A new fiber mixture and use thereof for improving gut microbiota

Abstract

The invention relates a fiber mixture comprising non-digestible oligosaccharides including galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS). The invention further relates to a synbiotic composition comprising the fiber mixture, and a nutritional composition comprising the fiber mixture or synbiotic mixture. The invention further relates to the use of the fiber mixture, synbiotic and nutritional compositions of improving or stimulating gut microbiota and / or treating or preventing dysbiosis.

Description

[0001] A NEW FIBER MIXTURE AND USE THEREOF FOR IMPROVING GUT MICROBIOTA

[0002] FIELD OF THE INVENTION

[0003] The invention relates a fiber mixture comprising non-digestible oligosaccharides including galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS). The invention further relates to a synbiotic composition comprising the fiber mixture, and a nutritional composition comprising the fiber mixture or synbiotic mixture. The invention further relates to the use of the fiber mixture, synbiotic and nutritional compositions for improving or stimulating gut microbiota and / or treating or preventing dysbiosis.

[0004] BACKGROUND OF THE INVENTION

[0005] The gut microbiome is a transducer of environmental signals and a contributory factor in ageing-related health loss. Upon ageing the gut microbiome undergoes age-related alterations which may be associated with ageing-linked disorders such as obesity, type 2 diabetes and inflammatory bowel disease. Additionally, the age-related alterations may lead to a disbalance in gut microbiome and dysbiosis.

[0006] Prebiotic fibres can be utilized to shape the human gut microbiota. However, there is great variability in the modulatory and reparatory activity of prebiotics, which is highly dependent on their inherent structural features. For instance, polymeric pectin with a complex structure has been described as a promoter of proliferation of their utilizing microbes (Cantu-Jungles & Hamakera, New View on Dietary Fiber Selection for Predictable Shifts in Gut Microbiota. mBio.

[0007] 2020 Feb 18;11(1):e02179-19. doi: 10.1128 / mBio.02179-19 - Erratum in: mBio. 2020 May 26;11(3): PMID: 32071263; PMCID: PMC7029134.), whereas some, but not all, oligomeric pectins have been reported to stimulate gut bifidogenic effects. Onumpai, Kolida, Bonnin, & Rastall (Microbial utilization and selectivity of pectin fractions with various structures. Appl Environ Microbiol. 2011 Aug 15;77(16):5747-54. doi: 10.1128 / AEM.00179-11. Epub 2011 Jul 1. PMID: 21724897; PMCID: PMC3165253.) reported that arabino-oligosaccharides (DP 2-11) and galacto-oligosaccharides (DP 2-9) components of pectin produced bifidogenic effect, while homogalacturonan-oligosaccharides (DP 1-23) and rhamnogalacturonan-oligo-saccharides (DP 2-19) do not.

[0008] Fermentability and prebiotic properties of pectin oligomers are thus neither yet fully explored nor can they be readily predictable.

[0009] WO2010 / 143947 discloses that there is a benefit for elderly from a nutritional treatment capable of increasing the energy levels and muscle strength, and improving the intestinal andimmunological functions, wherein the treatment involves the administration of a nutritional product comprising a very high content of whey protein that is supplemented with at least free leucine, preferably with added other free branched chain amino acids, probiotic bacteria and prebiotic dietary fibers selected from GOS, FOS and acidic oligosaccharides (AOS). It mentions that a mixture of galactooligosaccharides, fructooligosaccharides and acidic pectin hydrolysate improves natural killer cell activity. A preferred pectin, insofar as characterized, is a high methoxylated pectin, no other specifications provided.

[0010] W02009 / 067000 describes compositions comprising Lactobacillus and Bifidobacterium ssp. DN-173010, galactooligosaccharides, a pectin-derived product (galacturonic acid oligosaccharides) and inulin. The DP of the galacturonic acid oligosaccharide mentioned is in the range of 2 - 250. The pectin-derived fraction is described as useful to reduce adhesion of pathogenic microorganisms to the intestinal epithelial cells. There are no examples with galacturonic acid oligosaccharides provided.

[0011] The overall health of an individual is linked to an optimal gut microbiome. Considering that the gut microbiome undergoes age-related alterations it is thus relevant to support the gut microbiome upon ageing. In particular, there remains a need to provide a composition for promoting healthy ageing by supporting the gut microbiome. Additionally, there remains a need to improve the gut microbiota of subjects suffering from age-related diseases.

[0012] Pectins are often used for reasons other than supporting the microbiome, using their thickening properties under stomach conditions. US2003 / 118712 describes a liquid edible composition with pH > 6, the composition comprising at least 0.05 wt% LM pectin having a degree of methoxylation between 2 and 50 (LM), and / or alginate, for treatment or prevention of overweight or obesity. The intention is that the LM pectin or alginate is capable of forming a sufficiently rigid matrix at stomach pH, i.e. gelling behavior at acidic pH. Similarly, CN 107594483 describes a stable pectin solution where alginate is used to delay the degradation of pectin and improve the stability of the pectin solution. The pectin is a low methoxy pectin with a DE of 5 - 30% and a molecular weight of 10 - 100 kDa, with the intention for the pectin to thicken to an appropriate viscosity. Stability is addressed by controlling calcium interaction, as pectins tend to aggregate with calcium, as in fact demonstrated for LM pectins in example 5 herein. In line with the above, CN107518411 mentions a composition comprising low methoxy pectin having a DE of 10%, Bifidobacterium bifidum, carbohydrates, dietary fibre, vitamin C, vitamin E, having a pH of 6.4 to 6.8. The pectin may have a degree of esterification of less than 50% but is selected to maintain sufficient reactivity to calcium and tosafeguard sufficient thickening for tube feeding. The enteral nutritional composition relates to preventing intestinal gastrointestinal adverse reactions.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention overcomes the above problems and relates to a fiber mixture comprising non-digestible oligosaccharides selected from galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS) and a non-acidic pectin polysaccharide, wherein the non-acidic pectin polysaccharide has a pH between 4.5 and 8 when added in a concentration of 1 g / L in demineralized water at 25 °C. The benefits of the specific mixture are multiple, as detailed in the experimental parts herein:

[0015] Firstly, it was unexpectedly found that the combination of GOS, FOS and such a non-acidic pectin has an increased effect on the relative abundance (%) Anaerobutyricum, a known butyrate producer. Butyrate is a key anti-inflammatory metabolite and a supports the growth of beneficial gut bacteria. Increasing butyrate levels accordingly is associated with maintaining and improving a healthy gut microbiome. Moreover, Anaerobutyricum has a beneficial effect on improving glycemic variability which is linked to type 2 diabetes (Attaye et al., Oral Anaerobutyricum soehngenii augments glycemic control in type 2 diabetes. iScience, 2024, 27(8)).

[0016] Furthermore, the fiber mixture according to the invention also showed a decrease in the relative abundance (%) of Bilophila. Bilophila is a metabolic marker which has been attributed to metabolic syndrome which is a considered a marker for a disbalanced gut microbiome (Natividad et al., Bilophila wadsworthia aggravates high fat diet induced metabolic dysfunctions in mice. Nat Commun., 2018, 9, 2802).

[0017] Additionally, the effect of the fiber mixture according to the invention was explored on epithelial barrier function, and a significant increase in trans epithelial electrical resistance (TEER) was observed for the composition comprising the non-acidic pectin polysaccharide. An increase in TEER is indicative of an improvement of gut barrier function.

[0018] To date, hydrolysed pectin polysaccharides, also known as galacturonic acid oligosaccharides (AOS), have been used as prebiotics in nutritional compositions. Interestingly, fiber mixtures comprising AOS known in the art did not provide a decrease in the relative abundance of Bilophila. The inventors also tested these AOS in a composition comprising GOS, FOS and AOS. Considering that an increase in Anaerobutyricum and a decrease in Bilophila can belinked to improving or stimulating gut microbiota and / or treating or preventing dysbiosis, an increase in the ratio of Anaerobutyricum to Bilophila could be seen as marker for improving or stimulating gut microbiota and / or treating or preventing dysbiosis. The use of a fiber mixture with conventional AOS showed an increase in that ratio, but there is still potential for further improvement. The same applies to the increase in TEER. Hence, while fiber mixtures comprising AOS did not provide each of the above advantages or only to a limited extent, the fiber mixture comprising the non-acidic pectin according to the invention increases the ratio of Anaerobutyricum to Bilophila and thereby supports and improves the gut microbiome. The combination of GOS, FOS and non-acidic pectin has an improved effect on multiple factors contributing to an improved gut microbiome, in specific to factors related to ageing-related disorders and treating and preventing dysbiosis.

[0019] In a clinical study, 30 adults were provided with the synbiotic composition according to the invention comprising GOS, FOS, non-acidic pectin and Bifidobacterium for 28 days. Fecal samples were collected before intervention and at 7, 14, and 28 days after intervention started for gut microbiome analysis, fecal metabolite analysis and determination of eosinophil cationic protein (ECP) levels. Surprisingly, the inventors found that the synbiotic composition according to the invention resulted in a significant increase in the relative abundance (%) of Bifidobacterium and Anaerostipes, a significant increase in total short-chain fatty acid (SCFA) levels and a significant decrease in ECP levels compared to baseline levels. An increase in SCFA was observed, and for all of butyrate, acetate and propionate.

[0020] SCFAs are metabolites produced by the gut microbiota upon fermentation of non-digestible carbohydrates. These metabolites play a central role in modulating the composition and function of the intestinal microbiome by supporting the growth of beneficial bacteria. Moreover, SCFAs exert immunomodulatory effects by stimulating the production of anti-inflammatory cytokines and contribute to the maintenance of the intestinal barrier integrity. The main SCFAs are butyrate, acetate and propionate. Acetate has been shown to have a beneficial effect on epithelial integrity and suppresses pro-inflammatory cytokines (Hosmer eta / . Bacterial acetate metabolism and its influence on human epithelia. Emerg Top Life Sci, 2024 doi: https: / / doi.org / 10.1042 / ETLS20220092). Propionate in turn plays a role in regulating appetite, immune responses and gut barrier function. Hence, the observed increase in SCFA production is indicative for the synbiotic composition according to the invention to treat and / or prevent gut dysbiosis and stimulate gut microbiota.

