Nutritional composition comprising bifidobacterium longum SSP. infantis r0033 and human milk oligosaccharides

Abstract

The invention relates to a nutritional composition comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and the mixture of HMOs comprises or consists essentially of 2'-fucosyllactose (2'-FL) and at least one additional HMO selected from an additional fucosylated HMO, a neutral core HMO, and / or a sialylated HMO. The invention further relates to the nutritional composition for use in supporting or improving gut health and / or a healthy immune system and / or cognitive development, such as motor skills, learning, language skills, spatial cognition ability, in a subject.

Description

[0001] NUTRITIONAL COMPOSITION COMPRISING BIFIDOBACTERIUM LONGUM SSP. INFANTIS R0033 AND HUMAN MILK OLIGOSACCHARIDES

[0002] FIELD

[0003] The present invention relates to the field of prebiotics and probiotics. In particular, the present invention relates to a nutritional composition comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I- 3424, and the mixture of HMOs comprises or consists essentially of 2’-fucosyl lactose (2’-FL) and at least one additional HMO selected from an additional fucosylated HMO, a neutral core HMO, and / or a sialylated HMO. The invention also relates to the nutritional composition for use in supporting or improving gut health, a healthy immune system and / or the development of cognitive functions in a subject.

[0004] DEPOSIT

[0005] Bifidobacterium longum ssp. infantis R0033 has been deposited at Collection Nationale de Cultures de Microorganismes (CNCM; 25 Rue du Docteur Roux F-75724 Paris Cedex 15, France), according to the Budapest Treaty on May 12, 2005 under the accession number CNCM I-3424.

[0006] BACKGROUND

[0007] 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.

[0008] GOS is a commonly used dietary fiber in early life nutrition and a large number of bacteria in the microbiota can grow on GOS, including a number of harmful bacteria such as Escherichia coli, Clostridium difficile, Shigella flexneri, Shigella dysenteriae and Salmonella strains. Contrary to GOS, HMOs are only able to feed certain beneficial bifidobacteria, such as for example Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, as well as Bacteroides fragilis, Bacteroides vulgatus, and Bacteroides thetaiotaomicron. The bacterial selectivity in terms of HMO metabolization, such as metabolization of fucosy I lactose and sialyllactose, can be advantageous in promoting beneficial microbes without supporting the growth of harmful pathogens in contrast to other less selective prebiotics, such as GOS and FOS (Salli et al., 2021 , J. Agric. Food Chem., 69:170-182). Garrido et al., 2015, Scientific Reports, 5:13517 describes the ability of different Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum strains to grow on different carbon sources.

[0009] Human milk oligosaccharides (HMOs) are a heterogeneous mixture of soluble glycans found in human milk, with more than 200 different structures identified in human milk to date. They are the third most abundant solid component after lactose and lipids in human milk with concentrations of 5-25 g / l. Certain HMOs are believed to be important for the development of the infant gut microbiota, in particular by favouring the predominance of bifidobacteria. This is viewed as beneficial for infants because some strains of Bifidobacterium species may have a positive effect on gut health.

[0010] Combinations of prebiotics and probiotics refer to as synbiotics or synbiotic compositions. Such synbiotics comprising Bifidobacterium spp. and specific HMOs have been described, primarily in the context of supplements for infant formula or for promoting certain Bifidobacteria spp. In gastrointestinal tract of infants, but also for preventing or treating disease in adults. Disclosures of various Bifidobacterium infantis strains that have been tested in combination with different prebiotics, such as HMOs, are listed below.

[0011] W02009 / 077352 describes the prevention of opportunistic infections in immune-compromised infants or young children by combining various Bifidobacterium spp. with a fucosylated oligosaccharide.

[0012] WO2022 / 161865 discloses a combination of bifidobacteria and a mixture of HMOs consisting of 2’-FL, DFL, LNT, 6’-SL, and 3’-SL, and its use in prevention or treatment of infections or allergy in infants or young children.

[0013] WO2023 / 098541 discloses a combination of Bifidobacterium infantis strains, in particular the specific strain deposited with CGMCC No. 21109, and HMOs to increase the resilience against S. aureus infection.

[0014] ON 113796545 describes an increase in short chain fatty acids (SCFAs) and a decrease in gas production in infants using a combination of 2’-FL and 3’-SL in a ratio of 7:1 with the probiotics Lactobacillus helveticus R0052, Bifidobacterium infantis R0033 or Bifidobacterium bifidum R0071.

[0015] WO2021 / 217803 describes reducing intestinal gas production in infants and young children using a combination of an HMO and a probiotic Bifidobacterium such as Bifidobacterium infantis R33, Bifidobacterium bifidum R71 and / or Bifidobacterium breve M-16V. CN116439377 discloses a number of probiotic species in combination with 2’-FL to alleviate the dysbiosis caused by early antibiotic exposure in infants.

[0016] CN 117337992 discloses a combination of Bifidobacterium infantis R0033 or Bifidobacterium bifidum R0071 and 2’-FL to decrease Salmonella in the gut.

[0017] OBJECTIVE

[0018] An objective of the present disclosure is to provide a nutritional composition comprising a synbiotic combination with Bifidobacterium longum ssp. infantis R0033 and mixtures of HMOs, such synbiotic combination being able to increase short chain fatty acids (SCFAs), lactic acid, putrescine, gamma-aminobutyric acid (GABA), and / or dopamine levels in the gut of a subject, therefore providing beneficial effects for the subject’s health. It is also an object to provide the nutritional composition for use in supporting or improving gut health, a healthy immune system and / or the development of cognitive functions in a subject.

[0019] SUMMARY

[0020] The present disclosure relates to a synbiotic combination, of Bifidobacterium longum ssp. infantis R0033 or closely related strains and mixtures of HMOs, which is capable of increasing the levels of short chain fatty acids (SCFAs), lactic acid, putrescine, gamma-aminobutyric acid (GABA), and / or dopamine in the gut of a subject upon administration of the synbiotic combination.

[0021] As will be described in detail below, SCFAs, lactic acid, and putrescine have been shown to support a healthy gut microbiome and have various beneficial effects on gut health, including antiinflammatory effects and positive effects on gut barrier function, while GABA and dopamine are regulators of neuronal activity which, inter alia, support the development of cognitive functions as well as emotional and behavioral development, such as stress and anxiety reduction. The invention provides for in situ production of these molecules in the gut, thereby providing a prolonged and naturally controlled production. As such, the synbiotic combination according to the invention may be useful in supporting or improving gut health and reducing symptoms of inflammatory diseases of the gut, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, and ulcerative colitis. Likewise, the synbiotic combination according to the invention may be useful in supporting or improving the development of cognitive functions, such as motor skills, learning, language skills, and / or spatial cognition ability.

[0022] A first aspect of the present invention relates to a nutritional composition comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO, wherein, if 3’-sialyllactose (3’-SL) is present in the mixture, the ratio (w / w) of 2’-FL:3’- SL is above 7.5:1.

[0023] In a second aspect, the present invention relates to the nutritional composition according to the first aspect of the invention or any embodiments thereof, for use in supporting or improving one or more of the following: a) gut health, including a healthy gut microbiome, and / or b) a healthy immune system, such as reducing risk of developing allergies, as well as reducing risk of inflammation, including preventing or reducing symptoms of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, and / or ulcerative colitis, and / or c) cognitive development, such as motor skills, learning, language skills, spatial cognition ability, and / or d) emotional and behavioral development, such as stress and anxiety reduction, in a subject.

[0024] In a third aspect, the present invention relates to a kit of parts comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO, wherein, if 3’-sialyllactose (3’-SL) is present in the mixture, the ratio (w / w) of 2’-FL:3’- SL is above 7.5:1 , and wherein the probiotic and the mixture of HMOs are not mixed together.

[0025] DETAILED DESCRIPTION

[0026] Bifidobacteria spp. have been demonstrated to predominate in the gut microbiota of breastfed infants, whereas the colonization of such species is less predominant in non-breastfed infants and infants delivered by C-section. Human milk oligosaccharides (HMOs) are the primary substrate for Bifidobacterium longum subspecies infantis (B. infantis). Several B. infantis strains have been subjected to clinical testing and have shown various benefits, including Bifidobacterium longum ssp. infantis R0033 (see for example Chichlowski et al., 2020, Nutrients, 12:1581). Definitions

[0027] A “nutritional composition”, as used herein, refers to any composition formulated from one or more separate ingredients which are suitable for human or animal consumption, such as an infant formula or a dietary supplement. It can contain sources of protein, lipids, vitamins, minerals and / or digestible carbohydrates and can be in dry, such as powdered, or liquid forms, preferably it is in a dry form. The composition can be designed to be the sole source of nutrition or a nutritional supplement. A synthetic nutritional composition is a composition where at least one of the ingredients is obtained by a biological process (e.g., enzymatic or fermentation) or chemical process. In the context of the present disclosure mother’s milk is not considered to be a synthetic nutritional composition. In preferred embodiments the nutritional composition is a synthetic nutritional composition.

[0028] A “probiotic”, as used herein, refers to bacteria, which, when ingested in adequate amounts, provide a benefit to the host (human or animal) by replenishing or otherwise supplementing its natural gastrointestinal flora or by eliminating undesired bacteria in its gastrointestinal (Gl) tract or by executing beneficial metabolic activities along its Gl tract or by stimulating its immune system.

[0029] 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 unit, 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. The HMOs falling into these groups are described in further detail in the section “Mixture of HMOs”.

[0030] “Gut” or “intestine” 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”.

[0031] 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. In some embodiments, the subject is a healthy individual. In some embodiments, the subject is an infant. In some embodiments, the subject is a non-infant individual. In preferred embodiments, the subject is a healthy non-infant individual. 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. In a preferred embodiment, the human is a non-infant, such as a child, an adult or an elderly individual. 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. In another preferred embodiment, the human is a healthy individual.

[0032] The terms “treat” or “treatment” or “treating” as used herein refer to both treatment of an existing disease {e.g., a disease, condition or disorder as referred to herein) or prevention of a disease, i.e., prophylaxis. Maintenance and / or promotion of health in an individual not suffering from a disease, but who may be susceptible to the development of an unhealthy condition, is considered non-medical treatment in the context of the present invention.

[0033] An "effective amount" of a composition of the present disclosure means an amount that renders a desired health benefit / treatment outcome in the subject it is administered to, when compared to a non-administered subject. An effective amount can be administered in one or more doses to achieve the desired treatment outcome.

[0034] “Enteral administration” means any conventional form for delivery of a composition to a subject that causes the deposition of the composition in the gastrointestinal tract (including the stomach). Methods of enteral administration include feeding through a naso-gastric tube or jejunum tube, oral, direct delivery to the gut, sublingual and rectal.

[0035] "Oral administration" means any conventional form of delivery of a composition to a subject, such as an infant or a non-infant, through the mouth. Accordingly, oral administration is a form of enteral administration.

[0036] Metabolites

[0037] Metabolites produced in the gut are small molecules generated as a result of the metabolic activities of the gut microbiota and host cells. These gut-derived metabolites include short-chain fatty acids (SCFAs), bile acids, vitamins, amino acids, and gases such as hydrogen and methane. They play crucial roles in maintaining gut health, regulating immune responses, influencing the gut-brain axis, and contributing to the overall metabolic homeostasis of the host. The gut metabolites are influenced by the nutrients consumed. Prebiotics and probiotics can for example enhance the growth of beneficial bacteria, leading to increased production of health-promoting metabolites. Any variation in metabolites (i.e. , increase or decrease) effected or induced by the administration of the nutritional composition described herein is assessed relatively to the same subject prior to the administration of the composition described herein (i.e., the “non-administered subject(s)”). Alternatively, the comparative parameters may also constitute two cohorts of individuals, one cohort receiving a composition described herein (i.e., the “administered subjects”) and one or more additional cohorts receiving a placebo, such as a maltodextrin or lactose or alternatively the same mixture of HMOs without the probiotic (i.e., the “non-administered subjects”). The term “cohort” in this respect is understood as groupings of individuals with common traits, such as age, social and health factors. The size of the cohorts needed for comparative studies depends on the statistical variation observed within a cohort.

