WO2025219496A1 - Composition nutritionnelle comprenant bifidobacterium longum ssp. infantis r0033 et des oligosaccharides de lait maternel - Google Patents

Composition nutritionnelle comprenant bifidobacterium longum ssp. infantis r0033 et des oligosaccharides de lait maternel

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Publication number
WO2025219496A1
WO2025219496A1 PCT/EP2025/060591 EP2025060591W WO2025219496A1 WO 2025219496 A1 WO2025219496 A1 WO 2025219496A1 EP 2025060591 W EP2025060591 W EP 2025060591W WO 2025219496 A1 WO2025219496 A1 WO 2025219496A1
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WIPO (PCT)
Prior art keywords
hmos
mixture
lnnt
nutritional composition
hmo
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PCT/EP2025/060591
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English (en)
Inventor
Ghislain Schyns
Leigh Morgan OUELLETTE
Sylvie BINDA
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Danstar Ferment AG
DSM IP Assets BV
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Danstar Ferment AG
DSM IP Assets BV
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Priority claimed from DKPA202430231A external-priority patent/DK202430231A1/en
Application filed by Danstar Ferment AG, DSM IP Assets BV filed Critical Danstar Ferment AG
Publication of WO2025219496A1 publication Critical patent/WO2025219496A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula

Definitions

  • the present invention relates to the field of prebiotics and probiotics.
  • 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.
  • 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.
  • 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.
  • FOS fructo-oligosaccharides
  • GOS galacto-oligosaccharides
  • HMOs human milk oligosaccharides Due to their ability to feed beneficial microorganisms and induce the growth or activity of these, certain oligosaccharides are referred to as prebiotics.
  • GOS is a 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.
  • 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 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.
  • HMOs Human milk oligosaccharides
  • human milk oligosaccharides 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.
  • 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.
  • W02009/077352 describes the prevention of opportunistic infections in immune-compromised infants or young children by combining various Bifidobacterium spp. with a fucosylated oligosaccharide.
  • 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.
  • 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.
  • SCFAs short chain fatty acids
  • 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.
  • CN 117337992 discloses a combination of Bifidobacterium infantis R0033 or Bifidobacterium bifidum R0071 and 2’-FL to decrease Salmonella in the gut.
  • An objective of the present disclosure is to provide a nutritional composition
  • 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.
  • SCFAs short chain fatty acids
  • GABA gamma-aminobutyric acid
  • dopamine levels in the gut of a subject
  • 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.
  • SCFAs short chain fatty acids
  • GABA gamma-aminobutyric acid
  • dopamine in the gut of a subject upon administration of the synbiotic combination.
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • Crohn’s disease Crohn's disease
  • a first aspect of the present invention relates to a nutritional composition
  • 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.
  • HMOs human milk
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • Crohn’s disease Crohn’s disease
  • 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.
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • HMOs Human milk oligosaccharides
  • 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.
  • 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.
  • 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.
  • lactose a disaccharide
  • 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”.
  • intestine 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”.
  • 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.
  • the subject is a healthy individual.
  • the subject is an infant.
  • the subject is a non-infant individual.
  • the subject is a healthy non-infant individual.
  • the subject is a human.
  • the human may be an infant.
  • 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.
  • the human is a non-infant, such as a child, an adult or an elderly individual.
  • a “child” is a human of 3 to 12 years of age
  • an “adult” is a human of at least 18 years of age
  • an "elderly individual” means a human of at least 60 years, preferably above 65 years, more preferably above 70 years of age.
  • the human is a healthy individual.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SCFAs short-chain fatty acids
  • 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.
  • 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.
  • Bifidobacterium longum ssp. infantis R0033 is a known producer of short chain fatty acids (SCFAs) and of SCFA precursors such as lactate/lactic acid.
  • SCFAs short chain fatty acids
  • 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.
  • 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
  • an indirect increase via the production of said metabolite(s) by the microbiota present in the gut of the subject 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.
  • Bifidobacterium longum ssp. infantis R0033 is a known producer of short chain fatty acids (SCFAs) and of SCFAs precursors such as lactate/lactic acid.
  • SCFAs short chain fatty acids
