Use of 3'-sialyllacto-n-tetraose, 6'-sialyllacto-n-neotetraose and / or 3'-sialyl-3-fucosyllactose to improve health in a subject

Abstract

The present disclosure relates to a non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3'-sialyllacto-N-tetraose (LSTa), 6'-sialyllacto-N-neotetraose (LSTc) and 3'-sialyl- 3-fucosyllactose (FSL), or a combination thereof, in supporting or improving one or more of brain health, immune system health, gut health, and metabolic health, in a subject, optionally by increasing the production of one or more metabolites selected from the group consisting of of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine, N-acetyl-L-glutamine, N- acetyl-L-aspartic acid, trigonelline, trans-4-hydroxyproline, 3-hydroxybutyric acid (BHB), 4- hydroxybenzoic acid, and N-acetyl-L-tryptophan, in the gastrointestinal tract of the subject.

Description

[0001] USE OF 3’-SIALYLLACTO-N-TETRAOSE, 6’ -SIALYLLACTO-N-NEOTETRAOSE AND / OR 3’- SIALYL-3-FUCOSYLLACTOSE TO IMPROVE HEALTH IN A SUBJECT

[0002] FIELD

[0003] The disclosure relates to the use, in particular non-therapeutic use, of 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) or 3’-sialyl-3-fucosyllactose (FSL), a combination of any thereof, or a synthetic composition comprising any one or more thereof, for supporting or improving health in a subject. In particular it is shown that LSTa, LSTc and / or FSL can promote the production of metabolites in situ in the gut that support one or more of brain health, immune system health, gut health, and metabolic health, in a subject.

[0004] BACKGROUND

[0005] The human gut microbiome is composed of bacteria, archaea, viruses, and eukaryotic microbes that reside inter alia in the gut. These microbes have tremendous potential to impact our physiology, both in health and in disease. The microbiota of the human intestine is a complex and very dynamic microbial ecosystem, which is considered to serve numerous important functions for its human host, including protection against pathogens, induction of immune regulatory functions, nutrient processing, and metabolic functions. These basic functions affect, directly or indirectly, most of our physiologic functions. Various gut bacteria, including certain Bifidobacterium spp., can feed on oligosaccharides such as fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and human milk oligosaccharides (HMOs), whereas these structures are indigestible by humans. Due to their ability to feed beneficial microorganisms and induce the growth or activity of these, certain oligosaccharides are referred to as prebiotics. GOS is a simple polymer structure composed of galactose units linked to a glucose molecule and may be derivable from cow milk. HMOs on the other hand are more complex structures composed of various monosaccharides such as glucose, galactose, fucose, N- acetylglucosamine, and sialic acid. Both GOS and HMOs support gut health, in particular by stimulating growth of beneficial bacteria. GOS and FOS are, however, less selective since they also may support the growth of harmful bacteria (Salli et al., 2021 , J. Agric. Food Chem., 69:170-182).

[0006] HMOs are the third most abundant solid component after lactose and lipids in human milk with concentrations of 5-25 g / l. To date, more than 200 different HMO structures have been identified in human milk. In addition to their role in the development of the infant gut microbiota. HMOs have additional roles in immune modulation and potentially brain development which are not yet fully elucidated.

[0007] 2’FL is the most abundant HMO in mother’s milk. It is so far the most studied HMO and is increasingly being added to infant formulas. 2’FL acts on the microbiome, resulting in the production of bacterial metabolites. Metabolites observed after 2’FL feeding of various microbiota communities include short chain fatty acids (SCFA’s), such as acetate, propionate and butyrate; some branched chain fatty acids, such as isobutyric acid, isovaleric acid and isocaproic acid; and other metabolites such as lactic acid, indole-3-lactic acid, acetylcholine, y- aminobutyric acid (GABA), glutathione, phosphocholine and serotonin (see for example Zhang et al 2023 Food Research International 173:113293 and Bajic et al 2024 Metabolites 14:239).

[0008] There is a need to further understand the potential benefits on metabolite production associated with more complex HMOs, in the gut of infants and adults, which can be correlated with positive effects on brain health, immune system health, gut health, and metabolic health.

[0009] SUMMARY

[0010] In a first aspect, this disclosure provides a non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’- sialyl-3-fucosyllactose (FSL), or a combination thereof, in supporting or improving one or more of brain health, immune system health, gut health and / or metabolic health, in a subject.

[0011] A second aspect is a non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’-sialyl-3- fucosyllactose (FSL), or a combination thereof, for increasing the production of one or more metabolites selected from the group consisting of pyridoxine (PY), pyridoxamine (PX), nicotinic acid (Nl), N-acetyl-L-glycine (NAGY), sarcosine (NMGY), N-acetyl-L-glutamine (NAGU), N- acetyl-L-aspartic acid (NAA), trigonelline (TRG), 3-hydroxybutyric acid (BHB), trans-4- hydroxyproline (T4-OHP), 4-hydroxybenzoic acid (4H), and N-acetyl-L-tryptophan (NAT), in the gastrointestinal tract of a subject.

[0012] In relation to brain health, the HMO, or combination of HMOs, supports one or more of the following functions as a result of the production of one or more of the metabolites indicated in parentheses: i. cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition (PY / PX) (NAA) (Nl) (NAGY) (NAT) (NAGU) (NMGY) (BHB); ii. memory function (PY / PX) (Nl) (NAT) (NAA) (NAGY) (NAGU) (TRG) (NMGY) (BHB); iii. mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement (PY / PX) (Nl) (NAGY) (NAT) (TRG) (NMGY); iv. increasing neurotransmitter levels, such as an increase in aspartate, glutamate, GABA, serotonin, norepinephrine and / or dopamine levels or synthesis (PY / PX) (Nl) (NAT) (NAGU) (NAGY) (TRG) (NMGY); v. increasing antioxidant levels (Nl) (NAGY) (NAGU) (NAT) (TTG) (4H) (NMGY) (BHB); vi. protection of neurons (PY / PX) (Nl) (NAGY) (NAT) (TRG) (NMGY) (BHB); and vii. sleep quality and regulation (NAGY) (NAT) (NMGY). In relation to immune system health, the HMO, or combination of HMOs, supports one or more of the following functions as a result of the production of one or more of the indicated metabolites: i. increasing antioxidant levels; (Nl) (NAGY) (NAGU) (NAT) (TRG) (4H) (NMGY) (BHB) (T4-OHP); ii. Stimulating one or more anti-inflammatory cytokines;; and iii. reducing one or more proinflammatory cytokines (PY / PX) (TRG) (NAGY) (NAT) (Nl) (NMGY) (BHB) (T4-OHP).

[0013] In relation to gut health, the HMO, or combination of HMOs, supports one or more of the following functions as a result of the production of one or more of the indicated metabolites: i. gut barrier function (NAGU) (NAT) (T4-OHP); and ii. gut motility (NAT).

[0014] In relation to metabolic health, the HMO, or combination of HMOs, supports one or more of the following functions as a result of the production of one or more of the indicated metabolites: i. bone strength (PY / PX) (TRG) (T4-OHP); ii. muscle mass and function, such as grip strength, gait speed and / or muscle mass, (PY / PX) (TRG) (BHB) (T4-OHP); iii. exercise endurance (BHB) iv. coenzyme levels, such as of pyridoxin-5'-phosphate (PNP), pyridoxamine-5'- phosphate (PMP), and / or nicotinamide adenine dinucleotide (NAD+) (PY / PX) (Nl); v. peripheral nervous system, such as protection of neurons (PY / PX) (Nl) (TRG); vi. anti-aging benefits, such as control of skin aging and / or skin pigmentation (Nl): vii. weight management (Nl) (NAGY) (TRG) (NAGU) (NMGY) (BHB); viii. cardiovascular health (Nl) (TRG) and ix. blood pressure regulation (NAGY) (TRG) (NMGY).

[0015] BRIEF DESCRIPTION OF THE FIGURE

[0016] Figure 1 shows the AUG values of metabolites produced after incubation with 2’-fucosyllactose (2’-FL), galacto-oligosaccharides (GOS), 3’-sialyl-3-fucosyllactose (FSL), 3’-sialyllacto- / \ / - tetraose (LSTa) or 6’-sialyllacto- / \ / -neotetraose (LSTc), in the experiment disclosed in Example 1. Results are shown for pyridoxine (PY) (A), pyridoxamine (PX) (B), nicotinic acid (Nl) (C), N- acetylglycine (NAGY) (D), N-acetylglutamine (NAGU) (E), N-acetylaspartic acid (NAA) (F), trigonelline (TRG) (G), 4-hydroxybenzoic acid (4H) (H), and N-acetyltryptophan (NAT) (I).

[0017] Figure 2 depicts the concentration of the individual metabolite determined in each arm quantified by the BioCrates MxP® Quant 500 method. Each dot represents the metabolite concentration in the fermentation supernatant from a single donor, with the column representing the mean. A) Concentration of sarcosine (ng / mL), also known as N-methylglycine (NMGY). B) Concentration of trans-4-hydroxyproline (T4-OHP) (ng / mL). C) Concentration of 3- hydroxybutyric acid (BHB) (ng / mL).

[0018] DETAILED DESCRIPTION

[0019] The present disclosure relates to the three complex sialylated human milk oligosaccharides (HMOs); 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’-sialyl-3- fucosyllactose (FSL). LSTa, LSTc and FSL all contain a sialyl moiety and at least three additional monosaccharide units. In the present disclosure, the inventors show that each of these three HMOs can increase levels of metabolites which are known to play beneficial roles in supporting or improving human health. This discovery enables the present disclosure which comprises using each of these three HMOs, or any combination thereof, in supporting or improving health in a subject, such as one or more of brain health, immune system health, gut health, and metabolic health.

[0020] Definitions

[0021] “Non-therapeutic” or “non-medical” use, as used herein, means an improvement of performance of the body in a normal state or maintaining the normal state longer than can be expected in view of a subjects age. Therefore, the purpose of the non-therapeutic use is to enhance performance and perception of well-being in a healthy mammal / individual. Non-therapeutic uses include maintaining healthy levels of various biomarkers, amino acids, hormones, co-factors, lipids, vitamins, neurotransmitters, salts and biological structures, such as, bone, cells, collagen, mucus, nerves and muscle. Non-therapeutic uses exclude uses or therapy with the purpose of restoring the organism from a pathological condition to an improved or original condition, e.g., restoring abnormal levels of various biomarkers, amino acids, hormones, co-factors, lipids, vitamins, neurotransmitters, salts or restoring biological structures, such as, bone, cells, collagen, mucus, nerves and muscle to normal levels. A non-therapeutic use is therefore considered as improving or maintaining a normal, healthy state in a subject.

[0022] A “human milk oligosaccharide” or “HMO”, as used herein, refers to a complex carbohydrate found in human breast milk. The HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl-lactosaminyl- and / or one or more beta-lacto-N-biosyl-units, and this core structure can be substituted by an alpha-L- fucopyranosyl and / or an alpha-N-acetyl-neuraminyl (sialyl) moiety. HMO structures are, e.g., disclosed by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72. In the context of the present disclosure, lactose (a disaccharide) is not regarded as an HMO species. HMOs can be non-acidic (or neutral) or acidic. Neutral HMOs are devoid of a sialyl residue while acidic HMOs have at least one sialyl residue in their structure. The non- acidic (or neutral) HMOs can be fucosylated or non-fucosylated (neutral-core) HMOs. Within the category of HMO’s the mono-sialylated or mono-fucosylated HMOs (2'-fucosyllactose (2’FL), 3- fucosyllactose (3FL), 3’-sialyllactose (3’SL), 6’-sialyllactose (6’SL)) as well as the neutral non- fucosylated core HMOs (lacto-N-triose II (LNT-II) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT)) are considered as basic HMOs, in part because these are easy to produce and reasonable well studied. In the context of the present disclosure complex HMOs are for example HMOs composed of at least four monosaccharide units of which at least two are selected from a fucosyl and / or a sialyl moiety, or HMOs composed of at least five monosaccharide units, preferably with at least one sialyl monosaccharide. Production of complex HMOs are for example described in WO 2024 / 042235 A1.

[0023] “LSTa” or “sialyllacto-N-tetraose A” or “3’-sialyllacto-N-tetraose” is a complex oligosaccharide, more precisely a sialylated pentasaccharide composed of N-acetylneuraminic acid, D- galactose, N-acetylglucosamine, D-galactose, and D-glucose units (Neu5Ac-a2-3Gaipi- 3GlcNAcpi-3Gaipi-4Glc). It is naturally present in human mother’s milk, however in the context of the present disclosure it is synthetically produced. Synthetic LSTa may comprise some byproduct LNT and 3’SL.

[0024] “LSTc” or “sialyllacto-N-neotetraose C” or “6’-sialyllacto-N-neotetraose” is a complex oligosaccharide, more precisely a sialylated pentasaccharide composed of N-acetylneuraminic acid, D-galactose, N-acetylglucosamine, D-galactose, and D-glucose (Neu5Ac-a2-6Gaipi- 4GlcNAcpi-3Gaipi-4Glc). It is naturally present in human mother’s milk, however in the context of the present disclosure it is synthetically produced. Synthetic LSTc may comprise some byproduct LNnT and 6’SL.

[0025] “FSL” or “3’-sialyl-3-fucosyllactose” is a complex oligosaccharide, more precisely a sialylated and fucosylated tetrasaccharide composed of N-acetylneuraminic acid, D-galactose, L-fucose, and D-glucose units (Neu5Ac-a2-3Gaipi-4(Fuca1-3)Glc). It is naturally present in human mother’s milk, however in the context of the present disclosure it is synthetically produced. Synthetic FSL may comprise some by-product 3FL and 3’SL

[0026] A "subject", as referred to herein, may be a human or a mammal, or an animal selected from domestic animals such as pets (cats, dogs, rodents, rabbits, avian species, reptiles, etc.), livestock and performance animals (pigs, poultry, goat, sheep and cows) and working animals (horses, oxen, camels, donkeys and elephants), with a gut microbiome. Preferably, the subject is a human. The human may be an infant. As used herein, the term “infant” in the context of a human means a human of less than 3 years of age. The infant may be a pre-term infant, meaning that it is delivered before 37 weeks of pregnancy. The infant may be delivered by C- section, which means it has not been exposed to the vagina’s natural microbiota of the delivering woman. As used herein in the context of a human, a “child” is a human of 3 to 12 years of age, an “adult” is a human of at least 18 years of age, and an "elderly individual" means a human of at least 60 years, preferably above 65 years, more preferably above 70 years of age.

[0027] “Cognitive function” refers to a person's thinking, reasoning, problem-solving, and decisionmaking abilities, including learning ability, memory skills, motor skills, language skills, and / or spatial recognition.

[0028] “Motor skills” relate to the cognitive process of coordinating various muscles and body parts to achieve a specific goal. Examples of motor skills include walking, running, jumping, throwing, and catching.

