Prebiotic composition

Abstract

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

Description

[0001] DESCRIPTION

[0002] Prebiotic composition

[0003] 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 a HMO non-responder; to a food, food supplement or beverage for use in a method for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is a HMO non-responder, comprising the prebiotic composition; and to an inulin type fructan (ITF) for use in a method for maintaining or improving the health of a subject in need thereof by administration of a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is a HMO non-responder. The present invention further relates to uses of said prebiotic composition as a food supplement, food or beverage for a HMO non-responder. 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, preferably wherein the subject in need thereof is a HMO non-responder.

[0004] Human milk oligosaccharides (HMOs) are unique, complex glycans found in high concentrations, in particular 5 - 25 g / L HMO in human breast milk (Kunz et al. Annual review of nutrition, 20, 2000, 699-722).

[0005] They are selective substrates for specific intestinal bacteria, mainly bifidobacteria, protect against infection by blocking pathogen attachment to epithelial cells, promote immunomodulatory activity, and improve gut barrier function (Bode and lantscher-Krenn, Advances in nutrition, Bethesda, Md., 3 (3), 2012, 383S-91S). The most abundant structural classes are fucosylated HMO, representing 35% - 50% of all HMO with its most abundant representative 2’-O- Fucosyllactose (2’FL) (Han et al., The lournal of nutrition, 151 (6), 2021, 1383-1393).

[0006] 2’FL has been identified as substrate for Bifidobacterium spp., thus, its supplementation leads to increases in bifidobacteria (Fonvig et al., ournal of pediatric gastroenterology and nutrition, 73 (3), 2021, 408-414; Elison et al., The British journal of nutrition, 116 (8), 2016, 1356-1368). Higher levels of bifidobacteria correlate with healthy status and longevity, whereas reduction in this genus has been associated with several conditions, including irritable bowel syndrome (IBS), inflammatory bowel diseases (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). Thus, 2’FL is often added to food products or used as dietary supplement in order to selectively increase bifidobacterial levels and thus achieve an improvement in health and physiological function. In particular, 2’FL is used in infant formula and foods for toddlers, children and adults.

[0007] Combinations of HMOs and inulin-type fructans have been used, for instance for improving cognitive performance and affecting olfactory bulb structural development in pigs (Fleming et al., Nutrients, 12 (7), 2020). Furthermore, it is known that adding inulin to 2’FL in vitro fermentations with infant faecal inoculum expedited the fermentation of 2’FL (Akkerman et al., Food & function, 12 (19), 2021, 9018-9029).

[0008] Depression is one of the biggest mental health disorders recorded worldwide costing health services several billions per year (Dieleman et al., Jama-Journal of the American Medical Association, 316(24), 2016, 2627-2646). As a result, there is an increasing demand to find novel treatments in order to not only treat the burden of disease, but to also reduce the ever-increasing pressure on the health system (Liu, Walsh and Sheehan, Neuroscience and Biobehavioral Reviews, 102, 2019, 13-23). While the mechanism by which depression is regulated is not well understood (Huang and Wu, Frontiers in Cell and Developmental Biology, 9, 2021) there is increasing interest in the bi-directional relationship that exists between the gut and the brain - term coined the gutbrain axis (Cryan et al., Lancet Neurology, 19(2), 2020, 179-194) and its involvement in neuronal development, brain function and cognitive performance via regulation of neurologic, immunologic, or endocrine pathways (Morais, Schreiber and Mazmanian, Nature Reviews Microbiology, 19(4), 2021, 241-255). Thus, it stands to reason one way to potentially treat or decrease the risk of depression may be through targeted manipulation of the gut-brain axis. Prebiotics are an attractive means to influence the gut-brain axis and recently, HMOs such as 2’FL have been shown to be effective stimulants of gut microbiota changes.

[0009] However, it is known that a large proportion of individuals does not respond to an intervention with HMOs such as 2’FL in terms of an increase in bifidobacteria abundance. It is known that even with a daily HMO dose of 10 g the responder rate was below 65 % (Iribarren et al., Neurogastroenterology and motility: the official journal of the European Gastrointestinal Motility Society 32 (10), 2020, el3920). As not every person responds to the supplementation with HMOs, such as 2’FL, it is suggested that those persons do not benefit from it in terms of health or physiological benefit (Healey et al., Nutrition reviews, 75 (12), 2017, 1059-1080).

[0010] One reason for the high proportion of non-responders could be the underlying microbiota composition. 2’FL is metabolized mainly by bifidobacteria. However, not all bifidobacteria are able to 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 could be influenced by factors occurring in infancy such as feeding type, in particular breast feeding versus formula feeding, and secretor status of the mother. Depending on their genetic background, mothers secrete differing sets of HMOs, which may or may not include 2’FL, having an influence on the gut microbiota composition and potentially function of their offspring (Smith-Brown et al., PloS one, 11 (9), 2016, e0161211). If these differences during infancy exert effects until adulthood is currently unknown but imaginable and would offer one explanation for the differences in response to supplementation with 2’FL in this population.

[0011] The technical problem underlying the present invention therefore is to overcome the aforementioned disadvantages.

[0012] In particular, the technical problem underlying the present invention is to provide means for maintaining or improving the health of a subject in need thereof which means encompass the administration of at least one HMO, in particular 2’FL, to the subject, wherein the subject in need thereof is a HMO non-responder. In particular, the technical problem underlying the present invention is also to provide means to decrease high non-responder rates to a dietary HM0-, in particular 2’FL-based intervention. In particular, the technical problem underlying the present invention is also to provide means for treating mood disorders including depression and anxiety, preferably improving the treatment of depression and / or anxiety, in particular in a group of subjects including HMO non-responders.

[0013] The technical problem is solved by the teaching of the present invention, in particular the teaching as specified in the independent and dependent claims as well as in the present description.

[0014] In particular, the technical problem is solved by a 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. 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 a HMO non-responder.

[0015] It was surprisingly found that such a prebiotic composition can be used for maintaining or improving the health of a subject in need thereof, wherein the subject in need thereof is a HMO non-responder. Preferably, the subject in need thereof is a healthy subject in need thereof. Thus, it was surprisingly found, that in a group of subjects including HMO non-responders, which were supplemented with a composition for use according to the present invention in comparison to a state-of-the-art composition, in particular a composition comprising as the only prebiotic a HMO, in particular 2’FL, in particular a composition consisting of a HMO, in particular 2’FL, the prebiotic non-responder rate decreases. Advantageously, according to the present invention subjects in need for maintaining or improving their health which are HMO non-responders still can be treated with HMO while simultaneously displaying a prebiotic response, namely an increased abundance of bifidobacteria. Thus, in a group of subjects desiring to consume a HM0-, in particular a 2’FL-containing diet, the number of subjects showing a prebiotic response is significantly increased.

[0016] Thus, with the composition for use according to the present invention it is possible to use targeted combinations of prebiotics to decrease non-responder rates to a dietary intervention.

[0017] The technical problem of the present invention is, thus, solved by the prebiotic composition according to the present invention comprising on the one hand at least one inulin type fructan (ITF) and on the other hand at least one human milk oligosaccharide (HMO). 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, which subject is a 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, in particular maintains or increases the health of a subject being a HMO non-responder even though the subject is a HMO non-responder. Preferably, the subj ect in need thereof is a healthy subj ect in need thereof. Without wishing to be bound by theory, the maintaining or increasing health of a subject being a HMO non-responder by using the prebiotic composition according to the present invention is due to an increase in its bifidobacteria abundance, preferably at least after 1 to 28 days of use of the prebiotic composition according to the present invention.