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • acetate produced by B. infantis becomes a carbon source that stimulates growth and function of butyrate-producing microbes, such as Faecalibacterium prausnitzii.
  • Lactic acid or lactate 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.
  • 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.
  • 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.
  • 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.
  • innate immunity the body's first line of defence, including physical barriers, including the gut barrier, phagocytes, and natural killer cells
  • adaptive immunity which involves highly specialized responses mediated by T cells and B cells.
  • 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.
  • 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.
  • GABA Gamma-Aminobutyric Acid
  • ADHD attention deficit hyperactivity disorder
  • 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.
  • 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.
  • innate immunity the body's first line of defence, including physical barriers, including the gut barrier, phagocytes, and natural killer cells
  • adaptive immunity which involves highly specialized responses mediated by T cells and B cells.
  • 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.
  • 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).
  • proinflammatory cytokines such as TNF-a, IL6 and IL8
  • MPK mitogen- activated protein kinase
  • 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.
  • ADHD attention deficit hyperactivity disorder
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to synbiotic compositions and/or combinations.
  • a first aspect of the present disclosure relates to a nutritional composition
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • HMOs human milk oligosaccharides
  • 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 or a strain having at least 97% average nucleotide identity with CNCM I-3424
  • 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.
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • 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.
  • the nutritional composition comprises at least one additional probiotic.
  • the additional probiotic microorganism is a species selected from Bifidobacterium longum, Bifidobacterium lactis and/or Bifidobacterium bifidum.
  • 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.
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • 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.
  • ANI average nucleotide identity
  • 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.
  • ANI average nucleotide identity
  • 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.
  • ANI nucleotide identity
  • ANI Average nucleotide identity
  • 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.
  • 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.
  • LHS 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
  • 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.
  • rhamnosus R0343 LHS
  • L. rhamnosus R1039 LHS
  • L. salivarius HA-118 LHS
  • L. lactis R1087 LHS
  • L. rhamnosus GG ATCC 53103
  • L. rhamnosus LGG DSM 33156, Chr. Hansen
  • 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.
  • 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
  • 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.
  • 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).
  • LHS E. faecium R0026
  • LHS E. faecium HA-127
  • 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).
  • LHS well-known strain P. acidilactici HA-524
  • 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).
  • LHS S. thermophilus R1018
  • LHS S. thermophilus R0083
  • LHS S. thermophilus HA-110
  • 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.
  • B. bifidum R0071 CNCM I-3426
  • B. bifidum HA-132 CNCM I-5898
  • 8. breve HA-129 CNCM I-5897
  • lactis LAFTI® B94 CBS 118529
  • 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.
  • 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).
  • B. infantis R0033 is provided in the form of viable cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ratio (w/w) of 2’-FL: 3’-SL is above 7.5:1 , such as above 8:1.
  • 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.
  • 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.
  • 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-lacto-N
  • the additional fucosylated HMO is selected from 3-fucosyllactose (3- FL) and difucosyllactose (DFL), or is a mixture thereof.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL and DFL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL and 3-FL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL and DFL.
  • 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).
  • LNT-II lacto-N- triose II
  • LNT lacto-N-tetraose
  • LNnT lacto-N-neotetraose
  • LNnH lacto-N-neohexaose
  • pLNnH para-lacto-N-neohexaose
  • pLNH para-lacto-N-hexao
  • 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.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL and LNnT.
  • the mixture of HMOs comprises or consists essentially of 2’-FL and LNT.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT and LNT.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and LNnT.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and LNnT.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and LNT.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNnT and LNT.
  • 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-N-neote
  • 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.
  • 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.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL and 6’-SL.