[0029] “Learning” relates to the cognitive process of acquiring new knowledge, skills, behaviours, or attitudes through experience, study, or instruction. It involves a change in behaviour or mental processes that results from experience or practice. Learning can occur through a variety of methods, including observation, trial and error, feedback, and instruction. The ability to learn or “learning ability” is developed early in life and continues to develop throughout the lifespan. Infants are born with the ability to learn and are constantly learning about their environment through their senses and experiences. As they grow, their ability to learn becomes more sophisticated and complex, allowing them to acquire new knowledge and skills.

[0030] “Language skills” refers to the ability to understand and use the language. Language development begins in infancy and continues through childhood and adolescence. Language development involves several key milestones, including learning to recognize and differentiate between speech sounds, developing a vocabulary of words, learning grammar and syntax, and developing the ability to use language for communication.

[0031] “Spatial recognition” relates to the ability to perceive, analyse, and understand spatial relationships between objects and the environment. This includes the ability to recognize and remember the location of objects in space, to mentally manipulate objects in space, and to navigate through the environment.

[0032] “Neurotransmitter” is a signalling molecule secreted by a neuron to affect another cell across a synapse.

[0033] The terms “intestine” and “gut” are used interchangeably herein and refer to the portion of the gastrointestinal tract consisting of the small intestine and the large intestine. The “large intestine” (intestinum crassum) is the lower part of the gastrointestinal tract and is also referred to herein as “colon”.

[0034] The term ‘skin’ as used herein refers to the external surfaces of the human body including the oral cavity, the skin as well as the scalp. The term ‘skin microbiome’ as used herein refers to the group of microbes which colonize a defined skin area of an individual, such as e.g., the forehead, the forearm, the cheek or the scalp, without being limited thereto. The terms "microbiota", "microflora” and "microbiome" are used interchangeably and refer to a community of living microorganisms that typically inhabits a bodily organ or part in an animal or human. Particularly, in the gastrointestinal organs of animals or humans, the microflora is termed the gastrointestinal or gut microbiome or microbiota. The most dominant members of the gastrointestinal microbiota in non-infant humans include microorganisms of the phyla of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota., at genus level Bacteroides, Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Colli nsella, Blautia, Coprococcus, Ruminococcus, Eubacterium, and Dorea, at species level Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Ruminococcus bromii, Dorea longicatena, Bacteroides caccae, Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus torques, Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Bacteroides vulgatus, and Roseburia intestinalis. The gastrointestinal microbiota includes the mucosa- associated microbiota, which is located in or attached to the mucus layer covering the epithelium of the gastrointestinal tract, and luminal-associated microbiota, which is found in the lumen of the gastrointestinal tract.

[0035] “Enteral administration” means any conventional form for delivery of a composition to a human that causes the deposition of the composition in the gastrointestinal tract (including the stomach). Methods of enteral administration include oral administration, feeding through a nasogastric tube or jejunum tube, oral, sublingual and rectal. In the context of administering an HMO or a mixture of HMOs or a composition comprising an HMO or a mixture of HMOs it is understood that the administration is done at an effective level / dose. I.e., at a level / dose that results in a measurable production of the desired metabolite(s) in the gut of the subject.

[0036] "Oral administration" means any conventional form for the delivery of a composition to a human through the mouth.

[0037] “Synthetic nutritional composition” means a composition which is artificially prepared and preferably means a composition containing at least one compound that is produced ex vivo chemically and / or biologically, e.g. by means of chemical reaction, enzymatic reaction or using a genetically manipulated organism, in particular a microorganism or a plant. The synthetic composition disclosed herein comprises an active component, selected from 3’-sialyllacto-N- tetraose (LSTa), 6’-sialyllacto-N-neotetraose (LSTc) and 3’-sialyl-3-fucosyllactose (FSL) or a mixture thereof. The active component is capable of enhancing the production of one or more desired metabolites when administered to the subject. In some embodiments, the synthetic composition may comprise one or more compounds or components other than HMOs that may have a beneficial effect on the microbiota of a human subject, e.g., non-digestible oligosaccharides or prebiotics. Also, in some embodiments, the synthetic compositions may comprise one or more nutritionally or pharmaceutically active components which do not affect adversely the efficacy of the above-mentioned compound. Some non-limiting embodiments of a synthetic composition of the disclosure are also described below.

[0038] Use of HMOs in supporting or improving health in a subject

[0039] In the present disclosure it was shown that 3’-sialyllacto-N-tetraose (LSTa), 6’-sialyllacto-N- neotetraose (LSTc) and 3’-sialyl-3-fucosyllactose (FSL), in a simulated colonic fermentation tests, were capable of increasing the levels of certain desired metabolites as compared to the levels achieved with the reference HMO 2’-fucosyllactose (2’-FL), as shown in Example 1 and 4. As such, incubation with each of these three complex sialylated HMOs lead to an increased level of the metabolites pyridoxine (PY), pyridoxamine (PX), nicotinic acid (Nl), N-acetylglycine (NAGY), sarcosine (NMGY), 3-hydroxybutyric acid (BHB) and trans-4-hydroxyproline (T4-OHP). Incubation with LSTa further lead to an increased level of N-acetyltryptophan (NAT). Incubation with LSTc further lead to an increased level of trigonelline (TRG). Incubation with FSL further lead to an increased level of 4-hydroxybenzoic acid (4H). Finally, incubation with either of FSL and LSTc further lead to an increased level of N-acetylglutamine (NAGU) and N-acetylaspartic acid (NAA) compared to the reference level after incubation with 2’-FL (see Table 1 and table 2).

[0040] 2’-FL is the most abundant HMO in mother’s milk and is increasingly used to supplement conventional infant formulas or as dietary supplement for adults.

[0041] The findings presented in the present disclosure allow for further improvement of such supplements and formulas by using of each of the three HMOs, individually or in combination, to dynamically modulate the human intestinal microbiota to produce the above-mentioned metabolites and thus contribute to a healthy body, as will be described below.

[0042] Pyridoxine and pyridoxamine (PY / PX)

[0043] Pyridoxine (PY) and pyridoxamine (PX) are two different forms of vitamin B6, an essential micronutrient for human health. PY and PX are converted into their active forms, pyridoxin-5'- phosphate (PNP) and pyridoxamine-5'-phosphate (PMP), which serve as coenzymes in numerous biochemical reactions. Vitamin B6-derived metabolites serve as interconverting cofactors (transaminases) for >140 enzymatic reactions and have been estimated to be essential for 4% of all enzyme activities in the human genome (Peterson et al Nutrients. 2020 12(11): 3380 doi: 10.3390 / nu12113380).

[0044] B vitamin deficiencies, including vitamin B6 has been shown to play a role in cognitive decline. Supplementation with B6 has shown to improve cognitive function and enhances long term memory (Mikkelsen etal., Maturitas 2016; 93:108-113, doi: 10.1016 / j.maturitas.2016.08.001). Vitamin B6 has generally been shown to play a role in neurodegenerative diseases, promotion of CNS development and myelin formation, mood regulation / enhancement (depression and anxiety symptom improvement, stress reduction), and protection and nerve / neuron regeneration including peripheral nerves (see for example Kirksey et al 1990 Annals of the New York Academy of Sciences 585(1):202-218, Paulionis etal. BMC Geriatr. 2005; 5: 16; Curtin and Johnston, J Diet Suppl. 2023;20(4):550-562 doi: 10.1080 / 19390211.2022.2030843; and Baltrusch, Biomed Res Int. 2021 ; 2021: 9968228 doi:10.1155 / 2021 / 9968228).

[0045] In some embodiments, it is of particular interest to increase the prevalence of PY and PX in the gut of an elderly subject.

[0046] PY and PX play important roles in the brain, where they are involved in synthesis of intermediates and coenzymes for the formation of the neurotransmitters serotonin, dopamine and gamma-aminobutyric acid (GABA) (see for example Mughram et al. Int J Mol Sci. 2023; 24(1):42 doi: 10.3390 / ijms24010642; and Mikkelsen et al., Curr Med Chem. 2016; 23(38):4317- 4337 doi: 10.2174 / 0929867323666160920110810).

[0047] Dopamine is one of the major neurotransmitters in reward-motivated behavior and is a precursor for other catecholamines like norepinephrine and epinephrine. Dopamine facilitates working memory, which is the ability to hold and manipulate information over short periods. It helps cognitive flexibility, allowing individuals to adapt their thinking and behavior in response to changing environments or rules. Likewise, GABA is essential for synaptic plasticity, which is important for learning and memory formation. GABA and dopamine are regulators of neuronal activity which, inter alia, support development of cognitive functions as well as support for emotional and behavioral development, such as stress and anxiety reduction. By promoting the production of GABA and dopamine, PY and PX therefore support development of cognitive functions and memory functions, as well as mood regulation.

[0048] PY and PX produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii) improved memory function; iii) mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement, iv) neurotransmitter levels, such as serotonin, dopamine and GABA; and v) protection of neurons.

[0049] Vitamin B6 also plays important roles in the immune system, such as in production of cytokines (e.g., reduction of NF-kB) and T cells and in regulation of immune responses which can prevent and reduce microbial infections as well as inflammation. B6 has also been shown to be inversely associated with levels of chronic inflammation (for more information see Stach et al., Nutrients, 2021 , 13(9), 3229 doi:10.3390 / nu13093229, Mikkelsen et al., Maturitas. 2017 96:58- 71 doi: 10.1016 / j.maturitas.2016.11.012 and Selhub et al., J Nutr Biochem. 201324(12):2138- 43 doi: 10.1016 / j.jnutbio.2013.08.005). PY has also been shown to have anti-inflammatory effects e.g., by downregulating a number of pro-inflammatory cytokines such as (I L)-6, I L-1 p, TNF-a, NF-KB (Mikkelsen et al., Biomedicines, 2023, 11 (9):2578, doi: 10.3390 / biomedicines11092578). PY and PX produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to immune system health, in particular in relation to anti-microbial and antiinflammatory effects, such as the reduction of one or more pro-inflammatory cytokines.

[0050] Finally, vitamin B6 is involved in skeletal muscle health, and low vitamin B6 levels have been associated with sarcopenia, which is an age-related progressive loss of muscle mass and strength and frailty (Kato et al., Nutrients, 2024, Nutrients 16(1): 177 doi:10.3390 / nu16010177). PY and PX can therefore be used to and support muscle mass and function, such as grip strength and / or gait speed. Additionally, low vitamin B6 levels are associated with osteoporosis, due to a role of vitamin B6 in cross-linking collagen formation (Welan et al., J. Bone Metab., 2023 30(2): 141-147 doi:10.11005 / jbm.2023.30.2.141). PY and PX can therefore also be used to support bone strength. PY and PX can consequently also be associated with healthy aging.

[0051] PY and PX produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to metabolic health, in particular bone and muscle strength formation of the co-enzymes PNP and PMP, and protection of peripheral neurons.

[0052] Thus, PY and PX promote health, such as brain health, immune system health, and / or metabolic health, and use of an HMO which increases production and / or abundance of PY and PX in the gut therefore promotes these beneficial effects in an individual.

[0053] Nicotinic acid (Nl)

[0054] Nicotinic acid (Nl), also known as niacin or vitamin B3, is an essential nutrient involved in numerous metabolic processes, including energy production and DNA repair. It can be synthesized in the gut from the amino acid tryptophan by gut microbiota.

[0055] Nl has important functions in the brain. The ability of Nl synthesized in the gut to reach the brain involves several steps, including absorption into the bloodstream, crossing the blood-brain barrier (BBB), and utilization by brain cells. Nl crosses the BBB via facilitated diffusion or active transport mechanisms involving specific carrier proteins. These transport systems ensure that adequate levels of Nl reach the brain. Nl is a precursor of the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are essential coenzymes in cellular energy metabolism. As such, sufficient transport of Nl to the brain is crucial for maintaining adequate levels of NAD+, which is essential for energy production, DNA repair, and overall neuronal health (Surjana et al. J Nucleic Acids. 2010; 2010: 157591 doi: 10.4061 / 2010 / 157591). NAD+ is involved in the synthesis of several neurotransmitters by providing energy and acting as co-factor in the synthesis of neurotransmitters, including serotonin which in is a key neurotransmitter that regulates mood, anxiety, and overall emotional well-being. Proper neurotransmitter levels are crucial for synaptic transmission, mood regulation, cognition, and overall brain function. Furthermore, NAD+ support synaptic plasticity, the ability of synapses to strengthen or weaken over time, which is essential for learning and memory (Zhao et al J of Neuroinflammation 2021 18:207 doi:10.1186 / s12974-021-02250-8). In addition Nl has been shown to support cognitive function in elderly (Morris et al. J Neurol Neurosurg Psychiatry 2004;75:1093-1099. doi:

[0056] 10.1136 / jnnp.2003.025858). Nl has also been shown to have in regeneration of neurons e.g. in relation to ischemic damage as well as neuron protection (Gasperi et al. Int. J. Mol. Sci. 2019, 20, 974; doi:10.3390 / ijms20040974).

[0057] Nl and its derivatives can contribute to the synthesis of glutathione, one of the brain's primary antioxidants. Glutathione helps protect neurons from oxidative stress and free radical damage and can increase the antioxidative capacity.

[0058] Nl produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii) improved memory function; iii) mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement, iv) neurotransmitter levels, such as acting as co-factor for production of serotonin; v) increasing antioxidants in particular glutathione and the overall antioxidant capacity, and vi) protection of neurons.

[0059] Furthermore, Nl appear to have a therapeutic effect on migraine headaches, tension-type headaches, and headaches of other aetiologies (Prousky et al. Nutr J. 200526;4:3 doi: 10.1186 / 1475-2891-4-3).

[0060] In addition to its effects in the brain, Nl has been shown to have anti-inflammatory effects e.g. by downregulating pro-inflammatory cytokines such as IL-6, IL-8, IL10, TNF-a and NF-kB (Digby et al., Arterioscler. Thromb. Vase. Biol., 2012, 32(3): 669-676 doi:

[0061] 10.1161 / ATVBAHA.111.241836; Wanders et al., PLoS One, 2013, doi:

[0062] 10.1371 / journal. pone.0071285). In addition, Nl, and its derivative nicotinamide and NAD+, have been shown to have antioxidant effects which decrease reactive oxygen species including free radical and which also contribute to anti-aging effects, including effects on skin aging and skin pigmentation (Boo et al., Antioxidants, 2021 , 10:1315 doi: 10.3390 / antiox10081315 and Choi et al. Mol Cells. 2015 38(10):918-24 doi: 10.14348 / molcells.2015.0168).

[0063] Nl produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to immune system health, in particular by increasing antioxidant levels or the antioxidant capacity and reducing inflammation, in particular by reducing pro-inflammatory cytokines.

[0064] Nl favourably influences all lipoprotein classes, including lipoprotein[a], and belongs to the most potent hypolipidemic drugs for increasing HDL-cholesterol levels (Zeman et al. Acta Pharm. 66 (2016) 449-469 DOI: 10.1515 / acph-2016-0043). Nl produced in-situ in the gut is therefore suitable for non-therapeutic management of a healthy cholesterol profile, with high HDL. Nl produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to metabolic health including increasing the co-enzyme NAD+, anti-aging effects on the skin as well as weight management.