[0038] Bifidobacterium longum ssp. infantis R0033 is a known producer of short chain fatty acids (SCFAs) and of SCFA precursors such as lactate / lactic acid. The present disclosure relates to a synbiotic combination with B. infantis R0033 or closely related strains and mixtures of HMOs, wherein the combination is capable of increasing the levels of various metabolites, such as short chain fatty acids (SCFAs), of SCFAs precursors such as lactate / lactic acid, putrescine, gamma- aminobutyric acid (GABA), and / or dopamine in the gut of a subject upon administration. The combination is a synbiotic combination or kit of parts of the present disclosure. An increase in metabolites is preferably assessed by measuring the specific metabolite(s) in the gut prior to the administration of the composition or kit of parts of the present disclosure. As an alternative to measuring the metabolite(s) in the gut of a subject, the ability of a subject to produce the metabolite(s) following administration of the composition of the present disclosure can be assessed by fermenting a stool sample of said subject in the presence of a desired synbiotic composition, as described in the Examples. The increased levels of metabolite(s) upon administration of the synbiotic composition of the present disclosure can either be a direct increase due to the production of said metabolite(s) by B. infantis R0033 (or by closely related strains), or an indirect increase via the production of said metabolite(s) by the microbiota present in the gut of the subject. Such indirect increase of the metabolite(s) can be promoted or induced by metabolite(s) produced by B. infantis R0033 (or by closely related strains) such as for example lactic acid which is utilized by species of the microbiota in the subject.

[0039] Bifidobacterium longum ssp. infantis R0033 is a known producer of short chain fatty acids (SCFAs) and of SCFAs precursors such as lactate / lactic acid. The present disclosure relates to a synbiotic combination, with Bifidobacterium longum ssp. infantis R0033 or closely related strains and mixtures of HMOs, which is capable of increasing the levels of short chain fatty acids (SCFAs), lactic acid, putrescine and / or dopamine in the gut of a subject upon administration of the synbiotic combination. Such increase is preferably assessed by measuring the metabolite(s) levels, in particular SCFA(s), lactic acid, putrescine and / or dopamine, in the gut prior to the administration of the composition of the present disclosure. As an alternative to measuring the metabolite(s) in the gut of a subject, the ability of a subject to produce the metabolite(s) following administration of the composition of the present disclosure can be assessed by fermenting a stool sample from said subject in the presence of a desired synbiotic composition, according to the method described in the Examples. The increased levels of metabolite(s) upon administration of the synbiotic composition of the present disclosure can either be a direct increase due to the production of said metabolite(s) by Bifidobacterium bifidum R0033 (or by closely related strains), or an indirect increase via the production of said metabolite(s) by the microbiota present in the gut of the subject. Such indirect increase of the metabolite(s) can be promoted or induced by metabolite(s) produced by Bifidobacterium bifidum R0033 (or by closely related strains) such as for example lactic acid which is utilized by species of the microbiota in the subject.

[0040] SCFAs, of which butyrate, propionate, and acetate are the most predominant in the human gut, can have several health benefits, including potential benefits for gut immune health. SCFAs have been shown to have anti-inflammatory effects in the gut, which can help to promote a healthy immune response and reduce the risk and / or symptoms of inflammatory conditions in the gut, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, and / or ulcerative colitis. Additionally, SCFAs can help to support the growth and function of gut immune cells, such as T cells and regulatory T cells, which play a key role in maintaining a healthy immune system. Furthermore, SCFAs have been shown to help maintaining the gut barrier integrity, which can help to prevent harmful substances from entering the bloodstream and then trigger an immune response. Maintaining the gut barrier integrity can help to reduce the risk of allergies and chronic inflammation and other health issues. Examples of allergies where the risk may be reduced are in particular food allergies and potentially also drug allergies.

[0041] Acetate produced by bacteria acts in vivo to promote the defence functions of host epithelial cells. Acetate has been shown to promote intestinal antibody immunoglobulin (lg)A responses in the gut via the G protein-coupled receptor GPR43. Furthermore, acetate produced by B. infantis becomes a carbon source that stimulates growth and function of butyrate-producing microbes, such as Faecalibacterium prausnitzii.

[0042] Lactic acid or lactate (as SCFAs precursor) can be utilized by particular species of the Firmicutes phylum to induce SCFAs production, as such bacteria are able to convert lactate into butyrate and propionate in the gut. In addition, intraluminal lactate has been shown to modulate the inflammatory environment in intestinal mucosa. Besides immunomodulation, it has been shown that luminal lactate can stimulate enterocyte proliferation in a murine model of hunger-feedback, contributing to maintain intestinal barrier functions. Lactate has also been shown to inhibit the growth of some pathogenic bacteria, including Escherichia coli, and can also reach high concentrations in the gut of healthy infants. In addition, lactate can also decrease gas production from carbohydrate fermentation. Putrescine is a polyamine that is naturally produced in the body and is also found in certain foods such as cheese, fermented soybeans, and some meats. Like SCFAs, putrescine has been shown to have anti-inflammatory effects in the gut (Lagishetty and Naik, 2008, Indian J. Pharmacol., 40(3):121-5). Putrescine supplementation in piglets has also been shown to improve the intestinal morphology and reduce diarrhea, e.g., non-infectious diarrhea due to a change in diet, potentially leading to improved intestinal development and gut health. In addition, putrescine supplementation in the piglets showed improved anti-inflammatory function and suppression of inflammatory responses and increased immunity, by decreasing inflammatory cytokines like TNF- a, IL6 and IL8 in the gut mucosa and increasing anti-inflammatory cytokines such as TGF-pi as well as components of humoral immunity such as IgM, hepcidin and p-defensin1 (Liu et al., 2019, Food Funct., 10:4134; and Liu etal., 2019, Animal Science and Biotechnology, 10:69). Putrescine may also have antioxidant properties (Zeynali et al., 2023, Chem. Biodiversity, 20:e2023010), which could potentially help protect the body against oxidative stress and inflammation.

[0043] All in all, both SCFAs, lactic acid, GABA and putrescine are therefore believed to be beneficial for gut health and able to prevent or reduce symptoms of inflammatory diseases in the gut.

[0044] Immune system development refers to the process by which the immune system matures and becomes capable of defending the body against pathogens. The immune system consists of innate immunity (the body's first line of defence, including physical barriers, including the gut barrier, phagocytes, and natural killer cells) and adaptive immunity which involves highly specialized responses mediated by T cells and B cells. Using a composition of the present disclosure to support the development of the immune system in a healthy individual is a nonmedical use.

[0045] The ability of acetate and putrescine to modulate certain immunoglobulins can support the balancing of immunoglobulins, thereby supporting maintenance of immunoglobulin homeostasis. Furthermore, the effect on cytokine responses mediated by putrescine can influence B cell activity and thereby affect immunoglobulin levels.

[0046] The gut-brain axis is the interplay between the brain and the gut microbiome and plays an important role in a wide range of physiological and psychological processes, including digestion, immune function, mood, and cognition (see for example Strandwitz 2018 Brain Research 1693:128-133). The effect of the microbiota of infants and the HMO composition in mother’s milk on cognitive abilities, such as motor skills, learning, language skills, spatial cognition ability, and anxiety reduction was studied by Cho et al. (2023 Front. Nutr. 10:1216327), which clearly indicates different benefits of certain HMOs and certain bacteria, for example in the modulation of, or increase in, the production of certain neurotransmitters.

[0047] Gamma-Aminobutyric Acid (GABA) is an important neurotransmitter that plays a crucial role in brain development and function. It is involved in regulating neuronal activity and is believed to have a calming effect on the brain. Studies have suggested that GABA may have a number of health benefits for children, including improving cognitive function, reducing anxiety and stress, and promoting healthy sleep patterns. GABA has also been shown to have a positive impact on certain developmental disorders, such as autism and attention deficit hyperactivity disorder (ADHD).

[0048] GABA can also be found in certain fermented foods, such as kimchi, yogurt, and kefir, and may be produced by some probiotic lactobacilli strains. Additionally, GABA has been shown to have potential benefits for gut health. For example, GABA may support gut barrier function and tight junctions and thereby contribute to ameliorating the effects caused by pathogens on the gut barrier (see for example Kaur et al., 2023, Microbial Cell Factories, 22:256). Additionally, GABA may help to regulate gut motility, which could help to alleviate symptoms of constipation or diarrhea (Kerr and Ong, 2015 GABA Outside the CNS GABA and Gut Motility, pp 29-44). Using a composition of the present disclosure to support the gut motility in a subject, such as a healthy individual, not suffering from constipation or diarrhea is a non-medical use.

[0049] Immune system development refers to the process by which the immune system matures and becomes capable of defending the body against pathogens. The immune system consists of innate immunity (the body's first line of defence, including physical barriers, including the gut barrier, phagocytes, and natural killer cells) and adaptive immunity which involves highly specialized responses mediated by T cells and B cells. Using a composition of the present disclosure to support the development of the immune system in a healthy individual is a nonmedical use.

[0050] Furthermore, GABA plays a role in developing the immune system via the GABA receptors that are expressed on various immune cells, including T cells, B cells, macrophages, and dendritic cells. Activation of these receptors can influence the activity and behaviour of these immune cells and thereby influence the development of the immune system. The ability of GABA to modulate cytokine responses can influence B cell activity and thereby affect immunoglobulin levels. GABA may therefore support the balancing of immunoglobulins of the different classes IgG, IgA, IgM, IgE, and IgD and thereby support maintaining immunoglobulin homeostasis. In particular, high levels of IgE can be associated with development of allergies.

[0051] Additionally, GABA may help to reduce inflammation in the gut, e.g., by reducing production of proinflammatory cytokines, such as TNF-a, IL6 and IL8, through downregulation of p38 mitogen- activated protein kinase (MAPK), which could be beneficial in treating or reducing symptoms in individuals with inflammatory bowel disease (IBD) or other gut-inflammatory conditions (see for example Aggarwal et al. 2018, J. Neurogastroenterol. Motil. 24(3): 422-430).

[0052] Dopamine is one of the major neurotransmitters in reward-motivated behavior and is a precursor for other catecholamines like norepinephrine and epinephrine. Dopamine is believed to play a role in the development of certain cognitive and motor skills in children. It is however important to maintain a balanced dopamine level, since too high dopamine levels are associated with, e.g., attention deficit hyperactivity disorder (ADHD), and too low levels are associated with Parkinson’s disease. Dopamine is a regulator of neuronal activity which, inter alia, supports development of cognitive functions.

[0053] In conclusion, SCFAs (and their precursor) and putrescine have been shown to have antiinflammatory effects in the gut. GABA may also positively affect gut barrier function, be beneficial for gut health, and able to prevent or reduce symptoms of inflammatory diseases in the gut. In addition, GABA and dopamine are regulators of neuronal activity which, inter alia, support development of cognitive functions. All these molecules may thus improve gut health, provide anti-inflammatory functions, and / or support cognitive development.

[0054] An advantage of the present invention is that the composition disclosed herein provides a way to produce SCFAs, lactic acid, GABA and putrescine in situ in the gut. The “in situ” production of such metabolites is of particular interest for putrescine and dopamine which does not have to be ingested and pass through the acidic environment of the stomach. Without being bound to any theory, the probiotic -of the composition according to the invention would become a member of the microbiota community, and as such, it would be able to stay longer in the gut while providing a prolonged and naturally controlled production of SCFAs, lactic acid, putrescine and dopamine as compared to orally administered SCFAs, lactic acid, putrescine and dopamine. Moreover, the combination of a prebiotic and a probiotic is easy to deliver, known to be safe to consume, and well accepted by the end-consumers or health care practitioners. Furthermore, the costs of such delivery are reasonable.

[0055] Synbiotic composition

[0056] As described above, the present disclosure relates to synbiotic compositions and / or combinations.

[0057] A first aspect of the present disclosure relates to a nutritional composition comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO.

[0058] If 3’-SL (a sialylated HMO) is present in the nutritional composition, the ratio (w / w) of 2’-FL: 3’-SL is above 7.5:1 , such as above 8:1. In embodiments the nutritional composition comprises a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO.

[0059] In embodiments the nutritional composition comprises a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a sialylated HMO, wherein if the sialylated HMO is 3’-SL it is present in the nutritional composition in a ratio (w / w) of 2’-FL: 3’-SL above 7.5:1 , such as above 8:1.

[0060] In embodiments the nutritional composition comprises at least one probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO.