[0021] ECP is primarily excreted by activated eosinophils - immune cells involved in host defense and inflammatory responses. Elevated ECP levels have been consistently observed inpatients with inflammatory bowel diseases (IBD) and is considered a biomarker for IBD (Abedin et al. Fecal Eosinophil Cationic Protein Is a Diagnostic and Predictive Biomarker in Young Adults with Inflammatory Bowel Disease. J Clin Med. 2019, doi: 10.3390 / jcm8122025). Due to its cytotoxic properties, ECP can induce intestinal epithelial cell damage, thereby leading to barrier dysfunction. A decrease in ECP levels is thus a positive marker for improving and / or stimulating gut microbiota, particularly in subjects suffering from an impaired gut microbiota. The findings in the clinical study thus show that the synbiotic composition according to the invention beneficially improves SCFA production and reduces pro-inflammatory biomarkers together indicative of beneficial effects on the gut microbiome and dysbiosis.

[0022] Based on the above findings, the present invention also relates to a synbiotic composition comprising the fiber mixture comprising non-digestible oligosaccharides selected from galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS) and a non-acidic pectin polysaccharide according to the invention, further comprising Bifidobacterium ssp., and for the use thereof in improving or stimulating gut microbiota and / or treating or preventing dysbiosis, particularly in a subject in (therapeutic) need thereof.

[0023] The invention also relates to a nutritional composition comprising the fiber mixture or synbiotic composition, macro ingredients selected from digestible carbohydrates, fats, proteins and preferably further comprising a therapeutically effective combination of vitamin C and / or vitamin E, including equivalents thereof

[0024] A further aspect of the invention is the use of the fiber mixture, synbiotic composition or nutritional composition (in the manufacture of a product) for improving or stimulating gut microbiota and / or treating or preventing dysbiosis.

[0025] LIST OF FIGURES

[0026] Figure 1. Comparison of relative abundance (%) of Anaerobutyricum family at 72h between treatments.

[0027] Figure 2. Comparison of relative abundance (%) of Bilophila family at 72h between treatments.

[0028] Figure 3. TEER values after 6h treatment of the Caco-2 cells with the fermented samples of GOS, FOS, non-acidic pectin in a 9:1:2 ratio and GOS, FOS, AOS in a 9:1:2 ratio.

[0029] Figure 4. Comparison of relative abundance (%) of Anaerobutyricum family at 72h between treatments.LIST OF PREFERRED EMBODIMENTS

[0030] 1. A fiber mixture comprising non-digestible oligosaccharides selected from galactooligosaccharides and / or fructooligosaccharides and a non-acidic pectin polysaccharide, wherein the non-acidic pectin polysaccharide has a pH between 4.5 - 8 when measured for a 1 g / L non-acidic pectin polysaccharide in water at 25 °C.

[0031] 2. The fiber mixture according to embodiment 1, wherein the non-acidic pectin polysaccharide has a degree of esterification below 40%, more preferably below 30%, more preferably below 25%, most preferably below 20%.

[0032] 3. The fiber mixture according to any of the preceding embodiments, wherein the non-acidic pectin polysaccharide added to the fiber mixture is obtainable by adding an amount of salt, preferably potassium and / or calcium salt, to an aqueous pectin polysaccharide composition, thus dissociating the hydrogen part from the carboxylic acid groups of the galacturonic acid residues, to obtain the non-acidic polysaccharide having a pH between 4.5 - 8.0, preferably between 4.5 - 7.0, more preferably between 5.0 - 6.5, most preferably between 5.0 and 6.0, when measured for a 1 g / L non-acidic pectin polysaccharide in water at 25 °C.

[0033] 4. The fiber mixture according to any of the preceding embodiments, wherein the non-acidic pectin polysaccharide has an average molecular weight between 2 and 13 kDa, more preferably between 3 and 10 kDa, even more preferably between 4 and 8 kDa, most preferably between 5 and 7 kDa.

[0034] 5. The fiber mixture according to any of the preceding embodiments, wherein the non-acidic pectin polysaccharide has a degree of polymerisation (DP) of between 15 - 65, more preferably between 20 - 55, more preferably between 28 - 50, most preferably between 30 -40.

[0035] 6. The fiber mixture according to any of the preceding embodiments, wherein it comprises galactooligosaccharides and fructooligosaccharides.

[0036] 7. The fiber mixture according to any of the preceding embodiments, wherein the weight ratio of the sum of non-digestible oligosaccharides to pectin polysaccharides is between 2:1 to 30:1, preferably 3:1 to 20:1.

[0037] 8. A synbiotic composition comprising the fiber mixture according to any of the preceding embodiments and Bifidobacterium ssp.

[0038] 9. The synbiotic composition according to embodiment 8, comprising:

[0039] • 0.5-2.5 x 108cfu / gram on dry basis of Bifidobacterium ssp.;

[0040] • at least 5wt.% of non-digestible oligosaccharides on dry basis, selected from galactooligosaccharides and / or fructooligosaccharides; and

[0041] • 0.5-4 wt.% on dry basis of a non-acidic pectin polysaccharides.10. The synbiotic composition according to embodiments 8 or 9, wherein the probiotic bacterial strain is selected from Bifidobacterium spp., preferably Bifidobacterium longum, more preferably Bifidobacterium longum BL999.

[0042] 11. The synbiotic composition according to any of embodiments 8 - 10, comprising 0.8 - 2.0 x 108cfu / gram on dry basis of Bifidobacterium spp.

[0043] 12. A nutritional composition comprising the fiber mixture according to any of embodiments 1 - 7 or the synbiotic mixture according to any of embodiments 8 - 11, wherein the nutritional composition comprises vitamin C and vitamin E.

[0044] 13. The fiber mixture according to any of embodiments 1 - 7, the synbiotic composition according to any of embodiments 8 - 11 and / or the nutritional composition according to embodiment 12,

[0045] for use in preventing or treating intestinal barrier disruption, or improving or stimulating gut microbiota in a subject suffering from an impaired gut microbiota; and / or treating or preventing dysbiosis in a subject,

[0046] preferably for use in:

[0047] • improving a recovery of a gut disease or gut health problem;

[0048] • preventing / reducing the occurrence risk of gut diseases or gut health problems;

[0049] • stimulating epithelial cell modulators and so to improve gut health; and / or

[0050] • stimulating intestinal barrier functions, stimulating integrity of the gut barrier.

[0051] 14. The use according to embodiment 13, wherein the subject suffers from chronic diseases, gastrointestinal disorders and / or a weakened immune system, preferably the subjects suffer from obesity, irritable bowel syndrome, inflammatory bowel disease, cardiovascular diseases, frailty and / or malnutrition.

[0052] 15. A non-therapeutic method comprising administration of the fiber mixture according to any of embodiments 1 - 7, the synbiotic composition according to any of embodiments 8 - 11 and / or the nutritional composition according to claim 12 for promoting healthy ageing in a healthy subject.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] In a first aspect, the invention relates to a fiber mixture comprising non-digestible oligosaccharides selected from galactooligosaccharides and / or fructooligosaccharides and a non-acidic pectin polysaccharide, wherein the non-acidic pectin polysaccharide has a pH between 4.5 and 8, preferably a pH in the range of 4.5 - 8.0, wherein the pH is measured by adding the non-acidic pectin polysaccharide to demineralized water at 25 °C in a 1 g / L concentration.In a second aspect, the invention further relates to a synbiotic composition comprising non-digestible oligosaccharides selected from galactooligosaccharides and / or fructooligosaccharides, a non-acidic pectin polysaccharide as described herein, and Bifidobacterium ssp.

[0055] In a third aspect, the invention also relates to a nutritional composition comprising the fiber mixture or synbiotic composition as described herein.

[0056] In a fourth aspect, the invention also relates to the fiber mixture, synbiotic composition and / or nutritional composition for use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject. In some aspect, the invention can be defined as the use of the fiber mixture, synbiotic composition and / or nutritional composition for the manufacture of a product for use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject. Worded differently, the invention may likewise be defined as the use of non-digestible oligosaccharides selected from galactooligosaccharides and / or fructooligosaccharides, and a non-acidic pectin polysaccharide, and optionally Bifidobacterium ssp, in the manufacture of a fiber mixture, synbiotic composition and / or nutritional composition for use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject.

[0057] The invention may also be defined as a (non-therapeutic) method of improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject, the method comprising administration of the fiber mixture, synbiotic composition and / or nutritional composition as described herein to a subject in need of improving or stimulating gut microbiota. The subject may be a healthy subject, such as a healthy human subject. Related therewith, the present invention also encompasses a (non-therapeutic) method comprising administering the fiber mixture, the synbiotic composition and / or the nutritional composition as described herein for promoting healthy ageing in a healthy subject. Ageing is a physiological process. Healthy ageing subjects do not suffer from disease-related impaired gut microbiota. The invention may also be worded as the use of the fiber mixture, synbiotic composition and / or nutritional composition for the manufacture of a product for use in promoting healthy ageing in a healthy subject, preferably a healthy ageing human as described herein.

[0058] Definitions

[0059] "Nutritional composition" means a substance or formulation that satisfies at least a portion of a subject's nutrient requirements. The terms "nutritional(s)", "nutritional formula(s)", "enteral nutritional(s)", and "nutritional supplement(s)" are used as non-limiting examples of nutritionalcomposition(s) throughout the present disclosure. Moreover, "nutritional composition(s)" may refer to liquids, powders, gels, pastes, solids, concentrates, suspensions, or ready-to-use forms of enteral formulas, oral formulas, formulas for infants, formulas for paediatric subjects, formulas for children, growing-up milks and / or formulas for adults. The nutritional composition is preferably intended for oral administration.

[0060] According to the present invention the term ‘fiber’ or ‘dietary fiber’ means the edible part of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete, partial or no fermentation in the large intestine.

[0061] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0062] Fiber mix

[0063] “Non-digestible oligosaccharides’’ (NDO)

[0064] The term “non-digestible oligosaccharides” (NDO) as used in the present invention refers to oligosaccharides which are not digested in the intestine by the action of acids or digestive enzymes present in the human upper digestive tract, e.g. small intestine and stomach, but which are preferably fermented by the human intestinal microbiota. For example, glucose, galactose, sucrose, lactose, maltose and maltodextrins are considered digestible.

[0065] The non-digestible oligosaccharide are selected from galactooligosaccharides and / or fructooligosaccharides, and combinations thereof. Preferably, the composition comprises non-digestible oligosaccharides with a degree of polymerization (DP) in the range of 2 to 250, more preferably 3 to 60.