[0065] Thus, Nl promotes health, such as brain health, immune system health, and / or metabolic health, and use of an HMO which increases production and / or abundance of Nl in the gut therefore promotes these beneficial effects in an individual.

[0066] N-acetylglycine (NAGY)

[0067] N-acetylglycine (NAGY) is a derivative of the amino acid glycine, where an acetyl group is attached to the amino group of glycine. NAGY can be converted to glycine in the body through a process known as deacetylation.

[0068] Glycine can crosse the blood-brain barierr. The transport of glycine across the blood-brain barrier is facilitated by specific transport mechanisms, such as the amino acid transporters. These transporters help regulate the concentration of glycine in the brain, ensuring it can perform its various physiological functions.

[0069] In the brain, glycine functions as an inhibitory neurotransmitter acting on glycine receptors (GlyRs) as well as Ionotropic glutamate receptors (iGluRs) with beneficial effects on neurodevelopment, cognitive function and memory and mood regulation (see for example Stroebel et al. Neuropharmacology. 2021 1 ; 193: 108631. doi:

[0070] 10.1016 / j.neuropharm.2021.108631. Epub 2021 May 28. PMID: 34058193; Ito, J Physiol Sci. 201666(5):375-9 doi: 10.1007 / s12576-016-0442-7 and Salceda, Front Neurosci. 2022 6:947563. doi: 10.3389 / fnins.2022.947563).

[0071] In terms of mood regulation, the interaction of glycine with the GlyRs has been shown to have a positive effect on mood regulation, in particular anxiety- and depression-like behaviours (Li et al. Neuropharmacology 2019 157:107688 doi: 10.1016 / j.neuropharm.2019.107688)

[0072] It has also been shown that orally administered glycine has a positive effect on sleep quality. Specifically, the non-REM sleep periods were increased, and the sleep onset was shortened as was the wake state upon sleep disturbance (Bannai and Kawai, J Pharmacol Sci. 2012;118(2):145-8. doi: 10.1254 / jphs.11r04fm).

[0073] Glycine has also been shown to confer neuronal protection both against oxidative stress as well as during the acute period of ischemic stroke. Specifically, the neuroprotective effects were observed with respect to neuroapoptosis, neuroinflammation, synaptic dysfunction and prevented memory impairment (see for example llllah et al., Journal of Neuroinflammation 2020 17:303 doi.org / 10.1186 / s12974-020-01989-w and Gusev et al. Cerebrovasc Dis. 2000 10(1):49- 60. doi: 10.1159 / 000016025). NAGY produced in situ in the gut in the subject, can be converted to glycine and cross into the blood stream and enter the brain. Thereby NAGY provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii) improved memory function; iii) mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement, iv) increased neurotransmitter levels, such as an increase in glycine; v) increasing antioxidant levels or antioxidant capacity in particular increased glutathione; vi) protection or regeneration of neurons; and vii) improving sleep quality and regulation.

[0074] Glycine is involved in the synthesis of the antioxidant glutathione and also functions as an antioxidant itself. As such, glycine has antioxidant functions in the brain as well as in other organs and tissues and therefore support an increase in antioxidant capacity (Fang YZ, Yang S, Wu G. Free radicals, antioxidants, and nutrition. Nutrition. 2002 10:872-9 doi: 10.1016 / s0899- 9007(02)00916-4).

[0075] Glycine has also been found to have anti-inflammatory and immunomodulatory effects for example by in colonic expression of the pro-inflammatory cytokines I L-1 b and TNFa, cytokine- induced neutrophil chemoattractant and macrophage inflammatory protein (for more information see for example Li et al., British Journal of Nutrition, 2007 98(2):237-252 doi:10.1017 / S000711450769936X and Li et al. Infect Immun. 200169(9):5883-91. doi: 10.1128 / IAI.69.9.5883-5891.2001).

[0076] NAGY produced in situ in the gut in the subject, can be converted to glycine, and thereby provide for multiple beneficial effects in relation to immune system health such as increasing antioxidant levels and / or antioxidant capacity, in particular increasing glutathione and reducing inflammation, e.g. by reducing pro-inflammatory cytokine production.

[0077] With regards to metabolic health, glycine has been shown to have positive effects on metabolic syndrome including obesity, diabetes hyperlipidemia and hypertension. Glycine has for example been shown to reduce plasma triglycerides and blood pressure in sucrose fed rats (see for example Wang et al Amino Acids 201345, 463-477 doi: 10.1007 / s00726-013-1493-1 ; El Hafidi et al. Am J Physiol Regul Integr Comp Physiol. 2004287(6):R1387-93 doi:

[0078] 10.1152 / ajpregu.00159.2004 and Alvarado-Vasquez et al. Comp Biochem Physiol C Toxicol Pharmacol. 2003 134(4):521-7 doi: 10.1016 / s1532-0456(03)00046-2 and Imenshahidi and Hossenzadeh, J Endocrinol Invest 2022 45, 927-939 Doi:10.1007 / s40618-021-01720-3).

[0079] NAGY produced in situ in the gut in the subject, can be converted to glycine, and thereby provide for beneficial effects in weight management as well as blood pressure regulation.

[0080] Thus, NAGY promotes health, such as brain health, immune system health, and / or metabolic health, and use of an HMO which increases production and / or abundance of NAGY in the gut therefore promotes these beneficial effects in an individual. Sarcosine (NMGY)

[0081] Sarcosine, also known as N-methylglycine (NMGY), is a naturally occurring amino acid derivative and an intermediate in the metabolism of glycine. Sarcosine can be converted into glycine by sarcosine dehydrogenase. Sarcosine therefore promotes similar health benefits as N-acetylglycine (NAGY) described above.

[0082] In addition, sarcosine is a glycine transporter type 1 inhibitor (GlyT1), which allows sarcosine to act as an NMDA receptor co-agonist. Sarcosine has been shown to improve depression-like behaviours in rodent models and in human depression (Huang et al., 2013 BIOL PSYCHIATRY 74:734-741). Furthermore, the NMDA receptor co-agonistic effects of sarcosine have been assessed in toluene-induced motor incoordination and recognition memory impairment where it showed the ability to reverse these symptoms (Chan et al., 2012 Toxicol Appl Pharmacol. 265(2): 158-165). The NDMA receptor function of sarcosine may further benefit cognitive function.

[0083] Sarcosine produced in situ in the gut, may therefore show benefits in mood regulation and in particular in mood enhancement as well as providing positive benefits to memory function, cognitive function and motor skills.

[0084] Tanas et al 2022 (Biomed Res I nt. 5467498 ) demonstrated that in in vitro studies sarcosine increased the percentage of viable neuroblastoma cells against aluminum induced neurotoxicity. The same study further showed that the total oxidative stress (TOS) was reduced, and the total antioxidant capacity (TAC) was increased in rats that received sarcosine.

[0085] Sarcosine produced in situ in the gut, may therefore positively affect neuroprotection as well as increasing the antioxidant capacity (levels).

[0086] In addition sarcosine produced in situ in the gut of a subject, can be converted to glycine, which provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii) improved memory function; iii) mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement, iv) increased neurotransmitter levels, such as an increase in glycine; v) increasing antioxidant levels in particular glutathione; vi) protection or regeneration of neurons; and vii) improving sleep quality and regulation, (see the section on N-acetylglycine (NAGY) above)

[0087] As described in relation to N-acetylglycine (NAGY) above glycine also provide for multiple beneficial effects in relation to immune system health such as increasing antioxidant levels and / or antioxidant capacity, in particular increasing glutathione and reducing inflammation, e.g. by reducing pro-inflammatory cytokine production.

[0088] With regards to metabolic health, glycine has been shown to have positive effects on metabolic syndrome including obesity, diabetes hyperlipidemia and hypertension. Sarcosine produced in situ in the gut in the subject, can be converted to glycine, and thereby provide for beneficial in weight management as well as blood pressure regulation.

[0089] Thus, Sarcosine (NMGY) promotes health, such as brain health, immune system health, and / or metabolic health, and use of an HMO which increases production and / or abundance of NMGY in the gut therefore promotes these beneficial effects in an individual.

[0090] N-acetyltryptophan (NAT)

[0091] N-Acetyl-L-tryptophan (NAT) is a derivative of the amino acid tryptophan, where an acetyl group is attached to the nitrogen atom of the tryptophan molecule. NAT may be converted to tryptophan in the body through removal of this acetyl group, a process known as deacetylation as has been observed for N-acetyl-L-glutamine (see below).

[0092] Tryptophan is a precursor to serotonin, a pathway that starts with the conversion of tryptophan to 5-hydroxytryptophan (5-HTP) and then to serotonin (5-HT). The gut-brain axis is a bidirectional system between the brain and gastrointestinal tract, linking emotional and cognitive centres of the brain with peripheral functioning of the digestive tract. Tryptophan is generally obtained via the diet or via the gut microbiota from where it is transported to the brain to be converted to serotonin. Serotonin is a neurotransmitter that plays a crucial role in regulating mood, anxiety, and overall well-being. Adequate levels of serotonin are associated with improved mood, reduced anxiety, and a general sense of well-being. Low levels of serotonin are linked to depression, anxiety disorders, and other mood disorders (see for example Jenkins et al. Nutrients. 201620;8(1):56. doi: 10.3390 / nu8010056).

[0093] Serotonin is involved in various cognitive processes, including learning and memory function. It modulates the activity of neural circuits that underpin cognitive functions, and adequate serotonin levels contribute to better decision-making, problem-solving abilities, and overall cognitive flexibility (see for example Jenkins et al. Nutrients. 2016 20;8(1):56. doi: 10.3390 / nu8010056).

[0094] NAT has also been shown to protect cholinergic nerve terminals from neurodegeneration due to aluminum and therefore have positive effects on memory and neuroprotection, potentially due to its ability to block substance P mediated neuroinflammation, reduction in oxidative stress and anti-apoptotic properties (Fernandes et al. Toxicology Mechanisms and Methods, 28(5), 328- 334. doi:10.1080 / 15376516.2017.1411412). This effect on substance P has also been shown to be effective in traumatic brain injury (Donkin et al. J Cereb Blood Flow Metab. 2009 29(8):1388- 98 doi: 10.1038 / jcbfm.2009.63). NAT has also been shown to have neuroprotective effects including reduction of neuroinflammatory pathways and lowering of the cognitive decline in Alzheimer’s patients (Satarker et al. Mol Neurobiol. 202461(7):4421-4440. doi: 10.1007 / s 12035-023-03844-4) . Tryptophan has been found to have sleep inducing effects. In addition, serotonin is a positive modulator of the sleep signaling hormone melatonin that regulates sleep-wake cycles as well as the circadian rhythm (Sutanto et al. Nutr Rev. 20220;80(2):306-316. doi:

[0095] 10.1093 / nutrit / nuab027).

[0096] NAT produced in situ in the gut in a subject, from where it can act on its own or be converted to tryptophan, and thereby provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii) improved memory function; iii) mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement, iv) neurotransmitter levels, such as an serotonin; v) increasing antioxidant levels and or antioxidant capacity; vi) protection of neurons; and vii) improving sleep quality and regulation.

[0097] In addition to its effects in the brain, NAT or tryptophan has been shown to have positive effects on inflammation (anti-inflammatory), antimicrobial function (indole a tryptophan metabolite) and allergy and as an antioxidant (Roager and Licht, Nat Commun. 2018 17;9(1):3294. doi: 10.1038 / S41467-018-05470-4 and Gostner et a / . Int Arch Allergy Immunol. 2016;169(4):203-15 doi: 10.1159 / 000445500).

[0098] Tryptophan and its metabolites possess strong antioxidant and anti-inflammatory properties through multiple mechanisms, such as the aryl hydrocarbon receptor (AhR), reduction of pro- inflammatory cytokines, such as TNF-a and IL-6, (Tan, Free Radic Biol Med. 2022 1 ;184:30-41 doi: 10.1016 / j.freeradbiomed.2022.03.025 and Nayak et al. J Complement Integr Med. 2016 13(2):129-36 doi: 10.1515 / jcim-2015-0051).

[0099] NAT produced in situ in the gut in the subject, from where it can act on its own or be converted to tryptophan, and thereby provide for multiple beneficial effects in relation to immune system health, such as increasing antioxidant levels and / or antioxidant capacity, preventing microbial infections and reducing or preventing inflammation, in particular by reducing pro-inflammatory cytokines.

[0100] Tryptophan, which can be obtained by demethylation of NAT, has also been shown to have positive effects on gastrointestinal motility as well as the intestinal epithelial barrier (Roager and Licht, Nat Commun. 2018 17;9(1):3294. doi: 10.1038 / s41467-018-05470-4)

[0101] NAT produced in situ in the gut in the subject, from where it can act on its own or be converted to tryptophan, and thereby provide for multiple beneficial effects in relation to gut health, in particular gut barrier function and motility.

[0102] Thus, NAT promotes health, such as brain health, immune system health, and / or gut health, and use of an HMO which increases production and / or abundance of NAT in the gut therefore promotes these beneficial effects in an individual. Trigonelline (TRG)

[0103] Trigonelline (TRG) is an N-methylated form of nicotinic acid and is found in various plants, including fenugreek seeds, and some other legumes. Trigonelline levels are known to be particularly high in radish and coffee beans.

[0104] Trigonelline has been shown to have positive effects on memory and spatial learning through anti-oxidative effects, regulation of pathways related to inflammation and neurotransmitter release such as dopamine, noradrenaline, and serotonin (Aktar et al. Geroscience. 202446(2):1671-1691 doi: 10.1007 / s11357-023-00919-x and Khalili et al. Int Immunopharmacol. 201861:355-362 doi: 10.1016 / j.intimp.2018.06.019).

[0105] Trigonelline supplementation has been shown in rodents to have neuroprotective effects which improve cognition and protect against Alzheimer’s disease (Fahanik-Babaei et al., Metab Brain Dis. 2019 34(1): 191 -201. doi: 10.1007 / s11011-018-0338-8). Furthermore, Zhou et al 2012 (Evidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 164219) show a positive effect of trigonelline on peripheral neurons in peripheral neuropathy. In the same study trigonelline was also shown to downregulates pro-inflammatory signalling via the p38 MAPK pathway.

[0106] Fenugreek extract has high levels of trigonelline and has been shown to repress depression in perimenopausal women (Khanna et al. J Food Biochem. 202044(12):e13507 doi:

[0107] 10.1111 / jfbc.13507).

[0108] Trigonelline produced in situ in the gut can thereby provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as spatial recognition; ii) improved memory function; iii) mood regulation, such as mood enhancement, iv) neurotransmitter levels, such as dopamine, noradrenaline, and serotonin; v) increasing antioxidant levels and / or antioxidant capacity and vi) protection of neurons.

