Prebiotic Composition

Abstract

The present invention relates to a prebiotic composition for use in a method for maintaining or improving the health of a subject in need thereof, comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is an HMO non-responder; a food, a dietary supplement or a drink comprising a prebiotic composition for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder; and an inulin-type fructan (ITF) for use in a method for maintaining or improving the health of a subject in need thereof by administering a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is an HMO non-responder.The present invention also relates to the use of said prebiotic composition as a dietary supplement, food or drink for prebiotic non-responders. The present invention also relates to a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for the treatment of a mood disorder, preferably depression or anxiety, wherein a subject in need thereof is an HMO non-responder.

Description

[Technical Field]

[0001] The present invention relates to a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder; a food, dietary supplement, or beverage comprising the prebiotic composition for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder; and an inulin-type fructan (ITF) for use in a method for maintaining or improving the health of a subject in need thereof by administering a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is an HMO non-responder. The present invention further relates to the use of the prebiotic composition as a dietary supplement, food, or beverage for HMO non-responders. The present invention also relates to a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for the treatment of a mood disorder, preferably depression or anxiety, wherein the subject in need thereof is an HMO non-responder. [Background technology]

[0002] Human milk oligosaccharides (HMOs) are unique complex glucans found in human breast milk at high concentrations, particularly at 5-25 g / L HMO (Kunz et al. Annual review of nutrition, 20, 2000, 699-722).

[0003] HMOs are selective substrates for specific intestinal bacteria, primarily bifidobacteria, and protect against infection by blocking pathogen binding to epithelial cells, promoting immunomodulatory activity, and improving intestinal barrier function (Bode and Jantscher-Krenn, Advances in Nutrition, Bethesda, Md., 3 (3), 2012, 383S-91S). The most abundant structural class is fucosylated HMOs, accounting for 35%-50% of all HMOs, the most abundant of which is 2'-O-fucosyllactose (2'FL) (Han et al., The Journal of Nutrition, 151 (6), 2021, 1383-1393).

[0004] 2'FL has been identified as a substrate for Bifidobacterium spp., and therefore its supplementation leads to an increase in bifidobacteria (Fonvig et al., Journal of pediatric gastroenterology and nutrition, 73 (3), 2021, 408-414; Elison et al., The British journal of nutrition, 116 (8), 2016, 1356-1368). Higher bifidobacterial levels correlate with health and longevity, while reductions in this genus have been linked to irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and metabolic disorders (Arboleya et al., Frontiers in Microbiology, 7, 2016, 1204; Bhattarai et al., Gastrointestinal and Liver Physiology, 312 (1), 2017, G52-G62). Therefore, 2'FL is often added to food products or used as a dietary supplement to selectively elevate bifidobacterial levels and thus achieve improved health and physiological function. In particular, 2'FL is used in infant formulas and foods for infants, children, and adults.

[0005] The combination of HMOs and inulin-type fructans has been used, for example, to improve cognitive performance and influence the structural development of the olfactory bulb in pigs (Fleming et al., Nutrients, 12 (7), 2020). Furthermore, it is known that adding inulin to in vitro 2'FL fermentation with infant fecal inoculum promoted the fermentation of 2'FL (Akkerman et al., Food & Function, 12 (19), 2021, 9018-9029).

[0006] Depression is one of the most serious mental health disorders recorded worldwide, costing health services billions per year (Dieleman et al., Jama-Journal of the American Medical Association, 316(24), 2016, 2627-2646). As a result, there is a growing demand to find novel treatments not only to address the burden of disease but also to reduce the ever-increasing pressure on health systems (Liu, Walsh and Sheehan, Neuroscience and Biobehavioral Reviews, 102, 2019, 13-23). Although the mechanisms by which depression is regulated are not fully understood (Huang and Wu, Frontiers in Cell and Developmental Biology, 9, 2021), attention has been focused on the bidirectional relationship between the gut and the brain, coining the term gut-brain axis (Cryan et al., Lancet Neurology, 19(2), 2020, 179-194), and its potential for improving neurodevelopment, brain function, and cognitive abilities through modulation of neurological, immunological, or endocrine pathways (Morais, Schreiber, and Mazmanian, Nature Reviews Microbiology, 19(4), 2021, 241-255). Therefore, it stands to reason that one potential way to treat or reduce the risk of depression may be through targeted manipulation of the gut-brain axis. Prebiotics are an attractive tool for influencing the gut-brain axis, and HMOs such as 2'FL have recently been shown to be effective stimulators of changes in the gut microbiota.

[0007] However, it is known that a large proportion of individuals do not respond to interventions using HMOs such as 2'FL in terms of increasing the abundance of bifidobacteria. Even with a daily HMO dose of 10g, the responder rate was known to be less than 65% (Iribarren et al., Neurogastroenterology and motility: the official journal of the European Gastrointestinal Motility Society 32 (10), 2020, e13920). Because not everyone responds to supplementation with HMOs such as 2'FL, it is suggested that these people do not benefit from it in terms of health or physiological benefits (Healey et al., Nutrition reviews, 75 (12), 2017, 1059-1080).

[0008] One reason for the high rate of non-responders could be the underlying microbiota composition. 2'FL is primarily metabolized by bifidobacteria. However, not all bifidobacteria can degrade HMOs to the same extent (Jackson et al., Gut. Microb., 3, 2022, 61). Furthermore, the ability of the adult gut microbiota to utilize 2'FL may be influenced by factors present during infancy, such as the type of feeding, particularly breastfeeding versus formula feeding, and the mother's secretory habits. Depending on their genetic background, mothers secrete different sets of HMOs that may or may not contain 2'FL, which has implications for the gut microbiota composition and potentially function of their children (Smith-Brown et al., PloS one, 11 (9), 2016, e0161211). Whether these differences during infancy continue into adulthood is currently unknown but is conceivable, and would offer one explanation for the differences in response to 2'FL supplementation in this population. [Prior art documents] [Non-patent literature]

[0009] [Non-licensed document 1] Kunz et al. Annual review of nutrition, 20, 2000, 699-722 [Non-licensed document 2] Bode and Jantscher-Krenn, Advances in nutrition, Bethesda, Md., 3 (3), 2012, 383S-91S [Non-licensed document 3] Han et al., The Journal of nutrition, 151 (6), 2021, 1383-1393

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Summary of the Invention

[0010] Therefore, the technical problem underlying the present invention is to overcome the above-mentioned drawbacks.

[0011] In particular, the technical problem underlying the present invention is to provide a means for maintaining or improving the health of a subject in need thereof, which comprises administering at least one HMO, particularly 2'FL, to the subject, wherein the subject in need thereof is an HMO non-responder. In particular, the technical problem underlying the present invention is also to provide a means for reducing the high non-responder rate to dietary HMO, particularly 2'FL-based interventions. In particular, the technical problem underlying the present invention is also to provide a means for treating mood disorders, including depression and anxiety, preferably for improving the treatment of depression and / or anxiety, in a group of subjects, including in particular HMO non-responders. [Means for solving the problem]

[0012] The technical problem is solved by the teachings of the present invention, in particular by the teachings as set out in the independent and dependent claims and in the description.

[0013] In particular, the technical problem is solved by the use of a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for the treatment of a mood disorder in a healthy subject in need thereof, preferably wherein the healthy subject in need thereof is a healthy HMO non-responder.

[0014] In particular, the technical problem is solved by a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder.

[0015] It has been surprisingly found that such prebiotic compositions can be used to maintain or improve the health of subjects in need thereof, where the subjects in need thereof are HMO non-responders. Preferably, the subjects in need thereof are healthy subjects in need thereof. Thus, it has been surprisingly found that the prebiotic non-responder rate is reduced in a group of subjects, including HMO non-responders, supplemented with a composition for use according to the present invention compared to state-of-the-art compositions, particularly compositions containing HMO, particularly 2'FL, as the only prebiotic, particularly compositions consisting of HMO, particularly 2'FL. Advantageously, according to the present invention, HMO non-responder subjects in need of maintaining or improving their health can still be treated with HMO while simultaneously exhibiting a prebiotic response, i.e., an increased abundance of bifidobacteria. Thus, in a group of subjects who wish to ingest a diet containing HMO, particularly 2'FL, the number of subjects exhibiting a prebiotic response is significantly increased.

[0016] Thus, the compositions for use according to the present invention allow for the use of targeted combinations of prebiotics to reduce non-responder rates to dietary interventions.

[0017] Therefore, the technical problem of the present invention is solved by a prebiotic composition according to the present invention, which comprises at least one inulin-type fructan (ITF) on the one hand and at least one human milk oligosaccharide (HMO) on the other hand.The prebiotic composition according to the present invention is used in a method for maintaining or improving the health of a subject in need thereof, and the subject is an HMO non-responder.Preferably, the subject in need thereof is a healthy subject in need thereof.

