3'-SL. In some embodiments, the HMOs in the combination consist of or are essentially 2'-FL, LNnT, 3'-FL, and 3'-SL. In certain embodiments, the combination includes, or is essentially 2'-FL (about 0.8 g / L to 1.2 g / L of the ingested composition), 3'-FL (about 0.4 g / L to 0.6 g / L of the ingested composition), LNnT (about 0.4 g / L to 0.6 g / L of the ingested composition), 3'-SL (about 0.08 g / L to 0.12 g / L of the ingested composition), and optionally LNT (about 0.24 g / L to 0.36 g / L of the ingested composition), or consists of or is essentially 2'-FL (about 0.8 g / L to 0.36 g / L of the ingested composition),

[0056] In some embodiments, the combination includes 2'-FL, LNnT, 3'-FL, and 6'-SL, which are HMOs. In some embodiments, the HMOs in the combination consist of or are essentially 2'-FL, LNnT, 3'-FL, and 6'-SL. In certain embodiments, the combination includes, or is essentially composed of, HMOs, or is HMO, consisting of 2'-FL (about 0.8 g / L to 1.2 g / L of the ingested composition), 3'-FL (about 0.4 g / L to 0.6 g / L of the ingested composition), LNnT (about 0.4 g / L to 0.6 g / L of the ingested composition), 6'-SL (about 0.16 g / L to 0.24 g / L of the ingested composition), and optionally LNT (about 0.24 g / L to 0.36 g / L of the ingested composition).

[0057] In some embodiments, the combination, preferably the nutritional composition, is, with respect to the total amount of HMOs in the combination, preferably the nutritional composition. i. 2'-FL of approximately 22% to 65% by weight, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight; ii. LNnT of approximately 11% to 33% by weight, preferably about 17% to 26% by weight, preferably about 22% by weight; iii. 3'-FL in an amount of about 11% to about 33% by weight, preferably about 17% to about 26% by weight, preferably about 22% by weight; and iv. 3'-SL in approximately 2% to 7% by weight; preferably 3'-SL in approximately 3% to 5% by weight, preferably 4% by weight; and 6'-SL in approximately 4% to 13% by weight, preferably 6'-SL in approximately 7% to 10% by weight, preferably 9% by weight. Includes.

[0058] In some embodiments, the combination, preferably the nutritional composition, is relative to the total amount of HMO. i. 2'-FL of approximately 22% to 65% by weight, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight; ii. LNnT of approximately 11% to 33% by weight, preferably about 17% to 26% by weight, preferably about 22% by weight; iii. 3'-FL in an amount of about 11% to about 33% by weight, preferably about 17% to about 26% by weight, preferably about 22% by weight; and iv. 3'-SL of approximately 2% to 7% by weight; preferably 3'-SL of approximately 4% to 6% by weight, preferably 3'-SL of approximately 5% by weight; or 6'-SL of approximately 5% to 14% by weight, preferably 6'-SL of approximately 7% to 11% by weight, preferably 6'-SL of approximately 9% by weight Includes.

[0059] In some embodiments, the present invention provides combinations of HMOs including 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT. In some embodiments, the HMOs in the combination consist of, or essentially consist of, 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT. In certain embodiments, the combinations include, essentially consist of, or are HMOs: 2'-FL (about 0.8 g / L to 1.2 g / L of the ingested composition), 3'-FL (about 0.4 g / L to 0.6 g / L of the ingested composition), LNnT (about 0.4 g / L to 0.6 g / L of the ingested composition); 3'-SL (about 0.08 g / L to 0.12 g / L of the ingested composition), 6'-SL (about 0.16 g / L to 0.24 g / L of the ingested composition), and LNT (about 0.24 g / L to 0.36 g / L of the ingested composition). It consists of.

[0060] In some embodiments, the combination, preferably the nutritional composition, is relative to the total amount of HMO. i. Approximately 19% to 58% by weight of 2'-FL, preferably approximately 31% to 46% by weight, preferably approximately 38% by weight of 2'-FL; ii. LNnT of approximately 10% to 29% by weight, preferably about 15% to 23% by weight, preferably about 19% by weight; iii. 3'-FL in an amount of about 10% to about 29% by weight, preferably about 15% to about 23% by weight, preferably about 19% by weight; and iv. Approximately 2% to 6% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; v. Approximately 54% to approximately 12% by weight of 6'-SL, preferably approximately 6% to approximately 9% by weight, preferably approximately 8% by weight of 6'-SL; and vi. LNT of approximately 6% to 17% by weight, preferably approximately 9% to 14% by weight, preferably approximately 12% by weight Includes.

[0061] In certain embodiments of the present invention, the nutritional composition comprises 2'-FL and LNnT in 2'-FL; the LNnT weight ratio is 1:10 to 12:1, for example 1:7 to 10:1, or 1:5 to 5:1, or 2:1 to 5:1, or 1:3 to 3:1, or 1:2 to 2:1, or 1:1 to 3:1, or 1:5 to 1:0.5; for example 2:1 or 10:1. In certain embodiments of the present invention, the nutritional composition comprises 2'-FL and LNnT in 2'-FL; the LNnT weight ratio is approximately 2:1.

[0062] The subjects may be administered any appropriate amount of HMO in any appropriate form and via any appropriate route of administration (e.g., via any form and route described herein).

[0063] Appropriate doses of human oligosaccharides are described, for example, in 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.

[0064] Appropriately, the HMO combination is administered to the subject in amounts of at least approximately 0.05 g / day, at least approximately 0.1 g / day, or at least approximately 0 g / day. 5 g / day, at least approximately 1 g / day, or at least approximately 2 g / day. Appropriately, the HMO combination is administered to the subject in amounts of approximately 10 g / day or less, approximately 8 g / day or less, or approximately 5 g / day or less. Appropriately, the HMO combination is administered to the subject in amounts of approximately 0.05 g / day to approximately 10 g / day, approximately 0.1 g / day to approximately 10 g / day, approximately 0.5 g / day to approximately 10 g / day, approximately 1 g / day to approximately 8 g / day, or approximately 2 g / day to approximately 5 g / day.

[0065] Appropriately, 2'-FL is administered to the subject at a dose of at least approximately 0.05 g / day, at least approximately 0.1 g / day, or at least approximately 0.2 g / day. At least approximately 0.5 g / day, at least approximately 0.8 g / day, or at least approximately 1 g / day. Appropriately, 2'-FL is administered to the subject at a dose of approximately 5 g / day or less, approximately 4 g / day or less, or approximately 3 g / day or less. Appropriately, 2'-FL is administered to the subject at a dose of approximately 0.5 g / day to approximately 5 g / day, approximately 0.8 g / day to approximately 4 g / day, or approximately 1 g / day to approximately 3 g / day.

[0066] Appropriately, LNnT is administered to subjects at a dose of at least approximately 0.05 g / day, at least approximately 0.2 g / day, at least approximately 0.3 g / day, or at least approximately 0.4 g / day. Appropriately, LNnT is administered to subjects at a dose of approximately 2.5 g / day or less, approximately 2 g / day or less, or approximately 1.5 g / day or less. Appropriately, LNnT is administered to subjects at a dose of approximately 0.2 g / day to approximately 2.5 g / day, approximately 0.3 g / day to approximately 2 g / day, or approximately 0.4 g / day to approximately 1.5 g / day.

[0067] Appropriately, 3'-FL is administered to the subject at a dose of at least approximately 0.05 g / day, at least approximately 0.1 g / day, at least approximately 0.15 g / day, or at least approximately 0.2 g / day. Appropriately, 3'-FL is administered to the subject at a dose of approximately 1 g / day or less, approximately 0.8 g / day or less, or approximately 0.6 g / day or less. Appropriately, 3'-FL is administered to the subject at a dose of approximately 0.1 g / day to approximately 1 g / day, approximately 0.15 g / day to approximately 0.8 g / day, or approximately 0.2 g / day to approximately 0.6 g / day.

[0068] Appropriately, 3'-SL is administered to the subject at a dose of at least approximately 0.05 g / day, at least approximately 0.1 g / day, at least approximately 0.15 g / day, or at least approximately 0.2 g / day. Appropriately, 3'-SL is administered to the subject at a dose of approximately 1 g / day or less, approximately 0.8 g / day or less, or approximately 0.6 g / day or less. Appropriately, 3'-SL is administered to the subject at a dose of approximately 0.1 g / day to approximately 1 g / day, approximately 0.15 g / day to approximately 0.8 g / day, or approximately 0.2 g / day to approximately 0.6 g / day.

[0069] Appropriately, 6'-SL is administered to the subject at a dose of at least approximately 0.05 g / day, at least approximately 0.1 g / day, at least approximately 0.15 g / day, or at least approximately 0.2 g / day. Appropriately, 6'-SL is administered to the subject at a dose of approximately 1 g / day or less, approximately 0.8 g / day or less, or approximately 0.6 g / day or less. Appropriately, 6'-SL is administered to the subject at a dose of approximately 0.1 g / day to approximately 1 g / day, approximately 0.15 g / day to approximately 0.8 g / day, or approximately 0.2 g / day to approximately 0.6 g / day.

[0070] Appropriately, LNT is administered to the subject at a dose of at least approximately 0.05 g / day, at least approximately 0.2 g / day, at least approximately 0.3 g / day, or at least approximately 0.4 g / day. Appropriately, LNT is administered to the subject at a dose of approximately 2.5 g / day or less, approximately 2 g / day or less, or approximately 1.5 g / day or less. Appropriately, LNT is administered to the subject at a dose of approximately 0.2 g / day to approximately 2.5 g / day, approximately 0.3 g / day to approximately 2 g / day, or approximately 0.4 g / day to approximately 1.5 g / day.

[0071] Appropriately, 2'-FL is administered to subjects at a dose of approximately 0.05 g / day to approximately 5 g / day; LNnT is administered to subjects at a dose of approximately 0.05 g / day to approximately 2.5 g / day; 3'-FL is administered to subjects at a dose of approximately 0.05 g / day to approximately 1 g / day; 3'-SL is administered to subjects at a dose of approximately 0.05 g / day to approximately 1 g / day; 6'-SL is administered to subjects at a dose of approximately 0.05 g / day to approximately 1 g / day; and / or LNT is administered to subjects at a dose of approximately 0.05 g / day to approximately 2.5 g / day.

[0072] As used herein, the term “essentially derived from” means that any additional unlisted component, element, or process does not substantially affect the properties of the claimed apparatus, composition, method, etc. Suitablely, a composition containing an HMO “essentially derived from” an enlisted HMO may contain trace amounts of unlisted HMOs that do not substantially affect the properties of the composition.

[0073] Nutritional composition The term "nutritional composition" refers to a composition that provides nutrients to a subject.

[0074] Nutritional compositions are typically taken orally and usually contain a lipid source, or a fat source and a protein source.

[0075] In certain embodiments, 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., a synthetic nutritional composition is not breast milk).

[0076] The nutritional composition of the present invention may be in solid form (e.g., powder) or in liquid form. The amounts of various components (e.g., oligosaccharides) can be expressed as g / 100g of composition on a dry weight basis if it is in solid form, e.g., powder, or as a concentration of g / 1L of composition if it refers to liquid form (liquid form also includes liquid compositions that can be obtained from powder after reconstitution in a liquid such as milk or water, e.g., reconstituted infant formula, or follow-on / follow-up formula, or growing-up milk, or infant cereal products, or any other formulations designed for infant nutrition). Preferably, the nutritional composition is in powder form and is reconstituted with water.

[0077] In preferred embodiments, the nutritional composition is for infants or toddlers. Infants may be, for example, 0 to 12 months old. Toddlers may be, for example, 1 to 3 years old. In particularly preferred embodiments, the nutritional composition is infant formula or toddler formula.

[0078] The term "infant formula" can refer to a food product for specific nutritional uses intended for infants in the first year of life, which, on its own, meets the nutritional requirements for humans in this category (as defined in European Commission Regulation (Ell) 2016 / 127 of September 25, 2015).

[0079] The term "infant formula" encompasses both "starter formula" and "follow-up formula," or "follow-on formula."

[0080] Follow-up formula or follow-on formula should be given from 6 months of age onward.

[0081] The infant formula of the present invention may be a hypoallergenic infant formula. The infant formula of the present invention may be an extensively hydrolyzed infant formula (eHF) or an amino acid-based infant formula (AAF). Alternatively, the infant formula may be a partially hydrolyzed infant formula (pHF).