[0109] Trigonelline contained in Sakurajima radish has been shown to contribute to the improvement of vascular endothelial function in humans, e.g. by inducing nitrogen oxide (NO) production in blood vessels which through its vasodilatory effects may regulate blood pressure. TRG has also been shown to reduce triglycerides which in turn can prevent atherosclerosis and improve overall cardiovascular health, (see for example Sasaki et al. Nutrients 2020, 12:1872; doi:10.3390 / nu12061872).

[0110] Trigonelline also improves lipid metabolism in white adipocytes by decreasing adipogenesis and lipogenesis as well as promotes lipolysis and fatty acid oxidation, and is therefore suitable for weight management (Choi et al. Phytother Res. 2021 35(2): 1113-1124. doi: 10.1002 / ptr.6892 and llavenil et al. Phytomedicine. 2014 15;21(5):758-65. doi: 10.1016 / j.phymed.2013.11.007)

[0111] Trigonelline improves mitochondrial respiration and biogenesis and reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation enhances muscle strength and prevents skeletal muscle loss and prevents fatigue during ageing (see for example WO2010 / 142750, WO2021 / 004922, W02021 / 004913 and Membrez et al. Nature Metabolism 20246:433-447 doi.org / 10.1038 / s42255-024-00997-x). Trigonelline has further been shown to affect bone strength both in relation to diabetes as well as in an osteoporosis inducing rat model (Folwarczna et al. Nutrients 2016, 8, 133; doi:10.3390 / nu8030133 and Rathi et al. Drug Res 2020 70(6):257-264 doi: 10.1055 / a- 1147-5724 and Rathi et al. Drug Res. 2020 70(6):257-264. doi: 10.1055 / a-1147-5724).

[0112] Trigonelline has showed effects on diabetes in multiple studies (Folwarczna et al. Nutrients 2016, 8, 133; doi:10.3390 / nu8030133 and Rathi et al. Drug Res 2020 70(6):257-264 doi: 10.1055 / a-1147-5724; Adams et al. Grit Rev Food Sci Nutr. 2014;54(10):1322-9 doi: 10.1080 / 10408398.2011.635816 and Zhou et al. Curr Med Chem. 2012;19(21):3523-31 doi: 10.2174 / 092986712801323171 ).

[0113] Trigonelline produced in situ in the gut can thereby provide for multiple beneficial effects in relation to metabolic health, including bone strength, muscle mass and function, weight management, cardiovascular health and improved vascular endothelial function, including blood pressure regulation.

[0114] Finally, trigonelline has also been shown to have antimicrobial properties against Pseudomonas Kar et al ACS Infect Dis. 2024 9; 10(2): 746-762 doi: 10.1021 / acsinfecdis.3c00617).

[0115] Trigonelline produced in situ in the gut can thereby provide for multiple beneficial effects in relation to immune system health, such as increasing antioxidant levels, inhibiting pro- inflammatory pathways thereby reducing pro-inflammatory cytokines, preventing microbial infections and reducing or preventing inflammation.

[0116] Thus, TRG promotes health, such as brain health, immune system health, and / or metabolic health, and use of an HMO which increases production and / or abundance of TRG in the gut therefore promotes these beneficial effects in an individual.

[0117] 3-Hydroxybutyric acid (BHB)

[0118] 3-Hydroxybutyric acid, is also known as beta-hydroxybutyrate (BHB).

[0119] Ketosis is a metabolic state that occurs when the body is using fat as its primary source of energy instead of carbohydrate. BHB is one of the three ketone bodies produced by the liver during ketosis and serves as alternative energy sources for the brain, heart, and skeletal muscles during nutrient deprivation and adherence to low carbohydrate diets.

[0120] BHB has been shown to have antioxidative properties. In the review by Rojas-Morales et al 2020 (Redox Biology 29 (2020) 101395) several studies relating to the antioxidative properties of BHB are mentioned. In vitro studies have shown that, in the first place, BHB functions as a direct antioxidant for hydroxyl radical (-OH) and in addition inhibits mitochondrial reactive oxygen species (ROS) production in stressed neurons by facilitating NADH oxidation.

[0121] Furthermore, Youm et al 2015 (Nat Med. 21(3):263-9) report that BHB suppresses activation of the NLRP3 inflammasome which controls the activation of caspase-1 and the release of the pro- inflammatory cytokines I L-1 p and IL-18 in macrophages7-11. BHB therefore also possess antiinflammatory effects by reducing pro-inflammatory cytokines.

[0122] BHB produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to immune system health, in particular by increasing antioxidant levels and reducing inflammation, in particular by reducing pro-inflammatory cytokines.

[0123] BHB may help with weight loss by promoting fat burning and reducing appetite. It can increase the body's ability to burn fat for energy. Katsuya et at 2023 (J Nutr Sci Vitaminol 69(2):121-128) conducted a study on healthy adults administering BHB by a daily ingestion and showed significant reduction in visceral fat, body weight and fat weight without dieting or increased exercise intervention.

[0124] Mitochondria are known to form a highly dynamic and interconnected network in adult skeletal muscle. Mitochondria can adjust their morphology upon different energetic challenges induced by a nutritional stimulus, such as nutrient excess or deprivation. Monsalves-Alvarez et al 2020 (Nutrients 12, 1930; doi:10.3390 / nu12071930) studied the effects of sustained BHB supplementation in endurance capacity in mice, focusing on mitochondrial morphology and function. The study showed that BHB supplementation induce an improvement in exercise endurance and grip strength.

[0125] BHB produced in situ in the gut can thereby provide beneficial effects in relation to metabolic health, in particular in relation to weight management, muscle strength such as grip strength and in exercise endurance. Supplementation with one or more of the HMOs FSL, LSTa or LSTc may therefore be beneficial for both athletes and elderly, where muscle strength and endurance is highly desired.

[0126] BHB is a molecule that is capable of crossing the blood-brain barrier where it serves as energy source under ketosis. Interestingly Huang et al 2022 (Stress Biology 2:57) shows that BHB is also capable of promoting neurogenesis and improvements in spatial recognition and memory function. Specifically, BHB was shown to reduce or prevent decrease of synaptophysin thereby improving memory by enhancing synaptic plasticity in heat stress-treated mice. In addition, BHB has been shown to have neuroprotective effects.

[0127] BHB produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to brain health, including improved cognitive function, in particular spatial recognition; improved memory function, antioxidative effects, and protection of neurons. Trans-4-hydroxyproline (T4-OHP)

[0128] Trans-4-hydroxyproline also often just known as hydroxyproline is integral to collagen synthesis. It stabilizes the collagen triple helix structure, enhancing the tensile strength of the collagen fibres. Hydroxyproline comprises approximately 13.5% of the protein in collagen. Cao et al 2022 (Frontiers in Physiology 13: 913800) investigated dietary supplementation with hydroxyproline in fish which showed increase collagen content in the muscle of the fish.

[0129] As collagen is also a major part of bone, it is expected that in situ production in the gut of hydroxyproline may be beneficial to both muscle strength and bone strength. In particular in subjects primarily living on a plant-based diet.

[0130] Hydroxyproline has also been shown to increase the integrity and functional performance of the intestinal barrier in early-weaned piglets (Tang et al 2024 International Immunopharmacology 134:112268). In addition it was shown that hydroxyproline improved mucosal immunity in the gut by increasing the amount of secretory IgA (slgA) and the ratio of CD4+ / CD8+ T lymphocytes and decreasing NF- KB phosphorylation which lead to decreased levels of the pro-inflammatory cytokines IL1 and TNF-alpha. Furthermore, the antioxidant capacity in the piglets was increased by raising total antioxidant capacity (TAC) and glutathione levels in the intestinal mucosa.

[0131] Hydroxyproline produced in situ in the gut in the subject can thereby provide for multiple beneficial effects in relation to metabolic health, immune system health and gut health, in particular by improving bone and muscle strength and increasing antioxidant levels and reducing inflammation, in particular by reducing pro-inflammatory cytokines in the gut. Furthermore, a benefit on the gut-barrier function can be provided by hydroxyproline.

[0132] 4-hydroxybenzoic acid (4H)

[0133] The phenolic compound 4-hydroxybenzoic acid (4H), also known as p-hydroxybenzoic acid, is a naturally occurring derivative of benzoic acid. Microorganisms produce benzoic acid and hydrobenzoic acids from phenylalanine, and these compounds are also found in plants

[0134] 4H has antimicrobial activities against different pathogens. For example, Cho et al. (Biosci Biotechnol Biochem. 199862(11 ):2273-6 doi: 10.1271 / bbb.62.2273) showed that 4H isolated from rice hull inhibited growth of a range of pathogenic bacteria and fungi such as Staphylococcus, lactobacillus, E. coli, Salmonella and Saccharomyces cerevisiae. Kosova et al. (J Clin Pharm Ther. 201540(4):436-40. doi: 10.1111 / jcpt.12285 demonstrated antimicrobial activity of 4H against both bacteria and fungi such as Fusarium, Staphylococcus, E. coli and Saccharomyces cerevisiae

[0135] Furthermore, 4H, as a member of the group of 4-hydroxybenzoates, is a natural antioxidant, acting by radical scavenging (Rice-Evans et al., Free Radic Biol Med. 1996;20(7):933-56. doi: 10.1016 / 0891-5849(95)02227-9; Tyrakowska et al., Free Radic Biol Med. 1999 Dec;27(11- 12): 1427-36. doi: 10.1016 / s0891 -5849(99)00192-6). 4H therefore contribute to an improved antioxidant capacity.

[0136] This was confirmed by Winter et al. (Oxid Med Cell Longev. 2017;2017:6297080. doi: 10.1155 / 2017 / 6297080.), who studied the ability of 4H to protect reduce neuroprotective effects by reducing oxidative damage.

[0137] Thus, 4H promotes health, such as immune system health, in particular anti- microbial and antioxidant effects such as the antioxidant capacity, and use of an HMO which increases production of 4H in the gut therefore promotes these beneficial effects in an individual.

[0138] N-acetylglutamine (NAGU)

[0139] N-acetylglutamine (NAGU) is a derivative of the amino acid glutamine, where an acetyl group is attached to the nitrogen atom of the glutamine molecule. This modification enhances its stability and solubility, potentially improving its bioavailability. Arnaud et al Clinical Nutrition (2004) 23, 1303-1312 illustrate that NAGU is converted into glutamine or glutamic acid in the gut of pigs and therefore N-acetyl-L-glutamine is a good candidate for glutamine fortification of enteral nutrition formulas. Glutamine can pass directly through the BBB and act as a central nervous system stimulant. NAGU, through its conversion to glutamine, serves as a crucial precursor for the synthesis of various amino acids, nucleic acids, nucleotides and metabolites including glutamate, the primary excitatory neurotransmitter in the brain (Yelamanchi et al J Cell Commun Signal. 2016 10(1):69-75 doi: 10.1007 / s12079-015-0315-5). Glutamate is the most abundant excitatory neurotransmitter in the central nervous system and is essential for synaptic transmission, learning, and memory (Rahn Current Medicinal Chemistry, 2012, 19, 1335-1345 doi: 10.2174 / 092986712799462649). Glutamine is also a precursor for GABA, the main inhibitory neurotransmitter (Struzynska and Sulkowski Journal of Inorganic Biochemistry 2004 98:951-958). By contributing to the production of both excitatory and inhibitory neurotransmitters (glutamate and GABA), NAGU helps to maintain the balance necessary for optimal cognitive function, including learning and memory. Proper neurotransmitter balance is essential for synaptic plasticity, which underlies learning and long-term memory formation. During brain development, glutamine is involved in the proliferation and differentiation of neural progenitor cells. This process is essential for the formation of a functional neural network.

[0140] Glutamine also contributes to the synthesis of glutathione, one of the brain’s most important antioxidants. Glutathione protects neurons from oxidative stress and free radical damage and therefore provides neuroprotective effects and support an improved antioxidant capacity (Amores-Sanchez and Medina Molecular Genetics and Metabolism 199967:100-105).

[0141] NAGU produced in situ in the gut in the subject, where it can act on its own or be converted to glutamine, and thereby provide for multiple beneficial effects in relation to brain health, including i) improved cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii) improved memory function; iii) neurotransmitter levels, in particular glutamine and GABA and iv) increasing antioxidant levels or antioxidant capacity.

[0142] The increased antioxidant levels will also be beneficial in relation to immune system health.

[0143] In the gastrointestinal tract, glutamine supports the gut barrier function by protecting the intestinal epithelial tight junction integrity as well as gut mucosal integrity (for more information see Rao and Samak, J. Epithel. Biol. Pharmacol., 2012, 5:47-54 doi : 10.2174 / 1875044301205010047).

[0144] NAGU produced in situ in the gut in the subject, where it can act on its own or be converted to glutamine, and thereby provide for multiple beneficial effects in relation to gut health, in particular improving the gut barrier function and motility of the gut.

[0145] It has also been shown that glutamine supplementation can improve insulin sensitivity in obese rats and reduce waist circumference in obese humans (Abboud et al Nutrients 2019, 11 , 536; doi:10.3390 / nu11030536).

[0146] NAGU produced in situ in the gut in the subject, where it can act on its own or be converted to glutamine, and thereby provide for beneficial effects in relation to metabolic health, in particular in relation to weight management.

[0147] Thus, NAGU promotes health, such as brain health, gut health, and / or metabolic health, and use of an HMO which increases production and / or abundance of NAGU in the gut therefore promotes these beneficial effects in an individual.

[0148] N-acetylaspartic acid (NAA)

[0149] N-acetylaspartic acid (NAA) is a derivative of aspartic acid with an acetyl group attached to the nitrogen atom.

[0150] NAA is found in high concentrations in the brain, where it is synthesized in neurons from aspartic acid and acetyl-CoA.

[0151] It plays an important role during early postnatal CNS development, where its cleavage in oligodendrocytes provides acetate for use in the synthesis of fatty acids and steroids, which are then used as components in myelin lipid synthesis. Thus, NAA is involved in postnatal myelination and may also be involved in myelin lipid maintenance later in life (Moffett et al. Prog Neurobiol. 2007 Feb;81(2):89-131. doi: 10.1016 / j.pneurobio.2006.12.003).

[0152] In addition to its role in myelination, NAA has been suggested to be involved in energy metabolism and nitrogen balance in the brain as well as in neuronal osmoregulation and axon- glial signalling (Moffett et al.).

[0153] Several studies have found an association between reduced levels of NAA in specific brain regions and Alzheimer’s disease (reviewed in Moffett et al.). NAA is considered important for brain development, in particular myelination, and is therefore likewise important for cognitive function and memory.

[0154] Thus, NAA promotes health, such as brain health, and use of an HMO which increases production of NAA in the gut therefore promotes these beneficial effects in an individual.

[0155] Thus, in a first aspect, the disclosure provides a non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto-A / - neotetraose (LSTc) and 3’-sialyl-3-fucosyllactose (FSL), or a combination thereof, in supporting or improving health in a subject.