[0061] In embodiments the nutritional composition comprises at least one probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a sialylated HMO, wherein if the sialylated HMO is 3’-SL it is present in the nutritional composition the ratio (w / w) of 2’-FL: 3’-SL is above 7.5:1 , such as above 8:1. In embodiments the nutritional composition comprises at least one probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, preferably LNnT or LNT.

[0062] In embodiments the nutritional composition comprises at least one probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least two additional HMOs selected from i. an additional fucosylated HMO, preferably DFL or 3-FL; ii. a neutral core HMO, preferably LNnT and / or LNT, or iii. a sialylated HMO, preferably 3’-SL and / or 6’-SL.

[0063] In embodiments the nutritional composition comprises at least one probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and a neutral core HMO, preferably LNnT and / or LNT, and a sialylated HMO, preferably 3’-SL and / or 6’SL.

[0064] In embodiments the nutritional composition comprises at least one probiotic and a mixture of human milk oligosaccharides (HMOs), wherein a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs constitutes at least 95 wt%, such as at least 97wt%, of the total amount of HMOs in the nutritional composition, wherein the mixture is selected from i. 2’-FL and DFL, or ii. 2’-FL and 3-FL, or iii. 2’-FL and 3’-SL, or iv. 2’-FL and 6’-SL, or v. 2’-FL and LNnT, or vi. 2’-FL and LNT, or vii. 2’-FL, LNnT and 3’-SL, or viii. 2’-FL, 6’-SL, LNnT and 3’-SL, or ix. 2’-FL, DFL, LNnT, 6’-SL and 3’-SL, or x. 2’-FL, 3-FL, LNT, 6’-SL and 3’-SL, or xi. 2’-FL, DFL, LNT, LNnT, 6’-SL and 3’-SL, or xii. 2’-FL, 3-FL, LNT, LNnT, 6’-SL and 3’-SL, or xiii. 2’-FL, DFL, LNT, 6’-SL and 3’-SL.

[0065] In preferred embodiments the nutritional composition of the present disclosure comprises an HMO mixture selected from i) or v) or vii) or viii) or ix) or x) or xi) or xii) above.

[0066] In a further embodiment the nutritional composition comprises at least one additional probiotic. Preferably, the additional probiotic microorganism is a species selected from Bifidobacterium longum, Bifidobacterium lactis and / or Bifidobacterium bifidum.

[0067] The constitutive elements of the nutritional compositions are described in more detail in the sections below. It is understood that these elements can be integrated in the above aspect and embodiments.

[0068] The nutritional synbiotic composition may be a combination in the form of a kit of parts, wherein the probiotic and the mixture of HMOs are not mixed together. Alternatively, the nutritional synbiotic composition is a blend of a probiotic and a mixture of human milk oligosaccharides (HMOs), i.e. , the probiotic and the HMO mixture are in the same container.

[0069] Probiotics

[0070] The probiotic according to the first aspect of the invention is the Bifidobacterium longum ssp. Infantis (B. infantis) R0033 strain, which is sold as a probiotic under the name B. infantis Rosell®- 33 (Lallemand Health Solutions). The strain has been deposited at Collection Nationale de Cultures de Microorganismes (CNCM) under the accession number CNCM I-3424, as described under ‘Deposit’ above.

[0071] The probiotic may also be a closely related strain having at least 97% average nucleotide identity (ANI) with CNCM I-3424, such as 97.1%, such as 97.2%, such as 97.3%, such as 97.4%, such as 97.5%, such as 97.6%, such as 97.7%, such as 97.8%, such as 97.9%, such as 98.0%, such as 98.1 %, such as 98.2%, such as 98.3%, such as 98.4%, such as 98.5%, such as 98.6%, such as 98.7%, such as 98.8%, such as 98.9%, such as 99.0%, such as 99.1%, such as 99.2%, such as 99.3%, such as 99.4%, such as 99.5%, such as 99.6%, such as 99.7%, such as 99.8%, and such as 99.9%, ANI.

[0072] The probiotic may also be a closely related strain having at least 98% average nucleotide identity (ANI) with CNCM I-3424, such as 98.1 %, such as 98.2%, such as 98.3%, such as 98.4%, such as 98.5%, such as 98.6%, such as 98.7%, such as 98.8%, such as 98.9%, such as 99.0%, such as 99.1 %, such as 99.2%, such as 99.3%, such as 99.4%, such as 99.5%, such as 99.6%, such as 99.7%, such as 99.8%, and such as 99.9%, ANI.

[0073] The probiotic may also be a closely related strain having at least 99% average nucleotide identity (ANI) with CNCM I-3424, such as 99.1 %, such as 99.2%, such as 99.3%, such as 99.4%, such as 99.5%, such as 99.6%, such as 99.7%, such as 99.8%, and such as 99.9%, ANI.

[0074] Average nucleotide identity (ANI) is an in-silico method which can be used to determine genetic relatedness among bacterial strains. It is based on pairwise comparisons of all sequences shared by two strains and can be calculated using publicly available tools, such as OrthoANI with usearch (Yoon etal., 2017, Antonie van Leeuwenhoek, 110:1281-1286); ANI Calculator, JSpecies (Richter and Rossello-Mora, 2009, Proc Natl Acad Sci USA, 106:19126-19131); and JSpeciesWS (Richter et al., 2016, Bioinformatics, 32:929-931).

[0075] In some embodiments, the synbiotic composition of the present disclosure can further comprise at least one additional probiotic microorganism. The synbiotic composition can use, as the at least one additional probiotic microorganism, a probiotic bacteria species from the Lactobacillus genus, Bacillus genus, Bifidobacteria genus, Enterococcus genus, Pediococcus genus and / or Streptococcus genus.

[0076] Lactobacillus species include, without any limitation, L. acidophilus, L. brevis, L. bulgaricus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius or L. paracasei. While it is not intended that the present disclosure be limited to any particular species of Lactobacillus, exemplary species and strains of Lactobacillus for the present disclosure includes, but are not limited to, the following well-known strains: L. helveticus / L. acidophilus HA-122 (sold by Lallemand Health Solutions ("LHS")), L. acidophilus R0418 (LHS), L. brevis HA-112 (LHS), L. casei HA-108 (LHS), L. casei R0215 (LHS), L. casei 431 (Chr. Hansen), L. delbrueckii bulgaricus HA-137 (LHS), L. fermentum HA-179 (LHS), L. helveticus HA-128 (LHS), L. helveticus HA-501 (LHS), L. helveticus R0052 (LHS), L. helveticus Lafti L10 (LHS), L. paracasei HA-196 (LHS), L. paracasei HA-274 (LHS), L. paracasei Lafti L26 (LHS), L. plantarum R0403 (LHS), L. plantarum R0202 (LHS), L. plantarum R1012 (LHS), L. plantarum TIFN101 , L. reuteri HA-188 (LHS), L. reuteri (DSM12246, dsm-firmenich) L. rhamnosus HA-114 (LHS), L. rhamnosus HA-500 (LHS), L. rhamnosus R0011 (LHS), L. rhamnosus R0049 (LHS), L. rhamnosus R0343 (LHS), L. rhamnosus R1039 (LHS), L. salivarius HA-118 (LHS), L. salivarius R0078 (LHS), L. bulgaricus R0440 (LHS), L. lactis R1087 (LHS), L. rhamnosus GG (ATCC 53103) and / or L. rhamnosus LGG (DSM 33156, Chr. Hansen).

[0077] Bacillus species include, without any limitation, B. coagulans or B. subtilis. While it is not intended that the present disclosure be limited to any particular species of Bacillus, exemplary species and strains of Bacillus for the present disclosure includes, but are not limited to, the following well- known strains: B. subtilis R0179 (LHS) and / or B. subtilis HA-124 (LHS). Bifidobacteria species include, without any limitation, B. bifidum, B. breve, B. infantis, B. lactis or B. longum. While it is not intended that the present disclosure be limited to any particular species of Bifidobacteria, exemplary species and strains of Bifidobacteria for the present disclosure includes, but are not limited to, the following well-known strains: B. bifidum R0071 (CNCM I-3426, LHS), B. bifidum HA-132 (CNCM I-5898, LHS), B. breve R0070 (LHS), B. breve HA-129 (LHS), B. breve M-63 (NITE BP-02623, Morinaga), Bifidobacterium breve M-16V (LMG 23729, Morinaga), B. infantis HA-116 (LHS), B. lactis LAFTI® B94 (LHS), B. lactis HA-194 (LHS), B. lactis BB-12 (DSM 15954, Chr. Hansen), B. longum R0175 (CNCM I-3470, LHS), B. longum HA- 135 (LMG S-24801 , LHS), B. longum BB 536 (ATCC BAA-999, Morinaga), B. longum DSM 32946, B. Infantis BB-02 (DSM 33361 , Istilos, Chr. Hansen), B. Infantis LMG11588 (ATCC 17930), B. infantis EVC001 (PTA-125180), B. infantis Bi-26 (ATCC SD6720), B. infantis M-63 (NITE BP-02623), B. infantis (ATCC 15697), B. infantis (NCTC 11817), B. infantis (NCTC 13219), B. infantis BT1 (KCTC 11859BP), B. infantis UBBI-01 , B. infantis IN-F29, B. infantis TPY 12-1, B. infantis 1888B and / or B. infantis EK3.

[0078] Enterococcus species include, without any limitation, E. faecium. While it is not intended that the present disclosure be limited to any particular species of Enterococcus, exemplary species and strains of Enterococcus for the present disclosure includes, but are not limited to, the following well-known strains: E. faecium R0026 (LHS) and / or E. faecium HA-127 (LHS).

[0079] Pediococcus species include, without any limitation, P. acidilactici. While it is not intended that the present disclosure be limited to any particular species of Pediococcus, exemplary species and strains of Pediococcus for the present disclosure includes, but are not limited to, the following well-known strain P. acidilactici HA-524 (LHS).

[0080] Streptococcus species include, without any limitation, S. thermophilus. While it is not intended that the present disclosure be limited to any particular species of Streptococcus, exemplary species and strains of Streptococcus for the present disclosure includes, but are not limited to, the following well-known strains: S. thermophilus R1018 (LHS), S. thermophilus R0083 (LHS) and / or S. thermophilus HA-110 (LHS).

[0081] In a preferred embodiment, the synbiotic composition can use, as the at least one additional probiotic microorganism, one or more of the following probiotic species: B. bifidum R0071 (CNCM I-3426), B. bifidum HA-132 (CNCM I-5898), 8. breve HA-129 (CNCM I-5897), 8. lactis LAFTI® B94 (CBS 118529), L. helveticus R0052 (CNCM 1-1722), L. rhamnosus HA-114 (LMG S-24117, CNCM I-5765), L rhamnosus R0011 (CNCM 1-1720), L. rhamnosus GG (ATCC 53103), 8. Infantis LMG11588 (ATCC 17930), 8. infantis EVC001 (PTA-125180), B. infantis Bi-26 (ATCC SD6720), 8. infantis M-63 (NITE BP-02623), B. infantis (ATCC 15697), B. infantis (NCTC 11817), B. infantis (NCTC 13219), B. infantis BT1 (KCTC 11859BP), B. infantis UBBI-01 , B. infantis IN- F29, B. infantis TPY 12-1, B. infantis 1888B and B. infantis EK3. In some embodiment, the probiotic bacteria of the present disclosure (8. infantis R0033 and / or potential added probiotic bacteria) can be provided in the form of viable cells, or in an inactivated form of non-viable cells (i.e., killed cultures). In a preferred embodiment, B. infantis R0033 is provided in the form of viable cells. In some other embodiment, B. infantis R0033 is provided in the form of viable cells, and potential additional probiotic bacteria are provided in the form of viable cells and / or in an inactivated form of viable cells. As such, in some embodiments, the population of probiotic bacteria can be subjected to a thermal (heat or cold) treatment, a pH treatment, a radiation (sonication) treatment and / or a pressure treatment to kill the viable cells of the population of probiotic bacteria.

[0082] In a further embodiment, the probiotic bacteria of the present disclosure (8. infantis R0033 and / or potential additional probiotic bacteria) can be provided in encapsulated form to protect them from moisture during storage as well as from the stomach acidic conditions. The probiotic encapsulation processes are well known in the art, and can therefore be easily adapted by the one with skills in the art.