[0066] Suitable galactooligosaccharides (GOS) include, but are not limited to, betagalactooligosaccharides and / or alpha galactooligosaccharides. The galactooligosaccharides preferably are beta-galacto oligosaccharides. In a preferred embodiment, the present nutritional composition comprises betagalacto-oligosaccharides ([galactose]n-glucose; wherein n is an integer ranging from 2 to 60, i.e. 2, 3, 4, 5, 6, ...., 59 ,60; preferably n is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10, wherein the galactose units are in majority linked together via a beta linkage. Beta-galactooligosaccharides are also referred to as trans-galacto-oligosaccharides (TOS). Preferably, the galactooligosaccharide has beta-(1,4), beta-(1,3) and / or beta-(1 ,6) glycosidic bonds and a terminal glucose. In a preferred embodiment, galactooligosaccharides comprise at least 80% beta-1,4 and beta-1,6 linkages based on total linkages, more preferably at 10 least 90%. In another preferred embodiment, the galactooligosaccharides comprise at least 50% beta-1,3 linkages based on total linkages, more preferably at least 60% based on total linkages. According to one embodiment, the composition comprises non-digestible galacto oligosaccharide comprising beta-(1,4) linkages, in particular beta-(1,4) linkages between the galactose units, and having a degree of polymerization of at least 4, preferably with a degree of polymerization from 2 to 10, more preferably of 3 to 7. A commercially available source of GOS is VivinalOGOS from FrieslandCampina Domo (Amersfoort, The Netherlands) or Bimuno® GOS (Clasado Biosciences, Berkshire, United Kingdom).

[0067] The composition according to the invention comprises fructo-oligosaccharides (FOS). Fructooligosaccharides are non-digestible oligosaccharides (NDO) comprising a chain of beta-linked fructose units with a degree of polymerization (DP) or average DP of 2 to 250, more preferably 2 to 100, even more preferably 10 to 60. Fructo-oligosaccharide according to the invention includes inulin, levan and / or a mixed type of polyfructan. An especially preferred fructooligosaccharide is inulin. Fructooligosaccharide suitable for use in the compositions is also commercially available, e.g., Raftiline®HP (Orafti, Beneo GmbH, Mannheim Germany. Preferably the fructo-oligosaccharide has an average DP above 20.

[0068] Preferably, the fiber mixture comprises galactooligosaccharides and fructooligosaccharides. Preferably the combination of GOS and FOS is present in a weight ratio of from 1:99 to 99:1, more preferably from 1:19 to 19:1, more preferably from 1:1 to 19:1, more preferably from 2:1 to 15:1, more preferably from 5:1 to 12:1, even more preferably from 8:1 to 10:1, even more preferably in a ratio of about 9:1. The sum of GOS and FOS in the fiber mixture is preferably 80 - 100 wt%, more preferably 90 - 100 wt% of the total weight of all non-digestible oligosaccharides. For sake of completeness, a pectin polysaccharide is not a NDO according to the invention.

[0069] Pectin polysaccharide

[0070] The pectin polysaccharide of the invention showing specific benefits in combination with the above NDO is a non-acidic pectin polysaccharide. The non-acidic pectin polysaccharide according to the invention can be characterized as not showing any substantial gelling or thickening behavior in a calcium environment, and / or when subjected to pH < 4 or stomach pH. The non-acidity is thus reflected by the pectin not showing any substantial calcium- oracid-induced thickening behavior; it can also be recognized in a nutritional matrix by not showing any gelling behavior which is so typical for pectin when brought into contact with substantive calcium levels or at lower pH. When the non-acidic pectin is incorporated in a nutritional matrix with calcium concentrations typical to induce gelation for LM pectins, and / or when the nutritional matrix has pH < 4, unlike its untreated counterparts, the non-acidic pectin intrinsically lacks any substantive gelling behavior. This is shown in example 5. The non-acidic pectin does not change behavior in a strong acid environment, and keeps it non-acidic features also when then returned to pH > 4.5.

[0071] There are two major classes of pectin polysaccharides, i.e. low-methoxyl pectin (LMP) and high-methoxyl pectin (HMP), wherein HMP is defined by having a degree of esterification (DE) > 50% and LMP having a DE < 50%. The DE refers to the percentage of galacturonic acid units esterified with methanol. The DE is the same as the degree of methylation (DM). The DM or DE is about a methyl ester on C6 carboxyl group; a low DE or DM pectin is susceptible to calcium gelling. DM refers to the percentage of galacturonic acid units in the pectin polysaccharide that has a methoxyl group. As the presence of methoxyl groups indicate esterification, DM is essentially another way to express DE. HMP and LMP have different physiochemical properties and thus diverse applications. One important difference between LMP and HMP is their behaviour in food applications. However, the stabilizing or thickening properties of the non-acidic pectin are of no relevance in the context of the present invention. The non-acidic pectin polysaccharide of the invention is preferably a LMP or LM pectin, that is a non-acidic pectin polysaccharide with a low degree of esterification (DE / DM < 50%). In a preferred embodiment, the non-acidic pectin polysaccharide has a DE below 40%, more preferably below 30%, even more preferably below 25%, most preferably below 20%. In an embodiment, the non-acidic pectin polysaccharide has a low degree of methylation (DM) [or DE as expressed above). The preferred DM is less than 30%, more preferably below 20%, more preferably below 15%.

[0072] Esterified galacturonic acid may also be esterified in the form of an acetyl ester; the degree of acetylation (DA) of pectin polysaccharides reflects acetyl groups on the C2 / C3 hydroxyl groups, and a higher DA typically inhibits or reduces gelation. The DA of the non-acidic pectin according to the present composition is typically very low; accordingly, the preferred DA is less than 3%, more preferably less than 2%, most preferably less than 1%.

[0073] In the context of the invention, an important physiological property of the pectin is the pH. Pectin generally has an acidic pH, around pH 4.The pectin salt is preferably obtainable by adding an amount of salt, preferably potassium and / or sodium salt, to an aqueous pectin composition, thus dissociating the hydrogen part from the carboxylic acid groups of the galacturonic acid residues, in exchange for the salt cations, wherein the amount of salt which is sufficient to dissociate the appropriate amount of carboxylic groups yielding a pectin salt having a pH in the aforementioned range above 4.5 or any of the preferred ranges herein, as determined in a test in which 1 g / l of the pectin is brought in demineralized water at 25 °C. In the context of the invention, the terms ‘pectin salt’, ‘non-acidic pectin polysaccharide’, ‘pectin polysaccharide according to the invention’ and ‘pectin polysaccharide having a pH > 4.5’ are used interchangeably, characterizing a pectin polysaccharide in salt form (with an increased pH), which is distinct from pectins which have not been subjected to salt treatment and have an acidic pH. Hence, in a preferred embodiment, the pectin according to the invention is in salt form, i.e. a pectin salt, meaning the pectin is in the form of a pharmaceutically acceptable salt. Preferably, the pectin salt is a potassium and / or sodium salt. In an embodiment, the pectin salt preferably has a salt percentage of at least 1 wt%, more preferably at least 3 wt%, most preferably at least 5 wt%, even more preferably at least 7 wt%, based on the total weight of the pectin salt. Pectin salts are known in the nutritional field, such as for example from Borzelleca etal. “Evaluation of the safety of sodium pedate as a food ingredient” Food and Chem. Toxicology, vol. 34(4) (1996) page 432ff, or Braudo et al. “The interaction of polyuronides with calcium ions. 1: binding isotherms of calcium ions with pectin substances” Carbohydrate Polymers vol. 18(3) (1992) 165-169, albeit that the advantages of those pectin salts forms and the advantages over (acidic) pectins and AOS as found by the inventors have not been hinted at.

[0074] Accordingly, in an embodiment the pectin polysaccharide added to the composition according to the invention, is a pectin salt having a pH of between 4.5 - 8, preferably 4.5 - 8.0, more preferably between 4.5 - 7.0, even more preferably between 5.0 - 6.5, most preferably between 5.0 and 6.0. Such non-acidic pectins are commercially available, such as the APC401 by DSM Andre (as used in the examples). It is considered to be within the skilled person’s skills to determine the appropriate amount of salt required to yield a pectin salt with a pH within the desired range. The pH of the pectin polysaccharide can be determined when measuring the pH when the pectin polysaccharide is added to demineralized water in a 1 g / L concentration at 25 °C.

[0075] While it was found that the key property of the pectin polysaccharide in the context of the invention is its (non-)acidity, the pectin polysaccharide according to the invention preferably has an average molecular weight between 2 - 15 kDa, more preferably between 3 - 10 kDa,even more preferably between 4 - 10 kDa. The average molecular weight may be determined using size exclusion chromatography (SEC).

[0076] Degree of polymerization (DP) as used herein refers to the number of monomeric saccharide units in a (poly- or oligo-)saccharide that are linked by glycosidic bonds. In a preferred embodiment, the pectin polysaccharide of the invention has a DP in the range of 15 - 65, more preferably 20 - 55, more preferably 25 - 50, most preferably 30 - 40.

[0077] Preferably, the pectin polysaccharides of the invention comprises more than 50% by weight of the polysaccharide of galacturonic acid residues, preferably more than 55%, more preferably more than 60%, most preferably more than 65% by weight of the polysaccharide of galacturonic acid residues.

[0078] Preferably, the pectin polysaccharides of the invention comprise less than 30% by weight of the polysaccharide of rhamnose residues, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, most preferably less than 4% by weight of the polysaccharide of rhamnose residues. Herein, the rhamnose residues are in the backbone of the pectin polysaccharides.

[0079] Preferred pectin polysaccharides according to the invention are derived from fruits, preferably derived from citrus, apple, grapes, berries , more preferably derived from citrus. Commercially available non-acidic pectin polysaccharides according to the invention include e.g., Pectin type APC401 by DSM Andre, China.

[0080] Preferably, the fiber mixture comprises a weight ratio of the sum of non-digestible oligosaccharides to pectin polysaccharides of between 2:1 to 30:1, more preferably 3:1 to 20:1, even more preferably between 4:1 to 10:1. In a preferred embodiment, the fiber mixture comprises a weight ratio of the sum of non-digestible oligosaccharides to pectin polysaccharides of between 5:1 and 40:1, more preferably between 10:1 and 30:1, most preferably between 15:1 and 25:1. In a further preferred embodiment, the non-digestible oligosaccharides are GOS and FOS in a relative weight ratio of 2:1 to 15:1, more preferably 5:1 - 12:1, wherein the weight ratio of the sum of GOS to FOS to pectin polysaccharides is in the range of 5:1 to 40:1, more preferably between 10:1 and 30:1, most preferably between 15:1 and 25:1. Even more preferred, the fiber mixture comprises the non-digestible oligosaccharides are GOS and FOS and the non-acid pectin polysaccharide in a weight ratio of 5 - 15 : 0.5 - 5 : 1 - 10, more preferably 5 - 13 : 0.7 - 2 : 1.5 - 4, most preferably 7 - 10 : 0.8 - 1.5 : 1.8 - 3. In one embodiment, the fiber mixture comprises GOS, FOS and the non-acidic pectin polysaccharide in a 20% weight variation, preferably a 10% weight variation around the weight ratio 9: 1 :2.