[0082] The term “highly hydrolyzed infant formula” or “eHF” can refer to infant formula containing highly hydrolyzed proteins. eHF may be a hypoallergenic infant formula that provides complete nutrition for infants who cannot digest unprocessed cow’s milk protein (CMP), or who are intolerant to or allergic to CMP.

[0083] The term “amino acid-based infant formula” or “AAF” may refer to an infant formula containing only free amino acids as the source of protein. AAF may not contain detectable peptides. AAF may be a hypoallergenic infant formula that provides complete nutrition for infants with food protein allergies and / or food protein intolerances. For example, AAF may be a hypoallergenic infant formula that provides complete nutrition for infants who cannot digest untreated CMP, or who are intolerant to or allergic to CMP, or who may have extremely severe or life-threatening symptoms and / or sensitization to multiple foods.

[0084] A "hypoallergenic" composition is one that is less likely to cause an allergic reaction, and the protein source is provided by highly hydrolyzed proteins or free amino acids. Hypoallergenic infant formulas 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).

[0085] Infants may be given only infant formula, or infant formula may be used as a supplement to human breast milk.

[0086] The term "infant formula" can refer to a food intended to partially meet the nutritional requirements of infants aged 1 to 3 years. The expression "infant formula" encompasses "infant milk," "growing-up milk," or "infant formula." The ESPGHAN Committee on Nutrition recently outlined infant formula (Hojsak, I. et al., 2018. Journal of pediatric gastroenterology and nutrition, 66(1), pp. 177-185). Appropriately, infant 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.

[0087] The infant formula of the present invention may be a hypoallergenic infant formula. The infant formula of the present invention may be a highly hydrolyzed infant formula or an amino acid-based infant formula. Alternatively, the infant formula may be a partially hydrolyzed young-child formula (pHF).

[0088] The infant formula or toddler formula of the present invention may be in powder or liquid form.

[0089] The liquid may be, for example, concentrated liquid infant formula or ready-to-feed formula. The formula may be in the form of reconstituted infant or toddler formula (i.e., liquid formula reconstituted from powder form). Concentrated liquid infant or toddler formula can preferably be diluted to a liquid composition suitable for infant or toddler, for example, by adding water.

[0090] In some embodiments, the infant or toddler formula is in powder form. The powder can be reconstituted into a liquid composition suitable for infants or toddlers, for example, by adding water.

[0091] When formulated as directed, the nutritional composition may have an energy density of approximately 60-72 kcal per 100 mL. Ideally, when formulated as directed, the nutritional composition may have an energy density of approximately 60-70 kcal per 100 mL.

[0092] The nutritional composition according to the present invention may be, for example, infant formula, infant starter formula, follow-on or follow-up formula, a fortifier such as a human milk fortifier, or a supplement. In some specific embodiments, the composition of the present invention is infant formula, infant formula, or a supplement. In one preferred embodiment, the nutritional composition of the present invention is infant formula.

[0093] In the context of the present invention, the term “fortifier” refers to a composition containing one or more nutrients that have nutritional benefits to an infant. The term “milk fortifier” means any composition used to fortify or supplement 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 being dissolved in human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients, or it can be administered as a standalone composition. When administered as a standalone composition, the human milk fortifier of the present invention may also be identified as 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 formula fortifier, such as a breast milk fortifier (e.g., a human breast milk fortifier) ​​or an infant formula fortifier or a follow-on / follow-up formula fortifier.

[0094] In some 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 a unit dose. The supplement may be in the form of, for example, tablets, capsules, lozenges, or liquids. The supplement may further contain protective hydrophilic colloids (such as gum, protein, or modified starch), binders, film-forming agents, capsule encapsulants / capsule encapsulation materials, wall / shell wall materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (such as oils, fats, waxes, or lecithin), adsorbents, carriers, fillers, compounds, dispersants, wetting agents, processing aids (solvents), flowing agents, taste masking agents, bulking agents, gelling agents, and gel-forming agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients, and diluents, including, but not limited to, water, gelatin of any origin, vegetable gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavorings, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, and fillers.

[0095] Furthermore, supplements may also contain vitamins, minerals, trace elements, and other micronutrients, as recommended by government agencies such as LISRDA, in addition to organic or inorganic carrier materials suitable for oral or parenteral administration.

[0096] Other product formats, such as beverages and powders (in sachets), can also be selected. In further embodiments, the nutritional composition is selected from the group consisting of beverage products, amino acid-based beverages, yogurt products, fermented milk, fruit juices, dried powders in sachets, or cereal bars. These nutritional compositions are well suited for administering plant phenols to, for example, humans, older children and adults.

[0097] In further embodiments, the nutritional composition is a specific medical food, such as a healthcare nutritional composition for oral nutritional supplementation, and / or a nutritional product for enteral or parental nutritional supplementation. In the latter case, it should contain only components suitable for parenteral nutritional supplementation. Components suitable for parenteral nutritional supplementation are known to those skilled in the art. The nutritional composition of the present invention may be in solid (e.g., powder) or liquid or gelatinous form.

[0098] protein The term "protein" includes peptides and free amino acids. The protein content of a nutritional composition can be calculated by any method known to those skilled in the art. Preferably, the protein content can be determined by the nitrogen-protein ratio method, for example, as described in Maubois, JL and Lorient, D. (2016) Dairy Science & Technology 96(1):15-25. Preferably, the protein content is calculated as nitrogen content × 6.25, as defined in European Commission Regulation (EU) 2016 / 127 of September 25, 2015. The nitrogen content can be determined by any method known to those skilled in the art. For example, the nitrogen content can be measured by the Kjeldahl method.

[0099] The protein content of the nutritional composition of the present invention, and more particularly the infant formula of the present invention, is preferably in the range of 1.6 g to 3.2 g of protein per 100 kcal. In some embodiments, the protein content of the nutritional composition is in the range of 1.8 g to 3.0 g of protein per 100 kcal.

[0100] Infant formulas such as eHF or AAF, which have lower protein content, may be safer and more tolerable, while also supporting the proper growth and development of infants with allergies.

[0101] Therefore, in some embodiments, the nutritional composition of the present invention, and in particular the infant formula of the present invention, may contain about 2.5 g or less of protein per 100 kcal. For example, the nutritional composition may contain about 2.3 g or less of protein per 100 kcal, 2.0 g or less of protein per 100 kcal, or about 1.9 g of protein per 100 kcal.

[0102] Appropriately, the nutritional composition of the present invention, and in particular the infant formula of the present invention, contains about 1.8 g or more of protein per 100 kcal. For example, the nutritional composition may contain about 1.86 g or more of protein per 100 kcal, 1.9 g or more of protein per 100 kcal, 2.0 g or more of protein per 100 kcal, or 2.1 g or more of protein per 100 kcal. In some embodiments, the nutritional composition contains about 1.86 g or more of protein per 100 kcal in accordance with the current EU regulations for infant formula (EFSA NDA Panel (2014) EFSA journal 12(7):3760).

[0103] In some embodiments, the nutritional composition of the present invention, and in particular the infant formula of the present invention, may contain 1.8g to 2.8g of protein per 100kcal, 1.86g to 2.4g of protein per 100kcal, 1.9g to 2.4g of protein per 100kcal, 2.0g to 2.4g of protein per 100kcal, 2.0g to 2.3g of protein per 100kcal, 2.1g to 2.3g of protein per 100kcal, or 2.15g to 2.25g of protein per 100kcal.

[0104] In some embodiments, the nutritional composition of the present invention, and in particular the infant formula of the present invention, may contain 2.24 g to 3.36 g of protein per 100 kcal, or 2.5 g to 3.1 g of protein per 100 kcal.

[0105] Protein source The protein source may be any source suitable for use in the nutritional composition. The protein source may consist of free amino acids as the sole protein source, or it may consist of animal and / or plant-derived proteins as the sole protein source.

[0106] In some embodiments, the protein source comprises or consists of a mixture of animal-derived protein and plant-derived protein. In one embodiment, the protein source comprises or consists of a mixture of milk protein, preferably highly hydrolyzed whey protein, and one or more hydrolyzed plant proteins, preferably pea protein.

[0107] In some embodiments, the protein is milk protein. In some embodiments, the protein is milk protein casein, whey, or a combination thereof.

[0108] In some embodiments, the nutritional composition does not contain milk protein. In some embodiments, the protein source is hydrolyzed plant protein, such as rice-based prepared milk, soy-based prepared milk, or pea-based prepared milk, or a combination of at least two of rice, soybeans, and peas.

[0109] In some embodiments, the nutritional composition does not contain milk protein. Therefore, in some embodiments, 100% by weight of the total protein is non-milk protein.

[0110] In some embodiments, the combination, preferably the nutritional composition, includes highly hydrolyzed milk proteins such as proteins derived from goat's milk, donkey's milk, camel's milk, or cow's milk. In some embodiments, the combination, preferably the nutritional composition, includes highly hydrolyzed milk proteins such as highly hydrolyzed casein or highly hydrolyzed whey.

[0111] Highly hydrolyzed whey-based formulas may be more palatable than highly hydrolyzed casein-based formulas, and / or subjects may be sensitized only to casein protein. Therefore, appropriately, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 100% of the protein is whey protein. Preferably, the protein source is whey protein.

[0112] The whey protein may be whey derived from cheese production, and in particular may be sweet whey, such as that obtained by the coagulation of casein with rennet; acidic whey, obtained by the coagulation of casein with acid or the acidification of a fermentation product; or even a mixed whey obtained by coagulation with acid and rennet. This starting material may be whey known as desalted whey protein (DWP), which has been desalted by ion exchange and / or electrodialysis.

[0113] The source of whey protein may be sweet whey from which caseino-glycomacropeptide (CGMP) has been completely or partially removed. This is called modified sweet whey (MSW). Removing CGMP from sweet whey yields a protein material with threonine and tryptophan content closer to that of human breast milk. The process for removing CGMP from sweet whey is described in European Patent No. 880902.

[0114] The whey protein may be a mixture of DWP and MSW.

[0115] 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 can be determined by any method known to those skilled in the art.

[0116] Degree of hydrolysis Hydrolyzed proteins can be characterized as "partially hydrolyzed" or "highly hydrolyzed" depending on the degree to which the hydrolysis reaction is carried out. Currently, there is a WAO-compliant agreement that highly hydrolyzed products, while not having reached a legal / clinical definition agreement in accordance with the WAG (International Association of Allergology and Immunology) guidelines for cow's milk protein allergy (CMPA), are widely used and have been proven useful as a protein source for infants suffering from CMA. In this invention, partially hydrolyzed proteins are those in which 60% to 70% of the protein / peptide population has a molecular weight of less than 1000 daltons, while highly hydrolyzed proteins are those in which at least 95% of the protein / peptide population has a molecular weight of less than 1000 daltons. For example, see BSACI guideline for the diagnosis and management of cow's milk allergy, Clinical & Experimental Allergy (2014) 44:642-672. Highly hydrolyzed proteins are, alternatively, industryly defined as proteins in which at least 95% of the protein / peptide population has a molecular weight of less than 1200 daltons. See Nutten et al., Allergy (2020) 75:1446-1518. Partially hydrolyzed proteins are generally considered hypoallergenic (HA), while highly hydrolyzed proteins are generally considered non-allergenic.

[0117] The hydrolyzed proteins of the present invention may have a degree of hydrolysis characterized by NPN / TN%. Non-protein nitrogen relative to total nitrogen is widely used as a measure of soluble peptides produced by enzymatic hydrolysis. NPN / TN% means the non-protein nitrogen divided by total nitrogen multiplied by 100. NPN / TN% can be measured as described in detail in Adler-Nissen J-, 1979, J.Agric.Food Chem., 27(6), 1256-1262. Generally, highly hydrolyzed proteins are characterized by having an NPN / TN% greater than 95%, while partially hydrolyzed proteins are characterized by having an NPN / TN% in the range of 75% to 85%. Partially hydrolyzed proteins may also be characterized by 60% to 70% of their protein / peptide population having a molecular weight of less than 1000 daltons.

