HMO mixture and its use in reducing c. difficile toxins

Abstract

The present disclosure relates to a mixture of HMOs with at least 85 wt% of LNFP-I, 2'FL and 3'SL of the total amount of HMOs in the mixture and the use of such a mixture to decrease Clostridium difficile and its toxins in a subject. Furthermore, the mixture of HMOs can increase microbial taxa that are diminished with aging in a mammal, including increasing Bifidobacteria such Bifidobacterium longum and / or Bifidobacterium adolescentis and Faecalibacterium such as Faecalibacterium prausnitzii in the gut of a subject, in particular an elderly.

Description

[0001] HMO MIXTURE AND ITS USE IN REDUCING C. DIFFICILE TOXINS

[0002] FIELD OF INVENTION

[0003] The present disclosure relates to a mixture of HMOs comprising or consisting essentially of LNFP-I, 2’FL and 3’SL and the use of such a mixture to decrease Clostridium difficile and its toxins in a subject. Furthermore, the mixture of HMOs can increase microbial taxa that are diminished with aging in a mammal, including increasing Bifidobacteria such Bifidobacterium longum in the gut of a subject.

[0004] BACKGROUND

[0005] The human intestine harbours an estimated 1013to 1014bacterial cells, and the number of bacteria outnumbers the total number of cells in the body by a factor of 10 (Gill et al, Science 312, 1355 (2006)). The microbiota of the human intestine is a complex and very dynamic microbial ecosystem, which is considered to serve numerous important functions for its human host, including protection against pathogens, induction of immune regulatory functions, nutrient processing and metabolic functions (Tojo et al, World J. Gastroenterol. 20, 15163 (2014)).

[0006] Human milk oligosaccharides (HMOs) play diverse and important roles in the development of infants starting with their prebiotic function, which supports the establishment and maintenance of a balanced gut microbiota. HMOs have also been shown to have beneficial effects in maintaining the gut microbiota in adults where they are known as prebiotics and are known to increase beneficial Bifidobacterium in the gut.

[0007] The gut may also harbor pathogenic microorganisms, such as Clostridium difficile, Escherichia coll, Clostridium perfringens, Salmonella, Staphylococcus aureus and Klebsiella. C. difficile forms spores, which helps the bacterium to resist antibiotic treatment. In a dysbiotic intestine (where the microbiota is imbalanced), the spores can germinate and C. difficile can colonize and grow. After colonization, C. difficile mediates its effect on disease development through two toxins, exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB). These toxins cause damage to the epithelium and increase the permeability and inflammation of the gut and is a common cause of diarrhea and the more severe pseudomembranous colitis. C. difficile is increasingly being recognized as a major cause of gastrointestinal infections worldwide, with 70%-80% of C. difficile infections (CDIs) occurring in adults aged 65 and older (Asempa and Nicolau 2017 Clinical Interventions in Aging. 12: 1799-1809). C. difficile infections is also a known cause of infectious diarrhea in infants.

[0008] Bosheva et al 2022 (Frontiers in Nutrition 9: Article 920362 doi: 10.3389 / fnut.2022.920362) investigated the effect of a five HMO blend consisting of 2’-fucosyllactose (2’FL), 2’,3-di- fucosyllactose (DFL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’SL), and 6’-sialyllactose (6’SL) on the gut microbiota of formula feed infants. The blend was shown to decrease the abundance of DNA from C. difficile species in the infants, there are no measurements of TcdA and TcdB.

[0009] Mixtures of the HMOs 2’FL and lacto-N-neotetraose (LNnT) has also been shown to have an effect on the C. difficile abundance in the infants (WO 2017 / 021476 and WO 2018 / 024870) and 2’FL and LNnT can inhibit the spores and viable C. difficile following antibiotic treatment (WO 2017 / 084673).

[0010] SUMMARY OF INVENTION

[0011] An aspect of the present application relates to a mixture of HMOs comprising the target HMOs 2’-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL) in an amount of at least 90 wt% of the total amount of HMOs in the mixture. Preferably, the amount of fucosylated HMOs constitute at least 35 wt% of the synthetic mixture.

[0012] In particular the mixture of HMOs comprises the target HMOs in the following amounts: a. 5 to 35 wt % of 2’FL, preferably 10 to 30 wt% of 2’FL b. 30 to 75 wt % of LNFP-I, preferably 35 to 70 wt% of LNFP-I c. 10 to 30 wt % of 3’SL, preferably 15 to 25 wt% of 3’SL.

[0013] The mixture of HMOs claimed herein may be used in a composition such as a pharmaceutical composition or medical nutritional composition as well as in non-medical nutritional compositions such as dietary supplement and infant nutrition, such as infant formula or baby food.

[0014] The nutritional composition comprising the mixtures of HMOs claimed herein may further comprise one or more of the following ingredients docosahexaenoic acid (DHA), arachidonic acid (ARA), Eicosapentaenoic acid (EPA), plant based protein such as algae protein, pea protein or rapeseed protein, riboflavin (vitamin B2), nicotinic acid (vitamin B3), resveratrol, coenzyme Q10, or a probiotic.

[0015] In particular resveratrol and coenzyme Q10 are known to be beneficial in healthy aging.

[0016] A further aspect of the present application relates to the use of a mixture of HMOs comprising at least 30 wt% LNFP-I, in particular a mixture of HMOs as claimed herein, in preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) as well as decreasing C. difficile abundance in a subject. In particular, in a subject with C. difficile-associated diarrhea and / or a C. difficile infection. Target subjects are for example human infants or older adults and elderly.

[0017] A further aspect of the present application is use of a mixture of HMOs comprising at least 30 wt% LNFP-I, in particular a mixture of HMOs as claimed herein, in increasing the abundance of microbial taxa that are diminished with aging in a subject. Preferably, the genera Bifidobacteria and Faecalibacterium is increased in the subject, such as the species Bifidobacterium longum and / or Bifidobacterium adolescentis and / or Faecalibacterium prausnitzii. Target subjects are for example human older adults and elderly.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] In human milk, the third largest solid component after lactose and lipids is a group of over 200 structurally diverse oligosaccharides known as human milk oligosaccharides (HMOs). They are associated with multiple health benefits and their benefits, in particular in infant formula, has been investigated intensely. The health benefits are also being investigated in adults, where at least 2’FL and LNnT have been shown to have effects on the gut microbiome composition and can relieve IBS symptoms and reduce C. difficile abundance in adults following antibiotic treatment.

[0020] In the present application effects of other combinations of HMOs were investigated for their ability to not only reduce C. difficile abundance, but also exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) produced by C. difficile.

[0021] The rationale for investigating the effect both the bacterial infection and the level of toxins present following the infection, is that the reduction of the C. difficile bacteria as such does not necessarily lead to an immediate reduction of the toxins that have been produced during the infection. Therefore, a successful composition used to prevent damages caused by a C. difficile infection should be able to both reduce the infection and the exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB), which is not necessarily the same mechanism.

[0022] The term “human milk oligosaccharide" or "HMO" in the present context means a complex carbohydrate found in human breast milk. The HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl- lactosaminyl and / or one or more beta-lacto-N-biosyl unit, and this core structure can be substituted by an alpha-L-fucopyranosyl and / or an alpha-N-acetyl-neuraminyl (sialyl) moiety. HMO structures are e.g., disclosed by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

[0023] In the context of the present disclosure, lactose is not regarded as an HMO species. In the context of the disclosure an HMO is an oligosaccharide sub-species where the term “oligosaccharide” means a saccharide polymer containing a number of monosaccharide units. The most abundant HMOs are oligosaccharides of three to ten monosaccharide units, i.e., trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides and so forth.