[0081] Synbiotic composition

[0082] The invention further relates to a synbiotic composition comprising the above-described fiber mixture, and Bifidobacterium ssp. In accordance with the terminology applied in the field, the term “synbiotic composition” as used herein refers to a combination of probiotics and prebiotics, suitable for promoting healthy gut microbiome. All embodiments applicable to the fiber mixture and its components are thus applicable to the synbiotic composition.

[0083] In a preferred embodiment, the synbiotic composition comprises at least 70 wt.% on dry basis of non-digestible oligosaccharides selected from galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS). The synbiotic composition may comprise for instance 70 - 98 wt.% on dry basis of non-digestible oligosaccharides, preferably 75 - 90 wt.% on dry basis of non-digestible oligosaccharides selected from galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS). Preferably, the composition comprises galactooligosaccharides and fructooligosaccharides. Preferably the combination of GOS and FOS is present in a weight ratio of from 1 :99 to 99: 1 , more preferably from 1 : 19 to 19: 1 , more preferably from 1 : 1 to 19: 1 , more preferably from 2:1 to 15:1, more preferably from 5:1 to 12:1, even more preferably from 8:1 to 10:1, even more preferably in a ratio of about 9:1.

[0084] Preferably, the synbiotic composition comprises at least 3 wt.% on dry basis pectin polysaccharides. The synbiotic composition may comprise for instance 3 - 25 wt.% on dry basis of pectin polysaccharides, more preferably 5 - 20 wt.% on dry basis of pectin polysaccharides. In a preferred embodiment, the fiber mixture comprises a weight ratio of the sum of non-digestible oligosaccharides to pectin polysaccharides of between 5:1 and 40:1, more preferably between 10:1 and 30:1, most preferably between 15:1 and 25:1. Even more preferred, the fiber mixture comprises the non-digestible oligosaccharides are GOS and FOS and the non-acid pectin polysaccharide in a weight ratio of 5 - 15 : 0.5 - 5 : 1 - 10, more preferably 5 - 13 : 0.7 -2 : 1.5 - 4, most preferably 7 - 10 : 0.8- 1.5 : 1.8 - 3.

[0085] Bifidobacterium strains

[0086] Bifidobacterium strains are herein provided as a probiotic bacterial strain in the compositions of the invention. The term “probiotic” as used herein refers to micro-organisms which, when orally administered in adequate amounts, provide a health benefit. As such, the amounts of Bifidobacterium ssp. strains according to the invention are effective for providing a health benefit. Preferably, the strains used herein are selected from viable micro-organisms, non-viable micro-organisms, fragments of micro-organisms and combinations thereof, preferably viable micro-organisms.

[0087] The synbiotic composition preferably comprises 1.0 x 107to 1.0 x 1012cfu / gram on dry basis of Bifidobacterium spp, more preferably 4.0 x 107to 1.0 x 1O10cfu / gram on dry basis of Bifidobacterium spp.

[0088] Preferably, the Bifidobacterium spp. is selected from the group consisting of B. longum, B. breve, B. infantis, B. animalis, B. lactis and B. bifidum, most preferably B. longum. In one preferred embodiment, the species is Bifidobacterium longum, even more preferably Bifidobacterium longum, official deposit no.: ATCC BAA-999; NCC No.: NCC 3001; “BL999”. B. longum (ATCC BAA-999), also referred to as BL999, may be obtained from Morinaga Milk Industry Co. Ltd. of Japan under the trade mark BB536 which is commercially available.

[0089] The synbiotic composition according to the invention may in a preferred aspect be used as a nutritional supplement, i.e., as an additive to a diet. The supplement, preferably for enteral application, may be a solid or liquid galenical formulation. Examples of solid galenical formulations are tablets, capsules (e.g. hard or soft shell gelatine capsules), pills, sachets, powders, granules and the like which contain the active ingredient together with conventional galenical carriers. Any conventional carrier material can be utilized. The carrier material can be organic or inorganic inert carrier material suitable for oral administration. Suitable carriers include water, gelatine, gum Arabic, lactose, starch, magnesium stearate, talc, vegetable oils, and the like. Additionally, additives such as flavouring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of nutritional and pharmaceutical compounding.

[0090] Typically, the synbiotic compositions of the invention are in powder form for reconstitution or in liquid form, preferably in powder from for reconstitution. The compositions are preferably for enteral administration, more preferably oral administration.

[0091] Nutritional composition

[0092] The nutritional composition of the invention comprises the fiber mixture or the synbiotic composition as described here above. All of the above embodiments characterizing the fiber mixture and the synbiotic composition are thus applicable to the nutritional composition. The nutritional composition further comprises one or more macronutrients selected from digestible carbohydrates, fats, proteins, and preferably further comprises a combination of vitamin C and vitamin E.In a preferred embodiment, a serving comprises between 20 and 30 gram of the synbiotic mixture or nutritional composition. Preferably, the daily dose of the synbiotic mixture or nutritional composition comprises between 40 and 60 gram, preferably provided in two servings of between 20 and 30 gram.

[0093] The energy provided by nutrients is calculated using the Atwater calculation factors of 9 kcal per g lipid, 4 kcal per gram protein or gram digestible carbohydrate, 2 kcal per gram fiber and zero kcal for the other components in the product.

[0094] In a preferred embodiment, the synbiotic composition or nutritional composition comprises at least 2 wt.% on dry basis of non-digestible oligosaccharides selected from galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS). The synbiotic composition or nutritional composition may comprise for instance 3- 15 wt.% on dry basis of non-digestible oligosaccharides, preferably 4 - 10 wt.% on dry basis of non-digestible oligosaccharides selected from galactooligosaccharides (GOS) and / or fructooligosaccharides (FOS). In one embodiment, based on calories, preferably the synbiotic composition or nutritional composition according to the invention may comprise from 0.25 to 6 grams GOS per 100 kcal, more preferably from 0.5 to 4 grams per 100 kcal, most preferably from 0.75 to 3 grams GOS per 100 kcal based on total energy content of the synbiotic composition or nutritional composition. In a preferred embodiment, the synbiotic composition according to the invention comprises based on dry weight 1.0 to 25 g GGS / 100 g, more preferably 2.0 to 17 g GGS / 100 g, most preferably 3.2 to 13 g GGS / 100 g. Preferably, the synbiotic composition or nutritional composition comprises 0.25 to 6.0 g GOS per serving, more preferably 0.5 to 4.25 GOS per serving, most preferably 0.8 to 3.0 g GOS per serving.

[0095] In another embodiment, based on calories, preferably the synbiotic composition or nutritional composition according to the invention may comprise from 0.01 to 2 grams FOS per 100 kcal, more preferably from 0.05 to 1.5 grams FOS per 100 kcal, most preferably from 0.075 to 0.75 grams FOS per 100 kcal based on total energy content of the synbiotic composition or nutritional composition. In a preferred embodiment, the synbiotic composition or nutritional composition according to the invention comprises based on dry weight 0.05 to 8.5 g FGS / 100 g, more preferably 0.2 to 6.5 g FGS / 100 g, most preferably 0.3 to 3.5 g FGS / 100 g. Preferably, the synbiotic composition or nutritional composition comprises 0.01 to 2.0 g FOS per serving, more preferably 0.05 to 1.6 FOS per serving, most preferably 0.08 to 1.0 g FOS per serving.In a further embodiment, based on calories, preferably the synbiotic composition or nutritional composition according to the invention may comprise from 0.25 to 6 grams non-digestible oligosaccharides per 100 kcal, more preferably from 0.5 to 4 grams per 100 kcal, most preferably from 0.75 to 3.5 grams non-digestible oligosaccharides per 100 kcal based on total energy content of the synbiotic composition or nutritional composition.

[0096] The synbiotic composition or nutritional composition according to the invention preferably comprises at least 0.1 grams of pectin polysaccharides per 100 kcal more preferably 0.1 to 2 g, even more preferably 0.2 - 1 g pectin polysaccharides per 100 kcal based on total energy content of the synbiotic composition or nutritional composition. Based on dry weight, the synbiotic composition or nutritional composition according to the invention preferably comprises at least 0.5 g pectin polysaccharides per 100 g, more preferably from 0.5 to 4 g, even more preferably from 0.75 to 2.5 g per 100 g. In a preferred embodiment, the synbiotic composition or nutritional composition preferably comprises, based on dry weight, at least 0.1 g pectin polysaccharide per serving, more preferably from 0.1 to 1 g per serving, more preferably 0.15 to 0.6 g pectin polysaccharide per serving.

[0097] Expressed in calories, the synbiotic composition or nutritional composition according to the invention preferably comprises from 0.5 x 109to 4 x 109cfu per 100 kcal, more preferably from 0.75 x 109to 2.0 x 109cfu per 100 kcal, most preferably from 1.0 x 109to 1.5 x 109cfu per 100 kcal based on total energy content of the synbiotic composition or nutritional composition.

[0098] The synbiotic composition or nutritional composition preferably comprises 1.0 x 109to 9.0 x 109cfu / 100 g on dry basis more preferably 2.5 x 109to 7.5 x 109cfu / 100g, most preferably 4.0 x 109to 6.0 x 109cfu / 100g on dry basis of synbiotic composition or nutritional composition.

[0099] In a preferred embodiment, the synbiotic composition or nutritional composition preferably comprises 2.5 x 108to 2.25 x 109cfu per serving, more preferably 6.3 x 108to 1.9 x 109cfu per serving, more preferably 1.0 x 109to 1.5 x 109cfu per serving.

[0100] The nutritional composition according to the invention may be used as a pharmaceutical product or preferably a nutritional product. In one embodiment, the present nutritional composition is a solid (typically a powder or tablet, preferably a powder) which is reconstitutable with a liquid, preferably with water, to obtain a liquid composition. Preferably the composition is administered orally.In one aspect, the nutritional composition according to the invention may be used as a pharmaceutical product comprising one or more pharmaceutically acceptable carrier materials. Any conventional carrier material can be utilized. The carrier material can be organic or inorganic inert carrier material suitable for oral administration. Suitable carriers include water, gelatine, gum Arabic, lactose, starch, magnesium stearate, talc, vegetable oils, and the like. Additionally, additives such as thickeners, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding. While the individual active ingredients are suitably administered in a single composition, they may also be administered in individual dosage units.