[0019] Preferably, the use of the prebiotic composition according to the present invention advantageously solves a further technical problem, namely the technical problem to decrease non-responder rates in HMO consuming subjects in comparison to the use of a state-of-the-art composition, in particular a composition consisting of a HMO, in particular 2’FL, by combining at least one HMO with at least one ITF, preferably in a ratio of 1:4, resulting in a decrease of prebiotic nonresponders. Thus, an advantage of the present invention is a decrease of non-responder rates in HMO consuming subjects, in particular to a value lower than a prebiotic non-responder rate of 35 % in relation to the total amount of prebiotic non-responders and responders. The present invention thus provides the advantage that, due to the use of the prebiotic composition according to the present invention the prebiotic non-responder rates decreases and therefore, more people benefit from the use of HMO containing compositions. Thus, the present invention maximizes the chance of beneficial effects due to supplementation with the 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 maintaining or improving the health is the treatment of a mood disorder, in particular depression or anxiety, in particular, wherein the subject in need thereof is a HMO non-responder.

[0021] Preferably, the subject in need thereof is a healthy subject in need thereof. Preferably, a mood disorder of a healthy subject in need thereof is thus a mood change, hereinafter also called a “mood fluctuation”, in the daily life of such subject reflecting internal or external stimuli to said mood which mood change is of reversible and temporary character and does not reach an extent requiring a therapeutic treatment.

[0022] Preferably, the subject in need thereof is a mentally ill subject in need thereof, in particular suffering from clinically recognizable forms of depression, such as major depression, persistent depressive disorder, bipolar disorder or of anxiety. A mood disorder of a mentally ill subject in need thereof is thus preferably a mood disease which is to be classified as a medical clinically manifested condition of a mood disorder and requires a therapeutic treatment.

[0023] It was surprisingly found and thus represents an advantageous and preferred embodiment of the present invention 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 for the treatment of mood disorders, including depression and anxiety, of a subject. The subjects may be healthy subjects or mentally ill subjects, in particular suffering from clinical forms 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 for improving such treatment in relation to the use of a state of the art prebiotic composition consisting of HMO, in particular 2’FL.

[0024] In particular, a 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, of a subject in need thereof, preferably a healthy or mentally ill subject.

[0025] In a preferred embodiment of the present invention, the subject in need thereof is a HMO nonresponder.

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

[0027] Without wishing to be bound by theory the improvement of the treatment of mood disorders including depression and anxiety by using a prebiotic composition according to the present invention relates to an increase in bifidobacteria abundance and thus correlating to the gut-brainaxis of the subject using the prebiotic composition according to the present invention.

[0028] In a preferred embodiment of the composition for use according to the present invention, the inulin type fructan is a short-chain ITF, in particular oligofructose (OF), preferably with a DP -AV (average degree of polymerisation) of 3 to 5, preferably 4, a long-chain ITF, in particular inulin, preferably with 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 present invention, the inulin type fructan is a short-chain ITF, in particular oligofructose (OF), preferably with a DP-AV (average degree of polymerisation) of 3 to 5, preferably 4, a fructan mixture comprising 25%- 30% GFnand Fmcompounds with a degree of polymerization (DP) >11 (dry matter, based on the total mass of carbohydrates) with each of n and m being at least 2, preferably being independently at least 2, 15-30 wt.-% of Fmcompounds with m = 2 to 9 (dry matter, based on the total mass of carbohydrates) and the fructan mixture having an DP -AV of 7 to 8 or a long-chain ITF, in particular inulin, preferably with a DP -AV of at least 8, in particular 8 to 100, preferably 20 to 90 or a mixture thereof.

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

[0031] In a preferred embodiment of the composition for use according to the present invention, the shortchain ITF has preferably a DP ranging mainly from 2 to 7, with minor amounts, preferably less than 5 %, more preferably less than 3 % inulin of DP 8 to 9 (% is wt% of total ITF).

[0032] In a preferred embodiment of the composition for use according to the present invention, the ITF is a fructan mixture comprising 25% to 30% GFnand Fmcompounds with a degree of polymerization (DP) >11 (dry matter, based on the total mass of carbohydrates) with each of n and m being at least 2, preferably being independently at least 2, 15 to 30 wt.-% of Fmcompounds with m = 2 to 9 (dry matter, based on the total mass of carbohydrates) and the fructan mixture having an DP -AV of 7 to 8.

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

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

[0035] In a preferred embodiment of the present invention, the present fructan mixture does not contain galactose and / or oligosaccharides or polysaccharides, which oligo- or polysaccharides contain at least one galactose moiety.

[0036] In a preferred embodiment of the present invention, the fructan mixture comprises at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 90%, GFn- and Fm-compounds and the remainder adding up to 100% with sucrose and / or monosaccharides, preferably glucose, fructose, sucrose or a mixture thereof (each based on weight of dry matter and total mass of carbohydrates). In a preferred embodiment of the composition for use according to the present invention, the long-chain ITF has preferably a DP -AV of at least 23.

[0037] The ITF may be from any suitable source, in particular a plant source such as chicory or agave or a bacterial source.

[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, which are all 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 patent US 5,314,810. A suitable ITF is an ITF from agave.

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

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

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

[0042] As meant herein, the determination of the average degree of polymerization (,DP-AV‘) in an ITF, in particular compositions containing inulin, is based on AO AC Official Method 997.08 (2013 version, AOAC International):

[0043] As is known, this method first requires the determination of non-inulin related glucose and noninulin related fructose in the composition: free glucose and fructose concentrations, as well as the concentrations of glucose and fructose deriving from sucrose, starch and / or maltodextrins present in the inulin-containing composition are determined.

[0044] An inulinase treatment of the inulin-containing composition will result in complete enzymatic hydrolysis of the inulin compounds into free glucose and free fructose. Subtracting the earlier-determined non-inulin related concentrations of glucose and fructose from the overall fructose and glucose content determined after complete enzymatic hydrolysis of the inulin composition to be analyzed provides the concentrations, in particular weight-percentages, of inulin-related fructose (Fi) and inulin-related glucose (Gi).

[0045] If the inulin in the composition consists of GFncompounds, DP -AV can be calculated via the formula as given in AO AC 997.08:

[0046] DP-AV = [ (Fi / Gi) + l ] (1)

[0047] If the inulin in the composition is an inulin product that had undergone a partial hydrolysis, for example by treatment with an endo-inulinase, it no longer consists essentially only of GFncompounds but also has a significant amount of Fmcompounds. Formula (1) is then no longer sufficiently accurate and a correction is needed. AO AC 997.08 provides some approximations for the said correction.

[0048] In a preferred embodiment of the composition for use according to the present invention, the HMO is a neutral HMO. A "neutral human milk oligosaccharide" or "neutral HMO" preferably means a complex carbohydrate found in human breast milk that is in neutral form. Neutral HMO may occur in form of non-fucosylated and fucosylated oligosaccharides. Non-fucosylated neutral HMO include Glc, Gal and GIcNAc and are devoid of fucose and sialic acid, such as lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), lacto-N-hexaose (LNH) and para-lacto-N-neohexaose (pLNnH). Fucosylated HMO are for instance 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-para-lacto-N-neohexaose (F-pLNnH), lacto-N-difucohexaose 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-para-lacto-N-neohexaose (F- pLNnH).

[0049] In a preferred embodiment of the composition for use according to the present 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.

[0050] In a preferred embodiment of the composition for use according to the present invention, the weight ratio of the ITF to the HMO is within 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 wherein the weight ratio is from 10: 1 to 1 : 1, preferably 4: 1 (each based on dry weight).

[0051] In a preferred embodiment of the composition for use according to the present invention the composition comprises OF and 2’FL.