[0018] In particular, the prebiotic composition comprising ITF and HMO according to the present invention solves the technical problem, and in particular maintains or increases the health of a subject who is an HMO non-responder, even if the subject is an HMO non-responder. Preferably, the subject in need thereof is a healthy subject in need thereof. Without wishing to be bound by theory, the maintenance or increase in the health of a subject who is an HMO non-responder by using a prebiotic composition according to the present invention is preferably due to an increase in the abundance of Bifidobacteria therein after use of the prebiotic composition according to the present invention for at least 1 to 28 days.

[0019] Preferably, the use of the prebiotic composition according to the present invention advantageously solves a further technical problem, namely, by combining at least one HMO with at least one ITF, preferably in a ratio of 1:4, compared with the use of state-of-the-art compositions, particularly compositions consisting of HMO, particularly 2'FL, thereby reducing the non-responder rate among HMO-consuming subjects, resulting in a reduction in prebiotic non-responders.Therefore, the advantage of the present invention is, in particular, a reduction in the non-responder rate among HMO-consuming subjects to a value less than 35% of the prebiotic non-responder rate relative to the total amount of prebiotic non-responders and responders.Therefore, the present invention provides the advantage that, due to the use of the prebiotic composition according to the present invention, the prebiotic non-responder rate is reduced, and therefore, more people can benefit from the use of HMO-containing compositions.Therefore, the present invention maximizes the chance of beneficial effects due to the supplementation of a prebiotic combination.

[0020] In particular, the technical problem is also solved by a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for maintaining or improving the health of a subject in need thereof, wherein the maintaining or improving the health is the treatment of a mood disorder, in particular depression or anxiety, and in particular wherein the subject in need thereof is an HMO non-responder.

[0021] Preferably, the subject in need thereof is a healthy subject in need thereof. Preferably, the mood disorder of a healthy subject in need thereof is therefore a mood change, hereinafter also referred to as "mood fluctuations", in the daily life of such a subject, reflecting internal or external stimuli on said mood, which mood change is of a reversible and temporary nature and does not reach an extent that requires therapeutic treatment.

[0022] Preferably, the subject in need thereof is a subject suffering from a psychiatric disorder in need thereof, particularly one suffering from a clinically recognizable form of depression or anxiety, such as major depression, persistent depressive disorder, bipolar disorder, etc. Thus, the mood disorder of a subject suffering from a psychiatric disorder in need thereof should preferably be classified as a medically clinically evident condition of a mood disorder, and is a mood disorder requiring therapeutic treatment.

[0023] It has surprisingly been found that a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) as disclosed herein can also be used to treat mood disorders, including depression and anxiety, in a subject, and therefore represents an advantageous and preferred embodiment of the present invention.The subject may be a healthy subject, or a subject suffering from a psychiatric disorder, particularly a clinical form of depression or anxiety.In a preferred embodiment, a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) can be used to improve such treatment compared to the use of state-of-the-art prebiotic compositions consisting of HMO, particularly 2'FL.

[0024] In particular, the technical problem of the present invention is solved by a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for the treatment of mood disorders, in particular depression or anxiety, in a subject in need thereof, preferably a healthy subject or a subject suffering from a psychiatric disorder.

[0025] In a preferred embodiment of the invention, the subject in need thereof is an HMO non-responder.

[0026] In a preferred embodiment of the invention, the subject in need thereof is a prebiotic responder.

[0027] Without wishing to be bound by theory, the improved treatment of mood disorders, including depression and anxiety, by using a prebiotic composition according to the invention is related to an increase in the abundance of bifidobacteria and therefore to the brain-gut connection in subjects using a prebiotic composition according to the invention.

[0028] In a preferred embodiment of the composition for use according to the invention, the inulin-type fructan is a short-chain ITF, in particular oligofructose (OF), preferably having a DP-AV (average degree of polymerization) of 3 to 5, preferably 4, or a long-chain ITF, in particular inulin, preferably having a DP-AV of at least 8, in particular 8 to 100, preferably 20 to 90, or a mixture thereof.

[0029] In a preferred embodiment of the composition for use according to the invention, the inulin-type fructans are preferably short-chain ITFs, in particular oligofructose (OF), with a DP-AV (average degree of polymerization) of 3 to 5, preferably 4, and 25% to 30% GFs with a degree of polymerization (DP) of 11 or more. n and F m compound (dry matter amount based on the total mass of carbohydrates) (each of n and m is at least 2, preferably independently at least 2), 15 to 30 wt% F mA fructan mixture comprising compounds (m=2-9) (dry matter based on the total mass of carbohydrates) as well as a fructan mixture having a DP-AV of 7-8, or preferably a long-chain ITF, in particular inulin, having a DP-AV of at least 8, in particular 8-100, preferably 20-90, or a mixture thereof.

[0030] In a preferred embodiment of the composition for use according to the invention, the inulin-type fructan is a short-chain ITF, in particular an oligofructose (OF), with a DP (degree of polymerization) in the range of 2-10, in particular 3-9.

[0031] In a preferred embodiment of the composition for use according to the invention, the short-chain ITF preferably has a DP mainly in the range of 2-7, with trace amounts, preferably less than 5%, more preferably less than 3%, of inulin with a DP of 8-9 (% being wt% of total ITF).

[0032] In a preferred embodiment of the composition for use according to the invention, the ITF comprises 25% to 30% GF having a degree of polymerization (DP) of 11 or more. n and F m compound (dry matter amount based on the total mass of carbohydrates) (each of n and m is at least 2, preferably independently at least 2), 15 to 30 wt% F m A fructan mixture containing compounds (m = 2-9) (dry matter based on the total mass of carbohydrates) as well as a fructan mixture with a DP-AV of 7-8.

[0033] In a preferred embodiment of the present invention, the fructan mixture comprises 80-100%, preferably 80-99%, preferably 82-98%, preferably 85-95%, preferably 87-92% GF n and F m Preferably, the fructan mixture comprises, preferably consists essentially of, preferably consists of, 0-20%, preferably 1-20%, preferably 2-18%, preferably 5-15%, preferably 8-13% monosaccharides and sucrose (each based on dry matter weight and total carbohydrate weight). Most preferably, the fructan mixture comprises 86-89 wt% GFn and F m compounds, as well as 11-14 wt% glucose, fructose, and sucrose (each based on dry matter weight and total carbohydrate mass).

[0034] In the context of the present invention, the monosaccharide is preferably glucose, fructose or a mixture thereof.

[0035] In a preferred embodiment of the invention, the fructan mixture of the invention does not contain galactose and / or an oligo- or polysaccharide comprising at least one galactose moiety.

[0036] In a preferred embodiment of the present invention, the fructan mixture is at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 90% GF n and F m The long-chain ITF preferably comprises a DP-AV of at least 23, with the remainder being sucrose and / or monosaccharides, preferably glucose, fructose, sucrose or mixtures thereof totaling 100% (each based on dry matter weight and total carbohydrate weight).

[0037] The ITF may be derived from any suitable source, particularly plant sources such as chicory or agave, or bacterial sources.

[0038] In a preferred embodiment of the composition for use according to the present invention, the short-chain ITF may be Orafti® L85, Orafti® L95, or Orafti® P95, all of which are oligofructose grades obtained by enzymatic hydrolysis of chicory inulin. Suitable oligofructose can also be obtained by enzymatic in vitro synthesis from sucrose by known methods, for example, according to U.S. Pat. No. 5,314,810. A suitable ITF is agave-derived ITF.

[0039] In a preferred embodiment of the composition for use according to the invention, the long-chain ITF has a DP greater than 20 and may contain up to 45% (% is wt% of total ITF) of inulin molecules with a DP of 10-20.

[0040] In long-chain ITF, the content of glucose, fructose and sucrose is preferably very low, in particular less than 2% (% being wt% relative to the total ITF).

[0041] In a preferred embodiment of the composition for use according to the invention, the long-chain ITF is chicory inulin Orafti® HP.

[0042] As intended herein, the determination of the average degree of polymerization ("DP-AV") in compositions containing ITF, particularly inulin, is based on AOAC Official Method 997.08 (2013 edition, AOAC International): As is known, this method first requires the determination of non-inulin-associated glucose and non-inulin-associated fructose in the composition: the concentrations of free glucose and fructose present in the inulin-containing composition, as well as the concentrations of glucose and fructose derived from sucrose, starch and / or maltodextrin, are determined.

[0043] Inulinase treatment of an inulin-containing composition will result in complete enzymatic hydrolysis of the inulin compound into free glucose and free fructose.