[0024] HMOs are either neutral or acidic. In this regard, the non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure. The non- acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of neutral non-fucosylated HMOs (also termed neutral core HMOs) include, but is not limited to, lacto-N-triose II (LNT-II) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N- neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).

[0025] Examples of neutral fucosylated HMOs include, but is not limited to, 2'-fucosyllactose (2’FL), 3- fucosyllactose (3FL), difucosyllactose (DFL or LDFT), lacto-N-fucopentaose I (LNFP-I), lacto-N- fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP- V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N-difucohexaose I (LNDFH-I), lacto-N- difucohexaose II (LNDFH-II) and lacto-N-difucohexaose III (LNDFH-III).

[0026] Examples of acidic HMOs include, but is not limited to, 3’-sialyllactose (3’SL), 6’-sialyllactose (6’SL), 3-fucosyl-3’-sialyllactose (FSL), 3’-sialyllacto-N-tetraose a (LST-a), 6’-sialyllacto-N- tetraose b (LST-b) 6’-sialyllacto-N-neotetraose (LST-c).

[0027] In the present disclosure the focus is primarily on commercially available HMOs including 2’FL, 3FL, DFL, LNT, LNnT, 3’SL, 6’SL and LNFP-I.

[0028] One aspect of the current application is a mixture of HMOs comprising the target HMOs 2’- fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL) in an amount of at least 85 wt%, such as at least 90 wt% of the total amount of HMOs in the mixture, preferably in an amount of at least 92 wt%, such as at least 94 wt%, such as at least 96 wt%, such as at least 98 wt%, such as at least 99 wt% of the total amount of HMOs in the mixture. Such a mixture may also be considered as a mixture of HMOs consisting essentially of 2’- fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL). Preferably, the mixture is a synthetic mixture consisting essentially of 2’FL, LNFP-I and 3’SL.

[0029] In embodiments the synthetic mixture of HMOs comprises the target HMOs 2’-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL) in an amount of at least 85 wt%, such as at least 90 wt% of the total amount of HMOs in the mixture and wherein the amount of fucosylated HMOs constitute at least 35 wt%, such as at least 40 wt%, such as at least 50 wt%, such as at least 60 wt% of the synthetic mixture.

[0030] In embodiments the synthetic mixture comprising the target HMOs 2’FL, LNFP-I and 3’SL contains at least 30 wt%, such as at least 35 wt%, such as at least 40 wt%, such as at least 50 wt%, such as at least 60 wt% of LNFP-I.

[0031] In embodiments the synthetic mixture comprising the target HMOs 2’FL, LNFP-I and 3’SL contains at least 35 wt%, such as at least 40 wt%, such as at least 50 wt%, such as at least 60 wt% of 2’FL and LNFP-I. In further embodiments the synthetic mixture comprising the target HMOs 2’FL, LNFP-I and 3’SL contains less than 40 wt% sialylated HMOs, such as less than 30%, such as less than 25% of sialylated HMO, such as 3’SL. In preferred embodiment the sialylated HMO is 3’SL.

[0032] In one embodiment the mixture of HMOs comprises the target HMOs in the following amounts: a. 5 to 35 wt % of 2’FL, preferably 10 to 30 wt% of 2’FL, and b. 30 to 75 wt % of LNFP-I, preferably 35 to 70 wt% of LNFP-I, and c. 10 to 30 wt % of 3’SL, preferably 15 to 25 wt% of 3’SL.

[0033] Preferably, the total amount of the target HMOs is at least 90 wt% of the total amount of HMOs in the mixture. Preferably in an amount of at least 92 wt%, such as at least 94 wt%, such as at least 96 wt%, such as at least 98 wt% of the total amount of HMOs in the mixture.

[0034] In one embodiment the mixture of HMOs, e.g., synthetic mixture, consists essentially of 2’FL, LNFP-I and 3’SL, wherein: a. 2’FL constitute 5 to 35 wt %, preferably 2’FL constitute 10 to 30 wt% of the mixture, and b. LNFP-I constitute 30 to 75 wt % of LNFP-I, preferably LNFP-I constitute 35 to 70 wt% of the mixture, and c. 3’SL constitute 10 to 30 wt %, preferably 3’SL constitute 15 to 25 wt% of the mixture, and, wherein the total amount of 2’FL, LNFP-I and 3’SL in the HMO mixture constitute at least 85 wt%, such as 90 wt% such as 92 wt%, such as 96 wt%, such as at least 98 wt% of all the HMOs present in the HMO mixture.

[0035] In embodiments the synthetic mixture of HMOs of the present disclosure constitutes less than 15 wt%, such as less than 10 wt%, of other HMOs where the other HMOs in the mixture do not individually exceed 5 wt%, such as such as does not exceed 4 wt%, such as does not exceed 3 wt% of the total amount of HMOs present in the mixture.

[0036] In embodiments the synthetic mixture of HMOs of the present disclosure does not contain a sialylated HMO selected from one of 6’SL, LST-b and / or LST-c. Preferably, the synthetic mixture of HMOs does not contain 6’SL. More preferably, the synthetic mixture of HMOs does not contain any of the sialylated HMOs 6’SL, LST-b and LST-c.

[0037] In further embodiments the synthetic HMO mixture contains less than 1 wt% 3FL, such as less than 0.5 wt% 3FL, less than 2 wt% DFL, such as less than 1 wt% DFL and less than 5 wt% LNT, such as less than 3 wt% LNT. Preferably, the only HMOs present in the synthetic HMO mixture are 2’FL, LNFP-I, 3’SL, 3FL, DFL and LNT. More preferably the only HMOs present in the synthetic HMO mixture are 2’FL, LNFP-I, 3’SL, DFL and LNT. HMOs present in an amount below 5 wt%, such as below 3 wt%, such as below 2 wt% of the mixture of HMOs are considered insignificant and can be disregarded in terms of functional effect.

[0038] In one embodiment the synthetic mixture of HMOs consists essentially of a. 15 to 35 wt % of 2’FL, and b. 30 to 75 wt % of LNFP-I, and c. 10 to 30 wt % of 3’SL, and d. 0 to 5 wt% LNT e. 0 to 2 wt% DFL f. 0 to 1 wt% 3FL with the total HMO content in the mixture adding up to 100%.

[0039] In one embodiment the synthetic mixture of HMOs consists essentially of a. 10 to 30 wt % of 2’FL, and b. 35 to 70 wt % of LNFP-I, and c. 15 to 25 wt % of 3’SL, and d. 0 to 3 wt% LNT e. 0 to 1 wt% DFL f. 0 to 0.5 wt% 3FL with the total HMO content in the mixture adding up to 100%.

[0040] In one embodiment the synthetic mixture of HMOs consists essentially of a. 20 to 30 wt % of 2’FL, and b. 40 to 50 wt % of LNFP-I, and c. 15 to 25 wt % of 3’SL, and d. 0 to 2 wt% LNT e. 0 to 0.75 wt% DFL f. 0 to 0.1 wt% 3FL with the total HMO content in the mixture adding up to 100%.