[0083] Mixture of HMOs

[0084] The mixture of HMOs according to the first aspect of the invention comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO.

[0085] If 3’-SL (a sialylated HMO) is present in the nutritional composition, the ratio (w / w) of 2’-FL: 3’-SL is above 7.5:1 , such as above 8:1.

[0086] In embodiments the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’- FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO.

[0087] In embodiments the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’- FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a sialylated HMO, wherein if the sialylated HMO is 3’-SL it is present in the nutritional composition in a ratio (w / w) of 2’-FL: 3’-SL above 7.5:1 , such as above 8:1.

[0088] In some embodiments, the additional fucosylated HMO is selected from the group consisting of 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP-I), lacto-N- fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP- V),lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N- hexaose II (FLNH-II), lacto-N-difucohexaose II (LNDFH-I I), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (F-pLNnH II), and fucosyl-lacto-N-neohexaose (FLNnH).

[0089] In preferred embodiments, the additional fucosylated HMO is selected from 3-fucosyllactose (3- FL) and difucosyllactose (DFL), or is a mixture thereof.

[0090] In some embodiments, the mixture of HMOs comprises or consists of 2’-FL and one, such as two, additional fucosylated HMOs selected from the list of neutral fucosylated HMOs above. In some embodiments the mixture of HMOs comprises or consists of at least two different fucosyllactoses, such as 2’-FL, 3-FL, and / or DFL.

[0091] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL and DFL.

[0092] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL and 3-FL.

[0093] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL and DFL.

[0094] In some embodiments, the neutral core HMO is selected from the group consisting of lacto-N- triose II (LNT-II) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH), and lacto-N-hexaose (LNH).

[0095] In preferred embodiments, the neutral core HMO is selected from lacto-ZV-triose II (LNT-II), lacto- / V-tetraose (LNT), and lacto-N-neotetraose (LNnT), or is any mixture thereof. In more preferred embodiments, the neutral core HMO is LNnT.

[0096] In some embodiments, the mixture of HMOs comprises or consists of 2’-FL and one, such as two, additional neutral core HMOs selected from the list of neutral core HMOs above.

[0097] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL and LNnT.

[0098] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL and LNT.

[0099] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT and LNT.

[0100] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, at least one additional fucosy I lactose selected from the list of fucosyllactoses above and at least one neutral core HMO selected from the list above.

[0101] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and LNnT.

[0102] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and LNT. In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNnT and LNT.

[0103] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and LNnT.

[0104] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and LNT.

[0105] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNnT and LNT. In some embodiments, the sialylated HMO is selected from the group consisting of 3’- sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), 3-fucosyl-3’-sialyllactose (FSL), 3’-O-sialyllacto-N- tetraose a (LST a), fucosyl-LST a (FLST a), 6’-O-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6’-O-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3’-O-sialyllacto-N- neotetraose (LST d), fucosyl-LST d (FLST d), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N- neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II), and disialyl-lacto-N-tetraose (DSLNT).

[0106] In preferred embodiments, the sialylated HMO is a sialyllactose, optionally selected from 3’- sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL), or is a mixture thereof. In more preferred embodiments, the sialyllactose is 3’-SL.

[0107] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL and 3’-SL, preferably in a weight (w / w) ratio of 2’-FL:3’-SL above 7.5:1 , such as above 8:1. In embodiments the ratio (w / w) of 2’-FL: 3’-SL is between 7.5-20:1 , such as 9.5-18:1 , such as 11-16:1 of 2’-FL:3’- SL.

[0108] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL and 6’-SL.

[0109] In some embodiments, the sialylated HMO(s) is present in an amount which is at least 7-fold lower than the amount of 2’-FL, such as at least 7.5 fold lower, such as at least 8 fold lower. In further embodiments, the sialylated HMO is 3’-SL, and the ratio (w / w) of 2’-FL:3’-SL is above 7:1 , such as above 7.5:1 , such as above 8:1. In embodiments the ratio (w / w) of 2’-FL: 3’-SL is between 7.5-20:1 , such as 9.5-18:1 , such as 11-16:1 , of 2’-FL:3’-SL.

[0110] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and 3’-SL.

[0111] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and 6’-SL.

[0112] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, 3’-SL and 6’SL. In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and 3’-SL.

[0113] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and 6’-SL.

[0114] In some embodiment, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, 3’- SL and 6’-SL.In some embodiments the mixture of HMOs contains 2’-FL and at least two additional HMOs, where one is selected from a neutral core HMO and the other is selected from a sialylated HMO.

[0115] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT and 3’-SL.

[0116] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNT and 3’-SL.

[0117] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT and 6’-SL.

[0118] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT, 3’- SL and 6’-SL.

[0119] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNT and 6’-SL.

[0120] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, LNT, 3’- SL and 6’-SL.

[0121] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNnT, 3’-SL and 6’-SL.

[0122] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNT, 3’-SL and 6’-SL.

[0123] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNnT, 3’-SL and 6’-SL.

[0124] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL.

[0125] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNnT, 3’-SL and 6’-SL.

[0126] In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNT, LNnT, 3’-SL and 6’-SL. In some embodiments, the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNT, LNnT, 3’-SL and 6’-SL.

[0127] All ratios between specific HMOs given herein are by weight.

[0128] In some embodiments, the total amount of fucosylated HMO constitutes between 60 to 100 wt% of the total mixture of HMOs, such as between 60 to 95 wt% of the total mixture of HMOs, such as between 75 to 96 wt%, or such as between 88 to 98 wt%.

[0129] In some embodiments, the mixture of HMOs constitutes at least 95 wt% of the total amount of HMOs in the nutritional composition, and the mixture is selected from: a) 2’-FL and DFL, in a ratio between 4-10:1 of 2’-FL:DFL, such as between 5-8:1 , or b) 2’-FL and 3-FL, in a ratio between 2-4:1 of 2’-FL:3-FL, such as 3:1 , or c) 2’-FL and 3’-SL, in a ratio between 7.5-20:1 of 2’-FL:3’-SL, such as between 9.5-18:1 of 2’- FL:3’-SL, such as between 11-16:1 , or d) 2’-FL and 6’-SL, in a ratio between 4-10:1 of 2’-FL:6’-SL, such as between 5.5-8:1 , or e) 2’-FL and LNnT, in a ratio between 2-8:1 of 2’-FL:LNnT, such as between 4-6.5: 1, or f) 2’-FL, LNT and LNnT, in a ratio between 4-8:1-3:1 of 2’-FL:LNT:LNnT, such as between 5- 7:1.5-2.5:1, or g) 2’-FL, LNnT and DFL, in a ratio between 6-11 :0.5-3:1 of 2’-FL:LNnT:DFL, such as between 7-9: 1-2:1 , or h) 2’-FL, LNT and DFL, in a ratio between 6-11:0.5-4:1 of 2’-FL:LNT:DFL, such as between 7- 9:1.5-3:1 , or i) 2’-FL, LNnT and 3’-SL, in a ratio between 10-18:1-5:1 of 2’-FL:LNnT:3’-SL, such as between 12-16:2-4:1 or j) 2’-FL, 6’-SL and LNnT, in a ratio between 3-9:0.5-2:1 of 2’-FL:6’-SL:LNnT, such as between 4-8:1-1.5:1 or k) 2’-FL, 6’-SL, LNnT and 3’-SL, in a ratio between 8-16:0.5-4: 0.5-4:1 of 2’-FL:6’-SL:LNnT:3’- SL, such as between 10-14:1-3:1-3:1 , or l) 2’-FL, LNT and 3’-SL, in a ratio between 8-16:2-6:1 of 2’-FL:LNT:3’-SL, such as between 10- 14:3-5:1 or m) 2’-FL, LNT and 6’-SL, in a ratio between 4-8:1-4:1 of 2’-FL:LNT:6’-SL, such as between 5- 7:1-3:1, or n) 2’-FL, LNT, 6’-SL and 3’-SL, in a ratio between 8-16:2-6:0.5-4:1 of 2’-FL:6’-SL:LNT:3’-SL, such as between 10-14:3-5:1-3:1 , or o) 2’-FL, DFL and 3’-SL, in a ratio between 6-10:0.5-3:1 of 2’-FL:DFL:3’-SL, such as between 7-9:1-2:1 , or p) 2’-FL, LNT, LNnT, and DFL in a ratio between 4-12:1-4:0.5-3:1 of 2’-FL:LNT:LNnT:DFL, such as between 6-8: 1.5-3.5: 1-2:1, or q) 2’-FL, DFL, LNT, 6’-SL and 3’-SL, in a ratio between 10-16:1-3:2-6:1-5:1 of 2’-FL:DFL:LNT:6’- SL:3’-SL, such as between 10-14:1-2:3-5:1-3:1 , or r) 2’-FL, DFL, LNnT, 6’-SL and 3’-SL, in a ratio between 8-16:1-3:0.5-4: 0.5-4:1 of 2’- FL:DFL:LNnT:6’-SL:3’-SL, such as between 10-14:1-2:3-5:3-5:1 , or s) 2’-FL, 3-FL, LNT, 6’-SL and 3’-SL, in a ratio between 10-16:2-6:2-6:1-5:1 of 2’-FL:3- FL:LNT:6’-SL:3’-SL, such as between 10-14:3-5:3-5:1-3:1 , or t) 2’-FL, DFL, LNT, LNnT, 6’-SL and 3’-SL, in a ratio between 10-16:0.5-4:2-6:1 :5:1-5:1 of 2’- FL:DFL:LNT:LNnT:6’-SL:3’-SL, such as between 10-14:3-5:3-5:1-3:1-3:1 , or u) 2’-FL, 3-FL, LNT, LNnT, 6’-SL and 3’-SL, in a ratio between 10-16:2-6:2-6:1 :5:1-5:1 of 2’- FL:3-FL:LNT:LNnT:6’-SL:3’-SL, such as between 10-14:3-5:3-5:1-3:1-3:1.

[0130] In preferred embodiments the nutritional composition of the present disclosure comprises an HMO mixture selected from a) or c) or i) or k) or r) or s) or t) or u) above.

[0131] In further preferred embodiments, wherein the mixture is a), the ratio of 2’-FL:DFL is 7.8:1. In more preferred embodiments, the ratio is 6.1 :1.

[0132] In further preferred embodiments, wherein the mixture is c), the ratio of 2’-FL:3’-SL is 16:1. In more preferred embodiments, the ratio is 11.5:1.

[0133] In further preferred embodiments, wherein the mixture is d), the ratio of 2’-FL:6’-SL is 8:1. In more preferred embodiments, the ratio is 5.8:1.

[0134] In further preferred embodiments, wherein the mixture is e), the ratio of 2’-FL:LNnT is 4:1. In more preferred embodiments, the ratio is 6.1 :1.

[0135] In further preferred embodiments, wherein the mixture is f), the ratio of 2’-FL:LNnT:3’-SL is 16:4:1.

[0136] In more preferred embodiments, the ratio is 12:2:1.