[0052] In a preferred embodiment of the composition for use according to the present invention, the composition comprises no more than 2 g HMO, in particular 2’FL.

[0053] In a preferred embodiment of the composition for use according to the present invention, the composition comprises 8 g OF and 2 g HMO, in particular 2’FL.

[0054] In a preferred embodiment of the composition for use according to the present invention, the composition consists of OF and 2’FL, in particular in a weight ratio of 4: 1 (based on dry weight), in particular consists of 8 g OF and 2 g 2’FL (each based on dry weight).

[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 treatment of disorders associated with disturbed composition or functionality of the intestinal microbiome, in particular for promoting the gut microbiota of the subject, in particular is a method for increasing the number of Bifidobacteria, in particular B. longum, B. longum subspecies, B. infantis, B. pseudocatenulatum, B. adolescentis, or a mixture thereof, in the gastrointestinal tract (GI tract), in particular the large intestine, of the subject. A disturbed composition or functionality of the intestinal microbiome is preferably characterised by bacterial overgrowth, dysbiosis and / or an impaired mucosal barrier.

[0056] 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 a level of butyrate; increasing a level of short chain fatty acids (SCFAs); or both in the GI tract, in particular in the large intestine, of the subject. SCFAs are 2-carbon to 5- carbon weak acids, including acetate (C2), propionate (C3), butyrate (C4), and valerate (C5).

[0057] 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 of constipation, increased blood cholesterol levels, distorted balance of intestinal bacterial flora, increased intestinal pH values, metabolic disorder, intestinal barrier dysfunction, inflammation of the GI tract, in particular inflammatory bowel diseases, Celiac disease; ulcerative colitis; Crohn's disease, depressive disorders, anxiety, mood impairments, sleep disorders; or any combination thereof.

[0058] In a particularly preferred embodiment 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 Mellitus, glucose intolerance, abnormal lipid metabolism, hyperglycemia, hepatic steatosis, dyslipidemia, high cholesterol and elevated triglycerides.

[0059] 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 which is artificially prepared and preferably means a composition containing at least one compound that is produced ex vivo chemically and / or biologically, for instance by means of chemical reaction, enzymatic reaction or recombinantly.

[0060] In a preferred embodiment of the composition for use according to the present invention, the composition is a nutritional composition.

[0061] In a preferred embodiment of the composition for use according to the present invention, the composition contains an effective amount of one or more HMO and one or more ITF.

[0062] The composition of the present invention comprising one or more HMO and one or more ITF may be in any suitable form. For example, the composition of the present invention can be in the form of a nutritional composition which contains other macronutrients such as proteins, lipids and / or other digestible or non-digestible carbohydrates. The composition of the present invention can also be an incomplete nutritional composition in unit dosage form or pharmaceutical composition.

[0063] The composition of the present invention preferably is in powdered or liquid form. The composition can be the sole source of nutrition or a nutritional supplement.

[0064] Preferably, the nutritional composition is lactose-reduced, in particular lactose-free.

[0065] In a preferred embodiment, the protein is a milk protein, soy protein, rice protein, pea protein and oat protein, or mixtures thereof. Milk proteins can be in the form of milk protein concentrates, whey protein or casein, or mixtures of both. Soy, rice, pea and oat protein are preferably in isolated form. The protein can be whole protein or hydrolysed protein, either partially hydrolysed or extensively hydrolysed. The protein can provide 5 % to 50 %, preferably 10 % to 30 %, of the energy of the nutritional composition.

[0066] In a preferred embodiment, suitable digestible carbohydrates include maltodextrin, hydrolysed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, tapioca, sucrose, and glucose, or mixtures thereof. Preferably, digestible carbohydrates provide 35 % to 75 %, preferably 45 % to 70 %, of the energy of the nutritional composition. Preferably, the digestible carbohydrate is free from lactose.

[0067] In a preferred embodiment, suitable lipids include rapeseed oil, sunflower seed oil, palm oil, soy oil, milk fat, corn oil and soy lecithin. Long-chain poly unsaturated fatty acids (LC-PUFA), especially omega-3 fatty acids such as docosahexaenoic acid (DHA), can be used as a lipid. Suitable sources of LC-PUFA are plant oils, marine plankton oils, fungal oils, and fish oils. The lipid can also be a medium chain triglyceride (MCT). Fractionated coconut oils are 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 %.

[0068] In a preferred embodiment, the composition of the present invention preferably also includes vitamins and minerals. Preferred vitamins include vitamins A, B- complex (such as Bl, B2, B6 and B12), C, D, E and K, niacin and acid vitamins such as pantothenic acid and folic acid and biotin.

[0069] Preferred minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium and boron.

[0070] In a preferred embodiment, the composition of the present invention can also include a carotenoid such as lutein, lycopene, zeaxanthin and beta-carotene.

[0071] In a preferred embodiment, the composition of the present invention can also contain various other ingredients such as preservatives, emulsifying agents, thickening agents, buffers, fibres and probiotics, preferably Lactobacillus casei strain Shirota, B. infantis 35624, B. animalis subsp. lactis BB-12, B. lactis Bi-07, L. rhamnosus GG, L. rhamnosusLc705, L. plantarum DSM 9843, L. plantarum CECT7484, L. plantarum CECT7485, L. acidophilusNCFM, L. fermentum CECT5716, B. breve Bb99, Propionib acterium freundenreichii ssp. ShermaniiJS, P. acidilactici CECET7483, Streptococcus faecium, antioxidant / anti-inflammatory compounds including tocopherols, carotenoids, ascorbate / vitamin C, ascorbyl palmitate, polyphenols, glutathione, and superoxide dismutase (melon), other bioactive factors (e.g. growth hormones, cytokines, TFG-B), colorants, flavours and stabilisers and / or lubricants.

[0072] In a preferred embodiment, the composition of the present invention can be in the form of a soluble powder, a liquid concentrate, or a ready-to-use formulation. Various flavours, fibres and other additives can also be present.

[0073] In a preferred embodiment, the composition of the present invention can be prepared by any commonly used manufacturing techniques for preparing nutritional compositions in solid or liquid form. For example, the composition can be prepared from various feed solutions. For instance, a protein-in-fat feed solution can be prepared by heating and mixing a lipid and then adding an emulsifier in particular lecithin, fat soluble vitamins, and at least a portion of an optional protein while heating and stirring. A carbohydrate feed solution is also prepared by optionally adding minerals, minerals, thickening or suspending agents to water while heating and stirring. The resulting solution is held for instance 10 minutes with continued heat and agitation before adding the carbohydrates, in particular the ITF and the HMO. The resulting feed solutions are then mixed while heating and agitating and the pH adjusted to 6,0 to 8,0, in particular 6.6-7.0, after which the composition is preferably subjected to high- temperature short-time processing during which the composition is heat treated, emulsified and homogenized, and then allowed to cool. Optional water soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range, if necessary, optional flavours are added, and water is added to achieve the desired total solid level.

[0074] In a preferred embodiment, the present composition is in form of a liquid product, which preferably is an aseptically packaged nutritional composition. Preferably, the nutritional composition of the present invention can be in ready -to-feed or concentrated liquid form. Preferably, the composition of the present invention can be spray dried and, preferably, processed and packaged as a reconstitutable powder.

[0075] In a preferred embodiment, the present composition is a ready-to-feed nutritional liquid, wherein the total concentration of the one or more HMO in the liquid, by weight of the liquid, is from 0.002 % to 3.0 %, including from 0.005 % to 2 %, including from 0.05 % to 1.0 % and the total concentration of the one or more ITF in the liquid, by weight of the liquid, is from 0.002 % to 3.0 %, including from 0.005 % to about 2 %, including from 0.05 % to about 1.0 %.