[0041] In embodiments the target HMOs can also be mixed according to ratios between the individual HMOs. In one embodiment the mixture of HMOs comprises or consists essentially of the target HMOs in the following ratios LNFP-I:2’FL:3’SL 1.5-2.5:0.75-1.25:0.3-1.1 , a preferred ratio is 2:1 :0.8 of LNFP-I:2’FL:3’SL.

[0042] In embodiments the target HMOs 2’FL, LNFP-I and 3’SL in the synthetic mixture of HMOs constitute at least 84 wt%, such as at least 85 wt%, such as at least 90 wt% of the total composition of the mixture, including by-product elements which are not HMOs and which are not considered to have a materially effect on the properties of the mixture of HMOs. Such elements are for example mono- and di-saccharides, sialic acid and non-HMO oligosaccharides which may be present in individual amounts below 3 wt%. Generally, it is understood that the by-product elements are remnants or impurities from the production of the target HMOs, in particular biological production such as fermentation, but also in vitro enzymatic production.

[0043] For a review on biological (n vivo) HMO production see for example Bych et al 2019 Current Opinion in Biotechnology 56:130-137. In a preferred embodiment, the target HMOs, such as LNFP-I, 2’FL and 3’SL, of the present disclosure may be a synthetic mixture of target HMOs. Synthetic mixtures of target HMOs, means that they are not naturally occurring HMO mixtures and they are not isolated from mammalian milk. Synthetic target HMOs are for example produced chemically and / or biologically, e.g., by means of chemical reaction, enzymatic reaction or in recombinant cell cultures. In one embodiment the mixture of 2’FL, LNFP-I and 3’SL are produced by microbial fermentation. They can be produced by separate fermentations from which they are purified and then subsequently mixed in the desired ratios. They may also be produced by a single microbial cell capable of producing 2’FL, LNFP-I and 3’SL in one fermentation. In a preferred embodiment 2’FL and LNFP-I is produced in the same fermentation, e.g. by a single cell with a fucosyl-transferase capable of producing both 2’FL and LNFP-I, and the 3’SL is produced in a separate fermentation. Biological production (fermentation) of LNFP-I is described in WO 2022 / 243312 or a biological production (fermentation) of a mixture of LNFP-I and 2’FL is described in WO 2022 / 243314 and a biological production (fermentation) of 3’SL is for example described in EP4239066.

[0044] The term “consisting essentially of” in relation to the mixture of target HMOs, such as a mixture of 2’FL, LNFP-I and 3’SL, described herein is to be understood such that the target HMOs, e.g., 2’FL, LNFP-I and 3’SL, constitute at least 85 wt%, such as at least 90 wt%, such as alt least 92 wt %, such as alt least 94 wt %, such as alt least 96 wt %, such as at least 97 wt %, such as alt least 98 wt %, such as at least 99 wt % of the total amount of HMOs in the HMO mixture. With respect to the mixture of target HMOs 2’FL, LNFP-I and 3’SL the following additional HMOs, 3FL, LNT-II, LNT, DFL, may be present in individual amounts of less than 5 wt% of the mixture of 2’FL, LNFP-I and 3’SL since they are potential by-products of the fermentation process. The total amount of by-product HMOs does not exceed 15 wt%, preferably not 10 wt%, preferably not 8 wt%, preferably not 6 wt%, preferably not 4 wt%, preferably not 3 wt%, preferably not 2 wt% of the total amount of HMOs in the mixture of 2’FL, LNFP-I and 3’SL. Preferably, the mixture of target HMOs does not constitute HMOs which are not by-products from the manufacturing process producing the target HMOs. In embodiments the synthetic mixture of target HMOs does not contain 6’SL, LST-b, LST-c, and / or LNFP-I I .

[0045] In addition, the mixture of 2’FL, LNFP-I and 3’SL may include other elements which are not HMOs and which as not considered to materially affect the properties of the mixture of 2’FL, LNFP-I and 3’SL, such as for example, mono- and di-saccharides (e.g., fucose, lactose and lactulose), sialic acid and non-HMO oligosaccharides such as LNFP-I fructose isomer, 2’- fucosyl-lactulose and 3’sialyl-lactulouse which are not removed entirely after purification from the fermentation. Alternatively, a mixture consisting essentially of 2’FL, LNFP-I and 3’SL, described herein can therefore be understood such that the target HMOs, e.g., 2’FL, LNFP-I and 3’SL, constitute at least 80wt%, such as at least 84 wt%, such as at least 86 wt%, such as at least 90 wt%, such as alt least 92 wt %, such as alt least 94 wt %, such as alt least 96 wt %, such as at least 97 wt %, such as alt least 98 wt %, such as at least 99 wt % of the total amount of the total elements in the composition, including non-oligosaccharide elements, such as for example, mono- and di-saccharides (e.g., fucose, lactose and lactulose), sialic acid.

[0046] The HMO mixtures described herein may be used in a pharmaceutical or nutritional composition. Nutritional compositions are for example, an infant formula, a rehydration solution, or a dietary maintenance or supplement, medical nutrition or supplement for elderly individuals or immunocompromised individuals. Macronutrients such as edible fats, carbohydrates and proteins can also be included in such anti-infective compositions. Edible fats include, for example, coconut oil, soy oil and monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolysed cornstarch. Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (e. g. calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and B complex) can also be included in such anti-infective compositions.

[0047] In embodiments of the present disclosure the nutritional composition comprising the mixtures of HMOs as claimed herein further comprises one or more of the following ingredients docosahexaenoic acid (DHA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), plant based protein such as algae protein, pea protein or rapeseed protein, riboflavin (vitamin B2), nicotinic acid (vitamin B3), resveratrol, coenzyme Q10, or a probiotic. Preferably, the probiotic is a Bifidobacterium sp and / or Lactobacillus sp.

[0048] In embodiments, the composition comprising the mixtures of HMOs described herein is a pharmaceutical composition.

[0049] In other embodiments, the nutritional composition comprising the mixtures of HMOs described herein is a dietary supplement and / or medical nutrition.

[0050] In other embodiments, the nutritional composition comprising the mixtures of HMOs described herein is infant nutrition, such as infant formula or baby food.

[0051] The HMO mixture or the nutritional composition disclosed in the present application may be provided as a powder, a dry composition, a suspension, a liquid concentrate, an emulsion or a gel. The formulation may be a ready to use formulation, such as a unit dosage form, i.e., a capsule, tablet, gummy or sachet / stick pack or a formulation that needs to be dissolved in a liquid prior to use.

[0052] The term “intestine” or “gut” are used interchangeably herein and refers to the portion of the gastrointestinal tract consisting of the small intestine and the large intestine. The “large intestine” (jntestinum crass urn) is the lower part of the gastrointestinal tract and is also referred to herein as “colon”. The term "Microbiota", "microflora” and "microbiome" are used interchangeably and refer to a community of living microorganisms that typically inhabits a bodily organ or part in an animal or human. Particularly, in the gastrointestinal organs of animals or humans the microflora is termed the gastrointestinal or gut microbiome or microbiota. The most dominant members of the gastrointestinal microbiota include microorganisms of the phyla of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota; at genus level Bacteroides, Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Collinsella, Blautia, Coprococcus, Ruminococcus, Eubacterium and Dorea; at species level Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Ruminococcus bromii, Dorea longicatena, Bacteroides caccae, Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus torques, Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Bacteroides vulgatus and Roseburia intestinalis. The gastrointestinal microbiota includes the mucosa-associated microbiota, which is located in or attached to the mucus layer covering the epithelium of the gastrointestinal tract, and luminal- associated microbiota, which is found in the lumen of the gastrointestinal tract.