[0137] In some embodiments, the mixture of HMOs constitutes at least 95 wt% of the total amount of HMOs in the nutritional composition, and the mixture is selected from: a) 80 wt% to 95 wt% of 2’-FL and 5 wt% to 20 wt% of DFL, such as between 85 wt% to 90 wt% of 2’-FL and 10 wt% to 15 wt% of DFL, or b) 65 wt% to 85 wt% of 2’-FL and 15 wt% to 35 wt% of 3-FL, such as between 70 wt% to 80 wt% of 2’-FL and 20 wt% to 30 wt% of 3-FL, or c) 88 wt% to 98 wt% of 2’-FL and 2 wt% to 12 wt% of 3’-SL, such as between 90 wt% to 95 wt% of 2’-FL and 5 wt% to 10 wt% of 3’-SL, or d) 75 wt% to 95 wt% of 2’-FL and 5 wt% to 25 wt% of 6’-SL, such as between 80 wt% to 90 wt% of 2’-FL and 10 wt% to 20 wt% of 6’-SL, or e) 75 wt% to 95 wt% of 2’-FL and 5 wt% to 25 wt% of LNnT, such as between 80 wt% to 90 wt% of 2’-FL and 10 wt% to 20 wt% of LNnT, or f) 60 wt% to 75 wt% of 2’-FL, 15 wt% to 25 wt% LNT and 5 wt% to 16 wt% of LNnT, such as between 65 wt% to 70 wt% of 2’-FL,18 wt% to 22 wt% LNT and 9 wt% to 13 wt% of LNnT, or g) 65 wt% to 85 wt% of 2’-FL, 5 wt% to 15 wt% DFL and 8 wt% to 16 wt% of LNnT, such as between 70 wt% to 80 wt% of 2’-FL, 7 wt% to 11 wt% DFL and 10 wt% to 14 wt% of LNnT, or h) 60 wt% to 80 wt% of 2’-FL, 5 wt% to 14 wt% DFL and 18 wt% to 26 wt% of LNT, such as between 65 wt% to 75 wt% of 2’-FL, 6 wt% to 11 wt% DFL and 20 wt% to 24 wt% of LNT, or i) 70 wt% to 85 wt% of 2’-FL, 10 wt% to 25 wt% of LNnT, and 3 wt% to 10 wt% of 3’-SL, such as between 75 wt% to 80 wt% of 2’-FL, 12 wt% to 20 wt% of LNnT, and 4 wt% to 8 wt% of 3’-SL or j) 65 wt% to 85 wt% of 2’-FL, 8 wt% to 16 wt% of LNnT, and 9 wt% to 17 wt% of 6’-SL, such as between 70 wt% to 80 wt% of 2’-FL, 10 wt% to 14 wt% of LNnT, and 11 wt% to 15 wt% of 6’- SL, or k) 60 wt% to 85 wt% of 2’-FL, 9 wt% to 15 wt% of 6’-SL, 8 wt% to 14wt% of LNnT and 3 wt% to 9 wt% 3’-SL, such as between 65 wt% to 75 wt% of 2’-FL, 10 wt% to 12 wt% of 6’-SL, 9 wt% to 12wt% of LNnT and 4 wt% to 8 wt% 3’-SL, or l) 60 wt% to 80 wt% of 2’-FL, 18 wt% to 28 wt% of LNT, and 3 wt% to 10 wt% of 3’-SL, such as between 65 wt% to 75 wt% of 2’-FL, 20 wt% to 23 wt% of LNT, and 4 wt% to 8 wt% of 3’-SL, or m) 60 wt% to 80 wt% of 2’-FL, 10 wt% to 30 wt% of LNT, and 5 wt% to 15 wt% of 6’-SL, such as between 62 wt% to 72 wt% of 2’-FL, 18 wt% to 25 wt% of LNT, and 9 wt% to 13 wt% of 6’- SL, or n) 55 wt% to 80 wt% of 2’-FL, 16 wt% to 24 wt% of LNT, 7 wt% to 14wt% of 6’SL and 3 wt% to 9 wt% 3’-SL, such as between, or 60 wt% to 70 wt% of 2’-FL, 18 wt% to 22 wt% of LNT, 9 wt% to 12 wt% of 6’SL and 4 wt% to 7 wt% 3’-SL, or o) 75 wt% to 95 wt% of 2’-FL, 5 wt% to 15 wt% of DFL, and 3 wt% to 10 wt% of 3’-SL, such as between 80 wt% to 90 wt% of 2’-FL, 8 wt% to 12 wt% of DFL, and 4 wt% to 8 wt% of 3’-SL, or p) 55 wt% to 70 wt% of 2’-FL, 5 wt% to 12 wt% of DFL, 15 wt% to 22 wt% of LNT, and 5 wt% to 15 wt% of LNnT, such as between 58 wt% to 65 wt% of 2’-FL, 6 wt% to 10 wt% of DFL, 18 wt% to 22 wt% LNT and 8 wt% to 12 wt% of LNnT, or q) 55 wt% to 75 wt% of 2’-FL, 5 wt% to 10 wt% DFL, 12 to 25 wt% of LNT, 5 wt% to 15 wt% 6’SL and 3 wt% to 8 wt% of 3’-SL, such as between 57 wt% to 62 wt% of 2’-FL, 6 wt% to 9 wt% DFL, 15 to 20 wt% of LNT, 8 wt% to 12 wt% 6’SL and 4 wt% to 6 wt% of 3’-SL, or r) 55 wt% to 75 wt% of 2’-FL, 5 wt% to 10 wt% DFL, 6 to 14 wt% of LNnT, 5 wt% to 15 wt% 6’SL and 3 wt% to 8 wt% of 3’-SL, such as between 60 wt% to 70 wt% of 2’-FL, 6 wt% to 9 wt% DFL, 8 to 12 wt% of LNnT, 9 wt% to 13 wt% 6’SL and 4 wt% to 6 wt% of 3’-SL, or s) 48 wt% to 60 wt% of 2’-FL, 12 wt% to 20 wt% 3-FL, 12 to 25 wt% of LNT, 5 wt% to 15 wt% 6’SL and 2 wt% to 8 wt% of 3’-SL, such as between 50 wt% to 60 wt% of 2’-FL, 14 wt% to 18 wt% 3-FL, 15 to 20 wt% of LNT, 7 wt% to 11 wt% 6’SL and 3 wt% to 5 wt% of 3’-SL, or t) 45 wt% to 65 wt% of 2’-FL, 4 wt% to 10 wt% DFL, 12 wt% to 21 wt% of LNT, 6 wt% to 14 wt% of LNnT, 5 wt% to 15 wt% 6’SL and 3 wt% to 8 wt% of 3’-SL, such as between 50 wt% to 60 wt% of 2’-FL, 5 wt% to 8 wt% DFL, 15 wt% to 20 wt% of LNT, 7 wt% to 10 wt% of LNnT, 7 wt% to 11 wt% 6’SL and 2 wt% to 6 wt% of 3’-SL, or u) 40 wt% to 60 wt% of 2’-FL, 10 wt% to 20 wt% 3-FL, 10 wt% to 20 wt% of LNT, 4 wt to 12 wt% of LNnT 4 wt% to 12 wt% 6’SL and 2 wt% to 8 wt% of 3’-SL, such as between 45 wt% to 55 wt% of 2’-FL, 12wt% to 18 wt% 3-FL, 12 wt% to 18 wt% of LNT, 6 wt to 10 wt% of LNnT, 6 wt% to 10 wt% 6’SL and 3 wt% to 6 wt% of 3’-SL.

[0138] In preferred embodiments the nutritional composition of the present disclosure comprises an HMO mixture selected from a) or c) or i) or k) or r) or s) or t) or u) above.

[0139] The production of HMOs is well known. See for example Bych et al., 2019, Current Opinion in Biotechnology, 56:130-137, for a review on HMO production. In a preferred embodiment, a HMO, e.g., 2’-FL or 3’-SL, may be produced synthetically, meaning it is produced ex vivo chemically and / or biologically, e.g., by means of a chemical reaction, an enzymatic reaction or from recombinant cell cultures. See, for example, the methods described in W02012 / 127410, WO2010 / 115934, WO2010 / 115935, WO2013 / 139344, WO2022 / 136337 or EP4239066.

[0140] The nutritional composition

[0141] In some embodiments, the nutritional composition is an infant formula, growing up formula, a dietary supplement or a medical nutrition product. The nutritional composition of the present disclosure is particular suitable for infant formula and growing up formula.

[0142] The nutritional composition may be provided as a powder, a dry composition or a gel comprising the probiotic(s) and the mixture of HMOs. 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. The composition may also be in the form of a kit of parts with the probiotic in one compartment and the mixture of HMOs in another compartment and an instruction describing the best intake form.

[0143] Thus, the third aspect of the invention relates to a kit of parts comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO, wherein, if 3’-sialyllactose (3’-SL) is present in the mixture, the ratio (w / w) of 2’-FL:3’- SL is above 7.5:1 , and wherein the probiotic and the mixture of HMOs are not mixed together.

[0144] The embodiments of the first aspect of the invention as defined herein apply mutatis mutandis to the third aspect. Therefore, any embodiment relating to the probiotic or the mixture of HMOs or any specific use of the nutritional composition of the first aspect applies mutatis mutandis to the kit of parts according to the third aspect.

[0145] 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. A low water content is preferred to avoid that the probiotic assimilates the HMOs if they are co-formulated in the composition.

[0146] For dietary supplements the dry composition may be formulated into a tablet, capsule or sachet / stick pack or a gummy.

[0147] The nutritional composition can additionally contain or be mixed with sources of protein, lipids, vitamins, minerals and / or digestible carbohydrates. The composition can be designed to be the sole source of nutrition or a nutritional supplement.

[0148] Probiotics / bacteria can be dried in multiple ways, e.g., spray drying, freeze drying (lyophilization), drum drying or fluid bed drying. It is advantageous to have the bacteria in a dry inactive state, since this prevent them from consuming carbohydrates they may be formulated together with, such as lactose, HMOs or GOS, e.g., in an infant formula.

[0149] The probiotics / bacterial cells can be regenerated or revived (also termed reconstitution) by rehydration either in the stomach or gut of the consumer or by suspending the nutritional formulation in a water containing liquid prior to consumption. Preferably, the bacterial cells are viable after the reconstitution. The viability of the bacteria following reconstitution may be assessed by spreading them on an agar plate with suitable growth medium and counting the number of colonies formed after incubation for a predefined time (plate counting). Alternatively, FACS analysis may be used.

[0150] Use of the synbiotic composition or combination

[0151] The present disclosure relates to one or more methods of providing a subject with a health benefit by administering the synbiotic composition disclosed herein. Furthermore, an effective amount of the synbiotic composition disclosed herein can be used in a method for treating a condition, disease, or disorder. A second aspect of the present disclosure relates to the nutritional composition according to the first aspect of the present disclosure or any embodiments thereof, for use in supporting or improving one or more of the following: a) gut health, including a healthy gut microbiome, and / or b) a healthy immune system, such as reducing risk of developing allergies, as well as reducing risk of inflammation, c) cognitive development, such as motor skills, learning, language skills, spatial cognition ability, and / or d) emotional and behavioral development, such as stress and anxiety reduction, in a subject.

[0152] In some embodiments, the subject is an infant, child, adult or elderly individual.

[0153] In some embodiments, the subject is a non-infant individual.

[0154] In some embodiments, the subject is a healthy individual.

[0155] In some embodiments, the composition increases short chain fatty acid, lactic acid, putrescine, gamma-aminobutyric acid (GABA), and / or dopamine formation in the gut.

[0156] 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 diarrhea. Using a composition of the present disclosure to support or maintain a healthy gut in a healthy individual not suffering from any diseases in the gut is a nonmedical use.

[0157] In some embodiments, the healthy gut microbiome is supported or improved in the subject by the provision of a nutritional composition as described herein and by promoting the growth of the probiotic in the composition and / or other beneficial bacteria in the gut, without promoting the growth of non-beneficial bacteria.

[0158] Put another way, in some embodiments the nutritional composition described herein is able to promote the B. infantis R0033 strain colonization in the gut of a subject when co-administered with the mixture of HMOs to said subject, and / or the nutritional composition is able to promote the growth in the gut of one or more individual beneficial bacteria already present in the gut of the individual, e.g. a Bifidobacterium species, such as a Bifidobacterium species selected from, without being limited to, B. adolescentis, B. angulatum, B. animalis, B. animalis subsp. animalis, B. animalis subsp. lactis, B. asteroides, B. biavatii, B. bifidum, B. breve, B. catenulatum, B. coagulans, B. longum, B. infantis, B. longum subsp. infantis, B. longum subsp. longum, B. magnum, B. coryneforme, B. dentium, B. gallicum, or B. subtile or a Bacteroides species such as Bacteroides fragilis, Bacteroides vulgatus, and Bacteroides thetaiotaomicron.

[0159] In embodiments the health of a gut microbiome can be assessed by the presence of beneficial bacteria such as the Bifidobacterium species disclosed above. In particular, an increase in the ratio of beneficial bacteria over pathogenic bacteria compared to said ratio prior to administration in of the nutritional composition is one way of assessing improved gut health. Examples of pathogenic microorganisms which may be present in the gut are Clostridium difficile, Escherichia coli, Clostridium perfringens, Shigella flexneri, Shigella dysenteriae, Salmonella, Staphylococcus aureus and Klebsiella.