  • 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.
  • 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.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and 3’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, and 6’-SL.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, 3’- SL and 6’-SL.
  • 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.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT and 3’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNT and 3’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNnT, 3’- SL and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNT and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, LNT, 3’- SL and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNnT, 3’-SL and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNT, 3’-SL and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, DFL, LNnT, 3’-SL and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL.
  • the mixture of HMOs comprises or consists essentially of 2’-FL, 3-FL, LNnT, 3’-SL and 6’-SL.
  • 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.
  • 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%.
  • 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
  • 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.
  • the ratio of 2’-FL:DFL is 7.8:1. In more preferred embodiments, the ratio is 6.1 :1.
  • the ratio of 2’-FL:3’-SL is 16:1. In more preferred embodiments, the ratio is 11.5:1.
  • the ratio of 2’-FL:6’-SL is 8:1. In more preferred embodiments, the ratio is 5.8:1.
  • the ratio of 2’-FL:LNnT is 4:1. In more preferred embodiments, the ratio is 6.1 :1.
  • the ratio of 2’-FL:LNnT:3’-SL is 16:4:1.
  • the ratio is 12:2:1.
  • 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’
  • 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.
  • HMO HMO
  • 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.
  • the nutritional composition is a mixture of the nutritional composition.
  • 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.
  • 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.
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • 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.
  • 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.
  • the dry composition may be formulated into a tablet, capsule or sachet/stick pack or a gummy.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject is an infant, child, adult or elderly individual.
  • the subject is a non-infant individual.
  • the subject is a healthy individual.
  • the composition increases short chain fatty acid, lactic acid, putrescine, gamma-aminobutyric acid (GABA), and/or dopamine formation in the gut.
  • GABA gamma-aminobutyric acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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.
  • the health of a gut microbiome can be assessed by the presence of beneficial bacteria such as the Bifidobacterium species disclosed above.
  • beneficial bacteria such as the Bifidobacterium species disclosed above.
  • 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.
  • 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.
  • the gut barrier function is improved or supported by an in situ increase in GABA levels in the gut.
  • 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.
  • 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.
  • 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.
  • the anti-inflammatory cytokines may comprise any one or more of TGF-pi , IL12, IL22, IL37, and IL38
  • 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.
  • 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.
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • Crohn’s disease Crohn’s disease
  • ulcerative colitis ulcerative colitis
  • the reduced risk of developing allergies is useful in preventing development of allergies such as food allergy and drug allergy.
  • 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.
  • 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.
  • 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, ashamedy, 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.
  • psychological tests for anxiety and stress such as various self-reporting questionnaires, including Beck Anxiety Inventory, State-Trait Anxiety Inventory or Perceived Stress Scale.
  • 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.
  • 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.
  • the cognitive development is improved or supported by an in situ increase in GABA and/or dopamine levels in the gut.
  • the emotional and behavioral development such as stress and anxiety reduction, is improved or supported by an in situ increase in GABA.
  • the behavioural development is development of social skills.
  • 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.
  • 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.
  • CFU colony forming units
  • the probiotic dose can be higher than T10 5 and lower than T10 12 CFU/day.
  • the probiotic dose can be of at least T10 5 , 1 -10 6 , 2-10 6 , 3- 10 6 , 4- 10 6 , 5- 10 6 , 6- 10 6 , 7- 10 6 , 8- 10 6 , 9- 10 6 , 1 ⁇ 10 7 , 2- 10 7 , 3- 10 7 , 4- 10 7 , 5- 10 7 , 6- 10 7 , 7- 10 7 , 8- 10 7 , 9- 10 7 , 1 ⁇ 10 8 ,
  • 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 2 nd , 3 rd , 4 th or 5 th day 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.
  • 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.
  • HMOs human milk oligosaccharides
  • 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.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel disease
  • Crohn’s disease Crohn’s disease
  • ulcerative colitis and/or c.