[0053] "Effective amount" means an amount of a composition that provides the mixture of target HMOs in a sufficient amount to render a desired benefit or treatment outcome. An effective amount can be administered in one or more doses to achieve the desired treatment outcome. In the present disclosure it is assumed that the mixture of target HMOs is administered in an effective amount for any desired use. Suggested effective amounts of the mixture of target HMOs described herein are generally in the range from about 0.5 g to about 20 g per day, in certain embodiments from about 1 g to about 15 g per day, from about 2 g to about 10 g per day, in certain embodiments from about 1 g to about 10 g per day. Appropriate dose regimes can be determined based on the present disclosure and / or on factors known to a person of ordinary skill in the art. Preferably, the subject, such as a human, is administered the mixture of target HMOs for a period of at least 1 week, more preferably for at least 2 weeks. For example, the human can be administered the mixture of target HMOs for a period of at least 4 weeks.

[0054] “Enteral administration” means any conventional form for delivery of a composition to a subject that causes the deposition of the composition in the gastrointestinal tract (including the stomach). Methods of enteral administration include feeding through a naso-gastric tube or jejunum tube, oral, direct delivery to the gut, sublingual and rectal.

[0055] "Oral administration" means any conventional form for the delivery of a composition to a noninfant through the mouth. Accordingly, oral administration is a form of enteral administration.

[0056] In the present description the term “subject” refers to a mammal including humans. Other preferred mammals with a gut microbiome beyond humans are domestic animals such as pets (cats, dogs, rodents, rabbits, avian species, reptiles, etc.), livestock and performance animals (pigs, poultry, goat, sheep and cows) and working animals (horses, oxen, camels, donkeys and elephants). Preferably, the subject is a human, such as a non-infant human. The term “noninfant human” or “non-infant” means a human of 3 years of age and older. A non-infant human can be a child, a teenager, an adult or an elderly. The term "elderly" in the context of a human means an age from birth of at least 60 years, preferably above 65 years, more preferably above 70 years, the term “aging” in relation to a subject or person is used interchangeably with elderly. The term "older adult" in the context of a human means an age from birth of at least 40 years, preferably above 50 years, more preferably above 55 years, and includes elderly individuals. The term “younger adult” in the context of a human means an age from birth of between 18 years to 39 years. In embodiments the subject is preferably an older adult or an elderly or an aging human.

[0057] C. difficile infection (GDI) and in particular the two toxins, exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) produced by C. difficile can cause damage to the epithelium in the gut of an infected subject and is a common cause of diarrhea, in particular infectious diarrhea. TcdA and TcdB are also known to increase in permeability of the gut, which may lead to leaky gut. Inflammation in the gut is also common consequences of increased levels of TcdA and TcdB. In ageing people low-level systemic inflammation also known as inflammaging may be caused by a loss of control over systemic inflammation resulting in chronic overstimulation of the innate immune system. The loss of epithelial integrity observed in C. difficile infection may accelerate the low-level systemic inflammatory process in elderly people, even in mild disease.

[0058] In the present disclosure it was found that LNFP-I or HMO mixtures with LNFP-I, was able to reduce C. difficile abundance (infection) and reduce exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB). In particular LNFP-I in a mixture of HMOs where the amount of LNFP-I is at least 30 wt%, such as at least 35 wt% of the total mixture of HMOs showed superior effect compared to HMO mixtures without LNFP-I.

[0059] A further aspect of the present disclosure is a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I for the use in preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) in a subject. In preferred embodiments the amount of LNFP-I in the total mixture of HMOs is between 30 wt% to 92 wt%, such as from 35 wt% to 90 wt%, such as from 35 wt% to 75 wt%, such as from 40 wt% to 50 wt% of the total amount of HMO in the mixture of HMOs.

[0060] An embodiment of the present application is a mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above. In particular a mixture of HMOs comprising the target HMOs 2’-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL) in an amount of at least 85 wt% of the total amount of HMOs in the mixture and wherein the amount of fucosylated HMOs constitute at least 35 wt% of the synthetic mixture, or an HMO mixture described in the embodiment section herein. A further embodiment is a nutritional composition comprising such an HMO mixture for use in preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) in a subject, such as a mammal. Preferably, a significant reduction of exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) can be observed 24 hours after the infection occurred.

[0061] In one embodiment the mixture of HMOs or the nutritional composition as described and / or claimed herein is used to prevent, alleviate or treat a subject with C. d / ffic / 7e-associated diarrhea or a C. difficile infection.

[0062] Embodiments of the present disclosure relates to a method for preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) comprising administering to a subject an effective amount of a synthetic mixture of HMOs or a nutritional composition comprising at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein. In embodiments the subject is in need of the administration, for example the subject has C. difficile-associated diarrhea or a C. difficile infection.

[0063] In embodiments the synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein is administered in a preventive manner, e.g. before any risk of C. difficile infection occur, such as before hospitalization or as a preventive measure for elderly living in a nursing home.

[0064] In one embodiment the mixture of HMOs or the nutritional composition as described and / or claimed herein is used to reduce gut permeability and / or prevent, alleviate or treat leaky gut in a subject, such as a mammal.

[0065] Embodiments of the present disclosure relates to a method for reduce gut permeability and / or preventing, alleviating or treating leaky gut comprising administering to a subject an effective amount of a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein. In embodiments the subject is in need of the administration. The subject may for example be an aging human, such as an aging human with a dysbiotic gastrointestinal microbiome, or a subject with C. difficile-associated diarrhea or a C. difficile infection.

[0066] In one embodiment the mixture of HMOs or the nutritional composition as described and / or claimed herein is used to prevent, alleviate or treat low-level systemic inflammation and / or inflammaging in a subject, in particular in an older adult or elderly subject.

[0067] Embodiments of the present disclosure relates to a method for preventing, alleviating or treating low-level systemic inflammation and / or inflammaging comprising administering to a subject an effective amount of a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein. In embodiments the subject is in need of the administration. The subject may for example be an aging human, such as an aging human with a dysbiotic gastrointestinal microbiome, or a subject with C. d / ffic / 7e-associated diarrhea or a C. difficile infection.

[0068] As already described above C. difficile infection is a significant problem in the elderly population. This may in part be due to the change in microbiota observed in the elderly population, which in some elderly individual lead to a dysbiotic gastrointestinal microbiome, also simply known as dysbiosis.

[0069] In particular depletion of taxa such as, including Bifidobacteria, Blautia, Dorea, Coprococcus, Clostridium clusters IV and Clostridium clusters XlVa, especially the genera Roseburia (Lachnospiraceae family) and Butyricicoccus (Clostridiaceae family) and Faecalibacterium, in particular Faecalibacterium prausnitzii, have been observed in elderly. Many of these bacteria have known anti-inflammatory effects, in that they produce metabolites with anti-inflammatory properties such as short chain fatty acids (SCFAs). SCFAs are also known to improve intestinal gut barrier function. Species of Faecalibacterium, such as Faecalibacterium prausnitzii and Roseburia, such as Roseburia hominis, are butyrate producers and acetate consumers and are associated with anti-inflammatory regulatory T cell production. Species of Dorea, Blautia, and Coprococcus are SCFA-producers. Bifidobacteria have been associated with the production of a number of potentially health promoting metabolites including short chain fatty acids, conjugated linoleic acid and bacteriocins.