[0101] In a preferred aspect, the nutritional composition according to the invention may be used as a nutritional product, for example as a nutritional supplement, e.g. as an additive to a normal diet, as a fortifier, to add to a normal diet, or as a complete nutrition. The nutritional product preferably comprises at least one component, preferably all components, selected from the group of fats, proteins, and carbohydrates. It may further contain ingredients such as minerals, vitamins, organic acids, and flavouring agents. Although the term "nutraceutical product" is often used in literature, it denotes a nutritional product with a pharmaceutical component or pharmaceutical purpose. Hence, the nutritional composition according to the invention may also be used in a nutraceutical product.

[0102] In one embodiment, the nutritional composition comprises a lipid fraction, wherein the lipid fraction provides between 15 and 50 energy% of the composition. The term ‘en%’ stands for the caloric amount provided by the component compared to the total caloric content provided by the composition, using the Atwater constants.

[0103] The weight percentages and caloric values of the synbiotic composition also apply to the nutritional composition.

[0104] The amount of lipid fraction can be determined by applying the methods known in the art for measuring fat content in the food matrix as applicable. For example, fat content for general foods is determined by applying AOAC(R) official method 983.23, while the Roese-Gottlieb method (AOAC(R) 932.06) is better applicable for products based on dried milk (Lehner, R., Estoppey, A., (1954) Mitt. Lebensmitteluntersuchung Hyg. 54:183-185). The amount of individual lipid components can be determined by applying methods specifically designed for measuring that specific component or by fractionating the fat fraction isolated from the extraction of the chloroform-ethanol fraction as given in the 983.23 method.Suitable sources of lipids for use in the nutritional composition include but are not limited to soybean oil, rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, coconut oil, high oleic safflower oil and olive oil. An alternative option are lipids obtained from milk from non-human mammals, preferably cow's milk, goat milk, sheep milk, horse milk, buffalo milk, yak milk, reindeer milk, donkey milk and camel milk, particularly cow's milk and / or goat milk. Milk lipid is sometimes also referred to as milk fat or butter fat.

[0105] The nutritional composition according to the invention thus preferably comprises a lipid fraction, preferably a lipid fraction suitable for nutrition as known in the art. Based on calories, preferably the nutritional composition according to the invention may comprise 1.5 to 5 g of lipid per 100 kcal, preferably 2.0 to 4.5 g per 100 kcal of lipid per 100 kcal based on the total energy content of the composition. The lipid fraction includes polyunsaturated fatty acids and optionally also mono-unsaturated fatty acids (MLIFAs) and saturated fatty acids (SFA).

[0106] The total protein that is present in the nutritional composition, i.e., the combination of all proteins present, may also be referred to as the “protein fraction” of the nutritional composition. Based on calories, preferably the nutritional composition according to the invention may comprise 3.0 to 7.0 g of protein fraction per 100 kcal, preferably 3.5 to 6.5 g of protein fraction per 100 kcal, based on the total energy content of the composition. Suitable protein sources may be based on cows' milk proteins such as whey, casein, and mixtures thereof and vegetable proteins such as those based on soy, potato, pea, rapeseed and the like.

[0107] The nutritional composition preferably further comprises a carbohydrate fraction. Based on calories, preferably the nutritional composition according to the invention may comprise 8.5 to 20 g of carbohydrates per 100 kcal, preferably 10 to 17.5 g per 100 kcal of carbohydrates based on the total energy content of the composition. Suitable carbohydrate sources include lactose, glucose, sucrose, fructose, galactose, maltose, starch, isomaltulose, and maltodextrin.

[0108] Administration of the nutritional composition occurs preferably at least one time per day, more preferably one or two times per day, although alternative dosage regimes can be determined from these numbers.

[0109] Vitamin C

[0110] The present nutritional composition may further comprise vitamin C. Vitamin C is an antioxidant that may provide beneficial characteristics. Vitamin C includes functionalequivalents thereof including sodium ascorbate and calcium ascorbate, and may be present in a therapeutically effective amount, preferably to provide a daily dosage in the range of 5 to 100 mg, preferably in the range of 10 to 70 mg, more preferably in the range of 15 to 50 mg per day.

[0111] The nutritional composition according the invention, preferably comprises a therapeutically effective amount of vitamin C, more preferably 10 to 90 mg vitamin C / 100 g, more preferably 30 to 70 mg vitamin C / 100 g, most preferably 40 to 60 mg vitamin C / 100 g based on dry weight of nutritional composition. In terms of calories, preferably the nutritional composition according to the invention may comprise 2.5 to 25 mg vitamin C per 100 kcal, preferably 7 to 17 mg vitamin C per 100 kcal, more preferably 9 to 14 mg vitamin C per 100 kcal based on the total energy content of the composition. The above numbers for vitamin C are based on the molar weight of ascorbic acid.

[0112] Vitamin E

[0113] The nutritional composition according to the invention may comprise vitamin E. Vitamin E refers to compounds having vitamin E activity as known in the art, typically tocopherol and / or an equivalent thereof. In a preferred embodiment, the vitamin E in the nutritional composition is alpha-tocopherol.

[0114] If included, vitamin E may be present in the nutritional composition in an amount to provide a daily dosage in the range of 0.5 to 10 mg, preferably in the range of 2.5 to 7.5 mg, more preferably in the range of 4 to 6 mg. Vitamin E may be provided in all kinds of equivalent forms, in which the amounts here are calculated in terms of the corresponding weight amount of alpha-tocopherol.

[0115] The nutritional composition according the invention, preferably comprises 2.5 to 35 mg vitamin E per 100 g, more preferably 5 to 25 mg vitamin E per 100 g, most preferably 7.5 to 17.5 mg vitamin E per 100 g nutritional composition.

[0116] Based on calories, preferably the nutritional composition according to the invention may comprise 0.5 to 8 mg vitamin E per 100 kcal, preferably 1.0 to 6.0 mg vitamin E per 100 kcal, more preferably 1.5 to 4.5 mg vitamin E per 100 kcal based on the total energy content of the nutritional composition.

[0117] The term “tocopherol and / or an equivalent thereof”, as used in this description, comprises tocopherols (e.g. alpha- and gamma-), tocotrienols, pharmaceutical and / or nutritionalacceptable derivatives thereof and any combination thereof. The above numbers are based on alpha-tocopherol equivalents (alpha-TE) and the molecular weight thereof, as recognized in the art.

[0118] The composition may be provided with additional vitamins and minerals according to the subject’s general needs.

[0119] Supplement

[0120] The nutritional composition according to the invention may in a preferred aspect be used as a nutritional supplement, i.e., as an additive to a diet. The supplement, preferably for enteral application, may be a solid or liquid galenical formulation. Examples of solid galenical formulations are tablets, capsules (e.g. hard or soft shell gelatine capsules), pills, sachets, powders, granules and the like which contain the active ingredient together with conventional galenical carriers. Any conventional carrier material can be utilized. The carrier material can be organic or inorganic inert carrier material suitable for oral administration. Suitable carriers include water, gelatine, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, and the like. Additionally, additives such as flavouring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of nutritional and pharmaceutical compounding.

[0121] Application

[0122] The fiber mix, synbiotic mixture and / or nutritional composition according to the invention relates to the use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject. In the context of the invention, the terminology ‘improving gut microbiota’ and ‘stimulating gut microbiota’ are considered interchangeable.

[0123] A wide variety of beneficial bacteria support overall health and contribute to the protection against diseases, regulation of the immune system, reducing the risk of infections and chronic inflammatory conditions and regulate metabolism. While not implied in healthy ageing, these type of chronic conditions are more often observed in elderly people suffering from a disbalance in their gut microbiota. Hence, the invention is particularly useful for healthy elderly subjects and elderly subjects suffering from a therapeutically impaired disbalance in their gut microbiota and suffering from infections and chronic inflammatory conditions associated with an impaired gut microbiota. An impaired disbalance in gut microbiota may also be called dysbiosis. Dysbiosis is a medical term which reflects an impaired imbalance in bacterial composition, changes in bacterial metabolic activities, or changes in bacterial distribution within the gut which all go well beyond physiological variations. The three types of dysbiosisare: 1) loss of beneficial bacteria, 2) overgrowth of potentially pathogenic bacteria (bacterial overgrowth), and 3) loss of overall bacterial diversity.

[0124] The inventors surprisingly found that the fiber mixture, synbiotic composition and / or nutritional composition according to the invention has a positive effect on improving or stimulating gut microbiota and / or treating or preventing dysbiosis. The fiber mixture was found to have a positive effect on the relative ratio of Anaerobutyricum over Bilophila. Both are markers for the state of the gut microbiome, and hence these findings are particularly useful in healthy ageing and for subjects in therapeutic need of an improved gut microbiota i.e. subjects having an impaired gut microbiota. The fiber mixture, synbiotic mixture and / or nutritional composition according to the invention increases the ratio of Anaerobutyricum to Bilophila and thereby results in a more balanced microbiota, and supports and improves the gut microbiome.

[0125] Additionally, it was found that the synbiotic composition according to the invention increased the relative abundance (%) of Bifidobacteria and Anaerostipes, and SCFA production. These findings are associated with improving the gut microbiome. Increased levels of SCFA are moreover related to enhanced intestinal barrier integrity and exert anti-inflammatory effects. An increase in the relative abundance (%) of Bifidobacteria and Anaerostipes, and SCFA production thus beneficially results in an improved gut microbiome and protects against dysbiosis. Upon intervention with the synbiotic composition according to the invention also a significant decrease in ECP levels was observed, a protein known to contribute to inflammation and potentially negatively alters the gut microbiome. Hence, a decrease in EPC levels is indicative of a healthier gut microbiome.

[0126] In an embodiment the invention relates to

[0127] the fiber mixture,

[0128] the synbiotic composition and / or

[0129] the nutritional composition

[0130] according to the invention for use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject. In other words, the invention preferably relates to the fiber mixture, the synbiotic composition and / or the nutritional composition according to the invention for use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject. A further aspect of the invention is the use of the fiber mixture, synbiotic composition or nutritional composition (in the manufacture of a product) for improving or stimulating gut microbiota and / or treating or preventing dysbiosis.The invention may also be defined as a (non-therapeutic) method of improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject, the method comprising administration of the fiber mixture, synbiotic composition and / or nutritional composition as described herein to a subject in need of improving or stimulating gut microbiota.

[0131] In an embodiment the invention relates to, the fiber mixture, the synbiotic composition and / or the nutritional composition according to the invention for use in improving or stimulating gut microbiota and / or treating or preventing dysbiosis in a subject, preferably for use in one or more of:

[0132] • improving a recovery of a gut disease or gut health problem;

[0133] • preventing / reducing the occurrence risk of gut diseases or gut health problems;

[0134] • stimulating epithelial cell modulators and so to improve gut health; and / or

[0135] • stimulating intestinal barrier functions, stimulating integrity of the gut barrier.