[0118] In suitable embodiments, the protein may have an NPN / TN% of more than 90%, more than 95%, or more than 98%. In preferred embodiments where a “highly” hydrolyzed protein is desired, the hydrolyzed protein of the present invention has an NPN / TN% in the range of more than 95%. Preferably, the protein may have an NPN / TN% of more than 90%, more than 95%, or more than 98%. These highly hydrolyzed proteins may also be characterized in that at least 95% of their protein / peptide population has a molecular weight of less than 1000 daltons.

[0119] The degree of hydrolysis can also be determined by the degree of hydrolysis. The degree of hydrolysis (DH) is defined as the percentage of peptide bonds cleaved in the protein hydrolysate and can be determined by any method known to those skilled in the art. Appropriately, the degree of hydrolysis is determined by pH stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), or formal titration. (Rutherfurd, SM (2010) Journal of AOAC International 93(5):1515-1522). The degree of hydrolysis (DH) of a protein may be, for example, greater than 90, greater than 95, or greater than 98.

[0120] The degree of hydrolysis can also be determined by the peptide molecular weight distribution. The peptide molecular weight distribution can be determined by high-performance size exclusion chromatography, optionally with UV detection (HPSEC / UV) (Johns, PW et al., (2011) Food Chemistry 125(3):1041-1050). For example, the peptide molecular weight distribution may be an estimate based on HPSEC peak area determined at 205 nm, 214 nm, or 220 nm. Appropriately, when the peptide molecular weight distribution is determined by HPSEC / UV, the "weight percentage of peptides" having a particular molecular weight can be estimated by the "fraction of peak area as a percentage of total peak area" having that molecular weight, determined at 205 nm, 214 nm, or 220 nm. Appropriately, the degree of hydrolysis can be determined by the method described in International Publication 2016 / 156077. Alternatively, the peptide molecular weight distribution can be determined by any method known to those skilled 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, in order to bind to membrane-bound immunoglobulin E (IgE), peptides must be larger than approximately 1500 Da (about 15 amino acids), and in order to crosslink IgE molecules and induce an immune response, they must be larger than approximately 3000 Da (about 30 amino acids) (Nutten (2018), EMJ Allergy Immunol 3(1):50-59).

[0121] Therefore, appropriately, at least about 95% by weight, at least about 98% by weight, at least about 99% by weight, or about 100% by weight of the peptides in eHF have a molecular weight of less than about 3000 Da. For example, no detectable peptides with a size of about 3000 Da or larger can be present in eHF.

[0122] Therefore, appropriately, at least about 95% by weight, at least about 98% by weight, at least about 99% by weight, or about 100% by weight of the peptides in the eHF have a molecular weight of less than about 1500 Da. Preferably, at least 99% by weight of the peptides have a molecular weight of less than about 1500 Da. For example, no detectable peptides with a size of about 1500 Da or larger can be present in the eHF.

[0123] Preferably, at least about 85% by weight, at least about 90% by weight, at least about 95% by weight, at least about 98% by weight, or at least about 99% by weight of the peptides in the eHF have a molecular weight of less than about 1200 Da. More preferably, at least 95% by weight or 98% by weight of the peptides in the eHF have a molecular weight of less than about 1200 Da.

[0124] Preferably, at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, or at least about 95% by weight of the peptides in the eHF have a molecular weight of less than about 1000 Da. Preferably, at least about 95% by weight of the peptides in the eHF have a molecular weight of less than about 1000 Da.

[0125] Preferably, the eHF does not contain any detectable peptides larger than about 3000 Da, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da.

[0126] A higher proportion of dipeptides and tripeptides can improve nitrogen (protein) absorption, even in patients with intestinal dysfunction. PEPT1 is a dedicated transport pathway that promotes the absorption of low-molecular-weight peptides (e.g., dipeptides and tripeptides). In the first few weeks of life, intestinal PEPT1 is important for nutrient intake, and thereafter it is important for food transition after weaning.

[0127] Therefore, at least about 30% by weight, at least about 40% by weight, or at least about 50% by weight of the peptides in eHF may be, for example, dipeptides and tripeptides. Preferably, at least about 45% by weight, at least about 50% by weight, 45% to 55% by weight, or 50% to 54% by weight of the peptides in eHF are dipeptides and tripeptides. More preferably, at least 51% to 53% by weight, or more preferably about 52% by weight of the peptides in eHF are dipeptides and tripeptides. Preferably, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, or at least 80% by weight of the peptides in eHF have a molecular weight of less than 600 Da. Preferably, at least about 45% by weight, at least about 50% by weight, at least 60% to 80% by weight, or at least 80% by weight of the peptides in eHF have a molecular weight of less than 600 Da. More preferably, at least about 80% by weight of the peptides in eHF have a molecular weight of 240 Da to 600 Da.

[0128] The main recognized milk allergens are alpha-lactalbumin (aLA), beta-lactoglobulin (bLG), and bovine serum albumin (BSA).

[0129] Therefore, appropriately, eHF may have an undetectable aLA content, e.g., about 0.010 mg / kg or less of aLA; eHF may have an undetectable bLG content, e.g., about 0.010 mg / kg or less of bLG; and / or eHF may have an undetectable BSA content, e.g., about 0.010 mg / kg or less of BSA. Preferably, eHF does not contain detectable amounts of aLA, bLG, and BSA. The aLA, bLG, and BSA content can be determined by any method known to those skilled in the art, e.g., ELISA.

[0130] Hydrolysis method The nutritional composition, preferably the protein for use in the infant formula of the present invention, can be hydrolyzed by any suitable method known in the art. For example, the protein can be enzymatically hydrolyzed using, for example, a protease. For example, the protein can be hydrolyzed using an alcalase (for example, with an enzyme:substrate ratio of about 1% to 15% by weight, for a duration of about 1 to 10 hours). The temperature may be in the range of about 40°C to 60°C, for example, about 55°C. The reaction time may be, for example, 1 to 10 hours, and the pH value before the start of hydrolysis may be in the range of, for example, 6 to 9, preferably 6.5 to 8.5, more preferably 7.0 to 8.0.

[0131] Porcine enzymes, particularly porcine pancreatic enzymes, may be used in the hydrolysis process. For example, International Publication No. 1993004593(A1) discloses a hydrolysis method using trypsin and chymotrypsin. This method involves a two-step hydrolysis reaction, with a thermal denaturation step between the two steps to ensure that the final hydrolysate is substantially free of untreated allergenic proteins. The trypsin and chymotrypsin used in these methods are preparations produced from porcine pancreatic extracts. International Publication No. 2016156077(A1) discloses a method for preparing milk protein hydrolysates, comprising hydrolysis of milk-based proteinaceous material with a combination of microbial alkaline serine proteases and proteases derived from bromelain, Aspergillus, and Bacillus.

[0132] Free amino acids The nutritional composition of the present invention may contain free amino acids. In some embodiments, free amino acids are the sole protein source.

[0133] The concentration of free amino acids may be selected to provide an amino acid profile sufficient for infant nutrition, particularly one that satisfies nutritional regulations (e.g., European Commission Directive 2006 / 141 / EC).

[0134] Free amino acids can be incorporated into the eHF of the present invention, for example, to supplement the amino acids contained in peptides.

[0135] Examples of free amino acids for use in the nutritional composition of the present 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.

[0136] Free amino acids provide an equivalent source of nitrogen as proteins (i.e., they contribute to nitrogen content). As mentioned above, a high proportion of dipeptides and tripeptides can improve nitrogen (protein) absorption even in patients with intestinal dysfunction. Therefore, a low proportion of free amino acids can also improve nitrogen (protein) absorption even in patients with intestinal dysfunction.

[0137] Therefore, appropriately, free amino acids in eHF may be present at concentrations of 50% by weight or less, 40% by weight or less, 30% by weight or less, or 25% by weight or less, based on the total weight of amino acids. Preferably, eHF contains 25% by weight or less of free amino acids, based on the total weight of amino acids. More preferably, free amino acids in eHF are present at concentrations of 20-25% by weight, 21-23% by weight, or about 22% by weight, based on the total weight of amino acids.

[0138] The free amino acid content can be determined by any method known to those skilled in the art. Preferably, the free amino acid content can be obtained by separation of free amino groups present in an aqueous sample extract by ion-exchange chromatography and photodetection after post-column derivatization with a ninhydrin reagent. The total amino acid content can be obtained by hydrolysis of the test portion under nitrogen in 6 mol / L HCl, and by separation of individual amino acids by ion-exchange chromatography as described above.

[0139] carbohydrates Carbohydrates can be any carbohydrate suitable for use in nutritional compositions.

[0140] The carbohydrate content of the nutritional composition of the present invention, and in particular the infant formula of the present invention, is preferably in the range of 9 g to 14 g of carbohydrates per 100 kcal.

[0141] In addition to lactose provided by the combination of the present invention, further examples of carbohydrates for use in nutritional compositions include saccharose, maltodextrin, and starch. The nutritional composition may preferably comprise a mixture of carbohydrates.

[0142] In some embodiments, the carbohydrate content includes maltodextrin. In some embodiments, at least about 20% by weight, at least about 25% by weight, at least about 30% by weight, at least about 35% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, 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.

[0143] In some embodiments, the carbohydrates include 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.

[0144] In some embodiments, approximately 100% of the total non-HMO carbohydrate content is lactose.

[0145] fat The lipid content of the nutritional composition of the present invention, and more particularly the infant formula of the present invention, is preferably in the range of 4.0 g to 6.0 g of lipids per 100 kcal.

[0146] Examples of lipids for use in the nutritional composition of the present 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 acid, high oleic acid sunflower oil and high oleic acid safflower oil, and microbial fermented oils containing long-chain polyunsaturated fatty acids.

[0147] Lipids can also be in the form of fractions derived from oils, such as palm olein, medium-chain triglycerides (MCTs), and fatty acid esters, such as esters of arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linolenic acid, oleic acid, lauric acid, capric acid, caprylic acid, and caproic acid.

[0148] Further examples of lipids include structured lipids (i.e., lipids that have undergone chemical or enzymatic modification to alter their structure). Preferably, the structured lipids are sn2 structured lipids, and include, for example, triglycerides in which the proportion of palmitic acid at the sn2 position of the triglyceride is increased. Structured lipids may or may not be included. Oils such as fish oil or microbial oil containing a large amount of pre-formed arachidonic acid (ARA) and / or docosahexaenoic acid (DHA) may also be added.

[0149] Long-chain polyunsaturated fatty acids, such as dihomo-γ-linolenic acid, arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid (DHA), may be added.

[0150] Oils containing large amounts of SOFA, such as acetate, propionate, or butyrate, or any other lipid products derived from microbial fermentation may also be added.

[0151] Medium-chain triglycerides (MCTs) High concentrations of MOT may impair early weight gain. MOT is not stored and does not support fat accumulation. For example, Borschel et al. reported that infants fed formula without MOT showed significantly greater weight gain between days 1 and 56 than infants fed formula containing 50% MCT-derived lipids (Borschel, M. et al., (2018) Nutrients 10(3):289).

[0152] Therefore, in the nutritional composition of the present invention, about 30% by weight or less of the lipids may be, for example, medium-chain triglycerides (MCTs).

[0153] In some embodiments, medium-chain triglycerides (MCTs) constitute approximately 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.5% by weight or less, or 0.1% by weight or less of the lipid.

[0154] In some embodiments, medium-chain triglycerides (MCTs) are present in amounts of 0% to 30% by weight, 0% to 25% by weight, 0% to 20% by weight, 0% to 15% by weight, 0% to 10% by weight, 0% to 5% by weight, 0% to 4% by weight, 0% to 3% by weight, 0% to 2% by weight, 0% to 1% by weight, 0% to 0.5% by weight, or 0% to 0.1% by weight of the lipid.

[0155] In some embodiments, the nutritional composition does not contain added MCTs. Preferably, about 0% by weight of lipids is MCT, and / or the composition does not contain detectable MCTs. Preferably, the nutritional composition does not contain MCTs.

[0156] Dietary butyrates The combination or nutritional composition of the present invention may further include butyrate, particularly dietary butyrate.

[0157] Butyrate may be present in the nutritional composition in amounts of approximately 1 mg / L to approximately 30 mg / L of the ingested composition. Butyrate may also be present in the nutritional composition in amounts of approximately 1.6 mg / L to approximately 2.4 mg / L, or approximately 8 mg / L to approximately 12 mg / L, or approximately 16 mg / L to approximately 24 mg / L of the ingested composition.