[0070] Consequently, health strategies towards elderly people may advantageously address this reduction in microbial taxa which may in turn lead to higher risk of adverse effects following a C. difficile infection by increasing the abundance of microbial taxa that are diminished with aging in a mammal, such as an elderly human.

[0071] A further aspect of the present application is mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I for use in increasing the abundance of microbial taxa that are diminished with aging in the gut of a subject, such as a mammal, such as an aging human. In preferred embodiments the amount of LNFP-I in the total mixture of HMOs is between 30 wt% to 95 wt%, such as between 35 wt% to 90 wt%, such as from 35 wt% to 75 wt%, such as from 40 wt% to 50 wt% of the total amount of HMO in the mixture of HMOs. In an embodiment the use is a non-medical use and the subject is preferably an older adult and / or an elderly human.

[0072] An embodiment of the present application is a mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above and / or a nutritional composition comprising such an HMO mixture for use in increasing the abundance of microbial taxa that are diminished with aging in a subject, such as a mammal, such as an aging human. Embodiments of the present disclosure relates to a method for increasing the abundance of microbial taxa that are diminished with aging, in the gut of a subject, such as an elderly subject, where in the method comprises administering to a subject an effective amount of a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein. The subject may for example be an aging or elderly human, such as a healthy elderly subject or an elderly subject with a dysbiotic gastrointestinal microbiome.

[0073] In embodiments the microbial taxa to be increased by the mixture of HMOs comprising LNFP-I, such as a mixture of the target HMOs 2’FL, LNFP-I and 3’SL as described above and a nutritional composition comprising such an HMO mixture, are selected from one or more of the following genus Bifidobacteria, Roseburia, Blautia, Dorea, Coprococcus, Clostridium clusters IV and Clostridium clusters XlVa and Faecalibacterium. Preferably, Bifidobacteria sp. and / or Faecalibacterium sp. are increased. Even more preferably species of Bifidobacterium longum and Faecalibacterium prausnitzii are increased. In a preferred embodiment the mixture of HMOs comprising the target HMOs 2’-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I) and 3’- sialyllactose (3’SL) in an amount of at least 85 wt% of the total amount of HMOs in the mixture and wherein the amount of fucosylated HMOs constitute at least 35 wt% of the synthetic mixture, is capable of increasing Bifidobacteria sp. and / or Faecalibacterium sp. such as Bifidobacterium longum and Faecalibacterium prausnitzii by promoting the growth of these species in the gut of the subject, wherein the species are naturally present, but in lower amount than the average in a younger adult. Generally, it is not contemplated to supplement the HMO mixture with probiotics of the species Bifidobacterium longum and / or Faecalibacterium prausnitzii to achieve this increase in the gut of the subject, it is rather the HMO mixture that increases the species already present in the gut.

[0074] In the present disclosure, it was found that LNFP-I, such as an HMO mixture with LNFP-I as disclosed or claimed herein, was able to increase the abundance of Bifidobacteria such as Bifidobacterium longum as compared to a subject which has not received a mixture of HMOs comprising LNFP-I.

[0075] An embodiment of the present application is the use, in particular the non-medical use, of a mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above and / or a nutritional composition comprising such an HMO mixture for modulating the microbiota by increasing the abundance of Bifidobacteria in the gut of a subject, such as a mammal, such as an ageing human. Beneficial Bifidobacteria species to be increased can be selected from, the following Bifidobacterium sp.: B. adolescentis, B. angulatum, B. animalis, B. animalis subsp. animalis, B. animalis subsp. lactis, B. asteroides, B. biavatii, B. bifidum, B. breve, B. catenulatum, B. coagulans, B. longum, B. infantis, B. longum subsp. infantis, B. longum subsp. longum, B. magnum, B. coryneforme, B. dentium, B. gallicum, and / or B. subtile. In a preferred embodiment at least one of the following Bifidobacteria species is increased: Bifidobacterium adolescentis, Bifidobacterium bifidum and / or Bifidobacterium longum, such as Bifidobacterium longum subsp. longum or Bifidobacterium longum subsp. infantis. In a preferred embodiment in particular the abundance of Bifidobacterium longum is increased.

[0076] Embodiments of the present disclosure relates to a method for increasing the abundance of beneficial Bifidobacteria species in the gut of a subject comprising administering to a subject an effective amount of a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein. It is advantageous to increase Bifidobacterium adolescentis, Bifidobacterium bifidum and / or Bifidobacterium longum, such as Bifidobacterium longum subsp. longum or Bifidobacterium longum subsp. Infantis.

[0077] In one embodiment described herein Bifidobacterium longum abundance is increased by at least 5 fold as compared to before the administration of the HMO mixture, such as at least 6 fold, such as at least 7 fold, such as at least 8 fold, such as at least 9 fold, such as at least 10 fold as compared to before the administration of the HMO mixture.

[0078] The significance of Bifidobacterium to human health can be appreciated from its early colonization of the neonatal gut, where Bifidobacterium longum represents the most abundant species. Research into the beneficial properties of B. longum has unveiled a range of mechanisms, including the production of bioactive molecules, such as short-chain fatty acids (SCFAs), polysaccharides, and serine protease inhibitors (see for example Mills et all 2023 Gut Microbes.15(1): 2186098). In addition, B. bifidum and B. longum have been widely studied for their immunomodulatory properties and protecting effect by suppressing pathogens such as E. coll and Salmonella. B. bifidum and B. longum also affect epithelial cell function by regulating immune gene expression and tight junctions.

[0079] The microbiota-gut-brain axis describes the communication pathways between the intestinal microbiota, microbial metabolites, and the nervous system. B. longum is intimately connected with the nervous system and influences mood, cognition, and brain health by producing gamma amino butyric acid (GABA), short-chain fatty acids (SCFAs), and regulating serotonin synthesis by enterochromaffin cells in the intestines.

[0080] Bifidobacterium adolescentis also contributes to the production of GABA, a neurotransmitter that plays a role in reducing stress and anxiety. Some B. adolescentis strains can also synthesize B vitamins, such as folic acid.

[0081] More recently, the potential benefits of HMOs in adult health are receiving increased attention (see for example WO2018 / 157900 and WO2018 / 207110), both in terms of increasing the prevalence of beneficial bacteria in the gut, which in turn produce metabolites such as short chain fatty acids (SCFAs), of which butyrate, propionate, and acetate are the most predominant in the human gut, where they play a significant role both in gut and metabolic health, but also as signaling molecules in the brain-gut axis.

[0082] Acetate has been shown to promote intestinal antibody immunoglobulin (lg)A responses in the gut via the G-protein coupled receptor GPR43. Acetate can also be used by butyrate-producing bacteria in the gut, such as Faecalibacterium prausnitzii, to produce butyrate. Propionate and butyrate are thought to lower lipogenesis, serum cholesterol levels, and carcinogenesis in other tissues. Butyrate is also used as an energy source by gut epithelial cells, in particular colonocytes, and is involved in several physiological functions including intestinal barrier function, immunity, and brain function.

[0083] An embodiment of the present application is the use, in particular the non-medical use, of a mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above and / or a nutritional composition comprising such an HMO mixture to increase the production of beneficial short-chain-fatty-acids (SCFAs) in the gut of a subject.