  • cognitive development such as motor skills, learning, language skills
  • 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.
  • the probiotic and the mixture of HMOs are to be administered separately to the subject.
  • 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.
  • 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.
  • gut health including a healthy gut microbiome and/or gut motility
  • immune system development optionally in a healthy individual
  • a healthy immune system such as reducing risk of developing allergies, as well as reducing risk of inflammation
  • 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
  • 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.
  • gut health including a healthy gut microbiome and/or gut motility
  • immune system development optionally in a healthy individual
  • a healthy immune system such as reducing risk of developing allergies, as well as reducing risk of inflammation
  • 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
  • the subject is preferably a healthy subject, preferably a healthy infant or child.
  • 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.
  • 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.
  • 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.
  • 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.
  • such non-medical use can be in the form of a dietary supplement.
  • 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 T10 7 CFU.
  • HMOs human milk oligosaccharides
  • GOS Galacto-oligosaccharides
  • Examples 5 to 7 the oligosaccharides were dosed in g/L according to Table 2 below.
  • 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.
  • 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).
  • 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.
  • 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.
  • the fecal slurry sample from three donors 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.
  • 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.
  • B. infantis probiotic strain Bifidobacterium longum ssp. infantis
  • R0033 CNCM I-3424
  • HMOs human milk oligosaccharides
  • Table 3 Acetic acid and lactic acid production in mixed microbial communities incubated with test ingredients as specified.
  • 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.
  • desired acids i.e. , acetic and lactic acids
  • 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.
  • Table 4 Putrescine production in mixed microbial communities incubated with test ingredients as specified.
  • 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.
  • 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.
  • the mixture of 3 HMOs 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.
  • Mother stool, blood, saliva, vaginal swab, nasal cavity, breastmilk.
  • Infant umbilical cord blood, stool, saliva, blood (finger or heel prick), urine, nasal cavity.
  • 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;
  • anti-inflammatory cytokines TGF-pi IL12, IL22, IL37, and IL38
  • pro-inflammatory cytokines TNF-a, TNF- , IL1 , IL6, IL8, IL11, IL17, IL18, IFN-a, IFN-p and IFN-y
  • Malondialdehyde (MDA) Glutathione
  • GSH Glutathione
  • 8-Hydroxydeoxyguanosine 8- OHdG
  • Tryptophan Kynurenine
  • Kynurenine Total antioxidant capacity
  • Biomarkers of nutrients absorption (Arginine, Threonine, polyamines (putrescine, cadaverine) in feces)
  • Fecal inflammatory mediators gastrointestinal function through the measurement of fecal inflammatory mediators
  • Fecal gut barrier function gastrointestinal function through the measurement of Gl barrier function markers.
  • Bio markers e.g., salivary slgA, alpha-amylase levels, cortisol, melatonin, gut neurotransmitters (e.g., levels of GABA, dopamine, serotonin in feces)
  • 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.
  • CBC Complete Blood Count
  • CMP Comprehensive Metabolic Panel
  • Adverse events AEs
  • SAEs Serious adverse events
  • 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.
  • Example 1 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 .
  • 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.
  • the dosage of the HMOs in the present example were reduced to levels closer to what is used in infant formula today.
  • 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.
  • 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.
  • 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.
  • GABA production 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.

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Abstract

L'invention concerne une composition nutritionnelle comprenant un probiotique et un mélange d'oligosaccharides de lait maternel (HMO), le probiotique équivalant à la souche Bifidobacterium longum ssp. infantis R0033 (CNCM I-3424) ou une souche possédant au moins 97 % d'identité nucléotidique moyenne avec CNCM I-3424, le mélange de HMO comprenant ou étant constitué essentiellement de 2'-fucosyllactose (2'-FL) et d'au moins un HMO supplémentaire sélectionné parmi un HMO fucosylé supplémentaire, un HMO à noyau neutre et/ou un HMO sialylé. L'invention concerne en outre la composition nutritionnelle destinée à être utilisée pour supporter ou améliorer la santé intestinale et/ou un système immunitaire sain et/ou le développement cognitif, comme les compétences motrices, l'apprentissage, les compétences linguistiques, la capacité de cognition spatiale, chez un sujet.
PCT/EP2025/060591 2024-04-19 2025-04-16 Composition nutritionnelle comprenant bifidobacterium longum ssp. infantis r0033 et des oligosaccharides de lait maternel Pending WO2025219496A1 (fr)

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