[0136] In an embodiment, the invention preferably relates to the fiber mixture, synbiotic composition and / or nutritional composition for use in preventing intestinal barrier disruption or treating intestinal barrier disruption.

[0137] The intestinal (gut) barrier is a critical interface of the human body - on the one hand it provides for a strong defence against foreign substances while on the other hand it regulates the absorption of essential nutrients, water and electrolytes. The intestinal barrier is composed of multiple layers. The outer one comprises the mucus layer, the commensal gut microbiota and defence proteins such as antimicrobial proteins (AMPs) and secretory immunoglobulin A (slgA). Intestinal epithelial cells (lECs) are the middle layer, while the inner part is composed of e.g. immune cells of innate and adaptive immunity.

[0138] The term “intestinal barrier disruption” as used herein refers to any damage or wounding of the intestinal barrier that interferes with proper functioning of the intestinal barrier and results in reduced intestinal barrier integrity.

[0139] The term “preventing intestinal barrier disruption” as used herein refers to promoting intestinal conditions wherein the intestinal barrier is in optimal condition and thereby less vulnerable to damage or wounding.The term “treating intestinal barrier disruption” as used herein refers to promoting intestinal conditions where the intestinal barrier can recover faster and / or more completely after being exposed to damage or wounding.

[0140] Preferably, the subject is a human adult, more preferably the subject is older than 35 years, more preferably older than 40 years. An elderly person is preferably a person older than 50 years of age. In one embodiment, the subject is a non-healthy subject i.e. suffers from chronic diseases, gastrointestinal disorders and / or a weakened immune system. For example, but not limited to, are subjects suffering from obesity, irritable bowel syndrome, inflammatory bowel disease, cardiovascular diseases, frailty and / or malnutrition.

[0141] Additionally, the inventors surprisingly found that the increase in the ratio between Anaerobutyricum and Bilophila promotes a healthy gut microbiota in healthy subjects, preferably healthy humans. In an embodiment, the invention preferably relates to a non-therapeutic method comprising administering the fiber mixture, the synbiotic composition and / or the nutritional composition according to the invention for promoting healthy ageing in a healthy (human) subject. In a preferred embodiment, the healthy (human) subject is older than 35 years, more preferably older than 40 years. The healthy (human) subject is preferably an elderly subject, preferably a person older than 50 years of age. The term ‘healthy ageing’ as used herein refers to the maintenance of a balanced and diverse gut microbiome that supports overall health and well-being as one ages.

[0142] The invention is further illustrated by the following non-limiting examples.

[0143] EXAMPLES

[0144] Example 1

[0145] To study the effect on gut microbiome, the gut microbiome composition was analysed by metagenomic sequencing. A number of compositions were tested in respect to the contribution of galacturonic acid oligosaccharides, using a composition comprising GOS and FOS in a 9:1 weight ratio as a base, and comparing the effect of non-acidic pectin polysaccharide and galacturonic acid oligosaccharides (AOS), in a weight ratio of 9: 1 :2.

[0146] GOS was VivinalOGOS from FrieslandCampina Domo, Amersfoort, The Netherlands.

[0147] FOS was oligofructose, Orafti®.

[0148] AOS was produced by hydrolysing pectin oligosaccharides (liquid pectin hydrolysates, Cargill) to obtain oligomers or monomers with a DP of < 10 and a pH of 3.9 (determined by adding theoligosaccharides in 1 g / L demineralized water at 25 °C). Since it was produced by hydrolysing pectin, it thus contained a percentage of monosaccharide units which is similar to that of pectin, but then present in either oligomers or monomers instead of polymers.

[0149] The non-acidic pectin salt was Pectin type APC401 by DSM Andre, China, MW~6 kDa. It had a pH of 5.5 as determined by adding the pectin in 1 g / L demineralized water at 25 °C. There was about 10 wt% potassium and sodium salt in the pectin salt provided.

[0150] Samples

[0151] Fecal samples of six healthy adult volunteers with an age between 40 - 60 years had been collected. Gut microbiome of the subjects enrolled in this study was analyzed using a model as described in Van den Abbeele et al. (Bridging preclinical and clinical gut microbiota research using the ex vivo SIFR® technology, Front. Microbiol., 2023, 14, p.1 -11) and Systemic Intestinal Fermentation Research (SIFR®) technology. SIFR® technology involves the use of sets of small individual bioreactors that are handled in a custom-built processing device allowing for temperature control as well as headspace control and monitoring. This technology minimizes the bias in microbiota composition between in v / Vo-derived microbiota and the one colonizing the bioreactors.

[0152] After collection, the fecal samples were preincubated with Amoxicillin 24 hours to induce dysbiosis. After preincubation, fecal samples were fermented with different compositions at a total concentration of 5 g / L of prebiotics:

[0153] 1. GOS, FOS, non-acidic pectin in a 9:1:2 ratio;

[0154] 2. GOS, FOS, AOS in a 9:1:2 ratio;

[0155] The control in all experiments was a blanc fermentation sample.

[0156] Material and methods

[0157] Fecal samples were fermented and collected after 24 hours of fermentation for quantitative shallow shotgun sequencing analysis.

[0158] DNA extraction and rRNA gene profiling were performed as previously described (Van den Abbeele, P. et al. Serum-Derived Bovine Immunoglobulin Stimulates SOFA Production by Specific Microbes in the Ex Vivo SIFR® Technology (Van den Abbeele, P. et al. Impact of two human milk oligosaccharides and lactose on the faecal microbiome of infants with probable cow’s milk allergy. Benef Microbes 1-15 (2024)). Briefly, DNA was extracted via the SPINeasyDNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to manufacturer’s instructions.

[0159] The relative abundance of Anaerobutyricum and Bilophila was measured by 3M total shotgun metagenomics DNA sequencing. The relative abundance derived from sequencing (%) were corrected for the total amount of cells present in each sample (detected via flow cytometry), allowing to obtain representative insights in the impact of interventions.

[0160] Results

[0161] The relative abundance (%) of the Anaerobutyricum and Bilophila was measured after 24 hours of fermentation. The results are shown in Figures 1 and 2 and Tables 1 and 2.

[0162] The combination of GOS, FOS and non-acidic pectin showed an improvement in abundance of Anaerobutyricum. Furthermore, the combination of GOS, FOS and non-acidic pectin showed a decrease in the abundance of Bilophila. The decrease in Bilophila abundance for the composition comprising GOS, FOS and AOS was less pronounced. The fiber mixture according to the invention (GOS, FOS, non-acidic pectin) increased the ratio of Anaerobutyricum to Bilophila by 6.5 times compared to the control.

[0163] Table 1. Mean (S.D.) relative abundance of Anaerobutyricum family at 72 hours in the tested treatments.

[0164] Composition Condition Anaerobutyricum abundance (%)

[0165] Control 0.069 (0.036) Composition 1 GOS, FOS, non-acidic pectin 9:1:2 0.093 (0.055) Composition 2 GOS, FOS, AOS 9:1:2 0.071 (0.031)

[0166]

[0167] Table 2. Mean (S.D.) relative abundance of Bilophila family at 72 hours in the tested treatments.

[0168] Composition Condition Bilophila abundance (%) Control 0.101 (0.030) Composition 1 GOS, FOS, non-acidic pectin 9:1:2 0.024 (0.010) Composition 2 GOS, FOS, AOS 9:1:2 0.041 (0.021)

[0169]

[0170] ConclusionThese results demonstrate the ability of the composition according to the invention to increase the relative abundance of Anaerobutyricum while simultaneously decreasing the relative abundance of Biophilia. Both are indicators for improving or stimulating gut microbiota. The effect was more pronounced when using the non-acidic pectin according to the invention compared to when using AOS. It was only the specific combination of GOS, FOS and non-acidic pectin that increased the relative abundance in Anaerobutyricum and decreased the relative abundance of Bilophila.

[0171] Example 2

[0172] To study the effect of the composition on epithelial barrier function trans epithelial resistance (TEER) was measured. TEER is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers and thus gut barrier function, see for example Srinivasan et al., TEER measurement techniques for in vitro barrier model systems, J Lab Autom, 2015, 20(2), 107 -126.

[0173] Samples

[0174] A fecal sample of Example 1 was used in the TEER measurements. After the Amoxicillin treatment the fecal sample was fermented with compositions 1 and 2 as described in Example 1. Samples were collected after 24 hours of fermentation. For this experiment 50 pl of supernatant was diluted 1 / 10 with Caco-2 medium (DMEM) without FCS and filtered (0.2um) and subsequently was added to the apical upper left channel A1, 50 pl was added to apical channel A3. In the basolateral channel DMEM without FCS was added, 50 pl in C1 and 50 pl in C3.

[0175] Material and methods

[0176] Experiments were performed in the microfluidic OrganoPlate platform of Mimetas (Oegstgeest, Netherlands) and performed according to method as described in Nicolas et al., High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia, LAB CHIP, 2021, 21, 1676-1685. In the experiment, Caco-2 ready plates were provided by Mimetas. The plates contained two tubules grown directly against an extracellular matrix without the use of artificial membranes. The TEER was measured by a four-point measurement in a single chip with electrodes contacting the cell culture medium that addresses either the luminal or basil side of the epithelium. The epithelial tubules in the Caco-2 plates were seeded in the upper channel of the plate (day 0) as described in Nicolas et al. (2021). Upon arrival (day 4) the plates were incubated horizontally and static for 1 h at 37 °C, whereafter the transport medium was removed and fresh Caco-2 medium (DMEM withFCS) was added according to standard protocols provided by Mimetas and as described in Nicolas et al. (2021). The plates were placed on a rocker in the incubator for 24 h at 37 °C. On day 5 the medium was refreshed and on day 7 the supernatants were added to the plate.

[0177] 50 pl of the supernatant was added to the top perfusion inlet wells (left and right channel A1 and A3). In the basolateral channels (C1 and C3) 50 pl of CacO-2 medium was added as described in the protocol by Mimetas and in Nicolas etal., (2021).

[0178] The sample was added in five different chips on the plate (n=5) and TEER was measured over time according to the protocols of Mimetas as described in Nicolas etal., (2021).

[0179] Results

[0180] The effect on TEER values after 6 hours of Caco-2 treatment is shown in Figure 3.