[0084] Embodiments of the present disclosure relates to a method for increasing the production of beneficial short-chain-fatty-acids (SCFAs) in the gut of a subject comprising administering to a subject an effective amount of a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt%, such as at least 35 wt% of LNFP-I, such as a mixture of HMOs or a nutritional composition as described and / or claimed herein.

[0085] An embodiment of the present application is the use, in particular the non-medical use, of a mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above and / or a nutritional composition comprising such an HMO mixture, in healthy aging. In preferred embodiments the mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL is in combination with one or more of the following additional ingredients riboflavin (vitamin B2), nicotinic acid (vitamin B3), resveratrol, coenzyme Q10 and / or a probiotic. Preferably, the subject for the non-medical use is an older adult or an elderly.

[0086] In relation to any of the uses or methods of administration described herein the preferred route of administration is enteral administration, in particular oral administration.

[0087] In relation to any of the uses described herein, which result in a benefit, such as an increase, a reduction, a prevention, an alleviation or a treatment, it is understood that this is compared to the same subject prior to the administration of the mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above or the nutritional composition comprising such an HMO mixture. Alternatively, the comparative parameters may also constitute two cohorts of individuals one receiving the mixture of HMOs comprising the target HMOs 2’FL, LNFP-I and 3’SL as described above or the nutritional composition comprising such an HMO mixture and another cohort receiving a placebo, such as a maltodextrin or lactose. Potentially, further comparative cohorts may receive a known mixture of HMOs, such as a mixture of 2’FL and LNnT or a mixture of 2’FL, DFL, LNT, 3’SL and 6’SL. The term cohort in this respect is understood as groupings of individuals with common traits, such as age, social and health factors. The size of a cohorts needed for comparative studies depends on the statistical variation observed within a cohort.

[0088] EMBODIMENTS

[0089] The following embodiments of the present invention may be used in combination with any other embodiments described herein.

[0090] 1 . A synthetic mixture of HMOs comprising the target HMOs 2’-fucosyllactose (2’FL), lacto-N- fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL) in an amount of at least 85 wt% of the total amount of HMOs in the mixture.

[0091] 2. A synthetic mixture of HMOs consisting essentially of 2’-fucosyllactose (2’FL), lacto-N- fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL).

[0092] 3. The synthetic mixture of HMOs according to embodiment 1 or 2, wherein the amount of fucosylated HMOs constitute at least 35 wt%, such as at least 40 wt%, such as at least 50 wt%, such as at least 60 wt% of the synthetic mixture.

[0093] 4. The synthetic mixture of HMOs according to any one of embodiments 1 to 3, wherein the composition comprises at least 30 wt% LNFP-I, such as at least 35 wt% LNFP-I.

[0094] 5. The synthetic mixture of HMOs according to any one of embodiments 1 to 4, wherein the amount of sialylated HMO, such as the amount of 3’SL, constitute less than 40 wt%, such as less than 35 wt%, such as less than 30 wt%, such as less than 25 wt% of the synthetic mixture.

[0095] 6. The synthetic mixture of HMOs according to any one of embodiments 1 to 5, wherein the target HMOs consists essentially of: a. 5 to 35 wt % of 2’FL, preferably 10 to 30 wt% of 2’FL b. 30 to 75 wt % of LNFP-I, preferably 35 to 70 wt% of LNFP-I c. 10 to 30 wt % of 3’SL, preferably 15 to 25 wt% of 3’SL.

[0096] 7. The synthetic mixture of HMOs according to any one of embodiment 2 to 6, wherein the total amount of 2’FL, LNFP-I and 3’SL in the HMO mixture constitute at least 85 wt%, such as 90 wt% such as 92 wt%, such as 96 wt% of all the HMOs present in the HMO mixture.

[0097] 8. The synthetic mixture of HMOs according to any one of embodiments 1 to 7, wherein other HMOs in the mixture do not individually exceed 5 wt%, such as does not exceed 4 wt%, such as does not exceed 3 wt%, such as does not exceed 2 wt% of the total amount of HMOs present in the mixture. 9. The synthetic mixture of HMOs according to any one of embodiments 1 to 8, wherein the HMO mixture contains less than 1 wt% 3FL, less than 2 wt% DFL and less than 5 wt% LNT, such as less than 0.5 wt% 3FL, less than 1 wt% DFL and less than 3 wt% LNT.

[0098] 10. The synthetic mixture of HMOs according to any one of the preceding embodiments, wherein the synthetic mixture of HMOs does not contain a sialylated HMO selected from 6’SL, LST-b and LST-c.

[0099] 11 . The synthetic mixture of HMOs according to any one of the preceding embodiments, wherein the synthetic mixture of HMOs consists essentially of a. 10 to 30 wt % of 2’FL, and b. 35 to 70 wt % of LNFP-I, and c. 15 to 25 wt % of 3’SL, and d. 0 to 5 wt% LNT, preferably 0 to 3 wt% LNT e. 0 to 2 wt% DFL, preferably 0 to 1 wt% DFL f. 0 to 1 wt% 3FL, preferably 0 to 0.5 wt% 3FL with the total HMO content in the mixture adding up to 100%.

[0100] 12. The synthetic mixture according to any of the preceding embodiment, wherein the 2’FL, LNFP-I and 3’SL are produced by microbial fermentation.

[0101] 13. The synthetic mixture according to embodiment 12, wherein the 2’FL and LNFP-I is produced in the same fermentation.

[0102] 14. A nutritional composition comprising the synthetic mixture according to any one of embodiments 1 or 13.

[0103] 15. The nutritional composition according to embodiment 14, wherein the composition further comprises one or more of the following ingredients docosahexaenoic acid (DHA), arachidonic acid (ARA), Eicosapentaenoic acid (EPA), carotenoids, such as lutein and zeaxanthin, plant based protein such as algae protein, pea protein or rapeseed protein, riboflavin (vitamin B2), nicotinic acid (vitamin B3), resveratrol, coenzyme Q10 and / or a probiotic.

[0104] 16. The nutritional composition according to embodiment 14 or 15, wherein the nutritional composition is selected from the group consisting of infant formula, baby food, dietary supplements and medical nutrition.

[0105] 17. A synthetic mixture of HMOs comprising at least 30%, such as at least 35 wt% of LNFP-I for the use in preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) in a subject.

[0106] 18. The synthetic mixture of HMOs for the use according to embodiment 17, wherein the composition further comprises 2’FL and 3’SL. 19. The synthetic mixture of HMOs for the use according to embodiment 17 or 18, wherein the HMO mixture is selected from a mixture according to any one of embodiment 6 to 13.

[0107] 20. A nutritional composition according to any one of embodiments 14 to 16 for use in preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) in a subject.

[0108] 21 . The synthetic mixture or the nutritional composition for the use according to any one of embodiments 17 to 20, wherein the mammal has C. d / ffic / 7e-associated diarrhea or a C. difficile infection.

[0109] 22. A synthetic mixture according to any one of embodiments 1 to 13 or the nutritional composition according to any one of embodiments 14 to 16 for use in reducing gut permeability and / or to prevent, alleviate or treat leaky gut in a subject.

[0110] 23. A synthetic mixture according to any one of embodiments 1 to 13 or the nutritional composition according to any one of embodiments 14 to 16 for use in preventing, alleviating or treating low-level systemic inflammation and / or inflammaging in a subject.