[0158] Preferably, butyrate is present in the nutritional composition in some specific amount. In one embodiment of the present invention, butyrate is present in the nutritional composition in an amount of about 30 mg / L of the ingested composition. Butyrate may be present in the nutritional composition in an amount of about 10 mg / L to 30 mg / L of the ingested composition, for example, about 13 mg / L to 27 mg / L, or 16 mg / L to 24 mg / L, or 18 mg / L to 22 mg / L of the ingested composition. In another specific embodiment, butyrate is present in the nutritional composition in an amount of about 20 mg / L of the ingested composition.

[0159] In one embodiment of the present invention, butyrate is present in the nutritional composition in an amount of up to approximately 23 mg per 100 g of composition, based on the dry weight of the composition. In another embodiment of the present invention, butyrate may be present in the nutritional composition in an amount of 12 mg to 18 mg per 100 g of composition, based on the dry weight of the composition. The amount of butyrate may also be approximately 15 mg per 100 g of composition, based on the dry weight of the composition.

[0160] Dietary butyrate may be in the form disclosed, for example, in International Publication No. 2019 / 228851 or International Publication No. 2020 / 127642. The corresponding amount of the butyrate source in the nutritional composition is sufficient to provide butyrate in the nutritional composition in an amount of up to approximately 30 mg / L of the composition to be ingested.

[0161] The butyrate source may be present in the nutritional composition in an amount sufficient to provide butyrate in the nutritional composition at an amount of approximately 1 mg / L to approximately 30 mg / L of the ingested composition. The butyrate source may be present in the nutritional composition in an amount sufficient to provide butyrate in an amount of approximately 1.6 mg / L to approximately 2.4 mg / L of the ingested composition, or approximately 8 mg / L to approximately 12 mg / L of the ingested composition, or approximately 16 mg / L to approximately 24 mg / L of the ingested composition.

[0162] 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 mg / L to 30 mg / L, such as about 13 mg / L to 27 mg / L, or 16 mg / L to 24 mg / L, or 18 mg / L to 22 mg / L, of the composition ingested. In another specific 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 ingested.

[0163] Suitably, the combination or nutritional composition has the formula:

Chemical formula

[0164] In one embodiment, each of R 1 , R 2 , R 3 , R 4 , R 5 , and / or R 6 is an unsaturated fatty acid, preferably a monounsaturated fatty acid. In a specific embodiment, each of R 1 , R 2 , R 3 , R4 , R 5 , and R 6 Each of these is a long-chain fatty acid having 18 carbon atoms, and long-chain fatty acids having 18 carbon atoms are monounsaturated. In one embodiment, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 Each of these is oleic acid.

[0165] In one embodiment, the combination of lactose and HMO described herein is given by formula: [ka] The butyrate source may be in the form of one or more compounds having the same properties, or a combination thereof. In one embodiment, the nutritional composition described herein contains at least one of compounds (5), (6), (7), and (8) in about 100 mg / L to be ingested. In one embodiment, the nutritional composition described herein contains at least one of compounds (5), (6), (7), and (8) in about 100 mg, which corresponds to about 20 mg / L of dietary butyrate in the composition to be ingested.

[0166] Alternative sources of dietary butyrates may be triglycerides composed of a mixture of C4 fatty acids, such as butyric acid (C4:0), and C8 fatty acids, such as caprylic acid (C8:0). Triglycerides containing caprylic and butyric acid can be prepared by inter-esterification of a mixture of triglycerides containing either butyric acid (C4:0) or caprylic acid (C8:0). A commercially available source for BBB is triplylin (e.g., available from Sigma-Aldrich). A commercially available source for CCC is tricaprylin (e.g., Neobee 895, available from Stepan Specialty).

[0167] Further ingredients The nutritional compositions of the present invention, particularly infant formula or toddler formula, may also contain all vitamins and minerals that are understood to be nutritionally essential in substantial amounts in the daily diet. Minimum requirements have been established for certain vitamins and minerals. Examples of vitamins, minerals, and other nutrients for use in the nutritional compositions of the present invention, particularly infant formula, include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in the form of their salts.

[0168] The nutritional composition may contain one or more carotenoids.

[0169] The nutritional composition may also contain at least one probiotic. The term “probiotic” refers to a preparation of microbial cells or a component of microbial cells that has beneficial effects on the health or well-being of the host. Specifically, probiotics can improve intestinal barrier function.

[0170] Examples of probiotic microorganisms for use in the nutritional composition of the present invention include yeasts such as Saccharomyces, Debaromyces, Candida, Pichia, and Torulopsis, as well as bacteria such as Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, and Lactobacillus. Preferably, the combinations described herein, preferably the nutritional composition, contain one or more bacteria of the Bifidobacterium and / or Lactobacillus genera.

[0171] Preferred probiotics are those that are safe overall, are L(+) lactic acid-producing cultures, and have an acceptable shelf life for products that need to remain stable and effective for up to 24 months, such as Bifidobacterium, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, and Lactobacillus.

[0172] Specific examples of suitable probiotic microorganisms include Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium loingum subsp. infantis, Bifidobacterium longum subsp. longum, Enterococcus faecium, and Enterococcus faecalis. Lactobacillus faecalis), Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbrudkii subsp. lactis.Lactobacillus lactis, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus sake, Lactococcus lactis, Micrococcus valians, Pediococcus acidilactici, Pediococcus pentosaceus *Pediococcus pentosaceus*, *Pediococcus acidilactici*, *Peciococcus halophilus*, *Streptococcus faecalis*, *Streptococcus thermophilus*, *Staphylococcus carnosus* and *Staphylococcus xylosus*, *Lacticaseibacillus rhamnosus*, *Lacticaseibacillus paracasei*, *Limosilactobacillia*, *Akkermansia muciniphylla* These include *Muciniphila*, *Clostridales*, and *Prevotella*.

[0173] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, such as prebiotics, lactoferrin, fiber, nucleotides, and nucleosides.

[0174] Manufacturing method The nutritional composition of the present invention can be prepared by any suitable method.

[0175] For example, the nutritional compositions described herein can be prepared by blending a protein source, a carbohydrate source, and a lipid source in appropriate proportions. If emulsifiers are to be used, they can be added at this stage. Vitamins and minerals may also be added at this stage, although vitamins are usually added later to avoid thermal decomposition. Any lipophilic vitamins, emulsifiers, etc., may be dissolved in the lipid source before blending. The mixture can then be mixed with water, preferably reverse-osmotic water, to form a liquid mixture. A commercially available liquefaction device can also be used to form the liquid mixture. The liquid mixture may then be homogenized.

[0176] Next, the liquid mixture can be heat-treated to reduce the bacterial content. This heat treatment can be carried out, for example, by steam injection, or using an autoclave or heat exchanger, such as a plate heat exchanger.

[0177] The liquid mixture may then be cooled and / or homogenized. The pH and solids content of the homogenized mixture can be adjusted at this point.

[0178] The homogenized mixture can then be transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, to convert it into a powder. If a liquid nutritional composition is preferred, the homogenized mixture may be sterilized and then aseptically filled into a suitable container, or it may be filled into a container first and then retorted.

[0179] Those skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the invention as disclosed herein.

[0180] subject The subject may be any suitable subject. Suitablely, the subject may be a mammal. In a preferred embodiment, the subject is a human. In other embodiments, the subject is an animal, preferably a pet. The 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.

[0181] In some embodiments, the subjects are young people, adolescents, and children. The term “young people” may refer to individuals who have not yet reached adulthood. The term “adolescents” may refer to individuals during the period from the onset of puberty to adulthood. The term “children” may refer to individuals in the stage between birth and puberty.

[0182] Ideally, the target age group is approximately 3 years and older. In some embodiments, the target age group is approximately 4 years and older, or approximately 5 years and older.

[0183] In some embodiments, the target age is approximately 10 years old or younger. In some embodiments, the target age is approximately 9 years old or younger, approximately 8 years old or younger, approximately 7 years old or younger, approximately 6 years old or younger, or approximately 5 years old or younger.

[0184] In some embodiments, the target age group is approximately 3 to 10 years old, approximately 3 to 9 years old, approximately 3 to 8 years old, approximately 3 to 7 years old, approximately 3 to 6 years old, or approximately 3 to 5 years old.

[0185] The subjects may be infants, toddlers, or children; preferably, the subjects are infants or toddlers. In some embodiments, the subjects are infants or toddlers who have or are suspected of having milk protein allergy (CMPA).

[0186] Appropriately, infants under 12 months of age may be included.

[0187] Appropriately, infants may be those aged 12 to 36 months (1 to 3 years).

[0188] Appropriately, children may be those aged 3 to 7 years.

[0189] The subjects may be infants or toddlers who are not breastfeeding or whose breastfeeding has been discontinued.

[0190] More accurately, the subject may be suffering from dysbiosis of the gut microbiota.

[0191] The subjects may have or be suspected of having milk protein allergy (CMPA).

[0192] CMPA can be defined as a reproducible immune-mediated allergic reaction to one or more proteins in milk and is one of the most common presentations of food allergies seen in early childhood. It can be classified according to the underlying immune mechanism: immunoglobulin (Ig) E-mediated food allergies typically result in immediate symptoms that can affect multiple organ systems within two hours of milk ingestion. Non-IgE-mediated food allergic reactions usually appear between two and 72 hours after milk ingestion. Mixed IgE and non-IgE allergic reactions are typically delayed. The diagnosis of IgE-mediated allergy is typically based on the presentation of symptoms such as urticaria, angioedema, itching, cough, hoarseness, wheezing, or shortness of breath after milk ingestion. The diagnosis of non-IgE-mediated allergy should be suspected if there is one or more symptoms such as gastroesophageal reflux disease, abdominal discomfort, constipation, diarrhea, or atopic dermatitis, especially if the symptoms are resistant to treatment.

[0193] Subjects having CMPA may show an increase in CMPA associated with the induction of oral tolerance after administration of the composition or nutritional composition defined herein. Oral tolerance may be associated with partial or complete oral desensitization in subjects with allergies, particularly milk protein allergies. In the context of the present invention, induction of oral tolerance is understood to mean that oral tolerance to an allergen, such as milk protein, is induced compared to oral tolerance before initiating administration of the combination or nutritional composition defined herein. Some embodiments of the combination or nutritional composition defined herein are intended for use in inducing oral tolerance in subjects with allergies, particularly milk protein allergies. In some embodiments, the combination or nutritional composition defined herein is intended for use in partial or complete oral desensitization in subjects with allergies, particularly milk protein allergies.

[0194] Diet management In the context of “dietary management,” the terms “manage,” “control,” and “control” as used herein may mean the prevention or reduction of the severity or frequency of one or more symptoms of a disease or condition, including improving one or more existing symptoms of a disease or condition, preventing one or more existing symptoms of a disease or condition, preventing one or more underlying causes of a disease or condition, improving one or more underlying causes 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 is understood that this also includes stabilizing the disease or condition and preventing the progression of the disease or condition.

[0195] Dysbiosis In one embodiment, the present invention provides a combination, preferably in the form of a nutritional composition, as defined herein, for use in the dietary management of dysbiosis of the intestinal microbiota in a subject.

[0196] In another embodiment, the present invention provides a combination of lactose and at least one of the HMOs 2'-FL, LNnT, 3'-FL, 3'-SL, or 6'-SL, optionally with LNT, for use in the treatment of dysbiosis of the gut microbiota in a subject. In another embodiment, the present invention provides a combination of lactose and the 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.

[0197] In a further embodiment, the present invention provides the use of a combination of lactose and at least one of the HMOs 2'-FL, LNnT, 3'-FL, 3'-SL, or 6'-SL, optionally with LNT, for dietary management of gut microbiota dysbiosis in a subject. In another embodiment, the present invention provides a combination of lactose and the HMOs 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT for use in dietary management of gut microbiota dysbiosis in a subject.

[0198] In one embodiment, the present invention provides a method for dietary management of intestinal microbiome dysbiosis in a subject, comprising administering a combination defined herein, preferably in the form of a nutritional composition, to the subject.

[0199] The present invention also provides a method for treating dysbiosis of the intestinal microbiota in a subject, comprising administering to the subject a combination defined herein, preferably in the form of a nutritional composition.

[0200] Dysbiosis of the gut microbiota is characterized by a disruption of the microbiota that results in imbalances, changes in their functional composition and metabolic activity, or shifts in their local distribution. The most typical features of dysbiosis are a decrease in microbiota diversity, loss of beneficial microbiota, or overgrowth of harmful microbiota.