[0076] When the nutritional product is a concentrated nutritional liquid, the total concentration of HMO in the liquid, by weight of the liquid, is from 0.004 % to 6.0 %, including from 0.01 % to 4.0 %, including from 0.1 % to 2.0 % and the total concentration of ITF in the liquid, by weight of the liquid, is from 0.004 % to 6.0 %, including from 0.01 % to 4.0 %, including from 0.1 % to 2.0 %.

[0077] In a preferred embodiment, the composition of the present invention comprises one or more additional components, such as diluents, excipients, antioxidants, lubricants, colorants, binder and / or disintegrants.

[0078] In a preferred embodiment, suitable diluents, excipients, lubricants, colorants, binders, and disintegrants include polyethylene, polyvinyl chloride, ethyl cellulose, acrylate polymers and their copolymers, hydroxyethyl-cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose, polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), or polyacrylamide (PA), carrageenan, sodium alginate, polycarbophil, polyacrylic acid, tragacanth, methyl cellulose, 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, lignan, coenzyme Q10 and glutathione.

[0079] In a preferred embodiment, the amount of the present composition of the present invention required to be administered to the subject, in particular patient, will vary depending upon factors such as the severity of the disease, the age of the patient, the form of the composition, and other nutrition and medications being administered to the patient. Preferably, the amount is in the range of a 10 mg to 50 g per day, preferably 50 mg to 40 g per day, preferably from 100 mg to 25 g per day, in particular from 500 mg to 20 g per day, preferably from 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 secondary intervention phase, the dosing can be reduced, for example, to 20 mg to 20 g per day, preferably 100 mg to 10 g per day, more preferably 500 g to 7.5 g per day, in certain embodiments 750 mg to 5 g per day.

[0080] In a preferred embodiment of the composition for use according to the present invention, the composition is for oral administration, preferably is for ingestion by the subject during a period of at least 3 days in an amount providing at least 0,01 g of the prebiotic composition per kg of bodyweight per day. 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 an amino acid; a peptide; a protein; a lipid; a vitamin; a carbohydrate; a flavouring agent; a mineral; an anabolic nutrient; an antioxidant; a probiotic microbial strain; a diluent, an excipient, an antioxidant, a lubricant, a colorant, a binder, a disintegrant, a colouring agent; a preservative, a filler; a pharmaceutical agent and any combination thereof.

[0081] In a particularly preferred embodiment of the composition for use according to the present invention, the probiotic microbial strain is in the form of viable micro-organisms, non- viable micro-organisms, fragments of micro-organisms and combinations thereof.

[0082] In a preferred embodiment of the composition for use according to the present invention, the composition is an ingredient of a food or of a beverage or of a food supplement or is in the form of a capsule, a tablet, a powder. In a preferred embodiment, the composition of the present invention can thus be in a unit dosage form such as a capsule, tablet or sachet, in particular a single serve sachet.

[0083] In a preferred embodiment of the composition for use according to the present invention the prebiotic composition is a food supplement, food or beverage or is a part of any thereof.

[0084] In a further aspect of the present invention, the present invention also relates to a food, food supplement or beverage for use in a method for maintaining or improving the health of a subject in need thereof, in particular as defined in the present invention, wherein the subject in need thereof is a HMO non-responder, comprising the prebiotic composition as defined in the present invention.

[0085] In a further aspect of the present invention, 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 administration of a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is a HMO non-responder. Thus, accordingly, the ITF confers the advantages of the present invention to a HMO containing composition.

[0086] In a further aspect of the present invention, the present invention also relates to a 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 food supplement, food or beverage for a HMO non-responder. In a further aspect of the present invention, the present 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 a HMO non-responder and wherein at least one inulin type fructan (ITF) and at least one human milk oligosaccharide (HMO) is orally administered to the 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 food supplement, food or beverage is orally administered to the subject in need thereof.

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

[0088] In a further aspect of the present invention, the present invention also relates to an inulin type fructan (ITF) for use in a method for the treatment of a mood disorder, in particular depression or anxiety, in a subject in need thereof by administration of a prebiotic composition comprising at least one human milk oligosaccharide (HMO). Thus, accordingly, the ITF confers the advantages of the present invention to a HMO containing composition.

[0089] In a further aspect of the present invention, the present invention also relates to a 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 food supplement, food or beverage for a subject, which suffers from a mood disorder, in particular depression or anxiety.

[0090] In a further aspect of the present invention, the present invention also relates to a method for the treatment of a mood disorder, in particular depression or anxiety, a subject in need thereof, wherein at least one inulin type fructan (ITF) and at least one human milk oligosaccharide (HMO) is orally administered to the 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 food supplement, food or beverage is orally administered to the subject in need thereof. In a preferred embodiment of the present invention, the subject in need for the treatment of a mood disorder, in particular depression or anxiety, is a HMO non-responder.

[0091] In a preferred embodiment of the present invention, the subject in need for the treatment of a mood disorder, in particular depression or anxiety, is a prebiotic responder.

[0092] 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 according to the present teaching.

[0093] In the context of the present invention, the term "HMO non-responder" is understood to refer to a subject, in particular a human or animal, which subject does not show a significant, in particular no, increase in the abundance of bifidobacteria, in particular in the gastrointestinal tract of the subject, in response to an intervention encompassing an oral consumption of a prebiotic composition comprising at least one HMO as the only prebiotic ingredient, in particular 2’FL as the only prebiotic ingredient, in the composition, or a composition consisting of HMO, in particular 2’FL. Thus, a HMO non-responder does not show a significant, in particular no, increase in the abundance of bifidobacteria in response to a dietary intervention in the form of a diet which either consists of HMO, in particular 2’F1, or a diet which contains HMO, in particular 2’F1 and at least one further ingredient, which at least one further ingredient is no prebiotic. Preferably, a HMO non-responder is a member of a group of subjects, in particular HMO non-responders and HMO responders.

[0094] In the context of the present invention, the term "prebiotic responder" is understood to refer to a subject, in particular a human or animal, which subject shows a significant increase in the abundance of bifidobacteria, in particular in the gastrointestinal tract of the subject, in response to a HMO-intervention encompassing an oral consumption of a prebiotic HMO containing composition, which in addition to the HMO contains at least one further prebiotic. In the context of the present invention, a “prebiotic responder” is in particular a subject characterized by a significant increase in bifidobacteria abundance after 28 days of a HMO-intervention based on the bifidobacterial abundance at day 0 of the intervention.

[0095] In the context of the present invention, a “HMO intervention” is an oral consumption of a prebiotic composition comprising at least one HMO, in particular one HMO, in particular 2’FL which in addition to the HMO contains at least one further prebiotic. In the context of the present invention, the term "prebiotic response" is understood to refer to a increase in the abundance of bifidobacteria, in particular in the gastrointestinal tract of a subject, in response to a HMO-intervention encompassing an oral consumption of a prebiotic HMO- containing composition, which in addition to the HMO contains at least one further prebiotic.

[0096] In the context of the present invention, a “significant increase in bifidobacteria abundance” is understood to mean an increase in bifidobacteria abundance by at least 100 %, preferably at least 120%, preferably at least 150% after 28 days based on the bifidobacteria abundance at day 0 of the intervention.

[0097] In the context of the present invention, a “constant bifidobacteria abundance” is understood to mean an equal bifidobacteria abundance or an increase of less than 100 % of the bifidobacteria abundance after 28 days based on the bifidobacteria abundance at day 0 of the intervention.