[0111] 24. A synthetic mixture according to any one of embodiments 1 to 13 or the nutritional composition according to any one of embodiments 14 to 16 for use in increasing the abundance of microbial taxa that are diminished with aging in a subject.

[0112] 25. The synthetic mixture or the nutritional composition for the use according to embodiment 24 wherein the microbial taxa are selected from one or more of the following genus Bifidobacteria, Roseburia, Blautia, Dorea, Coprococcus, Clostridium clusters IV and Clostridium clusters XlVa and Faecalibacterium.

[0113] 26. The synthetic mixture or the nutritional composition for the use according to embodiment 24 or 25, wherein the Bifidobacteria is of the species Bifidobacterium longum Bifidobacterium longum and / or Bifidobacterium adolescentis and the Faecalibacterium is Faecalibacterium prausnitzii.

[0114] 27. A non-medical use of a synthetic mixture according to any one of embodiments 1 to 13 or the nutritional composition according to any one of embodiments 14 to 16 in healthy aging.

[0115] 28. The non-medical use according to embodiment 27, wherein the synthetic mixture is further combined with one or more of the following additional ingredients riboflavin (vitamin B2), nicotinic acid (vitamin B3), resveratrol, coenzyme Q10.

[0116] 29. The synthetic mixture or the nutritional composition for the use according to any one of embodiments 17 to 26, wherein the subject is selected from human, domestic animals, pet animals, livestock or working animals.

[0117] 30. The synthetic mixture or the nutritional composition for the use according to any one of embodiments 17 to 29, wherein the subject is an older adult or an elderly. 31 . The synthetic mixture or the nutritional composition for the use according to any one of embodiments 17 to 30, wherein the subject is an elderly human.

[0118] 32. The synthetic mixture or the nutritional composition for the use according to any one of embodiments 17 to 31 , wherein the subject is an elderly with a dysbiotic gastrointestinal microbiome, or a subject with C. d / ffic / 7e-associated diarrhea or a C. difficile infection.

[0119] 33. Use of a synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt% LNFP-I such as at least 35 wt% of LNFP-I for modulating the microbiota by increasing the abundance of Bifidobacteria in the gut of a subject.

[0120] 34. The use of the synthetic mixture of HMOs or the nutritional composition according embodiment 33, wherein the synthetic mixture of HMOs is according to any one of embodiments 6 to 13 or wherein the nutritional composition is according to any one of embodiments 14 to 16 for modulating the microbiota by increasing the abundance of Bifidobacteria in the gut of a subject.

[0121] 35. The use according to embodiment 33 or 34, wherein the Bifidobacteria is of the species Bifidobacterium longum.

[0122] 36. Use of a synthetic mixture according to any one of embodiments 1 to 13 or the nutritional composition according to any one of embodiments 14 to 16 to increase the production of beneficial short-chain-fatty-acids in the gut of a subject.

[0123] 37. The use according to embodiment 33 to 36, wherein the subject is selected from human, domestic animals, pet animals, livestock and working animals.

[0124] 38. The use according to embodiment 33 to 37, wherein the subject is a human selected from the group of infants, older adults and elderly.

[0125] 39. The use according to embodiment 33 to 38, wherein the subject is an elderly human.

[0126] 40. The use according to embodiment 33 to 38, wherein the subject is an elderly with a dysbiotic gastrointestinal microbiome, or a subject with C. difficile-associated diarrhea or a C. difficile infection.

[0127] EXAMLES

[0128] Materials and methods

[0129] HMO mixtures

[0130] The HMO mixtures in Table 1 below were prepared using the commercial products

[0131] 2’FL: GlyCare 2FL 9000

[0132] 2’FL / DFL: GlyCare 2FL / DFL 8001

[0133] 3FL: Under development LNnT: GlyCare LNnT 9000

[0134] LNT: GlyCare LNT 8001

[0135] 3’SL: GlyCare 3SL 9001

[0136] 6’SL: GlyCare 6SL 9001

[0137] LNFP-I / 2’FL: GlyCare LNFP-I / 2FL 8001

[0138] The products were dosed in the indicated amounts. The target HMO in the products is not 100% pure, therefore the percentage of the target HMO’s in the 5 g of product is indicated as “% of product”. The weight % of the individual target HMOs are also indicated based on a 100% pure HMO composition only containing the target HMOs (wt% on total target HMO’s) to indicate the ratio between the target HMOs.

[0139] Table 1 - mixtures of a total of 5 g HMOs tested in the present application

[0140] *TP10 additionally contains the following non-target HMOs: 0.02 wt% 3FL, 0.53 wt% DFL and

[0141] 1 .22 wt% LNT

[0142] Methods for C. difficile viability

[0143] C. difficile counts were determined on a C. difficile agar base (CM0601 B) supplemented with taurocholic acid, defibrinated horse blood and additional supplements (D-cycloserine, Cefoxitin, Norfloxacin and Moxalactam), thus ensuring optimal selectivity.

[0144] Methods for C. difficile toxin production determination

[0145] C. difficile toxin A / B production was determined via a C. difficile toxin A / B ELISA assay kit (CDT35-K01), according to manufacturer's instructions (Eagle Biosciences). Toxin levels were expressed as OD levels. Fecal C. Difficile Toxin AB ELISA Assay Kit is a “sandwich” ELISA designed, developed and produced for the qualitative measurement of Toxin A and Toxin B in stool specimen. The assay utilizes the microplate-based enzyme immunoassay technique by coating highly purified antibody onto the wall of microtiter wells. Controls and extracted fecal specimen are added to microtiter wells of microplate that was coated with a highly purified monoclonal anti-Toxin A and Toxin B on its wall. During the assay, the Toxin A and B Antibodies will be bound to the antibody coated plate after an incubation period. The unbound material is washed away and another HRP-conjugated monoclonal antibody which specifically recognizes the protein of Toxin A & B is added for further immunoreactions. After an incubation period, the immunocomplex of “Anti-Toxin A & B Capture Antibody - Toxin A & B - HRP-conjugated Anti-Toxin A & B Tracer Antibody” is formed if toxins are present in the test sample. The unbound tracer antibody and other proteins in buffer matrix are removed in the subsequent washing step. HRP conjugated tracer antibody bound to the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the tracer antibody bound to Toxin A & B proteins captured on the wall of each microtiter well is directly proportional to the amount of Toxin A & B level in each test specimen.

[0146] Methods for quantification of beneficial microbial species

[0147] Upon DNA extraction, standardized Illumina library preparation was performed followed by 3M total DNA sequencing. Results were analysed at different taxonomic levels (species, family and phylum level). For taxonomic analysis, the proportional data derived from sequencing (%) were corrected for the total amount of cells present in each sample (detected via flow cytometry), allowing to obtain more representative insights in the impact of interventions on the gut microbiota.

[0148] Example 1 - Viability of C. difficile

[0149] In the present example the effect of the HMO mixtures in table 1 were tested in terms of their ability to reduce the C. difficile bacterial count in fermented feces samples.

[0150] The experimental setup was as follows. Feces samples were obtained from healthy elderly human adults (above 65 years old) (n = 6). Individual bioreactors were processed in parallel in a bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 ml of nutritional medium-faecal inoculum blend dosed with 5 g / L of a HMO mixture from table 1 as well as a control with no HMO added. The bioreactors were sealed individually, before being rendered anaerobic and incubated under continuous agitation (140 rpm) at 37 °C. After 24h the bioreactors were inoculated with 3.04 * 106CFU / mL of C. difficile (ATCC9686). Samples were taken 24 h after C. difficile “infection” (48 from fermentation start) and analysed for C. difficile viability as described in the methods above. For each of the tested HMO mixtures the average from the 6 individual feces samples was calculated.