[0201] Dysbiosis can be assessed and confirmed using, for example, the range of tests and criteria described in Wei et al., (2021; Applied and Environmental Microbiology; 87(11)). Approaches to determining dysbiosis may include, for example, 16S rRNA gene profiling or gene sequencing to apply one or more of the following: large-scale bacterial marker profiling, methods based on relevant taxa, neighbor classification, and combinations of alpha and beta diversity measures.

[0202] In infants and toddlers with CMPA, dysbiosis can be characterized by lower levels of Bifidobacteriaceae, as well as enrichment of Proteobacteria and other Gram-negative enterobacteria. Bifidobacteriaceae levels in approximately 10% of the microbiome in CMPA infants compared to approximately 80% in healthy infants.

[0203] Appropriately, in the context of the present invention, dysbiosis may refer to a state in which the proportion of HMO-assimilating Bifidobacteria in the gut microbiota is less than about 10%, less than about 5%, less than about 2%, or less than about 1%.

[0204] Appropriately, dysbiosis in the context of the present invention may refer to the abnormal proliferation of a pathogen or proteobacteria. In some embodiments, dysbiosis may refer to the abnormal proliferation of one or more of the Staphylococcus, Clostridaceae, and Enterococcusaceae families. In some embodiments, dysbiosis in the context of the present invention may refer to a relatively small number of Bifidobacteria and a relatively large number of Enterobacteria and / or Clostridaceae, taking into account the total number of bacteria in the sample. The abnormal proliferation of a pathogen or proteobacteria may be determined by any means known in the art. In one embodiment, the relative concentration of bacteria is determined by 16S rRNA sequencing (known in the art and as described in the examples) and / or shotgun metagenomic sequencing (as known in the art), preferably by 16S rRNA sequencing. Both methods are commercially available; see, for example, WorldWideWeb microbiomeinsights.com.

[0205] Appropriately, dysbiosis in the context of the present invention may relate to proteolytic fermentation by the gut microbiota. Proteolytic fermentation may relate to the formation of metabolites such as phenols and indoles that have adverse effects on health.

[0206] Dysbiosis of the gut microbiota can be associated with the pathogenesis of both intestinal and extraintestinal disorders. Examples of intestinal disorders include inflammatory bowel disease, irritable bowel syndrome (IBS), and celiac disease, while examples of extraintestinal disorders include allergies, asthma, metabolic syndrome, cardiovascular disease, and obesity.

[0207] Appropriately, the intestinal disorder may be one or more of the following: inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and celiac disease.

[0208] Appropriately, extraintestinal disorders are one or more of the following: allergies, asthma, atopic dermatitis, metabolic syndrome, cardiovascular disease, obesity, and type 2 diabetes.

[0209] 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 diseases or disorders related to the metabolism and / or accumulation of BCFAs in a subject. In another aspect, the present invention provides a combination comprising lactose and the HMOs 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT, for use in the dietary management of diseases or disorders related to the metabolism and / or accumulation of BCFAs in a subject.

[0210] In another aspect, the present invention provides combinations as defined herein, preferably in the form of nutritional compositions, for use in the treatment of diseases or disorders related to the metabolism and / or accumulation of BCFAs in a subject. In another aspect, the present invention provides combinations comprising lactose and HMOs, 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT, for use in the treatment of diseases or disorders related to the metabolism and / or accumulation of BCFAs in a subject.

[0211] In one embodiment, the present invention provides a method for dietary management of a disease or disorder related to the metabolism and / or accumulation of BCFAs in a subject, comprising administering a combination as defined herein to the subject, preferably in the form of a nutritional composition.

[0212] In a further embodiment, the present invention provides a method for treating a disease or disorder related to the metabolism and / or accumulation of BCFAs in a subject, comprising administering a combination as defined herein to the subject, preferably in the form of a nutritional composition.

[0213] In another aspect, the present invention provides combinations as defined herein, preferably in the form of nutritional compositions, for use in reducing BCFAs in a subject.

[0214] In one embodiment, the present invention provides a method for reducing BCFAs in a subject, comprising administering a combination defined herein to the subject, preferably in the form of a nutritional composition.

[0215] In one embodiment, the present invention provides a combination, preferably in the form of a nutritional composition, as defined herein, for use in the dietary management of malnutrition in a subject.

[0216] In another aspect, the present invention provides combinations as defined herein, preferably in the form of nutritional compositions, for use in treating malnutrition in a subject.

[0217] In one embodiment, the present invention provides a method for dietary management of malnutrition in a subject, comprising administering a combination defined herein, preferably in the form of a nutritional composition, to the subject.

[0218] In another aspect, the present invention provides a method for treating malnutrition in a subject, comprising administering a combination defined herein to the subject, preferably in the form of a nutritional composition.

[0219] Appropriately, BCFAs may include isobutyrate, isovalerate, isocaproate, and / or 2-methylbutyrate.

[0220] Appropriately, BCFAs may include isobutyrate, isovalerate, and / or isocaproate.

[0221] Appropriately, this combination reduces BCFA levels compared to infants who have not been administered the combination as defined herein.

[0222] Appropriately, this combination reduces BCFA levels compared to infants administered a nutritional composition containing lactose but without one or more of the HMOs in this combination.

[0223] BCFAs produced by the microbiome include isovaleric acid, isobutyric acid, isocaproic acid, and 2-methylbutyric acid, which are produced by the fermentation of branched-chain amino acids (BCAAs) leucine, valine, and isoleucine, respectively.

[0224] BCFAs exhibit proteolytic fermentation by the gut microbiota and are associated with the formation of metabolites such as phenols and indoles. Furthermore, isovaleric acid can inhibit Na+,K+-ATPases, which are important enzymes involved in maintaining the ground potential membrane necessary for normal neurotransmission, providing insights into the involvement of BCFAs in gut-brain axis communication.

[0225] Appropriately, diseases or disorders related to the metabolism of BCFAs may include one or more of the following: malnutrition, Rett syndrome, colorectal cancer, anorexia nervosa, and depression.

[0226] Increased BCFA levels have been demonstrated in several medical conditions. High levels of fecal BCFA associated with changes in the microbiome have been reported in children with stunted growth and malnutrition (Li et al., 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 acid, isobutyric acid, and isovaleric acid have been found to be higher in colorectal cancer patients than in healthy individuals (Wang et al., 2017; Exp Ther Med. 2017; 13(6): 2848-54, and Nicolai et al., 2019; World J Gastroenterol. 25(36): 5543-58), and differences may exist between colorectal cancer patients and adenomatous polyp patients from healthy controls based on fecal SCFA fingerprinting (Niccolai et al., see above). Higher abundance of isovaleric acid and isobutyric acid has been found in patients with hypercholesterolemia, and isobutyric acid has also positively correlated with lipid parameters showing unfavorable profiles (Granado-Serrano et al., 2019; Sci Rep. 2019; 9(1): 1772). Intestinal dysbiosis and high concentrations of propionic acid and isobutyrate in feces have been reported in non-alcoholic fatty liver disease (Da Silva et al., 2018; Sci Rep. 2018; 8: 1466), while isovaleric acid, along with propionic acid and butyrate, has been shown to be a possible predictor of the gut microbiota for obesity in African American men (Barengolts et al., 2019; Microorganisms; 7(9): 320). Rett syndrome is a rare X-linked neurodevelopmental disorder that affects women, and changes in the composition of the gut microbiota may contribute to some of the symptoms associated with this condition. Significantly high levels of BCFAs, along with increased levels of microbial genes encoding propionate, butyrate, and amino acid metabolism, are associated with Rett syndrome (Borghi et al., 2017; Int J Mol Sci. 2017; 18(2): 344).High levels of isovaleric acid in feces have been found to be associated with human depression and high cortisol levels in adults (Szczesniak et al., Nutr Neurosci. 2016;19(7):279-83).

[0227] Dietary management of colonization / infection by intestinal pathogens In one embodiment, 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 embodiment, 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 enteropathogen, which is one or more of bacteria, viruses, or protozoan pathogens. In one embodiment, 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 enteropathogen, which is a viral pathogen, and the viral pathogen is norovirus and / or rotavirus.

[0228] In one embodiment, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of pathogenic bacteria in a subject. In one embodiment, the present invention provides a combination as defined herein, preferably in the form of a nutritional composition, for use in the dietary management of pathogenic bacteria in a subject, wherein the pathogenic bacteria are one or more C. difficile, C. perfringens, Campylobacter, or Escherichia coli. In one embodiment, 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 embodiment, 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.

[0229] In one embodiment, the present invention provides a method for dietary management of at least one pathogen in a subject, comprising administering a combination as defined herein to the subject, preferably in the form of a nutritional composition.

[0230] Appropriately, the pathogen may be associated with dysbiosis in the target organism.

[0231] For example, the present invention may be applied to the dietary management of C. difficile and / or C. perflingens.

[0232] In one embodiment, 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, the subject being an infant, toddler, or child who is not breastfed or has discontinued breastfeeding. In one embodiment, 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. perflingens, the subject being an infant, toddler, or child who is not breastfed or has discontinued breastfeeding. In one embodiment, 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. perflingens, the dietary management reduces the abundance of C. difficile and / or C. perflingens, the subject being an infant, toddler, or child who is not breastfed or has discontinued breastfeeding.

[0233] A decrease in the abundance of C. difficile and / or C. perflingens can be determined by analysis of the whole gut microbiota.

[0234] In one embodiment, a decrease in the abundance of C. difficile and / or C. perfringens may be determined by the resolution or reduction of one or more symptoms associated with the abnormal proliferation of C. difficile and / or C. perfringens. In one embodiment, resolution or reduction of at least one of diarrhea and fever.

[0235] In one embodiment, the present invention provides a method for dietary management of at least one pathogen in a subject, comprising administering a combination as defined herein to the subject, preferably in the form of a nutritional composition, wherein the subject is an infant, toddler, or child who is not breastfed or has discontinued breastfeeding.

[0236] Protein breakdown and fermentation In addition to BCFAs, proteolytic fermentation leads to the production of compounds such as ammonia, phenol, amines, hydrogen sulfide, and p-cresol, which can be harmful to host health. Therefore, high rates of colonic proteolytic fermentation accompanied by low levels of sugar-digesting fermentation are considered harmful to host health.

[0237] Molecules involved in proteolytic fermentation can cause cellular damage to the gut environment (e.g., they are toxic to colon cells) (Aguirrre et al., 2016; Res. Microbiol. 167, 114-125), and can cause inflammatory conditions (Yao et al., 2016; Aliment Pharmacol Ther, 2016. 43(2): p. 181-96, and Popkov et al., 2022; Int J Mol Sci, 23(1)), and have adverse effects on health (Nowak et al., 2006; Anaerobe 12, 80-84). For example, uremic toxins 4-cresol and 2- or 3-hydroxyphenylacetic acid, formed by bacterial tyrosine and phenylalanine fermentation, can have negative effects on mucous membranes and the entire body (Evenepoel et al., 2009; Kidney Int Suppl, 2009(114): p.S12-9; and Zheng et al., 2021; Front Neurosci, 2021.15: p.738220). Therefore, reducing toxic metabolites in feces may contribute to maintaining the systemic immune balance.

[0238] In one embodiment, the present invention provides a combination, preferably in the form of a nutritional composition, as defined herein, for use in reducing proteolytic fermentation by the intestinal microbiota in a subject.

[0239] In one embodiment, the present invention provides a method for reducing proteolytic fermentation by the intestinal microbiota in a subject, comprising a combination defined herein, preferably in the form of a nutritional composition, in the subject. [Examples]

[0240] The present invention will be further described with reference to the following embodiments. It will be understood that the claimed invention is not intended to be limited in any way by these embodiments.

[0241] Example 1 - The combination of HMO blend and lactose reduces branched-chain fatty acid production in the gut microbiota in an ex vivo colon fermentation model. The combination of four HMO blends (M2, M4.1, M4.2, and M6) with lactose (L) significantly reduced BCFA production in an ex vivo colonic fermentation model (see Figure 1). This effect was not observed for individual test products.