[0098] In the context of the present invention, the “abundance” of bifidobacteria preferably is observed in faecal samples.

[0099] In the context of the present invention, the term "prebiotic non-responder" is understood to refer to a subject, in particular a human or animal, which subject does not show a significant, in particular no, increase in the abundance of bifidobacteria, in particular in the gastrointestinal tract of the subject, in response to a HMO-intervention encompassing an oral consumption of a prebiotic HMO containing composition, which in addition to the HMO contains at least one further prebiotic. In the context of the present invention, a “prebiotic non-responder” is in particular a subject characterized by a constant bifidobacteria abundance or a decrease in bifidobacteria abundance after 28 days of a HMO-intervention based on the bifidobacterial abundance at day 0 of the intervention. Preferably, a prebiotic non-responder is a member of a group of subjects, in particular prebiotic non-responders and prebiotic responders.

[0100] In the context of the present invention, subjects were defined as non-responders (NR) and responders (RES) based on differential changes seen in bifidobacterial response to a HMO- intervention, which response is recognized by comparing the abundance of bifidobacteria prior to the intervention with the abundance of bifidobacteria subsequently to the HM0-, in particular 2’FL-, intervention.

[0101] By definition RES are classed as subjects who show a prebiotic response to an intervention, which is an increase in abundance, while, in contrast, NR are classed as those subjects who show no significant, in particular no, prebiotic response., which is either a decreased or constant abundance of bifidobacterial observed subsequently to the HMO intervention in comparison to the abundance prior to the intervention.

[0102] In a preferred embodiment of the composition for use, the use and a method of use according to the present invention, the subjects, in particular the responders and / or non-responders are humans or animals, in particular humans, in particular adult humans or animals, in particular adult humans.

[0103] Preferably, a “mood disorder” may be a mood disorder of a mentally healthy, in particular healthy, subject, or of a mentally ill subject.

[0104] Preferably, a “mood disorder of a healthy subject” is a mood change or a mood fluctuation in the daily life of such subject reflecting internal or external stimuli to said mood which mood change is of reversible and temporary character and does not reach an extent requiring a therapeutic treatment. A “mood disorder of a healthy subject” does preferably not show any clinically manifested symptoms of mental illness. Thus, a treatment of a “mood disorder of a healthy subj ecf ’ is preferably a non-therapeutic treatment. A treatment of a mood disorder of a healthy subject in need thereof is thus resulting in a good mood or temper and may preferably also be termed to “cheer up” the subject.

[0105] Preferably, a “mood disorder of a mentally ill subject” is a mood disease which is to be classified as a medical and clinically manifested condition of a mood disorder and requires a therapeutic treatment. Such a disorder is preferably a clinically recognizable form of depression, such as major depressive disorder, persistent depressive disorder, bipolar disorder or of anxiety. Thus, a treatment of a “mood disorder of a mentally ill subject” is preferably a therapeutic treatment aiming to cure an ill subject and resulting in a mentally healthy state.

[0106] In the context of the present invention, a “decrease in non-responder rate” is understood to refer to a reduction of the number of “prebiotic non-responders” in a first group of subjects having been subjected to a dietary HMO-intervention encompassing an oral consumption of a prebiotic HMO- containing composition, which in addition to the HMO contains at least one further prebiotic, compared to the number of “prebiotic non-responders” in a second group of subjects having been subjected to a comparative dietary HMO-intervention, in particular encompassing an oral consumption of a prebiotic composition comprising HMO as the only prebiotic, in particular 2’FL as the only prebiotic, or a composition consisting of HMO, in particular 2’FL. In the context of the present invention, a “prebiotic” is a substance or mixture of substances which is non-digestible and resistant to breakdown by stomach acid and enzymes in the human gastrointestinal tract, is fermented by microorganisms on or in the body and is stimulating the growth and activity of beneficial bacteria, in particular bifidobacteria. Specifically, ITF, HMO and galactans are substances which stimulate the activity and growth of beneficial bacterial colonies in the gut. An inulin type fructan (ITF) may be a short-chain ITF, in particular oligofructose (OF), preferably with a DP -AV (average degree of polymerisation) of 3 to 5 or a long-chain ITF, such as inulin, while galactans comprise of galactooligosaccharides. Other prebiotics are resistant starch, pectin, beta-glucans and xylooligosaccharides.

[0107] In the context of the present invention, the term "oral administration" preferably means any conventional form for the oral delivery of a composition to a patient that causes the deposition of the composition in the gastrointestinal tract of the patient. Accordingly, oral administration includes swallowing of composition by the patient or enteral feeding through a naso-gastric tube.

[0108] In the context of the present invention, the term "effective amount" preferably means an amount of a composition that provides the composition of the present invention in a sufficient amount to render a desired treatment outcome in a patient. An effective amount can be administered in one or more doses to the patient to achieve the desired treatment outcome.

[0109] In the context of the present invention, the term “use” being referred to in the context of a therapeutic method of the present composition and / or the ITF itself means the offer, in particular, advertising, marketing, teaching, or prescription or sale or administration, in particular ingestion, of the present composition and / or the ITF or a method using any one of it to a patient, such as evidenced for instance by prescription by a doctor, packing, packing leaflets, medical expert’s information, expert’s or doctor’s or pharmacist’s counselling, expert information in text or oral form, including in the internet, all of them also including off-label or cross-label use.

[0110] In preferred embodiments, “inulin” is considered to be a mixture of oligo- and polysaccharides which are composed of fructose (F) units and which oligo- and polysaccharides may or may not comprise a starting glucose moiety (G). Thus, in the context of the present invention, the term inulin refers to both GFn- (which is Glucose-Fructosenor Glu-Frun) and Fm- (which is Fructosemor Fnim) compounds, with n and m: number of fructose units in the oligo- or polysaccharide and each of n and m >2. Where quantitative indications, in particular percentages, of components of a product or composition are given in the context of the present invention, these add up to 100% of the composition and / or product together with the other explicitly stated or expertly apparent further components of the composition or product, unless explicitly stated otherwise or expertly apparent.

[0111] In the context of the present invention, the term “a“ or “the” is understood to refer to a single element or to a multitude of elements.

[0112] In the context of the present invention, the term "at least one" is understood to mean a quantity expressing a number of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 and so on. In a particularly preferred embodiment, the term "at least one" may represent exactly the number 1. In another preferred embodiment, the term "at least one" may also mean 2 or 3 or 4 or 5 or 6 or 7.

[0113] Where in the context of the present invention a "comprising", a "containing", an “encompassing”, a "having" or a "content" of a component is expressly mentioned or implied, this means that the respective component is present, in particular present in a measurable amount.

[0114] In the context of the present invention, the term “composition” refers to a substance (in case a composition consisting of X is referred to) or to a mixture of substances (in case a composition comprising or containing or having or encompassing X is referred to).

[0115] If in context of the present invention a "presence", a "containing" or a "content" of a component is expressed, mentioned or implied in an amount of 0 [unit], in particular mg / kg, pg / kg or wt.%, this means that the respective components are not present in a measurable amount, in particular are not present.

[0116] The number of decimal places indicated corresponds to the precision of the measurement method used in each case.

[0117] If, in the context of the present invention, the first and second decimal places or the second decimal place are / is not indicated for a number, the latter is / are to be set as zero.