[0160] The gut barrier function refers to the ability of the gut lining to act as a physical and functional barrier between the digestive system and the rest of the body. The strength of the tight junctions is another part of the gut barrier function. The tightness of the 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 be able to pass through the gut lining and into the bloodstream, resulting in higher levels of the molecule in the urine.

[0161] In some embodiments, the gut barrier function is improved or supported by an in situ increase in GABA levels in the gut.

[0162] 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.

[0163] In some embodiments, the immune system development is supported by an in situ increase in SCFA, GABA and / or putrescine levels in the gut, which causes anti-inflammatory effects.

[0164] In embodiments, a healthy immune system, such as a reduced risk of inflammation, 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 in of the nutritional composition. In further embodiments, the anti-inflammatory cytokines may comprise any one or more of TGF-pi , 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. 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.

[0165] 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.

[0166] In some embodiments, the reduced risk of developing allergies is useful in preventing development of allergies such as food allergy and drug allergy.

[0167] Cognitive development refers to the progressive growth and development of a person's thinking, reasoning, problem-solving, and decision-making abilities. This process starts from infancy and continues throughout adulthood.

[0168] 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.

[0169] 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.

[0170] 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).

[0171] Emotional development begins in infancy, with the development of basic emotions such as happiness, sadness, and fear. As children grow, they develop more complex emotions such as empathy, jealousy, and pride, and they learn to regulate their emotions in response to social cues and expectations. 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. 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 kit of parts 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.

[0172] Behavioural development involves the development of social skills and appropriate behaviours in different contexts. This includes the development of social skills such as communication, cooperation, and conflict resolution, as well as the ability to follow rules and norms of behaviour.

[0173] Embodiments of the use (in particular the non-medical use) of the present disclosure may for example be the use of the nutritional composition or a kit of parts of the present disclosure to support or improve cognitive, emotional and behavioral development by increasing GABA and / or dopamine in the gut and / or brain compared to its measured level prior to administration of the nutritional composition or the kit of parts.

[0174] In some embodiments, the cognitive development is improved or supported by an in situ increase in GABA and / or dopamine levels in the gut.

[0175] In some embodiments, the emotional and behavioral development, such as stress and anxiety reduction, is improved or supported by an in situ increase in GABA.

[0176] In some embodiments, the behavioural development is development of social skills.

[0177] The proper dosage of the nutritional composition of the present invention 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.

[0178] The required amount of the mixture of HMOs would generally be in the range from about 0.5 g to about 25 g per day, in certain embodiments 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.

[0179] 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. The dosage of the probiotic within the nutritional composition is expressed as colony forming units (CFU) / day. As used herein, CFU means the number of viable cells (i.e., probiotic cells which are able to multiply via binary fission under controlled conditions).

[0180] In some embodiments, the probiotic dose can be higher than T105and lower than T1012CFU / day.

[0181] In some other embodiments, the probiotic dose can be of at least T105, 1 -106, 2-106, 3- 106, 4- 106, 5- 106, 6- 106, 7- 106, 8- 106, 9- 106, 1 ■ 107, 2- 107, 3- 107, 4- 107, 5- 107, 6- 107, 7- 107, 8- 107, 9- 107, 1 ■ 108,

[0182] 2-108, 3-108, 4-108, 5-108, 6- 108, 7-108, 8- 108, 9- 108, 1 ■ 109, 2- 109, 3- 109, 4- 109, 5- 109, 6- 109, 7- 109, 8-109, 9-109, T1O10, 2-1O10, 3-1O10, 4-1O10, 5-1O10, 6-1O10, 7-1O10, 8-1O10, 9-1O10, T1011, 2-1011,

[0183] 3-1011, 4-1011, 5-1011, 6-1011, 7-1011, 8-1011, 9-1011, or at least 1 -1012, CFU / day.

[0184] 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.

[0185] A fourth aspect of the present disclosure relates to a kit of parts comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein: a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO, wherein, if 3’-sialyllactose (3’-SL) is present in the mixture, the ratio (w / w) of 2’-FL:3’-SL is above 7.5:1 , for use, such as non-medical use, in supporting or improving one or more of the following: a. gut health, including a healthy gut microbiome, and / or b. a healthy immune system, such as reducing risk of developing allergies, as well as reducing risk of inflammation, including preventing or reducing symptoms of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, and / or ulcerative colitis, and / or c. cognitive development, such as motor skills, learning, language skills, spatial cognition ability in a subject.

[0186] The use of the term “kit of parts” here has the same meaning as the term “combination” which is sometimes used. It indicates that each of the components - i.e., the probiotic and the mixture of HMOs, may be acquired as separate “parts” or compositions for later admixture before administration to the subject, or that each of the components may be administered separately to the subject for mixing in the gut. It is understood that a kit of parts comprising the same ingredients as the synbiotic compositions described herein can be applied for the same purposes / uses.

[0187] In some embodiments, the probiotic and the mixture of HMOs are to be administered separately to the subject.

[0188] For example, the probiotic can be administered 1 to 5 hours before the mixture of HMOs to allow the probiotic to start propagating in the gut prior to administering the mixture of HMOs.

[0189] The embodiments described above for the 1staspect of the present disclosure, relating to the probiotics and to the mixture of HMOs, respectively, apply mutatis mutandis to the 4thaspect of the present disclosure.

[0190] Furthermore, the embodiments described above for the 2ndaspect of the present disclosure also apply mutatis mutandis to the 4thaspect of the present disclosure.

[0191] A fifth aspect of the present disclosure relates to non-medical use of a nutritional composition according to the first aspect of the present disclosure or any embodiments thereof, for supporting or improving one or more of the following: a) gut health, including a healthy gut microbiome and / or gut motility, and / or b) immune system development, optionally in a healthy individual, and / or c) a healthy immune system, such as reducing risk of developing allergies, as well as reducing risk of inflammation, and / or d) cognitive development, such as motor skills, learning, language skills, spatial cognition ability, and / or e) emotional and behavioural development, such as stress and anxiety reduction, in a subject.

[0192] A sixth aspect of the present disclosure relates to non-medical use of the kit of parts according to the third aspect of the present disclosure or any embodiments thereof, for supporting or improving one or more of the following: a) gut health, including a healthy gut microbiome and / or gut motility, and / or b) immune system development, optionally in a healthy individual, and / or c) a healthy immune system, such as reducing risk of developing allergies, as well as reducing risk of inflammation, and / or d) cognitive development, such as motor skills, learning, language skills, spatial cognition ability, and / or e) emotional and behavioural development, such as stress and anxiety reduction, in a subject.

[0193] For the non-medical uses, the subject is preferably a healthy subject, preferably a healthy infant or child.

[0194] In the context of the non-medical use, it is not intended to treat any diseases that affect the health of the gut.

[0195] Reducing the risk of developing allergies, as well as reducing risk of inflammation are considered non-medical uses in the context of the present disclosure in so far that the subject is free of allergies and inflammatory conditions at the time when the subject is provided with / uses a nutritional composition of the present disclosure.

[0196] Supporting cognitive development is considered a non-medical use in the context of the present disclosure in so far that the subject is free from diseases that affect cognitive abilities at the time when the subject is provided with / uses a nutritional composition of the present disclosure.

[0197] Embodiments of the use (in particular the non-medical use) of the present disclosure may for example be the use of the nutritional composition of the present disclosure to support or improve gut health by a) increasing the ratio of beneficial bacteria over pathogenic bacteria in the gut compared to said ratio prior to administration of the nutritional composition; and / or b) increasing SCFAs and / or lactate in the gut compared to their measured level prior to administration of the nutritional composition; and / or c) increasing putrescine in the gut compared to its measured level prior to administration of the nutritional composition to reduce risk of diarrhea, such as non-infectious diarrhea, and / or d) increasing GABA in the gut compared to its measured level prior to administration of the nutritional composition.

[0198] Embodiments of the use (in particular the non-medical use) of the present disclosure may for example be the use of the nutritional composition of the present disclosure to support or improve the immune system by a) increasing SCFAs in the gut compared to their measured levels prior to administration of the nutritional composition; and / or b) increasing putrescine in the gut compared to its measured level prior to administration of the nutritional composition; and / or c) increasing GABA in the gut compared to its measured level prior to administration of the nutritional composition; and / or d) increasing one or more anti-inflammatory cytokines selected from the group consisting of TGF-pi, IL12, IL22, IL37, and IL38 compared to their measured levels prior to administration of the nutritional composition; and or e) maintaining balanced levels of immunoglobulins.

[0199] Embodiments of the use (in particular the non-medical use) of the present disclosure may for example be the use of the nutritional composition of the present disclosure to support or improve cognitive, emotional and behavioral development by: a) increasing GABA in the gut and / or brain compared to its measured level prior to administration of the nutritional composition; and / or b) increasing dopamine in the gut and / or brain compared to its measured level prior to administration of the nutritional composition.

[0200] In particular, such non-medical use can be in the form of a dietary supplement.

[0201] The embodiments described above for the 1staspect of the present disclosure, relating to the probiotic and to the mixture of HMOs, respectively, apply mutatis mutandis to the 5thaspect of the present disclosure.

[0202] The embodiments described above for the 2ndaspect of the present disclosure apply mutatis mutandis to the 5thaspect of the present disclosure.

[0203] EXAMPLES

[0204] Materials and methods

[0205] Materials:

[0206] Table 1: PD media recipe The probiotic strain was the Bifidobacterium longum ssp. infantis (B. in fa nt is) R0033 (CNCM I- 3424) strain from Lallemand Health Solutions (LHS). The dose used in all the experiments was T107CFU.

[0207] Preparations of the following human milk oligosaccharides (HMOs) were used:

[0208] 2’-Fucosyllactose (2’-FL; GLYCARE® 2FL 9000, dsm-firmenich), 3-fucosyllactose (3-FL; GLYCARE®3FL 9001 , dsm-firmenich), lacto- / V-neotetraose (LNnT; GLYCARE® LNnT 9000, dsm- firmenich), DFL (GLYCARE® 2’FL / DFL 8001 , dsm-firmenich), lacto- / V-tetraose (LNT; GLYCARE® LNT 8001 , dsm-firmenich), 3’-silyllactose (3’SL; GLYCARE® 3’-SL 9001 , dsm-firmenich), and 6’- silyllactose (6’SL; GLYCARE® 6’SL 9001 , dsm-firmenich).

[0209] Galacto-oligosaccharides (GOS) was obtained from FrieslandCampina (Biotis™ GOS-P).

[0210] In Examples 1 to 3 the oligosaccharides were dosed at 10g / L.

[0211] In Examples 5 to 7 the oligosaccharides were dosed in g / L according to Table 2 below.

[0212] Table 2: Oligosaccharide amounts (g / L) used in Examples 5 to 7

[0213] All oligosaccharides were prepared as 20% (w / v) stocks in H2O fi tered with a Nalgene syringe with a 0.2 pm filter (Thermo cat#723-2520). After individual stocks were made, mixtures of human milk oligosaccharides were combined into master stocks to test combinations.

[0214] Singapore infant fecal samples were sourced by Sequential Skin, Ltd (Singapore) and stored as 10% w / v in PBS:glycerol from a collection kit (Norgen Biotek Corp).

[0215] Experimental description:

[0216] Sample preparation

[0217] The probiotic strain was streaked on MRS media plates (Anaerobe Systems, cat#AS-6429) and grown anaerobically in a Coy chamber (Coy Laboratory Products) at 37°C for two days until single colonies were observed. Colonies were picked and inoculated into 10 mL of PD media (Anaerobe Systems cat#5255) with 1 % (v / v) lactose and grown for six to eight hours until an ODeoo between 0.2-0.5.

[0218] At the same time, 10% fecal slurry infant sample from two to three donors was diluted 1 :10 into PD media with 0.25% lactose (v / v). All samples were grown anaerobically at 37°C. Anaerobic chamber protocol (used in Examples 1 to 3)

[0219] After eight hours, the fecal slurry sample was diluted 1 :2 into fresh PD media (no sugar added). Samples were then prepped in triplicate replicates in 98 deep-well plates (Axygen, cat#P-2ML- SQ-CS) consisting of: probiotic diluted 1 :20 (5% v / v final), HMO or GOS (diluted 1 :10 to a final 1% v / v), and the remaining volume was filled with fecal slurry culture to 1 mL. Several wells were left empty as contamination controls. The plate was then sealed with a plate cover (Sigma-Aldrich, cat Z380059) and placed in an incubator / shaker (Benchmark Scientific) in the anaerobic chamber for 24 hours at 37°C. The samples were tested with two technical replicates.