[0242] The lower panel of Figure 1 shows that, at 48 hours, the combination of HMO and lactose induces lower levels of BCFAs than lactose alone or HMO alone. The combinations of HMO and lactose, particularly the M.4.1+L, M4.2+L, and M6+L combinations, significantly reduced BCFA production beyond an additive effect.

[0243] Figure 2 provides further evidence that the combined effect of lactose and HMO is not simply additive, and that the combined effect of the HMO blend and lactose is different from the sum of the effects of lactose alone and HMO alone. This principal component analysis (PCA), which summarizes the effect on the microbial community composition of infant-derived microbial communities, shows that the addition of lactose to the HMO blend results in a different pattern compared to the HMO blend without lactose (see PCA1).

[0244] Figure 3 shows that the combination of lactose and HMO blend increased beneficial bacteria (specifically, Bifidobacterium species (Bifidobacterium breve, longum, bifidum) which are key to the development of a healthy biome in infants, and a decrease in Clostridium difficile, which causes diarrhea) and Ruminococcus gnavus (associated with IBD and inflammation). Despite interpersonal differences among CMPA infants, consistent effects on microbial metabolite production and microbial composition were observed for HMO and lactose blends. Therefore, the beneficial effects are thought to be enhanced by using the combination of lactose and HMO rather than HMO alone or in combination with lower levels of lactose.

[0245] Materials and 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 substrate-free control (NSC) at the doses shown in Table 1.

[0246] [Table 1]

[0247] The lactose dosage was based on an estimated daily intake of 70g of lactose, assuming 96% in vivo absorption along the small intestine.

[0248] [Table 2]

[0249] Fecal donor selection criteria Twelve infants suspected of having CMPA who met the following criteria were included in the study: 1 to 7 months of age; fed highly hydrolyzed or amino acid-based formula (as per medical recommendations) or breastfed; gestational age at birth ≥ 36 weeks; no antibiotics in the 30 days prior to study participation; no prior necrotizing enterocolitis or bowel surgery; and no probiotic or prebiotic intake in the 14 days prior to study participation (Lactobacillus reuteri (DSM17938), Lactobacillus rhamnosus LGG (ATCC) are commonly used in this population and are not known to consume 2'-FL, 3'-FL, 3'-SL, 6'-SL, and LNnT (see, for example, Thongaram et al., Journal of Dairy Science (2017) 100:7825-7833)). Bifidobacterium animalis subsp. lactis BB-12 was permitted. One infant was partially breastfed (infant 2), and one was exclusively breastfed (infant 12). Fecal samples were collected by parents / caregivers after providing informed consent for participation in the study. In total, donor samples from 10 males and 2 females were evaluated. The ages of the subjects ranged from 2 to 7 months, with a mean of 4.3 months (4.2 months for male infants; 4.8 months for female infants). This study was approved by the Ethics Committee of University Hospital Ghent (reference number BC-09977).

[0250] SIFR® Research Design Ex vivo SIFR® experiments were performed as described by Van den Abbeele et al., Front Microbiol (2023) 14:1131662. Individual bioreactors were processed using a bioreactor control system (Cryptobiotix, Ghent, Belgium). Each bioreactor containing 5 mL of a blend of nutrient medium (M0018, Cryptobiotix, Ghent, Belgium), infant fecal inoculum, and the test compound (i.e., no test product (substrate-free control (NSC)), HMO, L, or HMO+L) was individually sealed and then brought to anaerobics. After preparation, the bioreactors were incubated at 37°C for 48 hours under continuous stirring (140 rpm) (MaxQ 6000, Thermo Scientific, Merelbeke, Belgium).

[0251] Four study groups were tested for each infant: (i) test product (C) none, (ii) HMO, (iii) L, and (iv) HMO+L. Samples were collected at 0, 6, 24, and 48 hours for key fermentation parameters, non-target metabolite profiling, and / or microbial composition during headspace gas pressure measurement. Control samples were technically run in triplicate, and coefficients of variation of less than 3% for basic fermentation parameters pH, gas production, and the three major SCFAs (acetate, propionate, and butyrate) were considered acceptable, as previously established for SIFR® technology (Van den Abbeele et al., Front Microbiol (2023) 14:1131662).

[0252] Taxonomic analysis of the microbiome: Quantitative 16S rRNA gene profiling DNA was extracted using the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany) according to the manufacturer's instructions. Subsequently, library preparation and sequencing were performed on the Illumina MiSeq platform using v3 chemistry. The 16S rRNA gene V3-V4 hypervariable region was amplified using primers 341F (50-CCT ACG GGN GGC WGC AG-30) and 785Rmod (50-GAC TAC HVG GGT ATC TAA KCC-30). Pre-processing from amplicons and OTU (operational taxa) picking were performed using Mothur v1.35.1 (Schloss et al., Appl Environ Microbiol (2009) 75:7537-7541) with representative sequences annotated in NCBI blast v2.10.0 (Altschul et al., J Mol Biol (1990) 215:403-410). Results were analyzed at the phylum, family, and operational taxa (OUT) levels. To determine the total count, samples were diluted in anaerobic phosphate-buffered saline (PBS), and cells were then stained with SYTO 16 at a final concentration of 1 μM and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium). Data were analyzed using FlowJo, version 10.8.1. Quantitative insights were obtained by correcting the percentage (%; 16S rRNA gene profiling) with the total count (cells / mL; flow cytometry), and estimated cells / mL for different taxa were obtained.

[0253] BCFA analysis Branched-chain fatty acids (BCFAs) (total of isobutyrates, isovalerates, and isocaproates) were determined via a GC-FID (gas chromatography with flame ionization detection) approach.

[0254] Data Analysis All univariate and multivariate analyses were performed using GraphPad Prism (v9.3.1; www.graphpad.com), while regularized canonical correlation analysis (rCCA) was performed using the mixOmics package in R (4.1.1; WorldWideWeb r-project.org) with the shrinkage method (version 6.16.3) for estimating the penalty parameter. (Rohart et al., PLOS Computational Biology (2017) 13: e1005752). The significance of the replenishment effect compared to C was evaluated by repeated-measures ANOVA analysis (based on paired tests between 12 infants) using p-value correction according to Benjamini-Hochberg (Journal of the Royal Statistical Society: Series B (Methodological) (1995) 57: 289-300). For the analysis of the microbial composition, three measurements were taken. First, the above-described statistical analysis was performed on the log 10 transformed values. Second, values of a given taxon below the limit of detection (LOD) were considered equal to the overall LOD as recently described (Van den Abbeele et al., Front Microbiol (2023) 14: 1131662). Finally, a threshold was set to retain the 100 most abundant OTUs in the analysis to avoid excessive p-value correction.

[0255] Embodiments Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs.

[0256] A combination for use in dietary management of dysbiosis of the gut microbiota in a subject, comprising lactose and at least one of human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL), lacto-N-neotetraose (LNnT), 3-fucosyllactose (3'-FL), and 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL).

[0257] A combination for use according to paragraph 1, which is an HMO and comprises 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and lacto-N-tetraose (LNT).

[0258] A combination for use according to paragraph 1 or 2, wherein the dysbiosis comprises an increase in the production of branched-chain fatty acids (BCFAs) such as isobutyrate, isovalerate, and / or isocaproate.

[0259] A combination for use according to any one of paragraphs 1 to 3, wherein the dysbiosis of the gut microbiota is associated with an intestinal disorder and / or an extra-intestinal disorder.

[0260] A combination for use according to paragraph 4, wherein the intestinal disorder is one or more of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and celiac disease.

[0261] A combination for use according to paragraph 5, wherein the IBD is Crohn's disease (CD) or ulcerative colitis (UC).

[0262] A combination for use according to paragraph 4, wherein the extra-intestinal disorder is one or more of allergy, asthma, atopic dermatitis, metabolic syndrome, cardiovascular disease, obesity, and type 2 diabetes.

[0263] Embodiment 8 A combination for use described in any one of paragraphs 1 to 7, in which dysbiosis is diagnosed by the presence of a relatively small number of Bifidobacteria and a relatively large number of Enterobacteria and / or Clostridium, taking into account the total number of bacteria in the fecal sample of interest.

[0264] Embodiment 9 A combination of uses described in any one of paragraphs 1 to 8, wherein the subject has or is suspected of having milk protein allergy (CMPA).

[0265] Embodiment 10 A combination comprising lactose and at least one of the HMOs 2'-FL, LNnT, 3'-FL, and 3'-SL or 6'-SL, for use in the dietary management of diseases or disorders related to the metabolism and / or accumulation of BCFAs in a subject.

[0266] Embodiment 11: A combination for use as described in paragraph 10, comprising HMOs 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT.

[0267] Embodiment 12: A combination of BCFAs for use as described in paragraph 10 or paragraph 11, comprising isobutyrate, isovalerate, and / or isocaproate.

[0268] Embodiment 13 A combination of uses according to any one of paragraphs 10-12, wherein the disease or disorder related to the metabolism of BCFAs is an increase in proteolytic fermentation and / or a decrease in sugar fermentation.

[0269] Embodiment 14 A combination of uses according to any one of paragraphs 10-12, wherein the disease or disorder related to the metabolism of BCFAs is one or more of malnutrition, Rett syndrome, colorectal cancer, anorexia nervosa, and depression.

[0270] Embodiment 15 A combination of uses according to any one of paragraphs 10-12, wherein the disease or disorder related to the metabolism and / or accumulation of BCFAs is the accumulation of one or more of isobutyrate, isovalerate, and isocaproate.

[0271] Embodiment 16 A combination comprising lactose and at least one of the HMOs 2'-FL, LNnT, 3'-FL, and 3'-SL or 6'-SL, (a) Pathogens in the subject; (b) A pathogen in the subject that is one or more of the following: bacteria, viruses, or protozoan pathogens; (c) One or more viral pathogens, preferably rotavirus or norovirus, that are pathogens in the subject; (d) One or more bacterial pathogens, preferably C. difficile, C. perflingens, Campylobacter, or Escherichia coli, which are pathogens in the subject. A combination for use in dietary management.

[0272] Embodiment 17: A combination for use as described in paragraph 16, comprising HMOs 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT.

[0273] Embodiment 18: The combination for use described in paragraph 16 or paragraph 17, wherein the pathogens are C. difficile and C. perflingens.

[0274] Embodiment 19: A combination of uses described in paragraph 18, which has the effect of alleviating or reducing one or more symptoms associated with the abnormal proliferation of C. difficile and / or C. perflingens.

[0275] Embodiment 20: The combination for use described in paragraph 19, wherein the symptom is at least one of diarrhea and fever.

[0276] Combination for use according to any one of paragraphs 16 to 20, wherein the subject is an infant, toddler, or child who is not being breastfed or has had breastfeeding discontinued.

[0277] Combination for use according to any one of paragraphs 1 to 21, wherein the combination of lactose and HMO reduces the level of BCFA in the intestine of the subject.

[0278] Combination for use according to paragraph 22, wherein the BCFA comprises isobutyrate, isovalerate, and / or isocaproate.

[0279] Combination comprising lactose and at least one of 2'-FL, LNnT, 3'-FL, and 3'-SL or 6'-SL, which are HMOs, for use in reducing BCFA in a subject.

[0280] Combination for use according to paragraph 24, comprising 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT, which are HMOs.

[0281] Combination for use according to paragraph 24 or paragraph 25, wherein the BCFA is reduced in the gut microbiota of the subject.

[0282] Combination for use according to any one of paragraphs 24 to 26, wherein the BCFA comprises isobutyrate, isovalerate, and / or isocaproate.

[0283] Combination for use according to any one of paragraphs 24 to 27, wherein the decrease in BCFA is associated with a decrease in proteolytic fermentation. ;

[0284] Combination for use according to any one of paragraphs 1 to 28, provided in the form of a nutritional composition.

[0285] Embodiment 30 a. Infant formula, infant starter formula, infant follow-on or follow-up formula, growing-up milk, fortifiers or supplements, b. Beverage products, yogurt products, pudding products, fermented milk, fruit juices, bars, mousses, snack foods, chips, meals or meal replacements, c. Healthcare nutritional compositions for oral nutritional support, nutritional products for enteral nutritional support, or parenteral nutritional support products The nutritional composition for use as described in paragraph 29.

[0286] Embodiment 31: A nutritional composition for use as described in paragraph 29 or paragraph 30, which is an infant formula, an infant starter formula, an infant follow-on, or a follow-up formula.

[0287] Embodiment 32: A nutritional composition for use according to any one of paragraphs 29 to 31, which is an infant formula.