[0044] Inulin-associated fructose (F) was calculated by subtracting the previously determined non-inulin-associated concentrations of glucose and fructose from the overall fructose and glucose content determined after complete enzymatic hydrolysis of the inulin composition being analyzed. i ) and inulin-associated glucose (G i ) concentrations, particularly weight percentages, are provided.

[0045] Inulin in the composition is GF n When consisting of compounds, the DP-AV can be calculated via the formula as given in AOAC 997.08: DP-AV=[(F i / G i )+1] (1) If the inulin in the composition is an inulin product that has been subjected to partial hydrolysis, for example by treatment with endo-inulinase, it is no longer essentially GF. n It does not consist solely of compounds, but contains a significant amount of F m As a result, formula (1) is no longer accurate enough and requires modification. AOAC 997.08 provides some approximations for said modification.

[0046] In a preferred embodiment of the composition for use according to the present invention, the HMO is a neutral HMO. "Neutral human milk oligosaccharide" or "neutral HMO" refers to the complex carbohydrates found in human breast milk, preferably in neutral form. Neutral HMO can exist in the form of non-fucosylated and fucosylated oligosaccharides. Non-fucosylated neutral HMOs contain Glc, Gal, and GIcNAc and lack fucose and sialic acid, such as lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), lacto-N-hexaose (LNH), and paralacto-N-neohexaose (pLNnH). Fucosylated HMOs include, for example, 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose III (LNFP-III), fucosyl-paralacto-N-neohexaose (F-pLNnH), lacto-N-difucohexaose (F-pLNnH), and lacto-N-difucohexaose (F-pLNnH). The fucosyl-lacto-N-hexaose I (LNDFH-I), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-lacto-N-hexaose III (FLNH-III), and fucosyl-paralacto-N-neohexaose (F-pLNnH).

[0047] In a preferred embodiment of the composition for use according to the invention, the HMO is a neutral HMO, in particular a fucosylated lactose (FL), preferably 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL) or lactodifucotetraose (LDFT), or a mixture thereof, in particular 2'FL.

[0048] In a preferred embodiment of the composition for use according to the invention, the weight ratio of ITF to HMO is in the range of 20:1 to 1:20, preferably 20:1 to 1:1, preferably does not exceed 15:1, preferably does not exceed 12:1, more preferably does not exceed 10:1, preferably the weight ratio is 10:1 to 1:1, preferably 4:1 (each on a dry weight basis).

[0049] In a preferred embodiment of the composition for use according to the present invention, the composition comprises OF and 2'FL.

[0050] In a preferred embodiment of the composition for use according to the invention, the composition comprises not more than 2 g of HMO, in particular 2'FL.

[0051] In a preferred embodiment of the composition for use according to the invention, the composition comprises 8 g of OF and 2 g of HMO, in particular 2'FL.

[0052] In a preferred embodiment of the composition for use according to the invention, the composition consists of OF and 2'FL, in particular in a weight ratio of 4:1 (based on dry weight), in particular 8 g of OF and 2 g of 2'FL (each based on dry weight).

[0053] In a preferred embodiment of the composition for use according to the present invention, the method for maintaining or improving the health of a subject in need thereof is a method for treating a disorder associated with a disturbed composition or functionality of the intestinal microbiota, particularly for promoting the intestinal microbiota of the subject, and in particular for increasing the number of bifidobacteria, particularly B. longum, B. longum subsp., B. infantis, B. pseudocatenulatum, B. adolescentis, or a mixture thereof, in the gastrointestinal tract (GI tract), particularly the large intestine, of a subject. The disturbed composition or functionality of the intestinal microbiota is preferably characterized by bacterial overgrowth, dysbiosis, and / or an impaired mucosal barrier.

[0054] In a preferred embodiment of the composition for use according to the present invention, the method for maintaining or improving the health of a subject in need thereof is a method for increasing or stabilizing butyrate levels, increasing short chain fatty acid (SCFA) levels, or both, in the GI tract, particularly the large intestine, of the subject. SCFAs are two- to five-carbon weak acids, including acetate (C2), propionate (C3), butyrate (C4), and valerate (C5).

[0055] In a preferred embodiment of the composition for use according to the present invention, the method for maintaining or improving the health of a subject in need thereof is a method for the improvement of intestinal disorders, preferably constipation, elevated blood cholesterol levels, imbalanced intestinal flora, elevated intestinal pH, metabolic disorders, intestinal barrier dysfunction, inflammation of the GI tract, in particular inflammatory bowel disease, celiac disease; ulcerative colitis; Crohn's disease, depressive disorders, anxiety, mood disorders, sleep disorders; or any combination thereof.

[0056] In particularly preferred embodiments of the composition for use according to the present invention, the metabolic disorder is selected from overweight, obesity, metabolic syndrome, insulin deficiency or insulin resistance related disorders, diabetes, glucose intolerance, abnormal lipid metabolism, hyperglycemia, hepatic steatosis, dyslipidemia, high cholesterol and high triglycerides.

[0057] In a preferred embodiment of the composition for use according to the present invention, the composition is a synthetic composition. The term "synthetic composition" refers to a composition that is artificially prepared, preferably a composition that contains at least one compound that is produced chemically and / or biologically ex vivo, for example by chemical reaction, enzymatic reaction or recombinantly.

[0058] In a preferred embodiment of the composition for use according to the present invention, the composition is a nutritional composition.

[0059] In a preferred embodiment of the composition for use according to the present invention, the composition contains effective amounts of one or more HMOs and one or more ITFs.

[0060] The composition of the present invention, which comprises one or more HMOs and one or more ITFs, can be in any suitable form.For example, the composition of the present invention can be in the form of a nutritional composition that contains other macronutrients, such as protein, lipid and / or other digestible or indigestible carbohydrates.The composition of the present invention can also be a unit-dose incomplete nutritional composition or pharmaceutical composition.

[0061] The compositions of the present invention are also preferably in powdered or liquid form. The compositions can be the sole source of nutrients or a nutritional supplement.

[0062] Preferably, the nutritional composition is low in lactose, especially lactose-free.

[0063] In a preferred embodiment, the protein is milk protein, soy protein, rice protein, pea protein, oat protein, or a mixture thereof. The milk protein can be in the form of milk protein concentrate, whey protein, or casein, or a mixture of both. The soy, rice, pea, and oat proteins are preferably in isolated form. The protein can be a complete protein or a hydrolyzed protein, either partially hydrolyzed or extensively hydrolyzed. The protein can provide 5% to 50%, preferably 10% to 30%, of the energy of the nutritional composition.

[0064] In a preferred embodiment, suitable digestible carbohydrates include maltodextrin, hydrolyzed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, tapioca, sucrose, and glucose, or mixtures thereof. Preferably, the digestible carbohydrates provide 35% to 75%, preferably 45% to 70%, of the energy of the nutritional composition. Preferably, the digestible carbohydrates do not contain lactose.

[0065] In a preferred embodiment, suitable lipids include rapeseed oil, sunflower seed oil, palm oil, soybean oil, milk fat, corn oil, and soybean lecithin.Long-chain polyunsaturated fatty acids (LC-PUFA), particularly omega-3 fatty acids such as docosahexaenoic acid (DHA), can be used as lipids.Suitable sources of LC-PUFA are vegetable oils, marine plankton oils, fungal oils, and fish oils.The lipid can also be a medium-chain triglyceride (MCT).Fractional coconut oil is a suitable source of medium-chain triglycerides.The lipid source preferably provides about 5% to 25% of the energy of the nutritional composition; for example, 10% to 20%.

[0066] In a preferred embodiment, the compositions of the present invention also preferably contain vitamins and minerals. Preferred vitamins include vitamins A, B complex (such as B1, B2, B6 and B12), C, D, E and K, niacin and acid vitamins such as pantothenic acid and folic acid and biotin.

[0067] Preferred minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium and boron.

[0068] In a preferred embodiment, the compositions of the present invention may also include carotenoids such as lutein, lycopene, zeaxanthin and beta-carotene.

[0069] In a preferred embodiment, the compositions of the present invention also contain various other ingredients, such as preservatives, emulsifiers, thickeners, buffers, fibers and probiotics, preferably Lactobacillus casei strain Shirota, B. infantis 35624, B. animalis subsp. lactis BB-12, B. lactis Bi-07, L. rhamnosus GG, L. rhamnosus Lc705, L. plantarum DSM 9843, L. plantarum CECT7484, L. plantarum CECT7485, L. acidophilus NCFM, L. fermentum CECT5716, B. breve (B. breve Bb99, Propionibacterium freundenreichii ssp. Shermanii JS, P. acidilactici CECET7483, Streptococcus faecium, antioxidants / anti-inflammatory compounds including tocopherols, carotenoids, ascorbate / vitamin C, ascorbyl palmitate, polyphenols, glutathione, and superoxide dismutase (melon), other bioactive factors (e.g., growth hormone, cytokines, TFG-β), colorants, flavors, and stabilizers and / or lubricants.