[0220] After 24 hours the experiment was ended and samples were spun down at 3000 rpm for 10 minutes at 4°C. 200 pL of supernatant was removed from each well, added to a 0.2 pm filter plate (PALL cat#8582), and spun down at 1000 rpm for 5 minutes. The collected filtered supernatants were sent for LC-MS for short chain fatty acid analysis or for untargeted LC-MS using Hydrophilic interaction chromatography (HILIC) covering polar metabolites including organic acids, nucleic acids, amino acids, hydrophilic vitamins, phospholipids etc. and reverse phase LC-MS covering more non-polar metabolites, and lipids including mono-, di-, tri-acylglycerides, fatty acids, etc.

[0221] Biolector protocol (used in Examples 5 to 7)

[0222] After eight hours, the fecal slurry sample from three donors (not the same as used above) was diluted 1 :2 into fresh PD media (no sugar added). Samples were then prepped in triplicate replicates in 48 deep-well FlowerPlates (Beckman Coulter Life Sciences, cat#M2P-MTP-48- BOH1) consisting of: probiotic diluted 1 :20 (5% v / v final), HMO or GOS (diluted 1 :10 to final concentrations (see Table 2), and the remaining volume was filled with fecal slurry culture to 1 mL. Several wells were left empty as contamination controls. The plate was then sealed with a plate cover (Sigma-Aldrich, cat Z380059), covered with an anaerobic gassing lid (Beckman Coulter Life Sciences, Cat# E-AN-300), and placed in BioLector XT Microbioreactor (Beckman Coulter Life Sciences, cat# M2P-G-BLXT). Samples were fermented anaerobically for 24 hours at 37°C. The samples were tested with three technical replicates.

[0223] After 24 hours the experiment was ended and samples were spun down at 3000 rpm for 10 minutes at 4°C. 200 pL of supernatant was removed from each well, added to a 0.2 pm filter plate (PALL cat#8582), and spun down at 1000 rpm for 5 minutes. The collected filtered supernatants were sent for LC-MS for short chain fatty acid analysis or for targeted LC-MS using the MxP Quant 500 kit (Biocrates).

[0224] The metabolites were analysed from all three donors. Outliers for each metabolite were identified as samples that were greater than two standard deviations away from the mean of all data points relating to the specific metabolite. Outliers were removed and the average was calculated on the remaining data points from the data set. Based on outlier criteria, for some metabolites only data from two donors is represented. EXAMPLE 1

[0225] Testing composition with B. infantis R0033 and selected HMOs in mixed microbial communities to assess the effect on lactic acid and the short chain fatty acid (SCFA) acetic acid production

[0226] The purpose of this experiment was to test the effects of incubating infant fecal samples (mixed microbial communities) with compositions containing the probiotic strain Bifidobacterium longum ssp. infantis (B. infantis) R0033 (CNCM I-3424) and different human milk oligosaccharides (HMOs) on the production of lactic acid and the short chain fatty acid (SCFA) acetic acid.

[0227] The amount of acetic acid and lactic acid produced in infant fecal samples incubated with different compositions with B. infantis R0033 and different oligosaccharides is shown in Table 3.

[0228] Table 3: Acetic acid and lactic acid production in mixed microbial communities incubated with test ingredients as specified.

[0229] Addition of the acetic acid or lactic acid values of the single ingredients ’Difference to expected additive value: [(measured - expected) / expected] 100%

[0230] *T107CFU

[0231] As can be seen in Table 3, Mix3 and Mix4, comprising B. infantis R0033 and 2’-FL (Mix3), or B. infantis R0033 and 3-FL (Mix4), showed an acetic acid production which was respectively 68.7% and 41.3% higher than the expected additive amount. Thereby, these two combinations had a synergistic effect on acetic acid production. This effect was not shown for the combination of B. infantis R0033 with LNnT (Mix1) or B. infantis R0033 with GOS (Mix2), where the amount of acetic acid in the combination was actually decreased compared to the expected additive effect. When looking at the lactic acid production in Table 3, the compositions comprising B. infantis R0033 and 2’-FL (Mix3) or B. infantis R0033 and 3-FL (Mix4) showed a lactic acid production which was respectively 280% and 480% higher than the expected additive amount, an effect which was also not observed with GOS (Mix2) and LNnT (Mix3).

[0232] Fucosyllactose in combination with B. infantis R0033 therefore seems to provide synergistic effects for the production of desired acids (i.e. , acetic and lactic acids), which can be beneficial for the overall gut health and immune system.

[0233] EXAMPLE 2

[0234] Testing composition with B. infantis R0033 and selected HMOs in mixed microbial communities to assess effect on production of metabolites of interest

[0235] The purpose of this experiment was to test the effects of incubating infant fecal samples (mixed microbial communities) with compositions containing the probiotic strain Bifidobacterium longum ssp. infantis (B. infantis) R0033 (CNCM 1-3424) and different human milk oligosaccharides (HMOs). Interesting metabolites were analysed following untargeted LC-MS, and putrescine was identified as a metabolite which, when increased in situ in the gut following administration of the compositions disclosed herein, could lead to beneficial immunomodulatory effects.

[0236] The amount of putrescine produced in infant fecal samples incubated with different compositions with B. infantis R0033 and different oligosaccharides is shown in Table 4.

[0237] Table 4: Putrescine production in mixed microbial communities incubated with test ingredients as specified.

[0238] ’Change probiotic compared to no probiotic *T107CFU

[0239] As can be seen from Table 4, Mix6 and Mix7, comprising B. infantis R0033 and 2’-FL and B. infantis R0033 and 3-FL, respectively, had a significantly increased production of putrescine compared to the respective HMOs alone (Ref6 and Ref7, respectively). Mix5, comprising B. infantis R0033 and GOS, did not have the same pronounced effect over GOS alone (Ref8). EXAMPLE 3

[0240] Testing composition with B. infantis R0033 and selected HMOs in mixed microbial communities to assess effect on production of metabolites of interest - dopamine

[0241] The purpose of this experiment was to test the effects of incubating infant fecal samples (mixed microbial communities) with compositions containing the probiotic strain Bifidobacterium infantis R0033 (CNCM 1-3424) and different human milk oligosaccharides (HMOs). Interesting metabolites were analysed following untargeted LC-MS, and dopamine was identified as a metabolite which, when increased in situ in the gut following administration of the compositions disclosed herein, could lead to the development of cognitive functions.

[0242] The amount of dopamine produced in infant fecal samples incubated with various combinations of B. infantis R0033and / or different oligosaccharides is shown in Table 5.

[0243] Table 5: Dopamine production in mixed microbial communities incubated with test ingredients as specified. The results are based on the average of two donors with two technical replicates.

[0244] #T107CFU

[0245] *Change probiotic compared to no probiotic

[0246] ** The ratio of 2’-FL:LNnT:3’-SL is 16:4:1 total amount is the same as for the single HMOs

[0247] In the present example, the ability of the B. infantis R0033 strain to produce dopamine was not assessed. It can however be seen from Table 5 that the co-administration of B. infantis R0033 with fucosyllactose increased dopamine levels.

[0248] Compared to 2’-FL alone, its combination with B. infantis R0033 (Mix8) showed an 80% increase in the amount of dopamine produced. The same applied for the combinations involving B. infantis R0033 with 3-FL (Mix10) or 2’-FL, LNnT and 3’-SL (Mix11) where the dopamine levels were increased by 56 and 42%, respectively, when compared to the dopamine levels measured using HMOs alone. However, the combination of B. infantis R0033 with GOS (Mix8) showed only a very slight improvement in terms of dopamine production vs. GOS alone. It is worth mentioning that the mixture of 3 HMOs (Mix11) did not contain the same amount of 2’-FL as in the combination with 2’FL alone, which indicates that it is possible to adjust the dopamine levels by adjusting the amount of at least 2’-FL. This is considered an advantage since too high dopamine levels may negatively impact a child’s ability to concentrate, so mixtures of HMOs can potentially be tailored to produce the desired amount of dopamine.

[0249] EXAMPLE 4

[0250] Effects of a synbiotic formulation in gut health, in cognitive functions development and in overall health (i.e., immune system, etc.) in infants up to 24 months of age

[0251] Study design: randomized, double-blind, parallel, placebo-controlled trial

[0252] Phase: II

[0253] Sample size: 80 participants

[0254] 1. 40 participants on the claimed synbiotic composition, namely “2 g per day of a 12:1 [2’-FL : 3’-SL] HMO mixture + 2.109CFU per day of B. infantis R0033”,

[0255] 2. 40 participants on the placebo (i.e., base formula feeding).

[0256] Study length: 24 months

[0257] Visits: Baseline (Birth), 1 month, 3 months, 6 months, 12 months, 18 months and 24 months

[0258] Biological samples collected:

[0259] Mother: stool, blood, saliva, vaginal swab, nasal cavity, breastmilk.

[0260] Infant: umbilical cord blood, stool, saliva, blood (finger or heel prick), urine, nasal cavity.

[0261] Population (main inclusion criteria):

[0262] Healthy pregnant mothers and their healthy newborn;

[0263] Singleton pregnancy;

[0264] Birth weight of 2500g or more;

[0265] Gestational age 37-42 weeks (i.e., term pregnancy);

[0266] - Absence of diagnosed genetic disease or malformations during pregnancy.

[0267] Population (main exclusion criteria):

[0268] 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;

[0269] 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);

[0270] Chorioamnionitis, pre-eclampsia or hepatogestosis;

[0271] Suspected malformation or serious condition of the fetus / neonate.

[0272] Study Description:

[0273] It is hypothesized that the synbiotic formulation, consumed over a 12-month period, is able to increase the levels of short chain fatty acids (SCFAs) and lactic acid, of putrescine and of neurotransmitters, such as dopamine and GABA, in the gut of a subject, with beneficial effects for the subject’s health, when compared to a non-administered subject.

[0274] Study objectives:

[0275] Primary objective

[0276] To measure the levels of SCFAs and lactic acid in stools of infant participants, measured by: Change from baseline in stool SCFAs and lactic acid levels as measured by LC / MSMS.

[0277] Hypothesis’. The study participants who consume the synbiotic formulation have greater mean increase change in SCFA levels from baseline to 24 months of follow up than the study participants on the placebo.

[0278] Secondary objectives

[0279] Changes in inflammation and oxidative markers from blood and urine samples

[0280] Including, but not limited to, anti-inflammatory cytokines TGF-pi , 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)

[0281] Hypothesis’. The study participants who consume synbiotic 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.

[0282] Reduction in the incidence of colics

[0283] Comparison of the incidence of colics or inconsolable crying episodes between the intervention groups and placebo group.

[0284] Hypothesis: There is less incidence of colics or inconsolable crying episodes in the synbiotic groups than in the placebo group after 3 months of intervention.

[0285] Prevention of infections or allergic diseases

[0286] 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.)

[0287] 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 synbiotic group than in the placebo group after 24 months of follow up.

[0288] Metagenomics and metabolomics

[0289] Changes in microbiome profiles at baseline versus 3 months, 6 months, 12 months, 18 months and 24 months (composition, diversity, and metabolites of interest) o 16S rRNA gene sequencing and shotgun metagenomics o Strain specific qPCR assays (strain recovery in biological samples) o Targeted and untargeted metabolomics

[0290] Hypothesis’. There is, on average, greater presence of beneficial microbes and metabolites of interest, and / or support the establishment and development of the gut microbiome, in the synbiotic group compared to the placebo group after 24 months of follow up.

[0291] Gastrointestinal Health

[0292] 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)

[0293] Biomarkers of nutrients absorption (Arginine, Threonine, polyamines (putrescine, cadaverine) in feces)

[0294] Fecal inflammatory mediators: gastrointestinal function through the measurement of fecal inflammatory mediators

[0295] Fecal gut barrier function: gastrointestinal function through the measurement of Gl barrier function markers.

[0296] Hypothesis’. Participants in the synbiotic 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.