[0288] Embodiment 33 The amount of HMO in the nutritional composition is, relative to the total amount of HMO in the nutritional composition, i. a. 2'-FL of approximately 22% to 65% by weight, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight; b. LNnT of approximately 11% to 33% by weight, preferably about 17% to 26% by weight, preferably about 22% by weight; c. Approximately 11% to 33% by weight of 3'-FL, preferably approximately 17% to 26% by weight, preferably approximately 22% by weight of 3'-FL; d. Approximately 2% to 7% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; and e. 6'-SL of approximately 4% to 13% by weight, preferably approximately 7% to 10% by weight, preferably approximately 9% by weight HMOs including or consisting of 2'-FL, LNnT, 3'-FL, 3'-SL, and 6'-SL, ii. a. 2'-FL of approximately 22% to 65% by weight, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight; b. LNnT of approximately 11% to 33% by weight, preferably about 17% to 26% by weight, preferably about 22% by weight; c. Approximately 11% to 33% by weight of 3'-FL, preferably approximately 17% to 26% by weight, preferably approximately 22% by weight of 3'-FL; and d. 3'-SL of approximately 2% to 7% by weight; preferably 3'-SL of approximately 4% to 6% by weight, preferably 3'-SL of approximately 5% by weight; or 6'-SL of approximately 5% to 14% by weight, preferably 6'-SL of approximately 7% to 11% by weight, preferably 6'-SL of approximately 9% by weight HMOs including or consisting of 2'-FL, LNnT, 3'-FL, and one of 3'-SL or 6'-SL, iii. a. Approximately 19% to 58% by weight of 2'-FL, preferably approximately 31% to 46% by weight, preferably approximately 38% by weight of 2'-FL; b. LNnT of approximately 10% to 29% by weight, preferably about 15% to 23% by weight, preferably about 19% by weight; c. Approximately 10% to 29% by weight of 3'-FL, preferably approximately 15% to 23% by weight, preferably approximately 19% by weight of 3'-FL; and d. Approximately 2% to 6% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; e. 6'-SL in approximately 54% to 12% by weight, preferably 6% to 9% by weight, preferably 8% by weight; and f. LNT of approximately 6% to 17% by weight, preferably approximately 9% to 14% by weight, preferably approximately 12% by weight HMOs including or consisting of the following are 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT. A nutritional composition for use as described in any one of paragraphs 30 to 32, comprising or consisting of the following.

[0289] Embodiment 34 HMOs: 2'-FL (approximately 0.8 g / L to 1.2 g / L of the ingested composition), 3'-FL (approximately 0.4 g / L to 0.6 g / L of the ingested composition), LNnT (approximately 0.4 g / L to 0.6 g / L of the ingested composition); 3'-SL (approximately 0.08 g / L to 0.12 g / L of the ingested composition), 6'-SL (approximately 0.16 g / L to 0.24 g / L of the ingested composition), and LNT (approximately 0.24 g / L to 0.36 g / L of the ingested composition). A nutritional composition for use as described in any one of paragraphs 30-33, including the following:

[0290] Embodiment 35 HMOs: 2'-FL (approximately 1 g / L of the ingested composition), 3'-FL (approximately 0.5 g / L of the ingested composition), LNnT (approximately 0.5 g / L of the ingested composition); 3'-SL (approximately 0.1 g / L of the ingested composition), 6'-SL (approximately 0.2 g / L of the ingested composition), and LNT (approximately 0.3 g / L of the ingested composition). A nutritional composition for use as described in any one of paragraphs 30-34, including the following:

[0291] Embodiment 36 A nutritional composition for use according to any one of paragraphs 30 to 35, comprising lactose in an amount of approximately 23 g / L to 50 g / L, or 29 g / L to 44 g / L, of the composition to be ingested.

[0292] Embodiment 37 A nutritional composition for use according to any one of paragraphs 30 to 35, comprising lactose in an amount of about 37 g / L of the composition to be ingested.

[0293] Embodiment 38 A nutritional composition for use according to any one of paragraphs 30 to 35, comprising lactose in an amount of about 58 g / L to 88 g / L or 65 g / L to 80 g / L of the composition to be ingested.

[0294] Embodiment 39 A nutritional composition for use according to any one of paragraphs 30 to 35, comprising lactose in an amount of approximately 73 g / L of the composition to be ingested.

[0295] Embodiment 40 A nutritional composition for use according to any one of paragraphs 30 to 39, which is a highly hydrolyzed prepared milk (eHF).

[0296] Embodiment 41: A nutritional composition for use according to paragraph 40, wherein the protein source in highly hydrolyzed prepared milk (eHF) is highly hydrolyzed casein, whey, or a combination thereof.

[0297] Embodiment 42 The nutritional composition for use according to paragraph 40, wherein the protein source in highly hydrolyzed prepared milk (eHF) is highly hydrolyzed whey.

[0298] Embodiment 43: A nutritional composition for use according to any one of paragraphs 30-39, which is an amino acid-based prepared milk (AAF).

[0299] Embodiment 44 A nutritional composition for use according to any one of paragraphs 30 to 39, which is a partially hydrolyzed infant formula (pHF).

[0300] Embodiment 45: The nutritional composition for use as described in paragraph 44, wherein the protein source in the partially hydrolyzed preparation (pHF) is partially hydrolyzed casein, whey, or a combination thereof.

[0301] Embodiment 46: A nutritional composition for use according to paragraph 40, wherein the protein source in partially hydrolyzed prepared milk (pHF) is partially hydrolyzed whey.

[0302] Embodiment 47 A nutritional composition for use according to any one of paragraphs 40-42 and 44-46, wherein casein, whey, or a combination thereof is derived from goat's milk, donkey's milk, camel's milk, or cow's milk.

[0303] Embodiment 48 A nutritional composition for use according to any one of paragraphs 40-42 and 44-47, wherein casein, whey, or a combination thereof is derived from milk.

[0304] Embodiment 49 A nutritional composition for use according to any one of paragraphs 30 to 39, comprising plant protein.

[0305] Embodiment 50 is an infant formula, an infant starter formula, an infant follow-on or follow-up formula, (a) 1.6g to 3.2g of protein per 100kcal; (b) 9g to 14g of carbohydrates per 100kcal; and / or (c) 4g to 6g of fat per 100kcal A nutritional composition for use as described in any one of paragraphs 30-49, including:

[0306] Embodiment 51 A combination or nutritional composition for use according to any one of paragraphs 1 to 50, further comprising a butyrate in an amount of about 1 mg / L to about 30 mg / L of the composition to be ingested.

[0307] Embodiment 52 A combination or nutritional composition for use according to any one of paragraphs 1 to 51, further comprising butyrate in an amount of about 1.6 mg / L to about 2.4 mg / L of the ingested composition, or about 8 mg / L to about 12 mg / L of the ingested composition, or about 16 mg / L to about 24 mg / L of the ingested composition.

[0308] Embodiment 53 A combination or nutritional composition for use as described in paragraph 51 or paragraph 52, comprising butyrate in an amount of about 16 mg / L to about 24 mg / L of the ingested composition, or about 20 mg / L of the ingested composition.

[0309] Embodiment 54 A source of dietary butyrate, with formula: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 (These are long-chain fatty acids that independently have 16 to 20 carbon atoms.) Compounds having, or combinations thereof A combination or nutritional composition for use as described in any one of paragraphs 1 to 50, further comprising:

[0310] Embodiment 55 R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 A combination or nutritional composition for use as described in paragraph 54, wherein each of the is oleic acid.

[0311] Embodiment 56 A combination or nutritional composition for use as described in paragraph 55, comprising about 1 mg / L to about 30 mg / L of the equivalent of a dietary butyrate in the composition to be ingested.

[0312] Embodiment 57 A combination or nutritional composition for use as described in paragraph 55 or paragraph 56, comprising approximately 1.6 mg / L to approximately 2.4 mg / L of dietary butyrate in the ingested composition, or approximately 8 mg / L to approximately 12 mg / L of dietary butyrate in the ingested composition, or approximately 16 mg / L to approximately 24 mg / L of an equivalent of dietary butyrate in the ingested composition.

[0313] Embodiment 58 A combination or nutritional composition for use according to any one of paragraphs 55 to 57, comprising approximately 16 mg / L to approximately 24 mg / L of dietary butyrate in the composition to be ingested, or an equivalent of approximately 20 mg / L of dietary butyrate in the composition to be ingested.

[0314] Embodiment 59 A combination or nutritional composition for use as described in paragraph 55, comprising approximately 30 mg / L of the equivalent of dietary butyrate in the composition to be ingested.

[0315] Embodiment 60 A combination or nutritional composition for use as described in paragraph 55 or paragraph 59, comprising about 16 mg / L to 24 mg / L of the equivalent of dietary butyrate in the composition to be ingested.

[0316] Embodiment 61 A combination or nutritional composition for use according to any one of paragraphs 55, 59, and 60, comprising about 20 mg / L of the equivalent of dietary butyrate in the composition to be ingested.

[0317] Embodiment 62 A combination or nutritional composition for use according to any one of paragraphs 1 to 50, further comprising a triglyceride comprising a mixture of butyric acid (C4:0) and caprylic acid (C8:0) as a source of dietary butyrate.

[0318] Embodiment 63 A combination or nutritional composition for use as described in paragraph 62, comprising about 1 mg / L to about 30 mg / L of the equivalent of a dietary butyrate in the composition to be ingested.

[0319] Embodiment 64 A combination or nutritional composition for use as described in paragraph 62 or paragraph 63, comprising approximately 1.6 mg / L to approximately 2.4 mg / L of dietary butyrate in the ingested composition, or approximately 8 mg / L to approximately 12 mg / L of dietary butyrate in the ingested composition, or approximately 16 mg / L to approximately 24 mg / L of an equivalent of dietary butyrate in the ingested composition.

[0320] Embodiment 65 A combination or nutritional composition for use according to any one of paragraphs 62 to 64, comprising approximately 16 mg / L to approximately 24 mg / L of dietary butyrate in the composition to be ingested, or an equivalent of approximately 20 mg / L of dietary butyrate in the composition to be ingested.

[0321] Embodiment 66: The combination or nutritional composition for use described in paragraph 62, comprising about 30 mg / L of the equivalent of dietary butyrate in the combination or composition to be ingested.

[0322] Embodiment 67 A combination or nutritional composition for use as described in paragraph 62 or paragraph 66, comprising about 16-24 mg / L of the equivalent of dietary butyrate in the combination or nutritional composition to be ingested.

[0323] Embodiment 68 A combination or nutritional composition for use according to any one of paragraphs 62, 66, and 67, comprising about 20 mg / L of the equivalent of dietary butyrate in the combination or nutritional composition to be ingested.

[0324] Embodiment 69 The amount of HMO in the combination is, relative to the total amount of HMO in the combination, i. a. 2'-FL of approximately 22% to 65% by weight, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight; b. LNnT of approximately 11% to 33% by weight, preferably about 17% to 26% by weight, preferably about 22% by weight; c. Approximately 11% to 33% by weight of 3'-FL, preferably approximately 17% to 26% by weight, preferably approximately 22% by weight of 3'-FL; d. Approximately 2% to 7% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; and e. 6'-SL of approximately 4% to 13% by weight, preferably approximately 7% to 10% by weight, preferably approximately 9% by weight HMOs including or consisting of 2'-FL, LNnT, 3'-FL, 3'-SL, and 6'-SL, ii. a. 2'-FL of approximately 22% to 65% by weight, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight; b. LNnT of approximately 11% to 33% by weight, preferably about 17% to 26% by weight, preferably about 22% by weight; c. Approximately 11% to 33% by weight of 3'-FL, preferably approximately 17% to 26% by weight, preferably approximately 22% by weight of 3'-FL; and d. 3'-SL of approximately 2% to 7% by weight; preferably 3'-SL of approximately 4% to 6% by weight, preferably 3'-SL of approximately 5% by weight; or 6'-SL of approximately 5% to 14% by weight, preferably 6'-SL of approximately 7% to 11% by weight, preferably 6'-SL of approximately 9% by weight HMOs including or consisting of 2'-FL, LNnT, 3'-FL, and one of 3'-SL or 6'-SL, iii. a. Approximately 19% to 58% by weight of 2'-FL, preferably approximately 31% to 46% by weight, preferably approximately 38% by weight of 2'-FL; b. LNnT of approximately 10% to 29% by weight, preferably about 15% to 23% by weight, preferably about 19% by weight; c. Approximately 10% to 29% by weight of 3'-FL, preferably approximately 15% to 23% by weight, preferably approximately 19% by weight of 3'-FL; and d. Approximately 2% to 6% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; e. 6'-SL in approximately 54% to 12% by weight, preferably 6% to 9% by weight, preferably 8% by weight; and f. LNT of approximately 6% to 17% by weight, preferably approximately 9% to 14% by weight, preferably approximately 12% by weight HMOs including or consisting of the following are 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT. A combination for use described in any one of paragraphs 1-29 and 50-68, including or consisting of the above.