[0070] In preferred embodiments, the compositions of the present invention may be in the form of a soluble powder, a liquid concentrate, or a ready-to-use formulation. Various fragrances, fibers, and other additives may also be present.

[0071] In a preferred embodiment, the compositions of the present invention can be prepared by any commonly used manufacturing technique for preparing nutritional compositions in solid or liquid form. For example, the compositions can be prepared from various nutritional feed solutions. For example, a protein-in-fat nutritional feed solution can be prepared by heating and mixing lipids, followed by adding an emulsifier, particularly lecithin, a fat-soluble vitamin, and at least a portion of an optional protein, while heating and stirring. A carbohydrate nutritional feed solution can also be prepared by optionally adding minerals, thickeners, or suspending agents to water while heating and stirring. The resulting solution is maintained with continued heating and stirring for, for example, 10 minutes before adding carbohydrates, particularly ITF and HMO. The resulting nutritional feed solution is then mixed with heating and stirring, and the pH is adjusted to 6.0-8.0, particularly 6.6-7.0. The composition is then preferably subjected to high-temperature, short-time processing, during which the composition is heat-treated, emulsified, and homogenized, followed by cooling. Optional water soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range, any flavorings are added if needed, and water is added to achieve the desired total solids level.

[0072] In a preferred embodiment, the composition of the present invention is in the form of a liquid product, preferably a nutritional composition packaged aseptically.Preferably, the nutritional composition of the present invention can be in the form of a ready-to-use or concentrated liquid.Preferably, the composition of the present invention is spray-dried, and can be processed and packaged as a powder that can be reconstituted.

[0073] In a preferred embodiment, the composition of the present invention is a ready-to-feed nutritional solution, wherein the total concentration of one or more HMOs in the solution is 0.002% to 3.0%, including 0.005% to 2%, including 0.05% to 1.0%, by weight of the solution, and the total concentration of one or more ITFs in the solution is 0.002% to 3.0%, including 0.005% to about 2%, including 0.05% to about 1.0%, by weight of the solution.

[0074] Where the nutritional product is a concentrated nutrient liquid, the total concentration of HMOs in the liquid is 0.004% to 6.0%, including 0.01% to 4.0%, including 0.1% to 2.0%, by weight of the liquid, and the total concentration of ITFs in the liquid is 0.004% to 6.0%, including 0.01% to 4.0%, including 0.1% to 2.0%, by weight of the liquid.

[0075] In a preferred embodiment, the compositions of the present invention comprise one or more additional ingredients such as diluents, excipients, antioxidants, lubricants, colorants, binders and / or disintegrants.

[0076] In a preferred embodiment, suitable diluents, excipients, lubricants, colorants, binders, and disintegrants include polyethylene, polyvinyl chloride, ethylcellulose, acrylate polymers and their copolymers, hydroxyethylcellulose, hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), or polyacrylamide (PA), carrageenan, sodium alginate, polycarbophil, polyacrylic acid, tragacanth, methylcellulose, pectin, natural gums, xanthan gum, guar gum, karaya gum, hypromellose, magnesium stearate, microcrystalline cellulose, and colloidal silicon dioxide. Suitable antioxidants are vitamin A, carotenoids, vitamin C, vitamin E, selenium, flavonoids, polyphenols, lycopene, lutein, lignans, coenzyme Q10, and glutathione.

[0077] In a preferred embodiment, the amount of the composition of the present invention required to be administered to a subject, particularly a patient, will vary depending on factors such as the severity of the disease, the patient's age, the form of the composition, and other nutrients and medications being administered to the patient. Preferably, the amount is in the range of 10 mg to 50 g per day, preferably 50 mg to 40 g per day, preferably 100 mg to 25 g per day, particularly 500 mg to 20 g per day, preferably 1 g to 10 g per day. During an optional initial first intervention phase, the amount can be higher, for example, 90 mg to 60 g per day, preferably 500 mg to 45 g per day, more preferably 1 g to 30 g per day, preferably 2.5 g to 15 g per day. During a second intervention phase, the dosage can be reduced, for example, to 20 mg to 20 g per day, preferably 100 mg to 10 g per day, more preferably 500 mg to 7.5 g per day, or in certain embodiments, to 750 mg to 5 g per day.

[0078] In a preferred embodiment of the composition for use according to the present invention, the composition is for oral administration, preferably for ingestion by a subject in an amount providing at least 0.01 g of prebiotic composition per kg of body weight per day for a period of at least 3 days.

[0079] In a preferred embodiment of the composition for use according to the present invention, the composition additionally comprises at least one additive selected from the group consisting of amino acids; peptides; proteins; lipids; vitamins; carbohydrates; flavorings; minerals; anabolic nutrients; antioxidants; probiotic microbial strains; diluents, excipients, antioxidants, lubricants, colorants, binders, disintegrants, colorants; preservatives, bulking agents; pharmaceutical agents and any combination thereof.

[0080] In particularly preferred embodiments of the composition for use according to the present invention, the probiotic microbial strains are in the form of viable microorganisms, non-viable microorganisms, microbial fragments and combinations thereof.

[0081] In a preferred embodiment of the composition for use according to the present invention, the composition is an ingredient of a food or drink or dietary supplement, or is in the form of a capsule, tablet, powder.Thus, in a preferred embodiment, the composition of the present invention may be in a unit dosage form such as a capsule, tablet or sachet, in particular a single-dose sachet.

[0082] In a preferred embodiment of the composition for use according to the present invention, the prebiotic composition is a dietary supplement, a food or a drink, or is part of any of these.

[0083] In a further aspect of the present invention, the present invention also relates to a food, dietary supplement or drink as defined in the present invention, in particular for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder, comprising a prebiotic composition as defined in the present invention.

[0084] In a further aspect, the present invention also relates to an inulin-type fructan (ITF) for use in a method for maintaining or improving the health of a subject in need thereof by administering a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is an HMO non-responder, and thus the ITF confers the benefits of the present invention over HMO-containing compositions.

[0085] In a further aspect, the present invention also relates to the use of a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), as specified in the present invention, as a dietary supplement, food or drink for HMO non-responders.

[0086] In a further aspect of the invention, the invention also relates to a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder, wherein: - at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) are orally administered to a subject in need thereof; or - a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), as specified in the present invention, as a dietary supplement, food or drink, is orally administered to a subject in need thereof.

[0087] In a further aspect of the invention, the invention also relates to a food, dietary supplement or drink, in particular for use in a method for the treatment of a mood disorder, in particular depression or anxiety, in a subject in need thereof, as defined in the present invention, comprising a prebiotic composition as defined in the present invention.

[0088] In a further aspect, the present invention also relates to an inulin-type fructan (ITF) for use in a method for treating mood disorders, particularly depression or anxiety, in a subject in need thereof by administering a prebiotic composition comprising at least one human milk oligosaccharide (HMO), i.e., the ITF thus confers the advantages of the present invention over HMO-containing compositions.

[0089] In a further aspect, the present invention also relates to the use of a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), as specified in the present invention, as a dietary supplement, food or drink for subjects suffering from mood disorders, in particular depression or anxiety.

[0090] In a further aspect of the invention, the invention also relates to a method for the treatment of a mood disorder, in particular depression or anxiety, in a subject in need thereof, comprising: - at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) are orally administered to a subject in need thereof; or - a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), as specified in the present invention, as a dietary supplement, food or drink, is orally administered to a subject in need thereof.

[0091] In a preferred embodiment of the invention, the subject in need of treatment for a mood disorder, particularly depression or anxiety, is an HMO non-responder.

[0092] In a preferred embodiment of the present invention, the subject in need of treatment for a mood disorder, particularly depression or anxiety, is a prebiotic responder.

[0093] In the context of the present invention, the term "prebiotic composition of the present invention" relates to a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), preferably as further specified in accordance with the teachings herein.

[0094] In the context of the present invention, the term "HMO non-responder" is understood to mean a subject, particularly a human or animal, that does not show a significant increase, particularly no increase, in the abundance of bifidobacteria, especially in the subject's digestive tract, in response to an intervention that includes oral ingestion of a prebiotic composition that contains at least one HMO, particularly 2'FL, as the only prebiotic component in the composition, or a composition that consists of HMO, particularly 2'FL.Thus, an HMO non-responder does not show a significant increase, particularly no increase, in the abundance of bifidobacteria, in response to a dietary intervention in the form of a diet that consists of HMO, particularly 2'Fl, or a diet that contains HMO, particularly 2'Fl, and at least one additional component that is not a prebiotic.Preferably, an HMO non-responder is a subject, particularly a member of the group of HMO non-responders and HMO responders.