[0181] The dialysates derived from the fecal samples treated with a composition comprising GOS, FOS and non-acidic pectin polysaccharide in a 9:1:2 ratio resulted in a significant increase in TEER compared to dialysates derived from the fecal samples treated with the composition comprising GOS, FOS and AOS in a 9:1:2 ratio.

[0182] Conclusion

[0183] These results demonstrate the ability of the fiber mixture according to the invention (GOS, FOS and non-acidic pectin polysaccharide) to beneficially effect the production of microbial metabolites which upon incubation with epithelial tubules lead to an improved effect on gut barrier function thus illustrating a synergistic effect of the composition on improving gut barrier function.

[0184] Example 3

[0185] To study the effect on gut microbiomecomposition, fecal metabolites and eosinophil cationic protein (ECP) levels were tested in an interventional, self-controlled single arm clinical study.

[0186] Material and methods

[0187] To study the effect, the synbiotic composition according to the invention comprising GOS, FOS, non-acidic pectin and Bifidobacterium was tested. The GOS, FOS and non-acidic were those according to Example 1. The Bifidobacterium used was Bifidobacterium longum, BL999 (BB536 from Morinaga).30 participants were enrolled in the study, of which 28 completed the study. Fecal samples were collected at baseline (V1), day 7 (V2), day 14 (V3), and day 28 (V4) of the intervention. The gut microbiome composition, fecal metabolites and inflammation biomarkers (ECP) were analyzed in the fecal samples.

[0188] Fecal Metagenomic Analysis

[0189] DNA was extracted from samples using MagPure Stool DNA KF Kit B (MAGEN, Guangzhou, China) I Magnetic Bead Fecal and Soil Genome Extraction Kit (MAGEN, Guangzhou, China) according to manufacturer instructions, and stored at -80 °C until further use. Genomic DNA (1 pg) was fragmented using a Covaris ultrasonicator, followed by size selection of 200-400 bp fragments using magnetic beads. The selected fragments underwent end repair, A-tailing, and adapter ligation at optimized temperatures. The ligated products were then PCR-amplified and purified with magnetic beads. Subsequently, PCR products were denatured into singlestranded DNA and circularized, with residual linear DNA digested to obtain the final singlestranded circular library. After quality control through concentration measurement, metagenomic sequencing was performed on the DNBSEQ platform (MGI Tech, Shenzhen, China) with PE150 read length.

[0190] Bioinformatics Analysis

[0191] Raw sequencing data were quality-filtered using SOAPnuke (Chen et al., GigaScience, 2018, 7(1), doi.org / 10.1093 / gigascience / gix120), followed by host sequence removal via Bowtie2 (Langmead et al., Nat Methods, 2012, 9(4), doi: 10.1038 / nmeth.1923) alignment. Cleaned reads were assembled into contigs with MEGAHIT (Li etal., Bioinformatics, 2015, 31(10), doi: 10.1093 / bioinformatics / btv033), and genes were predicted using MetaGeneMark (Zhu et al. Nucleic Acids Res., 2010, 38(12), doi: 10.1093 / nar / gkq275). Non-redundant gene sets were generated with CD-HIT (Fu et al. Bioinformatics, 2012, 28(3), doi: 10.1093 / bioinformatics / bts565) and quantified via Salmon (Patro et al. Nat Methods., 2015, 12(1), doi: 10.1038 / nmeth.4197). Functional annotation was performed using DIAMOND (Buchfink et al. Nature Methods, 2015, 12(1), doi: 10.1038 / nmeth.3176) against eggnog (Huerta-Cepas et al. Nucleic Acids Res., 2019 8(47), doi: 10.1093 / nar / gky1085), KEGG (Kanehisa etal. Nucleic Acids Res., 200028(1), doi: 10.1093 / nar / 28.1.27), BacMet (Pal etal. Nucleic Acids Res., 2014, 42, doi: 10.1093 / nar / gkt1252), CARD (Jia et al. Nucleic Acids Res., 2017, 45, doi: 10.1093 / nar / gkw1004), COG (Galperin et al. Nuleic Acids Res., 2015, 43doi: 10.1093 / nar / gku1223), CAZy (Lombard et al. Nucleic Acids Res., 2014, 42, doi: 10.1093 / nar / gkt1178), and Swiss-Prot (Poux et al. Bioinformatics, 2017, 33(21), doi: 10.1093 / bioinformatics / btx439) databases. Taxonomic profiling was conducted with Kraken2 (Wood etal. Genome Biol. 2019, 20(1), doi: 10.1186 / s13059-019-1891-0) (custom NCBI NT+ UHGG (Almeida et al. Nat Biotechnol. 2012, 39(1), doi: 10.1038 / s41587-020-0603-3) databases) and refined with Bracken2 for abundance estimation. Downstream analyses included: (1) alpha / beta diversity metrics (Simpson et al. Nature, 1949, 30, doi: 10.1038 / 163688a0, A Mathematical Theory of Communication Claude E. Shannon, Whittaker et al. Ecological Monographs, 1960, 1(30), doi: 10.2307 / 1943563, Stat et al. Ecol. Evol. 2013, 3(5), doi: 10.1002 / ece3.556) (PCA (Jolliffe etal. Phil. Trans. R. Soc. A., 2016, 374, doi: 10.1098 / rsta.2015.0202), PCoA, NMDS); (2) differential abundance testing (Wilcoxon / Kruskal, LEfSe [LDA>2.0, a=0.05]); (3) KEGG pathway enrichment (ReportScore method); and (4) correlation studies integrating environmental / phenotypic data.

[0192] SCFA Analysis

[0193] Short-chain fatty acids were analyzed by UPLC-MS. 50 mg fecal samples were extracted with methanokacetonitrile (2:1), derivatized with 3-NPH / EDC, and analyzed by UPLC-MS (Waters ACQUITY / QTRAP 6500+). Chromatography used a BEH C18 column with 0.1% formic acid / acetonitrile gradient. MS detection employed negative ESI (-4500V) with MRM mode. Data were processed in MultiQuant for metabolite identification (retention time / MRM transitions) and quantification.

[0194] Eosinophil cationic protein (ECP) Analysis

[0195] ECP levels were analyzed using enzyme-linked immunosorbent assay (ELISA). Fecal ECP was detected using Human RNASE3 / ECP (Ribonuclease A3 / Esonophil cationic protein) ELISA kit according to manufacture instructions (AssayGenie, Dublin, Ireland).

[0196] Analysis

[0197] All analyses were performed using R software. The relative abundance of Bifidobacterium was statistically analyzed Kruskal-Wallis test, Dunn-test plus BH correction for pairwise comparison. Total SCFAs, butyrate, acetate, propionate, and ECP values were Box-Cox transformed and followed by parametric intergroup comparison (ANOVA) and post-hoc testing (Tukey HSD). Significant difference is claimed at P-value smaller than 0.05.

[0198] Results

[0199] Gut microbiome

[0200] The relative abundance (%) of the Bifidobacterium was measured at V1, V2, V3 and V4. The results are shown in Table 3. The combination of GOS, FOS, non-acidic pectin and BB536 shows a significant improvement in the abundance of Bifidobacterium after 7, 14 and 28 days of intervention. Overall, a 2.5 - 4.0 time increase in the relative abundance (%) of Bifidobacterium was observed compared to baseline levels (V1).Table 3. Mean (S.D.) relative abundance of Bifidobacterium at V1, V2, V3 and V4.

[0201] Time point N Relative abundance (%) Increase compared to V1

[0202] (times)

[0203] V1 30 0.0021 (0.0036)

[0204] V2 29 0.0053 (0.0071)* 2.5

[0205] V3 29 0.0084 (0.0116)** 4.0

[0206] V4 28 0.0063 (0.0118)* 3.0

[0207]

[0208] *Significant increase compared to V1, *P < 0.05, ** P < 0.01.

[0209] Short-chain fatty acids (SCFA)

[0210] The combination of GOS, FOS, non-acidic pectin and BB536 resulted in an increase in total SCFA production. The effect on total SCFA, butyrate, acetate and propionate levels is shown in Tables 4- 7.

[0211] Total SCFA levels were significantly increased by 10 - 12% compared to baseline levels. A 5 - 10% increase was observed in butyrate levels and acetate levels significantly increased by 8 - 10%. Propionate levels significantly increased by 14 - 16%.

[0212] Table 4. Mean (S.D.) total SCFA levels at V1, V2, V3 and V4.

[0213] Time point N Total SCFA levels (nmol / g) Increase compared to V1 (%) V1 30 42.73 (4.30)

[0214] V2 29 47.13 (3.88)* 10%

[0215] V3 29 47.68 (4.27)* 12%

[0216] V4 28 46.92 (4.10)* 10%

[0217]

[0218] *Significant increase compared to V1, *P < 0.05.

[0219] Table 5. Mean (S.D.) butyrate levels at V1, V2, V3 and V4.

[0220] Time point N butyrate levels (nmol / g) Increase compared to V1 (%) V1 30 18.15 (2.22)

[0221] V2 29 19.47 (2.02) 7%

[0222] V3 29 19.91 (3.11)* 10%

[0223] V4 28 19.05 (2.76) 5%

[0224]

[0225] *Significant increase compared to V1, *P < 0.05.

[0226] Table 6. Mean (S.D.) acetate levels at V1, V2, V3 and V4.Time point N acetate levels (nmol / g) Increase compared to V1 (%) V1 30 22.26 (1.99)

[0227] V2 29 24.19 (1.68)* 1.09 (9%)

[0228] V3 29 24.55 (1.89)* 1.10 (10%)

[0229] V4 28 24.09 (1.75)* 1.08 (8%)

[0230]

[0231] *Significant increase compared to V1, *P < 0.05.

[0232] Table 7. Mean (S.D.) propionate levels at V1 , V2, V3 and V4.

[0233] Time point N propionate levels (nmol / g) Increase compared to V1 (%) V1 30 194.7 (35.5)

[0234] V2 29 225.7 (34.54)* 16%

[0235] V3 29 224.7 (46.56)* 15%

[0236] V4 28 221.3 (38.82) 14%

[0237]

[0238] *Significant increase compared to V1, *P < 0.05.

[0239] Eosinophil cationic protein (ECP)

[0240] The combination of GOS, FOS, non-acidic pectin and BB536 resulted in a significant decrease in ECP levels. The effect on ECP levels is shown in Table 8.

[0241] ECP levels were significantly decreased by 32 - 41% after 7, 14, and 28 days of intervention.

[0242] Table 8. Mean (S.D.) ECP levels at V1, V2, V3 and V4.

[0243] Time point N ECP levels (pg / g) Decrease compared to V1 (%) V1 30 5.54 (1.31)

[0244] V2 29 3.77 (1.28)* 32%

[0245] V3 29 3.56 (1.46)* 36%

[0246] V4 28 3.29 (1.28) 41%

[0247]

[0248] *Significant increase compared to V1, *P < 0.001.