[0156] In some embodiments, the HMO is LSTa.

[0157] In some embodiments, the HMO is LSTc.

[0158] In some embodiments, the HMO is FSL.

[0159] In some embodiments, the combination of HMOs is LSTa and LSTc.

[0160] In some embodiments, the combination of HMOs is LSTa and FSL.

[0161] In some embodiments, the combination of HMOs is LSTc and FSL.

[0162] In some embodiments, the combination of HMOs is LSTa, LSTc and FSL.

[0163] In some embodiments, the health is selected from one or more of brain health, immune system health, gut health, and metabolic health.

[0164] Brain health

[0165] In some embodiments, the brain health is selected from one or more of: i. cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ii. memory function; iii. mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement; iv. increased neurotransmitter levels, such as an increase in aspartate, glutamate, glycine, GABA, serotonin, norepinephrine and / or dopamine synthesis; v. increasing antioxidant levels and / or antioxidant capacity; vi. protection or regeneration of neurons; and vii. sleep quality and regulation.

[0166] Cognitive function refers to a person's thinking, reasoning, problem-solving, and decisionmaking abilities. The development of cognitive function starts from infancy and continues throughout adulthood. Elements of cognitive function in relation to the present disclosure are for example learning ability, memory skills, motor skills, language skills, and / or spatial recognition. From birth to about 2 years old, infants learn about the world through their senses and motor actions. Motor actions or motor skills relate to the coordination of various muscles and body parts to achieve a specific goal. Examples of motor skills include walking, running, jumping, throwing, and catching. Development of spatial recognition starts in infancy and continues into adolescence and is connected to the motor skills. Spatial recognition relates to the ability to perceive, analyse, and understand spatial relationships between objects and the environment. This includes the ability to recognize and remember the location of objects in space, to mentally manipulate objects in space, and to navigate through the environment.

[0167] From about 2 to 7 years old, children develop symbolic thinking, language, and the ability to understand others' perspectives. Language development refers to the process by which children acquire the ability to understand and use language. This process begins in infancy and continues through childhood and adolescence. Language development involves several key milestones, including learning to recognize and differentiate between speech sounds, developing a vocabulary of words, learning grammar and syntax, and developing the ability to use language for communication.

[0168] The ability to learn is developed early in life and continues to develop throughout the lifespan. Infants are born with the ability to learn and are constantly learning about their environment through their senses and experiences. As they grow, their ability to learn becomes more sophisticated and complex, allowing them to acquire new knowledge and skills. Learning can be defined as the process of acquiring new knowledge, skills, behaviours, or attitudes through experience, study, or instruction. It involves a change in behaviour or mental processes that results from experience or practice. Learning can occur through a variety of methods, including observation, trial and error, feedback, and instruction. In addition, there are a number of psychological tests for evaluating cognitive functions in infants and young children such as Agpar score (newborns), Bayley Scales of Infant and Toddler Development (BSID), Cognitive Assessment of Young Children (CAYC).

[0169] Embodiments of the use of the present disclosure may for example be the use of the HMO, the combination of HMOs or the synthetic nutritional compositions disclosed herein to support or improve cognitive development, and / or mood regulation by increasing levels of desired metabolites in the gut which as described above can positively affect pathways, including neurotransmitter formation such as dopamine, serotonin and GABA, that improve brain health including cognitive function, memory function and / or mood regulation.

[0170] Stress and anxiety can have a significant impact on emotional development, as they can interfere with a person's ability to regulate their emotions and form healthy relationships with others. Stress- and anxiety-reduction is improved or supported by an in situ increase in GABA. Negative stress, also known as distress, is a type of stress that is characterized by a feeling of overwhelm, anxiety, or helplessness in response to a perceived threat or challenge. Anxiety is characterized by excessive worry, fear, or apprehension. Stress and anxiety reduction can for example be assessed by monitoring a decrease in for example cortisol and alpha-amylase levels in the saliva of the subject before and after the administration of the composition or kits of part of the present disclosure. In addition, there are a number of psychological tests for anxiety and stress such as various self-reporting questionnaires including, Beck Anxiety Inventory, State-Trait Anxiety Inventory or Perceived Stress Scale. In the context of the present disclosure stress and anxiety are not medical conditions but rather reflect a state of mind which can be eased or relieved.

[0171] Antioxidant capacity refers to the ability of a substance to neutralize or counteract the effects of free radicals and oxidative stress in the body. Free radicals are unstable molecules that can damage cells, proteins, and DNA, contributing to aging and various diseases. Antioxidants are compounds that can donate electrons to free radicals, stabilizing them and preventing further damage. Examples of free radicals are superoxide (O2“), hydroxyl radical (OH*) and nitric oxide (NO*)

[0172] The measurement of antioxidant capacity is often used to evaluate the potential health benefits of foods, supplements, and other substances. Various assays and methods, such as the Oxygen Radical Absorbance Capacity (ORAC) and Ferric Reducing Antioxidant Power (FRAP), are employed to quantify the antioxidant activity of different compounds.

[0173] Antioxidants play a crucial role in maintaining brain health by neutralizing harmful free radicals, which are unstable molecules that can damage cells through oxidative stress. By reducing oxidative damage, antioxidants can support overall cognitive function, including memory, learning, and mental clarity. Oxidative stress is also linked to the development of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease. Antioxidants may help mitigate the progression of these conditions. Securing the presence of antioxidants in the brain is therefore important for cognitive functions as well as in protecting neurons, in particular in adults and elderly subjects. Electroencephalography (EEG) can measure electrical activity in the brain and may provide indirect evidence of changes in neural circuitry associated with neurogenesis. Furthermore, PET scans using radioligands that bind to receptors or proteins associated with neural progenitor cells can provide indirect evidence of neurogenesis. Biomarkers, such as Brain-Derived Neurotrophic Factor (BDNF) and Doublecortin (DCX), which can be measured in the blood or cerebrospinal fluid samples may reflect neurogenesis-related processes in the brain.

[0174] Sleep quality and regulation are vital for brain health, impacting cognitive function, emotional well-being, and physical health. Poor sleep can lead to a range of negative outcomes, including impaired memory, increased stress, and a higher risk of neurodegenerative diseases Components of sleep quality are sleep duration (total amount of sleep), sleep continuity (degree of uninterrupted sleep), sleep depth (proportion of deep sleep (slow-wave sleep) and REM (Rapid Eye Movement) sleep) and sleep latency (time it takes to fall asleep). Improvement of sleep quality is characterized by improvement in any one of the above, but in particular improvement in sleep continuity is desired. Regulation of sleep can for example be achieved by adjusting or influencing the circadian rhythm also known as the body’s internal clock, which regulates the sleep-wake cycle over a 24-hour period. Hormones and neurotransmitters such as melatonin, serotonin, and GABA play crucial roles in initiating and maintaining sleep.

[0175] In some embodiments, the brain health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. pyridoxine (PY); ii. pyridoxamine (PX); iii. nicotinic acid (Nl); iv. N-acetylglycine (NAGY); v. Sarcosine (NMGY); vi. N-acetylglutamine (NAGLI); vii. N-acetylaspartic acid (NAA); viii. N-acetyltryptophan (NAT); ix. trigonelline (TRG); x. 3-hydroxybutyric acid (BHB) and / or xi. 4-hydroxybenzoic acid (4H).

[0176] In some embodiments, the brain health is cognitive function, for example learning ability, motor skills, language skills, and / or spatial recognition, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, Nl, NAA, NAGY, NMGY, NAT BHB, and / or NAGU.

[0177] In some embodiments, the brain health is memory function, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY / PX, Nl, NAA, NAT, NAGY, NMGY, NAGU, BHB and / or TRG.

[0178] In some embodiments, the brain health is mood regulation, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, NAGY, NMGY, NAT, Nl, and / or TRG.

[0179] In some embodiments, the brain health is increased neurotransmitter levels, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, NAT, NAGU NAGY, NMGY, Nl and / or TRG.

[0180] In some embodiments, the brain health is increased antioxidant levels and / or antioxidant capacity, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: Nl, NAGU, NAGY, NMGY, NAT, TRG, BHB and / or 4H. In some embodiments, the brain health is protection or regeneration of neurons, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, NAT, NAGY, NMGY, Nl, BHB and / or TRG.

[0181] In some embodiments, the brain health is sleep quality and regulation of sleep such as regulation of circadian rhythms, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or both metabolites selected from: NAGY, NMGY and / or NAT.

[0182] Immune system health

[0183] In some embodiments, the immune system health is selected from one or more of: i. increasing antioxidant levels and / or antioxidant capacity; ii. stimulating one or more anti-inflammatory cytokines, preferably selected from TGF-pi, IL12, IL22 and IL37; and iii. reducing one or more pro-inflammatory cytokines, preferably selected from TNF-a, TNF-p, IL1 , IL6, IL8, IL11 , IL17, IL18, IFN-a, IFN-p and IFN-y. iv. reducing inflammation.

[0184] A healthy immune system is characterized by its ability to protect the body from harmful pathogens, such as viruses and bacteria, while also recognizing and ignoring harmless substances, such as food and pollen. A healthy immune system is able to distinguish between self and non-self, and it responds appropriately to foreign invaders while leaving the body's own cells and tissues unharmed. Additionally, a healthy immune system is able to adapt and remember previous encounters with pathogens, allowing for a quicker and more effective response upon subsequent exposure.

[0185] In embodiments, a healthy immune system, such as a reduced risk of inflammation or the ability to maintain a non-inflammatory status can for example be assessed by measuring an increased production of anti-inflammatory cytokines and / or a decreased production of pro-inflammatory cytokines compared to the levels prior to administration of the nutritional composition. In further embodiments, the anti-inflammatory cytokines may comprise any one or more of TGF-pi, IL10, IL12, IL22, IL37, and IL38, and the pro-inflammatory cytokines may comprise any one or more of TNF-a, TNF-p, IL1 , IL6, IL8, IL11, IL17, IL18, IFN-a, IFN-p and IFN-y.

[0186] Furthermore, the levels of immunoglobulins can also be measured to determine if the immune system is balanced and therefore likely to be healthy. Balanced levels of immunoglobulins are preferably in the following ranges IgG: 700-1600 mg / dL, IgA: 70-400 mg / dL, IgM: 40-230 mg / dL and IgE: less than 100 lll / mL.

[0187] Antioxidant capacity refers to the ability of a substance to neutralize or counteract the effects of free radicals and oxidative stress in the body. Free radicals are unstable molecules that can damage cells, proteins, and DNA, contributing to aging and various diseases. Antioxidants are compounds that can donate electrons to free radicals, stabilizing them and preventing further damage. Examples of free radicals are superoxide (O2“), hydroxyl radical (OH*) and nitric oxide (NO*)

[0188] Antioxidants also have a positive impact on a healthy immune system. Antioxidants can also contribute to reduced inflammation. Antioxidants can also work as a barrier towards infection and positively contribute to the growth of immune cells such as phagocytes and T-cells.

[0189] In some embodiments, the reduced risk of inflammation is useful in preventing or reducing symptoms of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, and / or ulcerative colitis.

[0190] In some embodiments, the reduced risk of developing allergies is useful in preventing development of allergies such as food allergy and drug allergy.

[0191] In some embodiments, the immune system health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. pyridoxine (PY); ii. pyridoxamine (PX); iii. trigonelline (TRG); iv. 4-hydroxybenzoic acid (4H); v. nicotinic acid (Nl); vi. 3-Hydroxybutyric acid (BHB); vii. N-acetylglycine (NAGY); viii. sarcosine (NMGY); ix. trans-4-hydroxyproline (T4-OHP); and x. N-acetyltryptophan (NAT).

[0192] In some embodiments, the immune system health is increasing antioxidant levels and / or antioxidant capacity, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: Nl, BHB, NAGLI, NAGY, NMGY, NAT, TRG, T4-OHP and / or 4H.

[0193] In some embodiments, the immune system health is preventing or reducing microbial infections, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, NAT, 4H and TRG. PY and PX may for example stimulate cytokine production whereas trigonelline (TRG), 4H and indole derived from NAT contribute to a reduced viability of potential pathogens including viruses, fungi and some bacteria such as Fusarium, Salmonella, Staphylococcus, Streptococcus and Pseudomonas.

[0194] In some embodiments, the immune system health is reducing one or more pro-inflammatory cytokines, leading to the prevention of or reduced inflammation, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, NAGY, NMGY, NAT, BHB, T4-OHP and / or TRG.

[0195] Gut health

[0196] In some embodiments, the gut health is selected from one or both of: i. gut barrier function; and ii. gut motility.

[0197] Gut health refers to the overall health and function of the digestive system, including the stomach, small intestine, large intestine, and colon. A healthy gut is characterized by a balanced and diverse microbiome, the ability to absorb nutrients, regular bowel movements and a strong gut barrier function. In addition, a healthy gut is mostly free of bloating, excessive gas production, abdominal pain, and diarrhoea.

[0198] Gut barrier function refers to the ability of the gut to prevent the leakage of harmful substances, such as toxins, bacteria, and undigested food particles, from the gut lumen into the bloodstream. The gut barrier is composed of several different components, including the gut epithelium, which is the layer of cells that lines the gut, as well as the mucus layer, which acts as a physical barrier to prevent direct contact between the gut epithelium and the gut lumen. The gut barrier also includes the tight junctions between gut epithelial cells, which help to regulate the passage of substances across the gut epithelium. When the gut barrier is compromised, harmful substances can leak into the bloodstream and potentially trigger inflammation and immune responses. Several factors can contribute to the breakdown of the gut barrier, including poor diet, stress, infections, and medications such as nonsteroidal antiinflammatory drugs (NSAIDs).

[0199] The strength of the tight junctions can be assessed by measuring the permeability of the gut lining to certain molecules. This can be done by administering a solution containing a small molecule, such as lactulose or mannitol, and then measuring the concentration of the molecule in the urine. If the gut lining is permeable, more of the molecule will pass through the gut lining and into the bloodstream, resulting in higher levels of the molecule in the urine.

[0200] Gut motility refers to the movements of the digestive system that help in the passage of food. Tryptophan and its metabolites, such as serotonin, play a significant role in regulating gut motility. Serotonin, which is synthesized from tryptophan, is a key neurotransmitter in the gut that influences motility. Gut motility can be measured by several means such as monitoring the passage of for example radioisotopes (scintigraphy or radiopaque marker test), endoscopic techniques, imaging techniques (MRI or CT scans) and breath tests e.g. using lactulose or nonradioactive isotopes.

[0201] In some embodiments, the gut health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. N-acetylglutamine (NAGLI); ii. trans-4-hydroxyproline (T4-OHP); and iii. N-acetyltryptophan (NAT).

[0202] In some embodiments, the gut health is gut barrier function, in particular the tight junctions between gut epithelial cells, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of the metabolite NAGLI, T4-OHP and / or NAT.

[0203] In some embodiments, the gut health is gut motility, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of the metabolite NAT.