[0095] In the context of the present invention, the term "prebiotic responder" is understood to mean a subject, in particular a human or animal, that shows a significant increase in the abundance of bifidobacteria, in particular in the subject's digestive tract, in response to an HMO intervention involving oral ingestion of a prebiotic HMO-containing composition that contains at least one additional prebiotic in addition to an HMO. In the context of the present invention, a "prebiotic responder" is a subject that is characterized by a significant increase in the abundance of bifidobacteria after 28 days of HMO intervention, in particular based on the abundance of bifidobacteria on day 0 of the intervention.

[0096] In the context of the present invention, "HMO intervention" is the oral ingestion of a prebiotic composition comprising at least one HMO, in particular one HMO, in particular 2'FL, which contains at least one further prebiotic in addition to the HMO.

[0097] In the context of the present invention, the term "prebiotic response" is understood to mean an increase in the abundance of bifidobacteria, particularly in the digestive tract of a subject, in response to an HMO intervention involving oral ingestion of a prebiotic HMO-containing composition which contains at least one further prebiotic in addition to an HMO.

[0098] In the context of the present invention, a "significant increase in the abundance of bifidobacteria" is understood to mean an increase in the abundance of bifidobacteria of at least 100%, preferably at least 120%, preferably at least 150% after 28 days, based on the abundance of bifidobacteria on day 0 of the intervention.

[0099] In the context of the present invention, "constant bifidobacterial abundance" is understood to mean an equal bifidobacterial abundance or an increase in bifidobacterial abundance of less than 100% after 28 days based on the bifidobacterial abundance on day 0 of the intervention.

[0100] In the context of the present invention, the "abundance" of Bifidobacteria is preferably observed in a fecal sample.

[0101] In the context of the present invention, the term "prebiotic non-responder" is understood to mean a subject, particularly a human or animal, that does not show a significant increase in the abundance of bifidobacteria, particularly in the subject's digestive tract, in response to HMO intervention, which comprises the oral ingestion of a prebiotic HMO-containing composition that contains at least one additional prebiotic in addition to HMO.In the context of the present invention, "prebiotic non-responder" is a subject that is characterized by a constant bifidobacteria abundance or a decrease in bifidobacteria abundance after 28 days of HMO intervention, particularly based on the bifidobacteria abundance on day 0 of intervention.Preferably, prebiotic non-responder is a subject, particularly a member of the group of prebiotic non-responder and prebiotic responder.

[0102] In the context of the present invention, subjects were defined as non-responders (NR) and responders (RES) based on the differential changes seen in the bifidobacterial response to the HMO intervention, as recognized by comparing the abundance of bifidobacteria before the intervention with the abundance of bifidobacteria after the HMO, particularly 2'FL, intervention.

[0103] By definition, RES are classified as subjects who show a prebiotic response to the intervention that is an increase in abundance, whereas in contrast, NR are classified as subjects who show a less significant prebiotic response, with a decreased or constant abundance of Bifidobacteria observed after the HMO intervention compared to pre-intervention abundance, and in particular no prebiotic response.

[0104] In preferred embodiments of the compositions for use, uses and methods of use according to the present invention, the subject, in particular the responder and / or non-responder, is a human or animal, in particular a human, in particular an adult human or animal, in particular an adult human.

[0105] Preferably, the "mood disorder" may be a mood disorder in a mentally healthy, particularly healthy, subject, or in a subject suffering from a mental disorder.

[0106] Preferably, a "mood disorder in a healthy subject" is a mood change or mood fluctuation in the daily life of such a subject that reflects internal or external stimuli to said mood, which mood change is of a reversible and temporary nature and does not reach an extent that requires therapeutic treatment. A "mood disorder in a healthy subject" preferably does not show any clinically evident symptoms of psychosis. Therefore, treatment of a "mood disorder in a healthy subject" is preferably a non-therapeutic treatment. Therefore, treatment of a mood disorder in a healthy subject in need thereof results in a good mood or feeling, and may preferably be referred to as "encouraging" the subject.

[0107] Preferably, the "mood disorder in a subject suffering from a mental disorder" is a mood disorder that should be classified as a medically and clinically evident condition of mood disorder and requires therapeutic treatment. Such disorders are preferably clinically recognizable forms of depression or anxiety, such as major depression, persistent depressive disorder, bipolar disorder, etc. Therefore, the treatment of the "mood disorder in a subject suffering from a mental disorder" is preferably a therapeutic treatment aimed at curing the ill subject and bringing about a mentally healthy state.

[0108] In the context of the present invention, "reduced non-responder rate" is understood to mean a reduction in the number of "prebiotic non-responders" in a first group of subjects subjected to a dietary HMO intervention involving oral ingestion of a prebiotic HMO-containing composition containing at least one further prebiotic in addition to HMO, compared to the number of "prebiotic non-responders" in a second group of subjects subjected to a comparative dietary HMO intervention, which particularly involves oral ingestion of a prebiotic composition comprising HMO as the only prebiotic, particularly 2'FL as the only prebiotic, or a composition consisting of HMO, particularly 2'FL.

[0109] In the context of the present invention, "prebiotics" refer to substances or mixtures of substances that are non-digestible and resistant to degradation by gastric acid and enzymes in the human digestive tract, are fermented by microorganisms on or in the body, and stimulate the growth and activity of beneficial bacteria, particularly bifidobacteria. Specifically, ITFs, HMOs, and galactans are substances that stimulate the activity and growth of beneficial bacterial colonies in the intestine. Inulin-type fructans (ITFs) can be short-chain ITFs, particularly long-chain ITFs such as oligofructose (OF) or inulin, preferably with a DP-AV (average degree of polymerization) of 3 to 5, while galactans are composed of galactooligosaccharides. Other prebiotics include resistant starch, pectin, β-glucan, and xylooligosaccharides.

[0110] In the context of the present invention, the term "oral administration" preferably refers to any conventional form for oral delivery of a composition to a patient that results in placement of the composition in the patient's gastrointestinal tract. Oral administration therefore includes swallowing of the composition by the patient or enteral feeding via a nasogastric tube.

[0111] In the context of the present invention, the term "effective amount" preferably refers to an amount of a composition of the present invention that provides the composition in an amount sufficient to provide a desired therapeutic result in a patient. An effective amount can be administered to a patient in one or more doses to achieve the desired therapeutic result.

[0112] In the context of the present invention, the term "use" as referred to in the context of a method of treatment of the compositions and / or ITFs of the present invention themselves means, in particular, advertising, marketing, teaching presentations of the compositions and / or ITFs of the present invention, or prescribing or selling or administering, in particular ingestion, thereof, or methods of using any one of them to a patient, such as those made clear by, for example, prescription by a physician, packaging, packaging leaflets, information of a medical professional, expert or physician or pharmacist counseling, written or oral expert information, including on the internet, all of which include off-label or cross-label uses.

[0113] In a preferred embodiment, "inulin" is considered to be a mixture of oligosaccharides and polysaccharides composed of fructose (F) units, which may or may not contain a starting glucose moiety (G). Thus, in the context of the present invention, the term inulin refers to GF n (Glucose-fructose n or Glu-Fru n ) compound and F m (Fructose m or Fru m where n and m are the number of fructose units in the oligosaccharide or polysaccharide, and where each of n and m is greater than 2.

[0114] When quantitative indications of ingredients of a product or composition, in particular percentages, are given in the context of the present invention, these, together with other explicitly stated or technically obvious further ingredients of the composition or product, add up to 100% of the composition and / or product, unless expressly stated or technically obvious otherwise.

[0115] In the context of the present invention, the terms "a" or "the" are understood to mean a single element or multiple elements.

[0116] In the context of the present invention, the term "at least 1" is understood to mean a quantity that represents a number such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10. In a particularly preferred embodiment, the term "at least 1" can represent the exact number 1. In another preferred embodiment, the term "at least 1" can also mean 2 or 3 or 4 or 5 or 6 or 7.

[0117] In the context of the present invention, when "comprising," "containing," "including," "having," or "content" of an ingredient is expressly mentioned or implied, this means that the respective ingredient is present, and in particular present in a measurable amount.

[0118] In the context of the present invention, the term "composition" means a substance (when reference is made to a composition consisting of X) or a mixture of substances (when reference is made to a composition that comprises or contains or has or includes X).