[0118] In the context of the present invention, the terms "comprising", “encompassing” and "containing" are understood to mean that in addition to the elements explicitly covered by these terms, other elements not explicitly mentioned may be added. In the context of the present invention, it is also understood by these terms that only the explicitly mentioned elements are covered and that no further elements are present. In this particular embodiment, the meaning of the terms "comprising" and "containing" is synonymous with the term "consisting of, which term, that means “consisting of’, means that the element referred to is the only element present, thereby excluding the presence of further elements. Furthermore, the terms "comprising", “encompassing” and "containing" also cover compositions which, in addition to the explicitly mentioned elements, also contain further elements which are not mentioned but which are of a functional and qualitatively subordinate nature. In this embodiment, the terms "comprising" and "containing" are synonymous with the term "consisting essentially of'.

[0119] In the context of the present invention, the term "and / or" is understood to mean that all members of a group connected by the term "and / or" are represented both cumulatively with respect to each other in any combination, and alternatively with respect to each other. Exemplarily, for the expression "A, B and / or C", the following disclosure is to be understood thereunder: 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).

[0120] In the context of the present invention, individual components or constituents of a composition or of one of its components, determined quantitatively in relative form, in particular in percentages, preferably add up to 100% by weight of the respective composition referred to or of the composition or, if referred to, of a component thereof, unless otherwise stated.

[0121] Further preferred embodiments of the present invention are apparent from the dependent claims.

[0122] The present invention will be explained in more detail in the following examples and the accompanying figures both of which are not to be understood as limiting.

[0123] The figures show:

[0124] Figure 1 a study design of a human intervention study according to example 1,

[0125] Figure 2 three diagrams, wherein one diagram shows the results of Flow-FISH measurements of Bifidobacterium spp. specific probes of one of the three study groups,

[0126] Figure 3 a bar diagram of the non-responder rates in the placebo group, 2’FL group and the prebiotic composition according to the present invention group,

[0127] Figure 4 a diagram of the Beck’s Depression Inventory relating to the effect of intervention and responder status on depression scores of the study groups, Figure 5 BDI scores for all participants receiving placebo, 2’FL or a combination of 2’FL and P95,

[0128] Figure 6 State-Trait Anxiety Inventory (STAI) Y 1 (state) scores for all participants receiving placebo, 2’FL or a combination of 2’FL and P95, Figure 7 State-Trait Anxiety Inventory (STAI) Y2 (trait) scores for all participants receiving placebo, 2’FL or a combination of 2’FL and P95,

[0129] Figure 8 Effect of intervention and responder status on current anxiety scores (State-Trait Anxiety Inventory),

[0130] Figure 9: Effect of intervention and responder status on general anxiety scores (State-Trait Anxiety Inventory) and

[0131] Figure 10 the results of in vitro batch fermentation of different substrates either alone or in combination (1 :1 ratio) - effects on Bifidobacterium numbers expressed as change from baseline

[0132] Example 1 : Human intervention study: Reduction of non-responder rate by combining HMO and inulin-type fructan

[0133] 1.1 Study design and participants

[0134] A human intervention study was conducted to detect and compare changes in the non-responder and responder rate of human subjects supplemented with a composition consisting of 2’FL or a prebiotic composition according to the present invention. The design of the human trial was a parallel, placebo-controlled, double-blind, randomized control trial (RCT) lasting four weeks preceded by a one-week run-in period without any study products and a 4 weeks intake period as shown in Figure 1. Each study arm contained 23 participants.

[0135] 1.1.1 The study was given favourable ethical consent by the University of Reading’s Research Ethics Committee (21 / 43) and was conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent prior to study entry. Healthy adults, both males and females, were recruited. The inclusion criteria were participants aged 18-50, body mass index (BMI) > 18.5 and < 30 kg / m2, no evidence of gastrointestinal disease and possessed mild / moderately elevated levels of stress and anxiety as measured via 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 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)

[0136] The participants were free of food allergies and had a stool frequency of at least 3 bowel movements per week. Exclusion criteria were extreme diets, i.e., ketogenic, vegetarian, vegan, intermittent fasting, antibiotic treatment in the 4 months preceding the study, anaemia, chronic or acute diseases i.e., (pre)-diabetic. Potential participants were also excluded if they had been previously or currently diagnosed neurological or psychiatric disorders and if they undergone surgical resection of any part of the bowel, were current smokers and / or had a history of alcohol or drug misuse or if they were pregnant or lactating. Use of laxatives was also not permitted 4 weeks prior to beginning of the intervention. Use of anti-depressants medication including selective serotonin receptor inhibitors or Amitriptyline was not allowed 3 months prior to commencing the trial. 72 potential participants completed the screening visit. In total 69 participants completed the trial with an average BMI of 23.8 kg / m2, and an average age of 28 years.

[0137] 1.1.2 Randomisation and blinding

[0138] Intervention sachets were pre-packaged by research assistant not otherwise involved in the study with unique randomisation codes. Random sequence was created utilizing the software RandList Version 1.5. Randomisation of participants was stratified via initial PHQ-9, and GAD-7 scores and sex and participants were allocated into one of three groups using a ratio of approx. 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 participants and investigators remained blind until completion of the study and data analysis had been completed.

[0139] 1.1.3 Interventions

[0140] The ITF used was oligofructose (OF) (oligofructose with a DP 3-9, DP- AV 4) (hereinafter and in the figures also termed to be P95). The HMO used was 2’fucosyllactose (2’FL). 2’fucosyllactose is a human milk oligosaccharide (HMOs) produced commercially using metabolically engineered organisms. 2’fucosyllatose (96-98% pure, without any further prebiotic) is a fucosylated HMOs composed of L-fucose, D-galactose and D-glucose. 2-fucosyllactose was supplied by BENEO- Orafti, (Tienen, Belgium). The comparator, placebo, product was maltodextrin, a readily digestible carbohydrate, which consists of varying chains of D-glucose primarily linked by a-(l,4)-linkages of various chain length. In order to maintain blinding interventions were packaged in identical sachets and of equal weight; placebo: 11 g maltodextrin, prebiotic composition: 2 g 2’FL (2.3 g 2’FL containing product) + 8,0 g OF (8.7 g OF containing product), 2’FL: 2 g 2’FL (2.3 g 2’FL containing product) + 8.7 g maltodextrin. In total volunteers received 10 g of assigned intervention per day. 2 g of 2’FL was chosen based on EFSA documentation 258 / 97 stating that 3 g / day of 2’FL is the maximum intended daily intake from food supplements at which no risk of adverse events should occur (Agostoni et al., Efsa Journal, 13(7), 2015).

[0141] 1.1.4 Study Procedure

[0142] The study lasted five weeks and was segregated into a one-week run-in phase to capture day-to- day variations in bowel habits and four-week intervention phase. Stool samples were collected from participants as the 1st stool samples after waking at start (day 0) and the end (day 28) of the intervention phase. Details on samples collection and processing is detailed below. Participants were asked to complete self-reported mood-state questionnaires (Beck’s Depression Inventory (BDI) and State-Trait Anxiety Inventory (STAI)) at the start and again at the end of the 4-week intervention phase. Participants were asked to consume the study products once per day in the morning in water just after / or with breakfast resulting in a total daily intervention intake of 10 g. Compliance with consumption of intervention was assessed with completion of dairy. Participants were considered compliant if they consumed > 95% of the supplied supplements.

[0143] The first study arm of 23 participants, the placebo group, was supplemented a 10 g composition consisting of the placebo over a four-week period.

[0144] The second study arm of 23 participants, the 2’FL group, was supplemented a 10 g in total composition consisting of 2 g 2’FL and 8 g of the placebo compound over a four-week period.

[0145] The third study arm of 23 participants, the prebiotic composition according to the present invention group (PC group), was supplemented the prebiotic composition according to the present 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 four-week period. The participant distribution is shown in the Table.