[0204] Metabolic health

[0205] In some embodiments, the metabolic health is selected from one or more of: i. bone strength; ii. muscle strength and function, such as grip strength, gait speed and / or muscle mass, optionally prevention of sarcopenia and / or frailty; iii. exercise endurance; iv. coenzyme levels, such as of pyridoxin-5'-phosphate (PNP), pyridoxamine-5'- phosphate (PMP) and / or nicotinamide adenine dinucleotide (NAD+); v. peripheral nervous system, such as protection or regeneration of neurons; vi. anti-aging benefits, such as control of skin aging and / or skin pigmentation; vii. weight management; viii. cardiovascular health, including a healthy lipid profile, with high HDL and low triglycerides; and ix. improving vascular endothelial function, including blood pressure regulation.

[0206] Metabolic health in the context of the present disclosure refers to the optimal functioning of the body's metabolism, which includes all the biochemical processes involved in maintaining life, such as converting food into energy, building, and repairing tissues, and regulating various bodily functions. Good metabolic health is crucial for overall well-being and can reduce the risk of chronic diseases such as diabetes, cardiovascular disease, and stroke. Important tissues are bone and muscle as well as the peripheral nervous system controlling the muscles.

[0207] Cardiovascular health is an important part of the metabolic health and is affected by high blood pressure as well as cholesterol levels. Weight management is important in relation to the risk of developing type diabetes, as well as bone and joint health since overweight add additional stress to the joints. Skin aging is also largely affected by metabolic processes in that it is affected by reduced collagen and elastin production and keratinocyte renewal.

[0208] Bone strength is a measure of the ability of bones to withstand forces and resist fractures. The amount of bone minerals, such as calcium, phosphorous and magnesium involved in forming hydroxyapatite in the bone, expresses the bone density which can measured via Dual X-Ray Absorptiometry (DXA) and Bioelectric Impedance Analysis (BIA). Higher density generally indicates stronger bones. The structural integrity of the bone can also be used as an indication of bone strength which includes the quality of collagen and the presence of other minerals in the bone. Bone integrity can be assessed at the microscopic level. Furthermore, vitamins such as vitamin D facilitate calcium uptake, vitamin B6 is involved in collagen formation, and vitamin B12 is involved in osteoblast production, the cells that are responsible for bone formation. Bone strength is in particular important in the elderly, where reduced bone strength can result in fractures which decrease mobility and life quality. During infancy and early childhood, bones undergo rapid growth and development, laying the foundation for future skeletal health, so here it is also desired to promote bone strength. In particular the metabolites pyridoxine (PY) and pyridoxamine (PX), trigonelline (TRG) and trans-4-hydroxyproline (T4-OHP) have shown relevance in relation to bone strength. Muscle strength also supports and protect the bone.

[0209] Muscle strength refers to the ability of a muscle or group of muscles to exert force against resistance. It is a crucial component of overall physical fitness and plays a significant role in daily activities, athletic performance, and overall health. Muscle strength includes muscle mass and function, such as grip strength and gait speed, which are important parameters both during infancy and childhood development as well as well as in maintaining strength and functionality in adulthood and during agining. Sarcopenia is an age-related progressive loss of muscle mass and strength and frailty. The metabolites identified in the present disclosure have been shown to have particular benefits in relation sarcopenia and are therefore useful in maintaining muscle strength in elderly subjects, in particular in healthy elderly subjects. Muscle strength and balance can be assessed by handgrip muscle strength (HGS) via dynamometer or via functional tests such as push-ups, sit-ups 1RM test and balance testing. Improvements in physical activity can also be measured using Physical Activity Scale for the Elderly (PASE). In particular the metabolites pyridoxine (PY) and pyridoxamine (PX), trigonelline (TRG) and 3-hydroxybutyric acid (BHB) have shown relevance in relation to muscle strength and function.

[0210] Endurance during exercise or exercise capacity is another important parameter when looking at metabolic health. Endurance or exercise capacity is the ability of an individual to sustain physical activity over a period of time. It is often measured by the duration and intensity of exercise that a subject can perform before reaching a point of fatigue. Administration of 3- hydroxybutyric acid BHB has shown benefits in relation to exercise endurance.

[0211] Co-enzymes, such as of pyridoxin-5'-phosphate (PNP), pyridoxamine-5'-phosphate (PMP) (described in relation to pyridoxine (PY) and pyridoxamine (PX)) and / or nicotinamide adenine dinucleotide (NAD+) (described in relation to nicotinic acid (N I)) are important for a number of metabolic functions and are therefore highly relevant for metabolic health.

[0212] The peripheral nervous system (PNS) is a critical component of the overall nervous system, functioning alongside the central nervous system (CNS) to control and coordinate bodily activities. The peripheral nervous system is essential for sensory perception, voluntary and involuntary movements, and reflex actions. It connects the central nervous system to the rest of the body, ensuring proper communication and coordination. Functionality of the PNS can be tested using Electromyography measuring electrical activity in muscles, Nerve Conduction Studies assessing the speed and strength of electrical signals traveling through peripheral nerves, MRI and CT scans to visualize nerve damage / repair.

[0213] Anti-aging benefits are in particular affected by the co-enzyme NAD+ which has been shown to be involved in the control of skin aging including skin pigmentation. Skin agining can for example be assessed by measurements of skin hydration, elasticity, photoaging, skin barrier function (using methods such as Courage&Khazaka Skin Station, VisiaCR imaging system).

[0214] Weight management in the context of the present disclosure relates to the ability to maintain a body mass index (BMI) in the range of 18 to 30, preferably in the range of 18.5 to 25, which is considered normal weight. Prevention of severe overweight (BMI above 30) is important for overall health and well-being, as it helps prevent and manage various health conditions such as obesity, cardiovascular diseases, diabetes, and more. In addition to BMI assessment weight management can also be assessed by waist circumference which should preferably be below 102 cm for male and less than 88 cm for female humans.

[0215] Cardiovascular health refers to the well-being of the heart and blood vessels, which are crucial for maintaining overall health and preventing cardiovascular diseases (CVD). Cardiovascular diseases include conditions such as atherosclerosis, coronary artery disease, hypertension, heart failure, stroke, and peripheral artery disease. Maintaining good cardiovascular health is essential for reducing the risk of these conditions. Cardiovascular health can be assessed by multiple parameters e.g., BMI, waist circumference, blood pressure, fasting lipid profile (total cholesterol, HDL-C and LDL-C, triglycerides, non-HDL-C)), apolipoproteins, blood nitrogen oxide levels, serum insulin, HbA1c, HOMA-IR and changes in lipidomic and polar metabolites profiles (plasma). Preferably, the HDL levels are normal to high which is in the range of 1.0-1.5 mmol / L to above 1.5 mmol / L. Normal triglyceride levels are below 1.7 mmol / L. A normal blood pressure is a systolic pressure between 80 - 120 mm HG and a diastolic pressure between 50 - 80 mm HG. Normal nitrogen oxide levels are for NO320 - 40 microM and for NO2 0.1 - 0.5 microM.

[0216] In some embodiments, the metabolic health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. pyridoxine (PY); ii. pyridoxamine (PX); iii. nicotinic acid (Nl); iv. N-acetylglycine (NAGY); v. sarcosine (NMGY); vi. N-acetylglutamine (NAGLI); vii. 3-hydroxybutyric acid (BHB), viii. trans-4-hydroxyproline (T4-OHP) and ix. trigonelline (TRG).

[0217] In some embodiments, the metabolic health is bone strength, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, T4-OHP and / or TRG.

[0218] In some embodiments, the metabolic health is muscle mass and function, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, BHB, T4-OHP and / or TRG.

[0219] In some embodiments, the metabolic health is exercise endurance, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of BHB.

[0220] In some embodiments, the metabolic health is coenzyme levels, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: PY, PX, and / or Nl.

[0221] In some embodiments, the metabolic health is peripheral nervous system, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or both metabolites selected from: Nl, TRG, PY and / or PX.

[0222] In some embodiments, the metabolic health is anti-aging benefits such a control of skin aging and / or skin pigmentation, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of the metabolite Nl.

[0223] In some embodiments, the metabolic health is weight management, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: Nl, NAGY, NMGY, BHB, TRG, and / or NAGU.

[0224] In some embodiments, the metabolic health is cardiovascular health, including a healthy lipid profile, with high HDL and low triglycerides; and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of the metabolite Nl and / or TRG.

[0225] In some embodiments, the metabolic health is an improved vascular endothelial function, including blood pressure regulation, and it is supported or improved by increasing the level in the gastrointestinal tract of the subject of the metabolite TRG, NMGY and / or NAGY.

[0226] Use of HMOs for supporting healthy development

[0227] The present disclosure relates to one or more methods of providing a health benefit. The health benefit preferably relates to a non-medical / non-therapeutic benefit, but can also relate to treating a condition, disease, or disorder. The method of providing the health benefit is obtained by administering a composition comprising a human milk oligosaccharide (HMO) selected from 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’-sialyl-3- fucosyllactose (FSL), or a combination thereof.

[0228] In some embodiments, the non-therapeutic use of LSTa, LSTc, FSL or a combination thereof comprises supporting or improving the development of one or more of brain health, immune system health, gut health, and metabolic health, optionally wherein the subject is an infant or elderly.

[0229] In some embodiments a composition comprising LSTa, LSTc, FSL or a combination thereof is used for supporting or improving the development of one or more of brain health, immune system health, gut health, and metabolic health in a subject, preferably in an infant or elderly.

[0230] Use of HMOs for producing metabolites

[0231] In a second aspect, the present disclosure provides a non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto-A / - neotetraose (LSTc) and 3’-sialyl-3-fucosyllactose (FSL), or a combination thereof, for increasing the production of one or more metabolites selected from the group consisting of pyridoxine (PY), pyridoxamine (PX), nicotinic acid (Nl), N-acetyl-L-glycine (NAGY), sarcosine (NMGY), N- acetyl-L-glutamine (NAGU), N-acetyl-L-aspartic acid (NAA), trigonelline (TRG), 3-hydroxybutyric acid (BHB), trans-4-hydroxyproline (T4-OHP), 4-hydroxybenzoic acid (4H), and N-acetyl-L- tryptophan (NAT), in the gastrointestinal tract of a subject.

[0232] In some embodiments, the HMO is LSTa, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4-hydroxyproline, and / or N-acetyl-L- tryptophan, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0233] In some embodiments, the HMO is LSTc, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4-hydroxyproline, N-acetyl-L- glutamine, N-acetyl-L-aspartic acid, and / or trigonelline, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0234] In some embodiments, the HMO is FSL, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4-hydroxyproline, N-acetyl-L- glutamine, N-acetyl-L-aspartic acid, and / or 4-hydroxybenzoic acid, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0235] In some embodiments, the combination of HMOs is LSTa and LSTc, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4-hydroxyproline, N-acetyl-L-glutamine, N-acetyl-L-aspartic acid, trigonelline, and / or N- acetyl-L-tryptophan, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0236] In some embodiments, the combination of HMOs is LSTa and FSL, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4- hydroxyproline, N-acetyl-L-glutamine, N-acetyl-L-aspartic acid, 4-hydroxybenzoic acid, and / or N-acetyl-L-tryptophan, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0237] In some embodiments, the combination of HMOs is LSTc and FSL, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4- hydroxyproline, N-acetyl-L-glutamine, N-acetyl-L-aspartic acid, trigonelline, and / or 4- hydroxybenzoic acid, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0238] In some embodiments, the combination of HMOs is LSTa, LSTc and FSL, and the use is for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N-acetyl-L-glycine, sarcosine 3-hydroxybutyric acid, trans-4-hydroxyproline, N-acetyl-L-glutamine, N-acetyl-L-aspartic acid, trigonelline, 4- hydroxybenzoic acid, and / or N-acetyl-L-tryptophan, in the gastrointestinal tract of a subject, thereby supporting or improving health in the subject.

[0239] Formulation and mode of administration

[0240] In some embodiments, the subject is a healthy human, or a human in risk of developing a neurological, metabolic or immune disorder. Preferably, a human at risk has not developed any symptoms. In situations where the risk for example is due to age or lifestyle it is considered non- therapeutic. For example, maintaining or improving cognitive abilities in an elderly person which does not have a neurodegenerative disorder is non-therapeutic.

[0241] The proper dosage of the HMO composition of the present disclosure may be determined, at least in part, based upon factors such immune status, body weight and age. In some cases, the dosage of the HMO disclosed herein will be similar to that found for the specific HMO in human breast milk. The effective amount of HMO or HMO mixture (HMO composition) would generally be in the range from about 1 g to about 20 g per day, in certain embodiments from about 2 g to about 15 g per day, from about 3 g to about 10 g per day, in certain embodiments from about 1 g to about 10 g per day. Appropriate dose regimes can be determined based on the present disclosure and / or on factors known to a person of ordinary skill in the art. In some embodiments, the HMO composition, or combination thereof, is administered to the subject at a daily dosage of 1 g to 20 g, such as 2 g to 15 g, such as 3 g to 10 g.

[0242] The HMO composition may be provided as a powder, a dry composition or a gel comprising the HMO(s). The formulation may be a premix suitable for mixing with other ingredients. The formulation may be a ready to use formulation, such as a unit dosage form, i.e. , a capsule, tablet or sachet / stick pack or a formulation that needs to be dissolved in a liquid prior to use.

[0243] The synthetic nutritional composition

[0244] In some embodiments, the HMO, or combination thereof, is comprised in a synthetic nutritional composition. In further embodiments, the synthetic nutritional composition is an infant formula, growing up formula or milk, dietary supplement or food supplement.

[0245] The nutritional composition may be in the form of a powder, a dry composition or a gel or a liquid.

[0246] In some embodiments, the nutritional composition is a dry composition, such as a powder or granulate, which comprises less than 5 wt%, such as less than 3 wt%, and such as less than 1 wt%, water.

[0247] For dietary supplements the dry composition may be formulated into a tablet, capsule or sachet / stick pack or a gummy or drinkable liquid.

[0248] The nutritional composition can additionally contain or be mixed with sources of protein, lipids, vitamins, minerals and / or digestible carbohydrates. The nutritional composition may additionally comprise one or more ingredients selected from the group consisting of: i. carbohydrate such as lactose and / or Inositol ii. protein, preferably plant-based protein such as algae protein, pea protein or rapeseed protein, a portion of the protein may be hydrolyzed protein; iii. long chain polyunsaturated fatty acids (LCPUFAs), such as docosahexaenoic acid (DHA) and / or arachidonic acid (ARA); iv. probiotics, such as microbial cells from the Bifidobacterium or Lactobacillus genus. v. vitamins, such as one or more of vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, Thiamin (vitamin B1), riboflavin (vitamin B2), nicotinic acid (vitamin B3), vitamin B6, vitamin B12, folic acid (vitamin B9), pantothenic acid (vitamin B5) and / or choline; vi. salts, such as one or more of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, selenium, sodium, potassium, and / or chloride; vii. enzymes, such as one or more of biotin, coenzyme Q10, and / or lactoferrin; and viii. antioxidants, such as one or more of resveratrol, a-carotene, p-carotene, p- cryptoxanthin, lycopene, lutein, zeaxanthin, retinol and / or a-tocopherol.