[0119] In the context of the present invention, when the "presence", "containing" or "content" of an ingredient is expressed, mentioned or implied in an amount of 0 [units], in particular mg / kg, μg / kg or wt%, this means that the respective ingredient is not present, in particular absent, in any measurable amount.

[0120] The number of decimal places indicated corresponds to the precision of the measurement method used in each case.

[0121] In the context of the present invention, if the tenths and tenths or two decimal places are not indicated for a number, the latter shall be set to zero.

[0122] In the context of the present invention, the terms "comprising," "including," and "containing" are understood to mean that in addition to the elements explicitly covered by these terms, other elements not explicitly mentioned can be added. In the context of the present invention, these terms also mean that only the elements explicitly mentioned are covered and that no additional elements are present. In this particular embodiment, the meaning of the terms "comprising" and "containing" is synonymous with the term "consisting," which means that the referenced elements are the only elements present, thereby excluding the presence of additional elements. Furthermore, the terms "comprising," "comprising," and "containing" also cover compositions that, in addition to the elements explicitly mentioned, also contain additional elements not mentioned but that are of a functionally and qualitatively auxiliary nature. In this embodiment, the terms "comprising" and "containing" are synonymous with the term "consisting essentially of."

[0123] In the context of the present invention, the term "and / or" is understood to mean that all members of the groups connected by the term "and / or" are represented both cumulatively with respect to one another and alternatively with respect to one another in any combination. Illustratively, with regard to the expression "A, B and / or C", the following disclosure is to be understood accordingly: (i) (A or B or C), or (ii) (A and B), or (iii) (A and C), or (iv) (B and C), or (v) (A and B and C), or (vi) (A and B or C), or (vii) (A or B and C), or (viii) (A and C or B).

[0124] In the context of the present invention, the individual components or constituents of a composition or of one of its components, determined quantitatively in relative form, in particular as a percentage, preferably add up to 100% by weight of the respective composition or composition or, if referenced, of that component, unless otherwise specified.

[0125] Further preferred embodiments of the invention are evident from the dependent claims.

[0126] The present invention is explained in more detail in the following examples and the accompanying figures, both of which should not be understood as limiting. [Brief explanation of the drawings]

[0127] [Figure 1] FIG. 1 shows the study design of the human intervention study according to Example 1. [Figure 2] Three figures, each showing the results of Flow-FISH measurements of probes specific for Bifidobacterium species in one of the three study groups. [Figure 3] FIG. 1 shows a bar graph of non-responder rates in the placebo group, the 2′FL group and the prebiotic composition according to the invention group. [Figure 4] FIG. 1 is a diagram of responder status on Beck's Depression Inventory and depression scores of study groups in relation to the effect of the intervention. [Figure 5] FIG. 1 shows BDI scores for all participants who received placebo, 2′FL, or a combination of 2′FL and P95. [Figure 6] FIG. 1 shows State-Trait Anxiety Inventory (STAI) Y1 (state) scores for all participants who received placebo, 2′FL, or a combination of 2′FL and P95. [Figure 7] FIG. 1 shows State-Trait Anxiety Scale (STAI) Y2 (trait) scores for all participants who received placebo, 2′FL, or a combination of 2′FL and P95. [Figure 8] FIG. 1 shows the effect of intervention on current anxiety scores (State-Trait Anxiety Scale) and responder status. [Figure 9] FIG. 1 shows the effect of the intervention on general anxiety scores (State-Trait Anxiety Scale) and responder status. [Figure 10] FIG. 1 shows the results of in vitro batch fermentation of various substrates either alone or in combination (1:1 ratio)—effect on Bifidobacterium counts expressed as change from baseline. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0128] Example 1: Human intervention study: Reducing non-responder rates by combining HMO with inulin-type fructans 1.1 Study design and participants A human intervention study was conducted to detect and compare changes in non-responder and responder rates in human subjects supplemented with a composition comprising 2'FL or a prebiotic composition according to the present invention. The human clinical trial design was a parallel, placebo-controlled, double-blind, randomized controlled trial (RCT) lasting 4 weeks, beginning with a 1-week run-in period and a 4-week intake period without any test product, as shown in Figure 1. Each study group included 23 participants.

[0129] 1.1.1 The study was given positive ethical approval (21 / 43) by the University of Reading Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent prior to study participation. Healthy adults of both sexes were recruited. Inclusion criteria were age 18-50 years, body mass index (BMI) ≥ 18.5 and ≤ 30 kg / m 2Participants had no signs of gastrointestinal disease and had mildly / moderately elevated levels of stress and anxiety as measured via the Patient Health Questionnaire-9 (PHQ-9, K. Kroenke, R.L. Spitzer, J.B. Williams: The PHQ-9. Validity of a brief depression severity measure. In: J Gen Intern Med. 16, 2001, S. 606-613) and the Generalized Anxiety Disorder Scale-7 (GAD-7, R.L. Spitzer, K. Kroenke, J.W. Williams, B. Lowe: A brief measure for assessing generalized anxiety disorder: the GAD-7. In: Arch Intern Med. 166, 2006, S. 1092-1097) (PHQ-9 range: 7-15 and GAD-7 range: 8-16).

[0130] Participants had no food allergies and a bowel frequency of at least three bowel movements per week. Exclusion criteria included extreme diets (i.e., ketogenic diet, vegetarianism, veganism), intermittent fasting, antibiotic treatment within four months prior to the study, anemia, and chronic or acute illness (i.e., pre-diabetes). Potential participants were also excluded if they had a previous or current diagnosis of a neurological or psychiatric disorder, had undergone surgical resection of any part of the intestine, were current smokers and / or had a history of alcohol or drug abuse, or were pregnant or breastfeeding. Laxative use was also prohibited within four weeks prior to the start of the intervention. Use of selective serotonin receptor inhibitors or antidepressant medications, including amitriptyline, was prohibited within three months prior to the start of the clinical trial. Seventy-two potential participants completed the screening visit. A total of 69 participants completed the clinical trial, with a mean body weight of 23.8 kg / m. 2 The subjects had a mean BMI of 1.0 and a mean age of 28 years.

[0131] 1.1.2 Randomization and blinding Intervention sachets were pre-packaged with a unique randomization code by a research assistant who was not otherwise involved in the study. The software RandList version 1.5 was used to generate the random sequence. Participant randomization was stratified via initial PHQ-9 and GAD-7 scores and gender, with participants assigned to one of three groups using a ratio of approximately 3:1 (female:male). 69 adults were divided into three groups: placebo (17:6), prebiotic composition (17:6), and 2'FL (18:5). This meant that participants and investigators remained blind until study completion and data analysis was complete.

[0132] 1.1.3 Intervention The ITF used was oligofructose (OF) (oligofructose with DP 3-9 and DP-AV 4) (also referred to as P95 hereafter and in the figures). The HMO used was 2'fucosyllactose (2'FL). 2'fucosyllactose is a human milk oligosaccharide (HMO) commercially produced using metabolically engineered organisms. 2'fucosyllactose (96-98% pure, without any additional prebiotics) is a fucosylated HMO composed of L-fucose, D-galactose, and D-glucose. 2-Fucosyllactose was supplied by BENEO-Orafti (Tienen, Belgium). The comparative placebo product was maltodextrin, a readily digestible carbohydrate composed of different chains of D-glucose linked primarily by α-(1,4) bonds of various chain lengths. To maintain blinding, the interventions were packaged in the same sachet and were of equal weight: placebo: 11 g maltodextrin, prebiotic composition: 2 g 2'FL (product containing 2.3 g 2'FL) + 8.0 g OF (product containing 8.7 g OF), 2'FL: 2 g 2'FL (product containing 2.3 g 2'FL) + 8.7 g maltodextrin. Volunteers received a total of 10 g of their assigned intervention per day. The 2 g of 2'FL was selected based on EFSA Reference 258 / 97, which states that 3 g / day of 2'FL is the maximum intended daily intake from a dietary supplement that should not result in a risk of adverse events (Agostoni et al., Efsa Journal, 13(7), 2015).

[0133] 1.1.4 Study Procedures The study lasted 5 weeks and was divided into a 1-week run-in phase to record daily variations in bowel habits and a 4-week intervention phase. Fecal samples were collected from participants upon awakening at the beginning (day 0) and at the end of the intervention phase (day 28) as the first fecal sample. Details regarding sample collection and processing are detailed below. Participants were asked to complete self-report mood state questionnaires (Beck Depression Inventory (BDI) and State-Trait Anxiety Inventory (STAI)) at the beginning and again at the end of the 4-week intervention phase. Participants were asked to consume the study product once daily in the morning, immediately after / with breakfast in water, for a total daily intervention intake of 10 g. Adherence to the intervention was assessed using a daily diary. Participants were considered eligible if they consumed more than 95% of the provided supplement.