[0146] Table: Participant metrics - age, weight, height, and BMI mean (upper cells in one row), and min and max (lower cells in one row) segregated by intervention. 69 participants (n = 23 per group). 1.1.5 Sample collection

[0147] Stool samples and outcome assessments were collected before supplementation and at the end of the four-week intervention period. Fluorescence in situ hybridization (FISH) by flow cytometry was conducted on stool samples for bacterial enumeration.

[0148] Participants were provided with sterile stool sample pots for day 0 and day 28 collections. Freshly collected faecal samples were kept in 2.5L Oxoid™ AnaeroJar™ (Oxoid, Hampshire, United Kingdom) with Oxoid™ AnaeroGen™ 2.5L sachet (O2 <0.1%; CO2: 7-15%). Faecal samples were collected from the participants place of residence within 2 hours of voiding. Samples (1.5 g) for metabolic profiling were stored at -80 °C until the study had been completed. Another portion (3 g) of the same faecal sample was diluted 1 : 10 (w:w) in anaerobic phosphate-buffered saline (PBS, 0.1 mol 1-1; pH 7.4), then homogenised using a stomacher (260 paddle beats / min) for 2 min at room temperature. 20 mL of faecal slurry was then vortexed with 3 mm diameter glass beads for 30 s before being centrifuged at 1,500 x g for 3 min at room temperature. 750 pL and (75 pL (diluted in 675 pL PBS 1 : 100 dilution)) aliquots were then stored at -20 °C until cells could be fixed. 750 pL (1 : 100 dilution) were then centrifuged at 11,337 * g for 5 min and the supernatant was discarded. Pellets were then resuspended in 375 pL of 0.1 mol 1-1 PBS and fixed in 4% (w:v) paraformaldehyde (1,125 pL) for 4 h at 4 °C. Fixed cells were centrifuged at 11,337 * g for 5 min at room temperature. Samples were then washed with 1 mL PBS, pellets aspirated and centrifuged at 11,337 x g for 5 min. The washing process was repeated twice more. Samples were re-suspended in 150 pL PBS and stored in ethanol (1 : 1, v:v) at -20 °C until analysis via fluorescence in situ hybridisation (FISH).

[0149] 1.1.6 Enumeration of faecal microbial populations by fluorescence in situ hybridisation flow cytometry (FISH-FLOW)

[0150] FISH by flow cytometry was carried out as described by Grimaldi et al., 2017, FEMS Microbiology Ecology, 93(2). The probe (Bifl 64, which is the target region in the 16S rRNA molecule of bifidobacterium) used to identify Bifidobacterium spp. in this study is described in Langendijk et al., Applied and Environmental Microbiology, 61(8), 1995, 3069-3075. 3. The number of logio cells is presented as per gram of wet fresh faeces.

[0151] 1.2 Results

[0152] 69 participants provided stool samples at baseline, day 0, and end, day 28, of the intervention. Figure 2 reports changes in bifidobacterial sp. (Bif. 164) counts observed in each group at day 0 and day 28 of the intervention.

[0153] Figure 2 shows three diagrams of the bifidobacterial sp. (Bif 164) amounts in study participants of all three study groups, from left to right: placebo-group, 2’FL group and prebiotic composition according to the present invention group. The y-axis in each diagram shows the bifidobacterium sp. amounts in logiocells / g wet faeces. The x-axis in each diagram shows test day zero and day 28 on which the stool samples were taken and analysed. Each line connecting a data point at day 0 to a data point at day 28 represents the change or maintenance of the abundance of bifidobacteria in an individual participant. A comparison of the abundance of bifidobacteria in the samples collected at day 0 and day 28 allows to conclude whether the individual is a responder or a nonresponder.

[0154] Regarding the left diagram in figure 2, thus the diagram relating to the placebo group, no significant change in Bifidobacterium sp. (Bif 164) counts across the entire placebo group can be seen. Regarding the middle diagram in figure 2, thus the diagram relating to the 2’FL group, a slight increase in Bifl 64 counts can be detected across the entire 2’FL group going from 8.63 ± 0.14 (SE) to 8.93 ± 0.12 (SE) (P = .001). Regarding the right diagram in figure 2, thus the diagram relating to the prebiotic composition according to the present invention group, a significant increase in Bifl 64 counts can be detected across the entire prebiotic composition according to the present invention group going from 8.68 ± 0. 10 (SE) to 9.13 ± 0.13 (SE) (P = 0.001).

[0155] The responder rates, thus observed by an increase in bifidobacteria after 28 days of supplementation in the participants, are: 39.1 % in placebo group, 60.9 % in 2’FL group, and 91.3 % in the prebiotic composition according to the present invention group (each based on the number of participants in each study arm). Therefore, the non-responder rate in the PC group, thus, supplemented with the prebiotic composition according to the present invention significantly decreases in relation to the 2’FL group, thus, supplemented with the composition consisting of 2’FL. This can also be seen in Figure 3.

[0156] The data representation in Figure 3 is represented by a bar diagram, wherein each bar shows the percentage of non-responders within the three groups, bars from left to right: placebo group, 2’FL group and prebiotic composition according to the present invention group. Comparing the prebiotic composition according to the present invention group to the 2’FL group, non-responder rates are reduced by more than 70 % by the supplementation of 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 in relation to the 2’FL group, leading to a lower nonresponder rate.

[0157] Example 2: Human intervention study: HMO responders and benefits for mood

[0158] The Study design is the same as described in example 1. Mood was assessed by means of validated questionnaires. Depression was assessed with Beck’s depression inventory (BDI).

[0159] 2.1 Beck’s Depression Inventory

[0160] The Beck’s Depression Inventory (BDI) is a 21-question self-reported rating inventory in a multiple-choice format, Beck et al., JAMA Psychiatry, 4, 1961, 561-571. Within each inventory participant is asked to choose from one of 4 statements that best describes his situation in the past two weeks. Each inventory is scored 0, 1, 2 or 3. 0 represents normal or least depressive statement and 3 the most depressive statement. An overall score is calculated via summing individual scores for each inventory. Scores range from 0 to 63. Lower scores being associated with lower levels of depression.

[0161] 2.2 Results

[0162] Figure 4 presents the baseline and post-test BDI scores of the 2’FL group and the prebiotic composition according to the present invention group broken down to RES / NR status.

[0163] Figure 4 shows a diagram of the Becks depression inventory at day 0 and day 28 of responders (Res), first and second bars from left to right, and non-responders (NR), third and fourth bars from left to right, in the 2’FL group (2’FL), as well as responders, fifth and sixth bar from left to right, and non-responders, seventh and eights bar from left to right, in the prebiotic composition according to the present invention group (2’FL + P95). The y-axis shows the Beck’ Depression score. As shown in Figure 4, responders in the prebiotic composition according to the present invention group, thus increase in bifidobacteria, the mood parameters were significantly improved, thus the beck’s depression score declined, while in non-responders, no significant effects could be observed. Responders in the 2’FL group only slightly benefit in terms of depression outcomes. At completion of the intervention the segregation analysis revealed decreases in BDI scores in the Res group only (p < 0.001), wherein a significant decrease in BDI score in the Res group of the prebiotic composition according to the present invention group can be seen. Surprisingly, it can be seen that the BDI of the Res in the prebiotic composition according to the present invention group further significantly decreases in comparison to the BDI of the Res in the 2’FL group. Without wishing to be bound by theory this could be based on a higher in bifidobacteria abundance in the prebiotic composition according to the present invention group.

[0164] 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.