[0249] The composition can be designed to be the sole source of nutrition or a nutritional supplement. The proper dosage of the nutritional composition of the present disclosure may be determined, at least in part, based upon factors such as immune status, body weight and age. In some cases, the dosage of the mixture of HMOs will be similar to that found for the specific HMOs in human breast milk.

[0250] The proper dose is preferably one that results in a measurable production of the desired metabolite(s) in the gut of the subject, also considered to be an effective dose.

[0251] The required amount of the HMO or mixture of HMOs in the nutritional composition would generally be in the range from about 1 g to about 20 g per day, in certain embodiments from about 2 g to about 15 g per day, from about 3 g to about 10 g per day, in certain embodiments from about 1 g to about 10 g per day.

[0252] Appropriate dose regimes can be determined based on the present disclosure and / or on factors known to a person of ordinary skill in the art.

[0253] It is understood that an amount / dose per day is a dose equivalent to the indicated amount per day, meaning that the daily amount / dose can be combined to a dose every 2nd, 3rd, 4thor 5thday or to a weekly dose, alternatively a daily amount / dose can be split into multiple dosages and administered two, three or four times a day.

[0254] In some embodiments, the HMO, or combination thereof, or synthetic nutritional composition, is administered orally or enterally to the subject.

[0255] Medical use

[0256] In a third aspect, the present disclosure provides a human milk oligosaccharide (HMO) selected from 3’-sialyllacto- / \ / -tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’-sialyl-3- fucosyllactose (FSL), or a combination thereof, for use as a medicament.

[0257] The embodiments of the first and second aspects listed above apply mutatis mutandis to the third aspect.

[0258] In some embodiments, the HMO, the combination of HMOs or the synthetic nutritional composition is for use in treating a disease or pathological state selected from: i. inflammation; ii. depression; iii. osteoporosis; iv. sarcopenia v. headache / migraine; vi. obesity; vii. diabetes; viii. alzheimer’s disease; ix. allergy; x. metabolic syndrome; xi. atherosclerosis, xii. cardiovascular diseases, and xiii. microbial infection, such as viral, bacterial and / or fungal infections.

[0259] For the purposes of the third aspect, the synthetic nutritional composition may be a medical nutrition product.

[0260] EXAMPLE 1

[0261] A kinetic, ex vivo study was implemented, simulating the colonic fermentation of test products by the gut microbiota derived from healthy infants (n = 12). Individual bioreactors were processed in parallel in a bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 ml of nutritional medium with faecal inoculum, dosed with the test product and sealed individually before being rendered anaerobic. After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37 °C for 24 h. Upon gas pressure measurement in the headspace, liquid samples were collected for subsequent analysis. Test products were HMOs (2’-FL, LSTa, LSTc, and FSL) and galactooligosaccharides (GOS), each corresponding to a study arm, at the concentration 5 g / l, simulating intake of 5 g per day.

[0262] 12 samples per study arm were pooled to a single sample and fractioned into pellet and supernatant. The samples were analysed in technical triplicate (n = 3) using the metabolite analysis described below.

[0263] Metabolite analysis

[0264] This analysis aims to identify changes in metabolites of interest, in the different study arms compared to 2’-FL as reference, using an untargeted metabolite screening.

[0265] The metabolites in the supernatant samples were identified with LC-MS by retention times and accurate mass.

[0266] The LC-MS analysis was carried out using a Thermo Scientific Vanquish LC coupled to Thermo Q Exactive HF MS. An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed using a slightly modified version of the protocol described by Doneanu et al.

[0267] (https: / / www.waters.com / webassets / cms / library / docs / 720004042en.pdf; accessed on 30 August 2024). Peak areas were extracted using Compound Discoverer 3.1 (Thermo Scientific). In addition to the automatic compound extraction by Compound Discoverer 3.1 , a manual extraction of compounds included in an in-house library was performed using Skyline 21.1 (MacCoss Lab Software).

[0268] Figure 1 depicts the Area Under the Curve (AUC) values determined in each arm. AUC is a measure of the concentration of the compound it represents. This area value was integrated based on the peak of the metabolite in the chromatograms of the LC-MS analysis. The bold circle is the mean value of the triplicates in each measurement.

[0269] Results

[0270] Figure 1 A to I depicts the results from the metabolite analysis, showing that at least one of the test HMOs (FSL, LSTa and LSTc) increased the abundance of the metabolite when added to the microbiota of the pooled faecal samples. In most cases the HMOs also showed an improved effect over the conventional prebiotic compound, GOS.

[0271] Table 1 summarizes what metabolites were increased by which HMO. The plus-signs indicate the relative difference between the HMOs, and do not necessarily reflect the AUG measured for each metabolite; those can be seen in Figure 1.

[0272] Table 1: Overview of HMOs that increase levels of the indicated metabolite compared to 2’-FL

[0273] From this table it can be seen that FSL, LSTa and LSTc all were capable of increasing the metabolites pyridoxine, pyridoxamine, nicotinic acid and N-acetylglycine. It can therefore be concluded that the three HMOs all have potential to show benefits within immunity, brain health and metabolism. Furthermore, FSL and LSTc increased the metabolites N-acetylglutamine and N-acetylaspartic acid, which are both relevant for brain health. Finally, FSL increased 4- hydroxybenzoic acid which is beneficial for immunity, LSTa increased N-acetyltryptophan which is beneficial for brain health, and LST increases trigonelline which is beneficial for immunity.

[0274] EXAMPLE 2

[0275] Effects of a formulation containing LSTa, LSTc or FSL in gut health, in cognitive, emotional and behavioral development, and in overall health (immune system development and antiinflammatory support) in infants up to 24 months of age

[0276] Study design: randomized, double-blind, parallel, placebo-controlled trial.

[0277] Phase: II

[0278] Sample size: 160 participants

[0279] 1 . 40 participants on “2 g per day of HMO formulation LSTa”,

[0280] 2. 40 participants on “2 g per day of HMO formulation LSTc” 3. 40 participants on “2 g per day of HMO formulation FSL”

[0281] 4. 40 participants on the placebo (i.e. , base formula feeding).

[0282] Study length: 24 months.

[0283] Visits: Baseline (Birth), 1 month, 3 months, 6 months, 12 months, 18 months, and 24 months

[0284] Biological samples collected:

[0285] Mother: stool, blood, saliva, vaginal swab, nasal cavity, breastmilk.

[0286] Infant: umbilical cord blood, stool, saliva, blood (finger or heel prick), urine, nasal cavity.

[0287] Population (main inclusion criteria):

[0288] Healthy pregnant mothers and their healthy newborn;

[0289] Singleton pregnancy;

[0290] Birth weight of 2500g or more;

[0291] Gestational age 37-42 weeks (i.e., term pregnancy);

[0292] - Absence of diagnosed genetic disease or malformations during pregnancy.

[0293] Population (main exclusion criteria):

[0294] Obstetric or fetal complications known for current pregnancy such as preterm labor (premature rupture of membranes or preterm labor), chorioamnionitis, placenta previa, active vaginal bleeding, cervical cerclage, fetal distress or anomalies, corticosteroid therapy;

[0295] Risk factors for obstetric complications during current pregnancy such as gestational diabetes, type I / II diabetes and high blood pressure (systole >140 and diastole >90); Chorioamnionitis, pre-eclampsia or hepatogestosis;

[0296] Suspected malformation or serious condition of the fetus / neonate.

[0297] Study Description:

[0298] It is hypothesized that the HMO formulation, consumed over a 12-month period, is able to increase the levels of one or more of the following metabolites i. pyridoxine (PY); ii. pyridoxamine (PX); iii. nicotinic acid (Nl); iv. N-acetylglycine (NAGY); v. sarcosine (NMGY); vi. N-acetylglutamine (NAGLI); vii. N-acetylaspartic acid (NAA); viii. N-acetyltryptophan (NAT); ix. Trigonelline (TRG); x. 3-hydroxybutyric acid (BHB); xi. trans-4-hydroxyproline (T4-OHP) and xii. 4-hydroxybenzoic acid (4H) in the gut of a subject, with beneficial effects for the subject’s health, when compared to a nonadministered subject.

[0299] Study objectives:

[0300] Primary objective

[0301] To measure the levels of the indicated metabolites in stools of infant participants, measured by: Change from baseline in stool metabolite levels as measured by LC / MSMS.

[0302] Hypothesis: The study participants who consume the HMO formulation have greater mean increase change in metabolite levels from baseline to 24 months of follow up than the study participants on the placebo.

[0303] Secondary objectives

[0304] Changes in inflammation and oxidative markers from blood and urine samples

[0305] Including, but not limited to, anti-inflammatory cytokines TGF-pi, IL10, IL12, IL22, IL37, and IL38, and pro-inflammatory cytokines TNF-a, TNF- , IL1 , IL6, IL8, IL11 , IL17, IL18, IFN-a, IFN-p and IFN-y, Malondialdehyde (MDA), Glutathione (GSH), 8-Hydroxydeoxyguanosine (8-OHdG), Tryptophan, Kynurenine, Total antioxidant capacity, C-reactive protein (hsCRP)

[0306] Hypothesis'. The study participants who consume HMO formulation have a reduced mean concentration in inflammatory and / or oxidative markers after 24 months of follow up than the study participants on the placebo.

[0307] Reduction in the incidence of colics

[0308] Comparison of the incidence of colics or inconsolable crying episodes between the intervention group and placebo group.

[0309] Hypothesis: There is less incidence of colics or inconsolable crying episodes in the HMO groups than in the placebo group after 3 months of intervention.

[0310] Prevention of infections or allergic diseases

[0311] Comparison of rate / occurrence of infections or allergic diseases diagnosed during intervention (cold, flu, eczema, atopic dermatitis, diarrhea or gastroenteritis, food allergies, inflammatory bowel diseases, etc.)

[0312] Hypothesis'. There is, on average, less number of days of infections and less occurrence of infections and / or allergic diseases and / or inflammatory bowel diseases diagnosed in the HMO groups than in the placebo group after 24 months of follow up.

[0313] Gastrointestinal Health Changes in gastrointestinal health and associated quality of life throughout intervention using the capture of frequency (mean daily number of stools) and consistency of bowel movement (Amsterdam infant stool scale)

[0314] Fecal inflammatory mediators: gastrointestinal function through the measurement of fecal inflammatory mediators

[0315] Fecal gut barrier function: gastrointestinal function through the measurement of Gl barrier function markers.

[0316] Hypothesis’. Participants in the HMO group report, on average, less gastrointestinal symptoms (e.g., constipation, diarrhea) and better nutrients absorption and a better barrier function than participants in the placebo group, after 24 months of follow up.

[0317] Cognition and mental health

[0318] Changes in cognitive status throughout intervention assessed by: Agpar score (newborns), Bayley Scales of Infant and Toddler Development (BSID), Cognitive Assessment of Young Children (CAYC)

[0319] Changes in mental health state including stress, sleep quality and anxiety assessed by: Mother’s daily / weekly diary, and Biological markers (e.g., salivary slgA, alpha-amylase levels, cortisol, melatonin, gut neurotransmitters (e.g., levels of GABA, dopamine, serotonin in feces).

[0320] Hypothesis’. Participants in the HMO group have a better cognitive development than participants in the placebo group, after 24 months of follow up, when controlling fortheir baseline values.

[0321] Muscle Health and Growth Rate

[0322] Changes in muscle strength assessed by handgrip muscle strength (HGS) via dynamometer

[0323] - Antropometric measurements

[0324] Body composition (PEA POD)

[0325] Hypothesis’. Participants in the HMO group have, on average, better muscle strength and a normal growth rate and body composition compared to participants in the placebo group, after 24 months of follow up.

[0326] Exploratory

[0327] - Infant fecal bacteria oligosaccharide consumption and infant fecal sialic acid concentrations

[0328] Hypothesis’. Participants in the HMO group have an improved and selective consumption of HMO’s and higher levels of sialic acid concentration than participants in the placebo group, after 24 months of follow up, when controlling fortheir baseline values.

[0329] Safety objectives To assess the safety and tolerability of a synbiotic formulation in the study population, measured by:

[0330] Growth rate and anthropometric measurements

[0331] Changes (outside normal variation) in clinical hematology markers (e.g., Complete Blood Count (CBC) including hemoglobin, hematocrit, red blood cell indices, platelet count, leukocyte count and differential elicited after baseline and throughout intervention

[0332] Changes (outside normal variation) in clinical chemistry markers (Comprehensive Metabolic Panel (CMP) including glucose, calcium, albumin, protein, sodium, potassium, bicarbonate, chloride, blood urea nitrogen, creatinine, alkaline phosphatase, alanine amino transferase, aspartate amino transferase and bilirubin) elicited after baseline and throughout intervention

[0333] - Adverse events (AEs) and Serious adverse events (SAEs) until study completion: Incidence of Adverse Events and treatments gastrointestinal symptoms and related symptoms (diarrhea, vomiting, constipation, colic, irritability) after synbiotic supplementation will be determined and reported. General health status of the infant such as occurrence of any illness, health care visits for sickness, fever, antibiotic and medication use and parental assessments of infant's overall health will also be documented.

[0334] We do not expect a significant difference for AEs / SAEs or product tolerance between the HMO groups and placebo group.

[0335] Hypothesis’. Participants in the HMO groups have, on average, a normal growth rate and less values outside normal variation for markers associated with organ failure / tissue degeneration than participants in the placebo group.

[0336] EXAMPLE 3

[0337] Effects of a formulation containing LSTa, LSTc or FSL in healthy aging

[0338] Study design: randomized, double-blind, parallel, placebo-controlled trial

[0339] Phase: II

[0340] Sample size: 160 participants:

[0341] 1 . 40 participants on “5 g per day of HMO formulation LSTa”,

[0342] 2. 40 participants on “5 g per day of HMO formulation LSTc”

[0343] 3. 40 participants on “5 g per day of HMO formulation FSL”

[0344] 4. 40 participants on the placebo (i.e. , maltodextrin or dextrose).

[0345] Study length: 18 months

[0346] Visits: Baseline, 1 month, 3 months, 6 months, 12 months and 18 months

[0347] Biological samples collected: stool, saliva, blood, urine. Potential biopsy on a subgroup of participants (20) Population (main inclusion criteria):

[0348] Female and Male

[0349] 65 to 80 years old (inclusive)

[0350] BMI between 18.5 and 29.9 (inclusive)

[0351] Study Description:

[0352] It is hypothesized that the HMO formulation, consumed over an 18-month period, increases levels of one or more of the following metabolites i. pyridoxine (PY); ii. pyridoxamine (PX); iii. nicotinic acid (Nl); iv. N-acetylglycine (NAGY); v. sarcosine (NMGY); vi. N-acetylglutamine (NAGLI); vii. N-acetylaspartic acid (NAA); viii. N-acetyltryptophan (NAT); ix. Trigonelline (TRG); x. 3-hydroxybutyric acid (BHB); xi. trans-4-hydroxyproline (T4-OHP) and xii. 4-hydroxybenzoic acid (4H)

[0353] Additionally, it is hypothesized that the increase in one or more of the metabolite levels is associated with an overall maintenance of wellbeing, promotes a healthy aging, and prevents muscle, bone, visual and cognitive degeneration.