[0325] Embodiment 70 A combination for use as described in any one of paragraphs 1-29 and 50-69, comprising HMOs: 2'-FL (about 0.8 g / L to 1.2 g / L of the ingested combination), 3'-FL (about 0.4 g / L to 0.6 g / L of the ingested combination), LNnT (about 0.4 g / L to 0.6 g / L of the ingested composition); 3'-SL (about 0.08 g / L to 0.12 g / L of the ingested combination), 6'-SL (about 0.16 g / L to 0.24 g / L of the ingested combination), and LNT (about 0.24 g / L to 0.36 g / L of the ingested combination).

[0326] Embodiment 71 A combination for use as described in any one of paragraphs 1-29 and 50-70, comprising HMOs: 2'-FL (about 1 g / L of the ingested combination), 3'-FL (about 0.5 g / L of the ingested combination), LNnT (about 0.5 g / L of the ingested combination); 3'-SL (about 0.1 g / L of the ingested composition), 6'-SL (about 0.2 g / L of the ingested combination), and LNT (about 0.3 g / L of the ingested combination).

[0327] Embodiment 72 A combination for use as described in any one of paragraphs 1-29 and 50-71, comprising lactose in an amount of approximately 23 g / L to 50 g / L or 29 g / L to 44 g / L of the combination to be ingested.

[0328] Embodiment 73 A combination for use described in any one of paragraphs 1-19 and 50-72, comprising lactose in an amount of approximately 37 g / L of the combination to be ingested.

[0329] Embodiment 74 A combination for use as described in any one of paragraphs 1-29 and 50-71, comprising lactose in an amount of approximately 58 g / L to 88 g / L, or 65 g / L to 80 g / L, of the combination to be ingested.

[0330] Embodiment 75 A combination for use described in any one of paragraphs 1-29 and 50-74, comprising lactose in an amount of approximately 73 g / L of the combination to be ingested.

[0331] Embodiment 76 A combination or nutritional composition for use according to any one of paragraphs 1 to 75, wherein the subject is an infant, toddler, or child; preferably an infant or toddler.

[0332] Embodiment 77 A combination or nutritional composition for use according to any one of paragraphs 1 to 76, further comprising one or more bacteria from the genera Bifidobacterium and / or Lactobacillus.

[0333] Embodiment 78 A combination or nutritional composition described in any one of paragraphs 1 to 77 for use in the dietary management of milk protein allergy (CMPA).

[0334] Embodiment 79 A combination or nutritional composition for use according to any one of paragraphs 1 to 78, wherein dietary management is the reduction of the severity or frequency of one or more symptoms of a disease.

[0335] Embodiment 80: A combination or nutrient organism described in any one of paragraphs 1 to 77.

[0336] While the present invention has been described in examples, it should be understood that modifications and alterations may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist for certain features, such equivalents are incorporated as if specifically referred to herein.

Claims

1. A combination comprising lactose and at least one of the human milk oligosaccharides (HMOs) 2'-fucosyl lactose (2'-FL), lacto-N-neotetraose (LNnT), 3-fucosyl lactose (3'-FL), and 3'-sialyl lactose (3'-SL) or 6'-sialyl lactose (6'-SL), (a) Dietary management of gut microbiota dysbiosis in the subjects; (b) Dietary management of diseases or disorders related to the metabolism and / or accumulation of branched-chain fatty acids (BCFAs) in the subject; or (c) Decrease in BCFA in the subjects A combination for use in [location].

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 2, wherein the combination is for use in dietary management of dysbiosis of the gut microbiota, wherein the dysbiosis includes increased production of BCFAs 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 dietary management of dysbiosis of the gut microbiota in a subject, wherein the dysbiosis of the gut microbiota is related to intestinal disorders and / or extraintestinal disorders; optionally, the intestinal disorder is one or more of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and celiac disease, and / or the extraintestinal disorder is one or more of allergies, asthma, atopic dermatitis, metabolic syndrome, cardiovascular disease, obesity, and type 2 diabetes.

5. The aforementioned combination is intended for use in the dietary management of diseases or disorders related to the metabolism and / or accumulation of branched-chain fatty acids (BCFAs), (a) The BCFA comprises isobutyrate, isovalerate, and / or isocaproate; and / or (b) The disease or disorder related to the metabolism of BCFA is an increase in proteolytic fermentation and / or a decrease in sugar fermentation. A combination for use according to claim 1 or 2.

6. A combination comprising lactose and at least one of the HMOs 2'-FL, LNnT, 3'-FL, and 3'-SL or 6'-SL, (a) Pathogens in the subject; (b) A pathogen in the subject, which is one or more of bacteria, viruses, or protozoan pathogens; (c) One or more viral pathogens, preferably rotavirus or norovirus, that constitute the pathogen in the subject; or (d) One or more bacterial pathogens, preferably C. difficile, C. perfringens, Campylobacter, or Escherichia coli, which are pathogens in the subject. It is intended for use in dietary management; Preferably, the subject is an infant, toddler, or child who is not breastfeeding or has stopped breastfeeding. combination.

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

8. (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 there is a resolution or reduction of one or more symptoms associated with the abnormal proliferation of C. difficile and / or C. perfringens, preferably at least one of diarrhea and fever. A combination for use according to claim 6 or 7.

9. The combination of lactose and HMO reduces the level of BCFA in the intestine of the subject, and optionally the BCFA comprises isobutyrate, isovalerate, and / or isocaproate, the combination for use according to any one of claims 1 to 8.

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

11. The combination for use according to any one of claims 1 to 10, wherein the combination is provided in the form of a nutritional composition, the nutritional composition being infant formula, infant starter formula, infant follow-on or follow-up formula, or growing-up milk, preferably infant formula.

12. The amount of HMO in the aforementioned combination or nutritional composition is such that, relative to the total amount of HMO in the aforementioned combination or nutritional composition, i. a. Approximately 22% to 65% by weight of 2'-FL, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight of 2'-FL; b. LNnT of about 11% to about 33% by weight, preferably about 17% to about 26% by weight, preferably about 22% by weight; c. Approximately 11% to 33% by weight of 3'-FL, preferably about 17% to 26% by weight, preferably about 22% by weight of 3'-FL; d. Approximately 2% to 7% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; and e. Approximately 4% to 13% by weight of 6'-SL, preferably approximately 7% to 10% by weight, preferably approximately 9% by weight of 6'-SL The HMOs comprising or consisting of the following: 2'-FL, LNnT, 3'-FL, 3'-SL, and 6'-SL, ii. a. Approximately 22% to 65% by weight of 2'-FL, preferably approximately 35% to 52% by weight, preferably approximately 43% by weight of 2'-FL; b. LNnT of about 11% to about 33% by weight, preferably about 17% to about 26% by weight, preferably about 22% by weight; c. Approximately 11% to 33% by weight of 3'-FL, preferably about 17% to 26% by weight, preferably about 22% by weight of 3'-FL; and d. Approximately 2% to 7% by weight of 3'-SL; preferably approximately 4% to 6% by weight, preferably approximately 5% by weight of 3'-SL; or approximately 5% to 14% by weight of 6'-SL, preferably approximately 7% to 11% by weight, preferably approximately 9% by weight of 6'-SL. The HMOs include or consist of the following: 2'-FL, LNnT, 3'-FL, and one of 3'-SL or 6'-SL, iii. a. Approximately 19% to 58% by weight of 2'-FL, preferably about 31% to 46% by weight, preferably about 38% by weight of 2'-FL; b. LNnT in an amount of about 10% to about 29% by weight, preferably about 15% to about 23% by weight, preferably about 19% by weight; c. Approximately 10% to 29% by weight of 3'-FL, preferably about 15% to 23% by weight, preferably about 19% by weight of 3'-FL; d. Approximately 2% to 6% by weight of 3'-SL; preferably approximately 3% to 5% by weight, preferably approximately 4% by weight of 3'-SL; e. Approximately 54% to 12% by weight of 6'-SL, preferably about 6% to 9% by weight, preferably about 8% by weight of 6'-SL; and f. LNT in an amount of approximately 6% to approximately 17% by weight, preferably approximately 9% to approximately 14% by weight, preferably approximately 12% by weight. The HMOs include or consist of the following: 2'-FL, LNnT, 3'-FL, 3'-SL, 6'-SL, and LNT A combination for use according to any one of claims 1 to 10 or a nutritional composition for use according to claim 11, comprising or consisting of the above.

13. The HMOs mentioned above are 2'-FL (about 0.8 g / L to 1.2 g / L of the ingested composition), 3'-FL (about 0.4 g / L to 0.6 g / L of the ingested composition), LNnT (about 0.4 g / L to 0.6 g / L of the ingested composition); 3'-SL (about 0.08 g / L to 0.12 g / L of the ingested composition), 6'-SL (about 0.16 g / L to 0.24 g / L of the ingested composition), and LNT (about 0.24 g / L to 0.36 g / L of the ingested composition). A nutritional composition for use according to claim 12, comprising:

14. (a) lactose in an amount of about 23 g / L to 50 g / L, or 29 g / L to 44 g / L, of the combination or nutritional composition to be ingested; (b) lactose in an amount of about 37 g / L of the above combination to be ingested; (c) lactose in an amount of about 58 g / L to 88 g / L or 65 g / L to 80 g / L of the combination or composition to be ingested; or (d) lactose in an amount of approximately 73 g / L of the combination or composition to be ingested Including a combination for use according to any one of claims 1 to 10 or a nutritional composition for use according to claim 12 or 13.

15. (a) an amount of about 1 mg / L to about 30 mg / L of the aforementioned combination or nutritional composition to be ingested; (b) in amounts of about 1.6 mg / L to about 2.4 mg / L of the combination or nutritional composition to be ingested, or about 8 mg / L to about 12 mg / L of the combination or nutritional composition to be ingested, or about 16 mg / L to about 24 mg / L of the combination or nutritional composition to be ingested; (c) in an amount of about 16 mg / L to about 24 mg / L of the composition or nutritional composition to be ingested; or (d) an amount of about 20 mg / L of the composition or nutritional composition to be ingested A combination or nutritional composition for use according to any one of claims 1 to 14, further comprising a butyrate.

16. (a) As a source of dietary butyrate, formula: 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ and R ６ Each of these is oleic acid. Compounds having, or combinations thereof, (b) Triglycerides composed of a mixture of butyric acid (C4:0) and caprylic acid (C8:0) as a source of dietary butyrates A combination for use according to any one of claims 1 to 10, 14 and 15, or a nutritional composition for use according to any one of claims 12 to 15, further comprising:

17. (a) Approximately 1 mg / L to approximately 30 mg / L of the aforementioned combination or nutritional composition to be ingested; (b) about 1.6 mg / L to about 2.4 mg / L of the combination or nutritional composition to be ingested, or about 8 mg / L to about 12 mg / L of the combination or nutritional composition to be ingested, or about 16 mg / L to about 24 mg / L of the combination or nutritional composition to be ingested; (c) Approximately 30 mg / L of dietary butyrate in the combination or nutritional composition to be ingested; (d) about 16 mg / L to 24 mg / L of dietary butyrate in the combination or nutritional composition to be ingested; or (e) Approximately 20 mg / L of dietary butyrate in the combination or nutritional composition to be ingested A combination for use according to claim 16 or a nutritional composition for use according to claim 16, comprising an equivalent of the above.

18. A combination or nutritional composition for use according to any one of claims 1 to 17, further comprising one or more bacteria from the genera Bifidobacterium and / or Lactobacillus.

19. A combination or nutritional composition according to any one of claims 1 to 18 for use in dietary management of milk protein allergy (CMPA).

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