[0165] The study results in Figure 5 also show that both groups receiving prebiotic substrates (2’FL alone and P95(OF)+2’FL) showed significant amelioration of mood scores from baseline. However, the effect was more pronounced and significantly different from the placebo group in the group receiving the combination of 2’FL and P95 (Figure 5). The difference in effect was mainly due to differences in numbers of “responders”, that means participants who show an increase in bifidobacteria, who are thus causative for the beneficial effect on mood.

[0166] The same result can be observed for another aspect of mood, namely anxiety. Anxiety can be assessed with the State-Trait Anxiety Inventory (ST Al) questionnaire, which was used in the current study. The STAI questionnaire consists of 2 x 40 statements that can be answered on a 4- point scale from “not at all” to “very much so”. Lower scores indicate better mental health and less anxiety. The statements are answered for the current state (Yl) and for the longer-term situation or trait (Y2). Results are shown in Figures 6 and 7. It was observed that participants receiving the prebiotic combination had significant reductions in STAI scores, both Yl and Y2, compared to participants receiving a placebo.

[0167] Figures 8 and 9 show the effects of the intervention and responder status on current and general anxiety scores.

[0168] It is evident that significant reductions in anxiety scores were observed in responders toward an intervention with 2’FL and in particular and pronounced for the combination of 2’FL and P95 (Figures 8 and 9). This needs to be considered especially in light of the substantially higher proportion of responders in the combined intervention group (as shown in Figure 3).

[0169] Therefore, supplementation with the prebiotic composition according to the present invention lowers non-responder rates to the intervention. Further, the prebiotic composition of the present invention increases the effect of the intervention on depression and anxiety scores in the RES. Without wishing to be bound by theory, it surprisingly shows, that the supplementation with the prebiotic composition according to the present invention has, further to the effect of decreasing the NR rate, an increased effect on the gut-brain axis due to a decreased BDI score and anxiety score compared to the supplementation with a composition consisting of 2’FL.

[0170] 3. Summary of the results

[0171] In summary, the prebiotic composition according to the present invention decreases the nonresponder rate and the bifidogenic response is more uniform after consumption of the prebiotic composition according to the present invention. Furthermore, the prebiotic composition decreases (amelioration) mood-related disorders e.g. depression as demonstrated by a decrease in the Beck’s depression inventory score and anxiety as demonstrated in the State-Trait Anxiety Inventory (ST Al) questionnaire, both in relation to the composition consisting of 2’FL and the placebo. Thus, the prebiotic composition increases responder rates in terms of bifidobacteria increase, and these people benefit more from a prebiotic intervention in terms of better mood i.e. lower depression- related ratings.

[0172] Example 3 : Effects of different ITF in combination with HMO on Bifidobacterium abundance

[0173] In in vitro batch culture experiments preceding the human trial described in Example 1, different combinations of fructans (P95 or fructan mixture, each separately in combination with 2’FL) that means two prebiotic compositions of the present invention, 2’FL alone, fructan mixture alone and P95 alone were used in stool samples from three healthy human subjects to study the effects of said components on the number of Bifidobacterium spec.

[0174] To this end, freshly voided faecal samples were obtained from three healthy adults aged between 18 and 40, who had not taken antibiotics for at least four months prior to the experiment and had no history of gastrointestinal disorders, were not regular consumers of prebiotics or probiotics and who did not follow any restrictive diet. Faecal samples were diluted 1 in 10 (w / v) using 0.1mol / l anaerobically prepared phosphate buffered saline (PBS), pH 7.4. Faecal samples were then homogenized. A volume of 15mL of faecal slurry was immediately used to inoculate each batch culture vessel. For each donor, one independent batch culture was run. For each donor, different substrates were added at 1% (w / v): P95, fructan mixture, 2’FL, P95+2’FL (1 :1), fructan- mixture+2’FL (1 :1). After 8 hours of incubation, a sample was removed from each vessel for Bifidobacterium analysis by fluorescent in situ hybridization flow cytometry (FISH-FLOW) according to the method described in example 1. One type of ITF used in this example was oligofructose (OF) (oligofructose with a DP 3 to 9, DP- AV: 4) (also termed to be P95), such as described in Example 1.

[0175] In addition, an ITF with shorter and longer chains was used a further ITF, namely a fructan mixture comprising 25% to 30% (dry matter, based on the total mass of carbohydrates)) of GFnand Fmcompounds having a DP >11 (degree of polymerization (DP)) with each of n and m being at least 2, 15 to 30 wt.-% (dry matter, based on the total mass of carbohydrates) of Fmcompounds with m = 2 to 9) and the fructan mixture having an average DP (DP- AV) of 7 to 8.

[0176] The HMO used was 2’fucosyllactose (2’FL) such as described in Example 1.

[0177] For the study, stool samples from three different donors were collected and substrates were added at 1% (w / v) to the fermentation medium. Combinations of substrates were done in equal amounts of each substrate. Fermentations were pH controlled and stirred and samples for microbial analysis were taken after 8 hours.

[0178] The results of this study are depicted in figure 10 (the term “fructan mix” refers to the fructan mixture described herein).

[0179] The results of the in vitro fermentations show that Donor 2 is a HMO non-responder. Donor 2 reacts to ITF (oligofructose P95 and fructan-mix) with a substantial increase in bifidobacteria (Figure 10). When 2’FL is used as fermentation substrate, the increase in bifidobacteria is only very small after 8 hours (y-axis, T8 difference to TO in loglO of Bifidobacterium count). However, combining oligofructose or fructan-mix with 2’FL led to substantial synergistic effects as demonstrated by an increase in bifidobacteria which is higher than with the individual substrate. This high increase was unexpected because the amount of fermentable substrate was still at 1% of the total medium (0.5% each).

[0180] The data show the effect of the individual substances and their combinations on bifidobacteria. Furthermore, the effect on bifidobacteria levels elicited by the combination of two ITFs (P95 and fructan-mix) with 2’FL in a HMO non-responder (Donor 2) is higher than expected by combining both substrates.

Claims

CLAIMS1. 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. The use according to claim 1, wherein the inulin type fructan is a short-chain ITF, in particular oligofructose (OF), preferably with a DP- AV (average degree of polymerisation) of 3 to 5, a fructan mixture comprising 25% to 30% GFnand Fmcompounds with a degree of polymerization (DP) >11 (dry matter, based on the total mass of carbohydrates) with each of n and m being at least 2, 15 to 30 wt.-% of Fmcompounds with m = 2 to 9 (dry matter, based on the total mass of carbohydrates) and the fructan mixture having an DP -AV (average degree of polymerisation) of 7-8, a long-chain ITF, in particular inulin, preferably with 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. The 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. The use according to any one of the preceding claims, wherein the weight ratio of the ITF to the HMO is within 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 wherein the weight ratio is from 10: 1 to 1 : 1, preferably 4: 1 (each based on dry weight).

5. The use according to any one of the preceding claims, wherein the composition comprises OF and 2’FL.

6. The use according to any one of the preceding claims, wherein the composition is for oral administration, preferably is for ingestion by the subject during a period of at least 3 days in an amount providing at least 0,01 g of the prebiotic composition per kg of bodyweight per day.

7. The use according to any one of the preceding claims, which composition additionally comprises at least one additive selected from the group consisting of an amino acid; a peptide; a protein; a lipid; a vitamin; a carbohydrate; a flavouring agent; a mineral; an anabolic nutrient; an antioxidant; a probiotic microbial strain; a diluent, an excipient, an antioxidant, a lubricant, a colorant, a binder, a disintegrant, a colouring agent; a preservative, a filler; a pharmaceutical agent and any combination thereof.

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

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

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

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

12. 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 a 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 administration of a prebiotic composition comprising at least one human milk oligosaccharide (HMO), wherein the subject in need thereof is a HMO non-responder.

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