[0354] Study objectives:

[0355] Primary objective

[0356] To measure the increase of levels of the listed metabolites in fasces, blood and / or brain of elderly participants, measured by:

[0357] Change from baseline in metabolite levels in fasces

[0358] Change from baseline in in metabolite levels in serum

[0359] Change from baseline in brain NAD+, dopamine, serotonin and / or GABA levels as measured by31P MRI

[0360] Hypothesis’. The study participants who consume the HMO formulation have greater mean increase change in metabolite levels from baseline to 18 months of intervention than the study participants on the placebo.

[0361] Secondary objective

[0362] Changes in inflammation and oxidative markers from blood samples Including, but not limited to, anti-inflammatory cytokines TGF-pi , IL10, IL12, IL22, IL37, and IL38, and pro-inflammatory cytokines TNF-a, TNF- , IL1 , IL6, IL8, IL11 , IL17, IL18, IFN-a, IFN-p and IFN-y, Malondialdehyde (MDA), Glutathione (GSH), 8-Hydroxydeoxyguanosine (8-OHdG), Tryptophan, Kynurenine, Total antioxidant capacity, C-reactive protein (hsCRP) Determination of CD38 activity (nicotinamide dinucleotide (NAD+) catabolic enzyme).

[0363] Hypothesis’. The study participants who consume HMO formulation have less mean concentration in inflammatory markers after 18 months of intervention than the study participants on the placebo.

[0364] Gastrointestinal Health

[0365] Changes in gastrointestinal health and associated quality of life throughout intervention using Gastrointestinal Symptom Rating Scale (GSRS) and Digestive Association Quality of Life (DQLQ), capture of frequency and consistency of bowel movement (Bristol Stool Scale (BSS))

[0366] Hypothesis’. Participants in the HMO groups report, on average, better quality of life and less gastrointestinal symptoms (e.g., constipation) than participants in the placebo group, after 18 months of intervention.

[0367] Cognition and mental health

[0368] Changes in cognitive status throughout intervention assessed by:

[0369] Questionnaires (e.g., Mini-Mental State Exam (MMSE), Clock Drawing Test (CDT), etc.) Changes in mental health state including stress, sleep quality and anxiety assessed by: Questionnaires (e.g., Patient Health Questionnaire-9 (PHQ-9), General Anxiety Disorder-7 (GAD-7), Insomnia Severity Index (ISI), State-Trait Anxiety Inventory (STAI), Profile of Mood States (POMS) or Pittsburgh Sleep Quality Index (PSQI))

[0370] Biological markers (e.g., cortisol, melatonin and salivary secretory IgA in saliva)

[0371] Actigraphy (sleep patterns)

[0372] Magnetic Resonance Imaging (MRI) to assess changes in brain structure and function throughout intervention

[0373] Tests (e.g., CNS Vital Signs (CNSVS) or Electroencephalography (EEG) to capture brain spontaneous activity at rest (with two conditions of eyes open and closed), hedonic function, and go / no-go tasks.

[0374] Hypothesis’. Participants in the HMO groups have beneficial mean, a better protection against cognition degeneration and mental health degradation than participants in the placebo group, after 18 months of intervention, when controlling fortheir baseline values.

[0375] Muscle and Bone Health

[0376] Changes in muscle strength and balance assessed by handgrip muscle strength (HGS) via dynamometer and balance testing Changes in bone density and body composition via Dual X-Ray Absorptiometry (DXA) and Bioelectric Impedance Analysis (BIA)

[0377] Changes in concentration of Vitamin D - 25(OH)D3 from serum samples

[0378] Measure of physical activity using Physical Activity Scale for the Elderly (PASE)

[0379] Hypothesis’. Participants in the HMO groups have, on average, better muscle strength and balance along with a greater bone density than participants in the placebo group, after 18 months of intervention.

[0380] Exercise endurance

[0381] Changes in time-to-exhaustion assessed submaximal treadmill test (VChmax)

[0382] Hypothesis’. Participants in the HMO groups have, on average, a longer endurance time during submaximal treadmill exercise and a better recuperation than participants in the placebo group, after 18 months of intervention.

[0383] Cardiovascular health

[0384] Changes in cardiometabolic parameters (e.g., BMI, waist circumference, fasting lipid profile (total cholesterol, HDL-C and LDL-C, triglycerides, non-HDL-C)), apolipoproteins, serum insulin, HbA1c, HOMA-IR)

[0385] Changes in lipidomic and polar metabolites profiles (plasma)

[0386] Hypothesis’. Participants in the HMO groups have beneficial mean and a better protection against metabolic degeneration than participants in the placebo group, after 18 months of intervention, when controlling for their baseline values.

[0387] Skin Health

[0388] Changes in skin health throughout intervention assessed by:

[0389] Objective measurements of skin hydration, elasticity, photoaging, skin barrier function (using methods such as Courage&Khazaka Skin Station, VisiaCR imaging system) Subjective assessment via self-evaluation questionnaires.

[0390] Hypothesis’. Participants in the HMO groups have a better protection against signs of skin aging and a better skin condition (hydration, elasticity), on average, than participants in the placebo group, after 18 months of intervention, when controlling fortheir baseline values.

[0391] Immunity

[0392] Compare the number of bacterial and viral infections throughout intervention

[0393] Hypothesis: Participants in the HMO groups will have a lower number of self-reported sick days and a shorter duration ofcold / flu episode, on average, than participants in the placebo group, after 18 months of intervention.

[0394] Safety objectives To assess the safety and tolerability of a synbiotic formulation in older populations, measured by:

[0395] Changes (outside normal variation) in clinical hematology markers (e.g., Complete Blood Count (CBC) including hemoglobin, hematocrit, red blood cell indices, platelet count, leukocyte count and differential elicited after baseline and throughout intervention Changes (outside normal variation) in clinical chemistry markers (Comprehensive Metabolic Panel (CMP) including glucose, calcium, albumin, protein, sodium, potassium, bicarbonate, chloride, blood urea nitrogen, creatinine, alkaline phosphatase, alanine amino transferase, aspartate amino transferase and bilirubin) elicited after baseline and throughout intervention

[0396] - Adverse events (AEs) and Serious adverse events (SAEs) until study completion

[0397] We do not expect a significant difference for AEs / SAEs or product tolerance between the HMO groups and placebo groups.

[0398] Participants in the HMO groups have, on average, less values outside normal variation for markers associated with organ failure / tissue degeneration than participants in the placebo group.

[0399] Example 4

[0400] An ex vivo study very similar to the one conducted in Example 1 was independently performed, simulating the colonic fermentation of test products by the gut microbiota derived from healthy infants (n = 6) (different from those used in example 1). Individual bioreactors were processed in parallel. Each bioreactor was handled under anaerobe conditions throughout the preparations and fermentation. Each bioreactor contained 5 ml of anaerobic nutritional medium with faecal inoculum, dosed with the test product and sealed individually before incubation at 37 °C for 24 h (no agitation). Test products were HMOs (2’-FL, LSTa, LSTc, and FSL) and galactooligosaccharides (GOS), each corresponding to a study arm, at the concentration 5 g / l.

[0401] After incubation, the 6 samples were centrifuged and fractioned into pellet and supernatant. The 6 sample supernatants were analysed using the metabolite analysis described below.

[0402] Metabolite analysis

[0403] The metabolites in the supernatant samples were quantified using a targeted metabolomics approach based on the Biocrates MxP® Quant 500 kit (Biocrates Life Sciences AG, Innsbruck, Austria) with a coverage of up to 630 metabolites from 26 biochemical classes. Samples were processed according to the manufacturer’s instructions. Analysis of sample extracts as well as of reference standards and quality controls was carried out by LIHPLC and flow injection analysis, both coupled to tandem mass spectrometry. An Agilent 1290 series LIHPLC system coupled to a 6500 + QTrap mass spectrometer equipped with an Ion-Drive Turbo V® ESI source (both Sciex, Foster City, CA, USA) was used for the analysis. Chromatographic and mass spectrometric parameters were set as indicated by the manufacturer of the kit. Data analysis was carried out using the Biocrates WeblDQ software.

[0404] Results

[0405] From the targeted metabolomics analysis three metabolites, sarcosine (NMGY), trans-4- hydroxyproline (T4-OHP) and 3-hydroxybutyric acid (BHB), were identified as having a higher increase in abundance when the test product was LSTa, LSTc or FSL as compared to the references HMO 2’FL and the conventional prebiotic compound GOS.

[0406] Figure 2 A to C depicts the concentration of the individual metabolite (ng / mL) determined in each arm quantified by the BioCrates MxP® Quant 500 method. From the figures it can be seen that all the test HMOs (FSL, LSTa and LSTc) increased the abundance of the metabolite when added to the microbiota of the pooled faecal samples more than bot 2’FL and GOS.

[0407] The effects of the individual target HMOs are summarized in table 2 below, indicating what metabolites are increased by which HMO. The plus-signs indicate the relative difference between the HMOs and do not necessarily reflect the concentrations measured for each metabolite; those can be seen in Figure 2A-C.

[0408] Table 2: Overview of HMOs that increase levels of the indicated metabolite compared to 2’-FL

[0409] From this table it can be seen that FSL, LSTa and LSTc were all capable of increasing the metabolites sarcosine (NMGY), trans-4-hydroxyproline (T4-OHP), and 3-hydroxybutyric acid (BHB). It can therefore be concluded that the three HMOs can all show benefits within cognition, digestive health, and immunity.

Claims

CLAIMS1. A non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3’-sialyllacto-A / - tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’-sialyl-3-fucosyllactose (FSL), or a combination thereof, in supporting or improving one or more of brain health, immune system health, gut health and / or metabolic health, in a subject.

2. The non-therapeutic use according to claim 1 , wherein the brain health is related to supporting one or more of: viii. cognitive function, such as motor skills, learning ability, language skills, and / or spatial recognition; ix. memory function; x. mood regulation, such as reduced stress, reduced anxiety, and / or mood enhancement; xi. increasing neurotransmitter levels, such as an increase in aspartate, glutamate, glycine, GABA, serotonin, norepinephrine and / or dopamine levels or synthesis; xii. improved antioxidant capacity / levels; xiii. protection of neurons; and xiv. sleep quality and regulation.

3. The non-therapeutic use according to claim 1 or 2, wherein the brain health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. pyridoxine (PY); ii. pyridoxamine (PX); iii. nicotinic acid (Nl); iv. N-acetylglycine (NAGY); v. sarcosine (NMGY); vi. N-acetylglutamine (NAGLI); vii. N-acetylaspartic acid (NAA); viii. N-acetyltryptophan (NAT); ix. 3-hydroxybutyric acid (BHB); x. trigonelline (TRG); and xi. 4-hydroxybenzoic acid (4H).

4. The non-therapeutic use according to claim 1 , wherein the immune system health is selected from one or more of: i. increasing antioxidant levels; ii. stimulating one or more anti-inflammatory cytokines; andiii. reducing one or more pro-inflammatory cytokines.

5. The non-therapeutic use according to claim 1 or 4, wherein the immune system health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. pyridoxine(PY); ii. pyridoxamine (PX); iii. trigonelline (TRG); iv. 4-hydroxybenzoic acid (4H); v. nicotinic acid; (Nl); vi. N-acetylglycine (NAGY); vii. sarcosine (NMGY); viii. N-acetylglutamine (NAGLI); ix. trans-4-hydroxyproline (T4-OHP); x. 3-hydroxybutyric acid (BHB), and xi. N-acetyltryptophan (NAT).

6. The non-therapeutic use according to claim 1 , wherein the gut health is selected from one or both of: i. gut barrier function; and ii. gut motility.

7. The non-therapeutic use according to claim 1 or 6, wherein the gut health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. N-acetylglutamine (NAGLI); ii. trans-4-hydroxyproline (T4-OHP); and iii. N-acetyltryptophan (NAT).

8. The non-therapeutic use according to claim 1 , wherein the metabolic health is selected from one or more of: i. bone strength; ii. muscle strength and function, such as grip strength, gait speed and / or muscle mass; iii. exercise endurance; iv. coenzyme levels, such as of pyridoxin-5'-phosphate (PNP), pyridoxamine-5'- phosphate (PMP), and / or nicotinamide adenine dinucleotide (NAD+); v. peripheral nervous system, such as protection of neurons;vi. anti-aging benefits, such as control of skin aging and / or skin pigmentation; vii. weight management; viii. cardiovascular health, including a healthy cholesterol profile, with high HDL; and ix. vascular endothelial function, including blood pressure regulation.

9. The non-therapeutic use according to claim 1 or 8, wherein the metabolic health is supported or improved by increasing the level in the gastrointestinal tract of the subject of one or more metabolites selected from: i. pyridoxine (PY); ii. pyridoxamine (PX); iii. nicotinic acid (Nl); iv. N-acetylglycine (NAGY); v. sarcosine (NMGY); vi. N-acetylglutamine (NAGLI); vii. trans-4-hydroxyproline (T4-OHP); viii. 3-hydroxybutyric acid (BHB), and ix. Trigonelline (TRG).

10. The non-therapeutic use according any one of the preceding claims, wherein the subject is a healthy human, preferably an infant or elderly.

11. A non-therapeutic use of a human milk oligosaccharide (HMO) selected from 3’- sialyllacto-ZV-tetraose (LSTa), 6’-sialyllacto- / \ / -neotetraose (LSTc) and 3’-sialyl-3- fucosyllactose (FSL), or a combination thereof, for increasing the production of one or more metabolites selected from the group consisting of pyridoxine, pyridoxamine, nicotinic acid, N- acetyl-L-glycine, sarcosine, N-acetyl-L-glutamine, N-acetyl-L-aspartic acid, trigonelline, trans- 4-hydroxyproline, 3-hydroxybutyric acid (BHB), 4-hydroxybenzoic acid, and N-acetyl-L- tryptophan, in the gastrointestinal tract of a subject.

12. The non-therapeutic use according to any one of the preceding claims, wherein the HMO, or combination thereof, is administered to the subject at a daily dosage of 1 g to 20 g, such as 2 g to 15 g, such as 3 g to 10 g.

13. The non-therapeutic use according to any one of the preceding claims, wherein the HMO, or combination thereof, is comprised in a synthetic nutritional composition.

14. The non-therapeutic use according to claim 13, wherein the synthetic nutritional composition is an infant formula or a dietary supplement.

15. The non-therapeutic use according to any one of the preceding claims, wherein the HMO, or combination thereof, or synthetic nutritional composition, is administered orally or enterally to the subject.

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