[0297] Cognition and mental health

[0298] Changes in cognitive status throughout intervention assessed by:

[0299] - Agpar score (newborns), Bayley Scales of Infant and Toddler Development (BSID), Cognitive Assessment of Young Children (CAYC)

[0300] Changes in mental health state including stress, sleep quality and anxiety assessed by: Mother’s daily / weekly diary

[0301] Biological markers (e.g., salivary slgA, alpha-amylase levels, cortisol, melatonin, gut neurotransmitters (e.g., levels of GABA, dopamine, serotonin in feces)

[0302] Hypothesis’. Participants in the synbiotic group have a better cognitive development than participants in the placebo group, after 24 months of follow up, when controlling fortheir baseline values.

[0303] Muscle Health and Growth Rate

[0304] Changes in muscle strength assessed by handgrip muscle strength (HGS) via dynamometer

[0305] - Antropometric measurements

[0306] Body composition (PEA POD) Hypothesis: Participants in the synbiotic 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.

[0307] Exploratory

[0308] - Infant fecal bacteria oligosaccharide consumption and infant fecal sialic acid concentrations

[0309] Hypothesis: Participants in the synbiotic 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 for their baseline values.

[0310] Safety objectives

[0311] To assess the safety and tolerability of a synbiotic formulation in the study population, measured by:

[0312] Growth rate and anthropometric measurements

[0313] 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

[0314] 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

[0315] - 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.

[0316] We do not expect a significant difference for AEs / SAEs or product tolerance between the synbiotic and placebo group.

[0317] Hypothesis: Participants in the synbiotic group 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.

[0318] EXAMPLE 5

[0319] Testing composition with B. infantis R0033 and selected HMO mixtures in mixed microbial communities to assess the effect on acetic acid production

[0320] The purpose of this experiment was to expand the knowledge obtained on individual HMOs in Example 1 to mixtures of different HMOs. In addition, the dosage of the HMOs in the present example were reduced to levels closer to what is used in infant formula today. It should also be noted that the fecal sample donors are different than in Example 1 , and therefore the microbial communities are different and the data in this example are therefore not directly comparable to the data in Example 1 .

[0321] In the present example the amount of acetic acid produced in infant fecal samples incubated with different compositions with B. infantis R0033 and different oligosaccharides is shown in Table 6.

[0322] Table 6: Acetic acid production in mixed microbial communities incubated with test ingredients as specified. Oligosaccharide amounts are in g / L. Amounts of the individual HMOs in the mixtures are specified in Table 2 in the method section.

[0323] #%change in sample with probiotic compared to non-probiotic [(mix - ref) / ref]- 100%

[0324] * T107CFU

[0325] As can be seen in Table 6, adding additional HMOs to 2’-FL (MixB, -C and -D) further increased the formation of acetic acid when combined with B. infantis R0033 as compared to 2’-FL (MixA), and all compositions with HMO and B. infantis R0033 are better than GOS and B. infantis R0033.

[0326] In particular the HMO blend with 5 HMOs in combination with B. infantis R0033 (MixD) resulted in a 13% increase in the amount of acetic acid produced compared to when the 5 HMO blend was used alone (Ref-V).

[0327] EXAMPLE 6

[0328] Testing composition with B. infantis R0033 and selected HMO mixtures in mixed microbial communities to assess effect on putrescine production

[0329] The purpose of this experiment was to expand the knowledge obtained on individual HMOs in Example 2 to mixtures of different fucosylated HMOs.

[0330] In addition, the dosage of the HMOs in the present example were reduced to levels closer to what is used in infant formula today. It should also be noted that the fecal sample donors are different than in Example 1 , and therefore the microbial communities are different. The data in this example are therefore not directly comparable to the data in Example 2. The amount of putrescine produced in infant fecal samples incubated with different compositions with B. infantis R0033 and different oligosaccharides is shown in Table 7.

[0331] Table 7: Putrescine production in mixed microbial communities incubated with test ingredients as specified. Oligosaccharide amounts are in g / L. Amounts of the individual HMOs in the mixtures are specified in Table 2 in the method section.

[0332] *T107CFU

[0333] As can be seen from Table 7, MixA and MixB, comprising B. infantis R0033 and 2’-FL alone or a combination of 2’-FL and DFL had an increase in the production of putrescine compared to the respective HMOs alone (Ref-ll and RefJII, respectively), and MixA and MixB appeared to produce slightly more putrescine than B. infantis R0033 in combination with GOS. Although the composition with B. infantis R0033 and 2’-FL and DFL (MixB) had a lower delta% relative to the HMO mix without B. infantis R0033 as compared to 2’-FL alone, it should be noted that the total putrescine product by MixB is higher than for MixA, which is also the case for the reference composition without B. infantis R0033. So, in conclusion the combination of 2’-FL and DFL with B. infantis R0033 seems to have a slight benefit over 2’-FL alone with B. infantis R0033.

[0334] EXAMPLE 7

[0335] Testing composition with B. infantis R0033 and selected HMO mixtures in mixed microbial communities to assess effect on production of metabolites of interest - gamma- aminobutyric acid (GABA)

[0336] The purpose of this experiment was to test the effects of incubating infant fecal samples with compositions containing the probiotic strain Bifidobacterium infantis R0033 (CNCM I-3424) and different mixtures of human milk oligosaccharides (HMOs). Selected metabolites were analysed using targeted LC-MS, and GABA was identified as a metabolite which, when increased in situ in the gut following administration of the compositions disclosed herein, could lead to the development of cognitive functions.

[0337] The amount of GABA produced in infant fecal samples incubated with various combinations of B. infantis R0033 and different oligosaccharide blends is shown in Table 8. Table 8: GABA production in mixed microbial communities incubated with test ingredients as specified. Oligosaccharide amounts are in g / L. Amounts of the individual HMOs in the mixtures are specified in Table 2 in the method section.

[0338] #T107CFU *%Change in probiotic compared to non-probiotic [(mix - ref) / ref]- 100%

[0339] As canbe seen from Table 8, the co-administration of B. infantis R0033 with any of the shown HMO mixtures increased the levels of GABA, whereas GOS in combination with B. infantis R0033 compared to GOS alone appeared to have a negative impact on GABA levels.

[0340] In particular the addition of LNnT to 2’-FL in combination with B. infantis R0033 (MixC and MixD) appeared to have a positive effect on GABA formation.

Claims

CLAIMS1 . A nutritional composition comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO, wherein, if 3’-sialyllactose (3’-SL) is present in the mixture, the ratio (w / w) of 2’-FL:3’- SL is above 7.5:1.

2. The nutritional composition according to claim 1 , wherein the additional fucosylated HMO is selected from 3-fucosyllactose (3-FL) and difucosyllactose (DFL), or is a mixture thereof.

3. The nutritional composition according to claim 1 or 2, wherein the total amount of fucosylated HMO constitutes between 60 to 100 wt% of the total mixture of HMOs, such as between 60 to 95 wt% of the total mixture of HMOs.

4. The nutritional composition according to any one of the preceding claims, wherein the neutral core HMO is selected from lacto-ZV-triose II (LNT-II), lacto-ZV-tetraose (LNT), and lacto-N- neotetraose (LNnT), or is any mixture thereof, preferably wherein the neutral core HMO is LNnT.

5. The nutritional composition according to any one of the preceding claims, wherein the sialylated HMO is a sialyllactose, optionally selected from 3’-sialyllactose (3’-SL) and 6’- sialyllactose (6’-SL), or is a mixture thereof, preferably wherein the sialyllactose is 3’-SL.

6. The nutritional composition according to any one of the preceding claims, wherein the mixture of HMOs constitutes at least 95 wt% of the total amount of HMOs in the nutritional composition, and wherein the mixture is selected from a) 2’-FL and DFL, or b) 2’-FL and 3-FL, or c) 2’-FL and LNnT, or d) 2’-FL, LNnT and 3’-SL, or e) 2’-FL, 6’-SL, LNnT and 3’-SL, or f) 2’-FL, DFL, LNT, 6’-SL and 3’-SL, or g) 2’-FL, 3-FL, LNT, 6’-SL and 3’-SL, orh) 2’-FL, DFL, LNT, LNnT, 6’-SL and 3’-SL, or i) 2’-FL, 3-FL, LNT, LNnT, 6’-SL and 3’-SL.

7. The nutritional composition according to any one of the preceding claims, wherein the mixture of HMOs constitutes at least 95 wt% of the total amount of HMOs in the nutritional composition, and wherein the mixture is selected from: a) 2’-FL and DFL, in a ratio (w / w) between 4-10:1 of 2’-FL:DFL, such as between 5-8:1 , or b) 2’-FL and 3-FL, in a ratio (w / w) between 2-4:1 of 2’-FL:3-FL, such as 3:1 , or c) 2’-FL and LNnT, in a ratio (w / w) between 8-2:1 of 2’-FL:LNnT, such as between 4-6.4: 1 , or d) 2’-FL, LNnT and 3’-SL, in a ratio (w / w) between 10-18:1-5:1 of 2’-FL:LNnT:3’-SL, such as between 12-16:2-4:1 , or e) 2’-FL, DFL, LNnT, 6’-SL and 3’-SL, in a ratio (w / w) between 8-16:1-3:0.5-4: 0.5-4: 1 of 2’- FL:DFL:LNnT:6’-SL:3’-SL, such as between 10-14:1-2:3-5:3-5:1 , or f) 2’-FL, 3-FL, LNT, 6’-SL and 3’-SL, in a ratio (w / w) between 10-16:2-6:2-6:1-5:1 , such as between 10-14:3-5:3-5:1-3:1 ; or g) 2’-FL, DFL, LNT, LNnT, 6’-SL and 3’-SL, in a ratio (w / w) between 10-16:0.5-4:2-6:1 :5:1- 5:1 of 2’-FL:DFL:LNT:LNnT:6’-SL:3’-SL, such as between 10-14:3-5:3-5:1-3:1-3:1 , or h) 2’-FL, 3-FL, LNT, LNnT, 6’-SL and 3’-SL, in a ratio (w / w) between 10-16:2-6:2-6:1 :5:1-5:1 of 2’-FL:3-FL:LNT:LNnT:6’-SL:3’-SL, such as between 10-14:3-5:3-5:1-3:1-3:1.

8. The nutritional composition according to any one of the preceding claims, wherein the nutritional composition is an infant formula or a dietary supplement.

9. The nutritional composition according to any one of the preceding claims, wherein 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.

10. The nutritional composition according to any one of the preceding claims for use in supporting or improving one or more of the following: a) gut health, including a healthy gut microbiome, and / or b) a healthy immune system, such as reducing risk of developing allergies, as well as reducing risk of inflammation, including preventing or reducing symptoms of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, and / or ulcerative colitis, and / or c) cognitive development, such as motor skills, learning, language skills, spatial cognition ability, and / or d) emotional and behavioral development, such as stress and anxiety reduction, in a subject.

11. The nutritional composition for the use according to claim 10, wherein the composition increases short chain fatty acid, lactic acid, putrescine, GABA and / or dopamine formation in the gut of the subject.

12. The nutritional composition for the use according to claim 10 or 11 , wherein the healthy gut microbiome is supported or improved in the subject by the provision of said composition and by promoting the growth of the probiotic in the composition and / or other beneficial bacteria, without promoting the growth of non-beneficial bacteria, in the gut of the subject.

13. The nutritional composition for the use according to claim 10 or 11, wherein the reduced risk of inflammation is supported by an increase in one or more anti-inflammatory cytokines selected from the group consisting of TGF-pi , IL12, IL22, IL37, and IL38.

14. The nutritional composition for the use according to any one of claims 10-13, wherein the subject is an infant, child, adult or elderly individual.

15. A kit of parts comprising a probiotic and a mixture of human milk oligosaccharides (HMOs), wherein a) the probiotic is the Bifidobacterium longum ssp. infantis R0033 strain (CNCM I-3424) or a strain having at least 97% average nucleotide identity with CNCM I-3424, and b) the mixture of HMOs comprises or consists essentially of 2’-fucosyllactose (2’-FL) and at least one additional HMO selected from i. an additional fucosylated HMO, and / or ii. a neutral core HMO, and / or iii. a sialylated HMO, wherein, if 3’-sialyllactose (3’-SL) is present in the mixture, the ratio (w / w) of 2’-FL:3’- SL is above 7.5:1 , and wherein the probiotic and the mixture of HMOs are not mixed together.

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