[0134] The first study group of 23 participants, the placebo group, was supplemented with 10 g of a composition consisting of placebo over a four-week period.

[0135] The second study group of 23 participants, the 2'FL group, was supplemented with 10 g of a total composition consisting of 2 g of 2'FL and 8 g of placebo compound over a 4-week period.

[0136] A third study group of 23 participants, the prebiotic composition according to the invention group (PC group), was supplemented with a prebiotic composition according to the invention comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO), in particular 2 g 2'FL and 8 g OF, over a period of 4 weeks.

[0137] The participant distribution is shown in the table.

[0138] [Table 1]

[0139] 1.1.5 Sample Collection Fecal samples and outcome assessments were collected before supplementation and at the end of the 4-week intervention period. Flow cytometric fluorescence in situ hybridization (FISH) was performed on fecal samples for bacterial enumeration.

[0140] Participants were provided with sterile fecal sample pots for collection on days 0 and 28. Freshly collected fecal samples were placed in Oxoid TM AnaeroGen TM 2.5L Oxoid with 2.5L sachets (O2 < 0.1%; CO2: 7-15%) TM AnaeroJar TM Stool samples were collected from participants' residences within 2 hours of defecation. Samples (1.5 g) for metabolic profiling were stored at -80°C until the completion of the study. Another portion (3 g) of the same stool sample was stored in anaerobic phosphate-buffered saline (PBS, 0.1 mol l -1 The fecal slurry was diluted 1:10 (w:w) in PBS (pH 7.4) and subsequently homogenized using a stomacher (260 paddle beats / min) for 2 minutes at room temperature. 20 mL of fecal slurry was then vortexed with 3 mm diameter glass beads for 30 seconds and then centrifuged at 1,500 × g for 3 minutes at room temperature. 750 μL and (75 μL (1:100 dilution in 675 μL PBS)) aliquots were then stored at −20°C until the cells could be fixed. 750 μL (1:100 dilution) was then centrifuged at 11,337 × g for 5 minutes and the supernatant was discarded. The pellet was then resuspended in 375 μL of 0.1 mol / L PBS. -1The cells were resuspended in PBS and fixed in 4% (w:v) paraformaldehyde (1,125 μL) for 4 hours at 4°C. The fixed cells were centrifuged at 11,337 × g for 5 minutes at room temperature. The samples were then washed with 1 mL PBS, the pellet was aspirated, and centrifuged at 11,337 × g for 5 minutes. The washing process was repeated two more times. The samples were resuspended in 150 μL PBS and stored in ethanol (1:1, v:v) at -20°C until analysis via fluorescence in situ hybridization (FISH).

[0141] 1.1.6 Enumeration of fecal microbial populations by fluorescence in situ hybridization flow cytometry (FISH-FLOW) Flow cytometric FISH was performed as described by Grimaldi et al., 2017, FEMS Microbiology Ecology, 93(2). The probe (Bif164, which is the target region in the 16S rRNA molecule of Bifidobacterium) used to identify Bifidobacterium species in this study is described in Langendijk et al., Applied and Environmental Microbiology, 61(8), 1995, 3069-3075. 3. log 10 Cell counts are presented as per gram of moist fresh feces.

[0142] 1.2 Results Sixty-nine participants provided fecal samples at baseline, day 0 of the intervention, and day 28, the end point.

[0143] Figure 2 reports the changes in Bifidobacterium species (Bif.164) counts observed in each group on days 0 and 28 of the intervention.

[0144] Figure 2 shows three graphs of the amount of Bifidobacterium species (Bif164) in study participants in all three study groups: the placebo group, the 2'FL group, and the prebiotic composition group according to the invention, from left to right. The y-axis in each graph is log 10The abundance of Bifidobacterium spp. in cells / g wet feces is shown. The x-axis in each figure indicates the study days, days 0 and 28, on which fecal samples were obtained and analyzed. Each line connecting a data point on day 0 to a data point on day 28 represents the change or maintenance of bifidobacterial abundance in an individual participant. Comparison of bifidobacterial abundance in samples collected on days 0 and 28 allows one to conclude whether an individual is a responder or non-responder.

[0145] Regarding the left panel of Figure 2, i.e., the panel relating to the placebo group, no significant change in Bifidobacterium species (Bif164) counts is observed among the placebo group as a whole. Regarding the center panel of Figure 2, i.e., the panel relating to the 2'FL group, a slight increase in Bif164 counts from 8.63 ± 0.14 (SE) to 8.93 ± 0.12 (SE) can be detected among the 2'FL group as a whole (P = 0.001). Regarding the right panel of Figure 2, i.e., the panel relating to the prebiotic compositions according to the invention, a significant increase in Bif164 counts from 8.68 ± 0.10 (SE) to 9.13 ± 0.13 (SE) can be detected among the prebiotic compositions according to the invention as a whole (P = 0.001).

[0146] Thus, the responder rates observed in participants with an increase in Bifidobacteria after 28 days of supplementation were 39.1% in the placebo group, 60.9% in the 2'FL group, and 91.3% in the prebiotic composition according to the invention group (each based on the number of participants in each study group). Thus, the non-responder rate in the PC group, i.e., the group supplemented with the prebiotic composition according to the invention, is significantly reduced relative to the 2'FL group, i.e., the group supplemented with a composition consisting of 2'FL. This can also be seen in Figure 3.

[0147] The data display in Figure 3 is represented by a bar graph, with each bar indicating, from left to right, the percentage of non-responders within three groups: placebo, 2'FL, and the prebiotic composition according to the present invention. When comparing the prebiotic composition according to the present invention with the 2'FL group, the non-responder rate is reduced by more than 70% by supplementation with the prebiotic composition according to the present invention. Without wishing to be bound by theory, this is due to a greater increase in bifidobacteria abundance compared to the 2'FL group, leading to a lower non-responder rate.

[0148] Example 2: Human Intervention Study: HMO Responders and Mood Benefits The study design was the same as that described in Example 1. Mood was assessed using a validated questionnaire. Depression was assessed using the Beck Depression Inventory (BDI).

[0149] 2.1 Beck Depression Scale The Beck Depression Inventory (BDI) is a 21-question self-report scale with multiple choice (Beck et al., JAMA Psychiatry, 4, 1961, 561-571). Within each inventory, participants are asked to choose one of four statements that best describes their state of affairs over the past two weeks. Each inventory is scored 0, 1, 2, or 3, with 0 representing normal or the lowest level of depression and 3 representing the highest level of depression. An overall score is calculated by summing the individual scores for each inventory. Scores range from 0 to 63. Lower scores are associated with lower levels of depression.

[0150] 2.2 Results FIG. 4 presents the baseline and post-test BDI scores of the 2′FL group and the prebiotic composition according to the invention group divided into RES / NR status.

[0151] Figure 4 shows diagrams of the Beck Depression Scale on days 0 and 28 for responders (Res) (bars 1 and 2) and non-responders (NR) (bars 3 and 4) in the 2'FL group (2'FL), and for responders (bars 5 and 6) and non-responders (bars 7 and 8) in the prebiotic composition group according to the invention (2'FL+P95). The y-axis represents the Beck depression score. As shown in Figure 4, the responders in the prebiotic composition group according to the invention, i.e., the increase in Bifidobacteria, significantly improved mood parameters, i.e., reduced Beck depression scores, while no significant effect was observed in the non-responders. The responders in the 2'FL group only benefited slightly in terms of depression outcomes. At the end of the intervention, segregation analysis revealed a decrease in BDI score only in the Res group (p<0.001), showing a significant decrease in BDI score in the Res group in the prebiotic composition according to the invention group. Surprisingly, it can be seen that the BDI of Res in the prebiotic composition according to the invention group is even more significantly decreased compared to the BDI of Res in the 2'FL group. Without wishing to be bound by theory, this could be due to a higher abundance of Bifidobacteria in the prebiotic composition according to the invention group.

[0152] Figure 3 clearly shows the reduced proportion of non-responders due to combined supplementation with P95 and 2'FL compared to 2'FL alone. The non-responders and responders in Figure 4 are the same as in Figure 3.

[0153] The study results in Figure 5 also show that both groups receiving the prebiotic substrates (2'FL alone and P95(OF) + 2'FL) showed significant improvements in mood scores from baseline. However, the effect was more pronounced in the group receiving the combination of 2'FL and P95, and was significantly different from the placebo group (Figure 5). The difference in effect was primarily due to differences in the number of "responders," meaning participants who exhibit an increase in bifidobacteria, which are responsible for the beneficial effects on mood.