[0249] Anaerostipes

[0250] The combination of GOS, FOS, non-acidic pectin and BB536 resulted in a significant increase in Anaerostipes, which is a known SCFA producer. The effects is shown in Table 9.

[0251] Table 9. Mean (S.D.) Anaerostipes (%) at V1, V2, V3 and V4.

[0252] Parameters V1 (n = 28) V2 (n = 28) V3 (n = 28) V4 (n = 28) ~Pi0.13 0.15 0.29 0.30 0.022 Anaerostipes, %

[0253] (0.09, 0.41) (0.08, 0.38) (0.14, 0.48) (0.11, 0.54)

[0254] P2— 0.279 0.033 0.086

[0255] The relative abundance of SCFA producer Anaerostipes sp. was increased over the study period.

[0256] Conclusion

[0257] The results of the clinical study demonstrate the ability of the synbiotic composition according to the invention (GOS, FOS, non-acidic pectin and BB536) to increase the relative abundance (%) of Bifidobacterium. Also, these results prove the ability of the synbiotic composition according to the invention to beneficially affect the production of SCFA, directly and by increasing the relative abundance of Anaerostipes. Of note, the numbers are significant at V3, after 14 days of intervention. For some parameters, a stabilization of the results was observed at V4, but this was a mere consequence of a clinical study with healthy subjects experiencing more difficulties adhering to the program conditions over longer time periods (going from day 14 (V3) to day 28 (V4)). There is no detrimental effect associated with extended intervention, most health effects and positive effects of the gut microbiota had however already been achieved in a relatively short period of time.

[0258] The increase in the relative abundance of Bifidobacterium, Anaerostipes and SCFA production are positive markers for improving gut microbiota. Moreover, the synbiotic composition according to the invention leads to a significant decrease in ECP levels. A decrease in ECP levels is positively related to a decrease in inflammatory responses. Together these findings provide evidence of the beneficial effects of the symbiotic composition according to the invention for preventing or treating intestinal barrier disruption, or improving or stimulating gut microbiota in a subject suffering from an impaired gut microbiota and / or treating or preventing dysbiosis in a subject.

[0259] Example 4

[0260] The effect of the synbiotic mixture according to the invention on the relative abundance of Anaerobutyricum was tested. Thereto, fecal samples were fermented with different compositions at a total concentration of 5 g / L of prebiotics, in addition to composition 1 and 2 as described in example 1, the following compositions were tested:

[0261] 3. GOS, FOS, non-acidic pectin in a 9:1:2 ratio and 2 x 107cfu BB536;

[0262] 4. GOS, FOS, AOS in a 9:1:2 ratio and 2 x 107cfu BB536.The GOS, FOS, non-acidic pectin and AOS were those already detailed in example 1. BB536 was the probiotic Bifidobacterium longum, BL999 (BB536 from Morinaga). The control in all experiments is a blanc fermentation sample.

[0263] Results

[0264] Reference is made to Figure 4. The synbiotic mixture (3) comprising GOS, FOS, non-acidic pectin and BB536 showed an effect on gut microbiota improved over the control and over (4). Specifically, an improved effect on relative abundance (%) of Anaerobutyricum was observed. The increase in Anaerobutyricum by the addition of BB536 to the composition comprising the non-acidic pectin salt according to the invention (1 vs 3) was more significant than when BB536 was added to the composition comprising AOS (2 vs 4).

[0265] Conclusion

[0266] These results demonstrate the ability of the synbiotic mixture according to increase the relative abundance of Anaerobutyricum, which is a positive marker for improving gut microbiota.

[0267] Example 5 - non-acidic pectin

[0268] In order to demonstrate that the non-acidity has its effect on the properties of the pectin, gelling behavior in presence of CaCh was studied before or after pH adjustment.

[0269] To this end, there was prepared 1 w / v% and 5 w / v% aqueous solutions with a non-acidic pectin as described in example 1 i.e. APC401 by DSM Andre, China, MW~6 kDa. There was about 5-8 wt% potassium and 2-5 wt% sodium salt, with a total of 10 wt% based on the total weight of the ingredient, in the pectin salt provided. As available, the non-acidic pectin had a pH of 5.5 as determined by preparing a pectin solution of 1 g / L demineralized water at 25 °C. The 1 w / v% solution had pH 5.5, the 5 w / v% solution had a pH 5.2.

[0270] The same was repeated with corresponding 1 and 5 w / v% LM pectin, which is an acidic pectin (Pectin from citrus peel- Galacturonic acid >74.0 % purity; Sigma-Aldrich P9135 CAS-No. 9000-69-5 ). As available, the LM pectin had a pH < 4.5 as determined by preparing a pectin solution of 1 g / L demineralized water at 25 °C. The 1 wt% solution had pH 3.2, the 5 w / v% solution had a pH 3.1.

[0271] There were also prepared corresponding aqueous solutions of the non-acidic and LM pectin which solutions were adjusted the pH to pH 3.5 using 3M hydrochloric acid, except for the 1 and 5 w / v% LM pectin for which 3M sodium hydroxide was used.All 1 and 5 w / v% solutions of non-acidic and LM pectins were enriched with CaCh to reach a final concentration of 10mM of CaCh.

[0272] After incubating samples overnight, rheological parameters like viscosity were measured at 20°C over time by performing a small-amplitude oscillatory shear test at 1 Hz frequency and 0.5% strain (within linear viscoelastic range). All samples were prepared in duplicates and within an hour, with pH adjusted immediately. Viscosity was analyzed the following day, after overnight incubation at room temperature.

[0273] Results

[0274] Calcium is known to induce gelation of pectins, so it would be anticipated to observe gelling at these calcium levels. However, gelling was only observed for the LM Pectin:

[0275] For the non-acidic pectin solutions at 1 and 5 w / v%, before acidification (i.e. adjustment to pH 3.5), viscosity remained below 15 cP. Even at these calcium levels, no gelling occurred. After acidification, viscosity results for these 1 and 5 w / v% non-acidic pectin slightly increased to between 100 and 150 cP, such values indicating still no gelling taking place.

[0276] In the case of LM pectin at concentrations of 1w / v% and 5 w / v%, initial viscosity measurements prior to pH adjustment already ranged between 150 and 3000 cP. At pH 3.5, a marked enhancement in gelling was observed, as evidenced by viscosities exceeding 3000 cP, with further increases noted thereafter.

Claims

CLAIMS1. A fiber mixture comprising non-digestible oligosaccharides selected from galactooligosaccharides and / or fructooligosaccharides and a non-acidic pectin polysaccharide, wherein the non-acidic pectin polysaccharide has a pH between 4.5 - 8 when measured for a 1 g / L non-acidic pectin polysaccharide in water at 25 °C.

2. The fiber mixture according to claim 1, wherein the non-acidic pectin polysaccharide has a degree of esterification below 40%, more preferably below 30%, more preferably below 25%, most preferably below 20%.

3. The fiber mixture according to any of the preceding claims, wherein the non-acidic pectin polysaccharide added to the fiber mixture is obtainable by adding an amount of salt, preferably potassium and / or calcium salt, to an aqueous pectin polysaccharide composition, thus dissociating the hydrogen part from the carboxylic acid groups of the galacturonic acid residues, to obtain the non-acidic polysaccharide having a pH between 4.5 - 8.0, preferably between 4.5 - 7.0, more preferably between 5.0 - 6.5, most preferably between 5.0 and 6.0, when measured for a 1 g / L non-acidic pectin polysaccharide in water at 25 °C.

4. The fiber mixture according to any of the preceding claims, wherein the non-acidic pectin polysaccharide has an average molecular weight between 2 and 13 kDa, more preferably between 3 and 10 kDa, even more preferably between 4 and 8 kDa, most preferably between 5 and 7 kDa.

5. The fiber mixture according to any of the preceding claims, wherein the non-acidic pectin polysaccharide has a degree of polymerisation (DP) of between 15 - 65, more preferably between 20 - 55, more preferably between 28 - 50, most preferably between 30 - 40.

6. The fiber mixture according to any of the preceding claims, wherein it comprises galactooligosaccharides and fructooligosaccharides.

7. The fiber mixture according to any of the preceding claims, wherein the weight ratio of the sum of non-digestible oligosaccharides to pectin polysaccharides is between 2:1 to 30:1, preferably 3:1 to 20:1.

8. A synbiotic composition comprising the fiber mixture according to any of the preceding claims and Bifidobacterium ssp.

9. The synbiotic composition according to claim 8, comprising:• 0.5-2.5 x 108cfu / gram on dry basis of Bifidobacterium ssp.;• at least 5wt.% of non-digestible oligosaccharides on dry basis, selected from galactooligosaccharides and / or fructooligosaccharides; and• 0.5-4 wt.% on dry basis of a non-acidic pectin polysaccharides.

10. The synbiotic composition according to claims 8 or 9, wherein the probiotic bacterial strain is selected from Bifidobacterium spp., preferably Bifidobacterium long urn, more preferably Bifidobacterium longum BL999.

11. The synbiotic composition according to any of claims 8 - 10, comprising 0.8 - 2.0 x 108cfu / gram on dry basis of Bifidobacterium spp.

12. A nutritional composition comprising the fiber mixture according to any of claims 1 - 7 or the synbiotic mixture according to any of claims 8 - 11, wherein the nutritional composition comprises vitamin C and vitamin E.

13. The fiber mixture according to any of claims 1 - 7, the synbiotic composition according to any of claims 8 - 11 and / or the nutritional composition according to claim 12,for use in preventing or treating intestinal barrier disruption, or improving or stimulating gut microbiota in a subject suffering from an impaired gut microbiota; and / or treating or preventing dysbiosis in a subject,preferably for use in:• improving a recovery of a gut disease or gut health problem;• preventing / reducing the occurrence risk of gut diseases or gut health problems;• stimulating epithelial cell modulators and so to improve gut health; and / or• stimulating intestinal barrier functions, stimulating integrity of the gut barrier.

14. The use according to claim 13, wherein the subject suffers from chronic diseases, gastrointestinal disorders and / or a weakened immune system, preferably the subjects suffer from obesity, irritable bowel syndrome, inflammatory bowel disease, cardiovascular diseases, frailty and / or malnutrition.

15. A non-therapeutic method comprising administration of the fiber mixture according to any of claims 1 - 7, the synbiotic composition according to any of claims 8 - 11 and / or the nutritional composition according to claim 12 for promoting healthy ageing in a healthy subject.

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