[0151] The results are shown in Table 2 as average cell count across all the donors. The results from the control is also included (C).

[0152] Table 2 - Effect of the HMO mixtures on C. difficile viability 24 h after infection

[0153] From the data, it can be seen that when no HMO is added to the fecal fermentation almost 7.5 million C. difficile colonies were viable 24 hours after infection. It can also be seen that when the HMO mixture containing LNFP-I (M3) was added to the fermentation the C. difficile viability was reduced by 82%, which is more than any of the other HMO mixtures can achieve in 24 hours.

[0154] Example 2 - C. difficile toxin reduction

[0155] From the samples taken in Example 1 the effect of the HMO mixtures in table 1 were also assessed with respect to their ability to reduce C. difficile toxins which are the cause of disease development observed in connection with a C. difficile infection.

[0156] Reduction of the C. difficile bacteria as shown in Example 1 does not necessarily lead to an immediate reduction of the toxins that have been produced during the infection. Therefore, a successful composition used to prevent damages caused by a C. difficile infection should be able to both reduce the infection (measured by cell count Example 1) and reduce the toxin amounts, which is not necessarily the same mechanism.

[0157] The toxin levels were measured as described in the materials and methods section. The results are presented in table 3 as relative values to the Control.

[0158] Table 3 - Effect of the HMO mixtures on C. difficile toxin relative to control

[0159] From the data, it can be seen that the HMO mixture containing LNFP-I is capable of reducing the C. difficile toxins TdcA and TdcB to a lower level than any of the other mixtures within 24 hours from the infection. Example 3 Increasing Bifidobacteria and other beneficial microbial species

[0160] The ability of the HMO mixtures to increase beneficial microbial species in the fecal samples upon incubation in the bioreactor was assessed prior to the infection with C. difficile.

[0161] In the experiment described in Example 1 samples were taken after 24 of incubation with the HMO mixtures just before the inoculation with C. difficile and the effect of the HMO mixtures in table 1 were also assessed with respect to their ability to increase the abundance of microbial taxa that are diminished with aging.

[0162] In particular, increasing Bifidobacteria species, such as Bifidobacterium longum, is relevant in terms of decreasing pathogenic bacteria such as C. difficile. An increased abundance of Bifidobacteria species will make it more difficult for the pathogen to proliferate since it makes the environment more competitive, and thereby prevent or reduce the C. difficile infection.

[0163] The beneficial microbial species were quantified as described in the materials and methods section. The results are presented in table 4.

[0164] Table 4 - Effect of the HMO mixtures on selected microbial species

[0165] The three samples with the highest amount of the indicated microbial species are marked with an asterisk (*) ranking from *** to *, where *** is the highest.

[0166] From the results in Table 4 it can be seen that the HMO mixture containing LNFP-I (M3) results in a more than 1 .4 fold increase in Bifidobacterium longum compared to the significantly more complex mixture of M5 and M4 and compared to the control the increase is 7.5 fold.

[0167] Bifidobacterium adolescentis was increased almost to the M3, M4 and M5 samples which was more than 6 fold compared to the control species

[0168] The HMO mixture containing LNFP-I (M3) was the second best mixture in increasing Faecalibacterium prausnitzii with the 3’SL and 6’SL (M2) mixture being better.

[0169] It can therefore be concluded that the mixture containing LNFP-I has potential to increase microbial taxa which are diminished with aging in a subject.

Claims

CLAIMS1 . A synthetic mixture of HMOs comprising the target HMOs 2’-fucosyllactose (2’FL), lacto-N- fucopentaose I (LNFP-I) and 3’-sialyllactose (3’SL) in an amount of at least 85 wt% of the total amount of HMOs in the mixture and wherein the amount of fucosylated HMOs constitute at least 35 wt% of the synthetic mixture.

2. The synthetic mixture of HMOs according to claim 1 , wherein the composition comprises at least 30 wt% LNFP-I.

3. The synthetic mixture of HMOs according to claim 1 or 2, wherein the target HMOs consists of: a. 5 to 35 wt % of 2’FL, preferably 10 to 30 wt% of 2’FL, and b. 30 to 75 wt % of LNFP-I, preferably 35 to 70 wt% of LNFP-I, and c. 10 to 30 wt % of 3’SL, preferably 15 to 25 wt% of 3’SL.

4. The synthetic mixture of HMOs according to any one of claims 1 to 3, wherein other HMOs in the mixture do not individually exceed 5 wt% of the total amount of HMOs present in the mixture.

5. The synthetic mixture according to any one of claim 1 to 4, wherein the 2’FL, LNFP-I and 3’SL are produced by microbial fermentation.

6. The synthetic mixture according to claim 5, wherein the 2’FL and LNFP-I is produced in the same fermentation.

7. A nutritional composition comprising the synthetic mixture according to any one of claims 1 to 6.

8. The nutritional composition according to claim 7, wherein the composition further comprises one or more of the following ingredients docosahexaenoic acid (DHA), arachidonic acid (ARA), Eicosapentaenoic acid (EPA), plant based protein such as algae protein, pea protein or rapeseed protein, riboflavin (vitamin B2), nicotinic acid (vitamin B3), resveratrol, coenzyme Q10 and / or a probiotic.

9. The nutritional composition according to claim 7 or 8, wherein the nutritional composition is selected from the group consisting of infant formula, baby food, dietary supplements and medical nutrition.

10. A synthetic mixture of HMOs or a nutritional composition comprising at least 30 wt% of LNFP-I for the use in preventing or reducing exotoxin toxin A (TcdA) and exotoxin toxin B (TcdB) in a subject.11 . The synthetic mixture of HMOs or the nutritional composition for the use according to claim 10, wherein the mixture or the composition further comprises 2’FL and 3’SL.

12. The synthetic mixture of HMOs or the nutritional composition for the use according to claim 10 or 11 , wherein the HMO mixture is according to any one of claim 3 to 6 or the nutritional composition is according to any one of claims 7 to 9.

13. The synthetic mixture or the nutritional composition for the use according to any one of claims 10 to 12, wherein the subject has C. d / ffic / 7e-associated diarrhea and / or a C. difficile infection.

14. The synthetic mixture according to any one of claims 1 to 6 or the nutritional composition according to any one of claims 7 to 9 for use in increasing the abundance of microbial taxa that are diminished with aging in a subject.

15. The synthetic mixture or the nutritional composition for the use according to claim 14 wherein the microbial taxa are selected from one or more of the following genus Bifidobacteria, Roseburia, Blautia, Dorea, Coprococcus, Clostridium clusters IV and Clostridium clusters XlVa and Faecalibacterium.

16. The synthetic mixture or the nutritional composition for the use according to claim 14 or 15, wherein the Bifidobacteria is of the species Bifidobacterium longum and / or Bifidobacterium adolescentis and the Faecalibacterium is Faecalibacterium prausnitzii.

17. The synthetic mixture or the nutritional composition for the use according to any one of claims 10 to 16, wherein the subject is selected from human, domestic animals, pet animals, livestock and working animals.

18. The synthetic mixture or the nutritional composition for the use according to any one of claims 10 to 17, wherein subject is an adult or an elderly human.

Images