[0154] The same results can be observed for another aspect of mood, namely anxiety. Anxiety can be assessed using the State-Trait Anxiety Scale (STAI) questionnaire used in this study. The STAI questionnaire consists of 2 x 40 statements that can be answered on a 4-point scale ranging from "not at all true" to "very true." Lower scores indicate better mental health and less anxiety. Statements are answered regarding current conditions (Y1) and longer-term situations or characteristics (Y2). The results are shown in Figures 6 and 7. It was observed that participants who received the prebiotic combination had a significant reduction in STAI scores at both Y1 and Y2 compared to participants who received the placebo.

[0155] Figures 8 and 9 show the effect of the intervention and responder status on current and general anxiety scores.

[0156] It is clear that a significant reduction in anxiety scores was observed in responders toward intervention with 2'FL, and was particularly evident for the combination of 2'FL and P95 (Figures 8 and 9). This should be considered especially in light of the substantially higher proportion of responders in the combined intervention group (as shown in Figure 3).

[0157] Therefore, supplementation with the prebiotic composition according to the present invention reduces the rate of non-responders to intervention.Furthermore, the prebiotic composition of the present invention increases the effect of intervention on depression and anxiety scores in RES.Without wishing to be bound by theory, it is surprisingly shown that supplementation with the prebiotic composition according to the present invention has an increased effect on brain-gut interaction, due to the reduced BDI score and anxiety score, compared to supplementation with the composition consisting of 2'FL, in addition to the effect of reducing NR rate.

[0158] 3. Summary of results In summary, the prebiotic composition according to the present invention reduces the non-responder rate, and the bifidogenic response is more uniform after ingesting the prebiotic composition according to the present invention.Furthermore, the prebiotic composition reduces (improves) mood-related disorders, such as depression as demonstrated by a reduction in Beck Depression Scale scores and anxiety as demonstrated by the State-Trait Anxiety Inventory (STAI) questionnaire, compared with both a composition consisting of 2'FL and placebo.Therefore, the prebiotic composition increases the responder rate in terms of bifidobacteria increase, and these people benefit more from prebiotic intervention in terms of better mood, i.e., lower depression-related ratings.

[0159] Example 3: Effect of various ITFs in combination with HMO on Bifidobacterium abundance In an in vitro batch culture experiment prior to the human clinical trial described in Example 1, different combinations of fructans representing two prebiotic compositions of the present invention (P95 or fructan mixture, each combined with 2'FL separately), 2'FL alone, fructan mixture alone and P95 alone were used to study the effect of said components on the number of Bifidobacterium species in fecal samples from three healthy human subjects.

[0160] For this purpose, freshly voided fecal samples were obtained from three healthy adults aged 18–40 years who had not taken antibiotics for at least four months prior to the experiment, had no history of gastrointestinal disorders, were not regular users of prebiotics or probiotics, and were not on any restrictive diet. The fecal samples were diluted 1:10 (w / v) with 0.1 mol / L anaerobically prepared phosphate-buffered saline (PBS) pH 7.4. The fecal samples were then homogenized. A volume of 15 mL of fecal slurry was immediately used to inoculate each batch culture vessel. One independent batch culture was performed for each donor. Various substrates were added at 1% (w / v) for each donor: P95, fructan mixture, 2'FL, P95 + 2'FL (1:1), and fructan mixture + 2'FL (1:1). After 8 hours of incubation, samples were removed from each container for Bifidobacterium analysis by fluorescence in situ hybridization flow cytometry (FISH-FLOW) according to the method described in Example 1.

[0161] One type of ITF used in this example was oligofructose (OF) (oligofructose with DP 3-9, DP-AV:4) (also referred to as P95) such as that described in Example 1.

[0162] In addition, ITF with shorter and longer chains, i.e., GF with a degree of polymerization (DP) of 25% to 30% (dry matter based on the total mass of carbohydrates) ≥ 11. n and F m Compounds (where n and m are each at least 2), 15-30 wt% (dry matter based on the total mass of carbohydrates) of F m Fructan mixtures containing compounds (m = 2-9) as well as fructan mixtures with average DP (DP-AV) of 7-8 were used as further ITFs.

[0163] The HMO used was 2'fucosyllactose (2'FL) such as that described in Example 1.

[0164] For the study, fecal samples from three different donors were collected, and substrates were added to the fermentation medium at 1% (w / v). Substrate combinations were performed with equal amounts of each substrate. Fermentations were pH-controlled and stirred, and samples for microbial analysis were taken after 8 hours.

[0165] The results of this study are depicted in Figure 10 (the term "fructan mix" refers to the fructan mixture described herein).

[0166] In vitro fermentation results indicate that Donor 2 is an HMO non-responder. Donor 2 responds to ITF (oligofructose P95 and fructan mix) with a substantial increase in bifidobacteria (Figure 10). When 2'FL was used as the fermentation substrate, the increase in bifidobacteria was very small after 8 hours (y-axis, T8 to T0 difference in log10 of Bifidobacterium counts). However, combining oligofructose or fructan mix with 2'FL resulted in substantial synergy, as demonstrated by a higher increase in bifidobacteria than with the individual substrates. This high increase was unexpected, as the amount of fermentable substrate was still 1% of the total medium (0.5% each).

[0167] The data show the effect of individual substances and their combination on bifidobacteria. Furthermore, the effect on bifidobacteria levels caused by the combination of two ITFs (P95 and fructan mix) with 2'FL in an HMO non-responder (donor 2) was higher than expected by combining both substrates.

Claims

1. Use of a prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for the treatment of a mood disorder in a healthy subject in need thereof.

2. Inulin-type fructans are preferably short-chain ITFs with a DP-AV (average degree of polymerization) of 3 to 5, in particular oligofructose (OF), 25% to 30% GF with a degree of polymerization (DP) of 11 or more. n and F m Compound (dry matter based on total mass of carbohydrates) (n and m are each at least 2), 15-30 wt% F m 2. The use according to claim 1, comprising a fructan mixture comprising compounds (m=2 to 9) (dry matter amount based on the total mass of carbohydrates) and a fructan mixture having a DP-AV (average degree of polymerization) of 7 to 8, preferably a long-chain ITF, in particular inulin, having a DP-AV of at least 8, in particular 8 to 100, preferably 20 to 90, or a mixture of two or three thereof.

3. 3. Use according to claim 1 or 2, wherein the HMO is a neutral HMO, in particular a fucosylated lactose (FL), preferably 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL) or lactodifucotetraose (LDFT), or a mixture thereof, in particular 2'FL.

4. 4. Use according to any one of claims 1 to 3, wherein the weight ratio of ITF to HMO is in the range of 20:1 to 1:20, preferably 20:1 to 1:1, preferably does not exceed 15:1, preferably does not exceed 12:1, more preferably does not exceed 10:1, preferably the weight ratio is 10:1 to 1:1, preferably 4:1 (each on a dry weight basis).

5. The use according to any one of claims 1 to 4, wherein the composition comprises OF and 2'FL.

6. 6. The use according to any one of claims 1 to 5, wherein the composition is for oral administration, preferably for ingestion by a subject in an amount providing at least 0.01 g of the prebiotic composition per kg of body weight per day, for a period of at least 3 days.

7. 7. The use according to any one of claims 1 to 6, wherein the composition additionally comprises at least one additive selected from the group consisting of amino acids; peptides; proteins; lipids; vitamins; carbohydrates; flavorings; minerals; anabolic nutrients; antioxidants; probiotic microbial strains; diluents, excipients, antioxidants, lubricants, colorants, binders, disintegrants, coloring agents; preservatives, bulking agents; pharmaceuticals and any combination thereof.

8. 8. The use according to claim 7, wherein the probiotic microbial strain is in the form of viable microorganisms, non-viable microorganisms, fragments of microorganisms and combinations thereof.

9. The use according to any one of claims 1 to 8, wherein the subject in need thereof is an HMO non-responder.

10. 10. The use according to any one of claims 1 to 9, wherein the prebiotic composition is a dietary supplement, a food or a drink, or is part of any of these.

11. Use of an inulin-type fructan (ITF) in a prebiotic composition comprising at least one human milk oligosaccharide (HMO) for the treatment of mood disorders, particularly depression or anxiety, in a healthy subject in need thereof, wherein preferably the subject in need thereof is an HMO non-responder.

12. A prebiotic composition comprising at least one inulin-type fructan (ITF) and at least one human milk oligosaccharide (HMO) for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is an HMO non-responder.

13. An inulin-type fructan (ITF) for use in a method for maintaining or improving the health of a subject in need thereof by administering a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is an HMO non-responder.

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